DOT1L activity limits transcription elongation velocity and favors RNAPII pausing to facilitate mutagenesis by AID

Domain: biology
Published: 2026-01-12
Editorial outcome: major_revision

Abstract
Activation-induced deaminase (AID) mutates immunoglobulin genes to initiate antibody diversification by class switch recombination and somatic hypermutation but can also target non-immunoglobulin loci with oncogenic consequences. The mechanisms determining gene susceptibility to AID remain unclear. Here, we show that the H3K79 histone methyltransferase DOT1L is proximal to nuclear AID and promotes both class switch recombination and off-target AID activity, including Igh-cMyc translocations in mouse B cells. AID-mutated genes display high DOT1L activity. In the absence of DOT1L, nascent transcription largely increases despite reduced RNA Polymerase II (RNAPII) occupancy. Integrative genomic analyses reveal that DOT1L locally restricts transcription elongation velocity proportionally to H3K79me2/3 levels and extends RNAPII pausing. These transcriptional conditions enhance AID occupancy and thereby its activity. Our findings provide a harmonizing explanation for the bidirectional gene expression changes observed in DOT1L-deficient cells, and link attenuated transcriptional elongation velocity and prolonged RNAPII pausing to productive AID engagement at target loci.

Introduction
Upon activation, B cells modify the immunoglobulin (Ig) genes to enable the production of high-affinity antibodies with distinct effector functions. The variable exons (IgV) encoding the antigen-binding domain accumulate point mutations through the process of somatic hypermutation (SHM), thereby modulating antibody affinity1. The antibody heavy chain IgH locus additionally undergoes class switch recombination (CSR) to replace the constant region that defines the isotype, thus diversifying its effector function2.

SHM and CSR are initiated by Activation-Induced Deaminase (AID), which deaminates cytosine to uracil in single-stranded DNA exposed during transcription1. SHM results from uracil processing at the IgV1. Transcription from the main IgH promoter upstream of IgV enables SHM and produces transcripts encoding IgM by default. The IgH locus contains a tandem array of independent germline transcription units (GLTUs), each comprising a cytokine-inducible promoter, a repetitive intronic switch (S)-region, and the constant region exons of each isotype2. GLTUs produce sterile transcripts that enable AID activity. Uracil processing leads to DNA double-strand breaks (DSBs) simultaneously at two S-regions, triggering CSR1. While Sµ, the first S-region downstream from the IgV is constitutively transcribed, transcription of the other S-regions is controlled by cytokine signaling, enabling the B cell microenvironment to influence isotype switching3. GLTU transcription interplays with cohesin-mediated chromatin loop extrusion to bring S-regions into proximity, facilitating recombination4. We hypothesized that proteins enabling AID can also introduce mutations and DSBs in a few hundred genes in normal B cells5–11. AID-induced DSBs can lead to oncogenic chromosomal translocations involving the Ig, such as IgH-cMyc fusions12. Notably, AID occupancy is not sufficient to predict mutagenesis, indicating that additional regulatory steps determine its activity6,13,14. Genes susceptible to AID activity share transcriptional features with the Ig, including super-enhancer regulation, antisense transcription, and high occupancy of RNA polymerase II (RNAPII) and its co-factor SPT57–9,15,16. We have proposed that AID activity requires a licensing step linked to the transition of RNAPII into productive elongation13, although the underlying mechanism remains unresolved.

Histone modifications may facilitate AID activity. However, the histone marks correlating with AID activity, such as H3K4me3, H3K27ac, H3K36me3, and H3K79me2, are associated with active transcription9,17–19. Dissecting whether these modifications actively promote AID activity or reflect the transcriptional state of targeted genes is not trivial.

DOT1L is the only methyltransferase producing H3K79me1, me2, and me3 via a distributive mechanism, while demethylation is thought to occur largely by nucleosome dilution20–22. B cell-specific ablation of Dot1l in mice has pleiotropic effects, hampering B cell development, germinal center formation, and humoral responses, with reduced switched antibodies but also IgM23,24. Dot1l−/− mouse B cells stimulated ex vivo have reduced IgG1 switching, supporting a B cell-intrinsic role of DOT1L in CSR, albeit the effect depended on the stimulus, and the mechanism was not explored24. DOT1L ablation or inhibition in a human B cell lymphoma line reduced SHM25. Thus, while DOT1L has been implicated in CSR and SHM24,25, and AID off-target genes have H3K79me2 among other epigenetic marks typical of high transcription9,18, whether DOT1L contributes to off-target mutations, and the underlying mechanism remain unknown.

H3K79me2/3 are associated with actively transcribed genes26–28. However, DOT1L deficiency produces relatively few gene expression changes in stem cells, and results in at least as many upregulated as downregulated genes in somatic cells22,29–31, including B cells24. Although genes with higher elongation rates are associated with higher H3K79me2 levels27,28, some evidence suggests that DOT1L may limit transcription32,33. Moreover, DOT1L activity has been proposed to regulate transcription, either positively or negatively, at either initiation34, promoter-proximal pause escape32,33, and, indirectly, elongation32,33. Moreover, DOT1L might promote transcription by methylation-independent functions in defined contexts35–37. Consequently, interpreting the role of DOT1L in AID function remains challenging for lack of consensus on how DOT1L modulates transcription at the molecular level.

Here, we identify DOT1L and the super elongation complex (SEC) as nuclear AID-proximal factors. We find that the DOT1L-MLLT10 complex promotes CSR independently of SEC components. DOT1L catalytic activity is essential for this function, implicating H3K79 methylation. DOT1L also facilitates AID off-target activity with high local DOT1L activity, marking AID-targeted genes. Mechanistically, DOT1L deficiency causes transcriptional changes indicative of increased RNAPII elongation velocity over H3K79me2/3 regions and shorter pausing. These changes coincide with reduced AID occupancy and activity. Furthermore, because increased transcription elongation velocity increases or decreases transcriptional output depending on the gene context, it could explain expression changes caused by DOT1L deficiency. Our findings reveal transcription elongation effects by DOT1L and a link between RNAPII elongation dynamics and AID activity.

Results
DOT1L and the SEC are proximal to nuclear AID and promote CSR genome-wide

AID activity would be enriched in its vicinity in the nucleus. To identify such factors, we performed proximity-dependent biotinylation (BioID) using AID fused to the biotin ligase BirA* at either terminus (AID-BirA, BirA-AID) in Flp-In 293 TREx cells38. This approach yielded 382 preys significantly enriched over BirA-GFP and BirA controls (Supplementary Data 1).

To refine this list, we first excluded preys also identified in BioID experiments using APOBEC2, a structurally similar but functionally distinct AID paralog39. Second, we exploited the inability of BirA-AID to access the nucleus to exclude preferentially cytoplasmic interactions. AID shuttles between nucleus and cytoplasm, but large N-terminal fusions block its nuclear import40. Accordingly, both AID-BirA and BirA-AID were mainly cytoplasmic under steady state, but only AID-BirA accumulated into the nucleus upon treatment with drugs that prevent AID cytoplasmic retention and nuclear export (Supplementary Fig. 1a)40. These filters yielded 124 preys preferentially labeled by AID-BirA (Supplementary Data 1), including known AID partners (e.g., Exosome-associated NEXT complex41). We prioritized transcription-associated factors for further analysis, in particular the DOT1L complex (Fig. 1a, b). We also identified histone readers MLLT6 and MLLT10, which can associate with DOT1L29,42,43, and components of the super elongation complex (SEC), which regulates promoter-proximal pause release (Fig. 1a–c)44. The SEC consists of one scaffold protein (AFF1 or AFF4), one histone reader (MLLT1 or MLLT3), and transcription elongation factors, notably P-TEFb) (Fig. 1c)44. AFF3 forms a distinct SEC-like complex, which also recruits P-TEFb44 and supports CSR to select isotypes45. DOT1L can interact with MLLT1 or MLLT3 independently of the SEC or MLLT6/10 (Fig. 1c), suggesting multiple functionally distinct DOT1L complexes29,42,46–49.

To test the function of these complexes in CSR, we knocked down each factor in the CH12F3 mouse B cell line, which switches to IgA upon stimulation with anti-CD40, IL-4, and TGFβ-1 (CIT)50. This approach showed that DOT1L, MLLT10, MLLT1, AFF1, and AFF4 were each required for optimal CSR, without affecting cell division or AID mRNA levels, except for MLLT10 knockdown, which reduced AID by ~25% (Fig. 1d and Supplementary Fig. 1b, c). Apoptosis increased upon CIT stimulation after depletion of DOT1L, MLLT1, MLLT6, MLLT10, or AFF4 (Supplementary Fig. 1d), suggesting a protective role after acute stimuli. However, CSR was quantified in viable (DAPI-) cells and apoptosis did not correlate with CSR defects. Sµ or Sα GLT levels did not significantly change after depleting DOT1L or the SEC components contributing the most to CSR (Fig. 1e). We measured unspliced GLTs, which have <75 min half-lives and would reflect large transcriptional changes (Supplementary Fig. 1e).

We conclude that DOT1L, MLLT10, MLLT1, AFF1, AFF3, and AFF4 are proximal to nuclear AID and have non-redundant contributions to CSR.

DOT1L functions independently of the SEC in CSR

Because DOT1L and the SEC can function cooperatively, and MLLT1/3 can be part of either complex49, we performed epistasis analysis to determine whether DOT1L acts with or independently of the SEC in CSR. We inactivated Dot1l in two parental WT CH12F3 populations to obtain two independent clones with identical homozygous in-frame deletions in the DOT1L methyltransferase domain (Dot1lΔ/Δ-1 and Dot1lΔ/Δ-2), as well as a Dot1l−/− clone with frameshifting deletions that truncate the protein at amino acid ~100 (Supplementary Fig. 2a). Although no antibody detected mouse DOT1L, the protein variant potentially produced by Dot1lΔ/Δ clones had no effect on CSR when overexpressed in WT CH12F3 cells, excluding dominant-negative activity

Methods

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Subramani et al connects transcription regulation by DOT1L to the initiation of class switch recombination (CSR) of antibody genes and “off-target” effects of Activation-induced deaminase (AID). The authors identify DOT1L as a protein “proximal” to AID in Flp-In 293 TREx cells and then use the CH12F3 mouse cell line derived from B cell lymphoma for most of the subsequent analyses. Several DOT1L mutant derivatives of CH12F3 have been generated and experiments comparing WT (lymphoma) and DOT1L mutant conditions have been performed. shRNA knockdown of DOT1L was used as an additional alternative approach. The authors report diminished CSR frequency, reduced off-target AID-dependent mutations, and elevated Pol II pause-release upon DOT1L loss. Since Pol II pausing favors AID activity the major conclusion of the paper is that DOT1L affects AID through its regulation of transcription elongation.\n\nThe strength of this work is in using several DOT1L mutant alleles and revealing the effect of the mutations on CSR and off-target AID activity leading to gene fusions. Also, the effects of DOT1L on cohesin distribution are interesting. In this regard, does the downregulation of cohesin affect CSR?\n\nThe weaknesses:\n1. The focus on DOT1L singled out among the numerous AID prays is not well justified. DOT1L complexes specific for cells used in functional analyses are not investigated.\n2. Lack of direct connection between DOT1L and loci subject to CSR. Since DOT1L has a global effect on transcription, the indirect role of DOT1L in CSR cannot be excluded.\n3. Claiming the discovery of Pol II pause-release regulation by DOT1L, despite the pioneering C. elegans study in 2013. Notably, a study using an ESCs reprogramming system showed a functional role for DOT1L-regulated Pol II pause-release of housekeeping genes (Wille et al. 2022, BioRxiv), like the C. elegans work.\n4. Results, such as in Figure 3 “Dot1l regulates GLTs expression fidelity” and Figure 5 “Global alteration in transcription output caused by Dot1l loss” touch upon complex subjects that are not well integrated into the main storyline. Figure 3 results are related to the regulation of gene expression plasticity by several factors, including DOT1L, which was comprehensively described in the Xiao et al Open Biology 2019 publication. The Figure 5 subject is complex and not limited to the role of DOT1L in Pol II pause release. There is literature connecting DOT1L to enhancer regulation through effects on chromatin compaction, as well as studies connecting DOT1L to antisense transcription regulation. This was not properly referenced in the paper.\nI do not find conclusions that gene length affects the mode of transcription regulation by DOT1L (UP or DOWN) particularly convincing, this part should be removed from the model in Figure 8 (or from the manuscript altogether). Data analyses based on gene function might be more informative, especially if related to the main story of the manuscript.\nThe authors relate their Pol II pause-release findings to literature about known pausing factors and their inhibitors: Spt5, RN7SK, NELF, PAF1, etc. This is not justified by the results. Unless they do experiments and implicate these factors in CSR, they must instead focus on the similarity of their DOT1L findings to the C. elegans system that lacks NELF and the Wille et al. 2022, BioRxiv publication.\n5. The introduction gives no background about DOT1L and the current state of our understanding of its effect(s) on transcription and chromatin organization.\n\nSpecific issues:\nFigure 4A, C: How does total RNA Pol II distribution look in Dot1l +/- conditions? It is expected to change.\nFigure 4B, bottom: Why ChIP-qPCR at the P region is not shown?\nFigure 4C: Analyses using randomized groups of genes with the same # should be performed for control.\nFigure 4F: Show expression changes corresponding to each group of genes.\nFigure 6 A, C: The difference between H3K79me3 distribution in splenic B cells and CH12F3 cells is concerning and must be addressed. How do these profiles look in unstimulated cells?\nFigure S2 B: Why does CIT lead to H3K79me reduction?\nTriplicate replicates should be shown for all RT-qPCR and ChIP-qPCR data.", "reviewer_2": "In this paper, the authors investigate the mechanism of DOT1L promoting AID-induced class switch recombination (CSR) as well as “off-target” mutations. They applied BioID to identify nuclear AID-proximal proteins that potentially contribute to AID-induced mutations. From the results, they identified DOT1L and its associated proteins, as well as SEC as being specifically enriched in the AID interacting complex. To evaluate the function of DOT1L they perform knockdown and knockout experiments and find that CSR is inconsistently affected at germ line transcripts depending on locus and method of knockout. Since the local effects at IgH do not show a trend based on characteristics of accumulated chromatin marks, they then perform RNAPII ChIP-seq from which they conclude a correlation between promoter pause release and H3K79me3 enrichment. Finally they perform experiments on cohesion accumulation that suggest a link It has been reported that DOT1L is essential for B cell differentiation, but the authors provide insights into the potential mechanisms that may be relevant.\n\nMajor points:\n1. The first major claim of the manuscript is that DOT1L has a role in CSR ( Fig 1-3 )- which has been reported before (EMBO Rep. from the Leeuwen lab). In that prior paper it was shown that DOT1L promotes somatic hypermutation and lowered RNA Pol II occupancy at immunoglobulin variable region.\n1.1 The results in this manuscript are inconsistent with the prior published results (Fig 1E showed a different result at the same locations (Su)) and at different regions where shDOT1L can both increase or decrease RNAPII occupancy (Fig 1E).\n1.2 The effects of the DOT1L binding partner MLLT10 on class switching (Fig 1K) are undermined by the lack of substantial knockdown at 48 hours (Fig S1C). Further, In Fig 1K, shAID further reduced CSR I the background of DOT1L deficiency, indicating DOT1L is not necessary for AID function.\n1.3 The CH12F3 delta/delta lines and the CH12F3 delta/- lines of DOT1L deletion are different from each other – The delta/delta lines retain frame (Fig S2A)- which means there is a DOT1l truncation mutant protein that could act as a dominant negative. DOT1L KO lines need a DOT1L western blot to confirm the protein is deleted.\nThis is an important consideration because the results in Fig 2A are misinterpreted by pooling the samples from both kinds of deletions on CSR ( triangles and squares in the bar graphs).\n1.4 The effects of DOT1L deletion on GLTs are inconsistent and modest at different loci, between different kinds of DOT1L deletion and in resting and activated conditions ( for example In Fig 3C, expression of lghg2c is increased in stimulated condition but decreased in resting state upon DOT1L KO).\nFurther in Fig2A, 2H and 3A, GLT changes do not correlate with reduced CSR or transcription elongation defects. Then DOT1L inhibition upregulates unscheduled GLTs cannot be the mechanism of DOT1L regulates CSR.\nWhile the authors argue for complex regulation by DOT1L for CSR, the data could also support a minor role for DOT1L that is far downstream of other players. For example in the mass spectrometry in addition to DOT1L, the H3K4 methyltransferase is also specifically enriched. This result would suggest that that the proteins interacting with AID are proximal to the transcription start site and not specific to AID. AID-ChIP at GLT loci in DOT1L deletion lines could be a method to support DOT1L deletion affects CSR through interaction with AID.\n1.5 Statistical tests are inconsistently applied across Figs 1-3 ( for example between the two kinds of DOT1L deleted lines in Fig 1H), in several cases there are only 2 replicates- it is not indicated whether the replicates are technical or biological , and standard error of mean is used instead of std deviation from mean. There are missing error bars and significance testing ( Fig S3C is missing error bars in some timepoints and in certain axis. S3D needs significance test).\n2. The second major claim of the manuscript is that DOT1L acts by limiting pause release and not transcription elongation, and that leads to transcriptional upregulation.\n2.1 First it is argued that the total RNAPII CUT&RUN signal is lower in DOT1L KO cells because of the epitope being masked by high S2P-RNAPII, but it is also argued there are no global changes in cellular levels of RNAPII or S2P-PolII which is impossible to interpret because Fig. S4B does not have a loading control. Total Pol II CHIP PCR for other GLT locations in 4B in WT/KO lines would also help observations on pause-release differences related to gene expression changes from Fig 3 and H3K79me levels.\n2.2 At the locus specific level Fig 4B – the total RNAPII enrichment differences are modest, the S2P-RNAPII enrichment at the promoter is not presented- this leads to the question of whether there is a nonspecific increase of S2P-RNAPII everywhere . The intergenic region ( bottom panel of Fig 4B) does not serve as a control for this question because the enrichment at that location is a magnitude lower than that at the Sgamma3 location.\n2.3 Most disconcertingly it is argued that the RNAP C-terminal antibody is unreliable- yet it is the one used for calculating the pausing index (Fig 4E and 4F).\n2.4 The ChIP-seq of H3K79me3 could also include H3K79me2 location as in Fig4A, 4C, 6C, 6B, the signal of H3K79me2 and H3K79me3 show almost the same pattern. Further, between Fig 6B and S5A, H3K79me2 ChIP displays very different patterns between mouse splenic B cells and CH12F3 cell line.\nIn the clustering of H3K79me3 levels (Fig 4C) most genes with estimated traveling ratios are in the first 2 quintiles. It is unclear why H3K79me3 quantification at TSS site is used for correlation to the pausing index since this location is not representative of general H3K79me3 distributions in the gene body. The conclusion that pause-release is related to H3K79me3 levels, would be more convincing if other features such as H3K79me1/2 and gene expression level are not corelated with the pausing index.\n2.5 In Fig 5G & S4H H3K4me3 has 2 populations- and every pairwise comparison is statistically significant – if that is the case then the distribution of H3K4me3 cannot causal for transcriptional changes.", "reviewer_3": "The manuscript of Subramani and co-workers entitled \"DOT1L limits RNAPII promoter-proximal pause release thus enabling Activation Induced Deaminase activity genome-wide\" deals with the molecular underpinnings of class switch recombination and somatic hypermutation of antibody genes, which involves the action of Activation induced deaminase (AID). But the data extend towards genome-wide analysis with the intention to generalize some of the findings. The authors discover that AID interacts with DOT1L and they embarked to characterize how AID and DOT1L mediate CSR and SHM, and whether DOT1L is involved in AID off-target deamination.\nIn all this is very impressive and experimental rich work that proposes novel mechanisms of action for the histone methyltransferase DOT1L. My main concern about the data presentation in its present form is that it should worked out much clearer - especially for the broader scientific community - how the different proposed mechanisms are linked to either genome-wide and/or IgH-specific transcriptional control. In its present form the proposed mechanisms are not always presented in a connected form but rather independent from each other.\nMajor points:\n1) The authors show that it is DOT1L and not, indirectly, the super elongation complex that accounts for CSR, although the SEC was also part of the interactome.\nAs later in the manuscript the authors show increased S2P-RNAPolII in the gene bodies upon DOT1L deletion, a stronger activity of the SEC might be likely. It would be good, if this aspect could be validated as well. Along the same line, showing a concomitant decrease in S5P-RNAPolII as the pausing form would also be direct proof, that the switch from pausing to elongating is affected by the action of DOT1L (or its interaction partners).\nIn the same setting some experiments and their interpretation might need clarification:\n- is the SEC increased (expression levels? occupancy (esp. in regard to the S2P RNAPII accumulation shown in Fig. 4C)? interaction with AID?) upon DOT1L deletion? And why would this be? Is there an increase in Dot1L or the relevant SEC components as well after CIT? Is there increased interaction between DOT1L and AID in this condition?\n- Dot1L is as Mllt1 not depleted but rather increased in Fig. 1E for Su. Please comment.\n- In regard to the AID interactions with SEC or DOT1L: Do the components sit there and wait for AID to come in? Or is AID recruiting? Or is AID being recruited? What is the dynamics in the system?\n2) In regard to placing the action of DOT1L up- or downstream of break formation: How do the authors explain that increased NHEJ occurs upon deletion of the catalytic domain of DOT1L in the delta/delta-1 clones, but unchanged NHEJ upon use to of the inhibitor?\nThis observation is not leading to the (sole) conclusion given, that \"Dot1l acts at or upstream of the break formation step in CSR\". It rather suggests that the scaffolding function of DOT1L would be involved - and it would be interesting to learn what this function would be.\n3) The authors use H3K79me2 or me3 as readout for DOT1L activity, however it is not entirely clear that the mark itself is implicated in the process. Rather, the scaffolding function of DOT1L or activity towards other protein targets might also explain some of the observations. Thus, it is necessary to show that DOT1L is indeed present at the IgH loci itself (in the context of Fig. 2), not only the marks, which might be secondary or not necessary for the function. Also, it needs to be shown that the catalytic deletion protein localizes similarly or not - or to use point mutations for inactivation to dissect clearly whether it is the mark or the scaffold.\nThe same is true for the statement in regard to Fig. 3B and S3F, S5A - is Dot1l present at these loci in a defined, sequence specific manner (not at Sy1, Se but on Syb, Sy3, Sy2c and Sa) - or is this finding of different methylation levels a coincidence of a locus-wide general activity and deposition of the mark?\n4) The authors observed that the enzymatic activity would be important for CSR; and it seems that also the pausing of RNAPolII correlates to this observation. Thus, is it sufficient to inhibit DOt1L to observe similar effects as shown in Fig. 4? However, the statement \"we conclude that the H3K79 methylation limits promoter-proximal pause escape genome-wide including the IgH GLTUs\" seems correlative. For being substantiated, it would need further experimentation to support this claim: e.g. gradual loss of H3k79me3 towards me2, me1 which should then release RNA Poll pausing. Or locus-specific non-methylation using dCas9 constructs. And if this is a claim to be generalized in terms of DOT1L function - i.e. independent on the Ig-locus CSR - this correlation should be observable in other cell systems as well. Which has to be demonstrated.\nIn addition the Chip profiles focusing at the IgH GLTUs might be good to provide in 4C to support the claim \"including the IgH GLTUs\".\n5) The authors conclude from their study that \"DOT1L activity enables genome-wide AID off-target activity\" - however, for me it is not entirely clear which mechanism is responsible despite that multiple were proposed, including\n*paused Pol II release upon loss of Dot1l\n*H3K4me1/me3 levels\n*CTCF/cohesin loop extrusion\nAs the off-target activity of AID was suggested to be the main focus of the study, I would have expected to have a clearer picture on the mechanism in this regard.\n6) In the discussion the authors mention other inhibitors/deletions that alter RNAPII pausing. If indeed the pausing is the mechanism behind CSR, it would be interesting to know if these inactivations/deletions (of RN7SK, Spt5) also affect the CSR events observed here? Showing such correlation would be a strong claim towards the pausing as major mechanism.\nOn the other hand, the authors claim that \"H3K79 methylation might maintain the balance of these kinase and phosphatase activities in situ\". How should this happen - and would this not need to be studied in order to provide the necessary mechanistic insights?\nMinor points:\n1) Figures of quantifications vary in showing single data points or not - please add single data points in coherent way to all figures. Please also indicate the individual n numbers used for each experiment within the legends.\nIt seems that some experiments are displayed with a n=2 (i.e. S2E, F,H). Even if this data in included \"only\" in the supplemental data - is it sufficient to interpret the experiment without proper statistical testing?\nAlong this line comes Fig. 2A, 3D, which is problematic, as first of all two experiments with different cell lines are combined in one bar, and variable numbers of experiments are shown (even within the combination of different cell lines). It seems that for CD4 only two data points are shown (this is why the legend should contain all n numbers, as this can also be a graphical problem). Please separate the experiments and provide valid statistical evaluation.\n2) Fig. S2B: what is the reason for showing me2 for two loci and me3 for three? A coherent representation would be much more convincing. Along the line is the representation of the GLT exons: What is Sy1 and why is this not shown for me3? The Sy1 fragment is not mentioned in the text or legend, and I think the schemes are also not depicting the fragment explicitly (also relevant for Fig.3A, where other Sy's show up).\n3) Fig. 1G: show H3K79me3 as well\n4) In the context of Fig. 4: That Pinometostat is not influencing AID expression is not necessarily corroborated by the blots presented - a slight increase in protein levels might be present. The authors might want to quantify this effect (accompanied by a power analysis to determine the numbers of samples they would need to analyze - as the effect seems rather small) to support this claim, which is central to discriminate between the methylation activity towards H3 or acting as scaffold.\n5) The authors conclude in line 236/237 that \"determines a transcriptional environment that enables AID activity,...\". The authors might to make this statement more precise and describe how this environment would look like and how it impacts on AID activity. The link between transcription (other than presence of RNAPII) and AID activity is not easy to appreciate - even if it might be within the data.\n6) The authors cite that \"Loss of H3K27me3 has been proposed to explain gene upregulation in Dot1l-deficient cells, but this mark was absent from the Igh...\". Another paper showing EZH2/DOT1L crosstalk was recently published (Appiah et al. 2023, EmboRep), making this point important to substantiate in this system. If H3K27me3 is not present at the IgH locus, is H3K27ac maybe enriched? How is the epigenetic landscape changing, maybe also in regard to ATAC seq, in Dot1l deficient cells?\n7) Fig. 4B: The P side in SerP2 chip could serve as a negative control. Statistics is missing in the figure as well as representation of individual data points.\n8) Line 254: In the context of Fig. 4 the authors conclude \"Collectively, these results suggested increased promoter-proximal pause release in the Dot1l-deficient cells for those GLTUs that were open and preloaded with RNAPII.\" The openness of the locus was not experimentally addressed.\n9) Line 257: \"Since we identified DOT1L proximal to AID by BioID, we expected that any role of DOT1L found at the IgH might be conserved genome-wide\"\nI do not understand this rationale. Please make it clearer. For me it would still be possible that the AID/switching is locus specific (admittantly for me this is more plausible then a genome wide mechanism).\n10) For the definition of the quantiles according to H3K79me3: what is the rationale to use H3K79me3 and not H3K79me2?\nPlease make clear to the reader.\n11) The authors observe an increase of RNAPII-S2P in the gene bodies - the scaling in Fig. 4C is probably distance from TSS and goes up to 120kb - an accumulation in the gene body is hardly visible in this present form, as it is not indicated in the figure - isn't a representation that spans TSS to TES better suited to show the distributions? At least as an additional representation, as I understand that the drop at 40kb becomes relevant later.\n12) The experiments concerning distribution of RNAPII and its phosphorylated form by means of the two antibodies used, are a little confusing and difficult to perceive - this might need better explanation. Within Fig. S4C the enrichment is given as % of input and this is (much) lower for N20 then for 8WG16. Please explain this discrepancy.\nProbably the authors would like to point towards the delta between the two conditions - however this can be variable between experiments as for N20 a N=2 is displayed, and for 8WG16 the N number is not given.\nThus - in the present representation of the results, it might be a real effect that is observed here - and this might be in favor of a locus specific observation opposing the claim of a general genome-wide phenomenon?\n13) Fig. 4C indicates an increased/accumulated signal of S2P RNA PII in the 3' end of the genes. How is this explained?\nWhat is trapping S2P RNAPII at this location - is there a problem in transcriptional termination?\n14) Line 315: why does the conclusion not contain the K4me1/me3 ratio that suggests a similar high correlation than the paused PolII?\nThe statement on the altered ratio of the K4 methylation somehow comes out of the blue - there is no ChIP seq profiles or ChIP qPCRs that address this point of H3K4me1/me3. But this seems highly relevant as mechanism and probably contributing to the RNAPII S2P escape from pausing.\n15) Please clarify the molecular definition of the stripe in regard to the marks/factors shown in Fig. 6A; and indicate selection of super enhancers and or criteria in the selection.\nSpelling/phrasing and other mistakes:\nline 69: \"However, determining the underlying mechanisms or these histone marks are necessary for AID activity\"\nline 106: Please add a citation - and maybe make stronger the hypothesis that the complexes have been proposed as being mutually exclusive (\"We also identified several histone readers that can form a complex with DOT1L but are also components of the super elongation complex (SEC)\")\nline 132: \"and are participate in CSR\".\nline 198: Papers 47, 48 are not describing demethylation - the citation seems not appropriate. In addition - the cited papers explicitly excluded the study of human Dot1l which seems not to follow the behavior of lower eucaryotes.\nFig. S4F: DGE or DEG?", "reviewer_4": "Activation Induced Deaminase (AID) is an essential factor that drives somatic hypermutation and class switch recombination in B Cells leading to antibody diversification. It is well accepted that AID mutates single strand DNA (ssDNA) sequences to generate point mutations during SHM (V gene sequence mutations) and DNA double strand breaks (DSBs) to cause CSR. In the process of catalyzing CSR and SHM, AID also incorporates SHM all across the B Cell genome (although at a much lower frequency), these mutations are related with occurrence of germinal center derived B cell lymphomas. A puzzling and important question is how does AID identify various DNA target sequences, at V genes and its target DNA sequence across the B cell genome. Many past studies have related AID activity with transcription states both at sense and antisense direction, secondary DNA structures like R-loops and G4s (that form during transcription), epigenetics and genome architecture formation, etc. In this study, the Subramani et al. proposes that the H3K79me1/2/3 inducing enzymes DOT1L induces AID recruitment to the B cell genome. The authors also propose that DOT1L functions with a similar mechanism to cause CSR, DOT1L deficient CH12F3 lines are also deficient in CSR. The authors show that DOT1L induces RNA polII promoter proximal pausing to accumulate RNA polII at AID target DNA sequences to induce AID recruitment (AID binds to RNA polII robustly and thus this is the likely mechanism). In this regard, the authors show that in absence of DOT1L promoter proximal paused RNA polII levels decrease but S2 phosphorylated elongating RNA polII increases in the gene body. Thus, this study also makes the argument that S2 phosphorylated RNA polII is unlikely to recruit AID. Although some previous studies (cited in the manuscript) have investigated and shown DOT1L’s role in AID recruitment and CSR, neither of these two studies suggested mechanisms similar to that proposed in this manuscript. Please find below some suggestions that the authors could consider as they revise the manuscript for publication. Overall, I feel this manuscript advances the field of AID biology and also provide insights into transcription and promoter-proximal pausing mechanism.\n1. In figure 1, authors present a nice assay with AID-BirA approach for AID interacting proteins. Some highlights of AID interaction with the NEXT complex and MRN complex was not discussed in the text. NEXT subunits functionally interact with AID has been shown before and with regards to AID activity regulation (Lim et al, 2017). Similarly, the interaction with MRN is also relevant for discussion.\n2. The inability of AID to stimulate CSR is an important marker for functional aspect of its cofactors, in cells that are deleted/hypomorphic of cofactor activity. In Fig. 1A versus 1K, the authors show different efficiencies of CSR in DOT1L deleted cells (50% versus less that 30%). Some clarity will help since this assay does decide how important DOT1L is for AID activity (at least during CSR).\n3. This maybe a confusion on my part, but it is not clear to me why in Fig. 4A the H3K79me2/3 ChIP-seq data for DOTL1-/- cells were not shown. The parallel demonstration of the tracks will make it clear that the mutant cells are deprived of DOT1L activity and still undergo robust transcription (RNA polII levels) and elongation (S2PRNA polII levels).\n4. The most striking observation in this study is the effect of DOT1L inhibition on AID off-targeting and IgH-myc translocations (Fig. 7 and S6). The authors find with chemical inhibition of DOT1L (chemical inhibition is well characterized with all proper controls), the authors report decreased translocations (fig. 7E). Myc-translocations occur at promoters and also in gene body and the PVT1 locus downstream in humans. What translocations are the ones the authors are mapping, are these Myc promoter translocations, some details with sequencing data of the translocation site would be helpful. Fig. 7E could be done from two different mice (instead of B cells from one mouse). Pinemetostat treatment may decrease IgH-Myc translocations at promoters, but does the translocations move to a new site (some bands are seen in the gel).\n5. The authors discuss that H3K79me3 modifications occur 5 Kb away from the TSS (Fig. 7A), but SHM and promoter proximal pausing occurs in the first 1 kb and 100 bps, respectively. This could be clarified.\n6. The model in Fig. 8 and the associated discussion could be improved. Based on my understanding of this study, DOT1L restricts PolII residence time in a promoter proximal paused state, thereby recruiting AID and increasing SHM. Previous studies have supported the fact that bidirectionally transcribed regulatory DNA sequences (like in promoters and enhancers) accumulate AID activity and those studies support the conclusions of this manuscript (Pefanis et al, 2014; Meng et al, 2014; Qian et al, 2014). These DNA regulatory sequences have accumulated promoter proximal pausing and are most likely be regulated with DOT1L activity. Authors should consider them in the discussion. The authors could also described SHM (following DOT1L inhibition) at some of the off-target genes whose transcription was measured and shown to increase (Fig. S6). This study does highlight that increased transcript levels at AID target sites is not always proportional to mutagenic activity of the protein. These observations are consistent with previously published studies where it has been shown that increase in switch region transcripts or at AID off target genes does not always lead to increase AID mediated DNA mutagenesis. These published studies should be discussed and considered in the revision.", "reviewer_5": "This manuscript examines the role of the H3K79 methyltransferase Dot1L in transcription, class switch recombination (CSR), and AID mistargeting in the genome. The authors identify Dot1L and other factors including components of the super elongation complex as AID proximity factors in 293 cells. As the authors summarize, it was previously known that Dot1L contributes to efficient CSR and somatic hypermutation (SHM), but the mechanism of action was unknown. The authors also nicely summarize previous findings linking Dot1L to defects in transcription and point out the limitations in those findings. This study substantially extends previous work by showing that Dot1L regulates transcription by restraining Pol2 promoter proximal pause release (PPPR). The substantial data presented here and the similarities with findings from other factors that negatively regulate PPPR provide a strong argument that the catalytic activity of Dot1L (and likely H3K79me2/me3) help restrict PPPR. This and the other findings related to the effects of Dot1L on transcription are the main strength of the manuscript.\nThe authors also make the claim (last sentence of abstract) that their findings have established that regulated pause release is “a critical step for AID activity”. We do not agree. While the findings strengthen the link between Dot1L (and its catalytic activity) and CSR and AID targeting, they do not establish mechanism. Specifically, the findings establish a correlation between Dot1L-regulated Pol2 PPPR and CSR/AID targeting, not a causal link. One alternative explanation among others is that methylated H3K79 is the key contribution of Dot1L, not the regulation of PPPR. We feel that the mechanistic advance offered by this study regarding the role of Dot1L in CSR/AID targeting is significant but not definitive. This is the primary limitation of the study. We emphasize that we find their model attractive and plausible; it simply hasn’t been established. Is this required for publication in Nature Communications? That’s a judgement call, though overall we would support publication without a requirement for substantial additional experiments. At minimum, the manuscript needs to be rewritten to remove overly strong mechanistic claims and to address alternative hypotheses.\nIf the authors decide to try to strengthen the link between dysregulated PPPR and AID targeting, they might attempt to identify a factor(s) that reduces CSR/AID targeting and alters PPPR similarly to Dot1L but does not reduce levels of methylated H3K79—this would substantially strengthen the correlation and argue against a primary role for methylated H3K79. Their study identifies several candidates for study, e.g., SEC components.\nIn other regards, the study is rigorous, thorough, and well written (which we appreciated; a pleasure to read). The experiments are well designed and controlled, caveats are noted and addressed, and conclusions are carefully stated for the most part. The Discussion is well crafted to put the findings in the context of other work.\nWe were unable to identify many other weaknesses with this study. Minor points for consideration are:\n1. Line 76: it was unclear what the phrase “strongly correlating with normal H3K79 methylation levels” refers to. Please rewrite.\n2. Line 155: where are the data to support this statement?\n3. Line 191: “Since switching to IgA in CH12F3 cells required Dot1l activity”; this claim is overly strong, since it implies that CSR is completely eliminated in the absence of Dot1l activity. A similar inappropriate use of the word “required” occurs on line 26 (Abstract).\n4. Something wrong in the plot of H3K27Ac in S5A: big blocks of purple below the axis.\n5. Line 333 and this paragraph: it is hard to appreciate the changes in the vicinity of Vh and Emu given the scale of Fig. 6B; a blow-up of this region in a supplementary figure would be helpful.\n6. Grammatical errors, missing words, or typos were found on the following lines (thank you for numbering the lines): 69, 132, 190, 354, 399-400."}, "author_response": "Point by point response\nReviewer #1 (Remarks to the Author):\nThe manuscript by Subramani et al connects transcription regulation by DOT1L to the initiation of class\nswitch recombination (CSR) of antibody genes and “off-target” effects of Activation-induced deaminase\n(AID). The authors identify DOT1L as a protein “proximal” to AID in Flp-In 293 TREx cells and then use the\nCH12F3 mouse cell line derived from B cell lymphoma for most of the subsequent analyses. Several DOT1L\nmutant derivatives of CH12F3 have been generated and experiments comparing WT (lymphoma) and\nDOT1L mutant conditions have been performed. shRNA knockdown of DOT1L was used as an additional\nalternative approach. The authors report diminished CSR frequency, reduced off-target AID-dependent\nmutations, and elevated Pol II pause-release upon DOT1L loss. Since Pol II pausing favors AID activity the\nmajor conclusion of the paper is that DOT1L affects AID through its regulation of transcription elongation.\nThe strength of this work is in using several DOT1L mutant alleles and revealing the effect of the mutations\non CSR and off-target AID activity leading to gene fusions. Also, the effects of DOT1L on cohesin distribution\nare interesting.\nIn this regard, does the downregulation of cohesin affect CSR?\nThe role of chromatin loop extrusion by cohesin in facilitating CSR is well established (Thomas-\nClaudepierre et al, 2013; Zhang et al, 2019). Transcriptional activation of Igh germline transcript units\n(GLTUs) in response to cytokine signaling leads to increased cohesin occupancy across the Igh locus\n(Zhang et al, 2019; Costea et al, 2023).\nWe note that new data in the revised manuscript (TT-seq and additional ChIP-seq) indicate that DOT1L\nregulates transcriptional elongation velocity. Given the known influence of transcriptional activity on\ncohesin dynamics, the moderate change in cohesin distribution in DOT1L-deficient cells is most likely an\nindirect consequence of transcriptional changes at the H3K79me2/3-rich GLTUs. In turn, chromatin loop\nextrusion alterations can explain the modest activation of internal H3K79me2/3-poor unscheduled GLTUs,\nas originally proposed. We make this point succinctly in Fig. 4, S5.\nThe weaknesses:\n1. The focus on DOT1L singled out among the numerous AID prays is not well justified.\nWe appreciate the reviewer’s concern regarding the focus on DOT1L and have modified the text to better\nexplain the knowledge gaps on DOT1L and AID targeting that we are addressing.\nWhen we initially identified DOT1L as an AID-proximal protein, it had not yet been functionally implicated\nin antibody diversification. We chose to focus on DOT1L in part because prior studies had reported an\nenrichment of H3K79me2 at AID target loci (Wang et al, 2014; Álvarez-Prado et al, 2018). At the time,\nthis modification was interpreted as a mere marker of transcriptional elongation, and the potential\nmechanistic relevance of DOT1L in AID activity had not been explored.\nWhile our work was ongoing, other studies were published demonstrating a requirement for DOT1L in\nCSR and SHM. However, these reports neither tested whether DOT1L enabled AID off-target activity, nor\nprovided a mechanistic explanation for how DOT1L supports AID activity at the Ig loci. We sought to\nanswer these important outstanding questions.\nIn the revised manuscript, we modified the Introduction to eliminate the “discovery” aspect and better\njustify the rationale to study DOT1L based on the gaps in knowledge.\ncomponents which can interact with DOT1L. Once the epistasis analysis included in the manuscript\nshowed that SEC and DOT1L contribute independently to CSR, the focus remains on DOT1L. We are\ninvestigating the role and mechanism of the SEC, which is a story on its own.\nDOT1L complexes specific for cells used in functional analyses are not investigated.\nWhile we did not perform biochemical isolation and identification of DOT1L complexes in CH12F3 cells,\nour functional data does provide insight into their composition and relevance.\nSpecifically, our epistasis analysis indicates that DOT1L cooperates with MLLT10, but not with MLLT1 or\nAFF1, in promoting CSR in CH12 cells. This is further supported by our structure-function analyses, which\nare consistent with the same interpretation. These data provide indirect evidence regarding the functional\ncomplexes operating in this B cell line, i.e. DOT1L/MLLT10 on one side and the SEC acting independently\nof DOT1L. Thus, our data provide meaningful mechanistic insights about the DOT1L complex in CH12 cells\n2. Lack of direct connection between DOT1L and loci subject to CSR. Since DOT1L has a global effect on\ntranscription, the indirect role of DOT1L in CSR cannot be excluded.\nWe acknowledge this important point. The possibility of indirect effects is an inherent caveat when\nanalyzing transcription-associated chromatin modifiers and applies to prior studies linking DOT1L to CSR\nand SHM, which did not establish a direct mechanistic connection. The revised manuscript provides\nseveral lines of new evidence that strongly support a direct link between DOT1L activity and local AID\nactivity at the Igh locus for CSR, and at off-target genes.\ni. Correlation between DOT1L activity and AID activity: We show that AID target genes are distinctly\nenriched in H3K79me3 relative to equally transcribed non-target genes in both primary B cells and CH12\ncells (Fig. 3A–D). We also provide data showing that the level of AID activity at individual genomic loci in\nCH12 cells is approximately proportional to their H3K79me3 content (new Fig. 3D). As CDADC1 is a\ndistributive enzyme, higher order H3K79 methylation indicates locally higher DOT1L activity. These data\nsupport a locus-specific requirement for DOT1L activity that is more consistent with a direct than a merely\nglobal transcriptional effect.\nii. Functional occupancy of DOT1L at the Igh locus: We performed ChIP-qPCR experiments in CH12 cells\nreconstituted with either WT or catalytically inactive DOT1L. Both forms of DOT1L are recruited to the Igh\nlocus (new Fig. 2B), yet only WT DOT1L can complement the CSR defect (Fig. 2A), again consistent with\nlocal DOT1L catalytic activity supporting CSR, and ruling out potential scaffold functions.\niii. The CSR machinery remains intact in DOT1L-deficient cells: We systematically examined the\nexpression of genes encoding the CSR machinery in both primary B cells and CH12 cells lacking DOT1L and\nfound few differences in primary and no differences in CH12 cells (new Fig. S4E). AID protein levels were\nunaffected (Figs. 1F, 2D, 2G). Furthermore, we demonstrate that DOT1L-deficient CH12 cells can still\nundergo CSR when double-strand breaks are introduced by CRISPR targeting (Figs. S2G, S2I), indicating\nthat downstream components of the CSR pathway are functional. These results argue against a model in\nwhich DOT1L indirectly affects CSR via disruption of the general CSR machinery.\niv. Transcription dynamics explain loss of AID targeting: New data in the revised manuscript show that\nloss of DOT1L causes increased transcription elongation rate in a manner that is directly correlated with\nH3K79me2/3 levels (new Fig. 5). Moreover, AID target genes have not only high H3K79me2/3 (Fig. 3D),\n2\nbut the H3K79me2/3 peak overlapping the region where SHM accumulates (Fig. 3E), which is where\nvelocity changes the most in Dot1l-deficien cells (Fig. 5E-F).\nTogether, these data provide multiple converging lines of evidence that DOT1L directly facilitates AID\nactivity through a locus-specific, enzymatic effect on chromatin that modulates transcription dynamics.\nFaster elongation provides a mechanistic explanation for the reduced AID occupancy we find at the Ig and\noff targets upon DOT1L loss. These points are summarized in the revised Discussion.\n3. Claiming the discovery of Pol II pause-release regulation by DOT1L, despite the pioneering C. elegans\nstudy in 2013. Notably, a study using an ESCs reprogramming system showed a functional role for DOT1L-\nregulated Pol II pause-release of housekeeping genes (Wille et al. 2022, BioRxiv), like the C. elegans work.\nWe apologize if we appeared to claim more than was due about pause release. This was not our intention.\nWe have modified the manuscript to ensure fair recognition of previous work.\nHowever, additional data and analyses have clarified the effect of DOT1L on transcription and made us\nreconsider our initial model. Our new data indicates that the effect on pausing is secondary to an increase\nin transcription elongation rate over the H3K792/3-enriched intragenic regions. Our data can help\nreconciling papers that detected an effect of DOT1L loss on pause release (Cecere et al, 2013; Wille et al,\n2023), and others that did not (Wu et al, 2021; Cao et al, 2020), via traveling ratio calculations.\nWe realized that the genome wide S2P-RNAPII increase was an artifact of the spike-in normalization we\noriginally used for ChIP-seq data. After performing more extensive confirmatory ChIP-qPCR at the Igh and\nother H3K79me2/3-high loci, we failed to see any increase (Fig S6B, S7D) (except at the unscheduled\nGLTUs, which is a particular case). Moreover, western blotting did not show any quantitative changes in\nRNAPII, S2P-RNAPII, and S5-RNAPII between WT and DOT1L-deficient cells (new Fig. S6B). Thus, we ruled\nout a global change in S2P-RNAPII. Based on this, we reanalyzed ChIP-seq data using library size\nnormalization.\nIn addition, we have now replaced the RNAPII CUT&RUN data with RNAPII ChIP-seq, for a more accurate\ncalculation of pausing index, and performed TT-seq to measure nascent transcription, which offers a\ntemporal component to analyze kinetics. Combined with RNAPII ChIP-seq, TT-seq enabled the calculation\nof elongation rate and the pause duration at the promoter-proximal region (Gressel et al, 2017; Velychko\net al, 2024).\nWT and Dot1l-deficient cells showed no change in pausing index between when calculating traveling ratio\nfrom RNAPII ChIP-seq data (Fig. S6D), as observed by others in leukemic cells (Wu et al, 2021; Cao et al,\n2020). However, calculating pause duration from TT-seq and RNAPII data, shows reduced pausing time in\nDot1l-deficient cells (Fig. 5D), which is consistent with the findings mentioned by the reviewer (Cecere et\nal, 2013; Wille et al, 2023). Transcription elongation velocity offers an explanation to harmonize the\napparently different effect on pausing. We find that elongation velocity is increased in Dot1l-deficient cell.\nThis increase is proportional to and spatially overlaps with, H3K79me3 abundance (Fig. 5E), thus most\nlikely reflecting a direct effect of DOT1L. In contrast, since the pausing site is relatively poor in\nH3K79me2/3, we propose that shorter pause duration in DOT1L deficient cells is a consequence of faster\nelongation, leading to more frequent release. This indirect effect may sometimes produce a detectable\nreduction in traveling ratio, as in (Cecere et al, 2013; Wille et al, 2023), or not, as in (Wu et al, 2021; Cao\net al, 2020) and our Dot1l-deficient CH12F3 cells.\nWe make these points in the revised Discussion to contextualize our findings, which are complementary\nto those of Cecere et al and Wille et al.\n3\n4. Results, such as in Figure 3 “Dot1l regulates GLTs expression fidelity” and Figure 5 “Global alteration in\ntranscription output caused by Dot1l loss” touch upon complex subjects that are not well integrated into\nthe main storyline. Figure 3 results are related to the regulation of gene expression plasticity by several\nfactors, including DOT1L, which was comprehensively described in the Xiao et al Open Biology 2019\npublication.\nWe appreciate the suggestions. We have restructured the manuscript based on the new data and\nreviewers comments to improve the integration of all data.\nNote that the Igh locus contains multiple independently regulated transcriptional units (the GLTUs) that\nare part of a single gene. This configuration is not found in other loci, so these indirect effects on the\nintermediate GLTUs are unique to this locus. In normal B cells, GLTU expression is responsive to cytokine\nstimulation, which can be an example of gene expression plasticity regulated by the environment, as\ncovered in the Xiao et al review. However, we also show that the effects on the GLTUs are independent\nof cytokine stimulation.\nThe Figure 5 subject is complex and not limited to the role of DOT1L in Pol II pause release. There is\nliterature connecting DOT1L to enhancer regulation through effects on chromatin compaction, as well as\nstudies connecting DOT1L to antisense transcription regulation. This was not properly referenced in the\npaper.\nThis point is no longer applicable to the revised manuscript, where we report direct effects on\ntranscription elongation rate that overlap with H3K79me3 content. We do not analyze enhancers or\nchromatin compaction, but the TT-seq data shows that DOT1L loss has little or substantially smaller effect\non antisense than sense transcription (Fig. 5A).\nI do not find conclusions that gene length affects the mode of transcription regulation by DOT1L (UP or\nDOWN) particularly convincing, this part should be removed from the model in Figure 8 (or from the\nmanuscript altogether). Data analyses based on gene function might be more informative, especially if\nrelated to the main story of the manuscript.\nWe have removed this consideration from the model. The observation that downregulated genes tend to\nbe longer than upregulated genes has been reported (Cao et al, 2020) and we find the same in DOT1L-\ndeficient CH12 and primary B cells (Fig. S4C). We only briefly mention this but make clear we do claim the\ngene length affects how DOT1L regulates transcription but rather that long genes may be more sensitive\nto transcription deregulation after DOT1L loss, thus partly explaining their downregulation.\nThe authors relate their Pol II pause-release findings to literature about known pausing factors and their\ninhibitors: Spt5, RN7SK, NELF, PAF1, etc. This is not justified by the results. Unless they do experiments and\nimplicate these factors in CSR, they must instead focus on the similarity of their DOT1L findings to the C.\nelegans system that lacks NELF and the Wille et al. 2022, BioRxiv publication.\nThis was indeed not clearly written. SPT5, PAF1, SPT6, have been previously implicated in CSR. This was\nreferenced but not directly stated. Nonetheless, as the new data now indicates that the effect on pausing\nis secondary to the direct increase in transcription elongation speed upon DOT1L loss, this was removed\nfrom the Discussion.\n4\n5. The introduction gives no background about DOT1L and the current state of our understanding of its\neffect(s) on transcription and chromatin organization.\nWe agree with the reviewer. We wrote the original manuscript from our chronological perspective of\nfinding DOT1L by AID BioID. At this stage this is no longer relevant, and we provide background on DOT1L\nin the revised introduction.\nSpecific issues:\nFigure 4A, C: How does total RNA Pol II distribution look in Dot1l +/- conditions?\nIf the reviewer refers to Dot1l+/- heterozygous CH12F3 cells we did not analyze them. We obtained a single\nclone that did not show much decrease in H3K79me2 by WB suggesting Dot1l is not haploinsufficient in\nthis line.\nIf this refers to RNAPII distribution in WT vs KO cells we have now performed ChIP-seq (instead of\nCUT&RUN) with a different anti-RNAPII antibody that provided better enrichment. Total RNAPII\nmetagenes and screenshots are in Figs. 5 to 7 for different gene subsets.\nFigure 4B, bottom: Why ChIP-qPCR at the P region is not shown?\nWe have remade these analyses (Fig. 7F, S7F). We bring to the attention of the reviewer that S-regions\nshow atypical pausing, with a broad RNAPII enrichment through the gene, instead of a narrow promoter-\nproximal peak as most other genes (see Fig 7E). Since this experiment asks whether DOT1L deficiency\naffects AID occupancy, we focused on the segment of higher AID occupancy in the S-regions.\nFigure 4C: Analyses using randomized groups of genes with the same # should be performed for control.\nWe think there was a misunderstanding. This figure showed all expressed genes stratified in quintiles by\ntheir H3K79me3 (now Fig. S3F). The original figure also showed S2P-RNAPII heatmaps for the quintiles,\nwhich is now shown as metagenes and also includes all expressed genes (new Fig. 5A). As this is a\ndescriptive analysis of all genes, we are unsure of what would the randomization be controlling for.\nFigure 4F: Show expression changes corresponding to each group of genes.\nWe now show gene expression for each H3K79me3 gene quintile in Fig. 4A, S4A.\nFigure 6 A, C: The difference between H3K79me3 distribution in splenic B cells and CH12F3 cells is\nconcerning and must be addressed. How do these profiles look in unstimulated cells?\nThis was intriguing for us too, but we are confident that this is not a technical artifact. As we explain in\nthe methods, the antibody against H3K79me3 has some cross reactivity against H3K79me2 but it is 10-\nfold more selective for me3. We note that the ChIP-seq profiles in primary B cells were done with the\nexact same antibodies and show different profiles, further supporting its selectivity.\nSo, cytokine stimulation is unlikely to explain the difference in profiles between splenic B cells and CH12F3\ncells.\nFigure R1: Screenshots of H3K79 methylation at the IgH of mouse naïve B cells. Track 1 and 2: H3K79me2 and me3 in naïve\nsplenic B cells. Track 3 and 4: H3K79me2 and me3 in splenic B cells stimulated with LPS + IL-4, which activates Sγ1 GLTU. All\ntracks are in the same scale. It can be appreciated that both H3K79me2 and me3 increase in activated versus naïve, cells, but\nthey still keep distinct profiles in these primary B cells (the major change being the acquisition of a peak at Sγ1 GLTU, which is\nactivated by LPS and IL-4.\nChIP-seq in CH12F3 cells was done without CIT so it is not influenced by cytokines. We further show by\nChIP-PCR that there is no change in H3K79me3 after CIT at the Igh (Fig. S2C). Importantly, CIT does not\nchange GLTs transcription levels in CH12F3 cells (Zhang et al, 2019).\nWhile in CH12F3 the pattern of H3K79me2 and H3K79me3 are similar, they are not fully overlapping as\ncan be appreciated in the metagenes over the chromatin stripes (now Fig. 4I), and by a relatively large\nnumber of peaks having either H3K79me2 or H3K79me2 (Fig. S3D).\nGiven that DOT1L is distributive, the higher abundance of H3K79me3 in CH12 likely reflects higher DOT1L\nactivity in CH12F3 than primary B cells. We posit that this could be explained by the distinct differentiation\npotential of CH12F3 and primary B cells, consistent with work proposing DOT1L as a barrier to\ndifferentiation and noticing that embryonic stem cells have lower H3K79me2/3 compared to somatic cells\n(Wille & Sridharan, 2022; Wille et al, 2023b). Primary B cells (whether naïve or activated) have substantial\ndifferentiation potential; they can become memory B cells, and plasma cells at terminal differentiation.\nThe CH12F3 cell line has little differentiation potential and can accumulate H3K79 methylation.\nFigure S2 B: Why does CIT lead to H3K79me reduction?\nThis is not the case. Biological replicates show that there is no change in H3K79me3 upon CIT at the Igh\n(now Fig. S2C).\nTriplicate replicates should be shown for all RT-qPCR and ChIP-qPCR data.\nWe present triplicates for most RT-qPCR and ChIP-qPCR data. In two cases, the differences, or lack of\ndifference, were clear enough from two experiments to sufficiently establish the relevant result (i.e. Fig\nS2C to show the mark is present in the Igh independent of cytokines and absent in Dot1l ko cells, Fig S5I\nshowing the decrease in CTCF at 3’ CBE in Dot1l ko cell).\nIn this paper, the authors investigate the mechanism of DOT1L promoting AID-induced class switch\nrecombination (CSR) as well as “off-target” mutations. They applied BioID to identify nuclear AID-proximal\nproteins that potentially contribute to AID-induced mutations. From the results, they identified DOT1L and\nits associated proteins, as well as SEC as being specifically enriched in the AID interacting complex. To\nevaluate the function of DOT1L they perform knockdown and knockout experiments and find that CSR is\ninconsistently affected at germ line transcripts depending on locus and method of knockout. Since the local\neffects at IgH do not show a trend based on characteristics of accumulated chromatin marks, they then\nperform RNAPII ChIP-seq from which they conclude a correlation between promoter pause release and\nH3K79me3 enrichment. Finally they perform experiments on cohesion accumulation that suggest a link It\nhas been reported that DOT1L is essential for B cell differentiation, but the authors provide insights into\nthe potential mechanisms that may be relevant.\nWe thank the Reviewer for recognizing our original contributions. The revised version has adjusted and\nfurther improved the mechanistic insight.\nMajor points:\n1. The first major claim of the manuscript is that DOT1L has a role in CSR ( Fig 1-3 )- which has been reported\nbefore (EMBO Rep. from the Leeuwen lab). In that prior paper it was shown that DOT1L promotes somatic\nhypermutation and lowered RNA Pol II occupancy at immunoglobulin variable region.\nIt was not our intention to claim that we were the first to report the involvement of DOT1L in CSR.\nHowever, we recognize that the way we originally framed the manuscript, based on the chronology of our\nown findings, gave that impression.\nWe have revised the Introduction to clearly cite the prior work from the Van Leeuwen laboratory on CSR\nand from the Scharff laboratory on SHM. We now explicitly state their contributions to establishing the\nrole of DOT1L in antibody diversification. We also better highlight the novelty of our study:\ni) Reporting the general role of DOT1L in facilitating AID activity not only at the Igh locus but also at\noff-target genes.\nii) Providing mechanistic insights into DOT1L’s function through structure–function analyses and\nepistasis experiments.\niii) Defining how loss of DOT1L affects transcription elongation dynamics and how this compromises\nAID occupancy and, consequently, its activity, in Dot1l-deficient B cells. In addition, changes in\ntranscription kinetics can explain gene expression changes in Dot1l-deficient cell, which has not\nbeen elucidate yet.\n1.1 The results in this manuscript are inconsistent with the prior published results (Fig 1E showed a different\nresult at the same locations (Su)) and at different regions where shDOT1L can both increase or decrease\nRNAPII occupancy (Fig 1E).\nWe thank the reviewer for this observation. We have removed Fig. 1E. That ChIP-qPCR experiment was\nintended as \"quick and dirty\" comparison of the effects of different DOT1L and SEC subunits on RNAPII\noccupancy. However, as it lacked replicates, we agree that it was not sufficiently robust for publication.\nWe have now conducted new experiments focusing on the effect of DOT1L loss on transcription: We\nperformed ChIP-seq for RNAPII and TT-seq to measure nascent transcription genome-wide relative to\noccupancy at genes enriched for H3K79me3. Furthermore, our data also provides a cause for this apparent\ndecrease: it is a consequence of increased transcription elongation velocity following DOT1L loss (see\ndetailed response to Point 2).\nAt the Igh locus specifically, we also observe a very slight reduction in RNAPII occupancy in Dot1l-/- cells,\nby both ChIP-seq (revised Fig. 7E) and ChIP-qPCR (revised Fig. S7D). These findings are consistent with the\nobservations of the Scharff’s lab at the IgH of DOT1L-deficient Ramos cells (Duan et al, 2021). Thus, the\nrevised dataset is more rigorous and aligns with previously published findings.\nNote that RNAPII increases in the unscheduled GLTs that are upregulated, as would be expected, which is\na special case and likely an indirect effect caused by alterations in chromatin loop extrusion.\n1.2 The effects of the DOT1L binding partner MLLT10 on class switching (Fig 1K) are undermined by the\nlack of substantial knockdown at 48 hours (Fig S1C).\nWe understand the concern, the explanation lies in the kinetics of events leading to CSR.\nDepletion of MLLT10 by shRNA is progressively lost over time in CH12 cells (we achieve ~50% depletion\nafter selection of the transduced cells, which is reduced to 20% depletion 48 h later). This suggests that\ncells with reduced MLLT10 expression are at a selective disadvantage, consistent with our inability to\ngenerate MLLT10 knockout CH12 clones by CRISPR/Cas9, despite obtaining heterozygous mutants.\nThis kinetics should be evaluated in the context of the kinetics of the CSR assay in CH12 cells. CSR is\nmeasured by flow cytometry as the percentage of IgA+ cells 48 h after cytokine (CIT) stimulation. However,\nsurface IgA expression reflects recombination events that occur much earlier. Based on our previously\npublished kinetics (Cortizas et al, 2013), AID protein is robustly expressed by 8 h post-CIT, with maximal\noccupancy of S-regions by 16 hours. Thus, the DNA damage triggering the recombination events driving\nIgA expression at 48 h occur in the first 24 h after CIT stimulation. In our experiments, the 50% depletion\nby shMllt10 before CIT stimulation can explain why a CSR defect (i.e. surface IgA proportion) is detectable\ndespite the recovery of MLLT10 expression by 48 hours.\nWe further implicate MLLT10 together with DOT1L in CSR by the epistasis analysis (Fig. 1H).\nCollectively, these findings are consistent with our conclusion that DOT1L and MLLT10 act in the same\nfunctional pathway to promote CSR.\nFurther, In Fig 1K, shAID further reduced CSR I the background of DOT1L deficiency, indicating DOT1L is\nnot necessary for AID function.\nWe respectfully disagree with this interpretation of the data (now in Fig. 1H). AID is the initiating factor\nrequired for CSR and is, therefore, essential under all conditions. Its depletion will reduce CSR regardless\nof the presence or absence of other factors.\nThis is analogous to MSH2, which contributes substantially to CSR but is not absolutely required due to\nredundancy with UNG-mediated pathways; nevertheless, CSR is still reduced when AID levels are lowered\nin MSH2-deficient cells. Similarly, DOT1L facilitates but is not essential for AID activity, so the residual CSR\nobserved in DOT1L-deficient cells remains AID-dependent.\nThe purpose of including shAID in these assays was to establish the dynamic range of the CSR assay under\nthe experimental conditions. CSR levels in shAID-treated cells provide the background, while residual CSR\nin DOT1L-deficient cells defines the maximum signal. The difference provides sufficient dynamic range to\n8\ndetect the effects of additional perturbations. Thus, depletion of AFF1 and MLLT1 further reduced CSR in\nthe DOT1L-deficient background, supporting their DOT1L-independent contributions to CSR. In contrast,\nMLLT10 depletion did not cause an additional reduction, indicating that it is epistatic with DOT1L.\n1.3 The CH12F3 delta/delta lines and the CH12F3 delta/- lines of DOT1L deletion are different from each\nother – The delta/delta lines retain frame (Fig S2A)- which means there is a DOT1l truncation mutant\nprotein that could act as a dominant negative. DOT1L KO lines need a DOT1L western blot to confirm the\nprotein is deleted.\nWe were unable to detect endogenous mouse DOT1L by western blot, which is why we used tagged DOT1L\nconstructs in the complementation assays.\nTo experimentally test whether the Dot1lD alleles could produce a dominant-negative protein, we\noverexpressed a corresponding truncation variant in WT cells. This had no effect on CSR, demonstrating\nit lacks dominant-negative activity (new Fig. S2B). Furthermore, we performed ChIP for DOT1L in Dot1l-\ndeficient CH12 cells complemented with either WT or catalytically inactive DOT1L. While the inactive\nprotein occupies the Igh locus, it fails to rescue CSR (new Fig. 2B). This shows that DOT1L catalytic activity\nis essential and that even full length inactive DOT1L does not have dominant negative activity when at the\nIgh, as its overexpression does not further impair CSR despite occupying the locus (Fig. 2A)\nThis is an important consideration because the results in Fig 2A are misinterpreted by pooling the samples\nfrom both kinds of deletions on CSR ( triangles and squares in the bar graphs).\nWe originally pooled data from multiple independent Dot1l-deficient clones to illustrate the consistency\nof the CSR phenotype across different lines, rather than relying on one or two clones. While all clones\nshowed qualitatively similar results, we acknowledge that pooling introduced variation due to clonal\nheterogeneity (a common occurrence with CRISPR/Cas9 KO in cell lines). We have addressed this in the\nrevised manuscript. We now present results separately for two independent Dot1l-deficient clones, each\nbased on ≥3 biological replicates, with statistical analysis. The results are consistent in both clones and\nfully support our original conclusions regarding CSR and DOT1L structure-function analysis.\n1.4 The effects of DOT1L deletion on GLTs are inconsistent and modest at different loci, between different\nkinds of DOT1L deletion and in resting and activated conditions ( for example In Fig 3C, expression of lghg2c\nis increased in stimulated condition but decreased in resting state upon DOT1L KO). Further in Fig2A, 2H\nand 3A, GLT changes do not correlate with reduced CSR or transcription elongation defects. Then DOT1L\ninhibition upregulates unscheduled GLTs cannot be the mechanism of DOT1L regulates CSR. While the\nauthors argue for complex regulation by DOT1L for CSR, the data could also support a minor role for DOT1L\nthat is far downstream of other players. For example in the mass spectrometry in addition to DOT1L,\nthe H3K4 methyltransferase is also specifically enriched. This result would suggest that that the proteins\ninteracting with AID are proximal to the transcription start site and not specific to AID. AID-ChIP at GLT\nloci in DOT1L deletion lines could be a method to support DOT1L deletion affects CSR through interaction\nwith AID.\nWe realize these points were not sufficiently well explained and have now addressed them carefully.\nFirstly, we note that we do not claim that DOT1L interacts with AID. In terms of specificity, we only claim\nin so far as the assay enables us: BioID indicates that DOT1L coincided nearby AID in the nucleus more\nfrequently than APOBEC2 or BirA controls did. We do not think the proteins interact because we have\n9\nperformed several AP-MS experiments using AID as bait in the past and never co-purified DOT1L\n(Orthwein et al, 2010, 2012).\nWe make it clear in the manuscript that we consider DOT1L an enabler of AID by contributing to establish\nthe transcriptional environment that favors AID activity.\nSecondly, we have improved our explanation of the effect of DOT1L loss on unscheduled GLTUs.\ni- The upregulation of unscheduled GLTs upon DOT1L loss is clear and reproducible across systems. We\nobserve it in primary B cells (reanalyzed RNA-seq from others, now Fig. 4C), in primary cells treated with\nthe DOT1L inhibitor pinometostat (Fig. 4D, S5B), and in the two Dot1l-deficient CH12 clones (Fig. 4F, S5C).\nii- We agree that the specific GLTUs upregulated in each of these systems is different, albeit some are\ncommonly upregulated (Sg3, Sg2c). We do not interpret these differences as inconsistency. We believe\nthat the variations in GLT profiles across systems reflect indirect effects of DOT1L loss on chromatin loop\nextrusion. Increased transcription elongation velocity (see response to Point 2) would be expected to\nsubtly alter chromatin architecture in a stochastic manner, leading to different GLTs becoming\nupregulated in different experimental conditions.\nImportantly, even if they display some differences in the identity and magnitude of the upregulated GLTs\nbetween the two CH12 clones, in each case their upregulation is a direct consequence of DOT1-loss:\ncomplementation with WT DOT1L reverses the phenotype, whereas catalytically inactive DOT1L does not\n(Fig. 4H, S5E).\nIndeed, unscheduled GLTUs upregulation is high in fold-change but typically modest in absolute increase.\nAs this has become an ancillary point we have reorganized the manuscript to shortly convey report this\nfinding. We highlight the case of Sg2b in primary B cells (Fig. 4D-E). Control B cells show substantial levels\nof CSR to IgG2b, implying the Sg2b transcription is sufficient for CSR in those conditions. Inhibiting DOT1L\nincreases Sg2b GLT levels but reduces CSR. This provides a first observation dissociating the effect of\nDOT1L in CSR from simple gene expression levels.\nThis is then directly confirmed by TT-seq data in Dot1l-deficient CH12 cells showing increased transcription\nat Sµ and Sa, the S-regions directly implicated in CSR to IgA, yet CSR is impaired.\nOur data supports the claim that genes become less susceptible to AID-mediated deamination in the\nabsence of DOT1L, rather than transcription levels per se being the key determinant.\n1.5 Statistical tests are inconsistently applied across Figs 1-3 ( for example between the two kinds of DOT1L\ndeleted lines in Fig 1H), in several cases there are only 2 replicates- it is not indicated whether the replicates\nare technical or biological , and standard error of mean is used instead of std deviation from mean. There\nare missing error bars and significance testing (Fig S3C is missing error bars in some timepoints and in\ncertain axis. S3D needs significance test).\nWe agree and apologize for this inaccuracy. We have revised all data and provide statistical analysis using\nthe appropriate tests depending on the type of data and normality test results. We also indicate that all\ndata points are biological replicates, having increased the n wherever was necessary.\n2. The second major claim of the manuscript is that DOT1L acts by limiting pause release and not\ntranscription elongation, and that leads to transcriptional upregulation.\n1) We detected a spike-in normalization artifact in the S2P-RNAPII ChIP-seq data that had misled our\nconclusion about a general increase in S2P-RNAPII. Library size normalization, which is supported by the\nabsence of global changes in RNAPII levels and validated by ChIP-qPCR, shows there is no such increase.\n2) We replaced the RNAPII CUT&RUN with ChIP-seq using an antibody against the Rbp1 N-terminal region\nthat resulted in a better signal that improved travelling ratio and other calculations.\n3) We directly measured nascent transcription by TT-seq, which showed changes coinciding with the\nH3K79me3-enriched regions.\n4) TT-seq and RNAPII ChIP-seq allowed us to calculate kinetics parameters transcription elongation\nvelocity and pausing duration.\nWe now show that DOT1L loss primarily affects transcription elongation velocity, which we speculate\nindirectly impacts the duration of the pausing step, rather than directly regulating pause release as we\npreviously proposed. This is in line with reports showing that elongation rate regulates RNAPII pausing.\nSpecifically, it has already been shown that faster transcription elongation results in reduced promoter-\nproximal pausing (Leng et al, 2020a).\n2.1 First it is argued that the total RNAPII CUT&RUN signal is lower in DOT1L KO cells because of the epitope\nbeing masked by high S2P-RNAPII, but it is also argued there are no global changes in cellular levels of\nRNAPII or S2P-PolII which is impossible to interpret because Fig. S4B does not have a loading control. Total\nPol II CHIP PCR for other GLT locations in 4B in WT/KO lines would also help observations on pause-release\ndifferences related to gene expression changes from Fig 3 and H3K79me levels.\nWe thank the reviewer for pointing out this confusion, which also applies to the next point. We have\naddressed the issues by performing new western blot (WB) analyses and additional ChIP-qPCR\nexperiments, as well as by reanalyzing ChIP-seq data.\nSpecifically, we conducted new WBs in two independent DOT1L-deficient CH12 cell lines. These analyses\nconfirm that there are no global changes in total RNAPII, S2P-RNAPII, or S5P-RNAPII levels (Fig. S6C).\nThis re-evaluation prompted a reanalysis of our ChIP-seq data, during which we identified a spike-in\nnormalization artifact that previously led to the erroneous conclusion of a global increase in S2P-RNAPII\nat H3K79me2-enriched genes. Upon correction using library size normalization, and supported by\nconfirmatory ChIP-qPCRs, we now show that S2P-RNAPII exhibits only small, localized changes (new data\npresented in Fig. 5, with confirmatory ChIP-qPCRs in Fig. S6B). These changes are consistent with\nalterations in elongation velocity, as suggested by the TT-seq and RNAPII ChIP-seq data, rather than global\neffects on RNAPII occupancy.\nA similar pattern is observed at the IgH Sµ and Sα GLTUs involved in CSR (Fig. 7E, S7D).\n2.2 At the locus specific level Fig 4B – the total RNAPII enrichment differences are modest, the S2P-RNAPII\nenrichment at the promoter is not presented- this leads to the question of whether there is a nonspecific\nincrease of S2P-RNAPII everywhere . The intergenic region ( bottom panel of Fig 4B) does not serve as a\ncontrol for this question because the enrichment at that location is a magnitude lower than that at the\nSgamma3 location.\nAs detailed in our response to the previous comment, our new analyses rule out a nonspecific global\nincrease in S2P-RNAPII following DOT1L loss.\nCH12 cells, including TT-seq data to measure nascent transcription, validating ChIP-seq results by well\ncontrolled ChIP-qPCRs (Fig 7E, S7D-E). The data show that the H3K79me2/3-rich Sµ and Sα GLTUs are\nregulated by DOT1L at the level of transcription elongation velocity, similarly to other H3K79me2/3-\nenriched genes.\n2.3 Most disconcertingly it is argued that the RNAP C-terminal antibody is unreliable- yet it is the one\nused for calculating the pausing index (Fig 4E and 4F).\nWe were wrong. Our initial concerns regarding RNAPII C-terminal epitope masking were incorrect and\narose from a spike-in normalization artifact, which falsely suggested a global increase in S2P-RNAPII.\nWe have corrected this issue in the revised manuscript.\nNonetheless, in the original version, RNAPII occupancy was assessed by CUT&RUN, which provides\nsuboptimal coverage for accurate calculation of the pausing index.\nTo address this, we have generated new RNAPII ChIP-seq data that provided improved coverage. We used\nan antibody directed against the N-terminal domain of Rpb1 to avoid any potential concern related to CTD\nphosphorylation. With this new data, we recalculated the pausing index and show that there is no\nsignificant difference between WT and DOT1L-deficient CH12 cells (Fig. S6D).\n2.4 The ChIP-seq of H3K79me3 could also include H3K79me2 location as in Fig4A, 4C, 6C, 6B, the signal of\nH3K79me2 and H3K79me3 show almost the same pattern. Further, between Fig 6B and S5A, H3K79me2\nChIP displays very different patterns between mouse splenic B cells and CH12F3 cell line.\nWe appreciate the reviewer’s careful examination of the H3K79 methylation patterns.\nThere are two distinct aspects to this observation:\ni) In CH12 cells, the H3K79me2 and H3K79me3 patterns are similar.\nii) The H3K79me2/3 patterns at the Igh locus differ between CH12 cells and primary B cells.\nBoth observations are correct and can be explained as follows:\ni) The similarity between H3K79me2 and H3K79me3 in CH12 cells is not a technical artifact. As described\nin the Methods, the H3K79me3 antibody exhibits some cross-reactivity but shows ~10-fold selectivity for\nme3 over me2. Moreover, the same antibodies we used for the ChIP-seq in CH12 cells have been used for\nthe primary B cells datasets. The fact that H3K79me2 and H3K79me3 display a distinct pattern in primary\nB cells (Fig. S5A) supports the reliability of the antibody specificity using similar ChIP conditions.\nThe H3K79me2 and H3K79me3 profiles are a lot more similar in CH12 cells, but they are not fully\noverlapping. This is illustrated by metagene analyses across chromatin stripes (now Fig. 4I) and by the\nidentification of peaks that are selectively enriched for either H3K79me2 or H3K79me3 (Fig. S3D).\nii) The different H3K79me2/3 patterns at CH12 and primary B cells Igh reflect meaningful biological\ndifferences in their transcriptional activity. H3K79 methylation is associated with active transcription.\nThe Igh locus contains multiple germline transcription units (GLTUs) that are regulated in a cytokine-\ndependent manner to induce CSR. In naïve primary B cells, transcription is largely restricted to Sµ, while\nLPS/IL-4 stimulation induces transcription of both Sµ and Sγ1 GLTUs to induce CSR to IgG1. In contrast,\nCH12 cells constitutively transcribe Sµ and Sα, independent of stimulation, and does CSR to IgA. Thus, in\nprimary B cells, H3K79me3 enrichment is primarily observed at Sµ in naïve cells or at Sµ and Sγ1 in IL4/LPS-\nstimulated cells (Fig. S5A), whereas in CH12 cells, both H3K79me2/3 are enriched at Sµ and Sα (Fig. 4G).\nactivity. This is consistent with the limited differentiation potential of CH12 cells compared to primary B\ncells. It has been proposed that DOT1L acts as a barrier to differentiation, with lower H3K79 methylation\nlevels observed in pluripotent cells compared to differentiated somatic cells (Wille & Sridharan, 2022;\nWille et al, 2023b). Given that primary B cells retain significant differentiation potential (e.g., toward\nmemory or plasma cells), while CH12 cells are terminally differentiated and lack such plasticity, the\naccumulation of H3K79me3 in CH12 cells is consistent with their biological state.\nIn the clustering of H3K79me3 levels (Fig 4C) most genes with estimated traveling ratios are in the first 2\nquintiles. It is unclear why H3K79me3 quantification at TSS site is used for correlation to the pausing index\nsince this location is not representative of general H3K79me3 distributions in the gene body. The\nconclusion that pause-release is related to H3K79me3 levels, would be more convincing if other features\nsuch as H3K79me1/2 and gene expression level are not corelated with the pausing index.\nWe agree that the original presentation was unclear, partly due to a labeling error and insufficient\nexplanation in the Methods section that incorrectly stated that we correlated the mark at the TSS to\npausing index.\nTo clarify, H3K79me3 quintiles were defined based on the H3K79me3 signal from TSS to the end of the\nsecond intron, where the signal is primarily detected. This has been corrected in the revised manuscript.\nWe correlate AID activity to high DOT1L activity by using H3K79me3 as a proxy. This is justified because\nDOT1L acts in a distributive manner, dissociating from chromatin after each methylation event. Thus, the\npresence of high H3K79me3 at a locus reflects sustained or repeated DOT1L activity, which could arise\nfrom greater recruitment, more frequent activity, or longer stability of the methylation marks.\nNevertheless, while the original analysis emphasized pausing index, considering additional data (new\nChIP-seq and TT-seq experiments), the conclusion has changed. Our results are consistent with DOT1L\naffecting transcription elongation velocity, rather than pause release per se. The revised manuscript has\nbeen updated to reflect this change in focus.\n2.5 In Fig 5G & S4H H3K4me3 has 2 populations- and every pairwise comparison is statistically significant\n– if that is the case then the distribution of H3K4me3 cannot causal for transcriptional changes.\nWe thank the reviewer for this insightful comment. We have removed this data from the paper. They are\nno longer relevant, as those correlations have been superseded by direct data on transcription that can\nexplain transcriptional changes caused by DOT1L deficiency.\nThe manuscript of Subramani and co-workers entitled \"DOT1L limits RNAPII promoter-proximal pause\nrelease thus enabling Activation Induced Deaminase activity genome-wide\" deals with the molecular\nunderpinnings of class switch recombination and somatic hypermutation of antibody genes, which involves\nthe action of Activation induced deaminase (AID). But the data extend towards genome-wide analysis with\nthe intention to generalize some of the findings. The authors discover that AID interacts with DOT1L and\nthey embarked to characterize how AID and DOT1L mediate CSR and SHM, and whether DOT1L is involved\nin AID off-target deamination.\nIn all this is very impressive and experimental rich work that proposes novel mechanisms of action for the\nhistone methyltransferase DOT1L. My main concern about the data presentation in its present form is that\nit should worked out much clearer - especially for the broader scientific community - how the different\nproposed mechanisms are linked to either genome-wide and/or IgH-specific transcriptional control. In its\npresent form the proposed mechanisms are not always presented in a connected form but rather\nindependent from each other.\nWe thank the reviewer for their positive comments and for the suggestion regarding data presentation.\nWe agree that the original manuscript did not sufficiently articulate how the genome-wide and Igh-\nspecific transcriptional effects of DOT1L loss are mechanistically connected. This was partly due to a\nnormalization artifact spike-in controls, which led us to overemphasize changes in promoter proximal\npause-release, a phenomenon that turned out to be secondary.\nNew data demonstrate a function for DOT1L activity in controlling transcription elongation velocity. The\nrevised manuscript incorporates additional RNAPII ChIP-seq, TT-seq, and ChIP-qPCR analyses that clarify\ntranscriptional changes caused by DOT1L deficiency, both globally and at the Igh locus. These new findings\nallow for an integrated model, in which the abundance of H3K79me2/3 regulates transcription elongation\nvelocity genome-wide, including at activated Igh S-regions. This control has critical consequences for CSR\nby regulating the transcriptional environment required for AID occupancy at activated S-regions.\nWe believe that these revisions significantly improve the novelty, as well as clarity and accessibility of the\nmanuscript.\nMajor points:\n1) The authors show that it is DOT1L and not, indirectly, the super elongation complex that accounts for\nCSR, although the SEC was also part of the interactome.\nAs later in the manuscript the authors show increased S2P-RNAPolII in the gene bodies upon DOT1L\ndeletion, a stronger activity of the SEC might be likely. It would be good, if this aspect could be validated\nas well.\nWe apologize for any confusion in the original manuscript. We have now clarified that we do not claim\nthat DOT1L accounts for CSR instead of the SEC. Rather, as shown in Fig. 1D, we implicate both DOT1L and\nseveral SEC components in CSR. We then show by epistasis analysis that DOT1L acts independently of the\nSEC complex in CSR (Fig. 1H).\nAs mentioned in the previous point, we have revised our interpretation about S2P-RNAPII caused by the\nspike-in artifact. This correction shows there is no increase in S2P-RNAPII making the possibility of a direct\ncompensatory increase in SEC activity less compelling.\ninvestigation; however, characterization of the SEC's contribution constitutes a substantial body of work\nthat falls outside the scope of the current study.\nAlong the same line, showing a concomitant decrease in S5P-RNAPolII as the pausing form would also be\ndirect proof, that the switch from pausing to elongating is affected by the action of DOT1L (or its\ninteraction partners).\nWe thank the reviewer for this suggestion. In the revised manuscript, we show by ChIP-seq that S5P-\nRNAPII levels are decreased at the H3K79me2/3-rich genes, specially around the TSS, upon DOT1L deletion\n(Fig 5A-C). This decrease is clear at Sµ and Sα GLTs of the Igh (Fig. 7E) and AID off-target genes (Fig. 7D).\nThese findings are consistent with reduced RNAPII pausing. However, our data suggests that DOT1L-\ndeficiency does not affect the amount of promoter proximal pausing per se (as calculated by RNAPII\noccupancy at TSS/gene body, Fig. S6D), but rather it shortens of the duration of pausing (Fig. 5D), likely\nresulting from accelerated RNAPII elongation through H3K79me2/3-rich regions. This interpretation is\nsupported by TT-seq analysis, which indicates an increase in nascent transcription despite modestly\ndecreased RNAPII occupancy at H3K79me2/3-rich regions (Fig. 5A, and 7E for Igh). This is in line with\nreports showing that elongation rate regulates RNAPII pausing. Specifically, it has already been shown\nthat faster transcription elongation results in reduced promoter-proximal pausing (Leng et al, 2020a).\nIt is worth noting that the apparent discrepancy between pause duration and pause index is due to the\nfact that the latter is a signal ratio and does not consider kinetics. This is only achieved when the amount\nof paused RNAPII (ChIP-seq) is integrated with the corresponding transcriptional activity over a specific\ntime (which is enabled by TT-seq).\nIn the same setting some experiments and their interpretation might need clarification:\n- is the SEC increased (expression levels? occupancy (esp. in regard to the S2P RNAPII accumulation shown\nin Fig. 4C)? interaction with AID?) upon DOT1L deletion? And why would this be? Is there an increase in\nDot1L or the relevant SEC components as well after CIT? Is there increased interaction between DOT1L and\nAID in this condition?\nWe agree with the reviewer that this was a\npossibility in the original manuscript. However,\nas mentioned above, revising our analysis\nshows there is no S2P-RNAPII accumulation,\narguing against compensatory SEC activity in\nDOT1L-deficient cells.\nTranscriptomic data shows DOT1L deficiency\ndoes not result in widespread compensatory\nupregulation of SEC components. Apart from a\nmodest increase in Mllt6, expression levels of\ncore SEC genes remain unchanged in DOT1L-\ndeficient CH12 cells (Fig R2). Furthermore, CIT\nFigure R2: Heatmap of gene expression in WT and Dot1l-/- cells (from\nhas minor effects on the expression of a few\nour RNA seq data, GSE233975, DESEq2-normalized counts, average\ngenes encoding SEC and DOT1L components in\nfrom triplicates), or unstimulated (UNS) and CIT-stimulated CH12 B\nCH12 cells (Fig R2) (Delgado-Benito et al,\ncells (RNA-seq GSE118794, RPKM, average from triplicates).\n2018).\nthe results observed are consequence of DOT1L deficiency.\nWe have not observed a physical interaction between DOT1L and AID. Specifically, AP-MS with tagged AID\nin Ramos B cells, performed in two independent studies with different tags, did not detect co-purification\nof DOT1L (or the SEC) with AID (Orthwein et al, 2010, 2012). These findings argue against a stable or direct\ninteraction between the two proteins.\nWe specifically used BioID to identify proteins in the proximity of AID rather than strict physical\ninteractors. For example, the MRN complex appears as a prominent proximity hit (Fig. 1A), likely reflecting\nits rapid recruitment to AID-induced DNA damage sites rather than a direct AID-MRN interaction. We\ninterpret the proximity labeling of DOT1L similarly, as a chromatin-associated factor whose activity\nmodulates the transcriptional landscape required for AID function, rather than as a direct binding partner.\n- Dot1L is as Mllt1 not depleted but rather increased in Fig. 1E for Su. Please comment.\nThe original Fig. 1E presented RNAPII occupancy in CH12 cells with knockdown for different SEC subunits.\nHowever, this experiment was intended as a \"quick and dirty\" comparison, conducted without biological\nreplicates. Given the shift in focus of the manuscript towards a detailed mechanistic analysis of DOT1L in\ntranscription, we have removed this figure. As mentioned previously, the analysis of the SEC during CSR\nwill be addressed in a dedicated study.\nIn the revised manuscript, we now present new RNAPII ChIP-seq and TT-seq (Fig. 5, S6), which provide a\nmuch more rigorous and quantitative analysis of RNAPII dynamics in DOT1L deficient cells. These new\ndata reveal a small but consistent reduction in RNAPII occupancy at H3K79me3-enriched genes.\nImportantly, we identify the underlying cause: the decrease in RNAPII occupancy results from an increase\nin transcription elongation velocity upon DOT1L loss, as detailed above. Faster elongating RNAPII\nmolecules transit through genes more rapidly, leading to lower ChIP-seq signals.\n- In regard to the AID interactions with SEC or DOT1L: Do the components sit there and wait for AID to\ncome in? Or is AID recruiting? Or is AID being recruited? What is the dynamics in the system?\nThank you for raising this important point. Prior work from the Scharff laboratory has demonstrated that\nAID is dispensable for DOT1L activity at the Igh, indicating that DOT1L function is not a response to AID-\ninduced DNA damage (Duan et al, 2021). This finding is consistent with our ChIP-qPCR data showing that\nH3K79me2/3 levels at the Igh locus in CH12 cells remain unchanged before and after CIT stimulation (Fig.\nS2C), further supporting that AID expression is not required to establish DOT1L-dependent chromatin\nmodifications.\nIn contrast, ChIP-qPCR data demonstrates that AID occupancy at the Igh and selected off-target genes is\nreduced in Dot1l-deficient CH12 cells (new Fig. 7F-G, S7F-G). We do not interpret this as evidence that\nDOT1L or H3K79me2/3 directly recruits AID. H3K79me2/3 is present in many more genes than AID has\never been reported or can mutate. Our data better fits a model in which DOT1L acts by regulating the\ntranscriptional environment. Specifically, we show that loss of DOT1L leads to increased transcription\nelongation velocity, reducing intragenic pausing (as monitored by S5P-RNAPII) which would diminish the\nability of AID to stably associate with its target sites, thereby reducing the opportunity for deamination.\nThus, we posit DOT1L is an enabler rather than a recruiter, facilitating AID activity.\n16\n2) In regard to placing the action of DOT1L up- or downstream of break formation: How do the authors\nexplain that increased NHEJ occurs upon deletion of the catalytic domain of DOT1L in the delta/delta-1\nclones, but unchanged NHEJ upon use to of the inhibitor?\nThis observation is not leading to the (sole) conclusion given, that \"Dot1l acts at or upstream of the break\nformation step in CSR\". It rather suggests that the scaffolding function of DOT1L would be involved - and\nit would be interesting to learn what this function would be.\nWe agree that distinguishing between the catalytic and non-catalytic (e.g., scaffold) functions of DOT1L is\nimportant for interpreting its role in CSR.\nFirst, regarding whether DOT1L acts at or upstream of the break formation step in CSR: our conclusion\nwas originally supported by two lines of evidence. DOT1L-deficient CH12 cells display reduced AID\noccupancy at the Igh locus (improved analysis in Fig. 7H) and reduced mutation frequency at Sμ (Fig. 1I),\nboth of which serve as proxies for deamination activity, which is an obligate upstream step in DSB\nformation. These data indicate a defect in CSR initiation.\nSecond, to directly address the possibility of a scaffold function, we improved complementation\nexperiments using catalytically inactive DOT1L in DOT1L-deficient CH12 cells. We now show by ChIP-qPCR\nthat both WT and catalytically inactive DOT1L are similarly recruited to the Igh (new Fig. 2B). However,\nonly WT DOT1L rescues CSR, whereas the catalytically inactive form does not (Fig. 2A). This result strongly\nargues against a scaffold role for DOT1L at Igh.\nRegarding the observed increase in NHEJ in Dot1l KO CH12 clones but not in CH12 cells treated with the\nDOT1L inhibitor: we believe this difference reflects clonal selection. DOT1L supports homologous\nrecombination (HR) repair (Alvarez et al, 2019; Kari et al, 2019). Accordingly, DOT1L-deficient CH12 cells\nexhibit impaired HR as measured by a GFP-based reporter assay (Fig. S2G). This deficiency likely favors\ncompensatory upregulation of NHEJ during clonal growth and long-term cultures. In contrast, DOT1L\ninhibitor-treated CH12 cells are exposed for only ~4 days, which is insufficient to select for alternative\nrepair pathways. Thus, the observed increase in NHEJ likely reflects adaptive selection in DOT1L-null\nclones, rather than a direct mechanistic effect of DOT1L loss on end-joining.\n3) The authors use H3K79me2 or me3 as readout for DOT1L activity, however it is not entirely clear that\nthe mark itself is implicated in the process. Rather, the scaffolding function of DOT1L or activity towards\nother protein targets might also explain some of the observations. Thus, it is necessary to show that\nDOT1L is indeed present at the IgH loci itself (in the context of Fig. 2), not only the marks, which might be\nsecondary or not necessary for the function. Also, it needs to be shown that the catalytic deletion protein\nlocalizes similarly or not - or to use point mutations for inactivation to dissect clearly whether it is the mark\nor the scaffold.\nThe same is true for the statement in regard to Fig. 3B and S3F, S5A - is Dot1l present at these loci in a\ndefined, sequence specific manner (not at Sy1, Se but on Syb, Sy3, Sy2c and Sa) - or is this finding of\ndifferent methylation levels a coincidence of a locus-wide general activity and deposition of the mark?\nWe agree that it is important to establish whether the H3K79me2/3 marks directly contribute to CSR,\nand\nto better explain the consequences of DOT1L deficiency for the transcription of activated S-regions (Sμ\nand Sα) versus non-activated or unscheduled GLTs\nIn the revised manuscript, we provide evidence indicating that the histone mark, rather than a potential\nscaffold function of DOT1L, directly regulates transcription and thereby influences CSR.\nwe directly tested this by showing that WT and catalytically inactive DOT1L are similarly recruited to the\nIgh, yet only WT DOT1L rescues CSR (new Fig. 2B). \nSecond, genome-wide analyses integrating RNAPII ChIP-seq and TT-seq (Fig. 5E-F) reveal increased\ntranscription elongation velocity in Dot1l-deficient CH12 cells, which spatially and proportionally overlap\nwith the H3K79me3 mark distribution. This relationship holds both globally and at the H3K79me3-rich Sµ\nand Sa Igh (Fig. 7G), supporting direct role for H3K79me2/3 in modulating transcription elongation.\nIn contrast, we clarify that unscheduled S-regions (such as Sg3 or Sg2c in CH12 cells), are H3K79me3-poor,\nand that their upregulation is most likely an indirect effect of DOT1L loss. This secondary effect appears\nspecific to the Igh locus due to its unique structure, containing multiple GLTUs within a region\ncharacterized by high chromatin loop extrusion (a.k.a. a stripe).\nWe propose a model in which the increased transcription velocity at H3K79me3-rich Sµ and Sa alters loop\nextrusion dynamics (as supported by changes in cohesin distribution, Fig 4J). This alteration promotes\nsemi-stochastic upregulation of a subset of H3K79me3-poor GLTUs by facilitating their contact with\ntranscriptionally active regions. These contacts may favor acquisition of RNAPII and S2P-RNAPII, in\ncontrast to the opposite effect at H3K79me3-rich genes (Figs 5 and 6).\n4) The authors observed that the enzymatic activity would be important for CSR; and it seems that also the\npausing of RNAPolII correlates to this observation. Thus, is it sufficient to inhibit DOt1L to observe similar\neffects as shown in Fig. 4? However, the statement \"we conclude that the H3K79 methylation limits\npromoter-proximal pause escape genome-wide including the IgH GLTUs\" seems correlative. For being\nsubstantiated, it would need further experimentation to support this claim: e.g. gradual loss of H3k79me3\ntowards me2, me1 which should then release RNA Poll pausing. Or locus-specific non-methylation using\ndCas9 constructs. And if this is a claim to be generalized in terms of DOT1L function - i.e. independent on\nthe Ig-locus CSR - this correlation should be observable in other cell systems as well. Which has to be\ndemonstrated. In addition the Chip profiles focusing at the IgH GLTUs might be good to provide in 4C to\nsupport the claim \"including the IgH GLTUs\".\nThis concern is addressed by responses to point 3) and others above, where we detailed how we have\nrevised our claims based on new data to focus on transcription elongation velocity, the data indicating a\ndirect effect of DOT1L activity and H3K79 methylation on transcription velocity (Fig 5, Fig 7), as well as the\ndistinction of the indirect effects of DOT1L-deficiency on the H3K79me2/3-poor GLTUs, likely a\nconsequence of the unique organization of the Igh with multiple transcriptional units.\nChIP profiles of the Igh GLTUs are provided in Fig 7E, as requested.\n5) The authors conclude from their study that \"DOT1L activity enables genome-wide AID off-target\nactivity\" - however, for me it is not entirely clear which mechanism is responsible despite that multiple\nwere proposed, including *paused Pol II release upon loss of Dot1l *H3K4me1/me3 levels *CTCF/cohesin\nloop extrusion As the off-target activity of AID was suggested to be the main focus of the study, I would\nhave expected to have a clearer picture on the mechanism in this regard.\nWe appreciate the need for greater clarity regarding the mechanism by which DOT1L activity enables\ngenome wide AID off-target activity.\ninto a single section (Fig. 3) to improve clarity and focus. These data robustly support the conclusion that\nDOT1L activity is required for efficient AID activity.\nWe acknowledge that the mechanism underlying this phenomenon was insufficiently defined in the\noriginal version. To address this, we now include several new experiments, described in detail in earlier\nresponses, which clarify the underlying process. These data show that loss of DOT1L results in a global\nincrease in transcription elongation velocity, accompanied by a reduction in RNAPII pause duration. We\npropose that the latter is a secondary consequence of the former. This shift in transcription dynamics\nrenders chromatin less permissive to AID activity, which has been associated to stalled RNAPII complexes.\nAdditionally, we have restructured the manuscript to more clearly delineate the downstream (indirect)\nconsequences of altered transcription elongation, such as effects on chromatin loop extrusion that we\ninfer from changes in cohesin distribution. In turn this affects the unscheduled GLTs.\nIn summary, the revised manuscript now presents a unified model in which DOT1L, via H3K79 methylation,\nmodulates transcription elongation kinetics that contributes to an RNAPII state that is favorable for AID\noccupancy and activity at both on-target and off-target sites.\n6) In the discussion the authors mention other inhibitors/deletions that alter RNAPII pausing. If indeed the\npausing is the mechanism behind CSR, it would be interesting to know if these inactivations/deletions (of\nRN7SK, Spt5) also affect the CSR events observed here? Showing such correlation would be a strong claim\ntowards the pausing as major mechanism.\nOn the other hand, the authors claim that \"H3K79 methylation might maintain the balance of these kinase\nand phosphatase activities in situ\". How should this happen - and would this not need to be studied in\norder to provide the necessary mechanistic insights?\nBecause we have obtained data that suggests a different model, we have now removed these speculative\npoints from the discussion.\nMinor points:\n1) Figures of quantifications vary in showing single data points or not - please add single data points in\ncoherent way to all figures. Please also indicate the individual n numbers used for each experiment within\nthe legends.\nWe agree that there was some sloppiness in making the panels. We have revised the panels and figures\nfor coherence in display and statistics and clearly indicate the number of biological replicates per\nexperiment in the legends and/or the figure panels.\nIt seems that some experiments are displayed with a n=2 (i.e. S2E, F,H). Even if this data in included \"only\"\nin the supplemental data - is it sufficient to interpret the experiment without proper statistical testing?\nIndeed, those panels (now Fig S2F,G,I) show an n=2. These are not simply duplicates but experiments\nperformed twice independently. Based on the results we claim that Dot1l-deficient CH12 cells apoptosis\npost-CIT is AID-independent (Fig. S2F), and that these cells exhibit no defect in NHEJ (Fig. S2G,I). While we\nacknowledge that n=2 is below the threshold for formal statistical analysis, the results across independent\nexperiments are highly consistent and qualitatively robust. We therefore consider the data sufficient to\nsupport these specific claims. Furthermore, the conclusion regarding NHEJ is independently validated in\nthe overall interpretation.\nAlong this line comes Fig. 2A, 3D, which is problematic, as first of all two experiments with different cell\nlines are combined in one bar, and variable numbers of experiments are shown (even within the\ncombination of different cell lines). It seems that for CD4 only two data points are shown (this is why the\nlegend should contain all n numbers, as this can also be a graphical problem). Please separate the\nexperiments and provide valid statistical evaluation.\nThese figures were structure-function assays by complementation of Dot1l-deficient CH12 clones with\ndifferent DOT1L variants. We originally pooled data from multiple independent Dot1l-deficient clones to\nillustrate the consistency of the CSR phenotype across different lines, rather than relying on one or two\nclones. While all clones showed qualitatively similar results, we acknowledge that pooling introduced\nvariation due to clonal heterogeneity (a common occurrence with CRISPR/Cas9 KO in cell lines). We have\naddressed this in the revised manuscript. We now present results separately for two independent Dot1l-\ndeficient clones, each based on ≥3 biological replicates, with statistical analysis. The results are consistent\nacross clones and fully support our original conclusions regarding CSR, structure-function analysis, and\neffects on unscheduled GLTUs.\n2) Fig. S2B: what is the reason for showing me2 for two loci and me3 for three? A coherent representation\nwould be much more convincing. Along the line is the representation of the GLT exons: What is Sy1 and\nwhy is this not shown for me3? The Sy1 fragment is not mentioned in the text or legend, and I think the\nschemes are also not depicting the fragment explicitly (also relevant for Fig.3A, where other Sy's show up).\nThis panel showing occupancy of H3K79me2 and H3K79me3 has been revised (now presented as Fig. S2C).\nThe purpose of this experiment early in the manuscript is not to comprehensively map the distribution of\nH3K79 methylation across the Igh locus, which is provided later by ChIP-seq (Fig. 7G), but rather to\ndemonstrate that H3K79me2/3 levels at Igh are unaffected by CIT stimulation and lost in Dot1l KOs.\nThe reviewer is correct that inclusion of Sγ1 in this context was unnecessary. We have now focused the\nanalysis on the Sµ and Sα regions, using amplicons located downstream of the TSS where H3K79\nmethylation is enriched. For comparison, we also included the Sµ promoter region, which exhibits low\nlevels of the mark as expected. This streamlined and coherent representation now better supports the\nintended point.\n3) Fig. 1G: show H3K79me3 as well\nThe anti-H3K79me3 did not work for WB in our hands. Nevertheless, the purpose of this experiment (now\nFig. 1F) is to confirm that the DOT1L-deficient CH12 clones generated via CRISPR/Cas9 lack H3K79\nmethylation. Since DOT1L methylates H3K79 in a stepwise, distributive manner, the absence of H3K79me2\nimplies that H3K79me3 is also absent.\n4) In the context of Fig. 4: That Pinometostat is not influencing AID expression is not necessarily\ncorroborated by the blots presented - a slight increase in protein levels might be present. The authors\nmight want to quantify this effect (accompanied by a power analysis to determine the numbers of samples\nthey would need to analyze - as the effect seems rather small) to support this claim, which is central to\ndiscriminate between the methylation activity towards H3 or acting as scaffold.\nWe are unsure how to interpret this concern. The original Figure 4 presented genomic data from DOT1L-\ndeficient CH12 cells and did not include western blots. This figure has been eliminated and relevant data\nintegrated into Figs 5 and 7. The western WB assessing the effect of pinometostat on AID expression is\nperformed in primary B cells and presented in Figure 2. These results clearly demonstrate that AID protein\nlevels are not decreased in response to DOT1L inhibition, which was the primary purpose of the control,\nto exclude reduced AID expression as a cause of impaired CSR after pinometostat.\nWe respectfully disagree that a power analysis or further quantification is necessary to evaluate a possible\nslight increase in AID protein. Even if such an increase exists, it would not alter the conclusion that DOT1L\nactivity is required for efficient CSR. Furthermore, it is unclear how the possibility of a minor increase in\nAID expression would contribute to distinguishing between a catalytic versus scaffold role for DOT1L in\nCSR. We address this mechanistic distinction in detail in our response to this reviewer’s major point 2,\nwhere we present evidence supporting a direct role of DOT1L enzymatic activity and H3K79 methylation\nin regulating CSR\n5) The authors conclude in line 236/237 that \"determines a transcriptional environment that enables AID\nactivity,...\". The authors might to make this statement more precise and describe how this environment\nwould look like and how it impacts on AID activity. The link between transcription (other than presence of\nRNAPII) and AID activity is not easy to appreciate - even if it might be within the data.\nWe appreciate this comment and agree that the original phrasing was imprecise. In the revised\nmanuscript, we now present a clearer and better-defined model that describes how DOT1L shapes the\ntranscriptional environment to facilitate AID activity.\n6) The authors cite that \"Loss of H3K27me3 has been proposed to explain gene upregulation in Dot1l-\ndeficient cells, but this mark was absent from the Igh...\". Another paper showing EZH2/DOT1L\ncross-talk was recently published (Appiah et al. 2023, EmboRep), making this point important to substantiate in this\nsystem. If H3K27me3 is not present at the IgH locus, is H3K27ac maybe enriched? How is the epigenetic\nlandscape changing, maybe also in regard to ATAC seq, in Dot1l deficient cells?\nOur initial speculation aimed at explaining why loss of DOT1L led to upregulation of the unscheduled\nGLTUs. In the revised manuscript, we include data that directly link H3K79 methylation to transcriptional\nelongation velocity, offering a mechanistic model to account for the observed transcriptional changes.\nThe unscheduled GLTUs are characteristically poor in H3K79me2/3, and we propose their upregulation is\nan indirect effect of alterations in chromatin loop extrusion, as detailed in previous answers. Accordingly,\nwe have removed speculative discussion regarding a role for other epigenetic marks. While it is possible\n(likely) that even the modest upregulation of unscheduled GLTUs may result in histone modifications,\nlike\nH3K27ac and others introduced co-transcriptionally, these would likely be downstream effects. Due to\nthese considerations we have reduced the space of the manuscript devoted to the deregulation of\nunscheduled GLTUs.\n7) Fig. 4B: The P side in SerP2 chip could serve as a negative control. Statistics is missing in the figure as\nwell as representation of individual data points.\nbiological replicates.\n8) Line 254: In the context of Fig. 4 the authors conclude \"Collectively, these results suggested increased\npromoter-proximal pause release in the Dot1l-deficient cells for those GLTUs that were open and preloaded\nwith RNAPII.\" The openness of the locus was not experimentally addressed.\nThe answer to minor point 6 applies here as well.\n9) Line 257: \"Since we identified DOT1L proximal to AID by BioID, we expected that any role of DOT1L found\nat the IgH might be conserved genome-wide\" I do not understand this rationale. Please make it clearer.\nFor me it would still be possible that the AID/switching is locus specific (admittantly for me this is more\nplausible then a genome wide mechanism).\nWe apologize that this was unclear. We have modified this sentence to clarify that this was embedded in\nthe nature of BioID and the experimental design. BioID detects protein-protein proximity based on\nstatistical enrichment across the entire proteome versus a control protein. For proteins that have wide\ncellular distribution, as is the case of AID, BioID lacks the sensitivity to identify locus-specific interactions.\nIf DOT1L were proximal to AID only at the Igh locus, the interaction would be too spatially restricted to\nyield a detectable signal over the controls (GFP or APOBEC2). This feature is inherent to the method and\napplies broadly to any bait protein, not only AID. Moreover, we conducted the BioID experiments in 293\ncells, which do not have an active Igh locus. So it is implied in our experimental design that proximity\nbetween AID and DOT1L is generalizable, or genome-wide because DOT1L is in the chromatin, rather than\nspecifically at Igh.\n10) For the definition of the quantiles according to H3K79me3: what is the rationale to use H3K79me3 and\nnot H3K79me2? Please make clear to the reader.\nSince Dot1l methylates H3K79 in a distributive manner, quintiles were based on the trimethylated state\nof the mark as it would be a better indicator of Dot1l activity. In CH12-F3 cells, most genes are assigned\nto the same quintile, regardless of whether quintile is based on H3K79me3 or H3K79me2, as seen in the\ntable below.\nQuintile based on H3K79me3\nQ1 Q2 Q3 Q4 Q5\nQ1 3733 176 2 0 0\nQ2 178 3315 406 11 1\nQuintile based\nQ3 0 417 2814 657 22\non H3K79me3\nQ4 0 3 652 2673 583\nQ5 0 0 36 570 3292\n11) The authors observe an increase of RNAPII-S2P in the gene bodies - the scaling in Fig. 4C is probably\ndistance from TSS and goes up to 120kb - an accumulation in the gene body is hardly visible in this present\nform, as it is not indicated in the figure - isn't a representation that spans TSS to TES better suited to show\nthe distributions? At least as an additional representation, as I understand that the drop at 40kb becomes\nrelevant later.\n22\n12) The experiments concerning distribution of RNAPII and its phosphorylated form by means of the two\nantibodies used, are a little confusing and difficult to perceive - this might need better explanation. Within\nFig. S4C the enrichment is given as % of input and this is (much) lower for N20 then for 8WG16. Please\nexplain this discrepancy. Probably the authors would like to point towards the delta between the two\nconditions - however this can be variable between experiments as for N20 a N=2 is displayed, and for\n8WG16 the N number is not given.\nThus - in the present representation of the results, it might be a real effect that is observed here - and this might be in favor of a locus specific observation opposing the claim of a general genome-wide phenomenon?\n13) Fig. 4C indicates an increased/accumulated signal of S2P RNA PII in the 3' end of the genes. How is this\nexplained? What is trapping S2P RNAPII at this location - is there a problem in transcriptional termination?\nPoints 11 to 13 have the same response. We corrected an artifact that led us to incorrectly report a\ngenome wide S2P-RNA increase. We have also added new RNAPII and S5P-RNAPII ChIP-seq and ChIP-PCR\nexperiments, as well as TT-seq, that clarify these points. We have also improved the representation of the\nresults for clarity.\n14) Line 315: why does the conclusion not contain the K4me1/me3 ratio that suggests a similar high\ncorrelation than the paused PolII?\nThe statement on the altered ratio of the K4 methylation somehow comes out of the blue - there is no ChIP seq profiles or ChIP qPCRs that address this point of H3K4me1/me3. But this seems highly relevant as mechanism and probably contributing to the RNAPII S2P escape from pausing.\nWe have removed the speculation about K4me1/me3 ratio in favor of more direct explanations based on\nnew data.\n15) Please clarify the molecular definition of the stripe in regard to the marks/factors shown in Fig. 6A;\nand indicate selection of super enhancers and or criteria in the selection.\nWe have modified the Methods section to expand the explanation of how we determined stripes and\nsuper enhancers in CH12 cells. Briefly. the molecular definition of stripe is based on their chromatin loop\nextrusion activity. Stripes were detected from published Hi-C data using the stripe detection tool Stripenn\n(Yoon et al, 2022). CH12 super-enhancers were identified as described by (Whyte et al, 2013) with the\nsoftware package ROSE based on H3K27Ac ChIP-seq data from dataset GSE121355 (El Khattabi et al,\n2019).\nSpelling/phrasing and other mistakes: line 69: \"However, determining the underlying mechanisms or these histone marks are necessary for AID activity\"line 106: Please add a citation - and maybe make stronger the hypothesis that the complexes have been proposed as being mutually exclusive (\"We also identified several histone readers that can form a complex with DOT1L but are also components of the super elongation complex (SEC)\") line 132: \"and are participate in CSR\". line 198: Papers 47, 48 are not describing demethylation - the citation seems not appropriate. In addition - the cited papers explicitly excluded the study of human Dot1l which seems not to follow the behavior of lower eucaryotes. Fig. S4F: DGE or DEG?\nThank you for highlighting these points, which have been adjusted or corrected unless in the revised\nversion (note that the text has changed substantially so line numbers do not apply).\nActivation Induced Deaminase (AID) is an essential factor that drives somatic hypermutation and class\nswitch recombination in B Cells leading to antibody diversification. It is well accepted that AID mutates\nsingle strand DNA (ssDNA) sequences to generate point mutations during SHM (V gene sequence\nmutations) and DNA double strand breaks (DSBs) to cause CSR. In the process of catalyzing CSR and SHM,\nAID also incorporates SHM all across the B Cell genome (although at a much lower frequency), these\nmutations are related with occurrence of germinal center derived B cell lymphomas. A puzzling and\nimportant question is how does AID identify various DNA target sequences, at V genes and its target DNA\nsequence across the B cell genome. Many past studies have related AID activity with transcription states\nboth at sense and antisense direction, secondary DNA structures like R-loops and G4s (that form during\ntranscription), epigenetics and genome architecture formation, etc. In this study, the Subramani et al.\nproposes that the H3K79me1/2/3 inducing enzymes DOT1L induces AID recruitment to the B cell genome.\nThe authors also propose that DOT1L functions with a similar mechanism to cause CSR, DOT1L deficient\nCH12F3 lines are also deficient in CSR. The authors show that DOT1L induces RNA polII promoter proximal\npausing to accumulate RNA polII at AID target DNA sequences to induce AID recruitment (AID binds to RNA\npolII robustly and thus this is the likely mechanism). In this regard, the authors show that in absence of\nDOT1L promoter proximal paused RNA polII levels decrease but S2 phosphorylated elongating RNA polII\nincreases in the gene body. Thus, this study also makes the argument that S2 phosphorylated RNA polII is\nunlikely to recruit AID. Although some previous studies (cited in the manuscript) have investigated and\nshown DOT1L’s role in AID recruitment and CSR, neither of these two studies suggested mechanisms similar\nto that proposed in this manuscript. Please find below some suggestions that the authors could consider\nas they revise the manuscript for publication. Overall, I feel this manuscript advances the field of AID\nbiology and also provide insights into transcription and promoter-proximal pausing mechanism.\nWe thank the reviewer for the positive comments. While the revised version no longer supports reduced\npause release as the explanation, the additional data and new analyses support a stronger model based\non DOT1L regulating transcription elongation velocity and, therefore, the pausing duration, and AID\noccupancy. The revised manuscript is substantially better, as it can explain the effect of DOT1L deficiency\non gene expression (Fig. 5 and 6) as well as on AID activity by the same mechanisms (Fig. 7).\n1. In figure 1, authors present a nice assay with AID-BirA approach for AID interacting proteins. Some\nhighlights of AID interaction with the NEXT complex and MRN complex was not discussed in the text. NEXT\nsubunits functionally interact with AID has been shown before and with regards to AID activity regulation\n(Lim et al, 2017). Similarly, the interaction with MRN is also relevant for discussion.\nWe appreciate this comment. We now reference the NEXT and MRN complexes, which indicate that our\nscreening in 293 cells recalls complexes implicated in genome wide AID activity in B cells. However, we do\nnot go further into the description of the BioID data for the sake of space and to keep the focus of the\nwork on DOT1L.\n2. The inability of AID to stimulate CSR is an important marker for functional aspect of its cofactors, in cells\nthat are deleted/hypomorphic of cofactor activity. In Fig. 1A versus 1K, the authors show different\nefficiencies of CSR in DOT1L deleted cells (50% versus less that 30%). Some clarity will help since this assay\ndoes decide how important DOT1L is for AID activity (at least during CSR).\nIndeed, as shown in revised Fig. 1D (shRNA knockdown) and Fig 1G (CRISPR/Cas9 ko clones), the extent\nof CSR impairment varies between ~50% and ~30%, depending on the experimental system.\nthe expected clonal heterogeneity associated with CRISPR/Cas9-mediated genome editing in cell lines.\nWe further demonstrate this functional requirement by an orthogonal approach: the treatment of murine\nB cells with the DOT1L inhibitor pinometostat. This results in a reproducible and substantial CSR reduction\nthat accumulates over successive cell divisions (Fig. 2E, H), confirming the importance of DOT1L in primary\nB cells.\nThe consistent reduction in CSR efficiency by 30–50% regardless of the system supports the conclusion\nthat DOT1L is a non-redundant, rate-limiting cofactor for efficient AID-mediated CSR, even if not essential.\nThis consistency between models also justifies our use of CH12 KO clones as a mechanistic model to\ndissect the transcriptional and chromatin-level changes underlying DOT1L function in CSR.\n3. This maybe a confusion on my part, but it is not clear to me why in Fig. 4A the H3K79me2/3 ChIP-seq\ndata for DOTL1-/- cells were not shown. The parallel demonstration of the tracks will make it clear that\nthe mutant cells are deprived of DOT1L activity and still undergo robust transcription (RNA polII levels) and\nelongation (S2PRNA polII levels).\nWe thank the reviewer for this observation. We have revised the corresponding figure (now Fig. 7G) to\nbetter capture the transcriptional consequences of DOT1L loss revealed by the new data.\nWe did not perform H3K79me2/3 ChIP-seq in the Dot1l ko CH12 cells but used input DNA for ChIP as the\nnegative control. We agree that showing H3K79me2/3 ChIP-seq data from Dot1l-deficient CH12 cells\nwould have provided a visually better figure, but we demonstrate the absolute absence of H3K79me2 in\nthe Dot1l-deficient CH12 cells by WB (Fig. 1F) and specifically at the Igh by ChIP-qPCR (Fig. S2C). We have\nimproved the measurement of the RNAPII occupancy by replacing the CUT&RUN with higher coverage\nChIP-seq data and added TT-seq analysis that directly demonstrates the increase in transcription.\n4. The most striking observation in this study is the effect of DOT1L inhibition on AID off-targeting and IgH-\nmyc translocations (Fig. 7 and S6). The authors find with chemical inhibition of DOT1L (chemical inhibition\nis well characterized with all proper controls), the authors report decreased translocations (fig. 7E). Myc-translocations occur at promoters and also in gene body and the PVT1 locus downstream in humans. What\ntranslocations are the ones the authors are mapping, are these Myc promoter translocations, some details\nwith sequencing data of the translocation site would be helpful. Fig. 7E could be done from two different\nmice (instead of B cells from one mouse). Pinemetostat treatment may decrease IgH-Myc translocations\nat promoters, but does the translocations move to a new site (some bands are seen in the gel).\nThe translocation data, now Fig. 3G, was obtained from two different mice that are presented\ndisaggregated. Our assay picked up translocations that occur between Igh Sµ region and Myc gene body.\nWe do not observe a change in the location of break points with Dot1l inhibition (new Fig S3H).\n5. The authors discuss that H3K79me3 modifications occur 5 Kb away from the TSS (Fig. 7A), but SHM and\npromoter proximal pausing occurs in the first 1 kb and 100 bps, respectively. This could be clarified.\nWe thank the reviewer for raising this important point. The lack of overlap between the pausing window\nand the H3K79me3 mark was part of the evidence prompting additional experiments to understand in\ndetail the effect of DOT1L on transcription.\nchanges in transcription elongation velocity in Dot1l-deficient cells that correlate precisely with the\nregions enriched for H3K79me3 in WT cells (new Fig. 5). This supports a model where DOT1L-catalyzed\nH3K79me3 slows RNAPII elongation across gene bodies.\nThe distance from TSS to the peak of H3K79me2/3 is ~1.5kb in both CH12 and primary B cells (as shown\nin Fig. 3A-C). Nonetheless, the lack of overlap between H3K79me3 and the pausing site reinforces our\nconclusion that the observed shortening of pausing in Dot1l-deficient cells is an indirect consequence of\nincreased elongation velocity. This is consistent with previous studies showing that changes in\ntranscription elongation impact on RNAPII pausing, with that faster transcription elongation resulting in\nreduced RNAPII promoter-proximal pausing (Leng et al, 2020; Li et al, 2013).\n6. The model in Fig. 8 and the associated discussion could be improved. Based on my understanding of this\nstudy, DOT1L restricts PolII residence time in a promoter proximal paused state, thereby recruiting AID and\nincreasing SHM.\nWe have revised our model, now presented in Fig. 7H. We posit that in the presence of DOT1L, RNAPII\nelongation velocity is moderated across gene bodies enriched in H3K79me2/3, which indirectly results in\na longer residence time of RNAPII in the paused state near the promoter but also intragenically. \nAccordingly, loss of DOT1L leads to a global increase in RNAPII elongation velocity and shorter pause\nduration, as calculated from TT-seq and RNAPII ChIP-seq. This altered transcriptional dynamic impairs the\nstable association of AID to chromatin, as shown by AID ChIP signal (Fig 7F-G, S7F-G) and thereby reduces\nmutation frequency.\nPrevious studies have supported the fact that bidirectionally transcribed regulatory DNA sequences (like in\npromoters and enhancers) accumulate AID activity and those studies support the conclusions of this\nmanuscript (Pefanis et al, 2014; Meng et al, 2014; Qian et al, 2014). These DNA regulatory sequences have\naccumulated promoter proximal pausing and are most likely be regulated with DOT1L activity. Authors\nshould consider them in the discussion.\nAs we now show in Fig 5A, nascent antisense RNA over the gene body is very modestly affected in DOT1L\ndeficient cells. The effect is much higher in the sense RNA, which further supports a direct role of the\nH3K79me2/3 mark in regulating transcription, as antisense RNA starts at regions with low mark levels. We\nhave mentioned this point with appropriate references.\nThe authors could also described SHM (following DOT1L inhibition) at some of the off-target genes whose\ntranscription was measured and shown to increase (Fig. S6).\nWe appreciate the reviewer’s point. However, because Dot1l deficiency results in faster transcription\nelongation rate at both down- and up- regulated genes (Fig. 6), we anticipate a less stable binding of AID\nto its targets, and therefore impaired activity, even at loci exhibiting more transcriptional activity. \nAccordingly, we show reduced AID occupancy at cMyc, which is modestly downregulated, as well as at the\nIgh Sµ and Sa regions and Ly6e, which are upregulated (Fig 7F-G, S7F-G).\nThis study does highlight that increased transcript levels at AID target sites is not always proportional to\nmutagenic activity of the protein. These observations are consistent with previously published studies\n26\nwhere it has been shown that increase in switch region transcripts or at AID off target genes does not\nalways lead to increase AID mediated DNA mutagenesis. These published studies should be discussed and\nconsidered in the revision.\nWe thank the reviewer for this suggestion, but we do not recall or have been able to identify the\npublications that they are referring to.\nThis manuscript examines the role of the H3K79 methyltransferase Dot1L in transcription, class switch\nrecombination (CSR), and AID mistargeting in the genome. The authors identify Dot1L and other factors\nincluding components of the super elongation complex as AID proximity factors in 293 cells. As the authors\nsummarize, it was previously known that Dot1L contributes to efficient CSR and somatic hypermutation\n(SHM), but the mechanism of action was unknown. The authors also nicely summarize previous findings\nlinking Dot1L to defects in transcription and point out the limitations in those findings. This study\nsubstantially extends previous work by showing that Dot1L regulates transcription by restraining Pol2\npromoter proximal pause release (PPPR). The substantial data presented here and the similarities with\nfindings from other factors that negatively regulate PPPR provide a strong argument that the catalytic\nactivity of Dot1L (and likely H3K79me2/me3) help restrict PPPR. This and the other findings related to the\neffects of Dot1L on transcription are the main strength of the manuscript.\nThe authors also make the claim (last sentence of abstract) that their findings have established that\nregulated pause release is “a critical step for AID activity”. We do not agree. While the findings strengthen\nthe link between Dot1L (and its catalytic activity) and CSR and AID targeting, they do not establish\nmechanism. Specifically, the findings establish a correlation between Dot1L-regulated Pol2 PPPR and\nCSR/AID targeting, not a causal link. One alternative explanation among others is that methylated H3K79\nis the key contribution of Dot1L, not the regulation of PPPR. We feel that the mechanistic advance offered\nby this study regarding the role of Dot1L in CSR/AID targeting is significant but not definitive. This is the\nprimary limitation of the study. We emphasize that we find their model attractive and plausible; it simply\nhasn’t been established. Is this required for publication in Nature Communications? That’s a judgement\ncall, though overall we would support publication without a requirement for substantial additional\nexperiments. At minimum, the manuscript needs to be rewritten to remove overly strong mechanistic\nclaims and to address alternative hypotheses.\nIf the authors decide to try to strengthen the link between dysregulated PPPR and AID targeting, they\nmight attempt to identify a factor(s) that reduces CSR/AID targeting and alters PPPR similarly to Dot1L but\ndoes not reduce levels of methylated H3K79—this would substantially strengthen the correlation and\nargue against a primary role for methylated H3K79. Their study identifies several candidates for study,\ne.g., SEC components.\nIn other regards, the study is rigorous, thorough, and well written (which we appreciated; a pleasure to\nread). The experiments are well designed and controlled, caveats are noted and addressed, and\nconclusions are carefully stated for the most part. The Discussion is well crafted to put the findings in the\ncontext of other work.\nWe were unable to identify many other weaknesses with this study.\nWe thank the Reviewer for the positive comments.\nWe agree that the original manuscript did not establish the mechanism by which DOT1L contributed to\nAID activity. The revised manuscript incorporates substantial new data and corrections that support a\nmore detailed mechanistic model. The model directly links the H3K792m2/3 to regulating transcription\nelongation velocity, which can explain how DOT1L facilitates the activity of AID.\nWe now show that the loss of DOT1L leads to a global increase in RNAPII elongation velocity and shorter\npause duration, as calculated from new TT-seq and RNAPII ChIP-seq data (Fig. 5).\nThis altered transcriptional dynamic impairs the stable chromatin association of AID, as shown by reduced\nAID ChIP signal at the Igh, Myc and Bcl6e loci (Fig. 7). Based on these findings, we propose a mechanism\nwhereby DOT1L, by moderating RNAPII elongation velocity, favors more pausing and/or prevents\n28\nsuccessive polymerases from rapidly displacing AID from the DNA, thereby increasing its dwell time at\ntarget loci and increasing mutagenesis.\nMinor points for consideration are: 1. Line 76: it was unclear what the phrase “strongly correlating with normal H3K79 methylation levels” refers to. Please rewrite.\nThank you. This statement was eliminated.\n2. Line 155: where are the data to support this statement?\nThis data was in Fig. S2E, which is now renumbered as S2F and appropriately referred to in the text.\n3. Line 191: “Since switching to IgA in CH12F3 cells required Dot1l activity”; this claim is overly strong, since\nit implies that CSR is completely eliminated in the absence of Dot1l activity. A similar inappropriate use of\nthe word “required” occurs on line 26 (Abstract).\nWe agree with the reviewer. We have revised the text throughout the manuscript to accurately describe\nthat DOT1L is important to facilitate CSR but is not essential.\n4. Something wrong in the plot of H3K27Ac in S5A: big blocks of purple below the axis.\nThis was the position of the super enhancer. The blocks were eliminated\n5. Line 333 and this paragraph: it is hard to appreciate the changes in the vicinity of Vh and Emu given the\nscale of Fig. 6B; a blow-up of this region in a supplementary figure would be helpful.\nWe have modified the screen shots of genomics data and produced blow-ups of relevant regions to permit\nthe visualization of any changes mentioned in the text.\n6. Grammatical errors, missing words, or typos were found on the following lines (thank you for numbering\nthe lines): 69, 132, 190, 354, 399-400.\nWe have checked the revised manuscript for grammar and typos.\nÁlvarez-Prado ÁF, Pérez-Durán P, Pérez-García A, Benguria A, Torroja C, de Yébenes VG &\nRamiro AR (2018) A broad atlas of somatic hypermutation allows prediction of activation-\ninduced deaminase targets. J Exp Med 215: 761–771\nCao K, Ugarenko M, Ozark PA, Wang J, Marshall SA, Rendleman EJ, Liang K, Wang L, Zou L,\nSmith ER, et al (2020) DOT1L-controlled cell-fate determination and transcription\nelongation are independent of H3K79 methylation. Proc Natl Acad Sci U S A 117: 27365–\n27373\nCecere G, Hoersch S, Jensen MB, Dixit S & Grishok A (2013) The ZFP-1(AF10)/DOT-1\ncomplex opposes H2B ubiquitination to reduce Pol II transcription. Mol Cell 50: 894–907\nCostea J, Schoeberl UE, Malzl D, von der Linde M, Fitz J, Gupta A, Makharova M,\nGoloborodko A & Pavri R (2023) A de novo transcription-dependent TAD boundary\nunderpins critical multiway interactions during antibody class switch recombination. Mol\nCell 83: 681-697.e7\nDuan Z, Baughn LB, Wang X, Zhang Y, Gupta V, MacCarthy T, Scharff MD & Yu G (2021)\nRole of Dot1L and H3K79 methylation in regulating somatic hypermutation of\nimmunoglobulin genes. Proc Natl Acad Sci U S A 118: 2022\nGressel S, Schwalb B, Decker TM, Qin W, Leonhardt H, Eick D & Cramer P (2017) CDK9-\ndependent RNA polymerase II pausing controls transcription initiation. Elife 6\nLeng X, Ivanov M, Kindgren P, Malik I, Thieffry A, Brodersen P, Sandelin A, Kaplan CD &\nMarquardt S (2020) Organismal benefits of transcription speed control at gene boundaries.\nEMBO Rep 21: e49315\nLi J, Liu Y, Rhee HS, Ghosh SKB, Bai L, Pugh BF & Gilmour DS (2013) Kinetic competition\nbetween elongation rate and binding of NELF controls promoter-proximal pausing. Mol\nCell 50: 711–22\nThomas-Claudepierre A-S, Schiavo E, Heyer V, Fournier M, Page A, Robert I & Reina-San-\nMartin B (2013) The cohesin complex regulates immunoglobulin class switch\nrecombination. J Exp Med 210: 2495–2502\nWille CK, Zhang X, Haws SA, Denu JM & Sridharan R (2023) DOT1L is a barrier to histone\nacetylation during reprogramming to pluripotency. Sci Adv 9: eadf3980\nWu A, Zhi J, Tian T, Cihan A, Cevher MA, Liu Z, David Y, Muir TW, Roeder RG & Yu M\n(2021) DOT1L complex regulates transcriptional initiation in human erythroleukemic cells.\nProc Natl Acad Sci U S A 118: e2106148118\nZhang X, Zhang Y, Ba Z, Kyritsis N, Casellas R & Alt FW (2019) Fundamental roles of\nchromatin loop extrusion in antibody class switching. Nature 575: 385–389\n30"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The revised manuscript by Subramani et al, “DOT1L activity limits transcription elongation velocity and favors RNAPII pausing to enable mutagenesis by Activation Induced Deaminase”, is significantly improved and provides additional data and analyses convincingly supporting the main conclusions of the study. I am satisfied with the responses to my critiques. It is satisfying that the new finding of the effect of DOT1L-mediated methylation (likely of H3K79) on transcription elongation velocity can resolve some previously conflicting data. However, the newly found mechanism cannot explain all known cases of DOT1L regulating gene expression, especially those where the catalytic function does not play a role. I encourage the authors to leave room for such possibilities in the discussion. Some thoughts on how H3K79 methylation may slow down transcription elongation would also be interesting to include in the discussion.", "reviewer_2": "In this manuscript by Subramani et. al. the authors implicate DOT1L activity on AID activity for class switch recombination and somatic hypermutation. Several of the findings in the original manuscript have been removed, making this revision read like a new submission. The authors have significantly improved the readability of the manuscript and removed erroneous conclusions from the prior version that are mentioned in the rebuttal.\n\nMajor claims from the abstract\n1.DOT1L deficiency causes mostly increased nascent transcription, accompanied by a modest reduction in RNAPII occupancy.\nThe relation between absolute (not relative to DOT1L KO) steady state gene expression, nascent expression and H3K79me3 quintiles can be made clearer by using heatmaps for each quintile. Currently it is unclear what happens to the ~50% of genes in Q1 that have increased nascent transcription but no change in steady state transcription. i.e. why does a change in nascent transcription not lead to change in steady state transcription- do they have very high absolute expression that cannot increase further?\n2. DOT1L restricts the velocity of transcription elongation proportional to higher order H3K79 methylation (H3K79me2/3)\nThis effect of DOT1L on elongation velocity is known from other systems that they reference.\nThe data in Fig 6D that all genes irrespective of change in expression increase in elongation velocity upon DOT1L knockout is also consistent with the hypothesis that H3K79me3 is irrelevant to gene expression. Fig 6C (which is not referenced at all in the main text) would suggest that downregulated genes are limited by the amount of RNAPII that they can recruit, that is it is an RNAPII initiation problem. Therefore, the author’s claim of mechanistic insight into why some genes are downregulated upon DOT1L KO is somewhat premature.\n3. The relationship between DOT1L and AID association and mutagenesis at target loci remain unclear\nBy the authors’ own statement “AID occupies far more genomic loci than it mutates, indicating that additional regulatory steps determine its activity in situ”. Similarly, DOT1L mediated H3K79 methylation occupies almost the entire transcribed genome. Yet only ~200 genes in B cells and 78 genes in the CH12F3 cell line are AID off targets. Even among these AID off targets, the genes behave like the rest of the H3K79me3 enriched genome in increasing elongation velocity upon DOT1L KO- leading to the question of whether DOT1L functions in a regulatory capacity.\n\nMinor points\n1. I am not in favor of the term “nuclear neighbors” – without a distance limit, any two nuclear proteins can be considered nuclear neighbors.\n2. AID off-target genes exhibited significantly higher levels of H3K79me2 and H3K79me3 downstream of the transcription start site (TSS) compared to the control gene set (Fig. 3A, 3B).\nThese changes extremely small changes although statistically significant in B cells\n3. 3E is not very useful because H3K79me is not normally distributed, unlike other histone modifications.\n4. 3G lacks significance testing\n5. In figure 4 What is 1ry?\n6. Fig 4G CTCF is misspelled\n4. In 7B are red and black lines switched for WT and DOT1L KO?", "reviewer_3": "All my relevant points have been addressed by the authors and the manuscript improved in clarity and in its coherence. I do not have other points to raise.", "reviewer_4": "The authors have revised the manuscript adequately and have addressed concerns.", "reviewer_5": "The authors have added substantial data and significantly revised the manuscript, leading to a revised model for the role of DOT1L in AID action focusing on restraining transcription elongation velocity. Reduced pausing downstream of the TSS in the absence of DOT1L is now interpreted as a secondary effect. The findings are rigorous and well-presented and carefully discussed. While much more molecular detail remains to be discovered regarding the mechanism by which the action of AID is coordinated with Pol2 transcription, the model proposed by the authors is provocative, plausible, consistent with the data, and will serve as a useful guidepost for future studies. A distinct and particular strength of the revised manuscript is the substantial insights it provides regarding the role of DOT1L and H3K79me2/3 in regulating Pol2 transcription, providing a new conceptual framework that helps make sense of a previously confusing literature. The level of advance and insight provided is easily sufficient for publication. Because the paper proposes a new model for DOT1L function, it would be easy to ask for more detailed mechanism but this would not be appropriate given the scope of findings and insights provided already in this paper.\n\nMinor:\n1. Typos on lines 266 and 440.\n2. Line 464: I was puzzled by the phrase “despite only”, since increased elongation velocity and decreased Pol2 “push” in opposite directions. Wouldn’t “accompanied by” be better, since decreased Pol2 emphasizes and supports the finding of increased velocity?"}, "author_response": "Point-by-point responses\nReviewer #1 (Remarks to the Author):\nThe revised manuscript by Subramani et al, “DOT1L activity limits transcription elongation velocity\nand favors RNAPII pausing to enable mutagenesis by Activation Induced Deaminase”, is\nsignificantly improved and provides additional data and analyses convincingly supporting the main\nconclusions of the study. I am satisfied with the responses to my critiques.\nIt is satisfying that the new finding of the effect of DOT1L-mediated methylation (likely of H3K79)\non transcription elongation velocity can resolve some previously conflicting data.\nWe thank the Reviewer for the positive comments and for appreciating the novelty of our finding on\ntranscriptional velocity.\nHowever, the newly found mechanism cannot explain all known cases of DOT1L regulating gene\nexpression, especially those where the catalytic function does not play a role. I encourage the\nauthors to leave room for such possibilities in the discussion.\nThank you for this comment. We agree that there is data in the literature suggesting that DOT1L can\nhave some catalytic activity-independent functions. While our study focuses on the\nmethyltransferase-dependent effects on facilitating AID activity and transcription, it is indeed\npossible the non-catalytic functions of DOT1L could contribute to gene regulation in specific\ncontexts. We have added the following statement to the Discussion to reflect this possibility.\n“While our findings emphasize DOT1L’s catalytic role, particularly high order H3K79 methylation, in\nfacilitating AID activity and regulating gene expression via modulation of elongation velocity, DOT1L\nmay also regulate gene expression through non-enzymatic mechanisms in specific settings, which\nmerit further investigation”\nSome thoughts on how H3K79 methylation may slow down transcription elongation would also be\ninteresting to include in the discussion.\nWe have added a short speculation about this in the Discussion. “While the exact mechanism\nremains speculative, H3K79me2/3 may alter nucleosome dynamics to create a more resistant\nchromatin environment, increasing pausing or backtracking. Alternatively, it may affect recruitment\nor activity of other factors that indirectly modulate RNAPII velocity such as the H3K79me2 reader\nMenin”\nReviewer #2 (Remarks to the Author):\nIn this manuscript by Subramani et. al. the authors implicate DOT1L activity on AID activity for\nclass switch recombination and somatic hypermutation. Several of the findings in the original\nmanuscript have been removed, making this revision read like a new submission. The authors have\nsignificantly improved the readability of the manuscript and removed erroneous conclusions from\nthe prior version that are mentioned in the rebuttal.\nWe thank the Reviewer for appreciating the improvements in the manuscript and the efforts\ninvested in the revisions.\n1.DOT1L deficiency causes mostly increased nascent transcription, accompanied by a modest\nreduction in RNAPII occupancy.\nThe relation between absolute (not relative to DOT1L KO) steady state gene expression, nascent\nexpression and H3K79me3 quintiles can be made clearer by using heatmaps for each quintile.\nCurrently it is unclear what happens to the ~50% of genes in Q1 that have increased nascent\ntranscription but no change in steady state transcription. i.e. why does a change in nascent\ntranscription not lead to change in steady state transcription- do they have very high absolute\nexpression that cannot increase further?\nWe thank the Reviewer for the opportunity to clarify this\npoint.\nTo address the Reviewer’s question, we have quantified\ntotal nascent and steady-state RNA fragments mapped\nto genes across H3K79me3 quintiles using spike-in\nnormalization for both TT-seq and RNA-seq datasets;\nthe closest approximation to absolute values.\nThis analysis involves the comparison of five variables\n(TT-seq and RNA-seq reads for WT and KO, plus\ndirectionality of significant changes).\nFor simplicity, we focused on Q1 genes with\nsignificantly increased TT-seq signal in DOT1L KO\nversus WT cells to directly answer the Reviewer’s\nquestion, (Fig. R1). The heatmap confirms a modest\nincrease in nascent transcription (consistent with the\n~20% relative increase shown in Figure 5A of the\nmanuscript), and importantly, shows that genes with\nunchanged RNA-seq levels span the full range of\nabsolute expression. This data rules out the possibility Figure R1 (Q1 only) - Comparison of steady-\nstate RNA abundance (RNA-seq) and nascent\nthat high or “saturated” expression levels account for\ntranscription (TT-seq) for genes in the highest\nthe lack of change in steady-state RNA.\nH3K79me3 quintile (Q1) that display\nsignificantly increased nascent transcription\nThe comprehensive figure including all genes and\nin Dot1l-/- compared to CH12F3 WT cells. For\nquintiles is appended at the end of the rebuttal for\nmost of these genes, steady-state RNA levels\ncompleteness. It shows the same result for all quintiles, remain largely unchanged (second bar),\nbut it takes time to interpret because of the number of regardless of their absolute expression level,\nvariables.\nwhich is shown as heatmaps of WT and\nDot1l-/- RNA-seq counts.\nWe believe the apparent disconnect between increased nascent transcription and unchanged\nsteady-state RNA levels in a large subset of genes arises from two main factors:\n1) The relationship between nascent transcription and steady state RNA abundance is not\nlinear. Steady-state RNA levels are influenced not only by transcription but also by RNA stability,\nprocessing, and degradation. For transcripts with long half-lives, even substantial changes in\ntranscription may not result in detectable changes in RNA abundance. This underscores the\nimportance of nascent transcriptomics in capturing true transcriptional activity, as our data shows.\nmechanisms of mRNA buffering, in which alterations in transcription or decay trigger\ncompensatory responses that stabilize overall cellular RNA levels (Berry & Pelkmans, 2022;\nHartenian & Glaunsinger, 2019).\nThese additional factors reduce the apparent magnitude of changes in steady-state RNA, even\nwhen transcriptional activity is altered. Figure 5A in the manuscript shows that the relative increase\nin TT-seq signal is most pronounced in Q1 (~20%) and decreases progressively across higher\nH3K79me3 quintiles. This directional proportionality supports our model that H3K79me3 directly\nmodulates elongation velocity and nascent transcription. However, these changes are modest and\nmay not be sufficient to produce measurable differences in RNA-seq.\nTo better visualize these subtle\nshifts, we generated slope graphs\nusing the absolute read counts.\nReviewer Fig. 2, shows the analysis\nfor Q1 genes, with full dataset\nappended at the end. These graphs\nshow that genes identified as\nupregulated by DESeq2\nbioinformatic analysis exhibit larger\nTT-seq increases than those\nFigure R2 (Q1 only) – Comparison of steady-state RNA abundance (RNA-seq)\nclassified as unchanged,\nand nascent transcription (TT-seq) for genes in the H3K79me3 first quintile (Q1)\nhighlighting a limitation in reaching that show significantly increased nascent transcription in DOT1L KO CH12F3\ncells. For most genes, steady-state RNA levels remain largely unchanged (top\nsignificant changes in steady state\npanel) despite significantly higher nascent RNA levels (bottom panel).\nmRNA expression. However, note that the relatively few upregulated genes show a larger increase\nin TT-seq.\n2) Functional consequences of DOT1L loss. The observed increase in elongation velocity\nfollowing DOT1L loss represents a deviation from the normal state. It is therefore reasonable to\nassume that such a mechanism, if evolutionary selected, confers a functional benefit. As\ndiscussed in the manuscript, we propose a model in which the impact of increased elongation\nvelocity resulting from DOT1L loss varies depending on both gene expression levels and gene\nlength. Our study identifies this phenomenon but does not fully elucidate the underlying\nmechanism. Nevertheless, we provide evidence suggesting that increased nascent transcription in\nthe absence of DOT1L is not uniformly productive. This is evident in highly transcribed long genes\nthat show decreased TT-seq and are concomitantly downregulated.\nWe hypothesize that similar transcriptional inefficiencies may occur at other genes enriched in\nH3K79me2/3, thereby contributing to the observed disconnect between transcriptional activity and\nmRNA abundance. In genes with intermediate expression, faster elongation may increase TT-seq\nsignal, but this could reflect a mixture of productive and non-productive transcription, which might\noffset each other, mitigating changes in mature mRNA output. In contrast, for lowly expressed and\nshorter genes, the increase in velocity and thereby TT-seq appears sufficient to enhance mRNA\noutput, so they are detected as differentially upregulated.\nWe do not think that adding the two figures generated for the Reviewer will improve the manuscript,\nbut they will remain available for readers thanks to the transparent review process of the journal.\nNonetheless, prompted by this comment, we have modified a paragraph of the Discussion, stating\n3\nthat we are only proposing a model, and summarizing the points above (major changes are\nhighlighted):\n“The modest increase in elongation velocity in DOT1L-deficient cells represents a deviation from\nthe normal transcriptional regulatory state. It is therefore reasonable to assume that such a\nmechanism, if evolutionary selected, confers a functional benefit. One possibility is that\ntranscription in the absence of DOT1L might not always lead to fully mature mRNA and therefore\nthe TT-seq signal includes both productive and non-productive transcription. We propose a model\nto account for the consequences of increased elongation velocity resulting from DOT1L loss\naccording to gene expression levels and gene length. Genes significantly upregulated in DOT1L-\ndeficient cells tend to be shorter, have the lowest elongation velocities and steady state transcript\nabundance in WT cells, as well as the longest pausing duration. These features make it more likely\nthat increases in transcription, even if a proportion is unproductive, would result in detectable\nincreases in transcript abundance. Genes with unchanged expression typically fall within\nintermediate expression ranges, where modest gains in nascent transcription are insufficient to\nalter steady-state RNA levels. In these cases, productive and non-productive transcripts may offset\neach other, resulting in unchanged mRNA output. Additionally, cellular mRNA buffering\nmechanisms, whereby changes in transcription or decay trigger compensatory responses, may\nfurther stabilize RNA levels (Berry & Pelkmans, 2022). In contrast, genes that in WT cells show the\nhighest elongation velocities, short pausing durations, and elevated H3K79me3 enrichment, may\nalready be operating near the maximal transcriptional output and might not tolerate further\nincreases in elongation velocity upon DOT1L deficiency, thus resulting in reduced mRNA\nabundance.\nSee also next point.\n2. DOT1L restricts the velocity of transcription elongation proportional to higher order H3K79\nmethylation (H3K79me2/3)\nThis effect of DOT1L on elongation velocity is known from other systems that they reference.\nWe respectfully disagree about this point. While some works have suggested a role of DOT1L in\ntranscription elongation (Wille et al, 2013; Cecere et al, 2013, referenced in the manuscript),\nthese\nworks have not analyzed elongation velocity. Only one study (Wu et al. 2021, also referenced in the\nmanuscript) has measured transcription elongation rate in the presence and absence of DOT1L. In\nthis study, the authors measured elongation rate in WT and Dot1l-/- K562 cells using 4sUDRB-seq\ntechnique and found no global change in elongation rate. However, the authors did not examine\nvelocity changes along gene bodies, nor did they stratify genes by levels of H3K79 methylation,\nwhich is what allowed us to detect the changes. We show that the increase in elongation velocity\nspatially coincides with regions of higher-order H3K79 methylation, which is mostly concentrated\nin the region ~1-10kb downstream of the TSS. Moreover, the method used by Wu et al allows\ninference of elongation rate only in long genes (>30 kb), , which partly explains why they might have\nunderestimated elongation velocity changes that are maximal at the gene region decorated with\nH3K79me2/3. To the best of our knowledge, we are the first to report a genome-wide analysis of\nelongation velocity, and to report the spatial as well as quantitative correlation with H3K79me2/3.\nupon DOT1L knockout is also consistent with the hypothesis that H3K79me3 is irrelevant to gene\nexpression.\nWe have addressed this in the response to the previous point. We state that “Our model also\nprovides an explanation…”. Thus, we are proposing a model that is consistent with the data. A\nmodel in which elongation velocity affects gene expression, as we discuss in the paper, fits better\nwith our data than the null hypothesis. We acknowledge that further work is needed to validate this\nmodel, and we hope our findings will encourage future studies to explore this possibility in greater\ndetail.\nFig 6C (which is not referenced at all in the main text) would suggest that downregulated genes are\nlimited by the amount of RNAPII that they can recruit, that is it is an RNAPII initiation problem.\nThank you for pointing out the omission of referencing figure 6C, we have corrected this oversight\nby modifying the text as follows: “Metagene analyses confirmed the predominance of increased\nnascent transcription in DOT1L deficient cells and revealed distinct transcriptional profiles for up-\nand downregulated genes in WT cells (Fig. 6C).”\nWe interpret the apparent reduction in RNAPII in Dot1l-/- cells at the downregulated genes as a\nmanifestation of increased RNAPII elongation velocity (Fig. 6D). Although we have not explicitly\nruled out a potential decrease in initiation at these genes, the consistent increase in elongation\nvelocity across all categories, suggests that this is the primary effect of DOT1L deficiency. Any\npotential reduction in transcription initiation at downregulated genes is likely a downstream or\ncompensatory effect of the increased velocity.\nTherefore, the author’s claim of mechanistic insight into why some genes are downregulated upon\nDOT1L KO is somewhat premature.\nWe agree with the Reviewer that we do not elucidate the mechanism explaining how increased\ntranscription elongation velocity causes downregulation of certain genes. This was acknowledged\nin the Discussion indicating we were discussing potential mechanisms, which we have now\nmodified to clarify and include the point about initiation:\nWe have not addressed the mechanism by which increased elongation velocity leads to\ndownregulation of this small subset of highly transcribed genes in DOT1L-deficient cells. While we\ncannot formally rule out a defect in transcription initiation in these genes, we think is more likely\nthat it reflects the loss of a process favored by RNAPII slow down over the H3K79me2/3 marks. A\nfew non-mutually exclusive mechanisms are possible. One possibility is that DOT1L supports a\ntranscriptional checkpoint, potentially by influencing promoter-proximal pausing . While depletion\nof pause-release inhibitors can increase RNAPII release into elongation, it often reduces\nprocessivity or causes premature termination, disproportionally affecting longer genes . However,\nthis scenario is not consistent with our data, as pausing duration was unchanged in downregulated\ngenes in DOT1L-deficient CH12F3 cells. Alternatively, RNAPII deceleration where H3K79\nmethylation peaks downstream from the TSS may facilitate coupling with processivity or splicing\nfactors . Another possibility is that excess nascent RNA disrupts elongation dynamics in DOT1L-\ndefficient cells. While nascent RNA promotes elongation condensate formation up to an optimal\nthreshold, excessive RNA can trigger condensate dissolution. In both models, longer genes are\n5\nmore vulnerable to early termination or reduced processivity, consistent with findings in DOT1L-\ndeficient CH12F3, primary B cells, and embryonic stem cells .\n3. The relationship between DOT1L and AID association and mutagenesis at target loci remain\nunclear\nBy the authors’ own statement “AID occupies far more genomic loci than it mutates, indicating that\nadditional regulatory steps determine its activity in situ”. Similarly, DOT1L mediated H3K79\nmethylation occupies almost the entire transcribed genome. Yet only ~200 genes in B cells and 78\ngenes in the CH12F3 cell line are AID off targets. Even among these AID off targets, the genes\nbehave like the rest of the H3K79me3 enriched genome in increasing elongation velocity upon\nDOT1L KO- leading to the question of whether DOT1L functions in a regulatory capacity.\nWe appreciate the Reviewer’s comment and the opportunity to clarify our interpretation.\nWe respectfully disagree with the implication that our data fail to support a functional relationship\nbetween DOT1L and AID activity.\nWe do not claim that DOT1L is essential for AID targeting. Rather, our data support a facilitator\nrole for DOT1L, akin to other known cofactors like ELOF1. We show that loss of DOT1L, and\nconsequently H3K79me2/3, leads to a reduction in AID activity, which is accompanied by a\nlocal increase in transcription elongation velocity and reduced Pol II pausing. As Pol II pausing is\nknown to favor AID occupancy and activity, our data provides a plausible mechanistic link between\nDOT1L function and AID-mediated mutagenesis.\nImportantly, we do not claim that DOT1L activity is sufficient to confer specificity. We claim\nthat high DOT1L activity, as inferred from elevated levels of H3K79me2/3, characterizes loci\npermissive to AID mutagenesis. This is a consistent observation across both primary B cells and\nthe CH12F3 cell line. While it is true that both AID and H3K79 methylation are broadly distributed\nacross the genome, mutagenesis by AID is restricted to a subset of loci. This selective activity is a\nwell-established phenomenon and has been similarly observed for other cofactors such as SPT5,\nSPT6, and ELOF1, which are enriched at AID targets and contribute to its activity, yet are not\nexclusive to those sites\nIt must be noted that the current catalog of AID targets is likely incomplete. As detection methods\nimprove, additional targets are likely to be identified, which we predict will still correlate with high\nDOT1L activity.\nIn response to the reviewer’s concern, we have revised the manuscript to avoid the term “enabler,”\nwhich may imply necessity, and instead use “facilitator” or “contributor” to more accurately reflect\nthe role of DOT1L. We have made some additional text changes to better convey the messages\nsummarized above.\nMinor points\n1. I am not in favor of the term “nuclear neighbors” – without a distance limit, any two nuclear\nproteins can be considered nuclear neighbors.\nThank you for this comment. We have replaced this for the more accurate “factors proximal to\nnuclear AID”.\n6\n2. AID off-target genes exhibited significantly higher levels of H3K79me2 and H3K79me3\ndownstream of the transcription start site (TSS) compared to the control gene set (Fig. 3A, 3B).\nThese changes extremely small changes although statistically significant in B cells.\nWe agree the changes were small for H3K79me2 (1.4-fold in AID targets vs control group), although\nthey are larger for H3K79me3 in primary B cells (1.7-fold). Fold changes were added to Fig. 3A.\nThis increase is consistent with AID preferring to deaminate at sites with signs of high DOT1L activity.\n3. 3E is not very useful because H3K79me is not normally distributed, unlike other histone\nmodifications.\nWe agree with the reviewer that H3K79 methylation does not follow a normal distribution across\nthe genome. However, we believe Figure 3E remains informative, as it highlights that the peak\nlevels of H3K79me2/3 are maximal at AID-targeted regions. We have modified the text to express it\nin this way. In addition, this figure panel supports our interpretation that it is not merely the\npresence of the mark, but specifically elevated levels of H3K79 methylation that preferentially\nfacilitate AID activity.\n4. 3G lacks significance testing\nThe experiment shown in Figure 3G was performed using p53-deficient mice in two independent\nreplicates, each involving a separate mouse on different days. While the result is clear and\nreproducible, statistical testing is not applicable with only two data points. Unfortunately, the p53\nmouse colony has since been discontinued, and repeating the experiment would require importing\nmice and obtaining new ethical approval, which will take several weeks.\nHowever, we believe the consistency across the two independent experiments supports the\nconclusion regarding reduced off-target AID activity. Moreover, this finding is further corroborated\nby the data presented in Figure 3F, strengthening the overall interpretation.\n5. In figure 4 What is 1ry?\nThank you for pointing this out. It was our internal abbreviation for “Primary” and remained in the\nfigure, we have corrected this to indicate Mouse splenic B cells.\n6. Fig 4G CTCF is misspelled\nThank you for pointing this out. We have corrected this mistake.\n4. In 7B are red and black lines switched for WT and DOT1L KO?\nThank you for pointing this out. Yes, the legend was inverted and it has been corrected.\nReviewer #3 (Remarks to the Author):\nand in its coherence. I do not have other points to raise.\nThank you very much.\nReviewer #4 (Remarks to the Author):\nThe authors have revised the manuscript adequately and have addressed concerns.\nThank you very much.\nReviewer #5 (Remarks to the Author):\nThe authors have added substantial data and significantly revised the manuscript, leading to a\nrevised model for the role of DOT1L in AID action focusing on restraining transcription elongation\nvelocity. Reduced pausing downstream of the TSS in the absence of DOT1L is now interpreted as a\nsecondary effect. The findings are rigorous and well-presented and carefully discussed. While\nmuch more molecular detail remains to be discovered regarding the mechanism by which the\naction of AID is coordinated with Pol2 transcription, the model proposed by the authors is\nprovocative, plausible, consistent with the data, and will serve as a useful guidepost for future\nstudies. A distinct and particular strength of the revised manuscript is the substantial insights it\nprovides regarding the role of DOT1L and H3K79me2/3 in regulating Pol2 transcription, providing a\nnew conceptual framework that helps make sense of a previously confusing literature. The level of\nadvance and insight provided is easily sufficient for publication. Because the paper proposes a new\nmodel for DOT1L function, it would be easy to ask for more detailed mechanism but this would not\nbe appropriate given the scope of findings and insights provided already in this paper.\nWe are grateful for the positive comments on the revision. Thank you also for appreciating the\nmechanistic insights into DOT1L and H3K79 methylation, and for recognizing that the scope of our\nfindings is sufficient for this work.\nMinor:\n1. Typos on lines 266 and 440.\nThank you, the typos were corrected.\n2. Line 464: I was puzzled by the phrase “despite only”, since increased elongation velocity and\ndecreased Pol2 “push” in opposite directions. Wouldn’t “accompanied by” be better, since\ndecreased Pol2 emphasizes and supports the finding of increased velocity?\nThank you for this suggestion, “accompanied by” works better and we have made the change.\nWe also note that increased transcriptional velocity and reduced RNAPII ChIP-seq signal are not\nnecessarily contradictory. A faster-moving polymerase spends less time at any given locus, which\ncan result in lower occupancy and thus reduced ChIP signal, even in the context of increased\nelongation\nBerry, S., & Pelkmans, L. (2022). Mechanisms of cellular mRNA transcript homeostasis. Trends in Cell Biology, 32(8),\n655–668. https://doi.org/10.1016/j.tcb.2022.05.003\nHartenian, E., & Glaunsinger, B. A. (2019). Feedback to the central dogma: cytoplasmic mRNA decay and transcription\nare interdependent processes. Critical Reviews in Biochemistry and Molecular Biology, 54(4), 385–398.\nhttps://doi.org/10.1080/10409238.2019.1679083\nindividual genes in Dot1l-/- CH12F3 B cells across H3K79me3 quintiles (Q1–Q5). Genes stratified by quintiles of\nH3K79me3 enrichment (Q1–Q5) and classified by relative nascent transcription (TT-seq) in Dot1l-/- compared to WT cells\nas increased, decreased, or unchanged. For each nascent category, the corresponding steady-state RNA status (RNA-\nseq) is displayed as upregulated, downregulated, or unchanged. Heatmaps show WT and Dot1l-/- counts for TT-seq and\nRNA-seq, with absolute values represented by independent color scales.\nquintiles (Q1–Q5). Genes stratified by quintiles of H3K79me3 enrichment (Q1–Q5) and classified by relative nascent\ntranscription (TT-seq) in Dot1l-/- compared to WT cells as increased, decreased, or unchanged. For each nascent\ncategory, the corresponding steady-state RNA status (RNA-seq) is displayed as upregulated, downregulated, or\nunchanged. Slope graphs show distribution of WT and Dot1l-/- absolute values for each category, with the same gene\njoined by lines between the two genotypes. Box plots and means of the data are shown.\n11"}]

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