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

Abstract

On stably replicating episomes, transcriptional activation of the -globin promoter by the ␤-globin locus control region HS2 enhancer is correlated with an increase in nuclease sensitivity which is limited to the TATAproximal nucleosome (N1). To elucidate what underlies this increase in nuclease sensitivity and the link between chromatin modification and gene expression, we examined the nucleoprotein composition and histone acetylation status of transcriptionally active and inactive promoters. Micrococcal nuclease digestion of active promoters in nuclei released few nucleosome-like nucleoprotein complexes containing N1 sequences in comparison to results with inactive promoters. We also observed that N1 DNA fragments from active promoters are of a subnucleosomal length. Nevertheless, chromatin immunoprecipitation experiments indicate that histones H3 and H4 are present on N1 sequences from active promoters, with H3 being dramatically hyperacetylated compared with that from inactive promoters and vector sequences. Strikingly, H3 in the adjacent upstream nucleosome (N2) does not appear to be differentially acetylated in active and inactive promoters, indicating that the nucleosome modification of the promoter that accompanies transactivation by HS2 is highly directed and specific. However, global acetylation of histones in vivo by trichostatin A did not activate transcription in the absence of HS2, suggesting that HS2 contributes additional activities necessary for transactivation. N1 sequences from active promoters also contain reduced levels of linker histone H1. The detection of a protected subnucleosomal sized N1 DNA fragment and the recovery of N1 DNA sequences in immunoprecipitations using antiacetylated H3 and H4 antibodies argue that N1 is present, but in an altered conformation, in the active promoters.

Farnesyltransferase and geranylgeranyltransferase I inhibitors upregulate RhoB expression by HDAC1 dissociation, HAT association and histone acetylation of the RhoB promoter

“…Reactions were performed in a final volume of 400 ml in dilution buffer (0.01% SDS, 16.7 mM Tris-HCI (pH 8.1), 1.2 mM EDTA, 150 mM NaCl and 1% Triton X-100) plus 3 mg of the appropriate primary antibody and rotated overnight at 41C. HDAC1 and p300 antibodies were purchased from Santa Cruz Biotechnologies, anti-acetylatedhistone H3 monoclonal antibodies were purchased from Upstate Inc. (Chicago, IL, USA) (Benjamin and Jost, 2001;Chaya et al, 2001;Gui and Dean, 2001;Dey et al, 2003) whereas secondary anti-mouse antibody was purchased from Pierce Biotechnology Inc. (Rockford, IL, USA). Four hundred microliters of Protein A Sepharose beads (30 mg/ml in dilution buffer) was then added to each reaction and rotated for 2 h at 41C.…”

Retinoic Acid-Induced Chromatin Remodeling of Mouse κ Opioid Receptor Gene

“…P19 cells treated with vehicle (control) or 10 Ϫ6 M RA for the indicated days were cross-linked with 1% formaldehyde for 15 min, followed by the addition of 1.47 ml of 1 M glycine/10 ml culture to stop cross-linking. The procedures were performed with some modifications as described previously (Gui and Dean, 2001). The nuclei isolated from ϳ10 8 cells (four plates) were suspended in 1 ml of wash buffer (10 mM Tris-HCl, pH 7.4, 15 mM NaCl, 50 mM KCl, 0.15 mM spermine, 0.5 mM spermidine, and 8.5% sucrose) and digested with micrococcal nuclease (MNase; 30 -120 U; Worthington Biochemical, Freehold, NJ) or DNase I (2-30 mg/ml) for 6 min at 37°C.…”

Quantification of DNaseI-sensitivity by real-time PCR: quantitative analysis of DNaseI-hypersensitivity of the mouse β-globin LCR 1 1Edited by J. Karn

“…5 Recent studies have suggested that some DNaseI-hypersensitive sites, such as that of the human ε-globin promoter, may still be bound by a nucleosome but with an altered structure. 6 DNaseI-hypersensitivity is therefore a structural marker for the disruption of the regular nucleosomal array caused by the formation of specific nucleoprotein complexes by the co-operative binding of ubiquitous and tissue-specific transcription factors 7,8 to exclude or modify nucleosomes. The high-density of transcription factors makes it likely that such sequences will be cis-acting regulatory elements, such as promoters and enhancers.…”