Acetylation of a Specific Promoter Nucleosome Accompanies Activation of the ɛ-Globin Gene by β-Globin Locus Control Region HS2

Alterations of chromatin structure accompany the activation of gene transcription in vivo during development and in response to hormones and other stimuli. In the erythroid lineage, the five DNase I hypersensitive sites (HSs) (HS1 to -5), which comprise the locus control region (LCR) of the β-globin gene cluster, appear early in embryogenesis before transcription of the globin genes commences (19, 23, 41). The formation of these sites is temporally correlated with chromatin changes over the entire locus in erythroid cells characterized by an increase in general sensitivity to DNase I and a change in replication timing from early to late in S phase (17). Moreover, high-level expression of the individual members of the β-globin gene family during the process of development depends upon the LCR (3). During the developmental stages when the individual globin genes are being actively transcribed, a DNase I HS is detected in the promoter (21). The appearance of DNase I HSs at the promoters of transcribed genes is indicative of altered chromatin structure associated with the binding of transcription factors to these regions (13). However, the alterations in chromatin structure at such sites may not be identical in every case. An early study of the chick β-globin gene showed that a short histone-free restriction fragment could be released from the promoter of the actively transcribing gene (30). In contrast, histones are present at the DNase I HSs of the induced mouse mammary tumor virus (MMTV) promoter (7, 40) and transcription factors and nucleosomes occupy the active albumin enhancer simultaneously (11). These observations raise the possibility that DNase I HSs are not necessarily devoid of nucleosomes (37). An abundance of data implicates histone modification as a critical component in the control of gene expression through localized and higher-order alterations in chromatin structure. For example, there is a marked coincidence of hyperacetylation, as revealed by anti-acetyl-lysine antibodies, and nuclease sensitivity within the domain of the transcriptionally active chick β-globin locus (22). Other recent reports describe histone modification that is more localized. Hyperacetylated histones may be confined to the promoters of yeast genes activated by the acetyltransferase Gcn5, and increased H3 and H4 acetylation attributed to CREB-binding protein (CBP) (also called p300) was found in a region that could accommodate two or three nucleosomes surrounding the induced beta interferon promoter (26, 31). Conversely, deacetylation of H4 within a region potentially spanning two promoter nucleosomes accompanies repression of certain yeast genes (25, 27, 34). It is likely that modification of histones alters their interaction with each other and with DNA in vivo as it does in vitro, providing at least part of the basis for nuclease sensitivity (46). Previous work in our laboratory using minichromosomes stably maintained in human cells indicated a series of positioned nucleosomes over the ɛ-globin gene. Transcription activation by the LCR HS2 enhancer alters the chromatin structure of the TATA-proximal N1 nucleosome in the promoter of the gene, creating a nuclease-sensitive site (20). However, the mechanism by which HS2 produces these effects has not been elucidated. To ask whether the promoter-proximal nucleosome is absent or altered under conditions of active transcription, we employed nucleoprotein electrophoresis and chromatin immunoprecipitation (ChIP) assays for linker histone H1 and for core histones H3 and H4 along with their acetylated variants. We also investigated the effect of trichostatin A (TSA)-mediated global histone acetylation on transcriptional activity of the ɛ-globin gene. These experiments indicate that N1 nucleoprotein complexes likely have altered properties, have reduced H1 levels compared to those of inactive promoters, and contain a subnucleosome-sized DNA component. Furthermore, a very specific and directed acetylation of H3 in the TATA-proximal nucleosome accompanies transcriptional activation. This modification requires a functional enhancer. Acetylation of H4 appears to be more widespread and is detected at two promoter nucleosomes spanning almost 400 bp of the 5′ ɛ-globin flanking sequence. Global histone acetylation in the absence of the HS2 enhancer is not sufficient for transcriptional activation, suggesting that enhancer function includes the recruitment of a specific acetylase activity responsible for high-level modification of H3 at the proximal promoter.