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Abstract: Histone H3K27 is acetylated or methylated in the environment of nuclear chromatin. Here, to characterize the modification pattern of H3K27 in locus control region (LCR) and to understand the correlation of various H3K27 modifications with transcriptional activity of genes, we analyzed the human β-globin locus using the ChIP assay. The LCR of the human β-globin locus was enriched by H3K27ac and H3K27me1 in erythroid K562 cells. The highly transcribed globin genes were hyperacetylated at H3K27, but the repressed globin genes were highly dimethylated at this lysine in these cells. However, in non-erythroid 293FT cells, the β-globin locus was marked by a high level of H3K27me3. EZH2 and SUZ12, subunits of polycomb repressive complex 2, were comparably detected with the H3K27me3 pattern in K562 and 293FT cells. In addition, H3K27ac, H3K27me1 and H3K27me3 were established in an enhancer-dependent manner in a model minichromosomal locus containing an enhancer and its target gene. Taken together, these results show that H3K27 modifications have distinctive correlations with the chromatin state or transcription level of genes and are influenced by an enhancer. [Copyright &y& Elsevier]

Analysis of physiological mechanisms that control transcription often requires extrapolation of in vitro measurements into in vivo mechanisms. This extrapolation is complex, as mammalian genes are commonly organized into broad chromosomal domains, and such domains cannot be readily reconstituted in vitro. Thus, the nucleoprotein structure of chromosomes constitutes a considerable impediment to elucidating transcriptional mechanisms. The development of assays to measure protein–DNA interactions and chromatin structure in living cells has greatly facilitated progress in understanding physiological transcriptional mechanisms. Chromatin immunoprecipitation (ChIP) is a powerful approach that allows one to define the interaction of factors with specific chromosomal sites in living cells, thereby providing a snapshot of the native chromatin structure and factors bound to genes in different functional states. ChIP involves treating cells or tissue briefly with formaldehyde to crosslink proteins to DNA. An antibody against a protein suspected of binding a given cis-element is then used to immunoprecipitate chromatin fragments. Polymerase chain reaction analysis of the immunoprecipitate with primers flanking the cis-element reveals whether a specific DNA sequence is recovered in an immune-specific manner and therefore whether the protein contacted the site in living cells. The central focus of this review is the use of ChIP to study transcriptional activation over long distances on chromosomes. [Copyright &y& Elsevier]