Didier Trouche

High-resolution profiling of γH2AX around DNA double strand breaks in the mammalian genome

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 human cell line that permits induction of multiple DSBs randomly distributed and unambiguously positioned within the genome. Using this system, we have generated the first genome‐wide mapping of γH2AX around DSBs. We found that all DSBs trigger large γH2AX domains, which spread out from the DSB in a bidirectional, discontinuous and not necessarily symmetrical manner. The distribution of γH2AX within domains is influenced by gene transcription, as parallel mappings of RNA Polymerase II and strand‐specific expression showed that γH2AX does not propagate on active genes. In addition, we showed that transcription is accurately maintained within γH2AX domains, indicating that mechanisms may exist to protect gene transcription from γH2AX spreading and from the chromatin rearrangements induced by DSBs.

The chromatin remodeler p400 ATPase facilitates Rad51-mediated repair of DNA double-strand breaks

The chromatin remodeling enzyme p400 forms a complex with Rad51 and is required for its recruitment to double-strand breaks during DNA repair by homologous recombination.

A new isoform of the histone demethylase JMJD2A/KDM4A is required for skeletal muscle differentiation.

In proliferating myoblasts, muscle specific genes are silenced by epigenetic modifications at their promoters, including histone H3K9 methylation. Derepression of the promoter of the gene encoding the myogenic factor myogenin (Myog) is key for initiation of muscle differentiation. The mechanism of H3K9 demethylation at the Myog promoter is unclear, however. Here, we identify an isoform of the histone demethylase JMJD2A/KDM4A that lacks the N-terminal demethylase domain (ΔN-JMJD2A). The amount of ΔN-JMJD2A increases during differentiation of C2C12 myoblasts into myotubes. Genome-wide expression profiling and exon-specific siRNA knockdown indicate that, in contrast to the full-length protein, ΔN-JMJD2A is necessary for myotube formation and muscle-specific gene expression. Moreover, ΔN-JMJD2A promotes MyoD-induced conversion of NIH3T3 cells into muscle cells. ChIP-on-chip analysis indicates that ΔN-JMJD2A binds to genes mainly involved in transcriptional control and that this binding is linked to gene activation. ΔN-JMJD2A is recruited to the Myog promoter at the onset of differentiation. This binding is essential to promote the demethylation of H3K9me2 and H3K9me3. We conclude that induction of the ΔN-JMJD2A isoform is crucial for muscle differentiation: by directing the removal of repressive chromatin marks at the Myog promoter, it promotes transcriptional activation of the Myog gene and thus contributes to initiation of muscle-specific gene expression.

Physical interaction between the histone acetyl transferase Tip60 and the DNA double-strand breaks sensor MRN complex.

Chromatin modifications and chromatin-modifying enzymes are believed to play a major role in the process of DNA repair. The histone acetyl transferase Tip60 is physically recruited to DNA DSBs (double-strand breaks) where it mediates histone acetylation. In the present study, we show, using a reporter system in mammalian cells, that Tip60 expression is required for homology-driven repair, strongly suggesting that Tip60 participates in DNA DSB repair through homologous recombination. Moreover, Tip60 depletion inhibits the formation of Rad50 foci following ionizing radiation, indicating that Tip60 expression is necessary for the recruitment of the DNA damage sensor MRN (Mre11-Rad50-Nbs1) complex to DNA DSBs. Moreover, we found that endogenous Tip60 physically interacts with endogenous MRN proteins in a complex which is distinct from the classical Tip60 complex. Taken together, our results describe a physical link between a DNA damage sensor and a histone-modifying enzyme, and provide important new insights into the role and mechanism of action of Tip60 in the process of DNA DSB repair.

Histone demethylases in chromatin cross-talks

The ‘histone code’ hypothesis states that chromatin‐based regulation of nuclear processes such as transcription is brought about by the combination of distinct modifications (histone marks) at specific loci. Its correct establishment involves chromatin cross‐talks, ensuring an ordered and concerted deposition/removal of a particular set of modifications that act together to give the correct transcriptional outcome. Histone methylation on lysine residues can negatively or positively impact on gene transcription, depending on the residue and on its degree of methylation. Thanks to this complexity and given the number of chromatin ‘readers’ that can recognize methylated lysine residues, histone methylation plays a very special role in specifying the various chromatin states. The recent discovery of histone demethylases, which represent a large family of enzymes often containing histone modification binding modules, sheds new light on cross‐talk mechanisms involving methylated residues. In the present review, after a brief overview of the various families of histone demethylases, we describe the different mechanisms by which they participate in chromatin cross‐talks and how these mechanisms are integrated to achieve the mutual exclusion or the link between chromatin marks, leading to the establishment of the correct histone code.

The E1A-Associated p400 Protein Modulates Cell Fate Decisions by the Regulation of ROS Homeostasis

The p400 E1A-associated protein, which mediates H2A.Z incorporation at specific promoters, plays a major role in cell fate decisions: it promotes cell cycle progression and inhibits induction of apoptosis or senescence. Here, we show that p400 expression is required for the correct control of ROS metabolism. Depletion of p400 indeed increases intracellular ROS levels and causes the appearance of DNA damage, indicating that p400 maintains oxidative stress below a threshold at which DNA damages occur. Suppression of the DNA damage response using a siRNA against ATM inhibits the effects of p400 on cell cycle progression, apoptosis, or senescence, demonstrating the importance of ATM–dependent DDR pathways in cell fates control by p400. Finally, we show that these effects of p400 are dependent on direct transcriptional regulation of specific promoters and may also involve a positive feedback loop between oxidative stress and DNA breaks since we found that persistent DNA breaks are sufficient to increase ROS levels. Altogether, our results uncover an unexpected link between p400 and ROS metabolism and allow deciphering the molecular mechanisms largely responsible for cell proliferation control by p400.

Interplay between chromatin modifying enzymes controls colon cancer progression through Wnt signaling

Cancer progression is associated with epigenetic alterations, such as changes in DNA methylation, histone modifications or variants incorporation. The p400 ATPase, which can incorporate the H2A.Z variant, and the Tip60 histone acetyltransferase are interacting chromatin-modifying proteins crucial for the control of cell proliferation. We demonstrate here that Tip60 acts as a tumor suppressor in colon, since mice heterozygous for Tip60 are more susceptible to chemically induced preneoplastic lesions and adenomas. Strikingly, heterozygosity for p400 reverses the Tip60-dependent formation of preneoplastic lesions, uncovering for the first time pro-oncogenic functions for p400. By genome-wide analysis and using a specific inhibitor in vivo, we demonstrated that these effects are dependent on Wnt signaling which is antagonistically impacted by p400 and Tip60: p400 directly favors the expression of a subset of Wnt-target genes and regulators, whereas Tip60 prevents β-catenin acetylation and activation. Taken together, our data underline the physiopathological importance of interplays between chromatin-modifying enzymes in the control of cancer-related signaling pathways.

Control of genetic stability by a new heterochromatin compaction pathway involving the Tip60 histone acetyltransferase

A new compaction pathway of mammalian pericentric heterochromatin is identified, which relies on H4K12ac by Tip60, probably followed by recruitment of BRD2, and therefore chromatin compaction, which can contribute to genetic stability.

The R438W polymorphism of human DNA polymerase lambda triggers cellular sensitivity to camptothecin by compromising the homologous recombination repair pathway

The human DNA polymerase lambda (Polλ) is a DNA repair polymerase, which is believed not only to play a role in base excision repair but also to contribute to DNA double-strand break repair by non-homologous end joining. We described here that cellular expression of the recently described natural polymorphic variant of Polλ, Polλ(R438W), affects the homologous recombination (HR) pathway and sister chromatid exchange (SCE) events. We show that the HR defect provoked by this polymorphism enhances cellular sensitivity to the anticancer agent camptothecin (CPT), most of whose DNA damage is repaired by HR. All these effects were dependent on the DNA polymerase activity of Polλ(R438W) as the expression of a catalytically inactive Polλ(R438W) did not affect either the HR and SCE frequencies or the cellular sensitivity to CPT. These results suggest that sensitivity to CPT could result from cancer-related mutation in specialized DNA repair polymerases.

Control of alternative end joining by the chromatin remodeler p400 ATPase

Repair of DNA double-strand breaks occurs in a chromatin context that needs to be modified and remodeled to allow suitable access to the different DNA repair machineries. Of particular importance for the maintenance of genetic stability is the tight control of error-prone pathways, such as the alternative End Joining pathway. Here, we show that the chromatin remodeler p400 ATPase is a brake to the use of alternative End Joining. Using specific intracellular reporter susbstrates we observed that p400 depletion increases the frequency of alternative End Joining events, and generates large deletions following repair of double-strand breaks. This increase of alternative End Joining events is largely dependent on CtIP-mediated resection, indicating that it is probably related to the role of p400 in late steps of homologous recombination. Moreover, p400 depletion leads to the recruitment of poly(ADP) ribose polymerase (PARP) and DNA ligase 3 at DNA double-strand breaks, driving to selective killing by PARP inhibitors. All together these results show that p400 acts as a brake to prevent alternative End Joining-dependent genetic instability and underline its potential value as a clinical marker.