RESUMO
We applied a combinatorial indexing assay, sci-ATAC-seq, to profile genome-wide chromatin accessibility in â¼100,000 single cells from 13 adult mouse tissues. We identify 85 distinct patterns of chromatin accessibility, most of which can be assigned to cell types, and â¼400,000 differentially accessible elements. We use these data to link regulatory elements to their target genes, to define the transcription factor grammar specifying each cell type, and to discover in vivo correlates of heterogeneity in accessibility within cell types. We develop a technique for mapping single cell gene expression data to single-cell chromatin accessibility data, facilitating the comparison of atlases. By intersecting mouse chromatin accessibility with human genome-wide association summary statistics, we identify cell-type-specific enrichments of the heritability signal for hundreds of complex traits. These data define the in vivo landscape of the regulatory genome for common mammalian cell types at single-cell resolution.
Assuntos
Cromatina/química , Análise de Célula Única/métodos , Animais , Análise por Conglomerados , Epigênese Genética , Epigenômica , Regulação da Expressão Gênica , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos , Masculino , Mamíferos , Camundongos , Camundongos Endogâmicos C57BL , Fatores de TranscriçãoRESUMO
Facioscapulohumeral muscular dystrophy (FSHD) is caused by insufficient epigenetic repression of D4Z4 macrosatellite repeat where DUX4, an FSHD causing gene is embedded. There are two forms of FSHD, FSHD1 with contraction of D4Z4 repeat and FSHD2 with chromatin compaction defects mostly due to SMCHD1 mutation. Previous reports showed DUX4-induced gene expression changes as well as changes in microRNA expression in FSHD muscle cells. However, a genome wide analysis of small noncoding RNAs that might be regulated by DUX4 or by mutations in SMCHD1 has not been reported yet. Here, we identified several types of small noncoding RNAs including known microRNAs that are differentially expressed in FSHD2 muscle cells compared to control. Although fewer small RNAs were differentially expressed during muscle differentiation in FSHD2 cells compared to controls, most of the known myogenic microRNAs, such as miR1, miR133a and miR206 were induced in both FSHD2 and control muscle cells during differentiation. Our small RNA sequencing data analysis also revealed both DUX4- and SMCHD1-specific changes in FSHD2 muscle cells. Six FSHD2 microRNAs were affected by DUX4 overexpression in control myoblasts, whereas increased expression of tRNAs and 5S rRNAs in FSHD2 muscle cells was largely recapitulated in SMCHD1-depleted control myoblasts. Altogether, our studies suggest that the small noncoding RNA transcriptome changes in FSHD2 might be different from those in FSHD1 and that these differences may provide new diagnostic and therapeutic tools specific to FSHD2.
Assuntos
Proteínas Cromossômicas não Histona/genética , Proteínas de Homeodomínio/genética , Distrofia Muscular Facioescapuloumeral/genética , Pequeno RNA não Traduzido/genética , Estudos de Casos e Controles , Diferenciação Celular/genética , Regulação da Expressão Gênica , Humanos , MicroRNAs/genética , Fibras Musculares Esqueléticas/patologia , Fibras Musculares Esqueléticas/fisiologia , Mutação , Mioblastos/patologia , Mioblastos/fisiologia , RNA Ribossômico 5S/genética , RNA de Transferência/genética , Reprodutibilidade dos TestesRESUMO
CCCTC-binding factor (CTCF) is a conserved transcription factor that performs diverse roles in transcriptional regulation and chromatin architecture. Cancer genome sequencing reveals diverse acquired mutations in CTCF, which we have shown functions as a tumour suppressor gene. While CTCF is essential for embryonic development, little is known of its absolute requirement in somatic cells and the consequences of CTCF haploinsufficiency. We examined the consequences of CTCF depletion in immortalised human and mouse cells using shRNA knockdown and CRISPR/Cas9 genome editing as well as examined the growth and development of heterozygous Ctcf (Ctcf+/-) mice. We also analysed the impact of CTCF haploinsufficiency by examining gene expression changes in CTCF-altered endometrial carcinoma. Knockdown and CRISPR/Cas9-mediated editing of CTCF reduced the cellular growth and colony-forming ability of K562 cells. CTCF knockdown also induced cell cycle arrest and a pro-survival response to apoptotic insult. However, in p53 shRNA-immortalised Ctcf+/- MEFs we observed the opposite: increased cellular proliferation, colony formation, cell cycle progression, and decreased survival after apoptotic insult compared to wild-type MEFs. CRISPR/Cas9-mediated targeting in Ctcf+/- MEFs revealed a predominance of in-frame microdeletions in Ctcf in surviving clones, however protein expression could not be ablated. Examination of CTCF mutations in endometrial cancers showed locus-specific alterations in gene expression due to CTCF haploinsufficiency, in concert with downregulation of tumour suppressor genes and upregulation of estrogen-responsive genes. Depletion of CTCF expression imparts a dramatic negative effect on normal cell function. However, CTCF haploinsufficiency can have growth-promoting effects consistent with known cancer hallmarks in the presence of additional genetic hits. Our results confirm the absolute requirement for CTCF expression in somatic cells and provide definitive evidence of CTCF's role as a haploinsufficient tumour suppressor gene. CTCF genetic alterations in endometrial cancer indicate that gene dysregulation is a likely consequence of CTCF loss, contributing to, but not solely driving cancer growth.
Assuntos
Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Sobrevivência Celular/fisiologia , Neoplasias do Endométrio/genética , Edição de Genes , Animais , Sistemas CRISPR-Cas , Proliferação de Células/genética , Proliferação de Células/fisiologia , Sobrevivência Celular/genética , Feminino , Haploinsuficiência/genética , Haploinsuficiência/fisiologia , Humanos , Células K562 , Camundongos , RNA Interferente Pequeno/genéticaRESUMO
Facioscapulohumeral muscular dystrophy (FSHD) is caused by the aberrant expression of the DUX4 transcription factor in skeletal muscle. The DUX4 retrogene is encoded in the D4Z4 macrosatellite repeat array, and smaller array size or a mutation in the SMCHD1 gene results in inefficient epigenetic repression of DUX4 in skeletal muscle, causing FSHD1 and FSHD2, respectively. Previously we showed that the entire D4Z4 repeat is bi-directionally transcribed with the generation of small si- or miRNA-like fragments and suggested that these might suppress DUX4 expression through the endogenous RNAi pathway. Here we show that exogenous siRNA targeting the region upstream of the DUX4 transcription start site suppressed DUX4 mRNA expression and increased both H3K9 methylation and AGO2 recruitment. In contrast, similarly targeted MOE-gapmer antisense oligonucleotides that degrade RNA but do not engage the RNAi pathway did not repress DUX4 expression. In addition, knockdown of DICER or AGO2 using either siRNA or MOE-gapmer chemistries resulted in the induction of DUX4 expression in control muscle cells that normally do not express DUX4, indicating that the endogenous RNAi pathway is necessary to maintain repression of DUX4 in control muscle cells. Together these data demonstrate a role of the endogenous RNAi pathway in repeat-mediated epigenetic repression of the D4Z4 macrosatellite repeat, and show that enhancing the activity of this pathway by supplying exogenous siRNA oligonucleotides represents a potential therapeutic approach to silencing DUX4 in FSHD.
Assuntos
Proteínas Argonautas/metabolismo , Epigênese Genética , Inativação Gênica , Repetições de Microssatélites , Distrofia Muscular Facioescapuloumeral/genética , Distrofia Muscular Facioescapuloumeral/metabolismo , RNA Interferente Pequeno/genética , Ribonuclease III/metabolismo , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Regulação da Expressão Gênica , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Distrofia Muscular Facioescapuloumeral/terapia , Interferência de RNA , Sítio de Iniciação de TranscriçãoRESUMO
Each unit of the D4Z4 macrosatellite repeat contains a retrotransposed gene encoding the DUX4 double-homeobox transcription factor. Facioscapulohumeral dystrophy (FSHD) is caused by deletion of a subset of the D4Z4 units in the subtelomeric region of chromosome 4. Although it has been reported that the deletion of D4Z4 units induces the pathological expression of DUX4 mRNA, the association of DUX4 mRNA expression with FSHD has not been rigorously investigated, nor has any human tissue been identified that normally expresses DUX4 mRNA or protein. We show that FSHD muscle expresses a different splice form of DUX4 mRNA compared to control muscle. Control muscle produces low amounts of a splice form of DUX4 encoding only the amino-terminal portion of DUX4. FSHD muscle produces low amounts of a DUX4 mRNA that encodes the full-length DUX4 protein. The low abundance of full-length DUX4 mRNA in FSHD muscle cells represents a small subset of nuclei producing a relatively high abundance of DUX4 mRNA and protein. In contrast to control skeletal muscle and most other somatic tissues, full-length DUX4 transcript and protein is expressed at relatively abundant levels in human testis, most likely in the germ-line cells. Induced pluripotent (iPS) cells also express full-length DUX4 and differentiation of control iPS cells to embryoid bodies suppresses expression of full-length DUX4, whereas expression of full-length DUX4 persists in differentiated FSHD iPS cells. Together, these findings indicate that full-length DUX4 is normally expressed at specific developmental stages and is suppressed in most somatic tissues. The contraction of the D4Z4 repeat in FSHD results in a less efficient suppression of the full-length DUX4 mRNA in skeletal muscle cells. Therefore, FSHD represents the first human disease to be associated with the incomplete developmental silencing of a retrogene array normally expressed early in development.
Assuntos
Perfilação da Expressão Gênica , Proteínas de Homeodomínio/genética , Distrofia Muscular Facioescapuloumeral/genética , Splicing de RNA , Adulto , Animais , Western Blotting , Linhagem Celular , Cromossomos Humanos Par 4/genética , Feminino , Imunofluorescência , Regulação da Expressão Gênica , Células HCT116 , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Dados de Sequência Molecular , Células Musculares/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular Facioescapuloumeral/patologia , Sequências Repetitivas de Ácido Nucleico/genética , Retroelementos/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Background: The number and escape levels of genes that escape X chromosome inactivation (XCI) in female somatic cells vary among tissues and cell types, potentially contributing to specific sex differences. Here we investigate the role of CTCF, a master chromatin conformation regulator, in regulating escape from XCI. CTCF binding profiles and epigenetic features were systematically examined at constitutive and facultative escape genes using mouse allelic systems to distinguish the inactive X (Xi) and active X (Xa) chromosomes. Results: We found that escape genes are located inside domains flanked by convergent arrays of CTCF binding sites, consistent with the formation of loops. In addition, strong and divergent CTCF binding sites often located at the boundaries between escape genes and adjacent neighbors subject to XCI would help insulate domains. Facultative escapees show clear differences in CTCF binding dependent on their XCI status in specific cell types/tissues. Concordantly, deletion but not inversion of a CTCF binding site at the boundary between the facultative escape gene Car5b and its silent neighbor Siah1b resulted in loss of Car5b escape. Reduced CTCF binding and enrichment of a repressive mark over Car5b in cells with a boundary deletion indicated loss of looping and insulation. In mutant lines in which either the Xi-specific compact structure or its H3K27me3 enrichment was disrupted, escape genes showed an increase in gene expression and associated active marks, supporting the roles of the 3D Xi structure and heterochromatic marks in constraining levels of escape. Conclusion: Our findings indicate that escape from XCI is modulated both by looping and insulation of chromatin via convergent arrays of CTCF binding sites and by compaction and epigenetic features of the surrounding heterochromatin.
RESUMO
X chromosome inactivation (XCI) is a female-specific process in which one X chromosome is silenced to balance X-linked gene expression between the sexes. XCI is initiated in early development by upregulation of the lncRNA Xist on the future inactive X (Xi). A subset of X-linked genes escape silencing and thus have higher expression in females, suggesting female-specific functions. One of these genes is the highly conserved gene Kdm6a , which encodes a histone demethylase that removes methyl groups at H3K27 to facilitate gene expression. Here, we investigate the role of KDM6A in the regulation of Xist . We observed impaired upregulation of Xist during early stages of differentiation in hybrid mouse ES cells following CRISPR/Cas9 knockout of Kdm6a . This is associated with reduced Xist RNA coating of the Xi, suggesting diminished XCI potency. Indeed, Kdm6a knockout results in aberrant overexpression of genes from the Xi after differentiation. KDM6A binds to the Xist promoter and knockout cells show an increase in H3K27me3 at Xist . These results indicate that KDM6A plays a role in the initiation of XCI through histone demethylase-dependent activation of Xist during early differentiation.
RESUMO
PURPOSE OF REVIEW: In recent years, we have seen remarkable progress in our understanding of the disease mechanism underlying facioscapulohumeral muscular dystrophy (FSHD). The purpose of this review is to provide a comprehensive overview of our current understanding of the disease mechanism and to discuss the observations supporting the possibility of a developmental defect in this disorder. RECENT FINDINGS: In the majority of cases, FSHD is caused by contraction of the D4Z4 repeat array (FSHD1). This results in local chromatin relaxation and stable expression of the DUX4 retrogene in skeletal muscle, but only when a polymorphic DUX4 polyadenylation signal is present. In some cases (FSHD2), D4Z4 chromatin relaxation and stable DUX4 expression occur in the absence of D4Z4 array contraction. DUX4 is a germline transcription factor and its expression in skeletal muscle leads to activation of early stem cell and germline programs and transcriptional activation of retroelements. SUMMARY: Recent studies have provided a plausible disease mechanism for FSHD in which FSHD results from inappropriate expression of the germline transcription factor DUX4. The genes regulated by DUX4 suggest several mechanisms of muscle damage, and provide potential biomarkers and therapeutic targets that should be investigated in future studies.
Assuntos
Cromatina/patologia , Distrofia Muscular Facioescapuloumeral/patologia , Animais , Proteínas de Homeodomínio/genética , Humanos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular Facioescapuloumeral/genética , Distrofia Muscular Facioescapuloumeral/fisiopatologia , Transcrição GênicaRESUMO
BACKGROUND: KDM6A is a demethylase encoded by a gene with female-biased expression due to escape from X inactivation. Its main role is to facilitate gene expression through removal of the repressive H3K27me3 mark, with evidence of some additional histone demethylase-independent functions. KDM6A mutations have been implicated in congenital disorders such as Kabuki Syndrome, as well as in sex differences in cancer. METHODS: Kdm6a was knocked out using CRISPR/Cas9 gene editing in F1 male and female mouse embryonic stem cells (ES) derived from reciprocal crosses between C57BL6 x Mus castaneus. Diploid and allelic RNA-seq analyses were done to compare gene expression between wild-type and Kdm6a knockout (KO) clones. The effects of Kdm6a KO on sex-biased gene expression were investigated by comparing gene expression between male and female ES cells. Changes in H3K27me3 enrichment and chromatin accessibility at promoter regions of genes with expression changes were characterized by ChIP-seq and ATAC-seq followed by diploid and allelic analyses. RESULTS: We report that Kdm6a KO in male and female embryonic stem (ES) cells derived from F1 hybrid mice cause extensive gene dysregulation, disruption of sex biases, and specific parental allele effects. Among the dysregulated genes are candidate genes that may explain abnormal developmental features of Kabuki syndrome caused by KDM6A mutations in human. Strikingly, Kdm6a knockouts result in a decrease in sex-biased expression and in preferential downregulation of the maternal alleles of a number of genes. Most promoters of dysregulated genes show concordant epigenetic changes including gain of H3K27me3 and loss of chromatin accessibility, but there was less concordance when considering allelic changes. CONCLUSIONS: Our study reveals new sex-related roles of KDM6A in the regulation of developmental genes, the maintenance of sex-biased gene expression, and the differential expression of parental alleles.
Assuntos
Histona Desmetilases , Histonas , Anormalidades Múltiplas , Alelos , Animais , Cromatina , Face/anormalidades , Feminino , Doenças Hematológicas , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Masculino , Camundongos , Doenças VestibularesRESUMO
Deletion of a subset of the D4Z4 macrosatellite repeats in the subtelomeric region of chromosome 4q causes facioscapulohumeral muscular dystrophy (FSHD) when occurring on a specific haplotype of 4qter (4qA161). Several genes have been examined as candidates for causing FSHD, including the DUX4 homeobox gene in the D4Z4 repeat, but none have been definitively shown to cause the disease, nor has the full extent of transcripts from the D4Z4 region been carefully characterized. Using strand-specific RT-PCR, we have identified several sense and antisense transcripts originating from the 4q D4Z4 units in wild-type and FSHD muscle cells. Consistent with prior reports, we find that the DUX4 transcript from the last (most telomeric) D4Z4 unit is polyadenylated and has two introns in its 3-prime untranslated region. In addition, we show that this transcript generates (i) small si/miRNA-sized fragments, (ii) uncapped, polyadenylated 3-prime fragments that encode the conserved C-terminal portion of DUX4 and (iii) capped and polyadenylated mRNAs that contain the double-homeobox domain of DUX4 but splice-out the C-terminal portion. Transfection studies demonstrate that translation initiation at an internal methionine can produce the C-terminal polypeptide and developmental studies show that this peptide inhibits myogenesis at a step between MyoD transcription and the activation of MyoD target genes. Together, we have identified new sense and anti-sense RNA transcripts, novel mRNAs and mi/siRNA-sized RNA fragments generated from the D4Z4 units that are new candidates for the pathophysiology of FSHD.
Assuntos
Processamento Alternativo , Proteínas de Homeodomínio/genética , Distrofia Muscular Facioescapuloumeral/genética , RNA não Traduzido/metabolismo , Sequências Repetitivas de Ácido Nucleico , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Desenvolvimento Muscular , Distrofia Muscular Facioescapuloumeral/metabolismo , Distrofia Muscular Facioescapuloumeral/fisiopatologia , Mioblastos/química , Mioblastos/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA não Traduzido/química , RNA não Traduzido/genética , Peixe-ZebraRESUMO
Cohesin is required to prevent premature dissociation of sister chromatids after DNA replication. Although its role in chromatid cohesion is well established, the functional significance of cohesin's association with interphase chromatin is not clear. Using a quantitative proteomics approach, we show that the STAG1 (Scc3/SA1) subunit of cohesin interacts with the CCTC-binding factor CTCF bound to the c-myc insulator element. Both allele-specific binding of CTCF and Scc3/SA1 at the imprinted IGF2/H19 gene locus and our analyses of human DM1 alleles containing base substitutions at CTCF-binding motifs indicate that cohesin recruitment to chromosomal sites depends on the presence of CTCF. A large-scale genomic survey using ChIP-Chip demonstrates that Scc3/SA1 binding strongly correlates with the CTCF-binding site distribution in chromosomal arms. However, some chromosomal sites interact exclusively with CTCF, whereas others interact with Scc3/SA1 only. Furthermore, immunofluorescence microscopy and ChIP-Chip experiments demonstrate that CTCF associates with both centromeres and chromosomal arms during metaphase. These results link cohesin to gene regulatory functions and suggest an essential role for CTCF during sister chromatid cohesion. These results have implications for the functional role of cohesin subunits in the pathogenesis of Cornelia de Lange syndrome and Roberts syndromes.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Células 3T3 , Alelos , Sequência de Aminoácidos , Animais , Fator de Ligação a CCCTC , Proteínas de Ciclo Celular/genética , Cromatina/genética , Imunoprecipitação da Cromatina , Proteínas Cromossômicas não Histona/genética , Cromossomos Humanos/metabolismo , Proteínas de Ligação a DNA/genética , Impressão Genômica , Genômica , Humanos , Elementos Isolantes , Fator de Crescimento Insulin-Like II/genética , Células Jurkat , Espectrometria de Massas , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteômica , Proteínas Repressoras/genética , CoesinasRESUMO
At least 25 inherited disorders in humans result from microsatellite repeat expansion. Dramatic variation in repeat instability occurs at different disease loci and between different tissues; however, cis-elements and trans-factors regulating the instability process remain undefined. Genomic fragments from the human spinocerebellar ataxia type 7 (SCA7) locus, containing a highly unstable CAG tract, were previously introduced into mice to localize cis-acting "instability elements," and revealed that genomic context is required for repeat instability. The critical instability-inducing region contained binding sites for CTCF -- a regulatory factor implicated in genomic imprinting, chromatin remodeling, and DNA conformation change. To evaluate the role of CTCF in repeat instability, we derived transgenic mice carrying SCA7 genomic fragments with CTCF binding-site mutations. We found that CTCF binding-site mutation promotes triplet repeat instability both in the germ line and in somatic tissues, and that CpG methylation of CTCF binding sites can further destabilize triplet repeat expansions. As CTCF binding sites are associated with a number of highly unstable repeat loci, our findings suggest a novel basis for demarcation and regulation of mutational hot spots and implicate CTCF in the modulation of genetic repeat instability.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Instabilidade Genômica , Mutação , Sequências Reguladoras de Ácido Nucleico , Proteínas Repressoras/metabolismo , Ataxias Espinocerebelares/genética , Expansão das Repetições de Trinucleotídeos , Animais , Ataxina-7 , Sítios de Ligação , Fator de Ligação a CCCTC , Metilação de DNA , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Repressoras/genéticaRESUMO
Escape from X inactivation results in expression of genes embedded within inactive chromatin, suggesting the existence of boundary elements between domains. We report that the 5' end of Jarid1c, a mouse escape gene adjacent to an inactivated gene, binds CTCF, displays high levels of histone H3 acetylation, and functions as a CTCF-dependent chromatin insulator. CpG island methylation at Jarid1c was very low during development and virtually absent at the CTCF sites, signifying that CTCF may influence DNA methylation and chromatin modifications. CTCF binding sites were also present at the 5' end of two other escape genes, mouse Eif2s3x and human EIF2S3, each adjacent to an inactivated gene, but not at genes embedded within large escape domains. Thus, CTCF was specifically bound to transition regions, suggesting a role in maintaining both X inactivation and escape domains. Furthermore, the evolution of X chromosome domains appears to be associated with repositioning of chromatin boundary elements.
Assuntos
Cromatina/metabolismo , Ilhas de CpG/fisiologia , Proteínas de Ligação a DNA/metabolismo , Mecanismo Genético de Compensação de Dose , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas Repressoras/metabolismo , Acetilação , Animais , Sítios de Ligação , Fator de Ligação a CCCTC , Células Cultivadas , Metilação de DNA , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Embrião de Mamíferos , Fator de Iniciação 2 em Eucariotos/metabolismo , Histona Desmetilases , Humanos , Imunoprecipitação/métodos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese/fisiologia , Oxirredutases N-Desmetilantes , Proteínas/metabolismo , Cromossomo XRESUMO
Firre encodes a lncRNA involved in nuclear organization. Here, we show that Firre RNA expressed from the active X chromosome maintains histone H3K27me3 enrichment on the inactive X chromosome (Xi) in somatic cells. This trans-acting effect involves SUZ12, reflecting interactions between Firre RNA and components of the Polycomb repressive complexes. Without Firre RNA, H3K27me3 decreases on the Xi and the Xi-perinucleolar location is disrupted, possibly due to decreased CTCF binding on the Xi. We also observe widespread gene dysregulation, but not on the Xi. These effects are measurably rescued by ectopic expression of mouse or human Firre/FIRRE transgenes, supporting conserved trans-acting roles. We also find that the compact 3D structure of the Xi partly depends on the Firre locus and its RNA. In common lymphoid progenitors and T-cells Firre exerts a cis-acting effect on maintenance of H3K27me3 in a 26 Mb region around the locus, demonstrating cell type-specific trans- and cis-acting roles of this lncRNA.
Assuntos
Epigênese Genética , RNA Longo não Codificante/genética , Inativação do Cromossomo X/genética , Alelos , Animais , Sequência de Bases , Linhagem Celular , Núcleo Celular/genética , Cromatina/metabolismo , DNA Complementar/genética , Feminino , Deleção de Genes , Ontologia Genética , Loci Gênicos , Genoma , Histonas/metabolismo , Lisina/metabolismo , Masculino , Metilação , Camundongos Endogâmicos C57BL , Complexo Repressor Polycomb 2/metabolismo , RNA Longo não Codificante/metabolismo , Transgenes , Regulação para Cima/genética , Cromossomo X/genéticaRESUMO
BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is in most cases caused by a contraction of the D4Z4 macrosatellite repeat on chromosome 4 (FSHD1) or by mutations in the SMCHD1 or DNMT3B gene (FSHD2). Both situations result in the incomplete epigenetic repression of the D4Z4-encoded retrogene DUX4 in somatic cells, leading to the aberrant expression of DUX4 in the skeletal muscle. In mice, Smchd1 regulates chromatin repression at different loci, having a role in CpG methylation establishment and/or maintenance. METHODS: To investigate the global effects of harboring heterozygous SMCHD1 mutations on DNA methylation in humans, we combined 450k methylation analysis on mononuclear monocytes from female heterozygous SMCHD1 mutation carriers and unaffected controls with reduced representation bisulfite sequencing (RRBS) on FSHD2 and control myoblast cell lines. Candidate loci were then evaluated for SMCHD1 binding using ChIP-qPCR and expression was evaluated using RT-qPCR. RESULTS: We identified a limited number of clustered autosomal loci with CpG hypomethylation in SMCHD1 mutation carriers: the protocadherin (PCDH) cluster on chromosome 5, the transfer RNA (tRNA) and 5S rRNA clusters on chromosome 1, the HOXB and HOXD clusters on chromosomes 17 and 2, respectively, and the D4Z4 repeats on chromosomes 4 and 10. Furthermore, minor increases in RNA expression were seen in FSHD2 myoblasts for some of the PCDHß cluster isoforms, tRNA isoforms, and a HOXB isoform in comparison to controls, in addition to the previously reported effects on DUX4 expression. SMCHD1 was bound at DNAseI hypersensitivity sites known to regulate the PCDHß cluster and at the chromosome 1 tRNA cluster, with decreased binding in SMCHD1 mutation carriers at the PCDHß cluster sites. CONCLUSIONS: Our study is the first to investigate the global methylation effects in humans resulting from heterozygous mutations in SMCHD1. Our results suggest that SMCHD1 acts as a repressor on a limited set of autosomal gene clusters, as an observed reduction in methylation associates with a loss of SMCHD1 binding and increased expression for some of the loci.
Assuntos
Proteínas Cromossômicas não Histona/genética , Metilação de DNA , Loci Gênicos , Distrofia Muscular Facioescapuloumeral/genética , Células Cultivadas , Proteínas Cromossômicas não Histona/metabolismo , Ilhas de CpG , Feminino , Heterozigoto , Humanos , Família Multigênica , Distrofia Muscular Facioescapuloumeral/metabolismo , Mutação , Mioblastos/metabolismo , Ligação ProteicaRESUMO
CTCF is a widely expressed 11-zinc finger (ZF) transcription factor that is involved in different aspects of gene regulation including promoter activation or repression, hormone-responsive gene silencing, methylation-dependent chromatin insulation, and genomic imprinting. Because CTCF targets include oncogenes and tumor suppressor genes, we screened over 100 human tumor samples for mutations that might disrupt CTCF activity. We did not observe any CTCF mutations leading to truncations/premature stops. Rather, in breast, prostate, and Wilms' tumors, we observed four different CTCF somatic missense mutations involving amino acids within the ZF domain. Each ZF mutation abrogated CTCF binding to a subset of target sites within the promoters/insulators of certain genes involved in regulating cell proliferation but did not alter binding to the regulatory sequences of other genes. These observations suggest that CTCF may represent a novel tumor suppressor gene that displays tumor-specific "change of function" rather than complete "loss of function."
Assuntos
DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Mutação de Sentido Incorreto , Proteínas Repressoras , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Dedos de Zinco/genética , Sequência de Aminoácidos , Sequência de Bases , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Fator de Ligação a CCCTC , Proteínas de Ciclo Celular/genética , Feminino , Genes Supressores de Tumor , Globinas/genética , Humanos , Masculino , Dados de Sequência Molecular , Muramidase/genética , Regiões Promotoras Genéticas , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Conformação Proteica , Especificidade por Substrato , Tumor de Wilms/genética , Tumor de Wilms/metabolismoRESUMO
BACKGROUND: In mammals, X chromosome genes are present in one copy in males and two in females. To balance the dosage of X-linked gene expression between the sexes, one of the X chromosomes in females is silenced. X inactivation is initiated by upregulation of the lncRNA (long non-coding RNA) Xist and recruitment of specific chromatin modifiers. The inactivated X chromosome becomes heterochromatic and visits a specific nuclear compartment adjacent to the nucleolus. RESULTS: Here, we show a novel role for the lncRNA Firre in anchoring the inactive mouse X chromosome and preserving one of its main epigenetic features, H3K27me3. Similar to Dxz4, Firre is X-linked and expressed from a macrosatellite repeat locus associated with a cluster of CTCF and cohesin binding sites, and is preferentially located adjacent to the nucleolus. CTCF binding present initially in both male and female mouse embryonic stem cells is lost from the active X during development. Knockdown of Firre disrupts perinucleolar targeting and H3K27me3 levels in mouse fibroblasts, demonstrating a role in maintenance of an important epigenetic feature of the inactive X chromosome. No X-linked gene reactivation is seen after Firre knockdown; however, a compensatory increase in the expression of chromatin modifier genes implicated in X silencing is observed. Further experiments in female embryonic stem cells suggest that Firre does not play a role in X inactivation onset. CONCLUSIONS: The X-linked lncRNA Firre helps to position the inactive X chromosome near the nucleolus and to preserve one of its main epigenetic features.
Assuntos
Histona Desmetilases com o Domínio Jumonji/genética , RNA Longo não Codificante/genética , Proteínas Repressoras/genética , Inativação do Cromossomo X/genética , Animais , Sítios de Ligação , Fator de Ligação a CCCTC , Metilação de DNA/genética , Feminino , Masculino , Camundongos , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Cromossomo X/genéticaRESUMO
Epigenetic alterations, particularly in DNA methylation, are ubiquitous in cancer, yet the molecular origins and the consequences of these alterations are poorly understood. CTCF, a DNA-binding protein that regulates higher-order chromatin organization, is frequently altered by hemizygous deletion or mutation in human cancer. To date, a causal role for CTCF in cancer has not been established. Here, we show that Ctcf hemizygous knockout mice are markedly susceptible to spontaneous, radiation-, and chemically induced cancer in a broad range of tissues. Ctcf(+/-) tumors are characterized by increased aggressiveness, including invasion, metastatic dissemination, and mixed epithelial/mesenchymal differentiation. Molecular analysis of Ctcf(+/-) tumors indicates that Ctcf is haploinsufficient for tumor suppression. Tissues with hemizygous loss of CTCF exhibit increased variability in CpG methylation genome wide. These findings establish CTCF as a prominent tumor-suppressor gene and point to CTCF-mediated epigenetic stability as a major barrier to neoplastic progression.
Assuntos
Metilação de DNA , Genes Supressores de Tumor , Neoplasias/genética , Proteínas Repressoras/genética , Animais , Fator de Ligação a CCCTC , Linhagem Celular Tumoral , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Haploinsuficiência , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Neoplasias/metabolismo , Ligação Proteica , Proteínas Repressoras/metabolismo , Análise de SobrevidaRESUMO
Facioscapulohumeral Disease (FSHD) is a dominantly inherited progressive myopathy associated with aberrant production of the transcription factor, Double Homeobox Protein 4 (DUX4). The expression of DUX4 depends on an open chromatin conformation of the D4Z4 macrosatellite array and a specific haplotype on chromosome 4. Even when these requirements are met, DUX4 transcripts and protein are only detectable in a subset of cells indicating that additional constraints govern DUX4 production. Since the direction of transcription, along with the production of non-coding antisense transcripts is an important regulatory feature of other macrosatellite repeats, we developed constructs that contain the non-coding region of a single D4Z4 unit flanked by genes that report transcriptional activity in the sense and antisense directions. We found that D4Z4 contains two promoters that initiate sense and antisense transcription within the array, and that antisense transcription predominates. Transcriptional start sites for the antisense transcripts, as well as D4Z4 regions that regulate the balance of sense and antisense transcripts were identified. We show that the choice of transcriptional direction is reversible but not mutually exclusive, since sense and antisense reporter activity was often present in the same cell and simultaneously upregulated during myotube formation. Similarly, levels of endogenous sense and antisense D4Z4 transcripts were upregulated in FSHD myotubes. These studies offer insight into the autonomous distribution of muscle weakness that is characteristic of FSHD.
Assuntos
Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Distrofia Muscular Facioescapuloumeral/genética , Transcrição Gênica , Animais , Sequência de Bases , Sítios de Ligação/genética , Células Cultivadas , Cromatina/genética , Cromatina/metabolismo , Células-Tronco Embrionárias/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Haplótipos , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Repetições de Microssatélites , Dados de Sequência Molecular , Família Multigênica , Fibras Musculares Esqueléticas/metabolismo , Mutagênese Sítio-Dirigida , Mioblastos Esqueléticos/metabolismo , Regiões Promotoras Genéticas , RNA Antissenso/genética , RNA Antissenso/metabolismo , Sítio de Iniciação de TranscriçãoRESUMO
CTCF is a highly conserved, multifunctional zinc finger protein involved in critical aspects of gene regulation including transcription regulation, chromatin insulation, genomic imprinting, X-chromosome inactivation, and higher order chromatin organization. Such multifunctional properties of CTCF suggest an essential role in development. Indeed, a previous report on maternal depletion of CTCF suggested that CTCF is essential for pre-implantation development. To distinguish between the effects of maternal and zygotic expression of CTCF, we studied pre-implantation development in mice harboring a complete loss of function Ctcf knockout allele. Although we demonstrated that homozygous deletion of Ctcf is early embryonically lethal, in contrast to previous observations, we showed that the Ctcf nullizygous embryos developed up to the blastocyst stage (E3.5) followed by peri-implantation lethality (E4.5-E5.5). Moreover, one-cell stage Ctcf nullizygous embryos cultured ex vivo developed to the 16-32 cell stage with no obvious abnormalities. Using a single embryo assay that allowed both genotype and mRNA expression analyses of the same embryo, we demonstrated that pre-implantation development of the Ctcf nullizygous embryos was associated with the retention of the maternal wild type Ctcf mRNA. Loss of this stable maternal transcript was temporally associated with loss of CTCF protein expression, apoptosis of the developing embryo, and failure to further develop an inner cell mass and trophoectoderm ex vivo. This indicates that CTCF expression is critical to early embryogenesis and loss of its expression rapidly leads to apoptosis at a very early developmental stage. This is the first study documenting the presence of the stable maternal Ctcf transcript in the blastocyst stage embryos. Furthermore, in the presence of maternal CTCF, zygotic CTCF expression does not seem to be required for pre-implantation development.