LUMINIDEPENDENS orchestrates global
transcriptional repression in Arabidopsis

Clara Bergis-Ser, Qingyi Wang, Xiaoning He, Maherun Nisa, Vickie Kaiser, Christelle Mazubert, Jeannine Drouin-Wahbi, Rim Brik-Chaouche, Layla Chmaiss, Jelle van Leene, et al.

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To cite this version:

Clara Bergis-Ser, Qingyi Wang, Xiaoning He, Maherun Nisa, Vickie Kaiser, et al. LUMINIDEPENDENS orchestrates global transcriptional repression in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America, 2025, 122 (49), pp.e2527372122. 10.1073/pnas.2527372122. hal-05394413

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Distributed under a Creative Commons Attribution 4.0 International License
1 LUMINIDEPENDENS orchestrates global transcriptional repression in
2 Arabidopsis

3

4 Clara Bergis-Ser¹,*, Qingyi Wang¹, Xiaoning He¹, Maherun Nisa¹, Vickie Kaiser¹, Christelle Mazubert¹,

5 Jeannine Drouin-Wahbi¹, Rim Brik-Chaouche¹, Layla Chmaiss¹, Jelle Van Leene²,³, Geert De Jaeger²,³,

6 Jose Gutierrez-Marcos⁴, Catherine Bergounioux¹, Clara Bourbousse⁵,⁶, David Latrasse¹, Moussa

7 Benhamed¹,⁷,⁸ and Cécile Raynaud¹⁵

8

9

10 ¹Université Paris-Saclay, CNRS, INRAE, Univ Evry, Institute of Plant Sciences Paris-Saclay (IPS2), Orsay,
11 91405, France

12 ²Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium.

13 ³VIB Center for Plant Systems Biology, Ghent, Belgium.

14 ⁴School of Life Science, University of Warwick, Coventry CV4 7AL, UK

15 ⁵Sorbonne Université, CNRS, INSERM, Development, Adaptation and Ageing, Dev2A, F-75005 Paris,
16 France

17 ⁶Sorbonne Université, CNRS, Inserm, Institut de Biologie Paris-Seine, IBPS, F75005 Paris, France

18 ⁷Université de Paris Cité, Institute of Plant Sciences Paris-Saclay (IPS2), 91190, Gif-sur-Yvette, France

19 ⁸Institut Universitaire de France (IUF), Orsay, France.

20

21 * Present address: Université de Sherbrooke, Faculté de Médecine et des Sciences de la Santé,
22 Sherbrooke, Québec J1H 5N4 (Canada) clara.bergis.ser@usherbrooke.ca

23 ⁵Author for correspondence

24

25 **Classification:** Biological Sciences, Plant Biology

26 **Keywords:** Replication stress, transcription regulation, RNA polII

27
28

Abstract

Genomic integrity is constantly challenged by transcription/replication conflicts, a major source of replication stress and instability across all life forms. While extensive studies have elucidated mechanisms for resolving transcription/replication conflicts in animals, yeast, and prokaryotes, their counterparts in plants remain largely unexplored. Through a forward genetic screen, we identified LUMINIDEPENDENS (LD), previously known for its role in regulating the flowering repressor *FLC*, as a key factor in mitigating replication stress in plants. Notably, transcriptomic analyses reveal that LD loss results in the upregulation of over half of the Arabidopsis genes, placing LD as a global transcriptional repressor. Consistent with this role, LD directly binds a substantial portion of the Arabidopsis genome and interacts with the MED18 subunit of the Mediator complex to modulate RNA polymerase II phosphorylation. These findings uncover a fundamental function of LD in fine-tuning transcription at a genome-wide scale, with potentially an additional role in suppressing transcription-replication conflicts by locally dampening transcription and promoting replication fork progression. Our work highlights a previously unrecognized genome-protective strategy in plants, opening new avenues for understanding transcription-replication conflict management in eukaryotic systems.

43

**Significance statement:** In a genetic screen designed to identify novel regulators of the plant replication stress response, we identified the LUMINIDEPENDENS (LD) protein. By combining genetics, transcriptomic and epigenomics approaches, we show that loss of *ld* or its partner *fld* mitigate developmental defects caused by replication stress. Beyond this potential role during DNA replication, we show that these two proteins are general repressors of transcription, likely acting through their interaction with the MEDIATOR complex and modulating RNA polII activity.

50
## 51 Introduction

In all living organisms, nucleic acid transactions, including DNA replication and transcription, are major sources of DNA lesions due to replication errors, formation of DNA breaks, DNA-protein crosslinks, or torsional tensions induced by the replication or transcription process (1, 2). Consequently, Replication Stress (RS) caused by stalling of the replication machinery due to obstacles on the DNA template, is an important cause of genomic instability in proliferating cells. In mammals, RS is a hallmark of cancer cells triggering the accumulation of mutations while also rendering tumor cells sensitive to drugs targeting the DNA Damage Response (DDR) pathway (3). Moreover, RS is also increasingly recognized as a key determinant in aging (4). In plants, the spontaneous occurrence of replication stress has not been investigated in details. However, the DDR triggered by RS is highly conserved with that of animals as its response depends on the ATR kinase that activates the transcriptional regulator SOG1, a functional homolog of p53, which controls the expression of cell cycle regulators and DNA repair genes (5). In agreement with its role in RS response, ATR is essential to the viability of Arabidopsis *pol2a* mutants as they display constitutive RS due to the partial inactivation of the replicative DNA polymerase ε (6). By contrast, although SOG1 contributes to the RS response, it is not required for the viability of *pol2a* mutants, thus suggesting that other pathways function downstream of ATR and independently of SOG1 in plants (6). Indeed, E2F transcription factors act in concert with SOG1 to fine-tune the expression of RS-induced genes, and to activate the WEE1 kinase (7). The latter contributes to the RS response by phosphorylating various targets controlling proteolysis or alternative splicing, thereby leading to cell cycle arrest (8–10). Interestingly, under control conditions, the RS response seems to be dispensable for vegetative growth in Arabidopsis since *atr* null mutants show no developmental defects (11). By contrast, loss of ATR severely affects embryo development and growth in maize and barley (12, 13), suggesting that RS is ubiquitous in plants, like in mammalian cells. Thus, former studies indicate that plant RS responses are mediated by an intricate set of regulatory layers and signaling pathways, of which more actors likely remain to be identified.

RS can be largely regarded as an inevitable consequence of normal cellular activities. Indeed, encounters between the replisome and the transcription machinery, termed transcription-replication conflicts (TRCs), are considered major sources of genomic instability, particularly when they occur in the head-on (HO) orientation (14, 15). Various mechanisms have been described to limit or resolve TRCs in both procaryotic and eucaryotic genomes. First, genome structure has been shaped through evolution to co-orient transcription with the direction of DNA replication, thereby limiting HO-TRCs (16, 17), especially in the case of highly transcribed genes. In addition, transcription and replication are largely anti-correlated during S-phase (18, 19). Yet, transcription and replication cannot be mutually exclusive since replication origins are enriched in the TSS of transcriptionally active genes (20). Thus,
several mechanisms contribute to the resolution of TRCs, which act both on the replisome to allow
replication restart, and on RNA polymerases (RNAP) to reduce transcription locally through RNAP
removal, resolution of R-loops (tripartite 3D structures composed of an RNA:DNA hybrid and a
displaced single-stranded DNA), and modulation of chromatin organization (15, 21).

The mechanisms limiting TRCs in plants have been poorly investigated, but could involve the
modulation of histone modifications (22). As in other eucaryotes, transcriptional activity is promoted
by histone acetylation (23), whereas histone methylation either promotes or represses transcription
depending on the modified lysine. In euchromatin, H3K4me3 correlates with active transcription, and
defects in H3K4me3 deposition by the RNA Polymerase II-associated complex COMPASS result in
defects in gene expression (24). By contrast, H3K4me1 and H3K4me2 are thought to promote or
repress gene expression depending on the context (25). Whether impinging on these modifications
could play a role in mitigating RS by avoiding or resolving TRCs is to date unknown. Nevertheless,
several lines of evidence point to a dual role of the FLD/LD/SDG26 complex in the control of
transcription and replication fork progression. This complex was originally identified for its role in the
autonomous pathway controlling flowering time (26-28). FLD is a homolog of the human demethylase
LSD1, involved in the repression of the floral repressor FLC via the demethylation of H3K4me1 (28),
while LD is a putative, albeit atypical, TF of the homeobox family (HB) (26, 27, 29), and SDG26 is a
putative histone methyl-transferase, although whether its catalytic activity is required for FLC
regulation remains unclear (30). Together, these three proteins allow long-lasting FLC silencing by
promoting H3K4me1 removal and subsequent H3K27me3 deposition (31, 32). In addition, the genome-
wide function of LD and FLD was extended to the regulation of transcription at convergent genes:
according to this model, FLD removes the H3K4me1 mark at convergent transcription sites, thereby
slowing-down transcription (33). In parallel with this work, R-loop formation at the FLC locus has been
shown to interfere with the progression of the replication fork, suggesting that factors controlling R-
loop formation and FLC expression could play a role in mitigating TRCs (34). Consistently, loss of the
FCA or FY proteins involved in R-loop resolution, and acting upstream of LD and FLD at the FLC locus,
affects replisome progression genome-wide (34). However, the role of LD and FLD in this process has
not yet been examined.

Through a genetic screen aimed at identifying novel regulators of the replication stress response, we
identified LD as a suppressor of the *pol2a* mutant, which exhibits impaired cell cycle progression and
reduced growth due to constitutive replication stress. Chromatin binding analysis and transcriptomic
profiling unexpectedly revealed that LD functions as a global repressor of gene expression in
*Arabidopsis*. This regulatory function operates independently of replication stress, uncovering a
previously unrecognized role of the FLD–LD complex in the broad control of transcriptional activity.
119

# Results

120

## LD is involved in the replication stress response

121 To identify new factors involved in the DDR, we performed an EMS genetic screen to isolate
122 suppressors of the *pol2a* mutant, in which replication stress response is constitutively activated,
123 leading to reduced growth and early flowering (6, 35). We mutagenized seeds of *pol2a* mutants
124 expressing the DDR reporter *pCYCB1;1::CYCB1;1D-box-GUS* (36). This enabled us to then screen for
125 suppressors using two criteria: (i) suppression of the growth phenotype of *pol2a* plants and (ii)
126 reduction in the accumulation of CYCB1;1, thus limiting the identification of flowering time suppressors
127 only. One of these suppressors, named *soa225*, shows partially restored growth compared to *pol2a*
128 and rescues its early flowering phenotype (Fig. 1A, SI Appendix Fig. S1A). Furthermore, expression of
129 *CYCB1;1* is also reduced compared to *pol2a* in both root and shoot meristems (Fig. 1B, C). We identified
130 by whole genome sequencing five non-synonymous mutations that were genetically linked to the
131 *soa225* phenotype (Supplementary Table S1). Among those, only one introduced a premature stop
132 codon in the *LUMINIDEPENDENS* (*LD*) gene (SI Appendix Fig. S1B). To confirm that mutations in *LD*
133 induce replication stress suppression, we obtained a T-DNA insertion mutant (*ld-4*) (Fig. 1A and SI
134 Appendix Fig. S1C-D), which showed notable delays in flowering time (SI Appendix Fig. S1A). We then
135 generated *ld-4 pol2a* double mutants and found that, as in *soa225*, this new *LD* mutant allele partially
136 restored the growth defects typical of *pol2a* (Fig. 1A and SI Appendix Fig. S1A, E). Furthermore, crosses
137 between *soa225* and *ld-4 pol2a* mutants confirmed that *soa225* and *ld-4* mutations were allelic (SI
138 Appendix Fig. S1E). Finally, both the *ld* and the *ld pol2a* mutant could be rescued by a GFP-tagged LD
139 under the control of the native LD promoter (*pLD::LD-GFP*, SI Appendix Fig. S1E), further validating the
140 identification of *ld* as the causal mutation in the *soa225* mutant. Because the *soa225* mutant contains
141 other mutations, further analyses were carried out using the *ld-4* allele (hereafter referred to as *ld*).

142 LD was previously characterized for its role in controlling flowering time (26), and we found that the
143 early flowering phenotype of the *pol2a* mutant (35) is fully rescued by *ld* (SI Appendix Fig. S1A, E). To
144 further demonstrate the role of LD in replication stress responses, we tested the sensitivity of *ld* to
145 hydroxyurea (HU). We found that *ld* roots grown on 1mM HU were significantly longer than those of
146 WT plants, while being similar when grown in control medium (Fig. 1D), confirming that *ld* has a
147 reduced sensitivity to replication stress. Further phenotypic characterization confirmed that rosette
148 area was partially restored in the *ld pol2a* mutant (Fig. 1E). In addition, we tested the sensitivity of
149 *gigantea* (*gi*), another late flowering mutant, also shown to produce very large rosettes due to its late
150 flowering (37). As shown on Fig. S2, only *ld* showed tolerance towards HU, whereas *gi* was as sensitive
151 as the wild-type. Thus, although some of the effects of the *ld* mutation on the growth of the *pol2a*
152

mutant likely result from a biomass effect associated with flowering time, the specific sensitivity of *ld* to HU, that is not observed in *gi*, confirms that LD is specifically involved in replication stress.

153

154

Considering that LD accumulates at high levels in the nuclei of meristematic cells (Aukerman *et al.*, 1999) and that *ld* mutants grow better than WT plants under replication stress conditions, we hypothesized that LD could play a role in the control of the cell cycle. To test this hypothesis, we measured the size of root meristems in WT (Col-0), *pol2a*, *ld* and *ld pol2a* seedlings. We found that *ld pol2a* double mutants display partially restored meristem size compared to *pol2a* (Fig. 1F-G). We have previously shown that growth defects in *pol2a* mutants are associated with dramatically delayed cell cycle progression (6). We therefore measured the cell cycle and S-phase length using cumulative EdU incorporation (38) to test if cell cycle progression defects are rescued in *ld pol2a* mutants. As expected, we found the average cell cycle length to be 10h longer and the average S-phase length to be increased by almost 8h in *pol2a* mutants than in WT. Further, we found that the average length of the cell cycle was reduced by around 4 hours (Fig. 1H) and that the average S-phase length was also shortened by about 2 hours (Fig. 1I) in *ld pol2a* double mutants compared to *pol2a*. These results suggest that LD is involved in the induction of cell cycle checkpoints in response to replication stress. Taken together, these results support the view that the increased plant tolerance to replication stress in *ld* is mediated by the modulation of cell cycle progression.

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## LD is a bona fide repressor of gene expression

170

The LD protein harbors a putative Homeo-domain (27), suggesting it could function as a transcriptional regulator (29). We therefore attempted to identify its direct targets by chromatin immunoprecipitations. To this end, we complemented the *ld* mutant with the *pLD:LD-GFP* construct (SI Appendix Fig. S1E) and found that LD-GFP protein was constitutively expressed, albeit at lower levels in mature organs (SI Appendix Fig. S3). We generated data from two independent ChIP-seq experiments (SI Appendix Fig. S4A, B) and found over 8000 LD-targets (Fig. 2A, SI Appendix Fig. S4C, Supplementary Table S2). Most of LD-binding sites were found in gene promoters, accounting for 75% of its binding sites, despite promoter regions comprising only 9% of the *Arabidopsis* genome (Fig. 2B). Additionally, LD binds to both the 5' and 3' regions of its target genes (Fig. 2C). To analyze the relationship between LD binding and gene expression, we grouped genes in four quartiles based on their expression level, and plotted LD binding on each quartile (Fig. 2C), finding that LD binding correlates with gene expression. We then identified the sequence motives enriched at LD binding sites and found that instead of the typical homeobox (HB) motif other sequence motives, such as bHLH/bZIP/BES1/E-box/BZR/Trehelix motives, GAGA repeats, WRKY, TCP and HB-like, were associated with LD binding (SI Appendix Fig. S4D). This diversity suggests that LD possesses a low sequence

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185 specificity and/or that it can be recruited to the chromatin independently of its putative DNA-binding domain. Next, we carried out a transcriptome analysis on shoot apices from WT (Col-0), *pol2a*, *Id* and *Id pol2a* plants (Supplemental Table S3) and found that despite the very large number of genes associated with LD binding, only 200 were differentially expressed in *Id* and not significantly enriched amongst LD targets (SI Appendix Fig. S4E). Moreover, the majority of *pol2a* DEGs were also mis-regulated in the *Id pol2a* mutant, and the number of DEGs was actually higher in double mutant than in single mutant plants, indicating that LD loss of function does not restore wild-type gene expression levels in the *pol2a* mutant (SI Appendix Fig. S5A). Furthermore, because *pol2a* mutants show constitutive activation of DDR genes, (6, 7), we checked whether the *Id* mutation specifically restored the expression of these genes, but we did not observe significant restoration of DDR gene expression in *Id* plants (SI Appendix Fig. S5B).

196 The very large number of putative LD targets, the lack of specificity amongst enriched TF binding motifs, the poor overlap between LD genomic binding sites and DEGs in the *Id* mutant and the absence of correlation between changes in gene expression and phenotypic rescue of the *Id pol2a* mutant compared to the *pol2a* mutant, prompted us to hypothesize that LD may affect gene expression globally rather than being involved in the regulation of specific genes. However, standard RNA-seq analysis is not suitable to detect a global change in mRNA accumulation (39, 40). To address this we carried out a RNA-seq experiment including spike-in RNAs from drosophila S2 cells, which allows mRNA data normalization (41). We found that *pol2a* plants had similar numbers of genes showing an increase and decrease in mRNA accumulation, however, both *Id* and *Id pol2a* displayed only an increase in transcript accumulation (Fig. 2D, Supplementary Table S4, SI Appendix Fig. S6A-C). In both *Id* and *Id pol2a* plants the increase in transcript accumulation affected most genes in the genome (Fig. 2D, Supplementary Table S4, SI Appendix Fig. S6A-C), and the set of up-regulated genes was very similar between the two mutants (Fig. 2E, SI Appendix Fig. S6D). This correlation was even more obvious when focusing only on LD targets identified by ChIP-seq (SI Appendix Fig. S6E). Importantly, this increase in mRNA accumulation observed in *Id pol2a* cannot be attributed to higher levels of endoreduplication, since the increased endoreduplication observed in *pol2a* (6) was rescued by the *Id* mutation (SI Appendix Fig. S6F). This global increase in mRNA levels also holds true for DDR-related genes (SI Appendix Fig. S6G-H), suggesting that the partial rescue of the *pol2a* phenotype results from this global reprogramming of DDR gene expression, rather than from a rescue of DDR activation. Taken together, our data suggest that LD plays a pivotal role in global mRNA production.

216 **LD directly impinges on the transcription machinery**
We next set to investigate the mechanism(s) by which LD modulates gene expression by carrying out an Affinity Purification assay coupled with Mass Spectrometry (AP-MS) based on the GS<sup>Rhino</sup> tag (42, 43). These assays confirmed that LD interacts with FLD, SDG26, ELF2, ELF4, and APRF1 (30, 31, 44) (Table 1, Supplementary Table S5). Genetic analyses revealed that, similarly to *ld*, *fld* mutants could also suppress the growth defects and meristem length reduction commonly observed in *pol2a* plants (Fig. 3A-C). Moreover, *fld* mutants exhibited increased mRNA accumulation to the same extent as *ld* plants (Fig. 3D, SI Appendix Fig. S7). Furthermore, both mutations led to the up-regulation of highly similar gene sets (Fig. 3E, SI Appendix Fig. S7, Supplementary Table S6). It is worth noting that between independent replicates of the spike-in RNAseq, the fold change observed for up-regulated genes displayed some variations. Nevertheless, the set of up-regulated genes was highly reproducible between experiments, and this was further confirmed by performing a third independent replicate of spike-in RNA seq on the *ld* mutant (SI Appendix Fig. S8, Supplementary Table S7). Since loss of *ld* and *fld* have very similar consequences on mRNA accumulation, we took advantage of the publicly available ChIP-seq data for the FLD protein (33). As shown on Fig. 3F, genes targeted by LD also displayed FLD binding, and were up-regulated genes in *ld* and *fld*. Interestingly, LD was found to be enriched in the 5' region of the gene and to a lesser extent in the 3' region, whereas FLD showed clear enrichment in the 3' region (Fig. 3F, SI Appendix Fig. S9 and Supplementary Table S8), suggesting the presence of gene loops at these loci. Together, these findings suggest that LD and FLD act in concert to suppress transcriptional activity.

<caption>**Table 1: LD Protein partners identified through AP-MS. Affinity Enrichment Score is the product of the NSAF ratio vs the large dataset and the -log(p-value) of the t-test.**</caption>

Only previously known partners and interactors selected for further analysis are listed in this table. The full list of partners can be found in Supplementary Table S5A-B.

<table><thead><tr><td>Protein accession</td><td>Protein name</td><td># Identified with min 2 peptides (in 3 replicates)</td><td>Affinity Enrichment Score</td></tr></thead><tbody><tr><td>AT4G02560</td><td>LD | Homeodomain-like superfamily protein</td><td>3</td><td>156585</td></tr><tr><td>AT1G72630</td><td>ELF4-L2 | ELF4-like 2</td><td>3</td><td>116724</td></tr><tr><td>AT1G17455</td><td>ELF4-L4 | ELF4-like 4</td><td>3</td><td>107562</td></tr><tr><td>AT3G10390</td><td>FLD, RSI1 | protein FLOWERING locus D-like protein</td><td>3</td><td>71351</td></tr><tr><td>AT5G14530</td><td>APRF1 | Transducin/WD40 repeat-like superfamily protein</td><td>3</td><td>53587</td></tr><tr><td>AT1G76710</td><td>ASHH1, SDG26 | SET domain group 26</td><td>3</td><td>62452</td></tr><tr><td>AT2G22370</td><td>MED18 | mediator of RNA polymerase II transcription subunit</td><td>2</td><td>976</td></tr><tr><td>AT1G07950</td><td>MED22B | Surfeit locus protein 5 subunit 22 of Mediator complex</td><td>3</td><td>313</td></tr></tbody></table>
241 FLD is thought to modulate transcription speed at convergent genes by facilitating the removal of the
242 H3K4me1 histone mark (33), but this process affects around 2000 genes, which alone cannot account
243 for the global increase in mRNA accumulation observed in *Id* and *fld*. We therefore investigated
244 whether total H3K4me1 levels could be modified in *Id* mutants. Western-blot analysis revealed that
245 global H3K4me1 accumulation remained unchanged in *Id* (SI Appendix Fig. S10A). Furthermore, the
246 activating mark H3K9ac, which strongly correlates with gene activation, was present at similar levels
247 in *Id* and wild-type plants (SI Appendix Fig. S10B). Together, these findings suggest that LD could also
248 modulate transcription through mechanisms that are independent of histone modifications. Indeed,
249 in our AP-MS analysis, we identified several chromatin associated proteins among LD partners,
250 including chromatin readers, and a topoisomerase, consistent with the finding that the effect of LD
251 and FLD on the expression of convergent genes depends on Topoisomerase I (33). In addition, we
252 found several proteins directly involved in transcription (Supplementary Table S5), including MED18
253 and MED22, two sub-units of the Mediator complex known for its role in promoting transcription
254 initiation (45, 46). Bimolecular Fluorescence Complementation (BiFC) and yeast two-hybrid assays
255 further confirmed that LD interacts with MED18 (Fig. 4A, SI Appendix Fig. S11).

256 Since the Mediator complex is known to promote transcription initiation by facilitating RNA Pol II
257 phosphorylation (45), we performed ChIP-seq experiments to determine whether RNA Pol II loading
258 on chromatin is increased in *Id* mutants. Upon recruitment on gene promoters, RNA Pol II is initially
259 phosphorylated on Ser5 of its C-Terminal repeat Domain (CTD) enabling transcription initiation. During
260 elongation, Ser2 phosphorylation becomes predominant, and transcription termination is ultimately
261 carried out by Ser2P RNA Pol II, after removal of Ser5P (47). To investigate RNA Pol II distribution in
262 wild-type and *Id* we performed ChIP-seq using specific antibodies for Ser2P and Ser5P RNA Pol II. To
263 refine our analysis of Pol II occupancy, genes were categorized into five quantiles based on their
264 expression levels in the *Id* mutant, as quantified by RNA-seq (Supplementary Table S9). We observed
265 an increase of Ser5P RNA Pol II occupancy across all genes up-regulated in *Id*, with this effect becoming
266 more pronounced in genes exhibiting higher expression levels (Fig. 4B and SI Appendix Fig. S12). As
267 expected, the levels of Ser2P RNA Pol II correlated with transcription, and a clear increase in Ser2P
268 RNA Pol II occupancy was observed along the gene body of most highly expressed genes. These findings
269 suggest that LD modulates transcription through its interaction with the Mediator complex and its
270 influence on RNA Pol II phosphorylation.

271 Discussion

272 Transcriptional activity is intricately linked to genome integrity through multiple mechanisms. First,
273 Transcription-Coupled DNA repair (TCR) serves as a key pathway for repairing small DNA lesions within
transcribed regions (48). Second, RNA Damage sensing by the transcriptional machinery has recently emerged as a key element of the cellular response to genotoxic stress (49). Finally, the coordination of transcription and DNA replication is essential for minimizing transcription-replication conflicts, thereby ensuring efficient S-phase progression during the cell cycle (15). In this study, we identified a previously unrecognized function of LD in the replication stress response through a genetic screen. Specifically, *Id* mutants were isolated in a suppressor screen of the replicative stress mutants *pol2a*, and we further confirmed that LD loss enhances plant tolerance to replication stress (RS) using hydroxyurea treatment. Importantly, this effect was not observed in the *gi* mutant that also affects flowering time, confirming that the effect of LD on RS response is not linked to increased biomass production resulting from late flowering.

Genome-wide analysis of LD binding sites and gene expression changes in *Id* mutants revealed that LD functions not only in the RS response but also as a global negative regulator of transcription. More than 13,000 protein-coding genes were upregulated in the *Id* mutant, and LD was found to bind more than half of them. Since these analyses were conducted on whole Arabidopsis plantlets rather than exclusively on proliferating or S-phase cells, these findings suggest that LD limits transcription globally, independently of RS occurrence. In addition, genetic analyses confirmed that this transcriptional repressive function is shared by FLD, an LD interactor, suggesting that the LD-FLD, complex, along with its previously characterized partners (30, 31), plays a key role in transcriptional repression. The mechanisms by which the LD-associated complex limits transcriptional activity may be diverse. This complex has been shown to reduce H3K4me1 and H3K36me3 levels at the *FLC* locus (31), and FLD-mediated H3K4me1 removal has been proposed to slow transcription at convergent genes (33). However, we did not detect a global increase in H3K4me1 levels that would correspond to the increased mRNA accumulation observed in *Id* mutants. This suggests that LD-dependent transcriptional control involves additional mechanisms beyond histone modifications.

Transcription operates as a cyclical process comprising initiation, elongation, termination, and the recycling of RNA polymerase II (RNA Pol II) to restart the cycle, with each step tightly regulated (50). Through AP-MS analysis, we identified LD as a co-purifying partner of MED18 and MED22, two subunits of the Mediator complex. Furthermore, BiFC and yeast two-hybrid assays confirmed the LD-MED18 interaction. In Arabidopsis, the mediator complex consists of at least 28 subunits (51) organized into three distinct modules: the head module, which interacts directly with RNA Pol II; the tail module, which engages with specific transcription factors; and the middle module, which bridges the two extremities and transmits regulatory signals from the tail to the core transcription machinery (45). MED18 and MED22 belong to the head module (45), and Mediator is essential for efficient transcription initiation and elongation (52), suggesting that LD may function in close proximity to RNA
308 Pol II and modulate its activity. Consistently, ChIP-seq data reveal that LD binds mostly the promoter
309 region of its target, and to a lesser extent their 3' end, while FLD is mostly found in the 3' region of
310 their common targets. This suggests that LD and FLD play a role throughout the transcription cycle,
311 possibly by impinging on RNA polII recycling that is favored by gene looping (53), reinforcing their
312 potential function in transcriptional regulation beyond elongation.

313 Alongside its interaction with the Mediator complex, phosphorylation of the C-terminal repeat domain
314 (CTD) of RNA Pol II serves as a key regulatory mechanism. RNA Pol II is initially recruited to promoters
315 in its unphosphorylated form and phosphorylation at Ser5P enables transcription initiation, while Ser2
316 phosphorylation promotes elongation. Finally, Ser5P dephosphorylation facilitates transcription
317 slowdown and termination (47, 50). Here, we found that RNA Pol II Ser5P levels were increased in *Id*
318 mutants, suggesting that LD contributes to Ser5 dephosphorylation. Supporting this hypothesis, LD
319 was recently proposed as a functional homolog of Ref2/PNUTS, a component of the Cleavage and
320 Polyadenylation Specificity Factor (CPSF) (44). In yeast and metazoans, Ref2/PNUTS interacts with the
321 protein phosphatase PP1 to regulate RNA Pol II Ser5 phosphorylation, thereby modulating the
322 elongation rate (54). Until recently, no PNUTS homolog had been identified in *Arabidopsis*. However,
323 LD was found to share structural features with PNUTS, and Ser5P levels were dramatically increased
324 at the *FLC* locus in *Id* mutants (44).

325 Whether this LD-mediated regulation of Ser5P extended beyond *FLC* remained unclear. Here, we show
326 that the increase in Ser5P levels is widespread across most upregulated genes in *Id* mutants, suggesting
327 that LD regulates transcription at least in part through RNA Pol II phosphorylation. RNA Pol II activity
328 is governed by a network of highly interconnected factors during transcription elongation. Proteins
329 containing a TFIIS N-terminal domain interact with TND-interacting motif (TIM)-containing proteins,
330 forming a dynamic network that links the transcription machinery, including the Mediator complex
331 and the PNUTS/PP1 complex (55). Notably, LD harbors a TFIIS domain, further supporting its potential
332 role as PNUTS functional homolog. Under this model, LD recruitment to transcription sites via LD-
333 MED18 interaction would facilitate Ser5P dephosphorylation, likely through APRF1 activity, thereby
334 modulating transcription levels (Fig. 5A). However, two recent studies reported that PNUTS is essential
335 for transcription, as its acute depletion via targeted protein degradation resulted in a global
336 transcriptional decrease (56, 57). This contrasts sharply with the transcriptional upregulation observed
337 in *Id* loss-of-function mutants in *Arabidopsis*, highlighting a major functional distinction between LD
338 and PNUTS.

339 Along with MED proteins and APRF1, our IP-MS analysis revealed that LD could interact with several
340 chromatin modifiers such as SUVR2, an ATRX-like protein of unknown function, DEK4, and the GTE4
protein. SUVR2 is involved in the RdDM pathway of gene silencing, and its activity is thought to go through its interaction with chromatin remodeling complexes, as its catalytic residues are dispensable for its activity (58). By contrast, GTE4 and DEK4 function as transcriptional activators (59, 60). Thus, the role of LD in transcriptional regulation likely involves highly complex interactions with both positive and negative regulators of gene expression. It is worth noting that caution for false positives and false negatives is always needed when interpreting interactomics results. Nevertheless, the large number of chromatin-associated proteins potentially associated with LD suggests that, in addition to its interaction with the Mediator complex and its impact on RNA Pol II phosphorylation, LD could also function through the modulation of histone modifications or chromatin remodelling, as suggested by (33).

Beyond its role as a global transcriptional repressor, our genetic analysis indicates that LD also plays a role in the replication stress (RS) response. It may be recruited to transcription-replication conflict (TRC) sites to attenuate transcription and facilitate TRC resolution (Fig. 5B), although this hypothesis will require further investigation. Interestingly, a DNA topoisomerase was identified among LD partners, further supporting our hypothesis that it could play a role in TRC resolution. The *ld* mutation was identified as a suppressor of the *pol2a* mutant, in which the RS response is constitutively activated, and *ld* mutants exhibit increased tolerance to hydroxyurea (HU). These findings appear paradoxical given LD's function as a transcriptional repressor, as the resolution or avoidance of transcription-replication conflicts (TRCs) generally require transcriptional downregulation, and mutants deficient for these mechanisms are generally hypersensitive to replication stress. Indeed, in mammals, TRCs in rapidly proliferating cells are prevented by transcriptional repression through FOXD3 recruitment to highly expressed genes (61). More broadly, several mechanisms that facilitate RNA Pol II removal from the replication machinery are essential for limiting TRCs (62, 63), including PNUTS-mediated RNA Pol II degradation (64). However, unlike PNUTS-deficient cells, which exhibit replication defects even in the absence of exogenous RS (64), *ld* mutants do not display such defects, suggesting key functional differences between LD and PNUTS.

Two non-mutually exclusive mechanisms could explain the suppression of RS-induced defects in *ld* *pol2a* mutants. First, the global upregulation of gene expression, including that of cell cycle regulators, may shift the balance between cell cycle repressors and activators, thereby partially inactivating RS-triggered checkpoints. Second, we propose that RS leads to the downregulation of specific genes in S-phase cells via LD activation, and that LD loss prevents this repression, ultimately promoting improved plant growth. While future studies will be required to further elucidate LD's role in RS, we propose that LD primarily functions as a global transcriptional repressor, whose activity can be locally recruited under RS conditions to reinforce transcriptional silencing at TRC sites.
375 **Materials and Methods**

376 **Plant material and growth conditions**

377 We used Columbia-0 (Col0) lines for WT plants, SAIL_743_B07 (Id-4) from the SAIL collection and SALK_015053 (fld) from the SALK collection. The *gi-2* mutant has been described previously (37). All 378 mutants are in Col-0 background. Seeds were surface-sterilized in diluted Bayrochlore™ for about 25 379 minutes under agitation, then washed two times with sterile water and left in sterile water at 4°C in 380 the dark for 2 days. Seeds were then sown on ½ MS medium, with 1,1g of Murashige and Skoog (MS) 381 medium (Basalt salt Mixture M0221, Duchefa) and 4g of Agar (Agar HP696, Kalys) in 500ml of sterile 382 water. Plants were grown for 10 days *in vitro* and transferred to soil. For rosette area measurement, 383 plants were placed one week in short day conditions (8h light, 20°C) then transferred to long day 384 conditions (16h light, 21°C). For other experiments, plants were directly placed under long day 385 conditions in the greenhouse. 386

387 **Molecular cloning and plant transformation**

388 Constructs encompassing the *LD* cDNA expressed under the control of the *LD* promoter were obtained 389 using the GreenGate technology (65). The chosen LD promoter sequence corresponds to 1kb upstream 390 of the ATG. Similarly, GreenGate cloning was used to generate the *LD-GS*<sup>rhino</sup> fusion for AP-MS, 391 combining the RPS5a:XVE inducible promoter with the *LD* cDNA and the *GS*<sup>rhino</sup> tag (66). Clones 392 encompassing the 5' and 3' moieties of LD were purchased from Eurofins Genomics. After GreenGate 393 assembly, constructs were introduced in Agrobacterium (ASE strain containing the pSOUP plasmid), 394 and plants were transformed via the floral dip method (Clough and Bent, 1998). Primers used for 395 cloning are listed in Supplementary Table S10. For BiFC experiments, the *LD* and *MED18* cDNAs were 396 cloned in the pENTR3C and the pDONR221 vectors, respectively (Thermofisher Scientific™). Because 397 the MED18 protein does not include a Nuclear Localization Signal (NLS), a short fragment encoding a 398 NLS was introduced upstream of MED18 in the pDON221 vector. LD and MED18 were then introduced 399 in the pBIFP1 and pBIFP3 vectors, respectively. The same NLS sequence was cloned in the pENTR3C 400 vector, and nuclear-targeted YFP moieties were used as negative controls. 401

401 **EMS mutagenesis**

402 About 5000 seeds were surfaced sterilized with Bayrochlore™ and washed three times with sterile 403 water. They were imbibed overnight at 4°C in a 0.1% KCl solution (w/v). The next morning, EMS was 404 added to the solution (final concentration 0.3% w/v), and seeds were incubated for only 7.5h due to 405 the high sensitivity of the *pol2a* mutant to genotoxins. Seeds were next washed twice with sodium 406 thiosulfate (100mM) for 15 min, and twice with sterile water for 15 min. Seeds were finally sown on
407
408

MS medium and grown at 20°C for two weeks. 2000 plants were transferred to the greenhouse, and seeds were harvested by pools of 10 plants for subsequent phenotypic screening.

409

## Root growth experiments

410
411
412
413
414
415

Four-day-old plantlets were transferred to new plates of 1/2 MS medium and 1/2 MS medium supplemented with 1 mM HU and aligned at the top of a square petri dish. They were then placed vertically in a growth chamber under long day conditions. After about one week, Petri dishes were scanned, and root length was measured using the Fiji software (https://imagej.net/software/fiji/). At least 20 roots were measured in each experiment. Three independent biological replicates were measured. Statistical analysis was conducted on the R 4.2.1 version software.

416

## Rosette area measurement

417
418
419
420
421

Pots of different genotypes were randomized on trays in the growth chamber to avoid light or temperature bias. Rosette's pictures were taken for each genotype and analyzed in Fiji. Rosettes were filled with a homogeneous red color, and areas were selected with the wand tool. At least 40 rosettes were measured in each experiment. Three independent biological replicates were measured. Statistical analyses were conducted on the R 4.2.1 version software.

422

## GUS staining

423
424
425
426
427

Root and shoot meristems were placed in 100% cold acetone for 15 minutes, and GUS activity was as described in (Ni et al., 2009). Plantlets were incubated at 37°C for one hour and then washed with 70% ethanol. Then, they were fixed with 4% PFA for 20 minutes under vacuum and cleared with a chloral hydrate solution at RT overnight. (8g CCl3CH(OH)2 (Sigma), 2 ml of 50% glycerol anhydrous and 1 ml of sterile water). Images were taken with a NIKON, AZ100 and a video camera, Nikon R11.

428

## Confocal microscopy

429
430
431
432
433
434
435

We used 10-day-old meristems stained for about 5 minutes in a propidium iodide (10mM) bath. We used a laser with an excitation wavelength of 561nm on a Zeiss LSM 880 laser scanning confocal microscope and imaged them by acquiring the fluorescence of emission wavelength between 560 and 700nm, using an X20 lens. At least 20 meristems were measured per experiment. Three independent biological replicates were performed. For BiFC experiments, we used a laser with an excitation wavelength of 514nm. YFP fluorescence was acquired between 520 and 570nm, and chlorophyll autofluorescence was acquired between 620 and 720nm.

436

## EdU labelling
Two lines of about 40 seeds were sown on square petri dishes containing ½ MS medium, and germinated vertically for 5 days. Liquid ½ MS medium supplemented with 10 µM EdU was poured on top of the roots until each root tip was covered with the solution. Plantlets were fixed at a minimum of four different time points, separated by a minimum of two hours each, in 4% (w/v) PFA in 1X PME (0.5 M PIPES pH 6.9, 50 mM MgSO4, 10 mM EGTA) for 15 minutes inside Eppendorf tubes pierced on the cap, under a vacuum. Sample preparation and image acquisition were performed as described in (7). EdU marked nuclei were counted in Fiji. At least 15 images with an average of 300 nuclei were used. Four independent biological replicates were measured. Statistical analyses were conducted on the R 4.2.1 version software.

## Western blotting

Nuclear proteins were extracted, and separated on a Bolt 4-12% Bis-Tris Plus Wedgewell Gel under NuPage MES buffer conditions, with migration at 100V for 1 hour. Semi-dry transfer was performed using the iBlot® 2 Dry Blotting System. Molecular weight markers were established using the SeeBlue™ Plus2 pre-stained protein ladder, with sizes indicated on the left in kDa. Primary antibodies used were anti-H3 (ab1791, Rabbit, 1:20,000) and anti-H3K9ac (Milipore 07-352, Rabbit, 1:10,000), followed by a secondary anti-Rabbit IgG antibody conjugated with alkaline phosphatase (Invitrogen™ 65-6122, 1:20,000 for H3 and 1:10,000 for H3K4me1). Detection was performed with an exposure time of 285 seconds, and the H3 band is observed within the expected molecular weight range of 15-17 kDa.

## Bimolecular Fluorescence complementation

For transfection and protein expression, saturated cultures of *Agrobacterium tumefaciens* GV3.101 encompassing plasmids of interest were washed twice and resuspended in an infiltration medium containing 20 mM MES (pH 5.2), 20 mM MgCl₂, and 150 µM acetosyringone. The bacterial cultures were adjusted to an OD₆₀₀ of 0.3 and co incubated for 1h at room temperature, with shaking, before being infiltrated into 3-4-week-old *Nicotiana benthamiana* leaves. Fluorescence imaging was performed 3 days after agroinfiltration using a LSM880 ZEISS confocal microscope, equipped with a x20 objective. YFP was excited at 514 nm, and emission was detected between 515 and 580 nm. Chlorophyll autofluorescence was also detected between 670 and 735nm. Images were acquired and analyzed using the Zen software.

## RNA-seq experiments

For standard RNA-seq, total RNA was extracted from the meristematic zone and the first two leaves of thirty 7-day-old plantlets with the NucleoSpin RNA-Plus Kit (Macherey-Nagel) according to the manufacturer's instructions. For spike-in RNAseq, total RNA was extracted from 400K nuclei using the
469 TRIZol reagent (Invitrogen) according to the manufacturer's instructions. For both types of
470 experiments, libraries were prepared with 2 µg of total RNA thanks to the NEBNext Ultra II RNA Library
471 Prep Kit for Illumina according to the manufacturer's instructions. Differential Expression analysis was
472 performed as described in (67) for standard RNA-seq and in (41) for spike-in RNAseq. Detailed
473 procedure and analysis methods can be found in SI Appendix.

474 ChIP-seq experiments

475 ChIP-seq experiments and analyses were done as described in (67). Detailed procedure and analysis
476 methods can be found in SI Appendix.

477 Affinity purification and Mass Spectrometry analysis (AP-MS)

478 To do this experiment, we followed the protocol described in (42, 43). Detailed procedure and analysis
479 methods can be found in SI Appendix. For this project, an Arabidopsis PSB-D cell culture expressing C-
480 terminally GS<sup>rhino</sup> tagged LD under control of the estradiol-inducible XVE promoter was prepared.
481 Detailed procedure and analysis methods can be found in SI Appendix.

482 Data availability

483 ChIP-seq raw data are available at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE292381
484 (reviewer token: qnoteaekptovtyh). RNAseq raw data are available at
485 https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE292906 (reviewer token: orcnaeyajhylxop).

486 AP-MS results are available at the PRIDE repository (http://www.ebi.ac.uk/pride) with the accession
487 number PXD065507 (reviewer token: FLGI7w4Ny05m).

488

489 Acknowledgements

490 This work was supported by grants of Agence Nationale de la Recherche (21-CE20-0027), a PhD
491 fellowship from the University Paris-Saclay to Clara Bergis-Ser, Chinese Research Council Scolarships
492 for Qingyi Wang and Xiaoning He, and a grant from the Fondation pour la Recherche Médicale
493 (ECO201806006824) for Maherun Nisa. IPS2 benefits from the support of Saclay Plant Sciences-SPS
494 (ANR-17-EUR-0007). BIFC plasmids were a kind gift of Dr François Parcy (LPCV, Grenoble, France). We
495 thank Fredy Barnèche for stimulating discussions and critical reading of the manuscript. We thank the
496 VIB Proteomics Core and Dominique Eeckhout for the LC-MSMS analyses.

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647

648 **Figure Legends**

649 **Figure 1. LD is involved in replication stress response**

650 **A.** Phenotype of WT (Col0), *pol2a*, *Id*, *soa225* and *Id pol2a* plants. *Pol2a* mutants display reduced size, *soa225* is the suppressor of *pol2a* isolated in the genetic screen, showing partial restoration of the *pol2a* growth defects. The *Id* mutant corresponds to the SAIL line SAIL_743_B07 which harbors a T-DNA insertion in the *Id* gene. Plants were grown in vitro for 10 days, and on soil for 14 days. Bar = 1cm
651
652
653
654 for all panels. **B.** GUS-staining pattern obtained in the different mutant lines expressing the *CYCB1-1:GUS* construct. Red arrows indicate shoot meristems. GUS staining was increased in *pol2a* mutants,
655
656 and returned to lower levels in the *soa225* mutant. Bar = 50µm for all panels. **C.** Quantification of GUS activity (fluorescence unit/minute/µg of protein extract) in the indicated genotypes. Values are
657
658 average +/- SD of three biological replicates, different letters indicate significantly different values
659 (Wilcoxon sum-ranked test). **D.** Root length measurement of 10-day-old Col0 and *Id* plantlets grown
660
661 on ½ MS supplemented or not with hydroxyurea (HU, 1 mM). A Two-Way Anova test was performed
662
to compare genotypes, followed by a Tukey HSD test for multiple comparisons. Letters represent
663
statistically different groups (p-value<0.05, n>20). This result is representative of 3 independent
664 experiments. **E.** Rosette area measurement of 24-day-old plants of the indicated genotypes. A Kruskal-
665
Wallis test was performed to compare the genotypes, followed by a Wilcoxon rank sum exact test for
666
pairwise comparisons. Letters represent statistically different groups (p-value<0.05, n≥45). The partial
667
restoration of *Id pol2a* phenotype seen in this graph is representative of 3 independent experiments.
668
669
**F.** Confocal images of 10-day-old meristems. Cell walls were stained with propidium iodide (10µg/mL).
670
Red arrows show the end of the meristematic zone. Bar = 50µm for all panels. **G.** Meristem length
671
measurement of 10-day-old plantlets. A Two-Way Anova test was performed to compare genotypes,
672
followed by a Tukey HSD test for multiple comparisons. Letters represent statistically different groups
673
(p-value<0.05, n≥34). This result is representative of 3 independent experiments. **H.** Cell cycle length
674
and *I. S*-phase length in the indicated genotypes. The red dotted lines show cell cycle and S-phase
length duration in the wild-type (Col-0). The error bars represent the SD between two independent
experiments.
675

675

676 **Figure 2. LD is a global repressor of gene expression.**

677 **A.** Top: Metaplot showing average LD binding profile centered on the peak's maximum obtained in the
678 ChIP-seq analysis performed with *pLD::LD-GFP* lines. Bottom: Heatmap displaying ChIP signal at
679 individual loci. The color gradient shows the sequence coverage in the ChIP data. The input is shown
680 as a negative control. **B.** Schematic representation of loci and pie charts comparing the proportion of
681 promoter, and genic sequences in the total Arabidopsis genome (TAIR 10 genome) (left) and amongst
682 LD binding sites (right). **C.** Metaplot showing the distribution of LD on its target genes. Genes were
683 distributed between five quantiles of expression based on the number of read counts obtained in the
684 wild-type. **D.** Volcano plots summarizing the results of the spike-in RNAseq experiments. Significantly
685 mis-regulated genes (adj p-value <0.05) are shown as colored dots (blue = down-regulated genes, red
686 = up-regulated genes). The *pol2a* mutant shows roughly the same number of up and down-regulated
687 genes compared to the wild-type. By contrast, *Id* and *Id pol2a* mutants show a global increase in gene
expression. E. Venn diagram showing the overlap between up-regulated genes in *ld* and *ld pol2a* mutants. The number of common up-regulated genes is higher than expected by chance (Fisher's exact test *p* = 0) F. Venn diagram showing the overlap between LD targets identified by ChIP-seq and up-regulated genes in *ld*. The number of common up-regulated genes is higher than expected by chance (Fisher's exact test *p* < 8.737e-307). G. Heatmap showing LD binding (left) and the observed FC in *ld* on LD targets identified by ChIP-seq (right).

694

695 **Figure 3.** LD and FLD likely function together during replication stress response

696 **A.** Pictures showing the rosette size of the different mutants. Plants were grown in vitro for 10 days
697 and then on soil for three weeks. *fld pol2a* has a partially restored rosette size compared to *pol2a*, like
698 *ld pol2a*. Bar = 2 cm for all panels. **B.** Confocal images of 10-day-old meristems. Plantlets were stained
699 with propidium iodide (10µg/mL), Red arrows show the end of the meristem zone. Bar = 50 µm for all
700 panels. **C.** Meristem length measurement of 10-day-old plantlets. A Kruskal-Wallis test was performed
701 to compare the genotypes, followed by a Wilcoxon rank sum exact test for pairwise comparisons.
702 Letters represent statistically different groups (p-value < 0.05, n > 20). This result is representative of
703 2 independent experiments. **D.** Volcano plots summarizing the results of the spike-in RNA-seq
704 experiments. Significantly mis-regulated genes (adj p-value <0.05) are shown as colored dots. **E.** Venn
705 diagram showing the overlap between up-regulated genes in *ld* and *ld pol2a* mutants. The number of
706 common up-regulated genes is higher than expected by chance (Fisher's exact test *p* = 0). **F.**
707 Comparison of LD and FLD binding on genes identified as LD targets. Left: Metaplots (top) and
708 heatmaps (bottom) showing LD or FLD binding profile centered on the genes obtained in the ChIP-seq
709 data generated with *pLD:LD-GFP* lines and with *pFLD:FLD-flag* lines (Inagaki *et al*, 2021). The color
710 gradient shows the sequence coverage in the ChIP data. The input is shown as a negative control for
711 both experiments. Right: Heatmap showing the observed FC in *ld* and *fld* on the same genes.
712

712

713 **Figure 4.** LD modulates RNA Pol III activity through its ability to bind the MED18 sub-unit of the
714 mediator complex

715 **A.** Confocal microscopy images showing BiFC experiments between LD and MED18, GUS was used as
716 a negative control. **B.** Metaplots showing RNAPolIII S5P and RNAPolIII S2P distribution over genes up-
717 regulated in *ld*. Genes were distributed in 5 quantiles based on their expression in *ld*.

718 **Figure 5.** Model describing the role of LD in the regulation of transcription

719 **A.** A complex comprising LD and FLD, and most likely the APRF1 phosphatase is recruited at
720 transcription sites through the ability of LD to interact with the MED18 subunit of the mediator
721 complex. Dephosphorylation of S5P of RNA Pol II decreases transcription rate leading to controlled
722 transcription levels. **B.** Working model for the role of LD and FLD during replication stress: after
723 detection of collisions between the replication and transcription machinery, LD and FLD recruitment
724 may allow transcription inhibition, TRC resolution and replication restart.

725

726 **Supplementary Figures**
727 Supplementary Figure S1. Identification of *LD* as the gene mutated in the *soa225* line.

728 Supplementary Figure S2. The « biomass effect » linked to late flowering is not the cause for the

729 tolerance of the *ld* mutant to replication stress.

730 Supplementary Figure S3. LD is an ubiquitous protein expressed at higher levels in young tissues

731 Supplementary Figure S4. Identification of LD target genes

732 Supplementary Figure S5. LD is not a specific regulator of DDR genes

733 Supplementary Figure S6. LD is a global repressor of transcription

734 Supplementary Figure S7. LD and FLD function together to reduce transcription

735 Supplementary Figure S8. Reproducibility of spike-in RNAseq experiments

736 Supplementary Figure S9. LD and FLD target respectively the 5' and the 3' region of the same genes

737 Supplementary Figure S10. Total accumulation of histone modifications remains unchanged in the

738 *ld* mutant.

739 Supplementary Figure S11. LD and MED18 interact in the yeast two-hybrid system

740 Supplementary Figure S12. LD modulates RNA PolII phosphorylation levels

741

742

743

744

745

746

747

748
<figure><img src="image_3.png" /><figcaption>Figure 1. LD is involved in replication stress response</figcaption></figure>

A. Phenotype of WT (Col0), *pol2a*, *Id*, *soa225* and *Id pol2a* plants. *Pol2a* mutants display reduced size, *soa225* is the suppressor of *pol2a* isolated in the genetic screen, showing partial restoration of the *pol2a* growth defects. The *Id* mutant corresponds to the SAIL line SAIL_743_B07 which harbors a T-DNA insertion in the *Id* gene. Plants were grown in vitro for 10 days, and on soil for 14 days. Bar = 1cm for all panels. B. GUS-staining pattern obtained in the different mutant lines expressing the *CYCB1-1:GUS* construct. Red arrows indicate shoot meristems. GUS staining was increased in *pol2a* mutants, and returned to lower levels in the *soa225* mutant. Bar = 50µm for all panels. C. Quantification of GUS activity (fluorescence unit/minute/µg of protein extract) in the indicated genotypes. Values are average +/- SD of three biological replicates, different letters indicate significantly different values (Wilcoxon sum-ranked test). D. Root length measurement of 10-day-old Col0 and *Id* plantlets grown on ½ MS supplemented or not with hydroxyurea (HU, 1 mM). A Two-Way Anova test was performed to compare genotypes, followed by a Tukey HSD test for multiple comparisons. Letters represent statistically different groups (p-value<0.05, n>20). This result is representative of 3 independent experiments. E. Rosette area measurement of 24-day-old plants of the indicated genotypes. A Kruskal-Wallis test was performed to compare the genotypes, followed by a Wilcoxon rank sum exact test for pairwise comparisons. Letters represent statistically different groups (p-value<0.05, n≥45). The partial restoration of *Id pol2a* phenotype seen in this graph is representative of 3 independent experiments. F. Confocal images of 10-day-old meristems. Cell walls were stained with propidium iodide (10µg/mL). Red arrows show the end of the meristematic zone. Bar = 50µm for all panels. G. Meristem length measurement of 10-day-old plantlets. A Two-Way Anova test was performed to compare genotypes, followed by a Tukey HSD test for multiple comparisons. Letters represent statistically different groups (p-value<0.05, n≥34). This result is representative of 3 independent experiments. H. Cell cycle length and I. S-phase length in the indicated genotypes. The red dotted lines show cell cycle and S-phase length duration in the wild-type (Col-0). The error bars represent the SD between two independent experiments.
<figure><img src="image_4.png" /><figcaption>**Figure 2.** LD is a global repressor of gene expression.</figcaption></figure>

**A** Top: Metaplot showing average LD binding profile centered on the peak's maximum obtained in the ChIP-seq analysis performed with *pLD::LD-GFP* lines. Bottom: Heatmap displaying ChIP signal at individual loci. The colour gradient shows the sequence coverage in the ChIP data. The input is shown as a negative control. **B.** Schematic representation of loci and pie charts comparing the proportion of promoter, and genic sequences in the total Arabidopsis genome (TAIR 10 genome) (left) and amongst LD binding sites (right). Bar = 50µm for all panels. **C.** Metaplot showing the distribution of LD on its target genes. Genes were distributed between five quantiles of expression based on the number of read counts obtained in the wild-type. **D.** Volcano plots summarizing the results of the spike-in RNAseq experiments. Significantly mis-regulated genes (adj p-value <0.05) are shown as colored dots (blue = down-regulated genes, red = up-regulated genes). The *pol2a* mutant shows roughly the same number of up and down-regulated genes compared to the wild-type. By contrast, *Id* and *Id pol2a* mutants show a global increase in gene expression. **E.** Venn diagram showing the overlap between up-regulated genes in *Id* and *Id pol2a* mutants. The number of common up-regulated genes is higher than expected by chance (Fisher's p < 0.000e+00). **F.** Venn diagram showing the overlap between LD targets identified by ChIP-seq and up-regulated genes in *Id*. The number of common up-regulated genes is higher than expected by chance (Fisher's exact test p < 8.737e-307). **G.** Heatmap showing LD binding (left) and the observed FC in *Id* on LD targets identified by ChIP-seq (right).
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /><figcaption>Figure 3. LD and FLD likely function together during replication stress response</figcaption></figure>

A. Pictures showing the rosette size of the different mutants. Plants were grown in vitro for 10 days and then on soil for three weeks. *fld pol2a* has a partially restored rosette size compared to *pol2a*, like *ld pol2a*. Bar = 2 cm for all panels. B. Confocal images of 10-day-old meristems. Plantlets were stained with propidium iodide (10µg/mL), Red arrows show the end of the meristem zone. Bar = 50 µm for all panels. C. Meristem length measurement of 10-day-old plantlets. A Kruskal-Wallis test was performed to compare the genotypes, followed by a Wilcoxon rank sum exact test for pairwise comparisons. Letters represent statistically different groups (p-value < 0.05, n > 20). This result is representative of 2 independent experiments. D. Volcano plots summarizing the results of the spike-in RNA-seq experiments. Significantly mis-regulated genes (adj p-value <0.05) are shown as colored dots. E. Venn diagram showing the overlap between up-regulated genes in *ld* and *ld pol2a* mutants. The number of common up-regulated genes is higher than expected by chance (Fisher's exact test p = 0). F. Comparison of LD and FLD binding on genes identified as LD targets. Left: Metaplots (top) and heatmaps (bottom) showing LD or FLD binding profile centered on the genes obtained in the ChIP-seq data generated with *pLD::LD-GFP* lines and with *pFLD:FLD-flag* lines (Inagaki et al, 2021). The color gradient shows the sequence coverage in the ChIP data. The input is shown as a negative control for both experiments. Right: Heatmap showing the observed FC in *ld* and *fld* on the same genes.
<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>Figure 4. LD modulates RNA Pol II activity through its ability to bind the MED18 sub-unit of the mediator complex</figcaption></figure>

<caption>A. Confocal microscopy images showing BiFC experiments between LD and MED18, nuclear-targeted YFP moieties were used as a negative control. Bar = 20µm for all panels. B. Metaplots showing RNAPol II S5P and RNAPol II S2P distribution over genes up-regulated in *Id*. Genes were distributed in 5 quantiles based on their expression in *Id*.</caption>
A

<figure><img src="image_12.png" /></figure>

B

<figure><img src="image_13.png" /><figcaption>**Figure 5. Model describing the role of LD in the regulation of transcription**</figcaption></figure>

A. A complex comprising LD and FLD, and most likely the APRF1 phosphatase is recruited at transcription sites through the ability of LD to interact with the MED18 subunit of the mediator complex. Dephosphorylation of S5P of RNA Pol II decreases transcription rate leading to controlled transcription levels. B. Working model for the role of LD and FLD during replication stress: after detection of collisions between the replication and transcription machinery, LD and FLD recruitment may allow transcription inhibition, TRC resolution and replication restart.
<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /><figcaption>**Supplementary Figure S1. Identification of LD as the gene mutated in the soa225 line.**</figcaption></figure>

A. Phenotype of WT (Col0), *pol2a*, *Id-4*, *soa225* and *ldpol2a* plants. Plants were grown in vitro for 10 days, and on soil for 28 days. B. Top: Sequence of the *LD* wild-type and *soa225* allele presenting a G to A transition at position 657 from the start codon, middle: structure of the *LD* gene and position of the *soa225* mutation in the 4th exon. Boxes correspond to exons and lines to introns and the position of the mutation is indicated by an asterisk, bottom: the *LD* protein comprises a homeobox domain and a TFIIS domain, followed by a long intrinsically disordered region. The *soa225* mutation corresponds to a non-sense mutation at position 219. C. Top: Position of the T-DNA insertion (red flag) in the *SAIL_743_B07* line, at the end of the 10th exon and position of primers used for RT-qPCR analysis of *LD* expression in the mutant. Bottom: Position of the insertion in the protein sequence. D. RT-qPCR quantification of *LD* expression in the *Id-4* mutant. Position of primer pairs is shown on panel C, the *Id-4* mutant does not accumulate a full length cDNA for *LD*. E. Representative images of 28 day-old plants of the indicated genotype. *Id pLD::LD-GFP* corresponds to the *Id-4* mutant complemented with a construct encompassing a *LD-GFP* fusion under the control of the endogenous *LD* promotor. *F1: soa225 x Id pol2a* corresponds to the F1 of a cross between the *soa225* suppressor and the *Id-4 pol2a* double mutant, showing that the two mutations are allelic. *Id pol2a pLD::LD-GFP* corresponds to the *Id-4* mutant complemented with a construct encompassing a *LD-GFP* fusion under the control of the endogenous *LD* promotor.
<figure><img src="image_19.png" /><figcaption>Supplementary Figure S2. The « biomass effect » linked to late flowering is not the cause for the tolerance of the *ld* mutant to replication stress.</figcaption></figure>

Root length measurement of 10-day-old WT (Col0), *ld* and *gi-2* plantlets grown on ½ MS supplemented or not with hydroxyurea (HU, 0.5 or 1 mM). A Two-Way Anova test was performed to compare genotypes, followed by a Tukey HSD test for multiple comparisons. Letters represent statistically different groups (p-value<0.05, n>20). This result is representative of 2 independent experiments.
<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /><figcaption>**Supplementary Figure S3. LD is an ubiquitous protein expressed at higher levels in young tissues**</figcaption></figure>

15-day-old plantlets of the *ld* mutant complemented with the pLD:LD-GFP construct were used for confocal imaging. Laser intensity and gain were kept identical for all organs. Representative images of the indicated organs are shown. Bar = 10µm for all panels except for the plantlet image (bar = 1mm), and the root tip image, bar = 25µm).

The boxplot shows quantification of the fluorescence signal in the different organs, data are from 10 measurements obtained on the brightest nuclei in the picture.
<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /></figure>

D Known motives p-value < 1e-230

<table><thead><tr><th>Motif</th><th>Family</th><th>Enrichment</th></tr></thead><tbody><tr><td>CACGTG</td><td>bHLH/bZIP/BES1/E-box<br>BZR/Trihelix-like</td><td>2,4</td></tr><tr><td>CTCTCTCTCT<br>AGACAGATA</td><td>GAGA-repeat</td><td>1,8</td></tr><tr><td>CGTTGACCTT</td><td>WRKY</td><td>1,6</td></tr><tr><td>GGGCCC</td><td>TCP</td><td>3.0</td></tr></tbody></table>

De novo motives p-value < 1e-285

<table><thead><tr><th>Motif</th><th>Family</th><th>Enrichment</th></tr></thead><tbody><tr><td>GGGCCCT<br>ATAC</td><td>TCP-like</td><td>2,0</td></tr><tr><td>CACGTGCC<br>ATAC</td><td>bHLH/bZIP/BES1/E-box<br>BZR/Trihelix-like</td><td>2,6</td></tr><tr><td>TCGATTTAAT<br>ATAC</td><td>HB-like</td><td>1,2</td></tr><tr><td>CGTTGACCTT<br>ATAC</td><td>WRKY-like</td><td>1,4</td></tr></tbody></table>

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>

**Supplementary Figure S4. Identification of LD target genes**

A. Screenshot showing the two ChIP replicates in a region of chromosome one spanning genes AT1G09280 to AT1G09530, illustrating the high number of genes targeted by LD, and the high reproducibility between the two ChIP replicates. B. Graph showing the position of peaks identified in the two ChIP-seq replicates performed in pLD::LD-GFP lines. Each dot represents one peak. C. Overlap between the lists of targets identified in the two ChIP-seq replicates.

D. Enriched sequence motifs found in LD target sites using the Homer software. Both the search for know-motifs and de novo motif discovery identified a wealth of motifs with modest enrichment compared to the whole genome. E. Venn diagram comparing the list of LD targets (i.e., genes that were identified as LD targets in both ChIP-seq experiments) with DE genes in *Id* compared to the WT (Col0). LD targets are not enriched amongst genes up or down-regulated in *Id* (Fisher's exact test p >0.05).
A

down in *pol2a*
vs Col0

<figure><img src="image_26.png" /></figure>

B

down in *pol2a*
vs Col0

<figure><img src="image_27.png" /><figcaption>**Supplementary Figure S5. LD is not a specific regulator of DDR genes**</figcaption></figure>

RNA-seq analysis was performed on shoot apices of wild-type, *Id*, *pol2a* and *Id pol2a* mutants. **A.** Venn diagram comparing lists of DEGs in *pol2a* compared to the wild-type and in *Id pol2a* compared to the wild-type. The majority of DEGs in *pol2a* are also DE in *Id pol2a*. **B.** Venn diagram comparing lists of DDR genes DE in *pol2a* compared to the wild-type, and in *Id pol2a* compared to *pol2a*. Most DDR genes misregulated in *pol2a* were not DE in *Id pol2a* compared to *pol2a*, indicating that the *Id* mutation does not rescue the expression of DDR genes globally.
<figure><img src="image_28.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_29.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_30.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_31.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_32.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_33.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_34.png" /><figcaption>H</figcaption></figure>

<figure><img src="image_35.png" /><figcaption>DNA damage repair genes (n=311)</figcaption></figure>

**Supplementary Figure S6. LD is a global repressor of transcription**

**A-C:** Raw read counts (**A**), Size factors calculated using the Drosophila spike-in (**B**) and normalized read counts (**C**), obtained from the spike-in RNA-seq experiments. **D-E** scatter plots showing the correlation between FC observed in the *Id* and in the *Id pol2a* mutants for all genes (**D**) or only LD target genes identified by ChIP-seq (**E**). **F:** Flow-cytometry analysis of endoreduplication levels in 8-day-old plantlets of the indicated genotypes. The *pol2a* mutant shows a mild increase in endoreduplication that is partially rescued by the *Id* mutation. **G-H:** Heatmap showing clusters of deregulated genes with the GO term “Cell cycle” (**G**) or “DNA damage repair” (**H**). Genes with the indicated annotation were retrieved from the Panther database, and only genes that were significantly mis-regulated in at least one mutant line were kept for the clustering analysis. The color gradient represents the z-score of gene expression in each mutant compared to the other three. Red color shows up-regulated genes and blue down-regulated genes. The clusters are numbered and framed in black.
A

<figure><img src="image_36.png" /></figure>

B

<figure><img src="image_37.png" /></figure>

C

<figure><img src="image_38.png" /></figure>

<figure><img src="image_39.png" /></figure>

**Supplementary Figure S7. LD and FLD function together to reduce transcription**

**A-C:** Raw read counts (A), Size factors calculated using the Drosophila spike-in (B) and normalized read counts (C), obtained from the spike-in RNA-seq experiments. D-E scatter plots showing the correlation between FC observed in the *Id* and in the *Id pol2a* mutants for all genes (D) or only LD target genes identified by ChIP-seq (E).
A

<figure><img src="image_40.png" /></figure>

B

<figure><img src="image_41.png" /><figcaption>**Supplementary Figure S8. Reproducibility of spike-in RNAseq experiments**</figcaption></figure>

**A:** Volcano plots summarizing the results of the third spike-in RNAseq experiments. Significantly mis-regulated genes (adj p-value <0.05) are shown as colored dots (blue = down-regulated genes, red = up-regulated genes). **B:** Venn diagram comparing the list of up-regulated genes identified in the three independent spike-in RNAseq performed on the *ld* mutant.
A

<figure><img src="image_42.png" /></figure>

pLD::LD-GFP_rep1

pLD::LD-GFP_rep2

pFLD::FLD-Flag_rep1

pFLD::FLD-Flag_rep2

B

<figure><img src="image_43.png" /></figure>

<figure><img src="image_44.png" /></figure>

<figure><img src="image_45.png" /></figure>

C

<figure><img src="image_46.png" /></figure>

FLD
Binding Genes

*pvalue: 1.44e-260

(Fisher's exact test)

<caption>**Supplementary Figure S9.** LD and FLD target respectively the 5' and the 3' region of the same genes.</caption>

A: Screenshot showing ChIP-seq data for LD and FLD. LD and FLD target very similar sets of genes, although for many targets LD is more enriched in their 5' and FLD in their 3'.

B: Three magnifications of the same region showing LD specific peaks in the 5' of common targets (blue arrows), FLD specific peaks in the 3' of common targets (black arrows), and peaks bound by the two proteins (red arrows).

C: Venn diagram showing the overlap between LD and FLD target genes identified by ChIP-seq.
<figure><img src="image_47.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_48.png" /></figure>

**Supplementary Figure S10. Total accumulation of histone modifications remains unchanged in the *ld* mutant.**

A-B: Western blots showing H3K4me1 (A) and H3K9ac (B) levels in wild-type (WT) and *ld* mutant (Id) samples. Total H3 was used for normalization. Images are representative of three independent experiments. Quantification was performed using the Image J software, and results are summarized in the graphs below. Data are average +/- standard deviation (n=3).
<figure><img src="image_49.png" /><figcaption>Supplementary Figure S11. LD and MED18 interact in the yeast two-hybrid system.</figcaption></figure>

Transformed yeast cells were resuspended in water to a final OD<sub>600nm</sub> of 1. Serial dilutions were grown on control medium (-L-T) and on two selective media (-L-T-H-Ade and -L-T-H + 3AT 1mM).
<figure><img src="image_50.png" /><figcaption>Supplementary Figure S12. LD modulates RNA Pol II phosphorylation levels</figcaption></figure>

Snapshot showing ChIP-seq data for LD, S5P in the wild-type (Col-0) and *Id* mutant, and S2P in the wild-type (Col-0) and *Id* mutant. LD binding sites coincide with increased S5P levels in the *Id* mutant.
# SUPPLEMENTARY METHODS

## RNAseq experiments

### For shoot apices experiments

Shoot apices were dissected with a scalpel. Samples were ground in a mortar with liquid nitrogen and total RNA was extracted from the meristematic zone and the first two leaves of thirty 7-day-old plantlets with the NucleoSpin RNA-Plus Kit (Macherey-Nagel) according to the manufacturer's instructions. Libraries were prepared with 2 µg of total RNA thanks to the NEBNext Ultra II RNA Library Prep Kit for Illumina according to the manufacturer's instructions. DNA fragment length and library quantity were assessed on a 2100 Bioanalyzer (Agilent). Finally, libraries were sequenced on a NextSeq500 (Illumina) 75-bp single-end run. Three independent biological replicates were generated.

### For whole plantlets spike-in experiments

About 500, 8-day-old plantlets were ground in 1.5 ml ice-cold extraction buffer (0.25 M Sucrose, 10 mM Tris-HCl pH8, 10 mM MgCl₂, 5 mM β-Mercaptoethanol in sterile water) in gentleMACS™ M Tubes (Miltenyi Biotec) on a gentleMACS™ Octo Dissociator with a personalized program (see below). After grinding, the solution was filtered with 30 µm filters (MACS® SmartStrainers) and nuclei were counted by flow cytometry on a Cyflow SL3 (Partec-Sysmex) to estimate the number of cells of each genotype. 400K nuclei were kept for each sample by pipetting the appropriate volume into a new Eppendorf tube (Sterile and low bind). Trizol was added with an adjusted volume to have a final volume of 85% per sample. 5000 Drosophila S2 cells were added to each sample. RNAs were extracted following Invitrogen protocol for TRIzol Reagent extraction (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/trizol_reagent.pdf) starting at step 2 of the section "Lyse samples and separate phases". A few modifications were made in the "Isolate RNA" section at step 1.1, 1 µl Glycoblue was added as well as 1:10 NaOAc 3 M and 1:1 Isopropanol based on the volume of the aqueous phase pipetted previously. At step 2.4, pellets were left to dry 7 minutes. At step 3.1, pellets were resuspended in sterile water. Finally at step 3.2, tubes were incubated in a heatblock for 10 minutes at 55°C. After RNA extraction with Trizol, polyA libraries were prepared with 2 µg of total RNA thanks to the NEBNext Ultra II RNA Library Prep Kit for Illumina according to the manufacturer's instructions (with a number of PCR amplification cycles adjusted to 13). DNA fragment length and library quantity were assessed on a 2100 Bioanalyzer (Agilent). Finally, libraries were sequenced on a NextSeq500 (Illumina) 75-bp single-end run. Three independent biological replicates were generated.
GentleMACS™ Octo Dissociator personalized program

Loop 3X
Spin 200 rpm 1'
End loop
Loop 3X
Spin 200 rpm 1"
Spin 300 rmp 1"
Spin 400 rpm 3"
Spin -400 rpm 2"
Spin 400 rmp 3"
Spin -400 rpm 1'
Spin 200 rpm 4"
Spin -400 rpm 2"
Spin 400 rpm 3"
Spin 200 rpm 6"
Spin -200 rpm 2"
Spin 200 rpm 8"
End loop

RNA-seq data analysis

For shoot apices experiments

RNA-seq samples was sequenced on single-end that were trimmed using Trimmomatic-0.38 (1) with the following parameters: minimum length of 30 bp, mean Phred quality score greater than 30, and leading and trailing base removal with base quality < 5. Hisat 2.2.1 Aligner (2) was used for mapping to the TAIR10 genome assembly. Raw read counts were extracted with featureCounts v2.0.0 (3) utility from Subread software based on the TAIR10 annotations. To identify differentially expressed genes we used DiCoExpress package (4).

For whole plantlets spike-in experiments

RNA-seq samples were sequenced on single-end that were trimmed using Trimmomatic-0.38 (1) with default settings except for the parameter "ILLUMINACLIP: TruSeq3- SE.fa:2:30:10". Trimmed FASTQ files were aligned to a chimeric genome combining TAIR10 reference genome assembly (available at https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_000001735.3/) and Drosophila's (available at https://ftp.ensembl.org/pub/release-110/fasta/drosophila_melanogaster/dna/) using Hisat 2.2.1 (2), followed by featureCounts (3) to quantify the raw read counts. Then, Drosophila genes were used to normalize all samples. We used DESeq2 (5) to calculate size factors (sj) for each sample. Then, build a read count matrix containing Spike-in genes with an average number of reads of at least 5 and all Arabidopsis genes to conduct the CATE package used to adjust the matrix by removing the effect of
the variability of spike-in genes (according to the procedure described in (6)). After that, DESeq2 1.32.0 was conducted to calculate new size factors based on this adjusted matrix to further correct the read counts, and to identify the genes differentially expressed (p-adjust < 0.05).

## ChIP-seq experiments

ChIP-seq was done on 6g of 12-day-old plantlets expressing the LD-GFP fusion and 3g of 14-day-old Col0 and *Id* plantlets for RNA pol II and H3K9ac ChIPs. Plantlets were cross-linked in 1% (v/v) formaldehyde for 15 minutes. Then, cross-linking was quenched with 125 mM glycine for 5 min. Cross-linked plantlets were ground in liquid nitrogen, resuspended in 25 mL of extraction buffer 1 (0.4 M Sucrose, 10 mM Tris-HCl pH8, 10 mM MgCl2, 5 mM β-mercaptoethanol, 200µl of a stock [1 tablet of cOmplete™ Mini, EDTA free Protease inhibitor cocktail in 1 ml of sterile water] in 100 ml, in sterile water), filtered in 100 µm filters and centrifuged 10 minutes at 1500g. Pellets were then resuspended and centrifuged two times for 5 minutes at 1000g, in 5 mL of extraction buffer 2 (0.25 M Sucrose, 10 mM Tris-HCl pH8, 10 mM MgCl2, 1% Triton X-100, 5 mM β-Mercaptoethanol, 200µl of a stock [1 tablet of cOmplete™ Mini, EDTA free Protease inhibitor cocktail in 1 ml of sterile water] in 100 ml final, in sterile water). Nuclei were next resuspended in 500 µL of nuclei lysis buffer (0.1% SDS, 50 mM Tris-HCl pH 8, 10 mM EDTA pH 8). Chromatin was sonicated for 10 minutes using Covaris S220 (peak power, 175; cycles/burst, 200; duty factor, 20). The sonicated chromatin was then immunoprecipitated using 2 µl of anti-GFP antibodies (Abcam, ab290) per pLD:LD-GFP IP (400µl of sonicated chromatin + 600 µl of dilution buffer) and 3 µl of anti-pol II S5 (Abcam, ab5408), anti-pol II S2 (Abcam, ab238146) and H3K9ac (Millipore 07-352) for Col0 and *Id* samples. They were incubated at 4 °C overnight on a rotating wheel. Immunocomplexes were recovered with 40 µl of washed Dynabead protein A (Invitrogen, 10002D) for rabbit primary antibodies and with Dynabeads™ M-280 Sheep Anti-Mouse IgG (ref: 11201D) for mouse primary antibodies, and incubated for 3 h at 4 °C with rotation. Immunoprecipitated material was washed 9 times with ChIP dilution buffer (1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8, 167 mM NaCl, protease inhibitors), on a rotating wheel at 4°C for 5 minutes each. ChIPed DNA was eluted by two incubation steps for 15 minutes at 65 °C with 200 µl of freshly prepared elution buffer (1% SDS, 0.1 M NaHCO3). Chromatin was reverse cross-linked by adding 16 µl of 5 M NaCl and incubated overnight at 65 °C. The next day, chromatin was treated with 1 µl of RNaseA+T1 (Ambion cocktail) for 20 min followed by incubation with 4 µl Proteinase K, 8 µl 0.5 M EDTA and 16 µl of 1 M Tris-HCl pH 6.5 for 3 h at 50 °C. DNA was extracted with 450 µl of phenol:chloroform:IAA (25:24:1) pH8. 1 mL of 100% ethanol was used to precipitate DNA after the addition of 1:10 NaOAc and 1 µl of 15 mg/ml GlycoBlue (centrifugation 10 min, 16K g at 4°C), which was then resuspended in 10 µl of nuclease-free water. Libraries were then generated using a minimum of 2 ng of DNA with the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs). The
quality of the libraries was assessed on a 2100 Bioanalyzer (Agilent), and the libraries were subjected
to 75 bp high-throughput sequencing by NextSeq 500 (Illumina).

ChIP-seq analysis

ChIP-seq analysis were conducted following the protocol described in (7). With a few changes. For
Trimmomatic, minimum length of 30 bp. Reads were mapped to TAIR10 genome assembly using the
Chromap software with default parameter (8). Extended size 150, and narrow peak. For heatmap and
metaplot generation, we used deepTools 3.5.0 (9). The mapped BAM files were converted to BigWig
format using deepTools. We visualized them with IGB. For peak annotation, ChIPseeker 1.30.3 was
used (10).

Affinity purification and Mass Spectrometry analysis (AP-MS)

The transgenic culture was harvested after four hours of induction with 10 µM β-estradiol. Three pull-down experiments were performed, using 25 mg total protein per pull-down, as described (11). On-bead digested samples were analyzed on a Q Exactive (ThermoFisher Scientific) and co-purified proteins were identified with Mascot (version 2.8.2, Matrixscience) using standard procedures (11).

The identified protein list was filtered versus a large dataset of 755 AP-MS experiments, including nine different experiments with XVE-based estradiol induction and covering baits not known to be related to LD according to data present in the literature, similarly as described in (12, 13) to filter-out potential background noise. For each identified protein, a Normalized Spectral Abundance Factor (NSAF) is calculated. The Ln-transformed mean NSAF of all proteins identified in at least 2 replicates out of 3 are compared to the Ln-transformed mean NSAF of the same protein in the large dataset by a two-tailed t-test. Proteins that comply with the following criteria are considered significantly enriched vs the large dataset and are retained in the Filtered set: (i) two-peptide identifications present in at least two out of three replicates are significantly enriched if they were found with less than three bait-groups in the large control AP-MS dataset or if they were enriched with a mean NSAF ratio ≥10 AND a -Log10(p-value) ≥ 10, or with a mean NSAF ratio ≥20 AND a -Log10(p-value) ≥ 8, (ii) one-peptide identifications present in all three replicates, if they were found in the large control dataset with less than seven bait-groups and if they were significantly enriched with a mean NSAF ratio ≥10 AND a -Log10(p-value) ≥ 10.

Yeast two-hybrid assays

LD and MED18 cDNAs were cloned in Gateway-adapted pGADT7 and pGBKT7 vectors. Yeast cells were grown over-night in liquid YPDA medium. For each transformation, 1mL of culture was washed twice
with water, and incubated at 30°C for 30 min with 5µg of Salmon sperm DNA (Invitrogen), 5µg of each plasmid and 200µL of PEG 4000 (50% w/v dissolved in 0.1M Tris-HCl, 10 mM EDTA, 100mM LiAc). After addition of 20µL of DMSO, cells were incubated at 42°C for 15 min, washed twice with water, and plated on SD -L -T medium. After two days, several colonies were resuspended in water to a final OD₆₀₀nm of 1, and serial dilutions were deposited on control and selective medium.

1. A. M. Bolger, M. Lohse, B. Usadel, Trimmomatic: a flexible trimmer for Illumina sequence data. *Bioinforma. Oxf. Engl.* **30**, 2114–20 (2014).

2. D. Kim, J. M. Paggi, C. Park, C. Bennett, S. L. Salzberg, Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. *Nat. Biotechnol.* **37**, 907–915 (2019).

3. Y. Liao, G. K. Smyth, W. Shi, featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. *Bioinforma. Oxf. Engl.* **30**, 923–30 (2014).

4. I. Lambert, C. Paysant-Le Roux, S. Colella, M.-L. Martin-Magniette, DiCoExpress: a tool to process multifactorial RNAseq experiments from quality controls to co-expression analysis through differential analysis based on contrasts inside GLM models. *Plant Methods* **16**, 68 (2020).

5. M. I. Love, W. Huber, S. Anders, Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. *Genome Biol.* **15**, 550 (2014).

6. G. Schivre, et al., Genome-scale transcriptome augmentation during Arabidopsis thaliana photomorphogenesis. [Preprint] (2025). Available at: https://www.biorxiv.org/content/10.1101/2025.01.30.635720v1 [Accessed 5 February 2025].

7. Y. Huang, et al., HSFA1a modulates plant heat stress responses and alters the 3D chromatin organization of enhancer-promoter interactions. *Nat. Commun.* **14**, 469 (2023).

8. H. Zhang, et al., Fast alignment and preprocessing of chromatin profiles with Chromap. *Nat. Commun.* **12**, 6566 (2021).

9. F. Ramírez, et al., deepTools2: a next generation web server for deep-sequencing data analysis. *Nucleic Acids Res.* **44**, W160-165 (2016).

10. G. Yu, L.-G. Wang, Q.-Y. He, ChIPseeker: an R/Bioconductor package for ChIP peak annotation, comparison and visualization. *Bioinforma. Oxf. Engl.* **31**, 2382–2383 (2015).

11. F. Persyn, et al., A Nitrogen-specific Interactome Analysis Sheds Light on the Role of the SnRK1 and TOR Kinases in Plant Nitrogen Signaling. *Mol. Cell. Proteomics* **23**, 100842 (2024).

12. J. Van Leene, et al., Mapping of the plant SnRK1 kinase signalling network reveals a key regulatory role for the class II T6P synthase-like proteins. *Nat. Plants* **8**, 1245–1261 (2022).

13. J. Van Leene, et al., An improved toolbox to unravel the plant cellular machinery by tandem affinity purification of Arabidopsis protein complexes. *Nat. Protoc.* **10**, 169–187 (2015).
<figure><img src="image_51.png" /></figure>

<figure><img src="image_52.png" /></figure>

<figure><img src="image_53.png" /></figure>

<figure><img src="image_54.png" /></figure>

<figure><img src="image_55.png" /><figcaption>**Supplementary Figure S1. Identification of LD as the gene mutated in the soa225 line.**</figcaption></figure>

A. Phenotype of WT (Col0), *pol2a*, *Id-4*, *soa225* and *ldpol2a* plants. Plants were grown in vitro for 10 days, and on soil for 28 days. B. Top: Sequence of the *LD* wild-type and *soa225* allele presenting a G to A transition at position 657 from the start codon, middle: structure of the *LD* gene and position of the *soa225* mutation in the 4th exon. Boxes correspond to exons and lines to introns and the position of the mutation is indicated by an asterisk, bottom: the *LD* protein comprises a homeobox domain and a TFIIS domain, followed by a long intrinsically disordered region. The *soa225* mutation corresponds to a non-sense mutation at position 219. C. Top: Position of the T-DNA insertion (red flag) in the *SAIL_743_B07* line, at the end of the 10th exon and position of primers used for RT-qPCR analysis of *LD* expression in the mutant. Bottom: Position of the insertion in the protein sequence. D. RT-qPCR quantification of *LD* expression in the *Id-4* mutant. Position of primer pairs is shown on panel C, the *Id-4* mutant does not accumulate a full length cDNA for *LD*. E. Representative images of 28 day-old plants of the indicated genotype. *Id pLD::LD-GFP* corresponds to the *Id-4* mutant complemented with a construct encompassing a *LD-GFP* fusion under the control of the endogenous *LD* promotor. *F1: soa225 x Id pol2a* corresponds to the F1 of a cross between the *soa225* suppressor and the *Id-4 pol2a* double mutant, showing that the two mutations are allelic. *Id pol2a pLD::LD-GFP* corresponds to the *Id-4* mutant complemented with a construct encompassing a *LD-GFP* fusion under the control of the endogenous *LD* promotor.
<figure><img src="image_56.png" /><figcaption>Supplementary Figure S2. The « biomass effect » linked to late flowering is not the cause for the tolerance of the *ld* mutant to replication stress.</figcaption></figure>

Root length measurement of 10-day-old WT (Col0), *ld* and *gi-2* plantlets grown on ½ MS supplemented or not with hydroxyurea (HU, 0.5 or 1 mM). A Two-Way Anova test was performed to compare genotypes, followed by a Tukey HSD test for multiple comparisons. Letters represent statistically different groups (p-value<0.05, n>20). This result is representative of 2 independent experiments.
<figure><img src="image_57.png" /></figure>

<figure><img src="image_58.png" /><figcaption>**Supplementary Figure S3. LD is an ubiquitous protein expressed at higher levels in young tissues**</figcaption></figure>

15-day-old plantlets of the *ld* mutant complemented with the *pLD:LD-GFP* construct were used for confocal imaging. Laser intensity and gain were kept identical for all organs. Representative images of the indicated organs are shown. Bar = 10µm for all panels except for the plantlet image (bar = 1mm), and the root tip image, bar = 25µm).

The boxplot shows quantification of the fluorescence signal in the different organs, data are from 10 measurements obtained on the brightest nuclei in the picture.
<figure><img src="image_59.png" /></figure>

<figure><img src="image_60.png" /></figure>

D Known motives p-value < 1e-230

<table><thead><tr><th>Motif</th><th>Family</th><th>Enrichment</th></tr></thead><tbody><tr><td>CACGTG</td><td>bHLH/bZIP/BES1/E-box<br>BZR/Trihelix-like</td><td>2,4</td></tr><tr><td>CTCTCTCTCT<br>TAGACAGATA</td><td>GAGA-repeat</td><td>1,8</td></tr><tr><td>CGTTGACCTT</td><td>WRKY</td><td>1,6</td></tr><tr><td>GGGCCC</td><td>TCP</td><td>3.0</td></tr></tbody></table>

De novo motives p-value < 1e-285

<table><thead><tr><th>Motif</th><th>Family</th><th>Enrichment</th></tr></thead><tbody><tr><td>GGGCCCT<br>ATAC</td><td>TCP-like</td><td>2,0</td></tr><tr><td>CACGTGCC<br>ATCGT</td><td>bHLH/bZIP/BES1/E-box<br>BZR/Trihelix-like</td><td>2,6</td></tr><tr><td>TCGATTTAAT<br>ATAC</td><td>HB-like</td><td>1,2</td></tr><tr><td>CGTTGACCTT<br>ATAC</td><td>WRKY-like</td><td>1,4</td></tr></tbody></table>

<figure><img src="image_61.png" /></figure>

<figure><img src="image_62.png" /></figure>

Supplementary Figure S4. Identification of LD target genes

A. Screenshot showing the two ChIP replicates in a region of chromosome one spanning genes AT1G09280 to AT1G09530, illustrating the high number of genes targeted by LD, and the high reproducibility between the two ChIP replicates. B. Graph showing the position of peaks identified in the two ChIP-seq replicates performed in *pLD:LD-GFP* lines. Each dot represents one peak. C. Overlap between the lists of targets identified in the two ChIP-seq replicates.

D. Enriched sequence motifs found in LD target sites using the Homer software. Both the search for known-motifs and *de novo* motif discovery identified a wealth of motifs with modest enrichment compared to the whole genome. E. Venn diagram comparing the list of LD targets (i.e., genes that were identified as LD targets in both ChIP-seq experiments) with DE genes in *ld* compared to the WT (Col0). LD targets are not enriched amongst genes up or down-regulated in *ld* (Fisher's exact test p >0.05).
A

<figure><img src="image_63.png" /></figure>

B

<figure><img src="image_64.png" /><figcaption>**Supplementary Figure S5. LD is not a specific regulator of DDR genes**</figcaption></figure>

RNA-seq analysis was performed on shoot apices of wild-type, *Id*, *pol2a* and *Id pol2a* mutants. **A.** Venn diagram comparing lists of DEGs in *pol2a* compared to the wild-type and in *Id pol2a* compared to the wild-type. The majority of DEGs in *pol2a* are also DE in *Id pol2a*. **B.** Venn diagram comparing lists of DDR genes DE in *pol2a* compared to the wild-type, and in *Id pol2a* compared to *pol2a*. Most DDR genes misregulated in *pol2a* were not DE in *Id pol2a* compared to *pol2a*, indicating that the *Id* mutation does not rescue the expression of DDR genes globally.
<figure><img src="image_65.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_66.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_67.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_68.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_69.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_70.png" /><figcaption>G</figcaption></figure>

Cell cycle genes (n=556)

<figure><img src="image_71.png" /><figcaption>H</figcaption></figure>

DNA damage repair genes (n=311)

<figure><img src="image_72.png" /></figure>

**Supplementary Figure S6. LD is a global repressor of transcription**

**A-C:** Raw read counts (**A**), Size factors calculated using the Drosophila spike-in (**B**) and normalized read counts (**C**), obtained from the spike-in RNA-seq experiments. **D-E** scatter plots showing the correlation between FC observed in the *Id* and in the *Id pol2a* mutants for all genes (**D**) or only LD target genes identified by ChIP-seq (**E**). **F:** Flow-cytometry analysis of endoreduplication levels in 8-day-old plantlets of the indicated genotypes. The *pol2a* mutant shows a mild increase in endoreduplication that is partially rescued by the *Id* mutation. **G-H:** Heatmap showing clusters of deregulated genes with the GO term “Cell cycle” (**G**) or “DNA damage repair” (**H**). Genes with the indicated annotation were retrieved from the Panther database, and only genes that were significantly mis-regulated in at least one mutant line were kept for the clustering analysis. The color gradient represents the z-score of gene expression in each mutant compared to the other three. Red color shows up-regulated genes and blue down-regulated genes. The clusters are numbered and framed in black.
A

<figure><img src="image_73.png" /></figure>

B

<figure><img src="image_74.png" /></figure>

C

<figure><img src="image_75.png" /></figure>

<figure><img src="image_76.png" /></figure>

**Supplementary Figure S7. LD and FLD function together to reduce transcription**

A-C: Raw read counts (A), Size factors calculated using the Drosophila spike-in (B) and normalized read counts (C), obtained from the spike-in RNA-seq experiments. D-E scatter plots showing the correlation between FC observed in the *Id* and in the *Id pol2a* mutants for all genes (D) or only LD target genes identified by ChIP-seq (E).
A

<figure><img src="image_77.png" /></figure>

B

<figure><img src="image_78.png" /><figcaption>**Supplementary Figure S8. Reproducibility of spike-in RNAseq experiments**</figcaption></figure>

**A:** Volcano plots summarizing the results of the third spike-in RNAseq experiments. Significantly mis-regulated genes (adj p-value <0.05) are shown as colored dots (blue = down-regulated genes, red = up-regulated genes). **B:** Venn diagram comparing the list of up-regulated genes identified in the three independent spike-in RNAseq performed on the *ld* mutant.
A

<figure><img src="image_79.png" /></figure>

pLD::LD-GFP_rep1

pLD::LD-GFP_rep2

pFLD::FLD-Flag_rep1

pFLD::FLD-Flag_rep2

B

<figure><img src="image_80.png" /></figure>

<figure><img src="image_81.png" /></figure>

<figure><img src="image_82.png" /></figure>

C

<figure><img src="image_83.png" /></figure>

FLD
Binding Genes

*pvalue: 1.44e-260

(Fisher's exact test)

<caption>**Supplementary Figure S9.** LD and FLD target respectively the 5' and the 3' region of the same genes.</caption>

A: Screenshot showing ChIP-seq data for LD and FLD. LD and FLD target very similar sets of genes, although for many targets LD is more enriched in their 5' and FLD in their 3'.

B: Three magnifications of the same region showing LD specific peaks in the 5' of common targets (blue arrows), FLD specific peaks in the 3' of common targets (black arrows), and peaks bound by the two proteins (red arrows).

C: Venn diagram showing the overlap between LD and FLD target genes identified by ChIP-seq.
<figure><img src="image_84.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_85.png" /></figure>

**Supplementary Figure S10. Total accumulation of histone modifications remains unchanged in the *Id* mutant.**

A-B: Western blots showing H3K4me1 (A) and H3K9ac (B) levels in wild-type (WT) and *Id* mutant (Id) samples. Total H3 was used for normalization. Images are representative of three independent experiments. Quantification was performed using the Image J software, and results are summarized in the graphs below. Data are average +/- standard deviation (n=3).
<figure><img src="image_86.png" /><figcaption>Supplementary Figure S11. LD and MED18 interact in the yeast two-hybrid system.</figcaption></figure>

Transformed yeast cells were resuspended in water to a final OD<sub>600nm</sub> of 1. Serial dilutions were grown on control medium (-L-T) and on two selective media (-L-T-H-Ade and -L-T-H + 3AT 1mM).
<figure><img src="image_87.png" /><figcaption>Supplementary Figure S12. LD modulates RNA Pol II phosphorylation levels</figcaption></figure>

Snapshot showing ChIP-seq data for LD, S5P in the wild-type (Col-0) and *Id* mutant, and S2P in the wild-type (Col-0) and *Id* mutant. LD binding sites coincide with increased S5P levels in the *Id* mutant.
<table><caption>Table S1: EMS-induced mutations affecting protein-coding genes in the fourth chromosome of *A. thaliana*.</caption><thead><tr><td>Gene</td><td>Mutation</td><td>Position from ATG</td><td>AA change</td></tr></thead><tbody><tr><td>AT4G00750</td><td>C-&gt;T</td><td>227</td><td>Ala76-&gt;Val</td></tr><tr><td>AT4G01570</td><td>C-&gt;T</td><td>1949</td><td>Arg650-&gt;Cys</td></tr><tr><td>AT4G02560</td><td>G-&gt;A</td><td>1232</td><td>Trp219-&gt;Stop</td></tr><tr><td>AT4G02580</td><td>C-&gt;T</td><td>2023</td><td>Pro220-&gt;Leu</td></tr><tr><td>AT4G07410</td><td>G-&gt;A</td><td>2592</td><td>Ser540-&gt;Asn</td></tr></tbody></table>

<table><caption>Table S2: Table summarizing the distribution of genes up-regulated in *Id* between five quantiles of expression.</caption><thead><tr><td>Average counts</td><td>Gene number</td></tr></thead><tbody><tr><td>0-50</td><td>2498</td></tr><tr><td>50-200</td><td>4518</td></tr><tr><td>200-500</td><td>4146</td></tr><tr><td>500-1000</td><td>2081</td></tr><tr><td>&gt;1000</td><td>1752</td></tr><tr><td>Total</td><td>14995</td></tr></tbody></table>
<table><caption>Table S3: Primers used in this study</caption><thead><tr><th>Primer Name</th><th>Sequence 5'-&gt;3'</th></tr></thead><tbody><tr><td colspan="2"><strong>Primers used for cloning</strong></td></tr><tr><td>pLD_Anw</td><td>AACAGGTCTCAACCTGAACAGCAGTATTAA</td></tr><tr><td>Bsal_edit-fw</td><td>GAGAGAAAGAGAGACGACACCTATGAGAAG</td></tr><tr><td>Bsal-edit-rev</td><td>CCTTCTCATAGGTGTCGTCTCTCTTTCTCT</td></tr><tr><td>pLD_B_rev</td><td>AACAGGTCTCTTGTTGCTAGACTCAATACC</td></tr><tr><td>LD_5'_B</td><td>AACAGGTCTCAACAATGGACGCGTTCAAG</td></tr><tr><td>LD_5'_C_rev</td><td>AACAGGTCTCATGCCCCAATTTTAATGGAC</td></tr><tr><td>LD_3'_C</td><td>AACAGGTCTCAGGCTCAACAAAAGCGGCCA</td></tr><tr><td>LD_3'_D_rev</td><td>AACAGGTCTCTCTGAACGCCATCTTTTGTT</td></tr><tr><td>LD_Sall</td><td>GCGTCGACCATGGACGCGTTCAAGGAGGAG</td></tr><tr><td>LD_EcoRI_rev</td><td>GAGAATTCCGCCATCTTTTGTTTTGGTTCC</td></tr><tr><td>MED18_BamHI</td><td>TTCGGATCCATGTCAATGGAGTGTGTGGTT</td></tr><tr><td>MED18_Sall_rev</td><td>GAAGTCGACTACAATGTAGTACCTCCTCCA</td></tr><tr><td>NLS_XmnI</td><td>CATGCCTAAGAAGAAGAGGAAGGTG</td></tr><tr><td>NLS_Sall_rev</td><td>TCGACACCTTCCTCTTCTTCTTAGGCATG</td></tr><tr><td colspan="2"><strong>Primers used for qPCR</strong></td></tr><tr><td>LD_qPCR1FW</td><td>GATACAAGATGCTTCTGTGCTG</td></tr><tr><td>LD_qPCR1RV</td><td>TGATAACAAACCTTTTGCCCTG</td></tr><tr><td>LD_qPCR2FW</td><td>ATCCTGGAATGAGTGGATGGG</td></tr><tr><td>LD_qPCR2RV</td><td>CCAATGTGATCGAGTTACTGTA</td></tr><tr><td>LD_qPCR3FW</td><td>GCCGCCAATGTCTGCTGATGA</td></tr><tr><td>LD_qPCR3RV</td><td>CTGAAGGTCCATTGACGGGCTG</td></tr><tr><td>ActinFW</td><td>TGAGACCTTTAACTCTCCCG</td></tr><tr><td>ActinRV</td><td>GATGGCATGAGGAAGAGAGA</td></tr><tr><td colspan="2"><strong>Primers used for genotyping</strong></td></tr><tr><td>Lba1</td><td>GGTTCACGTAGTGGGCCATCG</td></tr><tr><td>fld LP</td><td>ATG CCC AAA GGT ATC GAG ATC</td></tr><tr><td>fld RP</td><td>CCA CAT TAT ATC CAA ATG GCG</td></tr><tr><td>SAIL LB1</td><td>CAGAAATGGATAAATAGCCTTG</td></tr><tr><td>LD_LP</td><td>GAGAGCAACCAAATATTCCACATCATG</td></tr><tr><td>LD_RP</td><td>CTCTGAACATCAGCCTCCTTG</td></tr><tr><td>dCAPS_abo-BstXI</td><td>GGTCATGTAGAGTGCCATGAAAGTG</td></tr><tr><td>abo4-1rev</td><td>CTGCAGCACTTCAGAGGAAACTAC</td></tr></tbody></table>
<figure><img src="image_88.png" /></figure>

<figure><img src="image_89.png" /></figure>

<figure><img src="image_90.png" /></figure>

<figure><img src="image_91.png" /></figure>

<figure><img src="image_92.png" /><figcaption>**Supplementary Figure S1. Identification of LD as the gene mutated in the soa225 line.**</figcaption></figure>

A. Phenotype of WT (Col0), *pol2a*, *Id-4*, *soa225* and *ldpol2a* plants. Plants were grown in vitro for 10 days, and on soil for 28 days. B. Top: Sequence of the *LD* wild-type and *soa225* allele presenting a G to A transition at position 657 from the start codon, middle: structure of the *LD* gene and position of the *soa225* mutation in the 4th exon. Boxes correspond to exons and lines to introns and the position of the mutation is indicated by an asterisk, bottom: the *LD* protein comprises a homeobox domain and a TFIIS domain, followed by a long intrinsically disordered region. The *soa225* mutation corresponds to a non-sense mutation at position 219. C. Top: Position of the T-DNA insertion (red flag) in the *SAIL_743_B07* line, at the end of the 10th exon and position of primers used for RT-qPCR analysis of *LD* expression in the mutant. Bottom: Position of the insertion in the protein sequence. D. RT-qPCR quantification of *LD* expression in the *Id-4* mutant. Position of primer pairs is shown on panel C, the *Id-4* mutant does not accumulate a full length cDNA for *LD*. E. Representative images of 28 day-old plants of the indicated genotype. *Id pLD::LD-GFP* corresponds to the *Id-4* mutant complemented with a construct encompassing a *LD-GFP* fusion under the control of the endogenous *LD* promotor. *F1: soa225 x Id pol2a* corresponds to the F1 of a cross between the *soa225* suppressor and the *Id-4 pol2a* double mutant, showing that the two mutations are allelic. *Id pol2a pLD::LD-GFP* corresponds to the *Id-4* mutant complemented with a construct encompassing a *LD-GFP* fusion under the control of the endogenous *LD* promotor.
<figure><img src="image_93.png" /><figcaption>Supplementary Figure S2. The « biomass effect » linked to late flowering is not the cause for the tolerance of the *ld* mutant to replication stress.</figcaption></figure>

Root length measurement of 10-day-old WT (Col0), *ld* and *gi-2* plantlets grown on ½ MS supplemented or not with hydroxyurea (HU, 0.5 or 1 mM). A Two-Way Anova test was performed to compare genotypes, followed by a Tukey HSD test for multiple comparisons. Letters represent statistically different groups (p-value<0.05, n>20). This result is representative of 2 independent experiments.
<figure><img src="image_94.png" /></figure>

<figure><img src="image_95.png" /><figcaption>**Supplementary Figure S3. LD is an ubiquitous protein expressed at higher levels in young tissues**</figcaption></figure>

15-day-old plantlets of the *ld* mutant complemented with the *pLD:LD-GFP* construct were used for confocal imaging. Laser intensity and gain were kept identical for all organs. Representative images of the indicated organs are shown. Bar = 10µm for all panels except for the plantlet image (bar = 1mm), and the root tip image, bar = 25µm).

The boxplot shows quantification of the fluorescence signal in the different organs, data are from 10 measurements obtained on the brightest nuclei in the picture.
<figure><img src="image_96.png" /></figure>

<figure><img src="image_97.png" /></figure>

D Known motives p-value < 1e-230

<table><thead><tr><th>Motif</th><th>Family</th><th>Enrichment</th></tr></thead><tbody><tr><td>CACGTG</td><td>bHLH/bZIP/BES1/E-box<br>BZR/Trihelix-like</td><td>2,4</td></tr><tr><td>CTCTCTCTCT<br>AGACAGATA</td><td>GAGA-repeat</td><td>1,8</td></tr><tr><td>CGTTGACCTT</td><td>WRKY</td><td>1,6</td></tr><tr><td>GGGCCC</td><td>TCP</td><td>3.0</td></tr></tbody></table>

De novo motives p-value < 1e-285

<table><thead><tr><th>Motif</th><th>Family</th><th>Enrichment</th></tr></thead><tbody><tr><td>GGGCCCT<br>ATAC</td><td>TCP-like</td><td>2,0</td></tr><tr><td>CACGTGCC<br>ATAC</td><td>bHLH/bZIP/BES1/E-box<br>BZR/Trihelix-like</td><td>2,6</td></tr><tr><td>TCGATTTAAT<br>ATAC</td><td>HB-like</td><td>1,2</td></tr><tr><td>CGTTGACCTT<br>ATAC</td><td>WRKY-like</td><td>1,4</td></tr></tbody></table>

<figure><img src="image_98.png" /></figure>

<figure><img src="image_99.png" /></figure>

Supplementary Figure S4. Identification of LD target genes

A. Screenshot showing the two ChIP replicates in a region of chromosome one spanning genes AT1G09280 to AT1G09530, illustrating the high number of genes targeted by LD, and the high reproducibility between the two ChIP replicates. B. Graph showing the position of peaks identified in the two ChIP-seq replicates performed in *pLD:LD-GFP* lines. Each dot represents one peak. C. Overlap between the lists of targets identified in the two ChIP-seq replicates.

D. Enriched sequence motifs found in LD target sites using the Homer software. Both the search for known-motifs and *de novo* motif discovery identified a wealth of motifs with modest enrichment compared to the whole genome. E. Venn diagram comparing the list of LD targets (i.e., genes that were identified as LD targets in both ChIP-seq experiments) with DE genes in *ld* compared to the WT (Col0). LD targets are not enriched amongst genes up or down-regulated in *ld* (Fisher's exact test p >0.05).
A

<figure><img src="image_100.png" /></figure>

B

<figure><img src="image_101.png" /><figcaption>**Supplementary Figure S5. LD is not a specific regulator of DDR genes**</figcaption></figure>

RNA-seq analysis was performed on shoot apices of wild-type, *Id*, *pol2a* and *Id pol2a* mutants. **A.** Venn diagram comparing lists of DEGs in *pol2a* compared to the wild-type and in *Id pol2a* compared to the wild-type. The majority of DEGs in *pol2a* are also DE in *Id pol2a*. **B.** Venn diagram comparing lists of DDR genes DE in *pol2a* compared to the wild-type, and in *Id pol2a* compared to *pol2a*. Most DDR genes misregulated in *pol2a* were not DE in *Id pol2a* compared to *pol2a*, indicating that the *Id* mutation does not rescue the expression of DDR genes globally.
<figure><img src="image_102.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_103.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_104.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_105.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_106.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_107.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_108.png" /><figcaption>H</figcaption></figure>

<figure><img src="image_109.png" /><figcaption>DNA damage repair genes (n=311)</figcaption></figure>

**Supplementary Figure S6. LD is a global repressor of transcription**

**A-C:** Raw read counts (**A**), Size factors calculated using the Drosophila spike-in (**B**) and normalized read counts (**C**), obtained from the spike-in RNA-seq experiments. **D-E** scatter plots showing the correlation between FC observed in the *Id* and in the *Id pol2a* mutants for all genes (**D**) or only LD target genes identified by ChIP-seq (**E**). **F:** Flow-cytometry analysis of endoreduplication levels in 8-day-old plantlets of the indicated genotypes. The *pol2a* mutant shows a mild increase in endoreduplication that is partially rescued by the *Id* mutation. **G-H:** Heatmap showing clusters of deregulated genes with the GO term “Cell cycle” (**G**) or “DNA damage repair” (**H**). Genes with the indicated annotation were retrieved from the Panther database, and only genes that were significantly mis-regulated in at least one mutant line were kept for the clustering analysis. The color gradient represents the z-score of gene expression in each mutant compared to the other three. Red color shows up-regulated genes and blue down-regulated genes. The clusters are numbered and framed in black.
A

<figure><img src="image_110.png" /></figure>

B

<figure><img src="image_111.png" /></figure>

C

<figure><img src="image_112.png" /></figure>

<figure><img src="image_113.png" /></figure>

**Supplementary Figure S7. LD and FLD function together to reduce transcription**

A-C: Raw read counts (A), Size factors calculated using the Drosophila spike-in (B) and normalized read counts (C), obtained from the spike-in RNA-seq experiments. D-E scatter plots showing the correlation between FC observed in the *Id* and in the *Id pol2a* mutants for all genes (D) or only LD target genes identified by ChIP-seq (E).
A

<figure><img src="image_114.png" /></figure>

B

<figure><img src="image_115.png" /><figcaption>**Supplementary Figure S8. Reproducibility of spike-in RNAseq experiments**</figcaption></figure>

**A:** Volcano plots summarizing the results of the third spike-in RNAseq experiments. Significantly mis-regulated genes (adj p-value <0.05) are shown as colored dots (blue = down-regulated genes, red = up-regulated genes). **B:** Venn diagram comparing the list of up-regulated genes identified in the three independent spike-in RNAseq performed on the *ld* mutant.
A

<figure><img src="image_116.png" /></figure>

pLD::LD-GFP_rep1

pLD::LD-GFP_rep2

pFLD::FLD-Flag_rep1

pFLD::FLD-Flag_rep2

B

<figure><img src="image_117.png" /></figure>

<figure><img src="image_118.png" /></figure>

<figure><img src="image_119.png" /></figure>

C

<figure><img src="image_120.png" /></figure>

FLD
Binding Genes

*pvalue: 1.44e-260

(Fisher's exact test)

<caption>**Supplementary Figure S9.** LD and FLD target respectively the 5' and the 3' region of the same genes.</caption>

A: Screenshot showing ChIP-seq data for LD and FLD. LD and FLD target very similar sets of genes, although for many targets LD is more enriched in their 5' and FLD in their 3'.

B: Three magnifications of the same region showing LD specific peaks in the 5' of common targets (blue arrows), FLD specific peaks in the 3' of common targets (black arrows), and peaks bound by the two proteins (red arrows).

C: Venn diagram showing the overlap between LD and FLD target genes identified by ChIP-seq.
<figure><img src="image_121.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_122.png" /></figure>

**Supplementary Figure S10.** Total accumulation of histone modifications remains unchanged in the *Id* mutant.

A-B: Western blots showing H3K4me1 (A) and H3K9ac (B) levels in wild-type (WT) and *Id* mutant (Id) samples. Total H3 was used for normalization. Images are representative of three independent experiments. Quantification was performed using the Image J software, and results are summarized in the graphs below. Data are average +/- standard deviation (n=3).
<figure><img src="image_123.png" /><figcaption>Supplementary Figure S11. LD and MED18 interact in the yeast two-hybrid system.</figcaption></figure>

Transformed yeast cells were resuspended in water to a final OD<sub>600nm</sub> of 1. Serial dilutions were grown on control medium (-L-T) and on two selective media (-L-T-H-Ade and -L-T-H + 3AT 1mM).
<figure><img src="image_124.png" /><figcaption>Supplementary Figure S12. LD modulates RNA Pol II phosphorylation levels</figcaption></figure>

Screenshot showing ChIP-seq data for LD, S5P in the wild-type (Col-0) and *Id* mutant, and S2P in the wild-type (Col-0) and *Id* mutant. LD binding sites coincide with increased S5P levels in the *Id* mutant.