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Biologically precise enhancer licensing by lineage-determining transcription factors enables activation of transcripts appropriate to biological demand and prevents deleterious gene activation. This essential process is challenged by the millions of matches to most transcription factor binding motifs present in many eukaryotic genomes, leading to questions about how transcription factors achieve the exquisite specificity required. The importance of chromatin remodeling factors to enhancer activation is highlighted by their frequent mutation in developmental disorders and in cancer. Here, we determine the roles of CHD4 in enhancer licensing and maintenance in breast cancer cells and during cellular reprogramming. In unchallenged basal breast cancer cells, CHD4 modulates chromatin accessibility. Its depletion leads to redistribution of transcription factors to previously unoccupied sites. During cellular reprogramming induced by the pioneer factor GATA3, CHD4 activity is necessary to prevent inappropriate chromatin opening. Mechanistically, CHD4 promotes nucleosome positioning over GATA3 binding motifs to compete with transcription factor-DNA interaction. We propose that CHD4 acts as a chromatin proof-reading enzyme that prevents unnecessary gene expression by editing chromatin binding activities of transcription factors.
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Cromatina , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2 , Femenino , Humanos , Sitios de Unión , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Reprogramación Celular/genética , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Elementos de Facilitación Genéticos , Factor de Transcripción GATA3/metabolismo , Factor de Transcripción GATA3/genética , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , Nucleosomas/metabolismo , Nucleosomas/genética , Unión Proteica , Factores de Transcripción/metabolismoRESUMEN
Wolffian duct (WD) maintenance and differentiation is predominantly driven by the androgen action, which is mediated by the androgen receptor (AR). It is well established that the mesenchyme indicates the fate and differentiation of epithelial cells. However, in vivo developmental requirement of mesenchymal AR in WD development is still undefined. By designing a mesenchyme-specific Ar knockout (ARcKO), we discovered that the loss of mesenchymal Ar led to the bilateral or unilateral degeneration of caudal WDs and cystic formation at the cranial WDs. Ex vivo culture of ARcKO WDs invariably resulted in bilateral defects, suggesting that some factor(s) originating from surrounding tissues in vivo might promote WD survival and growth even in the absence of mesenchymal Ar. Mechanistically, we found cell proliferation was significantly reduced in both epithelial and mesenchymal compartments; but cell apoptosis was not affected. Transcriptomic analysis by RNA sequencing of E14.5 mesonephroi revealed 131 differentially expressed genes. Multiple downregulated genes (Top2a, Wnt9b, Lama2, and Lamc2) were associated with morphological and cellular changes in ARcKO male embryos (ie, reduced cell proliferation and decreased number of epithelial cells). Mesenchymal differentiation into smooth muscle cells that are critical for morphogenesis was also impaired in ARcKO male embryos. Taken together, our results demonstrate the crucial roles of the mesenchymal AR in WD maintenance and morphogenesis in mice.
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Mesodermo , Receptores Androgénicos , Conductos Mesonéfricos , Receptores Androgénicos/metabolismo , Mesodermo/metabolismo , Conductos Mesonéfricos/crecimiento & desarrollo , Conductos Mesonéfricos/metabolismo , Animales , Ratones , Morfogénesis , Masculino , Femenino , Técnicas de CultivoRESUMEN
Breast cancer continues to be a major cause of death among women. The GATA3 gene is often overexpressed in breast cancer and is widely used to support a diagnosis. However, lower expression of GATA3 has been linked to poorer prognosis along with frequent gene mutations. Therefore, the role of GATA3 in breast cancer appears to be context specific. This study aims to identify a new downstream target of GATA3 to better understand its regulatory network. Clinical data analysis identified the prolyl 4-hydroxylase transmembrane protein (P4HTM) as one of the most highly co-expressed genes with GATA3. Immunohistochemical staining of breast tumors confirms co-expression between GATA3 and P4HTM at the protein level. Similar to GATA3, P4HTM expression levels are linked to patient prognosis, with lower levels indicating poorer survival. Genomics data found that GATA3 binds to the P4HTM locus, and that ectopic expression of GATA3 in basal breast cancer cells increases the P4HTM transcript level. These results collectively suggest that P4HTM is a novel downstream target of GATA3 in breast cancer and is involved in tumor progression.
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Biologically precise enhancer licensing by lineage-determining transcription factors enables activation of transcripts appropriate to biological demand and prevents deleterious gene activation. This essential process is challenged by the millions of matches to most transcription factor binding motifs present in many eukaryotic genomes, leading to questions about how transcription factors achieve the exquisite specificity required. The importance of chromatin remodeling factors to enhancer activation is highlighted by their frequent mutation in developmental disorders and in cancer. Here we determine the roles of CHD4 to enhancer licensing and maintenance in breast cancer cells and during cellular reprogramming. In unchallenged basal breast cancer cells, CHD4 modulates chromatin accessibility at transcription factor binding sites; its depletion leads to altered motif scanning and redistribution of transcription factors to sites not previously occupied. During GATA3-mediated cellular reprogramming, CHD4 activity is necessary to prevent inappropriate chromatin opening and enhancer licensing. Mechanistically, CHD4 competes with transcription factor-DNA interaction by promoting nucleosome positioning over binding motifs. We propose that CHD4 acts as a chromatin proof-reading enzyme that prevents inappropriate gene expression by editing binding site selection by transcription factors.
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The assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) probes deoxyribonucleic acid (DNA) accessibility using the hyperactive Tn5 transposase. Tn5 cuts and ligates adapters for high-throughput sequencing within accessible chromatin regions. In eukaryotic cells, genomic DNA is packaged into chromatin, a complex of DNA, histones, and other proteins, which acts as a physical barrier to the transcriptional machinery. In response to extrinsic signals, transcription factors recruit chromatin remodeling complexes to enable access to the transcriptional machinery for gene activation. Therefore, identifying open chromatin regions is useful when monitoring enhancer and gene promoter activities during biological events such as cancer progression. Since this protocol is easy to use and has a low cell input requirement, ATAC-seq has been widely adopted to define open chromatin regions in various cell types, including cancer cells. For successful data acquisition, several parameters need to be considered when preparing ATAC-seq libraries. Among them, the choice of cell lysis buffer, the titration of the Tn5 enzyme, and the starting volume of cells are crucial for ATAC-seq library preparation in cancer cells. Optimization is essential for generating high-quality data. Here, we provide a detailed description of the ATAC-seq optimization methods for epithelial cell types.
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Secuenciación de Inmunoprecipitación de Cromatina , Neoplasias , Cromatina/genética , ADN/genética , Epigénesis Genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Neoplasias/genética , Análisis de Secuencia de ADN/métodos , Factores de Transcripción/metabolismoRESUMEN
GATA3 is known to be one of the most frequently mutated genes in breast cancer. More than 10% of breast tumors carry mutations in this gene. However, the functional consequence of GATA3 mutations is still largely unknown. Clinical data suggest that different types of GATA3 mutations may have distinct roles in breast cancer characterization. In this study, we have established three luminal breast cancer cell lines that stably express different truncation mutants (X308 splice site deletion, C321 frameshift, and A333 frameshift mutants) found in breast cancer patients. Transcriptome analysis identified common and distinct gene expression patterns in these GATA3 mutant cell lines. In particular, the impacts on epithelial-to-mesenchymal transition (EMT) related genes are similar across these mutant cell lines. Chromatin localization of the mutants is highly overlapped and exhibits non-canonical motif enrichment. Interestingly, the A333 frameshift mutant expressed cells displayed the most significant impact on the GATA3 binding compared to X308 splice site deletion and C321fs mutants expressed cells. Our results suggest the common and different roles of GATA3 truncation mutations during luminal breast cancer development.
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Leptomeningeal carcinomatosis (LC) occurs when tumor cells spread to the cerebrospinal fluid-containing leptomeninges surrounding the brain and spinal cord. LC is an ominous complication of cancer with a dire prognosis. Although any malignancy can spread to the leptomeninges, breast cancer, particularly the HER2+ subtype, is its most common origin. HER2+ breast LC (HER2+ LC) remains incurable, with few treatment options, and the molecular mechanisms underlying proliferation of HER2+ breast cancer cells in the acellular, protein, and cytokine-poor leptomeningeal environment remain elusive. Therefore, we sought to characterize signaling pathways that drive HER2+ LC development as well as those that restrict its growth to leptomeninges. Primary HER2+ LC patient-derived ("Lepto") cell lines in coculture with various central nervous system (CNS) cell types revealed that oligodendrocyte progenitor cells (OPC), the largest population of dividing cells in the CNS, inhibited HER2+ LC growth in vitro and in vivo, thereby limiting the spread of HER2+ LC beyond the leptomeninges. Cytokine array-based analyses identified Lepto cell-secreted GMCSF as an oncogenic autocrine driver of HER2+ LC growth. LC/MS-MS-based analyses revealed that the OPC-derived protein TPP1 proteolytically degrades GMCSF, decreasing GMCSF signaling and leading to suppression of HER2+ LC growth and limiting its spread. Finally, intrathecal delivery of neutralizing anti-GMCSF antibodies and a pan-Aurora kinase inhibitor (CCT137690) synergistically inhibited GMCSF and suppressed activity of GMCSF effectors, reducing HER2+ LC growth in vivo. Thus, OPC suppress GMCSF-driven growth of HER2+ LC in the leptomeningeal environment, providing a potential targetable axis. SIGNIFICANCE: This study characterizes molecular mechanisms that drive HER2+ leptomeningeal carcinomatosis and demonstrates the efficacy of anti-GMCSF antibodies and pan-Aurora kinase inhibitors against this disease.
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Comunicación Autocrina , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Carcinomatosis Meníngea/secundario , Transducción de Señal , Animales , Línea Celular Tumoral , Proliferación Celular/genética , Supervivencia Celular , Modelos Animales de Enfermedad , Expresión Génica , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Humanos , Carcinomatosis Meníngea/diagnóstico , Ratones , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Receptor ErbB-2/metabolismo , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
During cellular reprogramming, the pioneer transcription factor GATA3 binds chromatin, and in a context-dependent manner directs local chromatin remodeling and enhancer formation. Here, we use high-resolution nucleosome mapping in human cells to explore the impact of the position of GATA motifs on the surface of nucleosomes on productive enhancer formation, finding productivity correlates with binding sites located near the nucleosomal dyad axis. Biochemical experiments with model nucleosomes demonstrate sufficiently stable transcription factor-nucleosome interaction to empower cryo-electron microscopy structure determination of the complex at 3.15 Å resolution. The GATA3 zinc fingers efficiently bind their target 5'-GAT-3' sequences in the nucleosome when they are located in solvent accessible, consecutive major grooves without significant changes in nucleosome structure. Analysis of genomic loci bound by GATA3 during reprogramming suggests a correlation of recognition motif sequence and spacing that may distinguish productivity of new enhancer formation.
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Ensamble y Desensamble de Cromatina/genética , Factor de Transcripción GATA3/química , Nucleosomas/química , Nucleosomas/genética , Secuencias de Aminoácidos/genética , Sitios de Unión , Secuenciación de Inmunoprecipitación de Cromatina , Microscopía por Crioelectrón , Elementos de Facilitación Genéticos , Factor de Transcripción GATA3/genética , Factor de Transcripción GATA3/metabolismo , Factor de Transcripción GATA3/ultraestructura , Histonas/metabolismo , Humanos , Nucleosomas/metabolismo , Nucleosomas/ultraestructura , Unión Proteica , Dedos de Zinc/genéticaRESUMEN
Estrogen receptors (ER) are activated by the steroid hormone 17ß-estradiol. Estrogen receptor alpha (ER-α) forms a regulatory network in mammary epithelial cells and in breast cancer with the transcription factors FOXA1 and GATA3. GATA3 is one of the most frequently mutated genes in breast cancer and is capable of specifying chromatin localization of FOXA1 and ER-α. How GATA3 mutations found in breast cancer impact genomic localization of ER-α and the transcriptional network downstream of ER-α and FOXA1 remains unclear. Here, we investigate the function of a recurrent patient-derived GATA3 mutation (R330fs) on this regulatory network. Genomic analysis indicates that the R330fs mutant can disrupt localization of ER-α and FOXA1. Loci co-bound by all three factors are enriched for genes integral to mammary gland development as well as epithelial cell biology. This gene set is differentially regulated in GATA3 mutant cells in culture and in tumors bearing similar mutations in vivo. The altered distribution of ER-α and FOXA1 in GATA3-mutant cells is associated with altered chromatin architecture, which leads to differential gene expression. These results suggest an active role for GATA3 zinc finger 2 mutants in ER-α positive breast tumors.
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Neoplasias de la Mama/genética , Receptor alfa de Estrógeno/metabolismo , Factor de Transcripción GATA3/genética , Factor de Transcripción GATA3/metabolismo , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Cromatina/metabolismo , Femenino , Humanos , Mutación , Transcripción GenéticaRESUMEN
The aim of the present study was to understand the biology of unintegrated HIV-1 DNA and reveal the mechanisms involved in its transcriptional silencing. We found that histones are loaded on HIV-1 DNA after its nuclear import and before its integration in the host genome. Nucleosome positioning analysis along the unintegrated and integrated viral genomes revealed major differences in nucleosome density and position. Indeed, in addition to the well-known nucleosomes Nuc0, Nuc1, and Nuc2 loaded on integrated HIV-1 DNA, we also found NucDHS, a nucleosome that covers the DNase hypersensitive site, in unintegrated viral DNA. In addition, unintegrated viral DNA-associated Nuc0 and Nuc2 were positioned slightly more to the 5' end relative to their position in integrated DNA. The presence of NucDHS in the proximal region of the long terminal repeat (LTR) promoter was associated with the absence of RNAPII and of the active histone marks H3K4me3 and H3ac at the LTR. Conversely, analysis of integrated HIV-1 DNA showed a loss of NucDHS, loading of RNAPII, and enrichment in active histone marks within the LTR. We propose that unintegrated HIV-1 DNA adopts a repressive chromatin structure that competes with the transcription machinery, leading to its silencing.
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Ensamble y Desensamble de Cromatina , ADN Viral/genética , Infecciones por VIH/genética , VIH-1/genética , Histonas/genética , Nucleosomas/genética , Integración Viral/genética , Regulación Viral de la Expresión Génica , Genoma Viral , Infecciones por VIH/virología , Humanos , Secuencias Repetidas Terminales , Transcripción GenéticaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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PURPOSE: Sifrim-Hitz-Weiss syndrome (SIHIWES) is a recently described multisystemic neurodevelopmental disorder caused by de novo variants inCHD4. In this study, we investigated the clinical spectrum of the disorder, genotype-phenotype correlations, and the effect of different missense variants on CHD4 function. METHODS: We collected clinical and molecular data from 32 individuals with mostly de novo variants in CHD4, identified through next-generation sequencing. We performed adenosine triphosphate (ATP) hydrolysis and nucleosome remodeling assays on variants from five different CHD4 domains. RESULTS: The majority of participants had global developmental delay, mild to moderate intellectual disability, brain anomalies, congenital heart defects, and dysmorphic features. Macrocephaly was a frequent but not universal finding. Additional common abnormalities included hypogonadism in males, skeletal and limb anomalies, hearing impairment, and ophthalmic abnormalities. The majority of variants were nontruncating and affected the SNF2-like region of the protein. We did not identify genotype-phenotype correlations based on the type or location of variants. Alterations in ATP hydrolysis and chromatin remodeling activities were observed in variants from different domains. CONCLUSION: The CHD4-related syndrome is a multisystemic neurodevelopmental disorder. Missense substitutions in different protein domains alter CHD4 function in a variant-specific manner, but result in a similar phenotype in humans.
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Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , Trastornos del Neurodesarrollo/genética , Anomalías Múltiples/genética , Adolescente , Adulto , Niño , Preescolar , Ensamble y Desensamble de Cromatina/genética , Discapacidades del Desarrollo/genética , Femenino , Estudios de Asociación Genética , Genotipo , Pérdida Auditiva/genética , Cardiopatías Congénitas/genética , Humanos , Lactante , Recién Nacido , Discapacidad Intelectual/genética , Masculino , Megalencefalia/genética , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Anomalías Musculoesqueléticas/genética , Mutación Missense/genética , Fenotipo , Síndrome , Factores de Transcripción/genéticaRESUMEN
The HTML and PDF versions of this Article were updated after publication to remove images of one individual from Figure 1.
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The original version of this Article contained an error in the spelling of the author Laurence Faivre, which was incorrectly given as Laurence Faive. This has now been corrected in both the PDF and HTML versions of the Article.
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DNA methylation is an essential epigenetic process in mammals, intimately involved in gene regulation. Here we address the extent to which genetics, sex, and pregnancy influence genomic DNA methylation by intercrossing 2 inbred mouse strains, C57BL/6N and C3H/HeN, and analyzing DNA methylation in parents and offspring using whole-genome bisulfite sequencing. Differential methylation across genotype is detected at thousands of loci and is preserved on parental alleles in offspring. In comparison of autosomal DNA methylation patterns across sex, hundreds of differentially methylated regions are detected. Comparison of animals with different histories of pregnancy within our study reveals a CpG methylation pattern that is restricted to female animals that had borne offspring. Collectively, our results demonstrate the stability of CpG methylation across generations, clarify the interplay of epigenetics with genetics and sex, and suggest that CpG methylation may serve as an epigenetic record of life events in somatic tissues at loci whose expression is linked to the relevant biology.
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Metilación de ADN/genética , Epigénesis Genética , Preñez/genética , Animales , Islas de CpG , Metilación de ADN/fisiología , Femenino , Masculino , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Embarazo , Preñez/fisiología , Factores Sexuales , Especificidad de la Especie , Secuenciación Completa del GenomaRESUMEN
Chromatin remodeling is of crucial importance during brain development. Pathogenic alterations of several chromatin remodeling ATPases have been implicated in neurodevelopmental disorders. We describe an index case with a de novo missense mutation in CHD3, identified during whole genome sequencing of a cohort of children with rare speech disorders. To gain a comprehensive view of features associated with disruption of this gene, we use a genotype-driven approach, collecting and characterizing 35 individuals with de novo CHD3 mutations and overlapping phenotypes. Most mutations cluster within the ATPase/helicase domain of the encoded protein. Modeling their impact on the three-dimensional structure demonstrates disturbance of critical binding and interaction motifs. Experimental assays with six of the identified mutations show that a subset directly affects ATPase activity, and all but one yield alterations in chromatin remodeling. We implicate de novo CHD3 mutations in a syndrome characterized by intellectual disability, macrocephaly, and impaired speech and language.
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ADN Helicasas/genética , Discapacidades del Desarrollo/genética , Trastornos del Lenguaje/genética , Megalencefalia/genética , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , Mutación Missense , Trastornos del Neurodesarrollo/genética , Dominios Proteicos/genética , Trastornos del Habla/genética , Adenosina Trifosfatasas , Preescolar , Ensamble y Desensamble de Cromatina , Femenino , Expresión Génica , Genotipo , Células HEK293 , Humanos , Discapacidad Intelectual/genética , Masculino , Modelos Moleculares , Fenotipo , Secuenciación Completa del GenomaRESUMEN
GATA3 is frequently mutated in breast cancer; these mutations are widely presumed to be loss-of function despite a dearth of information regarding their effect on disease course or their mechanistic impact on the breast cancer transcriptional network. Here, we address molecular and clinical features associated with GATA3 mutations. A novel classification scheme defines distinct clinical features for patients bearing breast tumors with mutations in the second GATA3 zinc-finger (ZnFn2). An engineered ZnFn2 mutant cell line by CRISPR-Cas9 reveals that mutation of one allele of the GATA3 second zinc finger (ZnFn2) leads to loss of binding and decreased expression at a subset of genes, including Progesterone Receptor. At other loci, associated with epithelial to mesenchymal transition, gain of binding correlates with increased gene expression. These results demonstrate that not all GATA3 mutations are equivalent and that ZnFn2 mutations impact breast cancer through gain and loss-of function.
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Neoplasias de la Mama/genética , Factor de Transcripción GATA3/genética , Animales , Neoplasias de la Mama/metabolismo , Reprogramación Celular , Femenino , Mutación del Sistema de Lectura , Factor de Transcripción GATA3/metabolismo , Edición Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , Neoplasias Mamarias Experimentales , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de Neoplasias , Receptores de Progesterona/metabolismo , Dedos de Zinc/genéticaRESUMEN
T helper 17 (TH17) cells are critically involved in host defence, inflammation, and autoimmunity. Transforming growth factor ß (TGFß) is instrumental in TH17 cell differentiation by cooperating with interleukin-6 (refs 6, 7). Yet, the mechanism by which TGFß enables TH17 cell differentiation remains elusive. Here we reveal that TGFß enables TH17 cell differentiation by reversing SKI-SMAD4-mediated suppression of the expression of the retinoic acid receptor (RAR)-related orphan receptor γt (RORγt). We found that, unlike wild-type T cells, SMAD4-deficient T cells differentiate into TH17 cells in the absence of TGFß signalling in a RORγt-dependent manner. Ectopic SMAD4 expression suppresses RORγt expression and TH17 cell differentiation of SMAD4-deficient T cells. However, TGFß neutralizes SMAD4-mediated suppression without affecting SMAD4 binding to the Rorc locus. Proteomic analysis revealed that SMAD4 interacts with SKI, a transcriptional repressor that is degraded upon TGFß stimulation. SKI controls histone acetylation and deacetylation of the Rorc locus and TH17 cell differentiation via SMAD4: ectopic SKI expression inhibits H3K9 acetylation of the Rorc locus, Rorc expression, and TH17 cell differentiation in a SMAD4-dependent manner. Therefore, TGFß-induced disruption of SKI reverses SKI-SMAD4-mediated suppression of RORγt to enable TH17 cell differentiation. This study reveals a critical mechanism by which TGFß controls TH17 cell differentiation and uncovers the SKI-SMAD4 axis as a potential therapeutic target for treating TH17-related diseases.
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Diferenciación Celular , Proteínas de Unión al ADN/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Proteína Smad4/metabolismo , Células Th17/citología , Células Th17/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Diferenciación Celular/genética , Femenino , Eliminación de Gen , Humanos , Interleucina-6/metabolismo , Masculino , Ratones , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/deficiencia , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Proteína Smad4/deficiencia , Proteína Smad4/genéticaRESUMEN
Transposable elements, including endogenous retroviruses (ERVs), constitute a large fraction of the mammalian genome. They are transcriptionally silenced during early development to protect genome integrity and aberrant transcription. However, the mechanisms that control their repression are not fully understood. To systematically study ERV repression, we carried out an RNAi screen in mouse embryonic stem cells (ESCs) and identified a list of novel regulators. Among them, Rif1 displays the strongest effect. Rif1 depletion by RNAi or gene deletion led to increased transcription and increased chromatin accessibility at ERV regions and their neighboring genes. This transcriptional de-repression becomes more severe when DNA methylation is lost. On the mechanistic level, Rif1 directly occupies ERVs and is required for repressive histone mark H3K9me3 and H3K27me3 assembly and DNA methylation. It interacts with histone methyltransferases and facilitates their recruitment to ERV regions. Importantly, Rif1 represses ERVs in human ESCs as well, and the evolutionally-conserved HEAT-like domain is essential for its function. Finally, Rif1 acts as a barrier during somatic cell reprogramming, and its depletion significantly enhances reprogramming efficiency. Together, our study uncovered many previously uncharacterized repressors of ERVs, and defined an essential role of Rif1 in the epigenetic defense against ERV activation.
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Cromatina/genética , Retrovirus Endógenos/genética , Proteínas de Unión a Telómeros/genética , Activación Viral , Animales , Línea Celular , Células Cultivadas , Cromatina/metabolismo , Metilación de ADN , Células Madre Embrionarias/metabolismo , Retrovirus Endógenos/fisiología , Células HEK293 , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Humanos , Metilación , Ratones , Interferencia de ARN , Proteínas de Unión a Telómeros/metabolismoRESUMEN
In eukaryotes, genomic DNA is compacted as chromatin, in which histones and DNA form the nucleosome as the basic unit. DMC1 and RAD51 are essential eukaryotic recombinases that mediate homologous chromosome pairing during homologous recombination. However, the means by which these two recombinases distinctly function in chromatin have remained elusive. Here we found that, in chromatin, the human DMC1-single-stranded DNA complex bypasses binding to the nucleosome, and preferentially promotes homologous pairing at the nucleosome-depleted regions. Consistently, DMC1 forms ternary complex recombination intermediates with the nucleosome-free DNA or the nucleosome-depleted DNA region. Surprisingly, removal of the histone tails improperly enhances the nucleosome binding by DMC1. In contrast, RAD51 does not specifically target the nucleosome-depleted region in chromatin. These are the first demonstrations that the chromatin architecture specifies the sites to promote the homologous recombination reaction by DMC1, but not by RAD51.