Multiple roles of class I HDACs in proliferation, differentiation, and development

Class I Histone deacetylases (HDACs) play a central role in controlling cell cycle regulation, cell differentiation, and tissue development. These enzymes exert their function by deacetylating histones and a growing number of non-histone proteins, thereby regulating gene expression and several other cellular processes. Class I HDACs comprise four members: HDAC1, 2, 3, and 8. Deletion and/or overexpression of these enzymes in mammalian systems has provided important insights about their functions and mechanisms of action which are reviewed here. In particular, unique as well as redundant functions have been identified in several paradigms. Studies with small molecule inhibitors of HDACs have demonstrated the medical relevance of these enzymes and their potential as therapeutic targets in cancer and other pathological conditions. Going forward, better understanding the specific role of individual HDACs in normal physiology as well as in pathological settings will be crucial to exploit this protein family as a useful therapeutic target in a range of diseases. Further dissection of the pathways they impinge on and of their targets, in chromatin or otherwise, will form important avenues of research for the futur

seqMINER: an integrated ChIP-seq data interpretation platform

In a single experiment, chromatin immunoprecipitation combined with high throughput sequencing (ChIP-seq) provides genome-wide information about a given covalent histone modification or transcription factor occupancy. However, time efficient bioinformatics resources for extracting biological meaning out of these gigabyte-scale datasets are often a limiting factor for data interpretation by biologists. We created an integrated portable ChIP-seq data interpretation platform called seqMINER, with optimized performances for efficient handling of multiple genome-wide datasets. seqMINER allows comparison and integration of multiple ChIP-seq datasets and extraction of qualitative as well as quantitative information. seqMINER can handle the biological complexity of most experimental situations and proposes methods to the user for data classification according to the analysed features. In addition, through multiple graphical representations, seqMINER allows visualization and modelling of general as well as specific patterns in a given dataset. To demonstrate the efficiency of seqMINER, we have carried out a comprehensive analysis of genome-wide chromatin modification data in mouse embryonic stem cells to understand the global epigenetic landscape and its change through cellular differentiation

Interconversion between active and inactive TATA-binding protein transcription complexes in the mouse genome

The TATA binding protein (TBP) plays a pivotal role in RNA polymerase II (Pol II) transcription through incorporation into the TFIID and B-TFIID complexes. The role of mammalian B-TFIID composed of TBP and B-TAF1 is poorly understood. Using a complementation system in genetically modified mouse cells where endogenous TBP can be conditionally inactivated and replaced by exogenous mutant TBP coupled to tandem affinity purification and mass spectrometry, we identify two TBP mutations, R188E and K243E, that disrupt the TBP–BTAF1 interaction and B-TFIID complex formation. Transcriptome and ChIP-seq analyses show that loss of B-TFIID does not generally alter gene expression or genomic distribution of TBP, but positively or negatively affects TBP and/or Pol II recruitment to a subset of promoters. We identify promoters where wild-type TBP assembles a partial inactive preinitiation complex comprising B-TFIID, TFIIB and Mediator complex, but lacking TFIID, TFIIE and Pol II. Exchange of B-TFIID in wild-type cells for TFIID in R188E and K243E mutant cells at these primed promoters completes preinitiation complex formation and recruits Pol II to activate their expression. We propose a novel regulatory mechanism involving formation of a partial preinitiation complex comprising B-TFIID that primes the promoter for productive preinitiation complex formation in mammalian cells

Structure-function analysis of TBP in murine embryonic fibroblasts

Partie 1 : étude structure-fonction de la TBP (TATA Binding Protein) dans les fibroblastes murins. Le domaine C-terminal conservé de la TBP interagit avec de multiples partenaires pour former des complexes nécessaires à la transcription par les ARN polyméPart 1 : structure-function analysis of TBP in murine embryonic fibroblasts. The conserved C-terminal domain of the TATA binding protein (TBP) interacts with multiple partners to form several complexes required for transcription by RNA Polymerases I, II

Part 1 : structure-function analysis of TBP in murine embryonic fibroblasts. Part 2 : study of the role of retinoic acid in neuronal differentiation of embryonic stem cells.

Partie 1 : étude structure-fonction de la TBP (TATA Binding Protein) dans les fibroblastes murins. Le domaine C-terminal conservé de la TBP interagit avec de multiples partenaires pour former des complexes nécessaires à la transcription par les ARN polymérases I, II et III. Pour analyser ces interactions dans les cellules de mammifères, nous avons utilisé des fibroblastes embryonnaires murins modifiés génétiquement. Dans ces cellules, la TBP endogène peut être remplacée par une TBP exogène sauvage ou portant des mutations (substitution d’un seul acide aminé dans le domaine C-terminal). Nous avons montré que de nombreux mutants de TBP peuvent complémenter la perte de la TBP endogène, mais provoquent une baisse de la prolifération. Des expériences d’immunoprécipitation couplées à des analyses par spectrométrie de masse ont permis d’identifier deux mutations, R188E et K243E, qui affectent l'interaction TBP/BTAF1 et la formation du complexe B-TFIID. L'analyse du transcriptome montre que la mutation R188E affecte l'expression de seulement 474 gènes. En accord avec ces résultats, l'analyse par ChIP-seq montre que cette mutation n’a pas d’effet général sur la distribution de TBP et de la Pol II dans le génome, mais perturbe le fonctionnement d’un nombre réduit de promoteurs par des mécanismes différents. Sur certains promoteurs, la mutation affecte le recrutement de TBP, alors que sur d’autres, elle induit le remplacement de B-TFIID par TFIID, ce qui se traduit par le recrutement de la Pol II et l’activation du promoteur. Ces données montrent que le complexe B-TFIID n'est pas essentiel pour la viabilité cellulaire, mais il est nécessaire à une prolifération normale. Ces données montrent également que B-TFIID n’est pas un régulateur global de la transcription, mais régule spécifiquement un ensemble réduit de gènes. Partie 2 : étude de l’action de l’acide rétinoïque dans la différenciation neuronale des cellules souches embryonnaires. Les cellules souches embryonnaires murines (ES) se différencient en neurones pyramidaux glutaminergiques in vitro suite au traitement par l’acide rétinoïque (AR). Nous avons utilisé la technique de RNA-seq pour identifier les gènes dont l’expression est modifiée après le traitement par l’AR durant la différenciation neuronale. Nous avons ainsi pu établir des profils d’expression génique qui caractérisent chaque stade de ce processus. Plusieurs facteurs de transcription font partie des gènes régulés par l’AR. Parmi ces facteurs se trouve notamment HES3 (Hairy and Enhancer of Split). Afin de mieux comprendre la fonction de ce facteur, nous avons réprimé son expression dans les cellules ES par la technique de shRNA. La perte d’expression de HES3 n’affecte pas la réponse précoce à l’AR, ni l’initiation de la différenciation. Nos résultats indiquent que HES3 agit à une étape plus tardive où il est essentiel à l’expression des marqueurs neurogéniques Brn2 et Mapt. Ces observations suggèrent que HES3 est nécessaire à la différenciation neuronale des cellules ES in vitro.Part 1 : structure-function analysis of TBP in murine embryonic fibroblasts. The conserved C-terminal domain of the TATA binding protein (TBP) interacts with multiple partners to form several complexes required for transcription by RNA Polymerases I, II and III. To analyse these interactions in mammalian cells we used genetically modified mouse embryonic fibroblasts where endogenous TBP can be replaced by an exogenous wild-type or mutant TBP with a single aminoacid substitution in the C-terminal domain. We show that many TBP mutants can complement loss of the endogenous TBP, but induce a slow growth phenotype. Tandem immunopurifications and mass-spectrometry analysis identify two TBP mutations, R188E and K243E, that completely disrupt the TBP/BTAF1 interaction and formation of the B-TFIID complex. Transcriptome analysis shows that the R188E mutation affects the expression of only 474 genes. In agreement with this, ChIP-seq analysis does not show major changes in genomic TBP and Pol II distribution in cells expressing TBPR188E. However, at affected promoters, mutations in TBP result either in its de novo recruitment or in an exchange of the B-TFIID by TFIID, thus promoting Pol II recruitment and gene activation in R188E mutant cells. These data show that B-TFIID complex is not essential for cell viability, but is required for normal proliferation. The B-TFIID is required for regulation of only a small subset of genes. Part 2 : study of the role of retinoic acid in neuronal differentiation of embryonic stem cells.Mouse embryonic stem cells (ES) can be differentiated into glutamatergic pyramidal neurons in vitro following retinoic acid (RA) treatment. We used RNA-seq to identify genes that are deregulated after RA treatment during neuronal differentiation. We established gene expression profiles that characterize each stage of this process. Several transcription factors are regulated by RA. HES3 (Hairy and Enhancer of split) is one of the most upregulated genes. To better understand its function, we repressed its expression using shRNA. The loss of HES3 expression does not affect the early RA response nor the initiation of differentiation. Our results indicate that HES3 acts at a later stage where it is essential for Brn2 and Mapt neurogenic markers expression. These observations suggest that HES3 is essential for neuronal differentiation of ES cells in vitro

Partie I, Étude structure-fonction de TBP dans les fibroblastes murins (Partie II, Étude de l action de l acide rétinoïque dans la différenciation neuronale des cellules souches embryonnaires)

Partie 1 : étude structure-fonction de la TBP (TATA Binding Protein) dans les fibroblastes murins. Le domaine C-terminal conservé de la TBP interagit avec de multiples partenaires pour former des complexes nécessaires à la transcription par les ARN polymérases I, II et III. Pour analyser ces interactions dans les cellules de mammifères, nous avons utilisé des fibroblastes embryonnaires murins modifiés génétiquement. Dans ces cellules, la TBP endogène peut être remplacée par une TBP exogène sauvage ou portant des mutations (substitution d un seul acide aminé dans le domaine C-terminal). Nous avons montré que de nombreux mutants de TBP peuvent complémenter la perte de la TBP endogène, mais provoquent une baisse de la prolifération. Des expériences d immunoprécipitation couplées à des analyses par spectrométrie de masse ont permis d identifier deux mutations, R188E et K243E, qui affectent l'interaction TBP/BTAF1 et la formation du complexe B-TFIID. L'analyse du transcriptome montre que la mutation R188E affecte l'expression de seulement 474 gènes. En accord avec ces résultats, l'analyse par ChIP-seq montre que cette mutation n a pas d effet général sur la distribution de TBP et de la Pol II dans le génome, mais perturbe le fonctionnement d un nombre réduit de promoteurs par des mécanismes différents. Sur certains promoteurs, la mutation affecte le recrutement de TBP, alors que sur d autres, elle induit le remplacement de B-TFIID par TFIID, ce qui se traduit par le recrutement de la Pol II et l activation du promoteur. Ces données montrent que le complexe B-TFIID n'est pas essentiel pour la viabilité cellulaire, mais il est nécessaire à une prolifération normale. Ces données montrent également que B-TFIID n est pas un régulateur global de la transcription, mais régule spécifiquement un ensemble réduit de gènes. Partie 2 : étude de l action de l acide rétinoïque dans la différenciation neuronale des cellules souches embryonnaires. Les cellules souches embryonnaires murines (ES) se différencient en neurones pyramidaux glutaminergiques in vitro suite au traitement par l acide rétinoïque (AR). Nous avons utilisé la technique de RNA-seq pour identifier les gènes dont l expression est modifiée après le traitement par l AR durant la différenciation neuronale. Nous avons ainsi pu établir des profils d expression génique qui caractérisent chaque stade de ce processus. Plusieurs facteurs de transcription font partie des gènes régulés par l AR. Parmi ces facteurs se trouve notamment HES3 (Hairy and Enhancer of Split). Afin de mieux comprendre la fonction de ce facteur, nous avons réprimé son expression dans les cellules ES par la technique de shRNA. La perte d expression de HES3 n affecte pas la réponse précoce à l AR, ni l initiation de la différenciation. Nos résultats indiquent que HES3 agit à une étape plus tardive où il est essentiel à l expression des marqueurs neurogéniques Brn2 et Mapt. Ces observations suggèrent que HES3 est nécessaire à la différenciation neuronale des cellules ES in vitro.Part 1 : structure-function analysis of TBP in murine embryonic fibroblasts. The conserved C-terminal domain of the TATA binding protein (TBP) interacts with multiple partners to form several complexes required for transcription by RNA Polymerases I, II and III. To analyse these interactions in mammalian cells we used genetically modified mouse embryonic fibroblasts where endogenous TBP can be replaced by an exogenous wild-type or mutant TBP with a single aminoacid substitution in the C-terminal domain. We show that many TBP mutants can complement loss of the endogenous TBP, but induce a slow growth phenotype. Tandem immunopurifications and mass-spectrometry analysis identify two TBP mutations, R188E and K243E, that completely disrupt the TBP/BTAF1 interaction and formation of the B-TFIID complex. Transcriptome analysis shows that the R188E mutation affects the expression of only 474 genes. In agreement with this, ChIP-seq analysis does not show major changes in genomic TBP and Pol II distribution in cells expressing TBPR188E. However, at affected promoters, mutations in TBP result either in its de novo recruitment or in an exchange of the B-TFIID by TFIID, thus promoting Pol II recruitment and gene activation in R188E mutant cells. These data show that B-TFIID complex is not essential for cell viability, but is required for normal proliferation. The B-TFIID is required for regulation of only a small subset of genes. Part 2 : study of the role of retinoic acid in neuronal differentiation of embryonic stem cells.Mouse embryonic stem cells (ES) can be differentiated into glutamatergic pyramidal neurons in vitro following retinoic acid (RA) treatment. We used RNA-seq to identify genes that are deregulated after RA treatment during neuronal differentiation. We established gene expression profiles that characterize each stage of this process. Several transcription factors are regulated by RA. HES3 (Hairy and Enhancer of split) is one of the most upregulated genes. To better understand its function, we repressed its expression using shRNA. The loss of HES3 expression does not affect the early RA response nor the initiation of differentiation. Our results indicate that HES3 acts at a later stage where it is essential for Brn2 and Mapt neurogenic markers expression. These observations suggest that HES3 is essential for neuronal differentiation of ES cells in vitro.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

TRF2 is recruited to the pre-initiation complex as a testis-specific subunit of TFIIA/ALF to promote haploid cell gene expression

Mammalian genomes encode two genes related to the TATA-box binding protein (TBP), TBP-related factors 2 and 3 (TRF2 and TRF3). Male Trf2(-/-) mice are sterile and characterized by arrested spermatogenesis at the transition from late haploid spermatids to early elongating spermatids. Despite this characterization, the molecular function of murine Trf2 remains poorly characterized and no direct evidence exists to show that it acts as a bona fide chromatin-bound transcription factor. We show here that Trf2 forms a stable complex with TFIIA or the testis expressed paralogue ALF chaperoned in the cytoplasm by heat shock proteins. We demonstrate for the first time that Trf2 is recruited to active haploid cell promoters together with Tbp, Taf7l and RNA polymerase II. RNA-seq analysis identifies a set of genes activated in haploid spermatids during the first wave of spermatogenesis whose expression is down-regulated by Trf2 inactivation. We therefore propose that Trf2 is recruited to the preinitiation complex as a testis-specific subunit of TFIIA/ALF that cooperates with Tbp and Taf7l to promote haploid cell gene expression

Cell-Specific Interaction of Retinoic Acid Receptors with Target Genes in Mouse Embryonic Fibroblasts and Embryonic Stem Cells▿ †

All-trans retinoic acid (RA) induces transforming growth factor beta (TGF-β)-dependent autocrine growth of mouse embryonic fibroblasts (MEFs). We have used chromatin immunoprecipitation to map 354 RA receptor (RAR) binding loci in MEFs, most of which were similarly occupied by the RARα and RARγ receptors. Only a subset of the genes associated with these loci are regulated by RA, among which are several critical components of the TGF-β pathway. We also show RAR binding to a novel series of target genes involved in cell cycle regulation, transformation, and metastasis, suggesting new pathways by which RA may regulate proliferation and cancer. Few of the RAR binding loci contained consensus direct-repeat (DR)-type elements. The majority comprised either degenerate DRs or no identifiable DRs but anomalously spaced half sites. Furthermore, we identify 462 RAR target loci in embryonic stem (ES) cells and show that their occupancy is cell type specific. Our results also show that differences in the chromatin landscape regulate the accessibility of a subset of more than 700 identified loci to RARs, thus modulating the repertoire of target genes that can be regulated and the biological effects of RA

Retinoic acid receptors recognise the mouse genome through binding elements with diverse spacing and topology

peer reviewedRetinoic Acid Receptors (RARs) heterodimerise with Retinoid X Receptors (RXRs) and bind to RA-response elements (RAREs) in the regulatory regions of their target genes. While previous studies on limited sets of RA-regulated genes have defined canonical RAREs as direct repeats of the consensus RGKTCA separated by 1, 2 or 5 nucleotides (DR1, DR2, DR5), we show that in mouse embryoid bodies or F9 embryonal carcinoma cells, RARs occupy a large repertoire of sites with DR0, DR8 and IR0 (inverted repeat 0) elements. Recombinant RAR-RXR binds these non-canonical spacings in vitro with comparable affinities to DR2 and DR5. Most DR8 elements comprise three half sites with DR2 and DR0 spacings. This specific half site organisation constitutes a previously unrecognised, but frequent signature of RAR binding elements. In functional assays, DR8 and IR0 elements act as independent RAREs, while DR0 does not. Our results reveal an unexpected diversity in the spacing and topology of binding elements for the RAR-RXR heterodimer. The differential ability of RAR-RXR bound to DR0 compared to DR2, DR5 and DR8 to mediate RA-dependent transcriptional activation indicates that half site spacing allosterically regulates RAR function