ABSTRACT
We have designed a zebrafish genomic microarray to identify DNA-protein interactions in the proximal promoter regions of over 11,000 zebrafish genes. Using these microarrays, together with chromatin immunoprecipitation with an antibody directed against tri-methylated lysine 4 of Histone H3, we demonstrate the feasibility of this method in zebrafish. This approach will allow investigators to determine the genomic binding locations of DNA interacting proteins during development and expedite the assembly of the genetic networks that regulate embryogenesis.
Subject(s)
Gene Expression Regulation, Developmental , Genes/genetics , Promoter Regions, Genetic/genetics , Zebrafish/embryology , Zebrafish/genetics , Animals , Chromatin Immunoprecipitation , Embryo, Nonmammalian/embryology , Embryo, Nonmammalian/metabolism , Genomics/methods , Histones/genetics , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain Reaction , Zebrafish/metabolismABSTRACT
Polycomb group proteins are essential for early development in metazoans, but their contributions to human development are not well understood. We have mapped the Polycomb Repressive Complex 2 (PRC2) subunit SUZ12 across the entire nonrepeat portion of the genome in human embryonic stem (ES) cells. We found that SUZ12 is distributed across large portions of over two hundred genes encoding key developmental regulators. These genes are occupied by nucleosomes trimethylated at histone H3K27, are transcriptionally repressed, and contain some of the most highly conserved noncoding elements in the genome. We found that PRC2 target genes are preferentially activated during ES cell differentiation and that the ES cell regulators OCT4, SOX2, and NANOG cooccupy a significant subset of these genes. These results indicate that PRC2 occupies a special set of developmental genes in ES cells that must be repressed to maintain pluripotency and that are poised for activation during ES cell differentiation.
Subject(s)
Carrier Proteins/metabolism , Gene Expression Regulation, Developmental , Stem Cells/physiology , Animals , Carrier Proteins/genetics , Cells, Cultured , Gene Expression Profiling , Humans , Multiprotein Complexes , Neoplasm Proteins , Nuclear Proteins , Oligonucleotide Array Sequence Analysis , Polycomb Repressive Complex 2 , Protein Subunits/genetics , Protein Subunits/metabolism , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , Signal Transduction/physiology , Stem Cells/cytology , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
Eukaryotic genomes are packaged into nucleosomes whose position and chemical modification state can profoundly influence regulation of gene expression. We profiled nucleosome modifications across the yeast genome using chromatin immunoprecipitation coupled with DNA microarrays to produce high-resolution genome-wide maps of histone acetylation and methylation. These maps take into account changes in nucleosome occupancy at actively transcribed genes and, in doing so, revise previous assessments of the modifications associated with gene expression. Both acetylation and methylation of histones are associated with transcriptional activity, but the former occurs predominantly at the beginning of genes, whereas the latter can occur throughout transcribed regions. Most notably, specific methylation events are associated with the beginning, middle, and end of actively transcribed genes. These maps provide the foundation for further understanding the roles of chromatin in gene expression and genome maintenance.
Subject(s)
Gene Expression Regulation, Fungal/genetics , Genome, Fungal , Histones/metabolism , Nucleosomes/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Acetylation , Chromosome Mapping/methods , Genes, Regulator/genetics , Histones/genetics , Methylation , Nucleosomes/genetics , Oligonucleotide Array Sequence Analysis , Transcriptional Activation/geneticsABSTRACT
Hormones and nutrients often induce genetic programs via signaling pathways that interface with gene-specific activators. Activation of the cAMP pathway, for example, stimulates cellular gene expression by means of the PKA-mediated phosphorylation of cAMP-response element binding protein (CREB) at Ser-133. Here, we use genome-wide approaches to characterize target genes that are regulated by CREB in different cellular contexts. CREB was found to occupy approximately 4,000 promoter sites in vivo, depending on the presence and methylation state of consensus cAMP response elements near the promoter. The profiles for CREB occupancy were very similar in different human tissues, and exposure to a cAMP agonist stimulated CREB phosphorylation over a majority of these sites. Only a small proportion of CREB target genes was induced by cAMP in any cell type, however, due in part to the preferential recruitment of the coactivator CREB-binding protein to those promoters. These results indicate that CREB phosphorylation alone is not a reliable predictor of target gene activation and that additional CREB regulatory partners are required for recruitment of the transcriptional apparatus to the promoter.
