PAXIP1 and STAG2 converge to maintain 3D genome architecture and facilitate promoter/enhancer contacts to enable stress hormone-dependent transcription

How steroid hormone receptors (SHRs) regulate transcriptional activity remains partly understood. Upon activation, SHRs bind the genome together with a co-regulator repertoire, crucial to induce gene expression. However, it remains unknown which components of the SHR-recruited co-regulator complex are essential to drive transcription following hormonal stimuli. Through a FACS-based genome-wide CRISPR screen, we functionally dissected the Glucocorticoid Receptor (GR) complex. We describe a functional cross-talk between PAXIP1 and the cohesin subunit STAG2, critical for regulation of gene expression by GR. Without altering the GR cistrome, PAXIP1 and STAG2 depletion alter the GR transcriptome, by impairing the recruitment of 3D-genome organization proteins to the GR complex. Importantly, we demonstrate that PAXIP1 is required for stability of cohesin on chromatin, its localization to GR-occupied sites, and maintenance of enhancer-promoter interactions. In lung cancer, where GR acts as tumor suppressor, PAXIP1/STAG2 loss enhances GR-mediated tumor suppressor activity by modifying local chromatin interactions. All together, we introduce PAXIP1 and STAG2 as novel co-regulators of GR, required to maintain 3D-genome architecture and drive the GR transcriptional programme following hormonal stimuli.</p

Décryptage des mécanismes de la répression génique par le facteur de transcription SPI1/PU.1 dans l’érythroleucémie

La transcription est un processus complexe qui intervient dans la différenciation et le développement. Le facteur de transcription (TF) est un facteur clé qui orchestre, avec les facteurs épigénétiques, le contrôle qualitatif et quantitatif de la transcription. Parmi les TF, certains sont déterminants pour la différenciation du lignage et leur dérégulation conduit au cancer. Le facteur de transcription SPI1/PU.1 est un régulateur majeur de l'hématopoïèse agissant de manière dose-dépendante dans des lignées hématopoïétiques distinctes et dont la dérégulation contribue à plusieurs types d’hémopathies. Bien qu'étant un activateur transcriptionnel, SPI1 est également capable de réprimer la transcription des gènes. Cependant, cette activité n'est pas bien décrite. La forte expression anormale de SPI1 dans les progéniteurs érythroïdes conduit à une inhibition de la différenciation et de l'apoptose avec pour conséquence une expansion des progéniteurs érythroïdes qui conduit au développement d’une érythroleucémie. L'objectif de ma thèse était de mieux caractériser les mécanismes moléculaires de la répression transcriptionnelle médiée par SPI1 dans le contexte de l'érythroleucémie. SPI1 ne présente pas une fonction répressive autonome mais interagit avec des facteurs épigénétiques répressifs en induisant des changements dans l'épigénome.Nous avons utilisé des approches de biologie cellulaire et moléculaire en combinaison avec des techniques de séquençage à haut débit pour disséquer les mécanismes moléculaires à la base de la répression transcriptionnelle médiée par SPI1. Nous avons développé deux paquets R disponibles gratuitement pour la normalisation et la visualisation des données ChIP-seq, respectivement CHIPIN (https://github.com/BoevaLab/CHIPIN) et Rseb (https://github.com/sebastian-gregoricchio/Rseb).En utilisant un modèle d'érythroleucémie murine TgSpi1, nous avons montré que SPI1 réprime les gènes en se liant principalement à leurs enhancers actifs, caractérisés par la co-présence de marques d'histones H3K27ac et H3K4me1. Par ChIP couplé à des expériences de spectrométrie de masse et de co-immunoprécipitation, nous avons identifié que la déacétylase répressive HDAC1 interagit avec SPI1 au niveau de la chromatine. Des expériences d'inhibition de HDAC1 et/ou de SPI1 ont montré que SPI1 et HDAC1 sont nécessaires et coopèrent pour réprimer la transcription en réduisant l’acétylation de H3K27 dans un sous-ensemble d'enhancers actifs, ce qui s'accompagne d'une diminution de l'occupation de l'ARN-pol II et de l'accessibilité à la chromatine au niveau du promoteur correspondant. De manière intéressante, nous avons identifié qu'au moins deux mécanismes distincts sont impliqués dans la répression génique par SPI1 : un mécanisme dépendant et un mécanisme indépendant de l'activité de HDAC1. Les enhancers dont la régulation dépend de HDAC1 présentent, à proximité de la liaison de SPI1, un enrichissement de liaison du facteur de transcription GATA1, tandis que ceux indépendants de HDAC1 sont enrichis en facteurs de transcription de la famille ETS.En plus de la régulation par HDAC1 et l'acétylation de H3, nous avons observé une augmentation de la marque répressive H3K27me3, déposée par PRC2, au niveau des promoteurs des gènes co-réprimés par SPI1 et HDAC1, spécifiquement lorsque SPI1 est lié aux enhancers. De plus, SPI1, en interagissant avec PRC2, augmente l'activité de PRC2. Ces données suggèrent un rôle de SPI1 dans la modulation de H3K27me3 qui renforce la répression génique exercée par SPI1 et HDAC1 dans les cellules érythroleucémiques.Finalement, par l'inhibition pharmacologique simultanée de HDAC1 et PRC2, nous avons identifié un effet synergique de HDAC1 et de PRC2 sur la répression des gènes cibles de SPI1 conduisant à l'arrêt de la prolifération et à l'induction de la mort cellulaire des cellules érythroleucémiques.Transcription is a complex process that is involved in differentiation and development. A transcription factor (TF) is a key factor that orchestrates, with epigenetic factors, qualitative and quantitative control of transcription. Among TFs, some are lineage-determinant, and their deregulation leads to cancer. The transcription factor SPI1/PU.1 is a key regulator of hematopoiesis acting in a dose-dependent manner in distinct hematopoietic lineages whose deregulation contributes to several hemopathies. Despite being a transcriptional activator, SPI1 is also able to repress gene transcription. However, this activity is not well described. Abnormal unrestrained expression of SPI1 in erythroid progenitors leads to erythroleukemia, in part through transcriptional deregulation. This deregulation leads to inhibition of differentiation and of apoptosis with a consequent expansion of erythroid progenitors that lead to leukemia development. The aim of my thesis was to better characterize the molecular mechanisms of transcriptional repression mediated by SPI1 in the context of erythroleukemia. SPI1 does not display an autonomous repressive function but interacts with repressive epigenetic factors inducing changes in the epigenome.We employed cellular and molecular biology approaches in combination with high-throughput sequencing techniques and bioinformatics to dissect the molecular mechanisms underlying the SPI1-mediated transcriptional repression in erythroleukemia via its interaction with epigenetic factors. We developed two free-available R packages for ChIP-seq data normalization and visualization, CHIPIN (https://github.com/BoevaLab/CHIPIN) and Rseb (https://github.com/sebastian-gregoricchio/Rseb), respectively. Using a murine TgSpi1 erythroleukemic model, we showed that SPI1 represses genes by binding mainly to their active enhancers, characterized by the co-presence of H3K27ac and H3K4me1 histone marks. By ChIP coupled to mass spectrometry and co-immunoprecipitation experiments, we identified that the repressive deacetylase HDAC1 interacts with SPI1 at the chromatin. HDAC1 and/or SPI1 inhibition experiments showed that SPI1 and HDAC1 are required and cooperate to repress transcription by reducing the H3K27ac at a subset of active enhancers accompanied by the decreased RNA-pol II occupancy and chromatin accessibility at the corresponding regulated promoter. Interestingly, we identified that at least two distinct mechanisms are involved in the SPI1-mediated gene repression: one dependent and one independent on HDAC1 activity. Enhancers whose regulation is dependent on HDAC1 display, in proximity of SPI1 binding, an enrichment of GATA1 transcription factor occupancy, while those independent of HDAC1 are enriched for ETS-family transcription factors.Besides regulation by HDAC1 and acetylation of H3, we observed an increase of the repressive mark H3K27me3, deposited by PRC2, at promoters of SPI1 and HDAC1 co-repressed genes, specifically when SPI1 was bound to enhancers. Moreover, SPI1, by interacting with PRC2, increases PRC2 activity. These data suggest a role of SPI1 in modulating H3K27me3 that reinforces the gene repression exerted by SPI1 and HDAC1 in erythroleukemic cells.Finally, by simultaneous pharmacological inhibition of HDAC1 and PRC2, we identified a synergistic effect of HDAC1 and PRC2 on SPI1-target gene repression leading to proliferation arrest and cell death induction of erythroleukemic cells

CHIPIN: ChIP-seq inter-sample normalization based on signal invariance across transcriptionally constant genes

Background Multiple studies rely on ChIP-seq experiments to assess the effect of gene modulation and drug treatments on protein binding and chromatin structure. However, most methods commonly used for the normalization of ChIP-seq binding intensity signals across conditions, e.g., the normalization to the same number of reads, either assume a constant signal-to-noise ratio across conditions or base the estimates of correction factors on genomic regions with intrinsically different signals between conditions. Inaccurate normalization of ChIP-seq signal may, in turn, lead to erroneous biological conclusions. Results We developed a new R package, CHIPIN, that allows normalizing ChIP-seq signals across different conditions/samples when spike-in information is not available, but gene expression data are at hand. Our normalization technique is based on the assumption that, on average, no differences in ChIP-seq signals should be observed in the regulatory regions of genes whose expression levels are constant across samples/conditions. In addition to normalizing ChIP-seq signals, CHIPIN provides as output a number of graphs and calculates statistics allowing the user to assess the efficiency of the normalization and qualify the specificity of the antibody used. In addition to ChIP-seq, CHIPIN can be used without restriction on open chromatin ATAC-seq or DNase hypersensitivity data. We validated the CHIPIN method on several ChIP-seq data sets and documented its superior performance in comparison to several commonly used normalization techniques. Conclusions The CHIPIN method provides a new way for ChIP-seq signal normalization across conditions when spike-in experiments are not available. The method is implemented in a user-friendly R package available on GitHub: https://github.com/BoevaLab/CHIPINISSN:1471-210

HDAC1 and PRC2 mediate combinatorial control in SPI1/PU.1-dependent gene repression in murine erythroleukaemia

International audienceAlthough originally described as transcriptional activator, SPI1/PU.1, a major player in haematopoiesis whose alterations are associated with haematological malignancies, has the ability to repress transcription. Here, we investigated the mechanisms underlying gene repression in the erythroid lineage, in which SPI1 exerts an oncogenic function by blocking differentiation. We show that SPI1 represses genes by binding active enhancers that are located in intergenic or gene body regions. HDAC1 acts as a cooperative mediator of SPI1-induced transcriptional repression by deacetylating SPI1-bound enhancers in a subset of genes, including those involved in erythroid differentiation.Enhancer deacetylation impacts on promoter acetylation, chromatin accessibility and RNA pol II occupancy. In addition to the activities of HDAC1, polycomb repressive complex 2 (PRC2) reinforces gene repression by depositing H3K27me3 at promoter sequences when SPI1 is located at enhancer sequences. Moreover, our study identified a synergistic relationship between PRC2 and HDAC1 complexes in mediating the transcriptional repression activity of SPI1, ultimately inducing synergistic adverse effects on leukaemic cell survival. Our results highlight the importance of the mechanism underlying transcriptional repression in leukemic cells, involving complex functional connections between SPI1 and the epigenetic regulators PRC2 and HDAC1