RESUMO
Epigenetic regulation is achieved at many levels by different factors such as tissue-specific transcription factors, members of the basal transcriptional apparatus, chromatin-binding proteins, and noncoding RNAs. Importantly, chromatin structure dictates the availability of a specific genomic locus for transcriptional activation as well as the efficiency with which transcription can occur. Chromatin immunoprecipitation (ChIP) is a method that allows elucidating gene regulation at the molecular level by assessing if chromatin modifications or proteins are present at a specific locus. Initially, the majority of ChIP experiments were performed on cultured cell lines and more recently this technique has been adapted to a variety of tissues in different model organisms. Using ChIP on mouse embryos, it is possible to document the presence or absence of specific proteins and chromatin modifications at genomic loci in vivo during mammalian development and to get biological meaning from observations made on tissue culture analyses. We describe here a ChIP protocol on freshly isolated mouse embryonic somites for in vivo analysis of muscle specific transcription factor binding on chromatin. This protocol has been easily adapted to other mouse embryonic tissues and has also been successfully scaled up to perform ChIP-Seq.
Assuntos
Imunoprecipitação da Cromatina/métodos , Embrião de Mamíferos/metabolismo , Animais , Epigênese Genética/genética , Feminino , Camundongos , Desenvolvimento Muscular/genética , Desenvolvimento Muscular/fisiologia , Proteína MyoD/genética , Miogenina/genética , GravidezRESUMO
The Myogenic Regulatory Factors (MRFs) Myf5, MyoD, myogenin and MRF4 are members of the basic helix-loop-helix family of transcription factors that control the determination and differentiation of skeletal muscle cells during embryogenesis and postnatal myogenesis. The dynamics of their temporal and spatial expression as well as their biochemical properties have allowed the identification of a precise and hierarchical relationship between the four MRFs. This relationship establishes the myogenic lineage as well as the maintenance of the terminal myogenic phenotype. The application of genome-wide technologies has provided important new information as to how the MRFs function to activate muscle gene expression. Application of combined functional genomics technologies along with single cell lineage tracing strategies will allow a deeper understanding of the mechanisms mediating myogenic determination, cell differentiation and muscle regeneration.
Assuntos
Diferenciação Celular/genética , Linhagem da Célula/genética , Desenvolvimento Muscular/genética , Músculo Esquelético/metabolismo , Fatores de Regulação Miogênica/genética , Regeneração/genética , Animais , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Músculo Esquelético/citologia , Músculo Esquelético/embriologia , Fatores de Regulação Miogênica/classificação , FilogeniaRESUMO
During organism development, a diversity of cell types emerges with disparate, yet stable profiles of gene expression with distinctive cellular functions. In addition to gene promoters, the genome contains enhancer regulatory sequences, which are implicated in cellular specialization by facilitating cell-type and tissue-specific gene expression. Enhancers are DNA binding elements characterized by highly sophisticated and various mechanisms of action allowing for the specific interaction of general and tissue-specific transcription factors (TFs). However, eukaryotic organisms package their genetic material into chromatin, generating a physical barrier for TFs to interact with their cognate sequences. The ability of TFs to bind DNA regulatory elements is also modulated by changes in the chromatin structure, including histone modifications, histone variants, ATP-dependent chromatin remodeling, and the methylation status of DNA. Furthermore, it has recently been revealed that enhancer sequences are also transcribed into a set of enhancer RNAs with regulatory potential. These interdependent processes act in the context of a complex network of chromatin interactions, which together contributes to a renewed vision of how gene activation is coordinated in a cell-type-dependent manner. In this review, we describe the interplay between genetic and epigenetic aspects associated with enhancers and discuss their possible roles on enhancer function.
Assuntos
Montagem e Desmontagem da Cromatina , Elementos Facilitadores Genéticos , Animais , Metilação de DNA , Epigênese Genética , Código das Histonas , Humanos , Regiões Promotoras Genéticas , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
Gene promoters are frequently insufficient to drive the spatiotemporal patterns of gene expression during cell differentiation and organism development. Enhancers convey these properties through diverse mechanisms, including long-distance interactions with target promoters via their association with specific transcription factors. Despite unprecedented progress in the knowledge of enhancer mechanisms of action, there are still many unanswered questions. In particular, the contribution of an enhancer's local chromatin configuration to its mechanism of action is not completely understood. Here we describe a novel regulatory element, the Upstream Enhancer Element (UEE), which modulates the activity of the chicken α-globin 3' enhancer by regulating its chromatin structure, specifically by positioning a nucleosome upstream of the core enhancer. This element binds nuclear factors and confers a more restricted activation on the α-globin 3' enhancer, suggesting a progressive/rheostatic model for enhancer activity. Our results suggest that the UEE activity contributes to the positioning of a nucleosome that is necessary for the α-globin 3' enhancer activation.
