Histone H3 thiol reactivity and acetyltransferases in chicken erythrocyte nuclei

Abstract

Chicken erythrocyte nuclei previously incubated separately with two novel mercury compounds (N-chloromercuribenzoyl)-biocytin and bis(p-(chloromercuribenzoyl))-[3H]lysine diamide) were digested with micrococcal nuclease and the digest products fractionated according to their solubility in 0.15 M NaCl and molecular size. The identity and quantitation of the chromatin fractions and proteins containing covalently bound mercury were determined by Western blotting, autoradiography, and scintillation counting. The most highly acetylated species of histone H3 in the 0.15 M NaCl-soluble polynucleosome fraction also contained the highest proportion of bound mercury. This fraction contains hyperacetylated core histones, is depleted in linker histones, and enriched in nonhistone proteins. Histone H3 in the 0.15 M NaCl-soluble mononucleosomes, which are unacetylated and lack linker histones, was 45% less labeled than histone H3 in the 0.15 M NaCl-soluble polynucleosome fraction. In the 0.15 M NaCl-insoluble polynucleosomes, which contain unacetylated histones and molar proportions of linker histones, histone H3 was 63% less labeled. Allowing for the differential abundance of these subfractions in the nucleus, the relative H3 reactivities are 50, 7, and 1 for 0.15 M NaCl-soluble polynucleosomes, mononucleosomes, and 0.15 M NaCl-insoluble polynucleosomes, respectively. Thus a gradation of reactivities exists which correlates with increasing hyperacetylation and linker histone depletion. High mobility group proteins 1 and 2, found in subnucleosome particles in the 0.15 M NaCl-soluble fraction, are extensively mercury-labeled. Distribution of histone acetyltransferase activity among salt- and size-resolved micrococcal nuclease produced fractions was almost 5-fold greater in the 0.15 M NaCl-soluble supernatant than in the 0.15 M NaCl-insoluble pellet. Furthermore, the acetyltransferase activity, which is tightly bound to undigested chromatin, is rapidly released by both micrococcal nuclease and DNase I. For short digestion times the enzyme is associated with the salt-soluble polynucleosomes, but at longer times of digestion the enzyme appears to be free from intact nucleosomes. The enzyme may be localized in the globin domain in erythrocytes and maintains that region in a hyperacetylated state which results in an altered linker histone binding reflected in a change in the reactivity of the usually inaccessible H3 cysteine 110.