Examining the role of ATRX-RNA interactions in ATRX and PRC2 localization and gene expression.
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ABSTRACT:
PROVIDER: PRJNA540279 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8986730_1.fastq.gz | Fastqsanger.gz | |||
| SRR8986730_2.fastq.gz | Fastqsanger.gz | |||
| SRR8986740_1.fastq.gz | Fastqsanger.gz | |||
| SRR8986740_2.fastq.gz | Fastqsanger.gz | |||
| SRR8986731_1.fastq.gz | Fastqsanger.gz |
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Project description:We disrupted the RNA binding region in endogenous ATRX and tested localization of ATRX, EZH2 and H3K27me3 by CUT&RUN. We show that ATRX-RNA binding is important to regulate ATRX and PRC2 localization to specific genes.
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Project description:Heterochromatin in the eukaryotic genome is rigorously controlled by the concerted action of protein factors and RNAs. Here, we investigate the RNA binding function of ATRX, a chromatin remodeler with roles in silencing of repetitive regions of the genome and in recruitment of the polycomb repressive complex 2 (PRC2). We identify ATRX RNA binding regions (RBRs) and discover that the major ATRX RBR lies within the N-terminal region of the protein, distinct from its PHD and helicase domains. Deletion of this ATRX RBR (ATRXRBR) compromises ATRX interactions with RNAs in vitro and in vivo and alters its chromatin binding properties. Genome-wide studies reveal that loss of RNA interactions results in a redistribution of ATRX on chromatin. Finally, our studies identify a role for ATRX-RNA interactions in regulating PRC2 localization to a subset of polycomb target genes.
2020-03-09 | PXD017806 | Pride
Project description:Heterochromatin in the eukaryotic genome is rigorously controlled by the concerted action of protein factors and RNAs. Here, we investigate the RNA binding function of ATRX, a chromatin remodeler with roles in silencing of repetitive regions of the genome and in recruitment of the polycomb repressive complex 2 (PRC2). We identify ATRX RNA binding regions (RBRs) and discover that the major ATRX RBR lies within the N-terminal region of the protein, distinct from its PHD and helicase domains. Deletion of this ATRX RBR (ATRXΔRBR) compromises ATRX interactions with RNAs in vitro and in vivo and alters its chromatin binding properties. Genome-wide studies reveal that loss of RNA interactions results in a redistribution of ATRX on chromatin. Finally, our studies identify a role for ATRX-RNA interactions in regulating PRC2 localization to a subset of polycomb target genes.
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Project description:In mammals, dosage compensation entails inactivation of an entire X chromosome in female cells. X-chromosome inactivation (XCI) depends on the long noncoding RNA Xist and its recruitment of Polycomb Repressive Complex 2 (PRC2). How PRC2 specifically interacts with Xist and other Polycomb targets remains unclear. Using XCI as a model, we take an unbiased proteomics approach to search for new Xist and PRC2 regulators. We identify ATRX, a chromatin remodeler associated with mental retardation, alpha thalassemia, and cancer in humans. We show that ATRX is a high-affinity RNA-binding protein. ATRX directly interacts with RepA/Xist RNA and is required for loading of PRC2 onto the RNA in vivo. The Xist gene is a hotspot of ATRX occupancy. Without ATRX, PRC2 cannot load onto Xist RNA nor spread in cis along the X-chromosome. Epigenomic profiling reveals a dependency of PRC2 on ATRX that extends beyond XCI. Depleting ATRX results in genome-wide disruptions to PRC2 localization and trimethylation of histone H3-lysine 27 (H3K27me3). PRC2 is displaced from genes and relocalized to intergenic space, causing reduced H3K27me3 and concomitant changes in expression of Polycomb targets. We conclude that ATRX is a required specificity determinant for PRC2 targeting and function.
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Project description:ATRX is a chromatin remodelling protein of the SWI/SNF family, mutations in which cause alpha thalassemia X-linked (ATRX) intellectual disability syndrome and are highly associated with a number of cancers characterized by alternative lengthening of telomeres. ATRX functions with the histone chaperone DAXX to facilitate deposition of the histone variant H3.3 at heterochromatic regions such as telomere and pericentric repeats, resulting in a unique form of heterochromatin characterized by both trimethylation of histone H3 at lysine 9 (H3K9me3) and H3.3. How ATRX is recruited to these regions remain unclear.To better understand it, we therefore used immunoprecipitation-coupled to mass spectrometry (IP-MS) to identify potential factors that interact with ATRX and may promote its localization and function. Based on our proteomics results, we identified known ATRX-interacting proteins, DAXX and MRE11. We also identified a number of proteins that have been genetically linked to G-quadruplex structures. Interestingly, we identified members of DNA replcation machinery MCM complex (e.g. MCM2, MCM4, MCM6, and MCM7) and the faciliates chromatin transcription (FACT) comeplex as novel ATRX-interacting proteins. We then verified the interaction of ATRX to MCM proteins and FACT subunits by co-immunoprecipition/immunoblot and proximity ligation assay.
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Project description:The SWI/SNF-family chromatin remodeling protein ATRX is a tumor suppressor in sarcomas, gliomas and other malignancies. Its loss of function facilitates the alternative lengthening of telomeres (ALT) pathway in tumor cells, while it also affects Polycomb repressive complex 2 (PRC2) silencing of its target genes. To further define the role of inactivating ATRX mutations in carcinogenesis, we knocked out atrx in our previously published p53/nf1-deficient zebrafish line that develops malignant peripheral nerve sheath tumors and gliomas. Complete inactivation of atrx using CRISPR-cas9 was lethal in developing fish and resulted in an alpha-thalassemia-like phenotype including reduced alpha-globin expression. In p53/nf1-deficient zebrafish neither peripheral nerve sheath tumors nor gliomas showed accelerated onset in atrx+/- fish, but these fish developed various tumors that were not observed in their atrx+/+ siblings, including epithelioid sarcoma, angiosarcoma, undifferentiated pleomorphic sarcoma and rare types of carcinoma. Most of these cancer types are included in the AACR Genie database of human tumors associated with mutant ATRX, indicating that our zebrafish model reliably reflects a role for ATRX-loss in the early pathogenesis of these types of human cancers. RNA-seq of p53/nf1- and p53/nf1/atrx-deficient tumors revealed that down-regulation of telomerase accompanied ALT-mediated lengthening of the telomeres in atrx-mutant samples. Moreover, inactivating mutations in atrx disturbed PRC2-target gene silencing, indicating a connection between ATRX loss and PRC2 dysfunction in cancer development.
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Project description:the nature of the interaction between RNA and Polycomb repressive complex 2 (PRC2) has been an area of intensive investigation. Although PRC2 is now established as a bona fide RNA-binding protein, specific motifs have not been fully elucidated, nor has the association of PRC2-RNA complexes with active genes been reconciled. Here we employ denaturing CLIP to identify RNA motifs demonstrating high-affinity binding to PRC2 (Kd 11-80 nM). Mutating the motifs diminishes binding affinity in vitro and attenuates PRC2’s repressive function in vivo. A large fraction of interactions occurs at promoter-proximal regions and associates with POL-II pausing. Furthermore, although PRC2-associated nascent transcripts are highly expressed, ablating PRC2 further increases their expression. Thus, PRC2-RNA interactions are rheostats that operate at the level of transcription elongation to fine-tune gene activity, explaining why they frequently occur within active genes. PRC2-RNA interactions also impact expression of neighboring genes, supporting the classical model that RNA targets PRC2 to gene in cis. Our study supports a model in which RNA specifically targets PRC2 for two repressive functions — (i) control of POL-II pausing, and (ii) marking chromatin with H3K27me3.
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