The establishment of epigenetic marks, such as methylation on histone tails, is mechanistically linked to RNA polymerase II within active genes. To explore the interplay between these modifications in transcribed noncoding as well as coding sequences, we analyzed epigenetic modification and chromatin structure at high resolution across 300 kb of human chromosome 11, including the ␤-globin locus which is extensively transcribed in intergenic regions. Monomethylated H3K4, K9, and K36 were broadly distributed, while hypermethylated forms appeared to different extents across the region in a manner reflecting transcriptional activity. The trimethylation of H3K4 and H3K9 correlated within the most highly transcribed sequences. The H3K36me3 mark was more broadly detected in transcribed coding and noncoding sequences, suggesting that K36me3 is a stable mark on sequences transcribed at any level. Most epigenetic and chromatin structural features did not undergo transitions at the presumed borders of the globin domain where the insulator factor CTCF interacts, raising questions about the function of the borders.The amino-terminal tails of histones are subject to various posttranscriptional modifications in eukaryotic cells, including acetylation, methylation, phosphorylation, and ubiquitinylation (5). These modifications have the potential to alter chromatin structure and affect chromatin function. They may also act in a concerted fashion to provide a code by which downstream events related to transcription activation and repression are initiated or maintained and epigenetic information is transmitted to subsequent cell generations (17, 62). The acetylation of lysine residues in histone tails is usually associated with active genes, while the methylation of lysine residues can be associated with active or repressed genes (30, 55). Furthermore, lysines residues can be mono-, di-, or trimethylated. The functional interplay of the different levels of histone methylation has been the focus of considerable recent interest (33,39).Methylation can occur on four lysine residues in the H3 tail: K4, K9, K27, and K36. The methylation of K4 and K36 marks active chromatin. In the yeast Saccharomyces cerevisiae, Set1 and Set2, which methylate H3K4 and K36, respectively, are associated with different phosphorylated forms of the carboxyterminal heptad repeat domain (CTD) of RNA polymerase II (pol II). Set1 associates with the initiation and early elongation-competent pol II phosphorylated at Ser5 (31, 44). Accordingly, K4me2 and me3 peak early in active genes; however, me2 is also found at inactive but potentially active genes (6, 42, 44, 51, 53, 54). K36me2 and me3 marks peak toward the 3Ј end of transcribed coding sequences, consistent with the association of Set2 with the processively elongating Ser2 phosphorylated form of pol II (2,32,36,66). Mammalian homologues of these methylases have been characterized, and the human H3 K36 methylase HYPB has been reported to interact with hyperphosphorylated pol II (14,60). In contrast to K4 and...

Gene activation requires alteration of chromatin structure to facilitate active transcription complex formation at a gene promoter. Nucleosome remodeling complexes and histone modifying complexes each play unique and interdependent roles in bringing about these changes. The role of distant enhancers in these structural alterations is not well understood. We studied nucleosome remodeling and covalent histone modification mediated by the ␤-globin locus control region HS2 enhancer at nucleosome-level resolution throughout a 5.5-kb globin gene model locus in vivo in K562 cells. We compared the transcriptionally active locus to one in which HS2 was inactivated by mutations in the core NF-E2 sites. In contrast to inactive templates, nucleosomes were mobilized in discrete areas of the active locus, including the HS2 core and the proximal promoter. Large differences in restriction enzyme accessibility between the active and inactive templates were limited to the regions of nucleosome mobilization, which subsumed the DNase I hypersensitive sites. In contrast to this discrete pattern, histone H3 and H4 acetylation and H3 K4 methylation were elevated across the entire active locus, accompanied by depletion of linker histone H1. The coding region of the gene differed from the regulatory regions, demonstrating both nucleosome mobilization and histone hyperacetylation, but lacked differences in restriction enzyme accessibility between transcriptionally active and inactive genes. Thus, although the histone modification pattern we observe is consistent with the spreading of histone modifying activity from the distant enhancer, the pattern of nucleosome mobilization is more compatible with direct contact between an enhancer and promoter.During gene activation, ATP-dependent nucleosome remodeling complexes and complexes that covalently modify histones, such as acetyl transferases (HATs) and methyl transferases, alter the repressive nucleosomal structure of chromatin and provide a permissive environment for transcription. Remodeling by ATP-dependent complexes such as yeast SWI/ SNF and ISWI results in a positional change (mobilization or sliding) and/or a conformational (structural) change in nucleosomes in vitro (54). Histone acetylation modifies lysine residues in the N-terminal tails of the core histones H3 and H4, decreasing the stability of histone-DNA interactions and loosening the compaction of nucleosomal arrays (7). The structural effects of histone methylation are less clear and can be associated with activation (H3 K4 methylation) or repression (H3

The mammalian ␤-globin loci each contain a family of developmentally expressed genes, and a far upstream regulatory element, the locus control region (LCR). In adult murine erythroid cells, the LCR and the transcribed ␤-globin genes exist within domains of histone acetylation and RNA polymerase II (pol II) is associated with them. In contrast, the silent embryonic genes lie between these domains within hypoacetylated chromatin, and pol II is not found there. We used chromatin immunoprecipitation and real-time PCR to analyze histone modification and pol II recruitment to the globin locus in human erythroid K562 cells that express the embryonic -globin gene but not the adult ␤-globin gene. H3 and H4 acetylation and H3 K4 methylation were continuous over a 17-kb region including the LCR and the active -globin gene. The level of modification varied directly with the transcription of the -globin gene. In contrast, this region in nonerythroid HeLa cells lacked these modifications and displayed instead widespread H3 K9 methylation. pol II was also detected continuously from the LCR to the -globin gene. These studies reveal several aspects of chromatin structure and pol II distribution that distinguish the globin locus at embryonic and adult stages and suggest that both enhancer looping and tracking mechanisms may contribute to LCR-promoter communication at different developmental stages. The human ␤-globin locus consists of a family of erythroid specific genes and a far upstream regulatory element termed the locus control region (LCR) (1). The genes are expressed sequentially during development, beginning with the -globin gene, which is closest to the LCR, and proceeding to the more distant genes. This organization is mirrored in the murine globin locus. Homologous recombination studies that deleted the LCR in its natural chromosomal context show that the LCR is required for high-level expression of all of the genes, thus fulfilling at a minimum the definition of an enhancer (2-4). There is a different spatial arrangement of the genes in the chicken globin locus, with the adult ␤-globin genes flanked by the two embryonically expressed genes and a strong bidirectional enhancer located internally within the locus between the adult ␤-globin gene and the embryonic -globin gene (5, 6).Current views of gene regulation incorporate the concept that two types of complexes participate in the establishment of a chromatin structure which is accessible for transcription factors and the RNA polymerase II (pol II) transcriptional machinery (7,8). Nucleosome remodeling complexes of the SWI͞SNF type use the energy of ATP hydrolysis to alter nucleosome structure and͞or move nucleosomes along the chromatin fiber (9). Other enzymatic complexes covalently modify the N-terminal tails of histones by acetylation, methylation, phosphorylation, and ubiquitinylation (10). Core histone H3 and H4 acetylation and H3 K4 methylation are modifications strongly associated with active chromatin (11-13). Conversely, H3 K9 methylation and the presence of ...