RESUMO
In mammals, generally it is assumed that the genes inherited from each parent are expressed to similar levels. However, it is now apparent that in non-sex chromosomes, 6-10% of genes are selected for monoallelic expression. Monoallelic expression or allelic exclusion is established either in an imprinted (parent-of-origin) or a stochastic manner. The stochastic model explains random selection while the imprinted model describes parent-of-origin specific selection of alleles for expression. Allelic exclusion occurs during X chromosome inactivation, parent-of-origin expression of imprinted genes and stochastic monoallelic expression of cell surface molecules, clustered protocadherin (PCDH) genes. Mis-regulation or loss of allelic exclusion contributes to developmental diseases. Epigenetic mechanisms are fundamental players that determine this type of expression despite a homogenous genetic background. DNA methylation and histone modifications are two mediators of the epigenetic phenomena. The majority of DNA methylation is found on cytosines of the CpG dinucleotide in mammals. Several covalent modifications of histones change the electrostatic forces between DNA and histones modifying gene expression. Long-range chromatin interactions organize chromatin into transcriptionally permissive and prohibitive regions leading to simultaneous regulation of gene expression and repression. Non-coding RNAs (ncRNAs) are also players in regulating gene expression. Together, these epigenetic mechanisms fine-tune gene expression levels essential for normal development and survival. In this review, first we discuss what is known about monoallelic gene expression. Then, we focus on the molecular mechanisms that regulate expression of three monoallelically expressed gene classes: the X-linked genes, selected imprinted genes and PCDH genes.
Assuntos
Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Impressão Genômica , Modelos Genéticos , Alelos , Animais , Repressão Epigenética , Desenvolvimento Fetal , Humanos , Processos Estocásticos , Inativação do Cromossomo XRESUMO
Precise control of transcriptional programmes underlying metazoan development is modulated by enzymatically active co-regulatory complexes, coupled with epigenetic strategies. One thing that remains unclear is how specific members of histone modification enzyme families, such as histone methyltransferases and demethylases, are used in vivo to simultaneously orchestrate distinct developmental gene activation and repression programmes. Here, we report that the histone lysine demethylase, LSD1--a component of the CoREST-CtBP co-repressor complex--is required for late cell-lineage determination and differentiation during pituitary organogenesis. LSD1 seems to act primarily on target gene activation programmes, as well as in gene repression programmes, on the basis of recruitment of distinct LSD1-containing co-activator or co-repressor complexes. LSD1-dependent gene repression programmes can be extended late in development with the induced expression of ZEB1, a Krüppel-like repressor that can act as a molecular beacon for recruitment of the LSD1-containing CoREST-CtBP co-repressor complex, causing repression of an additional cohort of genes, such as Gh, which previously required LSD1 for activation. These findings suggest that temporal patterns of expression of specific components of LSD1 complexes modulate gene regulatory programmes in many mammalian organs.
Assuntos
Regulação para Baixo/genética , Regulação da Expressão Gênica no Desenvolvimento , Oxirredutases N-Desmetilantes/metabolismo , Animais , Diferenciação Celular , Hormônio do Crescimento/genética , Histona Desmetilases , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Lactotrofos/metabolismo , Camundongos , Oxirredutases N-Desmetilantes/deficiência , Oxirredutases N-Desmetilantes/genética , Hipófise/citologia , Hipófise/metabolismo , Ativação Transcricional , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
The pituitary gland functions as a relay between the hypothalamus and peripheral target organs that regulate basic physiological functions, including growth, the stress response, reproduction, metabolism and lactation. The development of the pituitary gland has been studied extensively in mice, and has begun to be explored in zebrafish, an animal model system amenable to forward genetics. Multiple signaling molecules and transcription factors, expressed in overlapping but distinct spatial and temporal patterns, are required at various stages of pituitary development. Defects in this precisely regulated genetic program lead to diverse pituitary dysfunction. The animal models have greatly enhanced our understanding of molecular mechanisms underlying pituitary development in addition to congenital pituitary disorders in humans.
Assuntos
Hipopituitarismo/genética , Hipófise/embriologia , Hipófise/metabolismo , Animais , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Hipopituitarismo/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Chromatin immunoprecipitation (ChIP) exploits the specific interactions between DNA and DNA-associated proteins. It can be used to examine a wide range of experimental parameters. A number of proteins bound at the same genomic location can identify a multi-protein chromatin complex where several proteins work together to regulate gene transcription or chromatin configuration. In many instances, this can be achieved using sequential ChIP; or simply, ChIP-re-ChIP. Whether it is for the examination of specific transcriptional or epigenetic regulators, or for the identification of cistromes, the ability to perform a sequential ChIP adds a higher level of power and definition to these analyses. In this chapter, we describe a simple and reliable method for the sequential ChIP assay.
Assuntos
Imunoprecipitação da Cromatina , Sequenciamento de Nucleotídeos em Larga Escala , Cromatina/genética , Cromatina/metabolismo , Imunoprecipitação da Cromatina/métodos , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido NucleicoRESUMO
Loss of tumor suppressor proteins, such as the retinoblastoma protein (Rb), results in tumor progression and metastasis. Metastasis is facilitated by low oxygen availability within the tumor that is detected by hypoxia inducible factors (HIFs). The HIF1 complex, HIF1α and dimerization partner the aryl hydrocarbon receptor nuclear translocator (ARNT), is the master regulator of the hypoxic response. Previously, we demonstrated that Rb represses the transcriptional response to hypoxia by virtue of its association with HIF1. In this report, we further characterized the role Rb plays in mediating hypoxia-regulated genetic programs by stably ablating Rb expression with retrovirally-introduced short hairpin RNA in LNCaP and 22Rv1 human prostate cancer cells. DNA microarray analysis revealed that loss of Rb in conjunction with hypoxia leads to aberrant expression of hypoxia-regulated genetic programs that increase cell invasion and promote neuroendocrine differentiation. For the first time, we have established a direct link between hypoxic tumor environments, Rb inactivation and progression to late stage metastatic neuroendocrine prostate cancer. Understanding the molecular pathways responsible for progression of benign prostate tumors to metastasized and lethal forms will aid in the development of more effective prostate cancer therapies.
Assuntos
Biomarcadores Tumorais/genética , Diferenciação Celular , Hipóxia/genética , Células Neuroendócrinas/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteína do Retinoblastoma/metabolismo , Apoptose , Movimento Celular , Proliferação de Células , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Masculino , Invasividade Neoplásica , Células Neuroendócrinas/metabolismo , Neoplasias da Próstata/metabolismo , Proteína do Retinoblastoma/genética , Células Tumorais CultivadasRESUMO
Despite a genetic homogeneity, cells in multicellular organisms are structurally and functionally heterogeneous. The diversity of cell phenotypes exists due to differential transcriptional programs precisely regulated by specific nuclear factors and induced upon differentiation. The differences in gene expression programs arise during development and become heritable during cell proliferation. Over the last few years, research has focused on three molecular mechanisms that mediate epigenetic phenomena: DNA methylation, histone modification, and formation of specialized nuclear domains or territories. All of these processes are dynamic and tightly linked to the organism's development. Here we review advances in understanding the significance of epigenetic mechanisms in the establishment and maintenance of the specialized transcriptional program. We project the accumulated knowledge onto the delineation of the molecular mechanisms by which central nervous system-specific genes are expressed in the nervous system and repressed in other tissues.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neurônios/fisiologia , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Transcrição Gênica/fisiologia , AnimaisRESUMO
Regulatory elements for the mouse growth hormone (GH) gene are located distally in a putative locus control region (LCR) in addition to key elements in the promoter proximal region. The role of promoter DNA methylation for GH gene regulation is not well understood. Pit-1 is a POU transcription factor required for normal pituitary development and obligatory for GH gene expression. In mammals, Pit-1 mutations eliminate GH production resulting in a dwarf phenotype. In this study, dwarf mice illustrated that Pit-1 function was obligatory for GH promoter hypomethylation. By monitoring promoter methylation levels during developmental GH expression we found that the GH promoter became hypomethylated coincident with gene expression. We identified a promoter differentially methylated region (DMR) that was used to characterize a methylation-dependent DNA binding activity. Upon DNA affinity purification using the DMR and nuclear extracts, we identified structural maintenance of chromosomes hinge domain containing -1 (SmcHD1). To better understand the role of SmcHD1 in genome-wide gene expression, we performed microarray analysis and compared changes in gene expression upon reduced levels of SmcHD1 in human cells. Knock-down of SmcHD1 in human embryonic kidney (HEK293) cells revealed a disproportionate number of up-regulated genes were located on the X-chromosome, but also suggested regulation of genes on non-sex chromosomes. Among those, we identified several genes located in the protocadherin ß cluster. In addition, we found that imprinted genes in the H19/Igf2 cluster associated with Beckwith-Wiedemann and Silver-Russell syndromes (BWS & SRS) were dysregulated. For the first time using human cells, we showed that SmcHD1 is an important regulator of imprinted and clustered genes.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Cromossomos de Mamíferos/genética , Epigênese Genética , Hormônio do Crescimento/genética , Família Multigênica/genética , Sequência de Aminoácidos , Animais , Azacitidina/farmacologia , Sequência de Bases , Síndrome de Beckwith-Wiedemann/genética , Caderinas/genética , Linhagem Celular , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/deficiência , Proteínas Cromossômicas não Histona/genética , Metilação de DNA/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Humanos , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Síndrome de Silver-Russell/genética , Regulação para Cima/efeitos dos fármacosRESUMO
Localized hypoxia in solid tumors activates transcriptional programs that promote the metastatic transformation of cells. Like hypoxia-inducible hyper-vascularization, loss of the retinoblastoma protein (Rb) is a trait common to advanced stages of tumor progression in many metastatic cancers. However, no link between the role of Rb and hypoxia-driven metastatic processes has been established. We demonstrated that Rb is a key mediator of the hypoxic response mediated by HIF1α/ß, the master regulator of the hypoxia response, and its essential co-activator, the thyroid hormone receptor/retinoblastoma-interacting protein (TRIP230). Furthermore, loss of Rb unmasks the full co-activation potential of TRIP230. Using small inhibitory RNA approaches in vivo, we established that Rb attenuates the normal physiological response to hypoxia by HIF1α. Notably, loss of Rb results in hypoxia-dependent biochemical changes that promote acquisition of an invasive phenotype in MCF7 breast cancer cells. In addition, Rb is present in HIF1α-ARNT/HIF1ß transcriptional complexes associated with TRIP230 as determined by co-immuno-precipitation, GST-pull-down and ChIP assays. These results demonstrate that Rb is a negative modulator of hypoxia-regulated transcription by virtue of its direct effects on the HIF1 complex. This work represents the first link between the functional ablation of Rb in tumor cells and HIF1α-dependent transcriptional activation and invasion.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas Nucleares/metabolismo , Proteína do Retinoblastoma/metabolismo , Proteínas do Citoesqueleto , Regulação Neoplásica da Expressão Gênica/fisiologia , Técnicas de Silenciamento de Genes , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Células MCF-7 , Proteínas Nucleares/genética , RNA Interferente Pequeno/genética , Proteína do Retinoblastoma/genéticaRESUMO
Single cell electroporation (SCE), via microcapillary, is an effective method for molecular, transmembrane transport used to gain insight on cell processes with minimal preparation. Although possessing great potential, SCE is difficult to execute and the technology spans broad fields within cell biology and engineering. The technical complexities, the focus and expertise demanded during manual operation, and the lack of an automated SCE platform limit the widespread use of this technique, thus the potential of SCE has not been realized. In this study, an automated biomanipulator for SCE is presented. Our system is capable of delivering molecules into the cytoplasm of extremely thin cellular features of adherent cells. The intent of the system is to abstract the technical challenges and exploit the accuracy and repeatability of automated instrumentation, leaving only the focus of the experimental design to the operator. Each sequence of SCE including cell and SCE site localization, tip-membrane contact detection, and SCE has been automated. Positions of low-contrast cells are localized and "SCE sites" for microcapillary tip placement are determined using machine vision. In addition, new milestones within automated cell manipulation have been achieved. The system described herein has the capability of automated SCE of "thin" cell features less than 10 µm in thickness. Finally, SCE events are anticipated using visual feedback, while monitoring fluorescing dye entering the cytoplasm of a cell. The execution is demonstrated by inserting a combination of a fluorescing dye and a reporter gene into NIH/3T3 fibroblast cells.
Assuntos
Eletroporação/instrumentação , Eletroporação/métodos , Micromanipulação/instrumentação , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos , Animais , Citoplasma/fisiologia , Camundongos , Células NIH 3T3 , Robótica/instrumentação , TransfecçãoRESUMO
The activated AHR/ARNT complex (AHRC) regulates the expression of target genes upon exposure to environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Importantly, evidence has shown that TCDD represses estrogen receptor (ER) target gene activation through the AHRC. Our data indicates that AHR and ARNT act independently from each other at non-dioxin response element sites. Therefore, we sought to determine the specific functions of AHR and ARNT in estrogen-dependent signaling in human MCF7 breast cancer and human ECC-1 endometrial carcinoma cells. Knockdown of AHR with siRNA abrogates dioxin-inducible repression of estrogen-dependent gene transcription. Intriguingly, knockdown of ARNT does not effect TCDD-mediated repression of estrogen-regulated transcription, suggesting that AHR represses ER function independently of ARNT. This theory is supported by the ability of the selective AHR modulator 3',4'-dimethoxy-α-naphthoflavone (DiMNF) to repress estrogen-inducible transcription. Furthermore, basal and estrogen-activated transcription of the genes encoding cathepsin-D and pS2 are down-regulated in MCF7 cells but up-regulated in ECC-1 cells in response to loss of ARNT. These responses are mirrored at the protein level with cathepsin-D. Furthermore, knock-down of ARNT led to opposite but corresponding changes in estrogen-stimulated proliferation in both MCF7 and ECC-1 cells. We have obtained experimental evidence demonstrating a dioxin-dependent repressor function for AHR and a dioxin-independent co-activator/co-repressor function for ARNT in estrogen signalling. These results provide us with further insight into the mechanisms of transcription factor crosstalk and putative therapeutic targets in estrogen-positive cancers.
Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Estrogênios/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais , Translocador Nuclear Receptor Aril Hidrocarboneto/deficiência , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Receptor alfa de Estrogênio/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Fenótipo , Dibenzodioxinas Policloradas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
Vertebrate opsin genes often occur in sets of tandem duplicates, and their expression varies developmentally and in response to environmental cues. We previously identified two highly conserved regions upstream of the long-wave sensitive opsin (LWS) gene cluster in teleosts. This region has since been shown in zebrafish to drive expression of LWS genes in vivo. In order to further investigate how elements in this region control opsin gene expression, we tested constructs encompassing the highly conserved regions and the less conserved portions upstream of the coding sequences in a promoter-less luciferase expression system. A â¼4500 bp construct of the upstream region, including the highly-conserved regions Reg I and Reg II, increased expression 100-fold, and successive 5' deletions reduced expression relative to the full 4.5 Kb region. Gene expression was highest when the transcription factor RORα was co-transfected with the proposed regulatory regions. Because these regions were tested in a promoter-less expression system, they include elements able to initiate and drive transcription. Teleosts exhibit complex color-mediated adaptive behavior and their adaptive significance has been well documented in several species. Therefore these upstream regions of LWS represent a model system for understanding the molecular basis of adaptive variation in gene regulation of color vision.
Assuntos
Visão de Cores/genética , Opsinas dos Cones/genética , Sequência Conservada/fisiologia , Peixes/genética , Regulação da Expressão Gênica , Receptores do Ácido Retinoico/metabolismo , Animais , Visão de Cores/fisiologia , Opsinas dos Cones/metabolismo , Elementos Facilitadores Genéticos , Genes Reporter , Luciferases , Ligação Proteica/genética , Células Fotorreceptoras Retinianas Cones , Receptor alfa de Ácido Retinoico , Fatores de Transcrição/metabolismoRESUMO
Nuclear receptors undergo ligand-dependent conformational changes that are required for corepressor-coactivator exchange, but whether there is an actual requirement for specific epigenetic landmarks to impose ligand dependency for gene activation remains unknown. Here we report an unexpected and general strategy that is based on the requirement for specific cohorts of inhibitory histone methyltransferases (HMTs) to impose gene-specific gatekeeper functions that prevent unliganded nuclear receptors and other classes of regulated transcription factors from binding to their target gene promoters and causing constitutive gene activation in the absence of stimulating signals. This strategy, based at least in part on an HMT-dependent inhibitory histone code, imposes a requirement for specific histone demethylases, including LSD1, to permit ligand- and signal-dependent activation of regulated gene expression. These events link an inhibitory methylation component of the histone code to a broadly used strategy that circumvents pathological constitutive gene induction by physiologically regulated transcription factors.
Assuntos
Receptor alfa de Estrogênio/metabolismo , Regulação da Expressão Gênica , Histonas/metabolismo , Oxirredutases N-Desmetilantes/metabolismo , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Estradiol/metabolismo , Genoma Humano , Código das Histonas , Histona Desmetilases , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Ligantes , Metilação , Regiões Promotoras Genéticas , Ativação TranscricionalRESUMO
The temporal and spatial regulation of gene expression in mammalian development is linked to the establishment of functional chromatin domains. Here, we report that tissue-specific transcription of a retrotransposon repeat in the murine growth hormone locus is required for gene activation. This repeat serves as a boundary to block the influence of repressive chromatin modifications. The repeat element is able to generate short, overlapping Pol II-and Pol III-driven transcripts, both of which are necessary and sufficient to enable a restructuring of the regulated locus into nuclear compartments. These data suggest that transcription of interspersed repetitive sequences may represent a developmental strategy for the establishment of functionally distinct domains within the mammalian genome to control gene activation.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Hormônio do Crescimento/genética , Elementos Isolantes , Organogênese , Hipófise/embriologia , Elementos Nucleotídeos Curtos e Dispersos , Transcrição Gênica , Animais , Sequência de Bases , Imunoprecipitação da Cromatina , DNA Polimerase II/metabolismo , DNA Polimerase III/metabolismo , Histonas/metabolismo , Metilação , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Hipófise/metabolismo , Ativação TranscricionalRESUMO
The molecular mechanisms by which central nervous system-specific genes are expressed only in the nervous system and repressed in other tissues remain a central issue in developmental and regulatory biology. Here, we report that the zinc-finger gene-specific repressor element RE-1 silencing transcription factor/neuronal restricted silencing factor (REST/NRSF) can mediate extraneuronal restriction by imposing either active repression via histone deacetylase recruitment or long-term gene silencing using a distinct functional complex. Silencing of neuronal-specific genes requires the recruitment of an associated corepressor, CoREST, that serves as a functional molecular beacon for the recruitment of molecular machinery that imposes silencing across a chromosomal interval, including transcriptional units that do not themselves contain REST/NRSF response elements.