In mammals, many germline genes are epigenetically repressed to prevent their illegitimate expres... more In mammals, many germline genes are epigenetically repressed to prevent their illegitimate expression in somatic cells. To advance our understanding of the mechanisms restricting the expression of germline genes, we analyzed their chromatin signature and performed a CRISPR-Cas9 knockout screen for genes involved in germline gene repression using a Dazl-GFP reporter system in mouse embryonic stem cells (mESCs). We show that the repression of germline genes mainly depends on the polycomb complex PRC1.6 and DNA methylation, which function additively in mESCs. Furthermore, we validated novel genes involved in the repression of germline genes and characterized three of them: Usp7, Shfm1 (also known as Sem1) and Erh. Inactivation of Usp7, Shfm1 or Erh led to the upregulation of germline genes, as well as retrotransposons for Shfm1, in mESCs. Mechanistically, USP7 interacts with PRC1.6 components, promotes PRC1.6 stability and presence at germline genes, and facilitates DNA methylation deposition at germline gene promoters for long term repression. Our study provides a global view of the mechanisms and novel factors required for silencing germline genes in embryonic stem cells.
bioRxiv (Cold Spring Harbor Laboratory), Aug 4, 2019
The CHD4 subunit of the Nucleosome Remodelling and Deacetylation (NuRD) complex regulates express... more The CHD4 subunit of the Nucleosome Remodelling and Deacetylation (NuRD) complex regulates expression of PADI1 (Protein Arginine Deiminase 1) and PADI3 in multiple cancer cell types modulating citrullination of three arginines of the allosterically-regulated glycolytic enzyme pyruvate kinase M2 (PKM2). PKM2 citrullination lowers its sensitivity to the inhibitors Tryptophan, Alanine and Phenylalanine shifting the equilibrium towards the activator Serine bypassing normal physiological regulation by low Serine levels and promoting excessive glycolysis, lowered intracellular ATP and slowed proliferation. Our data provide unique insight as to how conversion of arginines to citrulline impacts key interactions within PKM2 adding another layer of complexity to the mechanisms that regulate the activity of this important enzyme. CHD3 and CHD4 are mutually exclusive ATPase subunits of the Nucleosome Remodelling and Deacetylation (NuRD) complex that regulates gene expression, acting in many contexts as a co-repressor (1) (2) (3). An shRNA dropout screen previously identified CHD4 as essential for growth of multiple patient derived melanoma xenografts and for breast cancer (4) (5). Mining public data sets showed up-regulation of CHD4 upon transition from benign nevi to metastatic melanoma (Fig. ). In melanomas, CHD4 expression was comparable in the different mutation status, although CHD3 was lowered in NRAS mutated melanomas (Fig. ). Single cell RNA-seq (6) showed higher CHD4 expression in melanoma tumour cells compared to infiltrating B and T lymphocytes (Fig. ). SiRNA-mediated CHD3 or CHD4 silencing in a collection of melanoma cells in vitro reduced clonogenic capacity, increased the proportion of slow or non-proliferating cells (Fig. ), but did not induce apoptosis (Fig. ).
In mammals, the time period that follows fertilization is characterized by extensive chromatin re... more In mammals, the time period that follows fertilization is characterized by extensive chromatin remodeling, which enables epigenetic reprogramming of the gametes. Major changes in chromatin structure persist until the time of implantation, when the embryo develops into a blastocyst, which comprises the inner cell mass and the trophectoderm. Changes in DNA methylation, histone variant incorporation, and covalent modifications of the histones tails have been intensively studied during pre-implantation development. However, modifications within the core of the nucleosomes have not been systematically analyzed. Here, we report the first characterization and temporal analysis of 3 key acetylated residues in the core of the histone H3: H3K64ac, H3K122ac, and H3K56ac, all located at structurally important positions close to the DNA. We found that all 3 acetylations occur during pre-implantation development, but with different temporal kinetics. Globally, H3K64ac and H3K56ac were detected throughout cleavage stages, while H3K122ac was only weakly detectable during this time. Our work contributes to the understanding of the contribution of histone modifications in the core of the nucleosome to the "marking" of the newly established embryonic chromatin and unveils new modification pathways potentially involved in epigenetic reprogramming.
The linker histone H1 plays an essential role in maintaining and establishing higher-order chroma... more The linker histone H1 plays an essential role in maintaining and establishing higher-order chromatin structure. As with core histones, histone H1 is also extensively covalently modified. We showed previously that phosphorylation of S27 in human histone H1.4 (H1.4S27-P), prevents binding of heterochromatin protein 1 (HP1) family members (officially known as chromobox protein homologs) to the neighboring dimethylated K26. Here, we present the first functional characterization of H1.4S27-P in vivo and in vitro. We show that H1.4S27 phosphorylation is cell-cycle-regulated and its levels peak on metaphase chromosomes. We identify further Aurora B as the kinase phosphorylating H1.4S27. We demonstrate that histone H1.4 is the only somatic linker histone variant targeted by Aurora B and that Aurora B exclusively phosphorylates S27. Adjacent K26 dimethylation can regulate Aurora B activity towards S27, uncovering a crosstalk between these modifications. Finally, our fluorescence recovery after photobleaching (FRAP) analysis on histone H1.4 mutants suggests a role of S27 phosphorylation in the regulation of histone H1.4 mobility and chromatin binding in mitosis.
In mammalian cells, the level of estrogen receptor α (ERα) is rapidly decreased upon estrogen tre... more In mammalian cells, the level of estrogen receptor α (ERα) is rapidly decreased upon estrogen treatment and this regulation involves proteasome degradation. Using different approaches, we showed that the Mdm2 oncogenic ubiquitin-ligase directly interacted with ERα and is involved in the regulation of ERα turn-over. We showed that Mdm2 overexpression decreased ERα stability (both in the absence or presence of estrogens) and deletion of the ubiquitin-ligase domain abolished the negative regulation of ERα expression. Interestingly, our results indicated that ligand-dependent receptor turn-over was not required for efficient transactivation. Moreover, several lines of evidence suggested that Mdm2 interact with ERα in a ternary complex with p53. In MCF-7 human breast cancer cells, various p53-inducing agents (such as UV irradiation) or treatment with RITA (which inhibits the interaction of p53 with Mdm2) stabilized ERα and abolished its E2-dependent turn-over. Finally, in addition to this early positive effect on ERα stability, we showed that cellular stress inhibited ERα mRNA accumulation leading to a strong delayed inhibition of receptor expression. Altogether, our results indicate that ERα expression is complexly regulated by cellular stress, acting both on gene transcription and protein stability by targeting the Mdm2 protein.
N-terminal tails of histones are easily accessible outside of the nucleosomal core particle and p... more N-terminal tails of histones are easily accessible outside of the nucleosomal core particle and post-translational modifications (PTMs) of these tails have been the focus of attention in the past 15-20 years. By recruiting (or excluding) specific readers, histone modifications can regulate chromatin dynamics and, by extension, DNA-dependent processes. However, until very recently, the direct impact of histone PTMs on nucleosome structure and thus on chromatin function has remained somewhat elusive. Recent findings of novel sites and types of histone PTMs located within the globular domain of histones and, in particular, on the lateral surface of the histone octamer have changed this. As a result of their structurally important location in close proximity to the DNA molecule, this new class of histone PTMs can have a direct impact on chromatin function. Depending on their precise position at the nucleosome lateral surface (e.g. near the DNA entry/exit sites or in the dyad region), histone PTMs can regulate nucleosome structure and/or stability differently. We review recent progress on how histone PTMs can influence DNA unwrapping and/or nucleosome disassembly and shed light on how these types of novel modifications contribute mechanistically to the regulation of transcriptional activity.
Histone lysine methylation can have positive or negative effects on transcription, depending on t... more Histone lysine methylation can have positive or negative effects on transcription, depending on the precise methylation site. According to the "histone code" hypothesis these methylation marks can be read by proteins that bind them specifically and then regulate downstream events. Hetero-chromatin protein 1 (HP1), an essential component of heterochromatin, binds specifically to methylated Lys 9 of histone H3 (K9/H3). The linker histone H1.4 is methylated on Lys 26 (K26/H1.4), but the role of this methylation in downstream events remains unknown. Here we identify HP1 as a protein specifically recognizing and binding to methylated K26/ H1.4. We demonstrate that the Chromo domain of HP1 is mediating this binding and that phosphorylation of Ser 27 on H1.4 (S27/ H1.4) prevents HP1 from binding. We suggest that methylation of K26/H1.4 could have a role in tethering HP1 to chromatin and that this could also explain how HP1 is targeted to those regions of chromatin where it does not colocalize with methylated K9/H3. Our results provide the first experimental evidence for a "phospho switch" model in which neighboring phosphorylation reverts the effect of histone lysine methylation.
The heat shock response is characterized by the transcriptional activation of both hsp genes and ... more The heat shock response is characterized by the transcriptional activation of both hsp genes and noncoding and repeated satellite III DNA sequences located at pericentric heterochromatin. Both events are under the control of Heat Shock Factor I (HSF1). Here we show that under heat shock, HSF1 recruits major cellular acetyltransferases, GCN5, TIP60 and p300 to pericentric heterochromatin leading to a targeted hyperacetylation of pericentric chromatin. Redistribution of histone acetylation toward pericentric region in turn directs the recruitment of Bromodomain and Extra-Terminal (BET) proteins BRD2, BRD3, BRD4, which are required for satellite III transcription by RNAP II. Altogether we uncover here a critical role for HSF1 in stressed cells relying on the restricted use of histone acetylation signaling over pericentric heterochromatin (HC).
Chromodomain helicase DNA binding protein 4 (CHD4) is an ATPase subunit of the Nucleosome Remodel... more Chromodomain helicase DNA binding protein 4 (CHD4) is an ATPase subunit of the Nucleosome Remodelling and Deacetylation (NuRD) complex that regulates gene expression. CHD4 is essential for growth of multiple patient derived melanoma xenografts and for breast cancer. Here we show that CHD4 regulates expression of PADI1 (Protein Arginine Deiminase 1) and PADI3 in multiple cancer cell types modulating citrullination of arginine residues of the allosterically-regulated glycolytic enzyme pyruvate kinase M2 (PKM2). Citrullination of PKM2 R106 reprogrammes cross-talk between PKM2 ligands lowering its sensitivity to the inhibitors Tryptophan, Alanine and Phenylalanine and promoting activation by Serine. Citrullination thus bypasses normal physiological regulation by low Serine levels to promote excessive glycolysis and reduced cell proliferation. We further show that PADI1 and PADI3 expression is up-regulated by hypoxia where PKM2 citrullination contributes to increased glycolysis. We provide insight as to how conversion of arginines to citrulline impacts key interactions within PKM2 that act in concert to reprogramme its activity as an additional mechanism regulating this important enzyme.
In mammals, many germline genes are epigenetically repressed to prevent their illegitimate expres... more In mammals, many germline genes are epigenetically repressed to prevent their illegitimate expression in somatic cells. To advance our understanding of the mechanisms restricting the expression of germline genes, we analyzed their chromatin signature and performed a CRISPR-Cas9 knock-out screen for genes involved in germline gene repression using a Dazl-GFP reporter system in mouse embryonic stem cells (mESCs). We show that the repression of germline genes mainly depends on the polycomb complex PRC1.6 and DNA methylation, which function additively in mESCs. Furthermore, we validated novel genes involved in the repression of germline genes and characterized three of them: Usp7, Shfm1 (also known as Sem1) and Erh. Inactivation of Usp7, Shfm1 or Erh led to the upregulation of germline genes, as well as retrotransposons for Shfm1, in mESCs. Mechanistically, USP7 interacts with PRC1.6 components, promotes PRC1.6 stability and presence at germline genes, and facilitates DNA methylation de...
The tails of histone proteins are central players for all chromatin-mediated processes. Whereas t... more The tails of histone proteins are central players for all chromatin-mediated processes. Whereas the N-terminal histone tails have been studied extensively, little is known about the function of the H2A C-terminus. Here, we show that the H2A Cterminal tail plays a pivotal role in regulating chromatin structure and dynamics. We find that cells expressing C-terminally truncated H2A show increased stress sensitivity. Moreover, both the complete and the partial deletion of the tail result in increased histone exchange kinetics and nucleosome mobility in vivo and in vitro. Importantly, our experiments reveal that the H2A C-terminus is required for efficient nucleosome translocation by ISWI-type chromatin remodelers and acts as a novel recognition module for linker histone H1. Thus, we suggest that the H2A C-terminal tail has a bipartite function: stabilisation of the nucleosomal core particle, as well as mediation of the protein interactions that control chromatin dynamics and conformatio...
L'oncoproteine mdm2 inhibe l'activite de p53, non seulement en masquant son domaine de tr... more L'oncoproteine mdm2 inhibe l'activite de p53, non seulement en masquant son domaine de transactivation, mais aussi en stimulant la degradation du suppresseur de tumeur grace a son activite e3-ubiquitine ligase. Mdm2 est egalement une des cibles transcriptionnelles de p53, ce qui cree l'opportunite d'un retro-controle negatif de l'activite de p53, essentiel durant le developpement precoce de la souris. Pourtant, le role physiologique precis de mdm2 au cours du developpement n'est pas clair, bien que des arguments existent en faveur de son implication dans le controle de processus essentiels tels que la differenciation et l'apoptose. Dans une premiere partie, nous avons donc etudie l'expression de mdm2 pendant l'embryogenese precoce par hybridation in situ whole mount et avons montre une expression preferentielle de mdm2 entre les jours 8 et 9 pc, notamment dans la crete neurale. Nous pouvons aussi montrer une correlation entre des regions riches en...
In this article, the Ponceau staining presented in Fig. 1b (right, bottom) does not follow best p... more In this article, the Ponceau staining presented in Fig. 1b (right, bottom) does not follow best practices for figure preparation since itinadvertently included duplications from the Ponceau staining presented in Supplementary Fig. 1b (for which the same preparation ofnucleosomes from HeLa cells had been used). A new Fig. 1b is provided in the Author Correction.
Post-translational modifications of proteins have emerged as a major mechanism for regulating gen... more Post-translational modifications of proteins have emerged as a major mechanism for regulating gene expression. However, our understanding of how histone modifications directly affect chromatin function remains limited. In this study, we investigate acetylation of histone H3 at lysine 64 (H3K64ac), a previously uncharacterized acetylation on the lateral surface of the histone octamer. We show that H3K64ac regulates nucleosome stability and facilitates nucleosome eviction and hence gene expression in vivo. In line with this, we demonstrate that H3K64ac is enriched in vivo at the transcriptional start sites of active genes and it defines transcriptionally active chromatin. Moreover, we find that the p300 co-activator acetylates H3K64, and consistent with a transcriptional activation function, H3K64ac opposes its repressive counterpart H3K64me3. Our findings reveal an important role for a histone modification within the nucleosome core as a regulator of chromatin function and they demon...
In mammals, many germline genes are epigenetically repressed to prevent their illegitimate expres... more In mammals, many germline genes are epigenetically repressed to prevent their illegitimate expression in somatic cells. To advance our understanding of the mechanisms restricting the expression of germline genes, we analyzed their chromatin signature and performed a CRISPR-Cas9 knockout screen for genes involved in germline gene repression using a Dazl-GFP reporter system in mouse embryonic stem cells (mESCs). We show that the repression of germline genes mainly depends on the polycomb complex PRC1.6 and DNA methylation, which function additively in mESCs. Furthermore, we validated novel genes involved in the repression of germline genes and characterized three of them: Usp7, Shfm1 (also known as Sem1) and Erh. Inactivation of Usp7, Shfm1 or Erh led to the upregulation of germline genes, as well as retrotransposons for Shfm1, in mESCs. Mechanistically, USP7 interacts with PRC1.6 components, promotes PRC1.6 stability and presence at germline genes, and facilitates DNA methylation deposition at germline gene promoters for long term repression. Our study provides a global view of the mechanisms and novel factors required for silencing germline genes in embryonic stem cells.
bioRxiv (Cold Spring Harbor Laboratory), Aug 4, 2019
The CHD4 subunit of the Nucleosome Remodelling and Deacetylation (NuRD) complex regulates express... more The CHD4 subunit of the Nucleosome Remodelling and Deacetylation (NuRD) complex regulates expression of PADI1 (Protein Arginine Deiminase 1) and PADI3 in multiple cancer cell types modulating citrullination of three arginines of the allosterically-regulated glycolytic enzyme pyruvate kinase M2 (PKM2). PKM2 citrullination lowers its sensitivity to the inhibitors Tryptophan, Alanine and Phenylalanine shifting the equilibrium towards the activator Serine bypassing normal physiological regulation by low Serine levels and promoting excessive glycolysis, lowered intracellular ATP and slowed proliferation. Our data provide unique insight as to how conversion of arginines to citrulline impacts key interactions within PKM2 adding another layer of complexity to the mechanisms that regulate the activity of this important enzyme. CHD3 and CHD4 are mutually exclusive ATPase subunits of the Nucleosome Remodelling and Deacetylation (NuRD) complex that regulates gene expression, acting in many contexts as a co-repressor (1) (2) (3). An shRNA dropout screen previously identified CHD4 as essential for growth of multiple patient derived melanoma xenografts and for breast cancer (4) (5). Mining public data sets showed up-regulation of CHD4 upon transition from benign nevi to metastatic melanoma (Fig. ). In melanomas, CHD4 expression was comparable in the different mutation status, although CHD3 was lowered in NRAS mutated melanomas (Fig. ). Single cell RNA-seq (6) showed higher CHD4 expression in melanoma tumour cells compared to infiltrating B and T lymphocytes (Fig. ). SiRNA-mediated CHD3 or CHD4 silencing in a collection of melanoma cells in vitro reduced clonogenic capacity, increased the proportion of slow or non-proliferating cells (Fig. ), but did not induce apoptosis (Fig. ).
In mammals, the time period that follows fertilization is characterized by extensive chromatin re... more In mammals, the time period that follows fertilization is characterized by extensive chromatin remodeling, which enables epigenetic reprogramming of the gametes. Major changes in chromatin structure persist until the time of implantation, when the embryo develops into a blastocyst, which comprises the inner cell mass and the trophectoderm. Changes in DNA methylation, histone variant incorporation, and covalent modifications of the histones tails have been intensively studied during pre-implantation development. However, modifications within the core of the nucleosomes have not been systematically analyzed. Here, we report the first characterization and temporal analysis of 3 key acetylated residues in the core of the histone H3: H3K64ac, H3K122ac, and H3K56ac, all located at structurally important positions close to the DNA. We found that all 3 acetylations occur during pre-implantation development, but with different temporal kinetics. Globally, H3K64ac and H3K56ac were detected throughout cleavage stages, while H3K122ac was only weakly detectable during this time. Our work contributes to the understanding of the contribution of histone modifications in the core of the nucleosome to the "marking" of the newly established embryonic chromatin and unveils new modification pathways potentially involved in epigenetic reprogramming.
The linker histone H1 plays an essential role in maintaining and establishing higher-order chroma... more The linker histone H1 plays an essential role in maintaining and establishing higher-order chromatin structure. As with core histones, histone H1 is also extensively covalently modified. We showed previously that phosphorylation of S27 in human histone H1.4 (H1.4S27-P), prevents binding of heterochromatin protein 1 (HP1) family members (officially known as chromobox protein homologs) to the neighboring dimethylated K26. Here, we present the first functional characterization of H1.4S27-P in vivo and in vitro. We show that H1.4S27 phosphorylation is cell-cycle-regulated and its levels peak on metaphase chromosomes. We identify further Aurora B as the kinase phosphorylating H1.4S27. We demonstrate that histone H1.4 is the only somatic linker histone variant targeted by Aurora B and that Aurora B exclusively phosphorylates S27. Adjacent K26 dimethylation can regulate Aurora B activity towards S27, uncovering a crosstalk between these modifications. Finally, our fluorescence recovery after photobleaching (FRAP) analysis on histone H1.4 mutants suggests a role of S27 phosphorylation in the regulation of histone H1.4 mobility and chromatin binding in mitosis.
In mammalian cells, the level of estrogen receptor α (ERα) is rapidly decreased upon estrogen tre... more In mammalian cells, the level of estrogen receptor α (ERα) is rapidly decreased upon estrogen treatment and this regulation involves proteasome degradation. Using different approaches, we showed that the Mdm2 oncogenic ubiquitin-ligase directly interacted with ERα and is involved in the regulation of ERα turn-over. We showed that Mdm2 overexpression decreased ERα stability (both in the absence or presence of estrogens) and deletion of the ubiquitin-ligase domain abolished the negative regulation of ERα expression. Interestingly, our results indicated that ligand-dependent receptor turn-over was not required for efficient transactivation. Moreover, several lines of evidence suggested that Mdm2 interact with ERα in a ternary complex with p53. In MCF-7 human breast cancer cells, various p53-inducing agents (such as UV irradiation) or treatment with RITA (which inhibits the interaction of p53 with Mdm2) stabilized ERα and abolished its E2-dependent turn-over. Finally, in addition to this early positive effect on ERα stability, we showed that cellular stress inhibited ERα mRNA accumulation leading to a strong delayed inhibition of receptor expression. Altogether, our results indicate that ERα expression is complexly regulated by cellular stress, acting both on gene transcription and protein stability by targeting the Mdm2 protein.
N-terminal tails of histones are easily accessible outside of the nucleosomal core particle and p... more N-terminal tails of histones are easily accessible outside of the nucleosomal core particle and post-translational modifications (PTMs) of these tails have been the focus of attention in the past 15-20 years. By recruiting (or excluding) specific readers, histone modifications can regulate chromatin dynamics and, by extension, DNA-dependent processes. However, until very recently, the direct impact of histone PTMs on nucleosome structure and thus on chromatin function has remained somewhat elusive. Recent findings of novel sites and types of histone PTMs located within the globular domain of histones and, in particular, on the lateral surface of the histone octamer have changed this. As a result of their structurally important location in close proximity to the DNA molecule, this new class of histone PTMs can have a direct impact on chromatin function. Depending on their precise position at the nucleosome lateral surface (e.g. near the DNA entry/exit sites or in the dyad region), histone PTMs can regulate nucleosome structure and/or stability differently. We review recent progress on how histone PTMs can influence DNA unwrapping and/or nucleosome disassembly and shed light on how these types of novel modifications contribute mechanistically to the regulation of transcriptional activity.
Histone lysine methylation can have positive or negative effects on transcription, depending on t... more Histone lysine methylation can have positive or negative effects on transcription, depending on the precise methylation site. According to the "histone code" hypothesis these methylation marks can be read by proteins that bind them specifically and then regulate downstream events. Hetero-chromatin protein 1 (HP1), an essential component of heterochromatin, binds specifically to methylated Lys 9 of histone H3 (K9/H3). The linker histone H1.4 is methylated on Lys 26 (K26/H1.4), but the role of this methylation in downstream events remains unknown. Here we identify HP1 as a protein specifically recognizing and binding to methylated K26/ H1.4. We demonstrate that the Chromo domain of HP1 is mediating this binding and that phosphorylation of Ser 27 on H1.4 (S27/ H1.4) prevents HP1 from binding. We suggest that methylation of K26/H1.4 could have a role in tethering HP1 to chromatin and that this could also explain how HP1 is targeted to those regions of chromatin where it does not colocalize with methylated K9/H3. Our results provide the first experimental evidence for a "phospho switch" model in which neighboring phosphorylation reverts the effect of histone lysine methylation.
The heat shock response is characterized by the transcriptional activation of both hsp genes and ... more The heat shock response is characterized by the transcriptional activation of both hsp genes and noncoding and repeated satellite III DNA sequences located at pericentric heterochromatin. Both events are under the control of Heat Shock Factor I (HSF1). Here we show that under heat shock, HSF1 recruits major cellular acetyltransferases, GCN5, TIP60 and p300 to pericentric heterochromatin leading to a targeted hyperacetylation of pericentric chromatin. Redistribution of histone acetylation toward pericentric region in turn directs the recruitment of Bromodomain and Extra-Terminal (BET) proteins BRD2, BRD3, BRD4, which are required for satellite III transcription by RNAP II. Altogether we uncover here a critical role for HSF1 in stressed cells relying on the restricted use of histone acetylation signaling over pericentric heterochromatin (HC).
Chromodomain helicase DNA binding protein 4 (CHD4) is an ATPase subunit of the Nucleosome Remodel... more Chromodomain helicase DNA binding protein 4 (CHD4) is an ATPase subunit of the Nucleosome Remodelling and Deacetylation (NuRD) complex that regulates gene expression. CHD4 is essential for growth of multiple patient derived melanoma xenografts and for breast cancer. Here we show that CHD4 regulates expression of PADI1 (Protein Arginine Deiminase 1) and PADI3 in multiple cancer cell types modulating citrullination of arginine residues of the allosterically-regulated glycolytic enzyme pyruvate kinase M2 (PKM2). Citrullination of PKM2 R106 reprogrammes cross-talk between PKM2 ligands lowering its sensitivity to the inhibitors Tryptophan, Alanine and Phenylalanine and promoting activation by Serine. Citrullination thus bypasses normal physiological regulation by low Serine levels to promote excessive glycolysis and reduced cell proliferation. We further show that PADI1 and PADI3 expression is up-regulated by hypoxia where PKM2 citrullination contributes to increased glycolysis. We provide insight as to how conversion of arginines to citrulline impacts key interactions within PKM2 that act in concert to reprogramme its activity as an additional mechanism regulating this important enzyme.
In mammals, many germline genes are epigenetically repressed to prevent their illegitimate expres... more In mammals, many germline genes are epigenetically repressed to prevent their illegitimate expression in somatic cells. To advance our understanding of the mechanisms restricting the expression of germline genes, we analyzed their chromatin signature and performed a CRISPR-Cas9 knock-out screen for genes involved in germline gene repression using a Dazl-GFP reporter system in mouse embryonic stem cells (mESCs). We show that the repression of germline genes mainly depends on the polycomb complex PRC1.6 and DNA methylation, which function additively in mESCs. Furthermore, we validated novel genes involved in the repression of germline genes and characterized three of them: Usp7, Shfm1 (also known as Sem1) and Erh. Inactivation of Usp7, Shfm1 or Erh led to the upregulation of germline genes, as well as retrotransposons for Shfm1, in mESCs. Mechanistically, USP7 interacts with PRC1.6 components, promotes PRC1.6 stability and presence at germline genes, and facilitates DNA methylation de...
The tails of histone proteins are central players for all chromatin-mediated processes. Whereas t... more The tails of histone proteins are central players for all chromatin-mediated processes. Whereas the N-terminal histone tails have been studied extensively, little is known about the function of the H2A C-terminus. Here, we show that the H2A Cterminal tail plays a pivotal role in regulating chromatin structure and dynamics. We find that cells expressing C-terminally truncated H2A show increased stress sensitivity. Moreover, both the complete and the partial deletion of the tail result in increased histone exchange kinetics and nucleosome mobility in vivo and in vitro. Importantly, our experiments reveal that the H2A C-terminus is required for efficient nucleosome translocation by ISWI-type chromatin remodelers and acts as a novel recognition module for linker histone H1. Thus, we suggest that the H2A C-terminal tail has a bipartite function: stabilisation of the nucleosomal core particle, as well as mediation of the protein interactions that control chromatin dynamics and conformatio...
L'oncoproteine mdm2 inhibe l'activite de p53, non seulement en masquant son domaine de tr... more L'oncoproteine mdm2 inhibe l'activite de p53, non seulement en masquant son domaine de transactivation, mais aussi en stimulant la degradation du suppresseur de tumeur grace a son activite e3-ubiquitine ligase. Mdm2 est egalement une des cibles transcriptionnelles de p53, ce qui cree l'opportunite d'un retro-controle negatif de l'activite de p53, essentiel durant le developpement precoce de la souris. Pourtant, le role physiologique precis de mdm2 au cours du developpement n'est pas clair, bien que des arguments existent en faveur de son implication dans le controle de processus essentiels tels que la differenciation et l'apoptose. Dans une premiere partie, nous avons donc etudie l'expression de mdm2 pendant l'embryogenese precoce par hybridation in situ whole mount et avons montre une expression preferentielle de mdm2 entre les jours 8 et 9 pc, notamment dans la crete neurale. Nous pouvons aussi montrer une correlation entre des regions riches en...
In this article, the Ponceau staining presented in Fig. 1b (right, bottom) does not follow best p... more In this article, the Ponceau staining presented in Fig. 1b (right, bottom) does not follow best practices for figure preparation since itinadvertently included duplications from the Ponceau staining presented in Supplementary Fig. 1b (for which the same preparation ofnucleosomes from HeLa cells had been used). A new Fig. 1b is provided in the Author Correction.
Post-translational modifications of proteins have emerged as a major mechanism for regulating gen... more Post-translational modifications of proteins have emerged as a major mechanism for regulating gene expression. However, our understanding of how histone modifications directly affect chromatin function remains limited. In this study, we investigate acetylation of histone H3 at lysine 64 (H3K64ac), a previously uncharacterized acetylation on the lateral surface of the histone octamer. We show that H3K64ac regulates nucleosome stability and facilitates nucleosome eviction and hence gene expression in vivo. In line with this, we demonstrate that H3K64ac is enriched in vivo at the transcriptional start sites of active genes and it defines transcriptionally active chromatin. Moreover, we find that the p300 co-activator acetylates H3K64, and consistent with a transcriptional activation function, H3K64ac opposes its repressive counterpart H3K64me3. Our findings reveal an important role for a histone modification within the nucleosome core as a regulator of chromatin function and they demon...
Uploads
Papers by Sylvain Daujat