Mechanisms of long-range transcriptional regulation: Histone acetylation and the beta-globin locus

This thesis investigates the role of histone acetylation in the regulation of β-globin gene expression. The goal has been to increase the understanding of the mechanisms of long-range transcriptional regulation of chromatin templates. The β-globin locus contains the developmentally regulated and tissue-specific β-globin genes and an upstream regulatory element, the locus control region (LCR), and has been used extensively as a model for transcriptional regulation. We hypothesized that istone acetylation, a key transcriptional regulatory mechanism, plays an important role in the control of β-globin gene expression.; Identification of histone acetylase (HAT) containing complexes in human erythroleukemia cells by gel filtration chromatography followed by a HAT activity assay revealed that HATs exist in a variety of protein complexes in erythroid cells. The HAT CBP/p300 can interact with the hematopoietic transcription factor NF-E2, and NF-E2 has been implicated in LCR function. We showed that NF-E2 binds directly to important regulatory elements within the β-globin LCR and proposed that NF-E2 activates β-globin gene expression by recruiting a CBP/p300-containing complex to the LCR. Thus, we assessed the influence of a CBP/p300 inhibitor, the adenoviral protein E1A, on LCR function. EIA inhibited LCR-mediated transactivation of β-globin promoters, providing strong evidence for a role of CBP/p300 in the control of β-globin gene expression.; To understand the consequences of HAT recruitment to the β-globin locus, we defined the histone acetylation pattern of the endogenous murine locus in definitive erythroid cells. Hyperacetylation was detected at the LCR and active promoters, whereas the inactive promoters resided in a ∼30 kb hypoacetylated subdomain. Histone deacetylase inhibition selectively increased acetylation at an inactive and hypoacetylated promoter, suggesting that active deacetylation contributes to silencing of this promoter. Analysis of the acetylation pattern at an earlier developmental stage revealed hyperacetylation of both active and inactive promoters. Thus, the acetylation pattern varied at different developmental stages. We propose that dynamic histone acetylation and deacetylation play an important role in the tissue-specific and developmental regulation of β-globin gene expression.