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1.
Cell ; 186(12): 2593-2609.e18, 2023 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-37209683

RESUMO

Here, we describe an approach to correct the genetic defect in fragile X syndrome (FXS) via recruitment of endogenous repair mechanisms. A leading cause of autism spectrum disorders, FXS results from epigenetic silencing of FMR1 due to a congenital trinucleotide (CGG) repeat expansion. By investigating conditions favorable to FMR1 reactivation, we find MEK and BRAF inhibitors that induce a strong repeat contraction and full FMR1 reactivation in cellular models. We trace the mechanism to DNA demethylation and site-specific R-loops, which are necessary and sufficient for repeat contraction. A positive feedback cycle comprising demethylation, de novo FMR1 transcription, and R-loop formation results in the recruitment of endogenous DNA repair mechanisms that then drive excision of the long CGG repeat. Repeat contraction is specific to FMR1 and restores the production of FMRP protein. Our study therefore identifies a potential method of treating FXS in the future.


Assuntos
Síndrome do Cromossomo X Frágil , Expansão das Repetições de Trinucleotídeos , Humanos , Estruturas R-Loop , Metilação de DNA , Síndrome do Cromossomo X Frágil/genética , Epigênese Genética , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo
2.
Cell ; 184(25): 6157-6173.e24, 2021 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-34856126

RESUMO

Chromosome loops shift dynamically during development, homeostasis, and disease. CCCTC-binding factor (CTCF) is known to anchor loops and construct 3D genomes, but how anchor sites are selected is not yet understood. Here, we unveil Jpx RNA as a determinant of anchor selectivity. Jpx RNA targets thousands of genomic sites, preferentially binding promoters of active genes. Depleting Jpx RNA causes ectopic CTCF binding, massive shifts in chromosome looping, and downregulation of >700 Jpx target genes. Without Jpx, thousands of lost loops are replaced by de novo loops anchored by ectopic CTCF sites. Although Jpx controls CTCF binding on a genome-wide basis, it acts selectively at the subset of developmentally sensitive CTCF sites. Specifically, Jpx targets low-affinity CTCF motifs and displaces CTCF protein through competitive inhibition. We conclude that Jpx acts as a CTCF release factor and shapes the 3D genome by regulating anchor site usage.


Assuntos
Fator de Ligação a CCCTC/metabolismo , Cromossomos/metabolismo , RNA Longo não Codificante/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Células-Tronco Embrionárias , Camundongos , Ligação Proteica
3.
Cell ; 170(1): 86-101.e16, 2017 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-28666128

RESUMO

Through an integration of genomic and proteomic approaches to advance understanding of long noncoding RNAs, we investigate the function of the telomeric transcript, TERRA. By identifying thousands of TERRA target sites in the mouse genome, we demonstrate that TERRA can bind both in cis to telomeres and in trans to genic targets. We then define a large network of interacting proteins, including epigenetic factors, telomeric proteins, and the RNA helicase, ATRX. TERRA and ATRX share hundreds of target genes and are functionally antagonistic at these loci: whereas TERRA activates, ATRX represses gene expression. At telomeres, TERRA competes with telomeric DNA for ATRX binding, suppresses ATRX localization, and ensures telomeric stability. Depleting TERRA increases telomerase activity and induces telomeric pathologies, including formation of telomere-induced DNA damage foci and loss or duplication of telomeric sequences. We conclude that TERRA functions as an epigenomic modulator in trans and as an essential regulator of telomeres in cis.


Assuntos
DNA Helicases/metabolismo , Proteínas Nucleares/metabolismo , Proteoma/metabolismo , RNA Longo não Codificante/metabolismo , Telômero/metabolismo , Animais , Ensaio de Desvio de Mobilidade Eletroforética , Camundongos , Motivos de Nucleotídeos , Células-Tronco/metabolismo , Telomerase/metabolismo , Proteína Nuclear Ligada ao X
4.
Nucleic Acids Res ; 50(21): 12217-12234, 2022 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-36440760

RESUMO

The genome consists of non-B-DNA structures such as G-quadruplexes (G4) that are involved in the regulation of genome stability and transcription. Telomeric-repeat containing RNA (TERRA) is capable of folding into G-quadruplex and interacting with chromatin remodeler ATRX. Here we show that TERRA modulates ATRX occupancy on repetitive sequences and over genes, and maintains DNA G-quadruplex structures at TERRA target and non-target sites in mouse embryonic stem cells. TERRA prevents ATRX from binding to subtelomeric regions and represses H3K9me3 formation. G4 ChIP-seq reveals that G4 abundance decreases at accessible chromatin regions, particularly at transcription start sites (TSS) after TERRA depletion; such G4 reduction at TSS is associated with elevated ATRX occupancy and differentially expressed genes. Loss of ATRX alleviates the effect of gene repression caused by TERRA depletion. Immunostaining analyses demonstrate that knockdown of TERRA diminishes DNA G4 signals, whereas silencing ATRX elevates G4 formation. Our results uncover an epigenetic regulation by TERRA that sequesters ATRX and preserves DNA G4 structures.


Assuntos
Quadruplex G , RNA Longo não Codificante , Animais , Camundongos , Cromatina/genética , Epigênese Genética , Telômero/genética , Telômero/metabolismo , RNA Longo não Codificante/metabolismo , DNA , Proteína Nuclear Ligada ao X/genética , Proteína Nuclear Ligada ao X/metabolismo
5.
Genome Res ; 25(8): 1170-81, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25986499

RESUMO

Polycomb group (PcG) complexes PRC1 and PRC2 are well known for silencing specific developmental genes. PRC2 is a methyltransferase targeting histone H3K27 and producing H3K27me3, essential for stable silencing. Less well known but quantitatively much more important is the genome-wide role of PRC2 that dimethylates ∼70% of total H3K27. We show that H3K27me2 occurs in inverse proportion to transcriptional activity in most non-PcG target genes and intergenic regions and is governed by opposing roaming activities of PRC2 and complexes containing the H3K27 demethylase UTX. Surprisingly, loss of H3K27me2 results in global transcriptional derepression proportionally greatest in silent or weakly transcribed intergenic and genic regions and accompanied by an increase of H3K27ac and H3K4me1. H3K27me2 therefore sets a threshold that prevents random, unscheduled transcription all over the genome and even limits the activity of highly transcribed genes. PRC1-type complexes also have global roles. Unexpectedly, we find a pervasive distribution of histone H2A ubiquitylated at lysine 118 (H2AK118ub) outside of canonical PcG target regions, dependent on the RING/Sce subunit of PRC1-type complexes. We show, however, that H2AK118ub does not mediate the global PRC2 activity or the global repression and is predominantly produced by a new complex involving L(3)73Ah, a homolog of mammalian PCGF3.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Complexo Repressor Polycomb 2/metabolismo , Transcrição Gênica , Animais , Drosophila melanogaster/metabolismo , Inativação Gênica , Genoma , Histonas/metabolismo , Metilação
6.
Nat Commun ; 13(1): 1550, 2022 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-35322029

RESUMO

RCOR1 is a known transcription repressor that recruits and positions LSD1 and HDAC1/2 on chromatin to erase histone methylation and acetylation. However, there is currently an incomplete understanding of RCOR1's range of localization and function. Here, we probe RCOR1's distribution on a genome-wide scale and unexpectedly find that RCOR1 is predominantly associated with transcriptionally active genes. Biochemical analysis reveals that RCOR1 associates with RNA Polymerase II (POL-II) during transcription and deacetylates its carboxy-terminal domain (CTD) at lysine 7. We provide evidence that this non-canonical RCOR1 activity is linked to dampening of POL-II productive elongation at actively transcribing genes. Thus, RCOR1 represses transcription in two ways-first, via a canonical mechanism by erasing transcriptionally permissive histone modifications through associating with HDACs and, second, via a non-canonical mechanism that deacetylates RNA POL-II's CTD to inhibit productive elongation. We conclude that RCOR1 is a transcription rheostat.


Assuntos
Cromatina , RNA Polimerase II , Acetilação , Cromatina/genética , Metilação , Processamento de Proteína Pós-Traducional , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Transcrição Gênica
7.
Nat Cell Biol ; 22(9): 1116-1129, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32807903

RESUMO

How allelic asymmetry is generated remains a major unsolved problem in epigenetics. Here we model the problem using X-chromosome inactivation by developing "BioRBP", an enzymatic RNA-proteomic method that enables probing of low-abundance interactions and an allelic RNA-depletion and -tagging system. We identify messenger RNA-decapping enzyme 1A (DCP1A) as a key regulator of Tsix, a noncoding RNA implicated in allelic choice through X-chromosome pairing. DCP1A controls Tsix half-life and transcription elongation. Depleting DCP1A causes accumulation of X-X pairs and perturbs the transition to monoallelic Tsix expression required for Xist upregulation. While ablating DCP1A causes hyperpairing, forcing Tsix degradation resolves pairing and enables Xist upregulation. We link pairing to allelic partitioning of CCCTC-binding factor (CTCF) and show that tethering DCP1A to one Tsix allele is sufficient to drive monoallelic Xist expression. Thus, DCP1A flips a bistable switch for the mutually exclusive determination of active and inactive Xs.


Assuntos
Endorribonucleases/metabolismo , RNA/metabolismo , Transativadores/metabolismo , Cromossomo X/metabolismo , Alelos , Animais , Fator de Ligação a CCCTC/metabolismo , Linhagem Celular , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Transcrição Gênica/fisiologia , Regulação para Cima/fisiologia , Inativação do Cromossomo X/fisiologia
8.
Genome Biol ; 9(1): R2, 2008 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-18171483

RESUMO

We present an approach for identifying condition-specific regulatory modules by using separate units of gene expression profiles along with ChIP-chip and motif data from Saccharomyces cerevisiae. By investigating the unique and common features of the obtained condition-specific modules, we detected several important properties of transcriptional network reorganization. Our approach reveals the functionally distinct coregulated submodules embedded in a coexpressed gene module and provides an effective method for identifying various condition-specific regulatory events at high resolution.


Assuntos
Redes Reguladoras de Genes/genética , Saccharomyces cerevisiae/genética , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica
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