# HNF1β bookmarking involves Topoisomerase 1 activation and DNA topology relaxation in mitotic chromatin

Alessia Bagattin, Salvina Laura Tammaccaro, Magali Chiral, Munevver Parla

Makinistoglu, Nicolas Zimmermann, Jonathan Lerner, Serge Garbay, Nicolas

Kuperwasser, Marco Pontoglio

► To cite this version:

Alessia Bagattin, Salvina Laura Tammaccaro, Magali Chiral, Munevver Parla Makinistoglu, Nicolas Zimmermann, et al.. HNF1β bookmarking involves Topoisomerase 1 activation and DNA topology relaxation in mitotic chromatin. Cell Reports, 2024, 43 (10), pp.114805. 10.1016/j.celrep.2024.114805. hal-05407801

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Distributed under a Creative Commons Attribution 4.0 International License
# HNF1β bookmarking involves Topoisomerase 1 activation and DNA topology relaxation in mitotic chromatin

## Graphical abstract

<figure><img src="image_2.png" /></figure>

## Authors

Alessia Bagattin,
Salvina Laura Tammaccaro,
Magali Chiral, ..., Serge Garbay,
Nicolas Kuperwasser, Marco Pontoglio

## Correspondence

abagattin@gmail.com (A.B.),
marco.pontoglio@inserm.fr (M.P.)

## In brief

Bagattin et al. developed a ChIP methodology that strongly improves the identification of HNF1β-bound mitotic regions. They demonstrate that HNF1β remains associated with mitotic chromatin and recruits TOP1 on bookmarked sites in order to resolve DNA topological constraints. An HNF1β mutation, found in MODY patients, prevents the interaction with TOP1.

## Highlights

* HNF1β mitotic site binding is preserved with a specific methanol/formaldehyde ChIP

* BTBD2, an HNF1β partner, mediates mitosis-specific interaction with TOP1

* HNF1β recruits TOP1 and induces DNA relaxation around bookmarked HNF1β sites

* An HNF1β mutation, found in MODY patients, disrupts the interaction with TOP1

<figure><img src="image_3.png" /></figure>
Resource

# HNF1β bookmarking involves Topoisomerase 1 activation and DNA topology relaxation in mitotic chromatin

Alessia Bagattin,<fnref n="1" />,<fnref n="2" />,<fnref n="6" />,* Salvina Laura Tammaccaro,<fnref n="1" />,<fnref n="3" />,<fnref n="6" /> Magali Chiral,<fnref n="1" /> Munevver Parla Makinistoglu,<fnref n="1" />

Nicolas Zimmermann,<fnref n="1" /> Jonathan Lerner,<fnref n="1" />,<fnref n="4" /> Serge Garbay,<fnref n="1" /> Nicolas Kuperwasser,<fnref n="1" />,<fnref n="5" /> and Marco Pontoglio<fnref n="1" />,<fnref n="7" />,*

## SUMMARY

HNF1β (HNF1B) is a transcription factor frequently mutated in patients with developmental renal disease. It binds to mitotic chromatin and reactivates gene expression after mitosis, a phenomenon referred to as bookmarking. Using a crosslinking method that circumvents the artifacts of formaldehyde, we demonstrate that HNF1β remains associated with chromatin in a sequence-specific way in both interphase and mitosis. We identify an HNF1β-interacting protein, BTBD2, that enables the interaction and activation of Topoisomerase 1 (TOP1) exclusively during mitosis. Our study identifies a shared microhomology domain between HNF1β and TOP1, where a mutation, found in “maturity onset diabetes of the young” patients, disrupts their interaction. Importantly, HNF1β recruits TOP1 and induces DNA relaxation around HNF1β mitotic chromatin sites, elucidating its crucial role in chromatin remodeling and gene reactivation after mitotic exit. These findings shed light on how HNF1β reactivates target gene expression after mitosis, providing insights into its crucial role in maintenance of cellular identity.

## INTRODUCTION

Mitosis is characterized by drastic modifications in subcellular structures, including the breakdown of the nuclear envelope, release and diffusion of soluble nuclear components into the cytoplasm, and condensation of chromatin into compacted mitotic chromosomes. All of these modifications are associated with strongly reduced levels of transcription in condensed mitotic chromatin.<fnref n="1" />,<fnref n="2" /> It is known that certain chromatin-binding proteins do not remain bound to chromosomes during mitosis. On the other hand, some factors, called bookmarking factors, have the ability to remain associated with mitotic chromatin and contribute to the maintenance of cell identity throughout mitosis.<fnref n="3" /> Recent studies have shown that the loss of specific bookmarking factors can lead to delayed<fnref n="4" />⁻<fnref n="7" /> or impaired⁸ re-expression of target genes after mitosis. However, relatively little is known about the mechanisms through which bookmarking transcription factors, by binding to mitotic chromatin sites, help ensure epigenetic inheritance that leads to the conservation of postmitotic gene re-expression programs. Our previous studies have demonstrated that hepatocyte nuclear factor 1β

(HNF1β) is a bookmarking factor.⁸,⁹ HNF1β (also known as vHNF1 or LF-B3) is a POU (Pit1/Oct1/UNC-86) homeobox transcription factor expressed in the epithelium of several organs, including the pancreas, liver, digestive tract, lungs, and kidneys.¹⁰⁻¹² Mutations in *HNF1B* were initially identified in patients affected with “maturity onset diabetes of the young 5” (MODY5)¹³ and subsequently found to be also predominantly associated with renal disease.¹⁴ Recent studies have demonstrated that *HNF1B* mutations are among the most frequent genetic causes for congenital abnormalities of the kidneys and urogenital tract.¹⁵,¹⁶ Heterozygous null mutations in *HNF1B* can lead to severe phenotypes, including kidney agenesis, renal dysplasia and/or multicystic dysplastic kidneys, pancreas hypoplasia, and genital tract malformations.¹⁷ We have shown previously that the loss of HNF1β in mice results in severe polycystic disease, which is accompanied by a dramatic loss of mitotic spindle orientation during perinatal tubular elongation.¹⁸ In this context, the development of cysts is caused by the impaired expression of several HNF1β target genes, which are often found to be mutated in patients suffering from renal cysts.⁸,¹⁹,²⁰ Interestingly, when *Hnf1b* is inactivated in murine quiescent renal

<figure><img src="image_4.png" /></figure>

<footnote n="1">¹Epigenetics and Development Laboratory, Growth and Signaling Department, Université Paris Cité, CNRS, INSERM, Institut Necker-Enfants Malades, 75015 Paris, France</footnote>

<footnote n="2">²Present address: Gustave Roussy Cancer Campus, INSERM, 94800 Villejuif, France</footnote>

<footnote n="3">³Present address: Tessellate Bio BV, Stevenage Bioscience Catalyst, Gunnels Wood Road, Stevenage, SG1 2FX Hertfordshire, UK</footnote>

<footnote n="4">⁴Present address: Midwestern University, Downers Grove Campus, Downers Grove, IL 60515, USA</footnote>

<footnote n="5">⁵Present address: Genome Editing and Targeting Platform, Structure Fédérative de Recherche Necker INSERM US24-CNRS UMS 3633, 75015 Paris, France</footnote>

<footnote n="6">⁶These authors contributed equally</footnote>

<footnote n="7">⁷Lead contact</footnote>

<footnote>*Correspondence: abagattin@gmail.com (A.B.), marco.pontoglio@inserm.fr (M.P.)</footnote>

<footnote>https://doi.org/10.1016/j.celrep.2024.114805</footnote>
<figure><img src="image_5.png" /><figcaption>**Figure 1.** Methanol fixation preserves the mitotic localization of HNF1β</figcaption></figure>

(A) Confocal imaging of Madin-Darby canine kidney (MDCK) cells expressing the Nter-HNF1β-GFP fusion protein. Cells were observed either in live conditions or after fixation with 1% formaldehyde, pure methanol, or 0.2% formaldehyde in methanol. DNA was stained with Hoechst 33342 (magenta). Scale bar: 10 µm.

(B) HNF1β ChIP-qPCR. ChIP of HNF1β was performed on chromatin from 786-O cells fixed using AQC (aqueous crosslinking) or NAQC (non-aqueous crosslinking) with increasing concentrations of formaldehyde in methanol. Results are shown as percentage of input of a representative experiment (means ± SD, n = 3 technical replicates) on a conserved HNF1 binding site (HNF1BS) on the *HNF4G* gene and two flanking sites with no HNF1 consensus. The genomic position for HNF1BS on human genome assembly hg19 is shown in base pairs.

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

cells in adult kidneys, the expression of cystic genes is maintained, and mice do not develop cysts, but these specific target genes are silenced when Hnf1β-deficient cells are forced to proliferate.⁸ Moreover, the study of naturally occurring *HNF1B* human mutations allowed us to demonstrate that the sudden inducible loss of DNA-binding activity during mitosis leads to an immediate loss of mitotic chromatin binding.⁹ Currently, the specificity of HNF1β mitotic chromatin binding (sequence versus non-sequence specific) and the mechanisms that enable HNF1β to act as a bookmarking factor and reprogram postmitotic expression of target genes are poorly understood. With the use of a non-aqueous crosslinking methodology, here we show that the vast majority of HNF1β interphase chromatin binding sites are maintained in mitotic chromatin. In this context, we identified an HNF1β-interacting protein, BTBD2, that provides a platform for cell-cycle-restricted mitotic binding to Topoisomerase 1 (TOP1). We showed that this interaction is prevented by a naturally occurring mutation. Finally, our results demonstrate that HNF1β enhances TOP1 occupancy around HNF1β target genes in mitosis and stimulates its enzymatic DNA-relaxing activity,

possibly resolving DNA topological constraints linked to mitotic exit chromatin remodeling.

## RESULTS

### Crosslinking with formaldehyde dissolved in pure methanol preserves HNF1β mitotic chromatin binding

Transcription factors drastically differ in the way they interact with mitotic chromatin. Many factors do not bind mitotic chromosomes, and at prometaphase, when the nuclear envelope is dissolved, they are released in the cytoplasm. Some others are bound to mitotic chromosomal barrels but predominantly bind mitotic chromatin in a

non-sequence-specific way, while few others interact with mitotic chromatin in a sequence-specific way. However, it has been shown that only a relatively small fraction of chromatin sites that are normally bound by transcription factors in interphase are retained and bound in mitosis.³,²¹ In the present study, we set out to identify which chromatin sites are bound by HNF1β in mitotic chromatin. However, as we have demonstrated previously,⁹ the use of formaldehyde, a crosslinking agent commonly employed for chromatin immunoprecipitation (ChIP) analyses, leads to a dramatic dissociation of HNF1β from mitotic chromatin (Figure 1A). This dissociation has been shown to be a common problem that affects the analysis of most bookmarking factors.²²,²³ In our previous study,⁹ we showed that fixation with methanol preserves the mitotic localization of HNF1β. Therefore, we reasoned that methanol could provide a permissive environment so that formaldehyde treatment would not lead to the dissociation of HNF1β from mitotic chromatin. In fact, our results showed that, when cells were crosslinked with formaldehyde in pure methanol, HNF1β binding was perfectly preserved in immunofluorescence (Figure 1A). In a second step, we determined
whether chromatin crosslinking with formaldehyde in a non-aqueous crosslinking (NAQC) environment could provide a way to perform ChIP of HNF1β in mitotic cells. To this end, we used the cell line 786-O, a human renal tubular cell line that endogenously expresses HNF1β. Our results showed that increasing concentrations of formaldehyde in pure methanol led to a progressive ChIP enrichment of a prototypical HNF1β site (Figure 1B). However, we noticed that the use of high concentrations of formaldehyde in pure methanol induced excessive crosslinking, which, in turn, led to poor chromatin solubilization yields. Eventually, the optimal concentration of formaldehyde in pure methanol was found to be between 0.1% and 0.2% for 15 min at 25°C. These results indicated that HNF1β could be efficiently immunoprecipitated from chromatin crosslinked in a NAQC environment.

We then employed a ChIP sequencing (ChIP-seq) approach using 786-O cells to identify chromatin sites bound by HNF1β in asynchronously growing cells and in mitosis. These cells could be blocked in mitosis with the use of a specific CDK1 inhibitor (RO-3306), followed by nocodazole treatment (Figure S1A), without any significant loss in viability. We were able to harvest mitotic cells by shake-off with a purity of 88%-95%. One of our aims was to characterize and compare the way HNF1β binds interphase versus mitotic chromatin. In particular, we wanted to verify whether the binding of HNF1β to chromatin is maintained and evaluate whether mitotic binding is sequence specific.

In general, considering the number of the peaks and their intensities obtained in ChIP-seq experiments, bookmarking factors are known to bind mitotic chromatin with a much weaker efficacy compared to their interphase targets.²¹ In order to precisely compare the efficiency of the different immunoprecipitation techniques in mitosis versus interphase, we carried out a calibrated ChIP approach through the use of an internal control in order to monitor for the efficiency of HNF1β immunoprecipitation in the different conditions. To this end, we adopted the calibration methodology proposed by Hu et al.²⁴ As a calibrating control, we used freely cycling cells from a mouse cell line (mIMCD3) that natively expresses HNF1β and used an antibody that similarly recognizes both mouse and human HNF1β.

We first tested our calibration protocol on NAQC crosslinking by mixing different proportions of asynchronous 786-O and mIMCD3 cells just before the crosslinking step. As shown in Figure S1B, the recovery of a human-specific prototypical chromatin binding site (estimated by real-time PCR as percentage of input) was linearly correlated with the proportion of 786-O cells, while the signal given by mIMCD3 cells remained constant. These data indicated that our ChIP procedure led to a linear estimation of the recovery of chromatin fragments. We then applied this calibration approach to our HNF1β ChIP-seq, admixing the same freely cycling mIMCD3 cell population (20% of the total number of cells) to either mitotically blocked or asynchronously growing 786-O cells. We verified that the admixture of mouse and human cells at the moment of crosslinking could easily be resolved during the step of sequence alignment to their respective genomes and found that less than 1% of reads were misaligned to the wrong genome. With the classic aqueous crosslinking (AQC) method, we could identify a consistent number of peaks (7,074 chromatin positions) in asynchronously growing

cells (Figures 2A and S1C), while in mitotic cells, the binding of HNF1β was apparently very poor, and we detected a very small number of peaks (170 in the whole genome). Even though all peaks in both conditions contained the classic consensus site for HNF1β (Figure 2A), we could not exclude that the low mitotic signal was due to the small contamination of interphase cells. In agreement with the observed rapid dissociation of HNF1β from mitotic chromosomes upon formaldehyde fixation, this conventional crosslinking showed little binding of HNF1β to mitotic chromatin sites. In this context, the chromatin from our internal control (freely cycling mIMCD3 cells admixed either to human asynchronous or mitotic cells) gave rise to a comparable number of reads in peaks in mitotic cells compared to asynchronously growing cells (Figure S1D), indicating that the lack of signal in the mitotic sample was not due to low immunoprecipitation efficiency. Interestingly, when cells were formaldehyde crosslinked in methanol (NACQ), subsequent ChIP resulted in a more robust signal (Figures 2A, 2B, S1C, and S1E), with 16,115 peaks identified in asynchronous cells. In mitotic cells, HNF1β binding was slightly attenuated but significantly and closely correlated with the intensity of binding observed in interphase (Pearson's r = 0.75, p = 2.2⁻¹⁶). These findings indicate that using a non-aqueous environment (pure methanol) during formaldehyde crosslinking (NACQ) significantly enhances the detection of HNF1β binding to the same specific regions both in interphase and mitotic chromatin. Similarly, NAQC ChIP-seq gave a strong mitotic signal in a mouse renal tubular cell line (KECs [kidney epithelial cells]) (Figures 2C, 2D, and S1C-S1F). A typical peak profile obtained next to a direct HNF1β target gene (Cys1) in different crosslinking conditions is shown in Figure 2E, demonstrating increased ChIP efficiency using NAQC. It is worth noting that the peaks that were not claimed by the peak-calling genome-wide event finding and motif discovery (GEM) algorithm in mitotic cells had an overall genomic distribution very similar to those that instead were robustly detected in both mitotic and asynchronous cells (Figure S1G). In addition, we performed assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) in order to monitor the degree of accessibility of these chromatin regions. Our results showed that the chromatin regions detected by ChIP-seq for HNF1β in a non-aqueous environment were overall promptly accessible to ATAC-seq, indicating that they were likely functional sites (Figure S2).

### NAQC ChIP-seq HNF1β sites are pertinent for gene expression

The use of this alternative chromatin crosslinking method led to the identification of a significantly larger number of HNF1β binding sites. In order to assess the biological pertinence of the HNF1β peaks identified by the different crosslinking methodologies, we performed a transcriptomic analysis with RNA sequencing (RNA-seq) in 786-O cells that were efficiently knocked down for HNF1β (Figure S3A). We then analyzed our ChIP-seq and RNA-seq results with the Cistrome package²⁵ and found that the set of peaks detected by NAQC was significantly enriched in genes whose expression was decreased in the absence of HNF1β (Figures S3C and S3D), indicating that HNF1β predominantly behaves as a positive transcriptional
<figure><img src="image_7.png" /><figcaption>**Figure 2. NAQC-ChIP-seq allows the identification of HNF1β mitotic sites**</figcaption></figure>

(A and C) Heatmaps of HNF1β peaks identified in asynchronous (Asy) or mitotic (Mit) 786-O (A) or KEC cells (C) fixed with AQC or NAQC. The heatmap order is according to the decreasing intensity of peaks in Asy NACQ with a distance of + or -5 kb from the center of the peak. Detected peaks present an HNF1 consensus, as shown under the heatmaps for each condition.

(B and D) Average binding profiles shown as counts per million (CPM) for the 4 conditions of 786-O (B) and KEC (D) HNF1β ChIP-seq.

(E) Example of HNF1β peak visualization with Integrative Genomics Viewer (IGV) in the promoter of *Cys1* in the 4 conditions in KECs, shown as CPM.
See also Figures S1–S3.

regulator of its target genes. In this respect, the enrichment for NAQC sites in genes with decreased expression was comparable to those obtained by the classic AQC condition (Figure S3E), indicating that the global biological relevance of NAQC HNF1β

sites is similar to that obtained with a classic approach (AQC). RNA-seq was also performed in a KEC cell line, in which we deleted HNF1β with CRISPR-Cas9 (Figure S3B). Cistrome analysis on KEC RNA-seq and ChIP-seq data paralleled the results
<figure><img src="image_8.png" /><figcaption>**Figure 3.** HNF1β interacts with TOP1 via BTBD2 specifically in mitosis</figcaption></figure>

(A) Experimental scheme for the co-immunoprecipitation experiment.

(B) Wild-type (WT) and BTBD2 knockout (KO) HEK293 cells were transfected with the Nter-HNF1β-GFP plasmid, split, and blocked with nocodazole in prometaphase or left growing asynchronously. GFP immunoprecipitation (IP) was performed on total cell extracts, and detection of TOP1, BTBD2, and GFP was done by western blot with specific antibodies. An asterisk indicates a nonspecific background band seen by BTBD2 antibody in WT and BTBD2 KO cells.

See also Figure S4.

obtained with 786-O cells: HNF1β sites identified by NAQC are biologically relevant, and HNF1β predominantly acts as a positive transcriptional regulator (Figure S3G).

**Identification of BTBD2 as an HNF1β interactor**

In order to understand the possible molecular function of HNF1β, we set out to identify proteins that interact with HNF1β in mitotic chromatin. Our previous studies have demonstrated that the N-terminal portion of HNF1β, corresponding to the first 313 amino acids, which includes the dimerization and DNA-binding domains, represents the minimal portion of the protein that is still capable of binding mitotic chromatin.⁸,⁹ To find possible interactors of this crucial portion of HNF1β, we performed a yeast 2-hybrid screen with a library of embryonic mouse renal cDNA. The results showed that more than 90% of the best 160 identified cDNA independent hits encoded for either PCBD1 or PCBD2 (also called DCoH1 and DCoH2), two co-factors that are well known to physically interact with HNF1β.²⁶,²⁷ This result encouraged us to consider other potential partners that were identified in the same screen. Among these, we found a cDNA sequence encoding for the C-terminal fragment (amino acids [aa] 395–523) of BTBD2 (BTB domain-containing 2), encoding for a PHR (Pam-Highwire-RPM1) protein-protein interaction domain. BTBD2 had been initially cloned and identified as a TOP1-interacting protein, and it is worth noting that the BTBD2 interaction with TOP1 is mediated by the same PHR C-terminal domain (aa 289–525).²⁸

It is known that BTBD2 forms dimers via its N-terminal BTB-POZ (broad-complex, tramtrack, and bric-à-brac/poxvirus and zinc-finger) domain,²⁹ and we hypothesize that, in its dimeric conformation, the two PHR domains of the complex might provide a possible simultaneous interaction of BTBD2 with both TOP1 and HNF1β.

In order to identify the possible interaction between TOP1, HNF1β, and BTBD2, we carried out co-immunoprecipitation experiments in HEK293 cells transfected with HNF1β-GFP (Figure 3A). One caveat is that BTBD2, together with a very close paralog (BTBD1), has been shown to be predominantly localized in cytoplasmic bodies²⁸,³⁰ and not in the nucleus, where HNF1β and TOP1 are normally confined. In agreement with this, we could not see any significant interaction between HNF1β and BTBD2 in asynchronously growing cells (Figure 3B). However, in mitotically blocked cells, when the nuclear/cytoplasm compartmentalization is lost, the interaction between HNF1β and BTBD2, as well as HNF1β and TOP1, was detected (Figure 3B). In order to see whether BTBD2 plays a role in mediating the association between HNF1β and TOP1, we generated cell lines carrying null mutations in BTBD2 induced by CRISPR-Cas9 gene editing. The BTBD2 mutant cell lines expressed a negligible residual amount of mRNA encoding for BTBD2 but maintained constant expression of BTBD1, a very close BTBD2 paralog (Figure S4A). On the other hand, western blot analysis showed that the major BTBD2 band seen in parental wild-type cells disappeared in mutant cells, whereas a residual nonspecific background band continued to be expressed (Figure S4B). Our results showed that the mitotic interaction between HNF1β and TOP1 was decreased by the loss of BTBD2 (Figures 3B and S4C). Given the potential redundancy between the very similar proteins BTBD1 and BTBD2, the residual presence of BTBD1 might have contributed to reducing the biological effect of BTBD2 inactivation. In order to further assess the existence of this complex in a more physiological context, we monitored the interaction between TOP1 and HNF1β with a proximity assay based on the BioID system *in vivo*.³¹ BTBD2-null HEK293 cells or wild-type controls were transfected with HNF1β-BirA* and then blocked in mitosis or left freely cycling. After biotin treatment, cells were lysed, and protein extracts were affinity purified on streptavidin beads in order to capture biotinylated proteins (Figure 4A). Our results indicated that, when the N-terminal part of HNF1β is expressed in fusion with BirA*, TOP1 is biotinylated specifically in mitotic cells and not significantly in freely cycling cells. In addition, the loss of BTBD2 tended to decrease the levels of biotinylated TOP1 (Figures 4B and 4C). All of these results indicated that BTBD2 favors the formation of a complex where HNF1β and TOP1 interact specifically and predominantly in mitosis.

**The microhomology between HNF1β and TOP1**

We were intrigued by the fact that both HNF1β and TOP1, two proteins with no overt overall sequence homology, could interact with the same (and small, ~130 aa) protein interface represented by the PHR protein-protein interaction domain of BTBD2. We therefore wondered whether HNF1β and TOP1 may carry micro-homologies (mimicry) that could account for the BTBD2-TOP1 and BTBD2-HNF1β interactions mediated by the PHR
A

<figure><img src="image_9.png" /></figure>

B

<figure><img src="image_10.png" /></figure>

C

<figure><img src="image_11.png" /></figure>

D

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

E

<figure><img src="image_13.png" /><figcaption>Figure 4. The HNF1β mutation K258E impairs the interaction with TOP1</figcaption></figure>

(A) Scheme of the BioID assay protocol. WT or K258E Nter-HNF1β-BirA-HA constructs were transfected in WT or BTBD2 KO HEK93 cells. The day after splitting, cells were blocked in prometaphase using nocodazole or kept in regular medium as a freely cycling control. In both cases, biotin was added 6 h before collection for streptavidin affinity purification and western blot analysis.

(B) Western blot analysis of a representative BioID assay.

(C) Quantification of the BioID assay. Biotinylated TOP1 is shown as the ratio between affinity-purified TOP1 and HA, normalized to their own inputs. Data are represented as means ± SEM. n = 5 independent experiments. Statistical significance was determined by two-way ANOVA (\*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.0001).

(D) Alignment of HNF1β (254–267) and TOP1 (317–330) amino acid sequences. Brighter green and magenta indicate amino acid identity. The distance (in angstroms) between the alpha carbon of the corresponding amino acid residues in the two crystal structures is shown in the alignment. The gray bar indicates the beginning of the second α helix of the homeodomain of HNF1β.

(E) 3D structures of the N-terminal part of HNF1β and TOP1. The stretch of similarity (HNF1β 254–267, TOP1 317–330) is depicted in bright green and magenta, respectively, while DNA is shown in blue.

domain of BTBD2. Interestingly, when the critical minimal portions of TOP1 (215–331²⁸) and HNF1β (1–313) that account for interaction with BTBD2 were scored for similarities, we could find a weak homology alignment of 14 aa residues between TOP1 (aa 317–330) and HNF1β (aa 254–267) with 65% similarity and 50% identity (Figure 4D). Despite the limited homology, the 3D conformation of these parts of the two molecules is highly superimposable with a root-mean-square deviation of the distance of alpha carbons of only 1.5 Å (Figure 4D). In this microhomology region, 10 aa residues are in an α-helical conformation, which might have provided a constraint for the observed 3D similarity. Nevertheless, the overall 3D structure of the region, including the non-α-helical loop of 4 aa, shows that the two proteins have a very similar structure (Figure 4E).

Interestingly, HNF1α, a very close paralog of HNF1β, is, along with TOP1, extremely well conserved in these positions across evolution. In particular, one of these highly conserved residues (the K258 in HNF1β, corresponding to K226 in HNF1α) was found mutated to a glutamic acid (E) in a family affected by MODY3 diabetes,³² indicating that this residue may play an important role in the function of both HNF1α and HNF1β. In fact, our results from BioID experiments showed that, when this K258 residue was mutated into E, HNF1β was no longer able to interact in vivo with TOP1 (Figures 4B and 4C).

### HNF1β activates TOP1 enzymatic activity

The interaction of HNF1β with TOP1 raises the possibility that the enzymatic activity of TOP1 might be modulated by HNF1β.

Locally increased activity of TOP1 around HNF1β target genes may provide facilitated chromatin remodeling upon mitotic exit in order to quickly reactivate gene expression of bookmarked genes. In order to test this hypothesis, we monitored the *in vitro* topological relaxation of a supercoiled plasmid DNA containing an array of 10 multimerized high-affinity HNF1β binding sites (Figure 5A). Our results showed that, when either very small amounts of TOP1 recombinant protein alone or N-terminal HNF1β protein alone are used in the test, the DNA relaxation remains undetectable (Figure 5B). However, in these conditions, the concomitant presence of N-terminal HNF1β, immunoprecipitated from transfected cells, along with the same limiting amounts of TOP1, led to a substantial extent of DNA relaxation, indicating that N-terminal HNF1β protein, pulled down from cell extracts, is able to significantly stimulate TOP1 activity. (Figure 5B). Remarkably, when the critical K258 residue of HNF1β was mutated to E, the activity of TOP1 was no longer stimulated, indicating that the actual physical interaction of HNF1β with TOP1 leads to a significant increase of TOP1 enzymatic activity (Figures 5B–5D). These results indicate that the interaction between HNF1β and TOP1, possibly transiently mediated by the cytoplasmic BTBD2, which may have become available in total cell extracts, may increase the enzymatic activity of TOP1.

### HNF1β and TOP1 tend to be enriched in mitotic chromatin

In order to verify whether TOP1 tends to colocalize and be enriched on mitotic chromatin sites occupied by HNF1β, we
<figure><img src="image_14.png" /><figcaption>**Figure 5.** HNF1β increases TOP1 enzymatic activity</figcaption></figure>

(A) Scheme of the TOP1 assay. Human recombinant TOP1 (5–10 ng) was pre-incubated with immunoprecipitated GFP-HNF1β or GFP control for 10 min on ice. The reaction buffer and plasmid (containing 10 multimerized high-affinity HNF1 binding sites) were added just before incubation at 37°C for 15–30 min, and the reaction was stopped by the addition of SDS and Proteinase K and further incubation at 37°C for 15 min.

(B) Electrophoretic separation of supercoiled DNA (SCD) and relaxed/nicked DNA (RD/ND).

(C) Representative western blot of immunoprecipitated GFP and GFP-HNF1β proteins used in the TOP1 assay. In every experiment, bead-immuno-complexes were normalized to WT GFP-HNF1β.

(D) Quantification of 3 independent TOP1 assay experiments. SCD bands were quantified and compared to SCD without TOP1, and values were then compared to GFP and are shown as relaxed fraction. Relaxation degree was calculated by subtracting the activity level of background.
Data are shown as means ± SEM. Statistical analysis was performed by unpaired t test (*p* < 0.05).

performed ChIP-seq of TOP1 in mitotic KECs and monitored the consequences of the loss of HNF1β. A careful evaluation of the degree of occupancy of TOP1 on the most prominent HNF1β peaks next to genes whose expression decreases upon the inactivation of HNF1β showed that the TOP1 signal was decreased slightly but significantly in mitosis in HNF1β-deficient cells compared to wild-type cells (Figures 6A and S5).

### HNF1β activates TOP1 enzymatic activity in mitotic chromatin

Given the fact that the interaction with HNF1β increased the enzymatic activity of TOP1, we wondered whether chromatin positions occupied by HNF1β contain DNA whose topology could be more relaxed by the presence of a possibly more active TOP1. Psoralens are planar aromatic molecules that are able to cross cell membranes and to intercalate into DNA. Upon UV (360 nm) irradiation, psoralens can crosslink DNA strands. It is known that their DNA intercalation propensity is increased when the DNA is wound with negative superhelical tension compared to a relaxed or positive supercoiled status.³³ We considered that, as demonstrated previously,³⁴ psoralen incorporation could provide an indirect estimation of the level of heli-

cal relaxation that may be modulated by active transcription or replication or by the enzymatic activity of topoisomerases.

By using low doses (20 µM) of biotinylated psoralen for short periods of time (5 min) in asynchronous and mitotic KECs, we were able to pull down biotinylated psoralen-crosslinked DNA and estimate the degree of torsional stress of specific chromatin sites. We particularly focused on *Wnt16*, an HNF1β target gene characterized by the proximity of a prominent HNF1β chromatin binding site located 7.5 kb upstream of the transcription start site (TSS). In asynchronously cycling cells, we observed that the inhibition of TOP1 activity with camptothecin did not result in a significant decrease of psoralen intercalation. On the other hand, in mitotic chromatin, the inhibition of TOP1 led to an overall stronger decrease of psoralen incorporation, indicating a much higher TOP1 enzymatic activity. In this context, psoralen incorporation tended to be more prominent on the HNF1β binding site (Figures 6B and S6). The increased activity of TOP1 during mitosis is in line with previous studies.³⁵ In order to further assess the presence of enzymatically actively engaged TOP1, specifically in proximity of HNF1β binding, we also adopted an approach based on RADAR (rapid approach to DNA adduct recovery)³⁵,³⁶ followed by ChIP-qPCR analysis. In this assay, TOP1 was first crosslinked to DNA by the action of camptothecin only in positions where TOP1 is actively engaged in its DNA relaxation activity. In a second step, ethanol-precipitated chromatin was subsequently resuspended and immunoprecipitated with TOP1 antibodies. In this way, the presence of specific DNA fragments could be assayed by qPCR. Our results showed that TOP1 activity was higher in mitotic cells. More importantly, the peak of activity coincided with the peak HNF1β binding site upstream of the *Wnt16* gene (Figure 6C).

### DISCUSSION

Bookmarking transcription factors play a crucial role in ensuring the continuity of gene expression programs across cell divisions and are vital for maintaining cellular identity and function. The mechanism of bookmarking transcription factors involves their ability to bind to specific and critical DNA sequences even as the chromatin undergoes dramatic structural changes during mitosis. In our previous studies, we have shown that HNF1β remains associated with mitotic chromatin and that it controls the post-mitotic re-expression of specific target genes.⁸ However, we lacked experimental evidence that HNF1β could remain specifically bound to transcriptional control regions of bookmarked target genes in a sequence-specific way. In order to assess this essential point, we developed a technique to overcome the technical obstacles associated with classic formaldehyde crosslinking, which notoriously leads to the dissociation of bookmarking factors from mitotic chromatin.²²,²³ Our crosslinking technique, based on the use of formaldehyde crosslinking in a non-aqueous (pure methanol) environment, demonstrated that HNF1β remains bound to its chromatin targets both in interphase and in mitosis.

Bookmarking transcription factors are known to be involved in the rapid re-establishment of active transcription of their target genes.³,³⁷ The mechanisms through which bookmarking transcription factors facilitate the reactivation of transcription remain an area of ongoing research. Our investigation has disclosed
<figure><img src="image_15.png" /><figcaption>Figure 6. HNF1β and TOP1 colocalize on mitotic chromatin</figcaption></figure>

(A) Quantification of TOP1 occupancy on HNF1β-target genes in mitotic KECs. The group of genes analyzed was selected by the presence of prominent HNF1β peaks on the gene or promoter region and reduced expression in HNF1β KO cells. Statistical analysis was performed with Wilcoxon test (*p* = 0.0003001).

(B) Biotin-psoralen incorporation assay in asynchronous and mitotic KECs treated with camptothecin (CPT) or vehicle (DMSO). The amount of streptavidin-purified DNA was quantified by qPCR at different chromosomal positions of the *Wnt16* gene and is shown as percentage of input as distance in kilobases from the TSS. HNF1BS, HNF1β binding site; TSS, transcription start site. Results are shown as means ± SEM. *n* = 3 independent experiments.

(C) RADAR-ChIP assay showing the presence of catalytically active TOP1 around the HNF1β binding site of the *Wnt16* promoter in mitotic KECs as in (B). Results are shown as means ± SEM. *n* = 2 independent experiments. See also Figures S5 and S6.

experimental evidence for an innovative aspect involving the
implication of TOP1 that is mediated by BTBD2, an interactor
of HNF1β. Remarkably, we have shown that BTBD2 is able to
orchestrate a cell-cycle-restricted interaction between HNF1β
and TOP1, specifically during mitosis. Interestingly, during inter-
phase, BTBD2 is predominantly localized to the cytoplasm.³⁰
This compartmentalization probably prevents BTBD2 from bind-
ing to HNF1β and TOP1, which are located within the nucleus.
Our results showed that, in mitosis, after the breakdown of the
nuclear envelope, BTBD2, TOP1, and HNF1β interact, leading
to significantly increased TOP1 enzymatic activity.

Remarkably, both HNF1β and TOP1 interact with the same
protein-protein interaction domain of BTBD2: the PHR domain.
This led us to uncover a microhomology between HNF1β and
TOP1 that folds in a very similar 3D conformation and that could
provide a possible interaction interface between BTBD2 and
both HNF1β and TOP1. In this region, we identified an amino
acid residue whose mutation in HNF1α, a close paralog of
HNF1β, leads to a loss-of-function phenotype in MODY3 pa-
tients.³² We demonstrated that this mutant is no longer able
to bind to TOP1. This observation suggests that the highly
conserved residue in both TOP1 and HNF1α/β may provide an
evolutionary advantage by granting the interaction/activation of
TOP1 on HNF1β-dependent bookmarked target genes.

Our results demonstrated that this interaction leads to a signif-
icant stimulation of TOP1 enzymatic activity. This enzyme plays a
pivotal role in gene expression control and maintenance of DNA
stability by alleviating DNA torsional stress. As an essential
enzyme, TOP1 modulates the topological state of DNA by intro-
ducing transient single-strand breaks, allowing the relaxation of
torsional strain that arises during processes such as DNA repli-
cation and transcription.³⁸ In the realm of gene expression,
TOP1 facilitates the unwinding of DNA strands, enabling efficient
progression of RNA polymerase and the transcription machinery
along the DNA template. Moreover, TOP1 has been described
recently to physically and functionally interact with RNA poly-
merase II.³⁹ This active participation in transcriptional dynamics
ensures accurate RNA synthesis and faithful expression of ge-
netic information.⁴⁰,⁴¹ TOP1 has also been shown to be involved
in mitotic chromosomal condensation⁴² and to be a component
of the mitotic chromosome.⁴³,⁴⁴ Emerging evidence suggests
that TOP1 may play a crucial role in the complex process of
mitotic exit.³⁵ As cells transition from mitosis to interphase, intri-
cate chromatin reorganization and decondensation must occur,
necessitating the careful management of DNA topology. TOP1,
with its ability to relieve DNA torsional stress by introducing tran-
sient single-strand breaks, may play a pivotal role in facilitating
the smooth and controlled unwinding of condensed chromatin
structures during mitotic exit. By strategically modulating DNA
supercoiling and tension, TOP1 may contribute to the re-estab-
lishment of gene expression patterns in conjunction with the ac-
tion of bookmarking factors. Additionally, the potential involve-
ment of TOP1 in resolving topological DNA constraints during
chromatin decondensation at mitotic exit could participate in
the process of post-mitotic gene expression reactivation. While
the precise mechanisms of this process remain an exciting
area of investigation, our results show that HNF1β leads to a sig-
nificant accumulation of TOP1 on bookmarked HNF1β sites. In
addition, our results showed that TOP1 is active at these sites,
indicating that the interaction between HNF1β and TOP1, medi-
ated by BTBD2, leads to a functional consequence.

In conclusion, our findings provide a significant advancement in our understanding of a mitosis-specific function for HNF1β due to its interaction with BTBD2 and TOP1 that is specifically restricted in mitosis. Moreover, the interaction between HNF1β and BTBD2, along with the impact of a specific human mutation affecting its interaction with the PHR domain of TOP1, highlights important aspects of bookmarking-mediated gene regulation. This study advances our knowledge on the bookmarking activity
of a transcription factor whose haploinsufficiency leads to severe disease conditions.

## Limitations of the study

In this study, we showed that the cytoplasmic BTBD2 protein mediates the interaction between two nucleus-localized proteins, HNF1β and TOP1, specifically in mitosis, when the nuclear compartmentalization is lost. On the other hand, we showed that the HNF1β-GFP fusion protein is able to stimulate the enzymatic activity of TOP1 from cell extracts of asynchronously growing cells, where the interaction with BTBD2 is negligible. We do not fully understand how this works. One possibility is that the formation of the ternary complex might be a slow, limiting process or linked to posttranslational modifications. In the total cell extracts, the cytoplasm-derived BTBD2 may have become available for the formation of the ternary complex but with limited efficiency, sufficient to increase the enzymatic activity of TOP1.

## RESOURCE AVAILABILITY

### Lead contact

Requests for further information, resources, and reagents should be directed to and will be fulfilled by the lead contact, Marco Pontoglio (marco.pontoglio@inserm.fr).

### Materials availability

Plasmids and cell lines generated in this study are available from the lead contact upon request via a materials transfer agreement.

### Data and code availability

* ChIP-seq, ATAC-seq, and RNA-seq data generated in this paper have been deposited in the GEO database and are publicly available as of the date of publication. Accession numbers are listed in the key resources table.

* This paper does not report original code.

* Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

## ACKNOWLEDGMENTS

We thank the GENOM'IC (Cochin Institut, Paris, France) and the Cell Imaging (SFR Necker, Paris, France) core facilities for technical assistance. We are grateful to Nicolas Dupont (INEM, Paris, France) for sharing KECs. This work was supported by "Fondation pour la Recherche Médicale" Equipe FRM EQU202003010703, Labex "Who am I?" and MSDAVENIR DS-2017-0051. The graphical abstract was created with BioRender.

## AUTHOR CONTRIBUTIONS

Conceptualization, A.B., S.L.T., and M.P.; methodology, A.B., S.L.T., M.C., M.P.M., J.L., and N.K.; software, S.G., N.Z., and M.P.; visualization, A.B., M.C., S.G., N.Z., and M.P.; writing, A.B., S.L.T., and M.P.; supervision, A.B. and M.P.; funding acquisition, M.P.

## DECLARATION OF INTERESTS

The authors declare no competing interests.

## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* KEY RESOURCES TABLE

* EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

* Cell culture

* METHOD DETAILS

* Plasmid constructs

* Immunofluorescence

* Mitotic synchronization

* Chromatin immunoprecipitation (ChIP)

* ChIP-seq

* ChIP-seq analysis

* ATAC-seq

* ATAC-seq analysis

* RNA extraction and analysis

* RNA-seq

* RNA-seq analysis

* HNF1B silencing

* CRISPR-CAS9 generation of stable KNOCK-OUT cell lines

* Yeast-2-hybrid screen

* Immunoprecipitation

* BioID proximity-dependent biotinylation assay

* Western Blot

* Topoisomerase 1 assay

* 3D structure analysis

* Biotin-Psoralen incorporation assay

* RADAR (rapid approach to DNA adduct recovery)-ChIP

* QUANTIFICATION AND STATISTICAL ANALYSIS

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.celrep.2024.114805.

Received: December 4, 2023

Revised: July 3, 2024

Accepted: September 12, 2024

Published: October 9, 2024

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55. Liao, Y., Smyth, G.K., and Shi, W. (2019). The R package Rsubread is easier, faster, cheaper and better for alignment and quantification of RNA sequencing reads. Nucleic Acids Res. 47, e47. https://doi.org/10.1093/nar/gkz114.

56. Love, M.I., Huber, W., and Anders, S. (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550. https://doi.org/10.1186/s13059-014-0550-8.

57. Gong, Y., Ma, Z., Patel, V., Fischer, E., Hiesberger, T., Pontoglio, M., and Igarashi, P. (2009). HNF-1beta regulates transcription of the PKD modifier gene Kif12. J. Am. Soc. Nephrol. 20, 41-47. https://doi.org/10.1681/ASN.2008020238.

58. Ran, F.A., Hsu, P.D., Wright, J., Agarwala, V., Scott, D.A., and Zhang, F. (2013). Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281-2308. https://doi.org/10.1038/nprot.2013.143.

59. Anzalone, A.V., Randolph, P.B., Davis, J.R., Sousa, A.A., Koblan, L.W., Levy, J.M., Chen, P.J., Wilson, C., Newby, G.A., Raguram, A., and Liu, D.R. (2019). Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149-157. https://doi.org/10.1038/s41586-019-1711-4.

60. Chen, B., Gilbert, L.A., Cimini, B.A., Schnitzbauer, J., Zhang, W., Li, G.-W., Park, J., Blackburn, E.H., Weissman, J.S., Qi, L.S., and Huang, B. (2013). Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155, 1479-1491. https://doi.org/10.1016/j.cell.2013.12.001.

61. Grandi, F.C., Modi, H., Kampman, L., and Corces, M.R. (2022). Chromatin accessibility profiling by ATAC-seq. Nat. Protoc. 17, 1518-1552. https://doi.org/10.1038/s41596-022-00692-9.

62. Lu, P., Rha, G.B., and Chi, Y.-I. (2007). Structural basis of disease-causing mutations in hepatocyte nuclear factor 1beta. Biochemistry 46, 12071-12080. https://doi.org/10.1021/bi7010527.

63. Staker, B.L., Hjerrild, K., Feese, M.D., Behnke, C.A., Burgin, A.B., and Stewart, L. (2002). The mechanism of topoisomerase I poisoning by a camptothecin analog. Proc. Natl. Acad. Sci. USA 99, 15387-15392. https://doi.org/10.1073/pnas.242259599.

64. Herrero-Ruiz, A., Martínez-García, P.M., Terrón-Bautista, J., Millán-Zambrano, G., Lieberman, J.A., Jimeno-González, S., and Cortés-Ledesma, F. (2021). Topoisomerase IIα represses transcription by enforcing promoter-proximal pausing. Cell Rep. 35, 108977. https://doi.org/10.1016/j.celrep.2021.108977.

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## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3">Antibodies</td></tr><tr><td>anti-beta-Actin antibody</td><td>Sigma-Aldrich</td><td>Cat# A1978; RRID: AB_476692</td></tr><tr><td>anti-HNF1beta</td><td>Santa Cruz Biotechnology</td><td>Cat# sc-22840; RRID: AB_2279595</td></tr><tr><td>anti-HNF1beta</td><td>ABclonal, this study</td><td>E9034</td></tr><tr><td>anti-TOP1</td><td>ABclonal, this study</td><td>E9036</td></tr><tr><td>anti-HA.11</td><td>BioLegend</td><td>Cat# 901502; RRID: AB_2565007</td></tr><tr><td>anti-GFP</td><td>Abcam</td><td>Cat# ab290; RRID: AB_303395</td></tr><tr><td>Anti-BTBD2</td><td>Thermo Fisher Scientific</td><td>Cat# PA5-112834; RRID: AB_2867568</td></tr><tr><td>Sheep Anti-Mouse IgG - Horseradish Peroxidase</td><td>GE Healthcare</td><td>Cat# NA931; RRID: AB_772210</td></tr><tr><td>Donkey Anti-Rabbit IgG, Whole Ab ECL Antibody, HRP Conjugated</td><td>GE Healthcare</td><td>Cat# NA934; RRID: AB_772206</td></tr><tr><td>Donkey anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488</td><td>Thermo Fisher Scientific</td><td>Cat# A21202; RRID: AB_141607</td></tr><tr><td>Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488</td><td>Thermo Fisher Scientific</td><td>Cat# A21206; RRID: AB_2535792</td></tr><tr><td colspan="3">Bacterial and virus strains</td></tr><tr><td>DH5alpha Subcloning Efficiency</td><td>Thermo Fisher Scientific</td><td>Cat# 18265017</td></tr><tr><td colspan="3">Chemicals, peptides, and recombinant proteins</td></tr><tr><td>cOmplete, EDTA-free Protease Inhibitor Cocktail</td><td>Roche</td><td>Cat# 4693132001</td></tr><tr><td>TRIzol</td><td>Thermo Fisher Scientific</td><td>Cat# 15596018</td></tr><tr><td>Blasticidin</td><td>InvivoGen</td><td>Cat# ant-bl-05</td></tr><tr><td>Zeocin</td><td>Thermo Fisher Scientific</td><td>Cat# R25001</td></tr><tr><td>Doxycycline</td><td>Sigma-Aldrich</td><td>Cat# D9891</td></tr><tr><td>Thymidine</td><td>Sigma-Aldrich</td><td>Cat# T1895</td></tr><tr><td>Nocodazole</td><td>Sigma-Aldrich</td><td>Cat# M1404</td></tr><tr><td>RO-3306</td><td>Sigma-Aldrich</td><td>Cat# SML0569</td></tr><tr><td>S-trityl-L-cysteine</td><td>Sigma-Aldrich</td><td>Cat# 164739</td></tr><tr><td>Camptothecin</td><td>Bio-Techne</td><td>Cat# 1100</td></tr><tr><td>Formaldehyde</td><td>Sigma-Aldrich</td><td>Cat# F8775</td></tr><tr><td>Methanol</td><td>Carlo Erba</td><td>Cat# 414819</td></tr><tr><td>Phenol:chloroform:isoamylalcohol mix (25:24:1)</td><td>Thermo Fisher Scientific</td><td>Cat# 15593031</td></tr><tr><td>Proteinase K Solution</td><td>Eurobio</td><td>Cat# GEXPRK01-B5</td></tr><tr><td>DMEM, high glucose, GlutaMAX supplement</td><td>Thermo Fisher Scientific</td><td>Cat# 31966047</td></tr><tr><td>RPMI 1640 Medium, GlutaMAX Supplement</td><td>Thermo Fisher Scientific</td><td>Cat# 61870044</td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma-Aldrich</td><td>Cat# F7524</td></tr><tr><td>HBSS, calcium, magnesium, no phenol red</td><td>Thermo Fisher Scientific</td><td>Cat# 14025100</td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>Thermo Fisher Scientific</td><td>Cat# 14190250</td></tr><tr><td>Penicillin-streptomycin</td><td>Thermo Fisher Scientific</td><td>Cat# 15140130</td></tr><tr><td>Trypsin-EDTA (0.05%), phenol red</td><td>Thermo Fisher Scientific</td><td>Cat# 25300054</td></tr><tr><td>Biotin</td><td>Sigma-Aldrich</td><td>Cat# B4639</td></tr><tr><td>Digitonin</td><td>Santa Cruz Biotechnology</td><td>Cat# sc-280675B</td></tr><tr><td>Digitonin solution-high purity</td><td>Millipore</td><td>Cat# CHR103</td></tr></tbody></table>

(Continued on next page)
Continued

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>EZ-Link Psoralen-PEG3-Biotin</td><td>Thermo Fisher Scientific</td><td>Cat# 29986</td></tr><tr><td>MyOne Streptavidin C1 Dynabeads</td><td>Thermo Fisher Scientific</td><td>Cat# 65001</td></tr><tr><td>MyOne Streptavidin T1 Dynabeads</td><td>Thermo Fisher Scientific</td><td>Cat# 65601</td></tr><tr><td>Streptavidine Dynabeads™ M-280</td><td>Thermo Fisher Scientific</td><td>Cat# 11206D</td></tr><tr><td>Hoechst 33342, Trihydrochloride, Trihydrate</td><td>Thermo Fisher Scientific</td><td>Cat# H3570</td></tr><tr><td>Ethidium Bromide</td><td>Eurobio</td><td>GEPBET02-AF</td></tr><tr><td>Glycine</td><td>Sigma-Aldrich</td><td>Cat# G8898</td></tr><tr><td>RNase</td><td>Roche</td><td>Cat# 10109142001</td></tr><tr><td>GFP-Trap® Magnetic Particles M-270</td><td>Chromotek</td><td>Cat# gtd-20</td></tr><tr><td>Fluoromount G</td><td>Thermo Fisher Scientific</td><td>Cat# 00-4958-02</td></tr><tr><td>Puromycin</td><td>InvivoGen</td><td>Cat# ant-pr-1</td></tr><tr><td>Micrococcal nuclease</td><td>Sigma-Aldrich</td><td>Cat# N3755</td></tr><tr><td>Dynabeads protein A</td><td>Thermo Fisher Scientific</td><td>Cat# 10002D</td></tr><tr><td>BS3</td><td>Thermo Fisher Scientific</td><td>Cat# A39266</td></tr><tr><td>Recombinant human TOP1</td><td>MyBioSource</td><td>Cat# MBS142447</td></tr><tr><td>Linear PEI</td><td>Polysciences</td><td>Cat# 23966</td></tr><tr><td>Lipofectamine 2000 Transfection Reagent</td><td>Thermo Fisher Scientific</td><td>Cat# 11668019</td></tr><tr><td>Lipofectamine RNAiMAX</td><td>Thermo Fisher Scientific</td><td>Cat# 13778030</td></tr><tr><td>Opti-MEM, Reduced Serum Medium</td><td>Thermo Fisher Scientific</td><td>Cat# 31985070</td></tr><tr><td>Tagmentase - loaded</td><td>Diagenode</td><td>Cat# C01070012</td></tr><tr><td>Tagmentation Buffer (2x)</td><td>Diagenode</td><td>Cat# C01019043</td></tr></tbody></table>

Critical commercial assays

<table><tbody><tr><td>Pierce BCA Protein Assay Kit</td><td>Thermo Fisher Scientific</td><td>Cat# 23225</td></tr><tr><td>Venor®GeM Classic: Mycoplasma detection kit for conventional PCR</td><td>CliniSciences</td><td>Cat# 11-1050</td></tr><tr><td>iPure kit v2</td><td>Diagenode</td><td>Cat# C03010015</td></tr><tr><td>DNeasy Blood & Tissue Kit</td><td>Qiagen</td><td>Cat# 69506</td></tr><tr><td>QIAquick PCR Purification Kit</td><td>Qiagen</td><td>Cat# 28104</td></tr><tr><td>MinElute PCR Purification Kit</td><td>Qiagen</td><td>Cat# 28004</td></tr><tr><td>High-Capacity cDNA Reverse Transcription kit</td><td>Thermo Fisher Scientific</td><td>Cat# 4368813</td></tr><tr><td>GoTaq® qPCR Master Mix</td><td>Promega</td><td>Cat# A6002</td></tr><tr><td>PowerTrack™ SYBR Green Master Mix</td><td>Thermo Fisher Scientific</td><td>Cat# A46112</td></tr><tr><td>ECL™ Prime Western Blotting System</td><td>Amersham</td><td>Cat# RPN2232</td></tr><tr><td>ECL™ Select Western Blotting System</td><td>Amersham</td><td>Cat# RPN2235</td></tr><tr><td>Qubit™ dsDNA HS Assay Kit</td><td>Thermo Fisher Scientific</td><td>Cat# Q32851</td></tr></tbody></table>

Deposited data

<table><tbody><tr><td>786-O ChIP-seq</td><td>This paper</td><td>GSE245086</td></tr><tr><td>786-O RNA-seq</td><td>This paper</td><td>GSE245071</td></tr><tr><td>KEC ChIP-seq</td><td>This paper</td><td>GSE245070</td></tr><tr><td>KEC RNA-seq</td><td>This paper</td><td>GSE245072</td></tr><tr><td>KEC ATAC-seq</td><td>This paper</td><td>GSE245087</td></tr></tbody></table>

Experimental models: Cell lines

<table><tbody><tr><td>HEK293</td><td>ATCC</td><td>Cat# CRL-1573</td></tr><tr><td>HEK293 BTBD2-KO</td><td>This paper</td><td>N/A</td></tr><tr><td>mlMCD-3</td><td>ATCC</td><td>Cat# CRL-2123</td></tr><tr><td>MDCK pcDNA6/TR; pcDNA4/TO/Nter-HNF1β-GFP</td><td>Lerner et al.<sup>9</sup></td><td>N/A</td></tr><tr><td>KEC</td><td>Gregory J Pazour</td><td>N/A</td></tr><tr><td>KEC HNF1β-KO</td><td>This paper</td><td>N/A</td></tr></tbody></table>

(Continued on next page)
<figure><img src="image_17.png" /></figure>

Continued

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>786-O</td><td>ATCC</td><td>Cat# CRL-1932</td></tr><tr><td colspan="3"><strong>Oligonucleotides</strong></td></tr><tr><td>List of primers</td><td>This paper</td><td>Table S1</td></tr><tr><td>ON-TARGETplus Human HNF1B siRNA</td><td>Dharmacon</td><td>Cat# L-009721-00-0020</td></tr><tr><td>ON-TARGETplus Non-targeting Pool</td><td>Dharmacon</td><td>Cat# D-001810-10-50</td></tr></tbody></table>

Recombinant DNA

<table><tbody><tr><td>pcDNA4/TO/Nter-HNF1β-GFP</td><td>Lerner et al.<sup>9</sup></td><td>11p40</td></tr><tr><td>HNF1β isoform A</td><td>Bach, and Yaniv<sup>45</sup></td><td>6p15</td></tr><tr><td>pEGFP-N1</td><td>Clontech</td><td>N/A</td></tr><tr><td>pcDNA3.1 MCS-BirA(R118G)-HA</td><td>Addgene</td><td>Cat# 36047</td></tr><tr><td>HNF1βNter-BirA-HA</td><td>This paper</td><td>18p13</td></tr><tr><td>K258E HNF1bNter-BirA-HA</td><td>This paper</td><td>18p44</td></tr><tr><td>pSpCas9(BB)-2A-Puro</td><td>Addgene</td><td>Cat# 62988</td></tr><tr><td>pSpCas9(BB)-2A-Puro_BTBD2_18cr17Guide</td><td>This paper</td><td>18p22</td></tr><tr><td>pU6-pegRNA-GG-acceptor</td><td>Addgene</td><td>Cat# 132777</td></tr><tr><td>pU6-pegRNA-GG-acceptor_HNF1B_exon1_guide1</td><td>This paper</td><td>–</td></tr><tr><td>pU6-pegRNA-GG-acceptor_HNF1B_exon1_guide2</td><td>This paper</td><td>–</td></tr><tr><td>pNL1.2</td><td>Promega</td><td>Cat# N1011</td></tr><tr><td>pNL1.2-Multimerized HNF1β binding sites</td><td>This paper</td><td>16p38</td></tr></tbody></table>

Software and algorithms

<table><tbody><tr><td>ImageJ</td><td>NIH</td><td>https://imagej.nih.gov/ij/</td></tr><tr><td>Image Lab</td><td>Bio-Rad</td><td>https://www.bio-rad.com/en-fo/product/image-lab-software?ID=KRE6P5E8Z</td></tr><tr><td>Adobe Illustrator CS5.1</td><td>Adobe</td><td>https://www.adobe.com/fr/products/illustrator.html</td></tr><tr><td>GraphPad Prism 9.4</td><td>GraphPad Software</td><td>https://www.graphpad.com/scientificsoftware/prism/</td></tr><tr><td>ICE Analysis</td><td>Synthego</td><td>https://ice.synthego.com/#/</td></tr><tr><td>MxPro 4.10</td><td>Agilent</td><td>https://www.agilent.com/en/product/real-time-pcr-%28qpcr%29/real-time-pcr-%28qpcr%29-instruments/mx3000-mx3005p-real-time-pcr-system-software/mxpro-qpcr-software-232751</td></tr><tr><td>Design & Analysis Software 2.6.0</td><td>Thermo Fisher Scientific</td><td>https://www.thermofisher.com/fr/fr/home/technical-resources/software-downloads/quantstudio-3-5-real-time-pcr-systems.html</td></tr><tr><td>Jmol</td><td>http://www.jmol.org/</td><td>N/A</td></tr><tr><td>Aozan</td><td>Perrin et al.<sup>46</sup></td><td>https://www.outils.genomique.biologie.ens.fr/aozan/</td></tr><tr><td>FastQC</td><td>https://www.bioinformatics.babraham.ac.uk/projects/fastqc/</td><td>N/A</td></tr><tr><td>BowTie 2</td><td>Langmead, and Salzberg<sup>47</sup></td><td>https://bowtie-bio.sourceforge.net/bowtie2/manual.shtml</td></tr><tr><td>Sambamba</td><td>Tarasov et al.<sup>48</sup></td><td>–</td></tr><tr><td>Bedtools intersect</td><td>https://bedtools.readthedocs.io/en/latest/content/tools/intersect.html</td><td>N/A</td></tr><tr><td>bamCoverage</td><td>Ramírez et al.<sup>49</sup></td><td>https://deeptools.readthedocs.io/en/develop/content/list_of_tools.html</td></tr><tr><td>SAMtools</td><td>Li et al.<sup>50</sup></td><td>http://www.htslib.org/</td></tr><tr><td>GEM</td><td>Guo et al.<sup>51</sup></td><td>https://groups.csail.mit.edu/cgs/gem/</td></tr><tr><td>Genomation</td><td>Akalin et al.<sup>52</sup></td><td>https://bioconductor.org/packages/release/bioc/html/genomation.html</td></tr></tbody></table>

(Continued on next page)
Continued

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>MEME-ChIP</td><td>Bailey et al.<sup>53</sup></td><td>https://meme-suite.org/meme/doc/meme-chip.html</td></tr><tr><td>Cistrome</td><td>Liu et al.<sup>25</sup></td><td>http://cistrome.org/ap/root</td></tr><tr><td>STAR</td><td>Dobin et al.<sup>54</sup></td><td>https://bioinformaticshome.com/tools/rna-seq/descriptions/STAR.html#gsc.tab=0</td></tr><tr><td>Picard Tools</td><td>Broad Institute</td><td>https://broadinstitute.github.io/picard/</td></tr><tr><td>Rsubread</td><td>Liao et al.<sup>55</sup></td><td>https://bioconductor.org/packages/release/bioc/html/Rsubread.html</td></tr><tr><td>DESeq2</td><td>Love et al.<sup>56</sup></td><td>https://bioconductor.org/packages/release/bioc/html/DESeq2.html</td></tr><tr><td>R</td><td>https://www.r-project.org/</td><td>N/A</td></tr><tr><td colspan="3">Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.</td></tr></tbody></table>

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Cell culture

Kidney epithelial cells (KECs), generated by G.J. Pazour (University of Massachusetts, Worcester MA, USA), were kindly provided by N. Dupont (INEM, Paris, France). mIMCD3, Hek293, MDCK and KEC cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1% penicillin/streptomycin (Thermo Fisher Scientific). MDCK cells stably expressing inducible Nter-HNF1β-GFP, generated in our previous study,⁹ were grown with blasticidin (5 µg/mL) (Sigma-Aldrich) and zeocin (400 µg/mL) (Thermo Fisher Scientific). 786-O cells were cultured in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1% penicillin/streptomycin (Thermo Fisher Scientific). All cell lines were periodically tested for mycoplasma infection using the VENOR Gem Classic Kit (MB Minerva Biolabs).

METHOD DETAILS

Plasmid constructs

The Nter-HNF1β-GFP plasmid (house name 11p40), containing the N-terminal part of the human HNF1β isoform A (amino acids 1-313), was described in.⁹

The plasmid containing the human cDNA of HNF1B isoform A (house name 6p15) was described in.⁴⁵

The supercoiled plasmidic DNA used in the TOP1 relaxation assay (house name 16p38) was generated by cloning 10 multimerized high affinity HNF1 binding sites (TGGGGTGGGTAAATCTTTAACTTGO)⁵⁷ in the NanoLuc plasmid pNL1.2 (Promega).

pSpCas9(BB)-2A-Puro (Addgene plasmid # 62988) was a gift from Feng Zhang.⁵⁸ The sgRNA guide 18cr17 (CACCAGG ATGCCCGACTGTG), selected using the online CRISPOR tool (http://crispor.tefor.net), was cloned into the pSpCas9(BB)-2A-Puro vector (house name 18p22).

pU6-pegRNA-GG-acceptor (Addgene plasmid #132777) was a kind gift from David Liu.⁵⁹ Two guides targeting exon 1 of murine HNF1β (5'-CGGAAAAAGAAGGGGTTGAG-3' and 5'-ACTGCCGTCCCCGAATTTCG-3') were cloned into pre-digested (with BsaI restriction enzyme, Thermo Fisher Scientific) vector pU6-pegRNA-GG-acceptor in order to incorporate scaffold modifications that improve guide RNA expression.⁶⁰ The annealing and ligation procedures were performed following the protocol from⁵⁹ except that there were no pegRNA additions.

pcDNA3.1 MCS-BirA(R118G)-HA (Addgene plasmid # 36047) was a gift from Kyle Roux.³¹ For the construction of Nter-HNF1β-BirA-HA plasmid (house name 18p13), the N-terminal part of HNF1β was amplified by polymerase chain reaction (PCR) from the 11p40 plasmid with the 17i211 and 17i212 oligos and cloned into the pcDNA3.1 MCS-BirA(R118G)-HA plasmid after digestion of both plasmids with EcoRI and BamHI restriction enzymes. The K258E Nter-HNF1β-BirA-HA plasmid (house name 18p44) was created by introducing the K258E mutation in the Nter-HNF1β-BirA-HA plasmid (18p13) through site-directed mutagenesis (QuickChange II protocol, Agilent) using the 18i25 and 18i26 oligos. Mutagenesis was verified by sequencing with the 16i138, 16i141 and T7 oligos.

Immunofluorescence

MDCK cells stably expressing doxycycline-inducible N-terminal HNF1β-GFP were plated on coverslips. 24 h later, doxycycline (1 µg/mL, Sigma-Aldrich) was added for 16 h to induce overexpression of HNF1β-GFP and cells were fixed with 4% formaldehyde
(Sigma-Aldrich) in phosphate buffered saline (PBS), methanol or 0.2% formaldehyde in methanol for 15 min at room temperature. After three washes in PBS, cells were counterstained with 5 µg/mL Hoechst 33342 (Thermo Fisher Scientific) and mounted with Fluoromount G (Thermo Fisher Scientific). For live-cell imaging, MDCK cells expressing HNF1β-GFP were grown on IBIDI dishes (u-DISH 35mm High), counterstained with Hoechst 33342 and imaged in a thermostated chamber kept at 37°C and with 5% CO2. All images were acquired with a Zeiss confocal spinning disk and processed with ImageJ (NIH) and Adobe Illustrator CS5.1 (Adobe).

## Mitotic synchronization

786-O cells were plated at a 40% confluency and treated with 9 µM RO-3306 (Sigma-Aldrich) for 20-22 h in order to obtain a G2-M phase block. After RO-3306 treatment, cells were rinsed twice with PBS and released in fresh medium containing 30 ng/mL Nocodazole (Sigma-Aldrich) for 2 h and 30 min. KECs were plated at 40% confluency and blocked in S-phase by a 24 h incubation with 2 mM Thymidine (Sigma-Aldrich). Cells were then washed twice in HBSS and released for 3 h in regular medium. Mitotic block was obtained by adding 5 µM STLC (S-trityl-L-cysteine, Sigma-Aldrich) for 8-14 h. In both cases mitotic cells were collected by gentle shake-off, counted and used for further analysis. A small aliquot of cells was fixed in 4% formaldehyde (Sigma-Aldrich) for 20 min at room temperature, washed in distilled water and plated on SuperFrost slides (Thermo Fisher Scientific). Once dried, cells were washed twice with distilled water, twice with PBS, stained with Hoechst 33342 (Thermo Fisher Scientific) for 20 min, washed 3 times in PBS and mounted with Fluoromount-G (Thermo Fisher Scientific). The mitotic index was quantified with a BX53 microscope (Olympus) by counting the number of mitotic cells in at least 10 different fields.

## Chromatin immunoprecipitation (ChIP)

Since we needed to compare mitotic cells (shaken-off from plates and collected in suspension) with free-cycling cells (growing attached to the surface of a cell culture dishes) we wanted to ensure that comparable experimental conditions could apply to the two conditions during fixation. For this reason, asynchronously growing 786-O, KEC and mIMCD3 cells were collected by trypsinization in 0.05% Trypsin-EDTA (Thermo Fisher Scientific) and put in suspension before fixation. Free-cycling mIMCD3 cells (20% of the total number of cells) were admixed either to interphase or mitotic 786-O cells just before the crosslinking step as a calibrating internal control. Asynchronous or mitotic cells were centrifuged for 5 min at 1500 rpm at room temperature, washed with PBS and crosslinked at room temperature in AQC solution (1% formaldehyde (Sigma-Aldrich) in PBS) for 7 min or in NAQC solution (0.1% Formaldehyde in Methanol for 786-O or 0.2% Formaldehyde in Methanol for KECs) for 12 min. Cells were then centrifuged for 3 min at 25 °C at 1000 rpm and the crosslinking was quenched by the addition of 125 mM Glycine (Sigma-Aldrich) in PBS supplemented with 4% BSA (Sigma-Aldrich) for 5 min. After quenching, cells were centrifuged for 10 min at 4°C, washed in 4% BSA-PBS, centrifuged for 15 min at 4°C and resuspended in ChIP lysis buffer (25 mM Tris-HCl pH 8.0, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.3% SDS, protease inhibitors (Roche)). Samples were lysed by potter homogenization (Eberbach Corporation) and sonicated on a Bioruptor Pico (Diagenode). After clearing by centrifugation, 10 to 30 µg of sonicated chromatin were used for immunoprecipitation in ChIP buffer adjusted to 0.1% SDS. 5-10 µg of anti-HNF1β (sc-22840, Santa Cruz, for ChIP-QPCR shown in Figures 1 and S1, or custom made ABclonal E9034 for ChIP-seq) or TOP1 (custom made ABclonal E9036) antibodies were added to the chromatin and incubated overnight at 4°C on a rocking wheel. The immune complexes were collected with Dynabeads protein A (Thermo Fisher Scientific) for 3-4 h at 4°C and washed with five successive different washing buffers as described in.⁹ For 786-O cells, elution and decrosslinking of DNA was performed in Elution Buffer (1% SDS, 0.1M NaHCO3, 100 mM NaCl) with addition of 20 µg/mL RNase A at 65°C overnight. DNA was then extracted with QIAquick PCR Purification Kit (Qiagen) after treatment with 100 µg/mL proteinase K at 55°C for 1-2 h. For KECs, DNA was eluted, de-crosslinked and extracted with the IPure kit v2 (Diagenode). ChIP-DNA was analyzed by qRT-PCR with the GoTaq qPCR Master Mix (Promega) on a MX3005P machine (Stratagene) or with PowerTrack SYBR Green Master Mix (Thermo Fisher Scientific) on a QuantStudio 5 machine (Thermo Fisher Scientific). Primer sequences are listed in Table S1.

## ChIP-seq

Immunoprecipitated DNAs and corresponding inputs were quantified with the Qubit fluorometer (Thermo Fisher Scientific) using the dsDNA HS Assay Kit (Thermo Fisher Scientific) and 1 ng of each sample was used to prepare libraries with the MicroPlex v2 kit (Diagenode). Libraries were purified using AMPure XP beads (Beckman Coulter) and quantified using the Qubit fluorometer (Thermo Fisher Scientific). Library size was assessed using the Bioanalyzer High Sensitivity DNA chip (Agilent Technologies). Libraries were normalized and pooled at 2 nM and spike-in PhiX Control v3 (Illumina) was added. Sequencing was performed on a NextSeq 500 instrument (Illumina) in paired-end read mode (2x40 cycles) (GENOM'IC, Cochin Institute, Paris, France).

## ChIP-seq analysis

After sequencing, a primary analysis was performed using the AOZAN software⁴⁶ in order to demultiplex and control the quality of the raw data. Quality control of fastq files was done with FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Reads obtained from 786-O and KECs ChIP-seq were aligned with Bowtie2⁴⁷ respectively to the hg19 human assembly and to the mm10 mouse assembly. Replicates were pooled, then sorted and indexed using SAMtools.⁵⁰ Peak detection on the pooled replicates was performed using GEM.⁵¹ Heatmaps were produced using Genomation.⁵² Motif search was done using the MEME-ChIP.⁵³ The
analysis of calibrated ChIP-seq was performed using the protocol described in.²⁴ ChIP-seq correlation analysis with RNA-seq data was performed using Cistrome.²⁵ The enrichment for TOP1 on HNF1β binding sites was calculated by considering the 106 best HNF1β peaks next to genes that were downregulated in the absence of HNF1β. TOP1 occupancy (ChIP-seq read coverage) was measured using ScoreMatrixBin from the Genomation R package on ChIP-seq data for TOP1 immunoprecipitated from mitotic chromatin in two replicates in HNF1β-KO cells and their parental controls (WT). Coverage was measured in a window of 500 nt centered on HNF1β peaks and by subtracting the corresponding average coverage calculated at a distance of 10 kb from both sides of each HNF1β peak on a stretch of 5 kb for each side.

## ATAC-seq

ATAC-Seq experiments were performed following the Omni-ATAC protocol described in.⁶¹ Briefly, 50,000 asynchronous or mitotic KECs were harvested by centrifugation, washed in PBS and resuspended in 50 µL of transposition mix (25 µL 2X Tagmentation Buffer (Diagenode), 2.5 µL Tn5 transposase (Diagenode), 5 µL nuclease-free water, 16.5 µL PBS, 0.5 µL 1% digitonin (Millipore, 0.01% final), 0.5 µL 10% Tween 20 (Sigma, 0.1% final). The transposition reaction was incubated at 37°C for 30 min in a thermomixer with 1,000 rpm mixing and DNA was immediately purified using a MinElute PCR Purification Kit (Qiagen). Transposed fragments were barcoded using the NEBNext Ultra II Q5 2× Master Mix (NEB) and a unique combination of i5 and i7 adapters (Table S1). After 3 cycles of amplification, an aliquot of each library was quantified by real-time PCR using the NEBNext Library Quant Kit (NEB) and the appropriate number of amplification cycles were added to the main reactions in order to obtain a final yield of 240 fmol after column-based cleanup. Libraries were purified with the MinElute PCR Purification Kit (Qiagen), quantified using the Qubit fluorometer (Thermo Fisher Scientific) and the dsDNA HS Assay Kit (Thermo Fisher Scientific) and sequenced on a NextSeq 500 instrument (Illumina) in paired-end read mode (2x75 cycles) (GENOM'IC, Cochin Institute, Paris, France).

## ATAC-seq analysis

Fastq sequences were aligned to the mm10 genome with bowtie2⁴⁷ and exported as bams. All alignments files were indexed with SAMtools⁵⁰ and filtered with Sambamba⁴⁸ to eliminate reads with multiple alignments, unmapped reads and duplicates. The resulting filtered alignments were further filtered by removing the blacklist regions with the Bedtools intersect software. Resulting filtered bams of the different replicates for each condition were then merged with SAMtools.⁵⁰ The merged bams were then converted to bigwigs containing CPM normalized values using the bamCoverage⁴⁹ software.

## RNA extraction and analysis

786-O and KEC cells were collected in TRIzol (Thermo Fisher Scientific) and RNA was extracted following the manufacturer's instructions. cDNA was synthesized with the High-Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific). Quantitative PCR was performed with the GoTaq qPCR Master Mix (Promega) on a MX3005P machine (Stratagene) using primers listed in Table S1.

## RNA-seq

Libraries were prepared from 250 ng of total RNA per sample using the Stranded mRNA Prep kit (Illumina). Samples were sequenced on a NextSeq 500 Sequencing System (Illumina), single-read (1x75 Bp) for 786-O and paired-end (2x75 Bp) for KECs (GENOM'IC, Cochin Institute, Paris, France).

## RNA-seq analysis

Raw data was first analyzed using the AOZAN software⁴⁶ in order to perform demultiplexing and quality control. Obtained Fastq files were then aligned to the reference genome using the STAR algorithm⁵⁴ and the quality control of the alignment was done with Picard Tools (Broad Institute). Reads were counted using Rsubread⁵⁵ and differential expression analysis was performed with the DESeq2 package.⁵⁶

## HNF1B silencing

HNF1B-siRNA or non-targeting-siRNA pools (Dharmacon) were transfected in 786-O cells using Lipofectamine RNAiMAX (Thermo Fisher Scientific) at a final concentration of 37.5 nM. Cells were collected 48 h after transfection, and the efficiency of gene knock-down was determined by western blot.

## CRISPR-CAS9 generation of stable KNOCK-OUT cell lines

The BTBD2-KO HEK293 cell line was created targeting the PHR domain of BTBD2 for CrisprCas9. 13 µg of pSpCas9(BB)-2A-Puro plasmid containing the BTBD2_18cr17 guide (18p22) or the empty vector 17p18 were transfected in HEK-293 cells using Lipofectamine 2000 (Thermo Fisher Scientific) in 10 cm dishes. 48 h after transfection, 5 µg/mL puromycin (Gibco) were added for 2 days and then cells were cultured in regular medium until colonies were visible. Single colonies were picked up with filtered tips and transferred into a 96 well plate. At confluency, clones were trypsinized and split in 24 well plates for characterization. DNA was extracted with the DNeasy kit (Qiagen) and PCR products obtained with the 18i67 and 18i68 oligos were sent for sequencing with the same oligos (Europeans). KO clones were selected after sequence analysis with the SYNTHEGO online tool (https://ice.synthego.com/#/). The loss of BTBD2 in the selected clones was verified by RT-qPCR and by Western Blotting.
The HNF1β knockout KEC cell line was created targeting exon 1 of HNF1β. The two guide-expressing constructs derived from pU6-pegRNA-GG-acceptor plasmid were co-transfected with pSpCas9(BB)-2A-Puro using linear polyethylenimine (PEI, 1 mg/mL, Worthington). Forty-eight hours after transfection, cells underwent a pulse of puromycin selection (1 µg/mL) for 48 h to enrich for transfected cells. The enriched cells were then subjected to flow cytometry and sorted into 96-well plates as single cells. After 2 weeks of growth, colonies were split into 2 96-well plates, and verified for loss of HNF1β expression by Western Blotting.

## Yeast-2-hybrid screen

The Yeast-2-hybrid screen (ULTImate Y2H screen) was performed by Hybrigenics (France) using the N-terminal part of the human HNF1β isoform A from plasmid 6p15⁴⁵ coding amino acids 1–313 as a bait and a mouse embryonic kidney library as prey.

## Immunoprecipitation

N-ter HNF1β-GFP and GFP plasmids (1 µg each) were transfected in subconfluent HEK293 cells in 6 well plates using Lipofectamine (Thermo Fisher Scientific). Two days after transfection, cells were collected in MN Lysis Buffer (50 mM Tris-HCl pH7.5, 150 mM NaCl, 1% Triton, 2 mM Calcium Chloride, 0.0025 U/µL micrococcal nuclease (Sigma-Aldrich) and protease inhibitors (Roche)). Cell extracts were prepared by potter homogenization and incubation at 37°C for 30 min. Micrococcal nuclease activity was blocked by the addition of 8 mM EGTA (Sigma-Aldrich) and the extracts were cleared by centrifugation at 15000g at 4°C for 10 min. GFP antibody (ab290, Abcam) was crosslinked to dynabeads protein A (Thermo Fisher Scientific) using BS3 (bis sulfosuccinimidyl suberate, Thermo Fisher Scientific). GFP proteins were immunoprecipitated using GFP-crosslinked dynabeads for 1 h at 4°C, washed 3 times in Lysis Buffer (50 mM Tris-HCl pH7.5, 150 mM NaCl, 1% Triton, 1 mM EDTA and protease inhibitors (Roche)) and stored in 20 mM Tris pH 7.5 with protease inhibitors at 4°C for Top1 assay or analyzed by western blotting.

For HNF1β-TOP1-BTBD2 co-IP analysis, 1 µg N-ter HNF1β-GFP plasmid was transfected in subconfluent WT and BTBD2-KO HEK293 cells in 10 cm tissue culture dishes. The day after transfection, cells were split at 50% confluency for mitotic block or at 80% confluency for asynchronous growth. Mitotic cells were collected after 14 h incubation with 100 ng/mL Nocodazole (Sigma-Aldrich). Cell extracts from asynchronous and mitotic cells were prepared as described above and GFP immunoprecipitation was performed using GFP-Trap Magnetic Beads M-270 (ChromoTek).

## BioID proximity-dependent biotinylation assay

Subconfluent HEK293 cells in 10 cm dishes were transfected with 8 µg of BirA-HA plasmids expressing N-terminal HNF1β WT (18p13) or K258E mutant (18p44) using Lipofectamine 2000 and Opti-Mem (Thermo Fisher Scientific). The day after transfection, cells were split at 50% confluency for mitotic block or at 80% confluency for asynchronous growth. Mitotic synchronization was obtained by treating cells with 100 ng/mL nocodazole (Sigma-Aldrich). After 16 h of nocodazole treatment, 50 µM of biotin (Sigma-Aldrich) was added to both asynchronous cells and mitotic cells for 6 h. After the biotin treatment, cells were washed twice with PBS and collected in BirA lysis buffer (50 mM Tris-HCl pH 7.5, 5 mM EDTA, 300 mM NaCl, 2% Triton X-, 0.5% SDS, protease inhibitors). Cell extracts were prepared by sonication (6–10 min with a 30 s ON, 30 s OFF cycle) on a Bioruptor Pico sonicator (Diagenode) followed by centrifugation at 14000g for 20 min at 4°C. Affinity purification of biotin-labeled proteins was performed by incubating 1 mg of cell extract (diluted at a final concentration of 50 mM Tris-HCl pH 7.5, 2.5 mM EDTA, 150 mM NaCl, 1% Triton X-, 0.125% SDS) with 80 µL of M-280 Streptavidin dynabeads (Thermo Fisher Scientific) at 4°C for 2–4 h on a rotating wheel. After 6 washes in the same buffer, biotinylated proteins were eluted by boiling 5 min in 2X Laemmli buffer supplemented with 1 mM biotin and separated on 4–20% mini-PROTEAN TGX gels (Bio-rad) for western blot analysis.

## Western Blot

For western blot analysis of endogenous HNF1β, TOP1 and BTBD2, cells were washed twice with PBS and collected in SDS-Lysis buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% SDS, protease inhibitors). Cell lysates were then boiled for 5 min and centrifuged at 14000 g for 10 min at 4°C. Proteins were quantified with the BCA assay (Thermo Fisher Scientific). Protein extracts or eluates (from immunoprecipitations or affinity purifications) were separated on 4–20% or 4–15% mini-PROTEAN TGX gels (Bio-rad) and transferred to nitrocellulose membranes with the Trans-Blot Turbo transfer system (Bio-rad). After blocking in 5% milk (in TBS-Tween 0.1%), membranes were incubated with the indicated primary antibodies in blocking buffer (3% BSA in TBS-Tween 0.1%) overnight at 4°C or at room temperature for 2 h. The following primary antibodies were used: TOP1 (ABclonal E9036, custom-made, 1/2000), BTBD2 (Thermo Fisher Scientific #PA5-112834, 1/1000), GFP (Abcam #ab290, 1/5000), HA (BioLegend #901502, 1/2000), HNF1β (ABclonal E9034, custom-made, 1/2000), beta-actin (Sigma-Aldrich # A1978, 1/5000). Custom-made TOP1 rabbit polyclonal antibody was produced using a synthetic peptide corresponding to the aa 5-23 (HLHNDSQIEADFRLNDSHK) of the human TOP1 protein. Custom-made HNF1β rabbit polyclonal antibody was raised against aa 48–64 LSPGSGAEPDTKPVFHT of the HNF1β protein. After 3 washes in TBS-Tween 0.1%, membranes were incubated with secondary antibodies (Sheep Anti-Mouse-HRP or Donkey Anti-Rabbit-HRP, GE Healthcare #NA931 and #NA934, 1/10000; Donkey anti-Mouse or Donkey anti-Rabbit Alexa Fluor 488, Thermo Fisher Scientific #A21202 and #A21206, 1/5000). Chemiluminescence and fluorescence detections were performed using a ChemiDoc XRS (Bio-Rad) and an ECL Prime or ECL Select kit (Amersham). Images were then analyzed using Image Lab (BioRad).
### Topoisomerase 1 assay

The DNA relaxation assay was performed as described in³⁹ with some modifications. Recombinant human TOP1 (MyBioSource) was diluted in TOP1 dilution buffer (50% glycerol, 20 mM Tris pH7.5, 5 µg/mL acetylated BSA) and incubated with immunoprecipitated GFP or GFP-HNF1β on ice for 10 min. The reaction was performed in TOP1 relaxation buffer (10 mM Tris-HCl pH 7.5, 10 mM MgCl₂ 150 mM NaCl, 0.1 mM EDTA, 15 µg/mL acetylated BSA, 0.2 mM DTT) with 300 ng of plasmidic DNA containing 10 HNF1 multimerized binding sites (16p38) for the indicated time at 37°C and stopped by the addition of 1% SDS and 50 µg/mL proteinase K and additional incubation at 37°C for 15 min. Supercoiled and relaxed DNAs were separated on a 1% agarose gel and quantified using a Bio-Rad Gel Doc XR + system after Ethidium Bromide (0.7 µg/mL) staining.

### 3D structure analysis

HNF1β (2H8R⁶²) and TOP1 (1K4S⁶³) 3D structures were downloaded from NCBI Structure database and analyzed with Jmol (http://www.jmol.org/).

### Biotin-Psoralen incorporation assay

Psoralen crosslinking was performed according to³⁵ with some modifications. KECs were either grown asynchronously or enriched in metaphase as described above. 2 million cells per condition were treated with 0.01% digitonin and 20 µM EZ-Link Psoralen-PEG3-Biotin (Thermo Fisher Scientific) in 1 mL growth medium for 5 min at 37°C. For TOP1 inhibition, cells were pretreated with 20 µM CPT for 5 min and CPT was kept during Psoralen incubation. Psoralen was crosslinked to DNA by irradiating cells with 40 kJ/m² of 360 nm UV light on ice using a Bio-Link BLX crosslinker (Vilber Lurmat). Cells were immediately centrifuged, resuspended in 0.5 mL TE buffer with 0.5% SDS and 200 µg/mL Proteinase K and incubated at 55°C on a thermomixer with continuous shaking over-night. DNA was purified with phenol-chloroform and isopropanol precipitation, then sequentially treated with 5 µg/mL RNase for 30 min at 37°C and 200 µg/mL Proteinase K at 55°C for 1 h and repurified by phenol-chloroform extraction and ethanol precipitation. The purified DNA was resuspended in TE buffer and sonicated to 200–1000 bp using a Bioruptor Pico (Diagenode). Biotinylated DNA was isolated with 80 µL Streptavidin MyOne C1 or T1 dynabeads (Thermo Fisher Scientific) for 1 h in TE buffer with 1M NaCl at room temperature. The beads were washed, as described in,⁶⁴ sequentially for 15 min at room temperature with Wash solution 1 (20 mM Tris pH 8, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.1% SDS), Wash solution 2 (20 mM Tris pH 8, 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% SDS) and Wash solution 3 (10 mM Tris pH 8, 0.25 M LiCl, 1 mM EDTA, 1% NP40, 1% deoxycholate) and twice with TE buffer. In order to elute and decrosslink psoralen-enriched DNA, beads were incubated in 50 µL 0.1 N KOH for 30 min at 90°C.⁶⁵ Eluted DNA was brought to 400 µL with 10 mM Tris pH 8 and analyzed by qPCR with the indicated oligos.

### RADAR (rapid approach to DNA adduct recovery)-ChIP

TOP1-DNA covalent cleavage complexes (TOP1cc) were detected as in³⁶ and³⁵ with some modifications. Asynchronous or mitotic KECs (4-6x10⁹) were treated with 20 µM CPT for 15 min, collected by centrifugation and resuspended in 1 mL M Buffer (5.7M guanidine thiocyanate, 9.5 mM Tris-HCl pH 6.8, 19 mM EDTA, 3.8% Triton X-100, 0.95% Sarkosyl, 1% DTT). DNA was precipitated by the addition of 1 mL 100% Ethanol, centrifuged at 14000 rpm for 10 min at 4°C and washed 3 times with 500 µL Wash Buffer (20 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM EDTA, 50% EtOH). Pellets were resuspended in 200–300 µL TE-0.1% SDS (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 0.1% SDS) and solubilized by gentle agitation for 30 min. DNA was then sonicated on a Bioruptor Pico (Diagenode) in order to obtain fragments smaller than 1 kb and 20–30 µg were used for immunoprecipitation with 50 µL Dynabeads protein A (Thermo Fisher Scientific) pre-coated with 10 µg TOP1 antibody (Abclonal E9036) over-night at 4°C. Beads were washed as described for the ChIP protocol, but always in presence of 0.1% SDS, and then incubated, along with the corresponding inputs, with 100 µL TE-0.5% SDS supplemented with 500 µg/mL proteinase K 4h at 65°C shaking at 1500 rpm. DNA was purified with QIAquick PCR Purification Kit (Qiagen) and analyzed by qRT-PCR as for ChIP.

### QUANTIFICATION AND STATISTICAL ANALYSIS

All statistical tests were performed on GraphPad Prism version 9 or using statistical functions in R. Quantifications of western blot and agarose gel bands were performed with Image Lab Software. Detailed information about the statistical test used is provided in figure legends. N represents the number of biological or technical replicates and is reported in the legend of each figure. For RNA-seq, ChIPseq and ATACseq, quantifications and statistics were derived from at least two independent experiments.