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Whole genome functional characterization of RE1 silencers using a modified massively parallel reporter assay

ABSTRACT: Both upregulation and downregulation by cis-regulatory elements help establish precise gene expression. Our understanding of how elements repress transcriptional activity is far more limited than activating elements. To address this gap, we characterized RE1, a group of transcriptional silencers bound by REST, on a genome-wide scale using an optimized massively parallel reporter assay (MPRAduo). MPRAduo empirically defined a minimal binding strength of REST required by silencer (REST m-value), above which multiple cofactors colocalize and act to directly silence transcription. We identified 1,500 human variants that alter RE1 silencing and found their effect sizes are predictable when they overlap with REST binding sites above the m-value. In addition, we demonstrate that non-canonical REST binding motifs exhibit silencer function only if they precisely align two half sites with specific spacer length. Our results show mechanistic insights into RE1 silencer which allows us to predict its activity and effect of variants on RE1, providing a paradigm for performing genome-wide functional characterization transcription factors binding sites.

ORGANISM(S): synthetic construct Homo sapiens

PROVIDER: GSE196171 | GEO | 2022/02/17

REPOSITORIES: GEO

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Whole genome functional characterization of RE1 silencers using a modified massively parallel reporter assay BRB-seq

Project description:Both upregulation and downregulation by cis-regulatory elements help establish precise gene expression. Our understanding of how elements repress transcriptional activity is far more limited than activating elements. To address this gap, we characterized RE1, a group of transcriptional silencers bound by REST, on a genome-wide scale using an optimized massively parallel reporter assay (MPRAduo). MPRAduo empirically defined a minimal binding strength of REST required by silencer (REST m-value), above which multiple cofactors colocalize and act to directly silence transcription. We identified 1,500 human variants that alter RE1 silencing and found their effect sizes are predictable when they overlap with REST binding sites above the m-value. In addition, we demonstrate that non-canonical REST binding motifs exhibit silencer function only if they precisely align two half sites with specific spacer length. Our results show mechanistic insights into RE1 silencer which allows us to predict its activity and effect of variants on RE1, providing a paradigm for performing genome-wide functional characterization transcription factors binding sites.

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human genome-wide silencers

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Mechanism of REST/NRSF Regulation of Clustered Protocadherin Alpha Genes

Project description:Repressor element-1 silencing transcription factor (REST) or neuron-restrictive silencer factor (NRSF) is a zinc-finger (ZF) containing transcriptional repressor that recognizes thousands of neuron-restrictive silencer elements (NRSEs) in mammalian genomes. How REST/NRSF regulates gene expression remains incompletely understood. Here, we investigate the binding pattern and regulation mechanism of REST/NRSF in the clustered protocadherin (PCDH) genes. We find that REST/NRSF directionally forms base-specific interactions with NRSEs via tandem ZFs in an anti-parallel manner but with striking conformational changes. In addition, REST/NRSF recruitment to the HS5-1 enhancer leads to the decrease of long-range enhancer-promoter interactions and downregulation of the clustered PCDH alpha genes. Thus, REST/NRSF represses PCDH alpha gene expression through directional binding to a repertoire of NRSEs within the distal enhancer and variable target genes.

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Expression data from NSCs treated with REST inhibitor X5050

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Expression data from NSCs treated with REST inhibitor X5050

Project description:REST has been initially described as a repressor of neuronal genes in non-neuronal cells by binding to its recognition sequence RE1. Over-activation of this factor has been shown in several diseases such as Huntington disease or central nervous system cancers. High-throughput screening of a library of 6,984 compounds with luciferase-assay measuring REST activity in neural derivatives of human embryonic stem cells led to the identification of one benzoimidazole-5-carboxamide derivative (X5050) that inhibited REST silencing in a RE1-dependent manner. Differential transcriptomic analysis revealed the upregulation of neuronal genes targeted by REST. RNA was extracted from 6 samples, corresponding to 3 independent cultures of NSC SA-01, each one treated either with DMSO (0.1 % final) or with X5050 (100 ÃÂµM final). RNA was isolated using RNeasy Mini kit with DNase I digestion and genome-wide gene expression profiling was performed by hybridization on Affymetrix microarrays

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A genome-wide screen identifies silencers with distinct chromatin properties and mechanisms of repression

Project description:Differential gene transcription enables development and homeostasis in all animals and is regulated by two major classes of distal cis-regulatory DNA elements (CREs), enhancers and silencers. While enhancers have been thoroughly characterized, the properties and mechansisms of silencers remain largely unknown. By an unbiased genome-wide functional screen in Drosophila melanogaster S2 cells, we discover a class of silencers that bind one of three transcription factors (TFs) and are generally not included in chromatin-defined CRE catalogs, as they mostly lack detectable DNA accessibility. The silencer-binding TF CG11247, which we term Saft, safeguards cell fate decisions in vivo and functions via a highly-conserved domain we term ZAC and the corepressor G9a, independently of G9a’s H3K9-methyltransferase activity. Overall, our identification of silencers with unexpected properties and mechanisms has important implications for the understanding and future study of repressive CREs, as well as the functional annotation of animal genomes.

2024-11-13 | PXD048161 | Pride

A genome-wide screen identifies silencers with distinct chromatin properties and mechanisms of repression

2024-11-13 | PXD047987 | Pride

TRF2 Inhibition-Mediated Degradation Derepresses the Neuronal Differentiation Program

Project description:Telomere binding factor 2 (TRF2), is a protein that plays a major role in the maintenance of telomere integrity. In mitotic normal and transformed cells, TRF2 inhibition triggers a rapid telomere DNA damage response that results in cell senescence or apoptosis. Here we provide evidence that TRF2 plays a role suppressing neuronal differentiation. TRF2 interacts with the RE1-silencing transcription factor (REST) in nuclear PML protein-containing compartments of neuronal cells in vivo. Inhibition of TRF2 function with a dominant-negative form of TRF2 elicits a telomeric DNA damage response, and disrupts the TRF2-REST complex resulting in proteasomal degradation of REST. Overexpression of REST impairs the ability of DN-TRF2 to induce neuronal differentiation, indicating that enhanced degradation of REST is sufficient to account for the differentiation-inducing effect of DN-TRF2. REST degradation derepresses RE1-regulated genes (L1CAM, BDNF, b3-tubulin, syntaxin and others) resulting in morphological and functional differentiation of neurons. Our findings identify a novel interaction between the telomeric protein TRF2 and REST which regulates the molecular differentiation program of neurons. Keywords: transfection and molecular inhibition

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C-terminal Domain Small Phosphatase 1 and MAP kinase reciprocally control REST stability and neuronal differentiation.

Project description:The RE1 Silencing Transcription Factor (REST) in stem cells represses hundreds of genes essential to neuronal function. During neurogenesis, REST is degraded in neural progenitors to promote subsequent elaboration of a mature neuronal phenotype. Prior studies indicate that part of the degradation mechanism involves phosphorylation of two sites in the C-terminus of REST that require activity of the E3 ubiquitin ligase, bTrCP. We identify a new proline-directed phosphorylation motif, at Serines 861/864 upstream of these sites, which is a substrate for the Peptidyl-prolyl cis-trans Isomerase, Pin1, as well as the ERK1/2 kinases. Mutation at S861/864 stabilizes REST, as does inhibition of Pin1 activity. Interestingly, we find that C-Terminal Domain Small Phosphatase1 (CTDSP1) is recruited by REST to neuronal genes, is present in REST immunocomplexes, dephosphorylates S861/864 and stabilizes REST. Expression of a REST peptide containing S861/864 in neural progenitors inhibits terminal neuronal differentiation. Together with previous work indicating that both REST and CTDSP1 are expressed to high levels in stem cells and down regulated during neurogenesis, our results suggest that CTDSP1 activity stabilizes REST in stem cells, and that ERK dependent phosphorylation combined with Pin1 activity promotes REST degradation in neural progenitors.

2014-09-15 | PXD001127 | Pride

Systematic identification of silencers in human cells

Project description:The majority of the human genome does not encode proteins. Many of these noncoding regions contain important regulatory sequences that control gene expression. To date, most studies have focused on activators such as enhancers, but regions that repress gene expression⎯silencers⎯have not been systematically studied. We have developed a system that identifies silencer regions in a genome-wide fashion based on silencer-mediated transcriptional repression of caspase 9. We found that silencers are widely distributed and may function in a tissue-specific fashion. These silencers harbor unique epigenetic signatures and are associated with specific transcription factors. Silencers also act at multiple genes, and at the level of chromosomal domains and long-range interactions. Deletion of silencer regions linked to the drug transporter genes ABCC2 and ABCG2 caused chemo-resistance. Overall, our study demonstrates that tissue-specific silencing is widespread throughout the human genome and likely contributes significantly to the regulation of gene expression and human biology.

2020-01-09 | GSE108536 | GEO

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