Abstract
We have examined salt-soluble chromatin released by micrococcal nuclease from a 15-day-old chicken embryo erythrocyte nuclei for histone acetyltransferase (HAT) activities. This chromatin is enriched in transcriptionally active sequences from within the active beta-globin locus and contains elevated levels of acetylated core histones. HAT activities present in this fraction target histones H4, H3, and H2A when the chromatin itself is used as the substrate. In gel HAT activity assay demonstrates that the salt-soluble chromatin fraction contains four acetyltransferase molecules distinguished by their different molecular masses (47, 33, 32, and 28 kDa). Further separation of the chromatin by centrifugation through sucrose gradients shows that the acetyltransferases segregate into chromatin-bound and chromatin-free populations. The 32- and 28-kDa HATs are associated with chromatin, whereas the 47- and 33-kDa HAT molecules are not. The chromatin-bound HAT activities predominantly target H4 to give the diacetyl and triacetyl species; some acetylation of H2A can also be seen. Our results suggest that the chromatin-associated acetyltransferases have a role in gene regulation.
Highlights
Acetyltransferase (HAT)1 activity [12] and could acetylate the core histones as the transcription complex passes along the template
We have examined salt-soluble chromatin released by micrococcal nuclease from a 15-day-old chicken embryo erythrocyte nuclei for histone acetyltransferase (HAT) activities
Salt-soluble Chromatin Contains HAT Activity—Nuclei were prepared from 15-day-old chicken embryo erythrocytes and subsequently digested with micrococcal nuclease and lysed with EDTA to release approximately 50% of the DNA content into the supernatant
Summary
Preparation of Chromatin—Nuclei were prepared from 15-day-old chicken embryos as described previously [4] but by using 10 mM NaCl in cell lysis and nuclear wash buffers. The pellet was resuspended in a lysis buffer (5 mM Na3EDTA, 10 mM Tris-HCl, pH 7.5, 10 mM sodium butyrate, 0.1 mM PMSF, 0.1 mM benzamidine), incubated on ice for 60 min, and centrifuged as above retaining supernatant S2. Salt-soluble chromatin was either dialyzed overnight against 10 mM Tris-HCl, pH 7.5, 10 mM sodium butyrate, 0.25 mM Na3EDTA, 0.1 mM PMSF, 0.1 mM benzamidine or was loaded directly onto a 5–25% linear sucrose gradient in the same buffer and centrifuged at 36,000 rpm in a Beckman SW 41ti rotor for 14 h at 4 °C. HAT Assays—To assay for chromatin-bound acetyltransferase activity in chromatin samples or across sucrose gradients, 50 l of each sample was taken and made into a final concentration of 50 mM TrisHCl, pH 7.9, 10 mM sodium butyrate To this buffer, 0.1 Ci of [3H]acetyl-CoA (2–10 Ci/mmol, Amersham Pharmacia Biotech) was added, and the sample was incubated at 37 °C for 60 min. Filters were blotted dry and exposed to PhosphorImager screens (Molecular Dynamics, Sunnyvale, CA)
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