RESUMO
Activation of incretin receptors by their cognate agonist augments sustained cAMP generation both from the plasma membrane as well as from the endosome. To address the functional outcome of this spatiotemporal signaling, we developed a nonacylated glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) receptor dual agonist I-M-150847 that reduced receptor internalization following activation of the incretin receptors. The incretin receptor dual agonist I-M-150847 was developed by replacing the tryptophan cage of exendin-4 tyrosine substituted at the amino terminus with the C-terminal undecapeptide sequence of oxyntomodulin that placed lysine 30 of I-M-150847 in frame with the corresponding lysine residue of GIP. The peptide I-M-150847 is a partial agonist of GLP-1R and GIPR; however, the receptors, upon activation by I-M-150847, undergo reduced internalization that promotes agonist-mediated iterative cAMP signaling and augments glucose-stimulated insulin exocytosis in pancreatic ß cells. Chronic administration of I-M-150847 improved glycemic control, enhanced insulin sensitivity, and provided profound weight loss in diet-induced obese (DIO) mice. Our results demonstrated that despite being a partial agonist, I-M-150847, by reducing the receptor internalization upon activation, enhanced the incretin effect and reversed obesity.NEW & NOTEWORTHY Replacement of the tryptophan cage (Trp-cage) with the C-terminal oxyntomodulin undecapeptide along with the tyrosine substitution at the amino terminus converts the selective glucagon-like peptide-1 receptor (GLP-1R) agonist exendin-4 to a novel GLP-1R and GIPR dual agonist I-M-150847. Reduced internalization of incretin receptors upon activation by the GLP-1R and GIPR dual agonist I-M-150847 promotes iterative receptor signaling that enhances the incretin effect and reverses obesity.
Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1 , Incretinas , Camundongos Endogâmicos C57BL , Obesidade , Animais , Obesidade/metabolismo , Obesidade/tratamento farmacológico , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Camundongos , Masculino , Incretinas/farmacologia , Incretinas/metabolismo , Transporte Proteico/efeitos dos fármacos , Controle Glicêmico/métodos , Camundongos Obesos , Receptores dos Hormônios Gastrointestinais/agonistas , Receptores dos Hormônios Gastrointestinais/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Humanos , Dieta Hiperlipídica/efeitos adversos , Glicemia/metabolismo , Glicemia/efeitos dos fármacos , Insulina/metabolismo , Exenatida/farmacologia , Transdução de Sinais/efeitos dos fármacos , AMP Cíclico/metabolismoRESUMO
Diabetes shortens the life expectancy by more than a decade, and the excess mortality in diabetes is correlated with the incidence of kidney disease. Diabetic kidney disease (DKD) is the leading cause of end-stage kidney disease. Macrophage accumulation predicts the severity of kidney injury in human biopsies and experimental models of DKD. However, the mechanism underlying macrophage recruitment in diabetes glomeruli is unclear. Elevated plasma growth hormone (GH) levels in type I diabetes and acromegalic individuals impaired glomerular biology. In this study, we examined whether GH-stimulated podocytes contribute to macrophage accumulation. RNA-seq analysis revealed elevated TNF-α signaling in GH-treated human podocytes. Conditioned media from GH-treated podocytes (GH-CM) induced differentiation of monocytes to macrophages. On the other hand, neutralization of GH-CM with the TNF-α antibody diminished GH-CM's action on monocytes. The treatment of mice with GH resulted in increased macrophage recruitment, podocyte injury, and proteinuria. Furthermore, we noticed the activation of TNF-α signaling, macrophage accumulation, and fibrosis in DKD patients' kidney biopsies. Our findings suggest that podocytes could secrete TNF-α and contribute to macrophage migration, resulting in DKD-related renal inflammation. Inhibition of either GH action or TNF-α expression in podocytes could be a novel therapeutic approach for DKD treatment.
Assuntos
Nefropatias Diabéticas , Monócitos , Podócitos , Fator de Necrose Tumoral alfa , Animais , Humanos , Camundongos , Monócitos/citologia , Podócitos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Diferenciação CelularRESUMO
The mechanisms that guide the clonally stable random mono-allelic expression of autosomal genes remain enigmatic. We show that (1) mono-allelically expressed (MAE) genes are assorted and insulated from bi-allelically expressed (BAE) genes through CTCF-mediated chromatin loops; (2) the cell-type-specific dynamics of mono-allelic expression coincides with the gain and loss of chromatin insulator sites; (3) dosage of MAE genes is more sensitive to the loss of chromatin insulation than that of BAE genes; and (4) inactive alleles of MAE genes are significantly more insulated than active alleles and are de-repressed upon CTCF depletion. This alludes to a topology wherein the inactive alleles of MAE genes are insulated from the spatial interference of transcriptional states from the neighboring bi-allelic domains via CTCF-mediated loops. We propose that CTCF functions as a typical insulator on inactive alleles, but facilitates transcription through enhancer-linking on active allele of MAE genes, indicating widespread allele-specific regulatory roles of CTCF.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Genes/genética , Genômica/métodos , Humanos , MitoseRESUMO
BACKGROUND: Proximity ligation based techniques, like Hi-C, involve restriction digestion followed by ligation of formaldehyde cross-linked chromatin. Distinct chromatin states can impact the restriction digestion, and hence the visibility in the contact maps, of engaged loci. Yet, the extent and the potential impact of digestion bias remain obscure and under-appreciated in the literature. RESULTS: Through analysis of 45 Hi-C datasets, lamina-associated domains (LADs), inactive X-chromosome in mammals, and polytene bands in fly, we first established that the DNA in condensed chromatin had lesser accessibility to restriction endonucleases used in Hi-C as compared to that in decondensed chromatin. The observed bias was independent of known systematic biases, was not appropriately corrected by existing computational methods, and needed an additional optimization step. We then repurposed this bias to identify novel condensed domains outside LADs, which were bordered by insulators and were dynamically associated with the polycomb mediated epigenetic and transcriptional states during development. CONCLUSIONS: Our observations suggest that the corrected one-dimensional read counts of existing Hi-C datasets can be reliably repurposed to study the gene-regulatory dynamics associated with chromatin condensation and decondensation, and that the existing Hi-C datasets should be interpreted with cautions.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Posicionamento Cromossômico , Genômica/métodos , Cromossomos Politênicos , Cromossomo X , Animais , Imunoprecipitação da Cromatina , Drosophila/genética , Epigenômica , Humanos , Camundongos , Análise de Sequência de DNARESUMO
The development of mammary gland as a lactogenic tissue is a highly coordinated multistep process. The epithelial cells of lactiferous tubules undergo profound changes during the developmental window of puberty, pregnancy, and lactation. Several hormones including estrogen, progesterone, glucocorticoids and prolactin act in concert, and orchestrate the development of mammary gland. Understanding the gene regulatory networks that coordinate proliferation and differentiation of HC11 Mammary Epithelial stem-like Cells (MEC) under the influence of lactogenic hormones is critical for elucidating the mechanism of lactogenesis in detail. In this study, we analyzed transcriptome profiles of undifferentiated MEC (normal) and compared them with Murine Embryonic Stem Cells (ESC) using next-generation mRNA sequencing. Further, we analyzed the transcriptome output during lactogenic differentiation of MEC following treatment with glucocorticoids (primed state) and both glucocorticoids and prolactin together (prolactin state). We established stage-specific gene regulatory networks in ESC and MEC (normal, priming and prolactin states). We validated the top up-and downregulated genes in each stage of differentiation of MEC by RT-PCR and found that they are comparable with that of RNA-seq data. HC11 MEC display decreased expression of Pou5f1 and Sox2, which is crucial for the differentiation of MEC, which otherwise ensure pluripotency to ESC. Cited4 is induced during priming and is involved in milk secretion. MEC upon exposure to both glucocorticoids and prolactin undergo terminal differentiation, which is associated with the expression of several genes, including Xbp1 and Cbp that are required for cell growth and differentiation. Our study also identified differential expression of transcription factors and epigenetic regulators in each stage of lactogenic differentiation. We also analyzed the transcriptome data for the pathways that are selectively activated during lactogenic differentiation. Further, we found that selective expression of chromatin modulators (Dnmt3l, Chd9) in response to glucocorticoids suggests a highly coordinated stage-specific lactogenic differentiation of MEC.
Assuntos
Células-Tronco Embrionárias/citologia , Animais , Ciclo Celular/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Redes Reguladoras de Genes/genética , Redes Reguladoras de Genes/fisiologia , Immunoblotting , Lactação/metabolismo , Lactação/fisiologia , Glândulas Mamárias Animais/citologia , Camundongos , Gravidez , Prolactina/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
OBJECTIVES: Understanding of transcriptional networks specifying HC11 murine mammary epithelial stem cell-like cells (MEC) in comparison with embryonic stem cells (ESCs) and their rewiring, under the influence of glucocorticoids (GC) and prolactin (PRL) hormones, is critical for elucidating the mechanism of lactogenesis. In this data note, we provide RNA sequencing data from murine MECs and ESCs, MECs treated with steroid hormone alone and in combination with PRL. This data could help in understanding temporal dynamics of mRNA transcription that impact the process of lactogenesis associated with mammary gland development. Further integration of these data sets with existing datasets of cells derived from various stages of mammary gland development and different types of breast tumors, should pave the way for effective prognosis and to develop therapies for breast cancer. DATA DESCRIPTION: We have generated RNA-sequencing data representing steady-state levels of mRNAs from murine ESCs, normal MECs (N), MECs primed (P) with hydrocortisone (HC) alone and in combination with PRL hormone by using Illumina sequencing platform. We have generated ~ 58 million reads for ESCs with an average length of ~ 100 nt and an average 115 million good quality mapped reads with an average length of ~ 150 nt for different stages of MECs differentiation.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/metabolismo , Células Epiteliais/metabolismo , Glucocorticoides/farmacologia , Lactação/metabolismo , Glândulas Mamárias Animais/citologia , Prolactina/farmacologia , Análise de Sequência de RNA , Transdução de Sinais/fisiologia , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Células-Tronco Embrionárias/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Feminino , Lactação/efeitos dos fármacos , Camundongos , Transdução de Sinais/efeitos dos fármacos , TranscriptomaRESUMO
Molecular signatures and their interactions behind the successful establishment of infection of Mycobacterium tuberculosis (Mtb) inside macrophage are largely unknown. In this work, we present an inter-system scale atlas of the gene expression signatures, their interactions and higher order gene functions of macrophage-Mtb environment at the time of infection. We have carried out large-scale meta-analysis of previously published gene expression microarray studies andhave identified a ranked list of differentially expressed genes and their higher order functions in intracellular Mtb as well as the infected macrophage. Comparative analysis of gene expression signatures of intracellular Mtb with the in vitro dormant Mtb at different hypoxic and oxidative stress conditions led to the identification of the large number of Mtb functional groups, namely operons, regulons and pathways that were common and unique to the intracellular environment and dormancy state. Some of the functions that are specific to intracellular Mtb are cholesterol degradation and biosynthesis of immunomodulatory phenolic compounds. The molecular signatures we have identified to be involved in adaptation to different stress conditions in macrophage environment may be critical for designing therapeutic interventions against tuberculosis. And, our approach may be broadly applicable for investigating other host-pathogen interactions.
Assuntos
Proteínas de Bactérias/genética , Interações Hospedeiro-Patógeno/genética , Mycobacterium tuberculosis/genética , Tuberculose/genética , Regulação Bacteriana da Expressão Gênica/genética , Humanos , Macrófagos/microbiologia , Macrófagos/patologia , Análise em Microsséries , Mycobacterium tuberculosis/patogenicidade , Transcriptoma/genética , Tuberculose/microbiologia , Tuberculose/patologiaRESUMO
Genome organization in 3D nuclear-space is important for regulation of gene expression. However, the alterations of chromatin architecture that impinge on the B cell-fate choice of multi-potent progenitors are still unclear. By integrating in situ Hi-C analyses with epigenetic landscapes and genome-wide expression profiles, we tracked the changes in genome architecture as the cells transit from a progenitor to a committed state. We identified the genomic loci that undergo developmental switch between A and B compartments during B-cell fate determination. Furthermore, although, topologically associating domains (TADs) are stable, a significant number of TADs display structural alterations that are associated with changes in cis-regulatory interaction landscape. Finally, we demonstrate the potential roles for Ebf1 and its downstream factor, Pax5, in chromatin reorganization and transcription regulation. Collectively, our studies provide a general paradigm of the dynamic relationship between chromatin reorganization and lineage-specific gene expression pattern that dictates cell-fate determination.
Assuntos
Diferenciação Celular/genética , Cromatina/metabolismo , Células Precursoras de Linfócitos B/metabolismo , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Sítios de Ligação/genética , Células Cultivadas , Cromatina/genética , Perfilação da Expressão Gênica/métodos , Camundongos Knockout , Células Precursoras de Linfócitos B/citologia , Ligação Proteica , Sequências Reguladoras de Ácido Nucleico/genética , Transativadores/genética , Transativadores/metabolismoRESUMO
Apoptosis is a physiological cell death process essential for development, tissue homeostasis, and for immune defense of multicellular animals. Inhibitors of apoptosis proteins (IAPs) regulate apoptosis in response to various cellular assaults. Using both genetic and pharmacological approaches we demonstrate here that the IAPs not only support opportunistic survival of intracellular human pathogens like Chlamydia pneumoniae but also control plasticity of iNOS+ M1 macrophage during the course of infection and render them refractory for immune stimulation. Treatment of Th1 primed macrophages with birinapant (IAP-specific antagonist) inhibited NO generation and relevant proteins involved in innate immune signaling. Accordingly, birinapant promoted hypoxia, angiogenesis, and tumor-induced M2 polarization of iNOS+ M1 macrophages. Interestingly, birinapant-driven changes in immune signaling were accompanied with changes in the expression of various proteins involved in the metabolism, and thus revealing the new role of IAPs in immune metabolic reprogramming in committed macrophages. Taken together, our study reveals the significance of IAP targeting approaches (Smac mimetic compounds) for the management of infectious and inflammatory diseases relying on macrophage plasticity.
RESUMO
TAL1 is an important regulator of hematopoiesis and its expression is tightly controlled despite complexities in its genomic organization. It is frequently misregulated in T-cell acute lymphoblastic leukemia (T-ALL), often due to deletions between TAL1 and the neighboring STIL gene. To better understand the events that lead to TAL1 expression in hematopoiesis and in T-ALL, we studied looping interactions at the TAL1 locus. In TAL1-expressing erythroid cells, the locus adopts a looping "hub" which brings into close physical proximity all known TAL1 cis-regulatory elements including CTCF-bound insulators. Loss of GATA1 results in disassembly of the hub and loss of CTCF/RAD21 from one of its insulators. Genes flanking TAL1 are partly dependent on hub integrity for their transcriptional regulation. We identified looping patterns unique to TAL1-expressing T-ALL cells, and, intriguingly, loops occurring between the TAL1 and STIL genes at the common TAL1/STIL breakpoints found in T-ALL. These findings redefine how TAL1 and neighboring genes communicate within the nucleus, and indicate that looping facilitates both normal and aberrant TAL1 expression and may predispose to structural rearrangements in T-ALL. We also propose that GATA1-dependent looping mechanisms may facilitate the conservation of TAL1 regulation despite cis-regulatory remodeling during vertebrate evolution.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Cromatina/genética , Regulação Neoplásica da Expressão Gênica , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteínas Proto-Oncogênicas/genética , Animais , Cromatina/química , Elementos Facilitadores Genéticos/genética , Fator de Transcrição GATA1/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Células Jurkat , Células K562 , Linfócitos/citologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos ICR , Núcleosídeo-Fosfato Quinase/genética , Regiões Promotoras Genéticas/genética , Conformação Proteica , Proteína 1 de Leucemia Linfocítica Aguda de Células TRESUMO
Chromosome conformation capture (3C) is a powerful technique for analyzing spatial chromatin organization in vivo. Technical variants of the assay ('4C') allow the systematic detection of genome-wide coassociations with bait sequences of interest, enabling the nuclear environments of specific genes to be probed. We describe enhanced 4C (e4C, enhanced chromosome conformation capture on chip), a technique incorporating additional enrichment steps for bait-specific sequences, and thus improving sensitivity in the detection of weaker, distal chromatin coassociations. In brief, e4C entails the fixation, restriction digestion and ligation steps of conventional 3C, with an optional chromatin immunoprecipitation (ChIP) step to select for subsets of chromatin coassociations, followed by bait enrichment by biotinylated primer extension and pull-down, adapter ligation and PCR amplification. Chromatin coassociations with the bait sequence can then be assessed by hybridizing e4C products to microarrays or sequencing. The e4C procedure takes approximately 1 week to go from tissue to DNA ready for microarray hybridization.
Assuntos
Imunoprecipitação da Cromatina/métodos , Cromatina/química , Cromossomos/química , Epigenômica/métodos , Conformação de Ácido Nucleico , Biotinilação , Cromatina/metabolismo , Cromossomos/metabolismo , Primers do DNA/genéticaRESUMO
Insulators help separate active chromatin domains from silenced ones. In yeast, gene promoters act as insulators to block the spread of Sir and HP1 mediated silencing while in metazoans most insulators are multipartite autonomous entities. tDNAs are repetitive sequences dispersed throughout the human genome and we now show that some of these tDNAs can function as insulators in human cells. Using computational methods, we identified putative human tDNA insulators. Using silencer blocking, transgene protection and repressor blocking assays we show that some of these tDNA-containing fragments can function as barrier insulators in human cells. We find that these elements also have the ability to block enhancers from activating RNA pol II transcribed promoters. Characterization of a putative tDNA insulator in human cells reveals that the site possesses chromatin signatures similar to those observed at other better-characterized eukaryotic insulators. Enhanced 4C analysis demonstrates that the tDNA insulator makes long-range chromatin contacts with other tDNAs and ETC sites but not with intervening or flanking RNA pol II transcribed genes.
Assuntos
Elementos Isolantes/genética , RNA de Transferência/genética , Animais , Linhagem Celular , Cromatina/genética , Cromossomos Humanos Par 17/genética , Biologia Computacional/métodos , DNA Fúngico/genética , DNA Fúngico/metabolismo , Elementos Facilitadores Genéticos/genética , Inativação Gênica , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Mamíferos/genética , Ligação Proteica , RNA Polimerase III/metabolismo , Schizosaccharomyces/genética , Alinhamento de Sequência , Sintenia , Fatores de Transcrição TFIII/metabolismo , Transcrição Gênica/genética , TransgenesRESUMO
The discovery of interchromosomal interactions in higher eukaryotes points to a functional interplay between genome architecture and gene expression, challenging the view of transcription as a one-dimensional process. However, the extent of interchromosomal interactions and the underlying mechanisms are unknown. Here we present the first genome-wide analysis of transcriptional interactions using the mouse globin genes in erythroid tissues. Our results show that the active globin genes associate with hundreds of other transcribed genes, revealing extensive and preferential intra- and interchromosomal transcription interactomes. We show that the transcription factor Klf1 mediates preferential co-associations of Klf1-regulated genes at a limited number of specialized transcription factories. Our results establish a new gene expression paradigm, implying that active co-regulated genes and their regulatory factors cooperate to create specialized nuclear hot spots optimized for efficient and coordinated transcriptional control.
Assuntos
Células Eritroides/metabolismo , Perfilação da Expressão Gênica , Redes Reguladoras de Genes/genética , Estudo de Associação Genômica Ampla/métodos , Animais , Imunoprecipitação da Cromatina , Células Eritroides/citologia , Imunofluorescência , Globinas/genética , Globinas/metabolismo , Humanos , Hibridização in Situ Fluorescente/métodos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Ligação ProteicaRESUMO
The genome is spatially organized inside nuclei, with chromosomes and genes occupying preferential positions relative to each other and to various nuclear landmarks. What drives this organization is unclear, but recent findings suggest there are extensive intra- and inter-chromosomal communications between various genomic regions that appear to play important roles in genome function. Here we review transcription factories, distinct sub-nuclear foci where nascent transcription occurs. We argue that the spatially restricted, limited number of transcription sites compels transcribed regions of the genome to dynamically self-organize into tissue-specific conformations, thus playing a major role in the three-dimensional interphase organization of the genome.
Assuntos
Cromatina/química , Cromatina/fisiologia , RNA Polimerases Dirigidas por DNA/fisiologia , Interfase/fisiologia , Animais , Cromatina/genética , RNA Polimerases Dirigidas por DNA/química , Humanos , Transcrição Gênica/genética , Transcrição Gênica/fisiologiaRESUMO
Asynchronous replication during S phase is a universal characteristic of genomically imprinted genes. Replication timing in imprinted domains is determined epigenetically, as it is parent of origin specific, and is seen in the absence of sequence divergence between the two alleles. At the imprinted H19/Igf2 domain, the methylated paternal allele replicates early while the CTCF-bound maternal allele replicates late during S phase. CTCF regulates the allele-specific epigenetic characteristics of this domain, including methylation, transcription and chromosome conformation. Here we show that maternal, but not paternal inheritance of a mutated H19 imprinting control region, lacking functional CTCF binding sites, underlies a late to early switch in replication timing of the maternal H19/Igf2 domain.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Impressão Genômica , Fator de Crescimento Insulin-Like II/genética , RNA não Traduzido/genética , Proteínas Repressoras/fisiologia , Fase S/fisiologia , Alelos , Animais , Sítios de Ligação , Fator de Ligação a CCCTC , Cromossomos de Mamíferos , Proteínas de Ligação a DNA/metabolismo , Padrões de Herança/fisiologia , Fator de Crescimento Insulin-Like II/química , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , RNA Longo não Codificante , RNA não Traduzido/química , Proteínas Repressoras/metabolismo , Fase S/genéticaRESUMO
Accumulating evidence converges on the possibility that chromosomes interact with each other to regulate transcription in trans. To systematically explore the epigenetic dimension of such interactions, we devised a strategy termed circular chromosome conformation capture (4C). This approach involves a circularization step that enables high-throughput screening of physical interactions between chromosomes without a preconceived idea of the interacting partners. Here we identify 114 unique sequences from all autosomes, several of which interact primarily with the maternally inherited H19 imprinting control region. Imprinted domains were strongly overrepresented in the library of 4C sequences, further highlighting the epigenetic nature of these interactions. Moreover, we found that the direct interaction between differentially methylated regions was linked to epigenetic regulation of transcription in trans. Finally, the patterns of interactions specific to the maternal H19 imprinting control region underwent reprogramming during in vitro maturation of embryonic stem cells. These observations shed new light on development, cancer epigenetics and the evolution of imprinting.
Assuntos
Cromossomos/química , Clonagem Molecular/métodos , Epigênese Genética/fisiologia , Regulação da Expressão Gênica/genética , Animais , Animais Recém-Nascidos , Sítios de Ligação , Fator de Ligação a CCCTC , Cromatina/química , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias , Impressão Genômica/fisiologia , Fígado/metabolismo , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Conformação de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Longo não Codificante , RNA não Traduzido/genética , Proteínas Repressoras/metabolismo , TransativadoresRESUMO
It is thought that the H19 imprinting control region (ICR) directs the silencing of the maternally inherited Igf2 allele through a CTCF-dependent chromatin insulator. The ICR has been shown to interact physically with a silencer region in Igf2, differentially methylated region (DMR)1, but the role of CTCF in this chromatin loop and whether it restricts the physical access of distal enhancers to Igf2 is not known. We performed systematic chromosome conformation capture analyses in the Igf2/H19 region over >160 kb, identifying sequences that interact physically with the distal enhancers and the ICR. We found that, on the paternal chromosome, enhancers interact with the Igf2 promoters but that, on the maternal allele, this is prevented by CTCF binding within the H19 ICR. CTCF binding in the maternal ICR regulates its interaction with matrix attachment region (MAR)3 and DMR1 at Igf2, thus forming a tight loop around the maternal Igf2 locus, which may contribute to its silencing. Mutation of CTCF binding sites in the H19 ICR leads to loss of CTCF binding and de novo methylation of a CTCF target site within Igf2 DMR1, showing that CTCF can coordinate regional epigenetic marks. This systematic chromosome conformation capture analysis of an imprinting cluster reveals that CTCF has a critical role in the epigenetic regulation of higher-order chromatin structure and gene silencing over considerable distances in the genome.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Impressão Genômica/fisiologia , Heterocromatina/química , Heterocromatina/metabolismo , Fator de Crescimento Insulin-Like II/genética , Proteínas/genética , RNA não Traduzido/metabolismo , Proteínas Repressoras/metabolismo , Animais , Fator de Ligação a CCCTC , Proteínas de Ligação a DNA/fisiologia , Epigênese Genética/fisiologia , Feminino , Inativação Gênica , Heterocromatina/genética , Fator de Crescimento Insulin-Like II/metabolismo , Masculino , Camundongos , Ligação Proteica/genética , Conformação Proteica , RNA Longo não Codificante , RNA não Traduzido/fisiologia , Proteínas Repressoras/fisiologiaRESUMO
Most of the transcription factors, RNA polymerases and enhancer binding factors are absent from condensed mitotic chromosomes. In contrast, epigenetic marks of active and inactive genes somehow survive mitosis, since the activity status from one cell generation to the next is maintained. For the zinc-finger protein CTCF, a role in interpreting and propagating epigenetic states and in separating expression domains has been documented. To test whether such a domain structure is preserved during mitosis, we examined whether CTCF is bound to mitotic chromatin. Here we show that in contrast to other zinc-finger proteins, CTCF indeed is bound to mitotic chromosomes. Mitotic binding is mediated by a portion of the zinc-finger DNA binding domain and involves sequence specific binding to target sites. Furthermore, the chromatin loop organized by the CTCF-bound, differentially methylated region at the Igf2/H19 locus can be detected in mitosis. In contrast, the enhancer/promoter loop of the same locus is lost in mitosis. This may provide a novel form of epigenetic memory during cell division.
Assuntos
Cromatina/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mitose , RNA não Traduzido/genética , Proteínas Repressoras/metabolismo , Animais , Sítios de Ligação , Fator de Ligação a CCCTC , Células Cultivadas , Cromatina/química , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/química , Humanos , Camundongos , Conformação Molecular , RNA Longo não Codificante , Proteínas Repressoras/química , Dedos de ZincoRESUMO
The differentially methylated imprinting control region (ICR) region upstream of the H19 gene regulates allelic Igf2 expression by means of a methylation-sensitive chromatin insulator function. We have previously shown that maternal inheritance of mutated (three of the four) target sites for the 11-zinc finger protein CTCF leads to loss of Igf2 imprinting. Here we show that a mutation in only CTCF site 4 also leads to robust activation of the maternal Igf2 allele despite a noticeably weaker interaction in vitro of site 4 DNA with CTCF compared to other ICR sites, sites 1 and 3. Moreover, maternally inherited sites 1 to 3 become de novo methylated in complex patterns in subpopulations of liver and heart cells with a mutated site 4, suggesting that the methylation privilege status of the maternal H19 ICR allele requires an interdependence between all four CTCF sites. In support of this conclusion, we show that CTCF molecules bind to each other both in vivo and in vitro, and we demonstrate strong interaction between two CTCF-DNA complexes, preassembled in vitro with sites 3 and 4. We propose that the CTCF sites may cooperate to jointly maintain both methylation-free status and insulator properties of the maternal H19 ICR allele. Considering many other CTCF targets, we propose that site-specific interactions between various DNA-bound CTCF molecules may provide general focal points in the organization of looped chromatin domains involved in gene regulation.