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ChIP-chip analysis of HTZ-1 and RNA Polymerase II in wildtype (N2) embryos

ABSTRACT: In all eukaryotes, histone variants are incorporated into a subset of nucleosomes to create functionally specialized regions of chromatin. One such variant, H2A.Z, replaces histone H2A and is required for viability in all metazoans tested to date. However, the function of H2A.Z in chromatin organization, transcription, and development remains controversialunclear. We mapped the genome-wide distribution of the C. elegans H2A.Z ortholog HTZ-1 during embryogenesis by Chromatin ImmunoPrecipitation on DNA microarrays (ChIP-chip). We find that H2A.Z is incorporated upstream of approximatelybout 25% of C. elegans genes, preferentially upstream of genes required for development and occupied by RNA polymerase II. Fewer sites of HTZ-1 localization occur on the X chromosome relative to autosomes, consistent with the lack of essential genes on X. The data provide evidence for unexpectedly widespread independent regulation of genes within operons. In 37% of operons, HTZ-1 is incorporated upstream of internally encoded genes. In conjunction with ChIP-chip, we used genetic mutation, RNAi, and microscopy to establish that HTZ-1 is present in every cell, and that maternally supplied HTZ-1 is essential for normal development. We interpret these results to indicate that C. elegans HTZ-1 functions in establishing or maintaining an essential chromatin state at promoters regulated dynamically during development. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Keywords: chip-chip

ORGANISM(S): Caenorhabditis elegans

PROVIDER: GSE10201 | GEO | 2008/07/28

REPOSITORIES: GEO

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Project description:H2A.Z is a highly conserved histone variant involved in several key nuclear processes. It is incorporated into promoters by SWR-C-related chromatin remodeling complexes, but whether it is also actively excluded from non-promoter regions is not clear. Here, we provide genomic and biochemical evidence that RNA polymerase II (RNAPII) elongation-associated histone chaperones FACT and Spt6 both contribute to restricting H2A.Z from intragenic regions. In the absence of FACT or Spt6, the lack of H2A.Z eviction, coupled to its pervasive incorporation by mislocalized SWR-C, alters chromatin composition and contributes to cryptic initiation. Thus, chaperone-mediated H2A.Z removal is crucial for restricting the chromatin signature of gene promoters, which otherwise may license or promote cryptic transcription. We profiled H2A.Z occupancy in S. cerevisiae by ChIP-chip on tiling arrays in different mutants for chromatin remodelers and histone chaperones. In most experiments, H2A.Z ChIP samples (Cy5-labeled) were performed using a polyclonal antibody against H2A.Z and hybridized against H2B ChIP samples (Cy3-labeled), also performed using a polyclonal antibody. Temperature-sensitive mutants for the histone chaperones Spt16 and Spt6 (spt16-197 and spt6-1004 respectively) showed strong H2A.Z localization defects so the role of these factors in H2A.Z localization was further analyzed. H2A.Z ChIP-chip experiments in spt16-197 and spt6-1004 were repeated using different experimental designs [normalized against Input DNA or Mock (IgG) ChIP samples]. The contribution of Spt16 and Spt6 on H2A.Z localization was also confirmed by nuclear depletion of Spt16 and Spt6 using the anchor-away system. H2A.Z ChIP-chip experiments were also performed in sic1Δ and spt16-197/sic1Δ cells in order to rule out any G1-arrest artifact. H2A.Z ChIP-chip experiments were repeated using spike-in controls for normalization, revealing widespread H2A.Z occupancy in Spt16 and Spt6 mutants. In addition, the localization of the chromatin remodeler SWR-C was determined in wild type cells as well as in spt16-197 and spt6-1004 cells. Finally, we also profiled histone H4 occupancy by ChIP-chip in wild type, spt16-197, spt6-1004, spt16-197/htz1Δ and spt6-1004/htz1Δ cells. All ChIP-chip experiments were done in duplicates. Each microarray was normalized using the Lima Loess and replicates were combined using a weighted average method as previously described (Pokholok et al., 2005).

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ChIP-chip analysis of HTZ-1 and RNA Polymerase II in wildtype (N2) embryos

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