Pierre Caron

• PhD
• Researcher CRCN, CNRS at Institute of Structural Biology

SRSF2 (serine/arginine-rich splicing factor 2) is a critical regulator of pre-messenger RNA splicing, which also plays noncanonical functions in transcription initiation and elongation. Although elevated levels of SRSF2 are associated with advanced stages of lung adenocarcinoma (LUAD), the mechanisms connecting SRSF2 to lung tumor progression remai...

The eukaryotic genome is assembled in a nucleoprotein complex called chromatin, whose organization markedly influences the repair of DNA lesions. For instance, compact chromatin states, broadly categorized as heterochromatin, present a challenging environment for DNA damage repair. Through transcriptional silencing, heterochromatin also plays a vit...

In response to DNA double-strand breaks (DSBs), chromatin modifications orchestrate DNA repair pathways thus safeguarding genome integrity. Recent studies have uncovered a key role for heterochromatin marks and associated factors in shaping DSB repair within the nucleus. In this review, we present our current knowledge of the interplay between hete...

Using a barcoded reporter introduced within a thousand different chromatin locations in human cells, (Schep et al., 2021) characterize repair outcomes of Cas9-induced DNA double-strand breaks (DSBs) and the relative use of DSB repair pathways depending on the local chromatin context.

Heterochromatin is a critical chromatin compartment, whose integrity governs genome stability and cell fate transitions. How heterochromatin features, including higher-order chromatin folding and histone modifications associated with transcriptional silencing, are maintained following a genotoxic stress challenge is unknown. Here, we establish a sy...

The repair of DNA double-strand breaks (DSBs) is essential for safeguarding genome integrity. When a DSB forms, the PI3K-related ATM kinase rapidly triggers the establishment of megabase-sized, chromatin domains decorated with phosphorylated histone H2AX (γH2AX), which act as seeds for the formation of DNA-damage response foci¹. It is unclear how t...

DNA Double-Strand Breaks (DSBs) repair is essential to safeguard genome integrity. Upon DSBs, the ATM PI3K kinase rapidly triggers the establishment of megabase-sized, γH2AX-decorated chromatin domains which further act as seeds for the formation of DNA Damage Response (DDR) foci. How these foci are rapidly assembled in order to establish a "repair...

DNA double-strand breaks (DSBs) elicit major chromatin changes. Using super-resolution microscopy in human cells, Ochs et al. unveil that the DSB response protein 53BP1 and its effector RIF1 organize DSB-flanking chromatin into circular micro-domains. These structures control the spatial distribution of DSB repair factors safeguarding genome integr...

Heterochromatin is a critical chromatin compartment, whose integrity governs genome stability and cell fate transitions. How heterochromatin features, including higher-order chromatin folding and histone modifications associated with transcriptional silencing, are maintained following a genotoxic stress challenge is unknown. Here, we establish a sy...

DNA double-strand breaks (DSBs) affect chromatin integrity and impact DNA-dependent processes such as transcription. Several studies revealed that the transcription of genes located in close proximity to DSBs is transiently repressed. This is achieved through the establishment of either a transient repressive chromatin context or eviction of the RN...

DNA double-strand breaks (DSBs) at RNA polymerase II (RNAPII) transcribed genes lead to inhibition of transcription. The DNA-dependent protein kinase (DNA-PK) complex plays a pivotal role in transcription inhibition at DSBs by stimulating proteasome-dependent eviction of RNAPII at these lesions. How DNA-PK triggers RNAPII eviction to inhibit transc...

Movie S3. 53BP1 Foci Cluster in 4OHT-Treated 53BP1-GFP DivA Cells—Example 1, Related to Figure 5 Same as Movie S1, except that acquisition was performed after 2 hr of 4OHT treatment and every 15 s. A magnification of 53BP1 foci ongoing clustering is presented. Related to Figure S5C bottom sequence.

Movie S4. 53BP1 Foci Cluster in 4OHT-Treated 53BP1-GFP DivA Cells—Example 2, Related to Figure 5 Same as Movie S1, except that acquisition was performed after 2 hr of 4OHT treatment and every 3 min. Related to Figure S5C top sequence.

Although both homologous recombination (HR) and nonhomologous end joining can repair DNA double-strand breaks (DSBs), the mechanisms by which one of these pathways is chosen over the other remain unclear. Here we show that transcriptionally active chromatin is preferentially repaired by HR. Using chromatin immunoprecipitation-sequencing (ChIP-seq)...

Recent advances in our understanding of the management and repair of DNA double-strand breaks (DSBs) rely on the study of targeted DSBs that have been induced in living cells by the controlled activity of site-specific endonucleases, usually recombinant restriction enzymes. Here we describe a protocol for quantifying these endonuclease-induced DSBs...

5′ strand resection at DNA double strand breaks (DSBs) is critical for homologous recombination (HR) and genomic stability. Here we develop a novel method to quantitatively measure single-stranded DNA intermediates in human cells and find that the 5′ strand at endonuclease-generated break sites is resected up to 3.5 kb in a cell cycle–dependent man...

Chromatin undergoes major remodeling around DNA double-strand breaks (DSB) to promote repair and DNA damage response (DDR) activation. We recently reported a high-resolution map of gammaH2AX around multiple breaks on the human genome, using a new cell-based DSB inducible system. In an attempt to further characterize the chromatin landscape induced...

SCC1 recruitment at DSBs does not increase over time. AsiSI-ER-U20S cells, either untreated or treated with 4OHT during 4H or 14H, were subjected to ChIP analyses using SCC1 (Abcam) or γH2AX antibodies as indicated. Enrichment was scored by Q-PCR in the vicinity of an AsiSI-induced DSBs (DSB1) and normalized to the signal observed on a genomic loca...

Profile of SMC3 in AsiSI-ER-U20S. A, Detailed view of the SMC3/input (black) in untreated AsiSI-ER-U20S cells and SCC1/input (grey) from HeLa cells, retrieved from [35]. ChIP-chip data, expressed as log2 are shown from selected areas of chromosome 1. B, The location and orientation of the 3072 genes located on chromosome 1 and 6 were used to subset...

The lack of cohesin spreading around DSBs is not due to a limited amount of soluble cohesin. A, Soluble (chromatin unbound) and insoluble (chromatin bound) fraction were prepared from AsiSI-ER U20S 4OHT-treated or untreated cells. Western blot against SMC3 and SCC1 showed that the soluble pool of cohesin is not depleted after DSB induction. B, ChIP...

Cohesin rich genes show a low level of γH2AX. The average Log2 (γH2AX/input) (y axis) and Log2 (cohesin/input) (x axis) were calculated over the entire length of each of the 359 genes encompassed within γH2AX domains, and plotted against each other. Results are shown for SMC3 (top panel), and SCC1 retrieved from the HeLa dataset [35] (bottom panel)...

Validation of SMC3 antibodies. A, The specificity of SMC3 antibodies (α-SMC3-A or α-SMC3-B as indicated) was analyzed by western blot with HeLa nuclear extracts. B, HeLa nuclear extracts were immunoprecipitated using either a α-SMC3-A or α-SMC3-B antibodies as indicated. Flowthrough and immunoprecipitated samples were analyzed by western blot probe...

Validation of SA1/SA2 antibodies. SA1 and SA2 antibodies were validated by western blot with HeLa nuclear extract (A), using control or SA1/SA2 siRNA transfected HeLa cells extracts (B), and in ChIP assay followed by Q-PCR (C) using a previously characterized cohesin binding site as a positive control [35], and the gapdh promoter as a negative cont...

Cohesin ChIP in synchronized cells. A, ChIP against SCC1 was performed in AsiSI-ER-U20S cells synchronized in G2 upon RO-3306 treatment (18H at 9 µM), before and after 4OHT, and analyzed by Q-PCR. Fold enrichment at two DSBs is shown relative to the negative locus (devoid of AsiSI sites). Note that 4OHT dependant recruitment of SCC1 at DSBs in G2 i...

SCC1 depletion leads to an increase of γH2AX as detected by immunofluorescence. AsiSI-ER-U2OS cells transfected with Control (CTRL) or SCC1 siRNA for 48H were treated with 4OHT for 4H and subjected to γH2AX immunofluorescence (Cell Signaling). Images were quantified and classified based on the percentage of their nucleus covered by γH2AX staining....

γH2AX increases on SCC1 rich domains upon SCC1 depletion. The averaged SCC1 signal over an 80 kb window around each AsiSI site was calculated (x-axis) and plotted against the γH2AX ratio in cells transfected with siRNA SCC1 versus siRNA CTRL(y axis). Pearson correlation and p value are indicated. (PDF)

Genes showing high changes in γH2AX between SCC1 siRNA and control cells, show higher levels of cohesin. As for Figure S19, for each gene encompassed in γH2AX domains, the ratio of γH2AX in SCC1 depleted versus control cells and the SMC3 signal (top panel) were averaged. The box plots show the difference in SMC3 (top panel) or SCC1 (bottom panel) b...

List of primers used in this study. (PDF)

Cohesin accumulation at DSBs is statistically significant and does not occur at random positions. Three independent random simulations were run with 100 sets of sites designed to be similar in size (single points), distribution (scattered uniformly across both chr 1 and 6) and number (24 sites per random run) to the actual AsiSI sites. Paired t-tes...

γH2AX is depleted at cohesin binding sites. Regions enriched in SCC1 were identified using the algorithm detailed in [10] and in the Material and Methods section (applied on SCC1 ChIP-chip data in HeLa cells [35]). Binding sites located within γH2AX domains were selected and the averaged SCC1 (top panel) and γH2AX (bottom panel) profiles around the...

Example γH2AX profiles around AsiSI sites upon SCC1 depletion. Detailed views, around selected AsiSI sites, indicated by arrows (upper and middle panels) and on two genomic regions devoid of AsiSI sites (lower panels). γH2AX enrichment over input in control (dark red) and in siRNA SCC1 (light red) transfected AsiSI-ER-U20S cells, are shown expresse...

SCC1 depletion does not change the cleavage efficiency of AsiSI sites. Genomic DNA was extracted from siRNA transfected AsiSI-ER-U20S cells treated or not with 4OHT for 4H and assayed for cleavage at AsiSI sites. Pulled down DNA was analyzed by quantitative PCR amplification using primers close to two cleaved AsiSI sites. The mean and standard devi...

List of γH2AX domain boundaries on chromosome 1 and chromosome 6 determined using the γH2AX signal in siRNA control transfected cells. All genomic coordinates are from the genome assembly NCBI Build 36.1. The boundaries positions were determined using the algorithm described in [10]. (PDF)

Profile of SCC1 in AsiSI-ER-U20S. A, Detailed view of the SCC1 distribution observed in HeLa cells retrieved from [35] (black, upper panel), and SCC1 observed in untreated AsiSI-ER-U20S cells (grey, lower panel). ChIP-chip data are presented as Log2 (Signal/input) on selected areas of the chromosome 1. B, The location and orientation of the 3072 ge...

SCC1 counteracts γH2AX. A, Detailed views of the γH2AX/H2AX signal (red) and the SCC1/input signal (black) around an AsiSI site (arrow), expressed as log2 and smoothed using a 500 probes sliding window. B, The average Log2 (γH2AX/input) (y axis) and Log2 (SCC1/input) (x axis) were calculated over the entire length of each of the 359 genes encompass...

γH2AX increases around DSBs in SCC1 depleted cells. A, The average Log2 (γH2AX/input) was calculated over a 4 kb window (left panel) or an 80 kb window (right panel) surrounding AsiSI sites, in cells transfected with control or SCC1 siRNA as indicated. The box plots represent the distribution of the values obtained for the 24 AsiSI sites. The γH2AX...

γH2AX increases at TSS upon SCC1 depletion. The average Log2 (γH2AX/input) upstream to promoters (−2000 to −1600 bp) (left panel), at promoters (−200 to +200 bp) (middle panel) and downstream of promoters (+1600 to −2000 bp) (right panel) for the 359 genes encompassed in γH2AX domains were calculated, in Control and SCC1 siRNA transfected cells as...

γH2AX increases on SCC1 rich genes upon SCC1 depletion. For each gene encompassed in γH2AX domains, the SCC1 signal was averaged and plotted against the ratio of γH2AX in SCC1 depleted versus control cells. Pearson correlation and p value are indicated. (PDF)

Detailed views of the areas analyzed by Q-PCR with SCC1 siRNA. Detailed views of the SMC3/input (black) and γH2AX/H2AX (red) ChIP-chip data, on five cohesin-bound regions (A–E) and two cohesin-unbound regions (F–G). Positions of the primer pairs used for the Q-PCR analysis presented in Figure 4 are shown (arrows and grey boxes), as well as the posi...

Expression fold changes upon 4OHT treatment and SCC1 depletion. AsiSI-ER-U20S cells were transfected with the indicated siRNAs. After 48 h, cells were treated or not with 4OHT as indicated. Total RNAs were extracted and reverse transcribed. The amount of each cDNA was measured by quantitative real-time PCR, divided by the amount of P0 cDNA and calc...

Cohesin binding does not correlate with boundary position. A, Boundaries of γH2AX domains were aligned and overlaid (right and mirror left borders are combined). Data are shown over a 200 kb window centered on domain boundaries and averaged using a 10 kb window size. Profiles are shown for γH2AX (red) and for SMC3 (black) in control cells. Note tha...

4OHT-induced γH2AX increases in SCC1 depleted cells compared to control cells. AsiSI-ER-U20S cells were transfected with Control (CTRL) or SCC1 siRNA for 48 hours. Untreated or 4OHT treated cells were subjected to ChIP analyses against γH2AX. Enrichment was scored by Q-PCR within 5 γH2AX domains. Distances of the primers from the DSB are indicated....

Comparison of γH2AX boundaries with chromosomal domain transitions. Hi-C realized with the human lymphoblastoid cell line GM06990, led to the identification of chromosomal domains at various scales, from the nucleus scale (such as the open and closed chromatin compartments) to a megabase scale. Chromosomal domains could be easily visualized using a...

Averaged interaction matrix around γH2AX domain boundaries. The Hi-C interaction matrix [52] located from −1 MB to +1 MB around each identified γH2AX domain boundary (23 AsiSI domains i.e. 46 boundaries) were retrieved and averaged (left and mirror right boundaries were combined). The averaged boundary (the 0 position) correlates with a chromosomal...

List of cleaved AsiSI sites on chromosome 1 and chromosome 6. All genomic coordinates are from the genome assembly NCBI Build 36.1. The AsiSI sites efficiently cleaved were determined thanks to our previous analysis using both the γH2AX signal and the cleavage signal [10]. (PDF)

DNA double strand breaks (DSBs) are among the most deleterious forms of lesions and deciphering the details of the chromatin landscape induced around DSBs represents a great challenge for molecular biologists. Chromatin Immunoprecipitation, followed by microarray hybridisation (ChIP-chip) or high-throughput sequencing (ChIP-seq), are powerful techn...

Chromatin acts as a key regulator of DNA-related processes such as DNA damage repair. Although ChIP-chip is a powerful technique to provide high-resolution maps of protein–genome interactions, its use to study DNA double strand break (DSB) repair has been hindered by the limitations of the available damage induction methods. We have developed a hum...

Rituximab (RTX), a monoclonal antibody directed against the CD20 protein, is a drug commonly used in the treatment of B-cell-derived lymphoid neoplasias and of antibody-mediated autoimmune diseases. In addition to cell- and complement-mediated B-cell depletion, RTX is thought to inhibit B-cell survival and proliferation through negative regulation...