Micronuclei and the cytoplasm of growing Tetrahymena contain a histone acetylase activity which is highly specific for free histone H4.

Abstract

Abstract. Salt extracts prepared from purified micronuclei and the cytoplasm of growing Tetrahymena contain a histone acetylase (also referred to as histone acetyltransferase) activity which is highly specific for H4 when tested as a free historic. With both extracts, H4 is acetylated first at position 4 (monoacetylated) or positions 4 and I1 (diacetylated), sites diagnostic of deposition-related acetylation of newly synthesized H4 in vivo. As the concentration of cytosolic extract is decreased in the in vitro reactions, acetylation of H3 is also observed. Neither activity acetylates histone in a chromatin form.These activities are distinct from a macronuclear acetylase which acetylates H3 and H4 (macro-or micronuclear) equally well as free histones and which acetylates all four core histones when mononucleosomes are used as substrate. As well, the micronuclear and cytoplasmic activities give similar thermalinactivation profiles which are different from that of the macronuclear activity. In situ enzyme assays demonstrate a macronuclear-specific activity which acetylates endogenous macronuclear chromatin and an independent micronuclear-cytosolic activity which is able to act upon exogenously added free H4. These results argue strongly that an identical acetylase is responsible for the micronuclear and cytoplasmic activity which is either modified or altogether distinct from that in macronuclei.TUDIES in vivo have demonstrated that at least two general systems of historic acetylation exist in the ciliated protozoan, Tetrahymena. One system, which we will refer to as "transcription-related" acetylation, is postsynthetic and affects all of the inner (non-H1) histones in transcriptionally active macronuclei. This system does not appear to exist in transcriptionally inactive micronuclei (Vavra et al., 1982;. In contrast, a second system, which we will refer to as "deposition/replicationrelated" acetylation, requires protein synthesis and affects histones H3 and H4, regardless of whether they are destined for macro-or micronuclei (Allis et al., 1985).In an attempt to understand further the biological function of histone acetylation as it relates to both transcription and chromatin assembly, we have begun to analyze the enzyme system(s) responsible for carrying out histone acetylation in Tetrahymena. Recently, we described results which suggest that a single histone acetylase (also referred to as histone acetyltransferase) extracted from purified macronuclei catalyzes both transcription-and deposition-related acetylation, depending upon the substrate used in the reaction (Chicoine et al., 1987). When mononucleosomes are used as a substrate for this activity, all four core histones are acetylated in a manner indistinguishable from postsynthetic transcriptionrelated acetylation. However, when free histories are used as a substrate, the same activity acetylates mainly H3 and H4 in a manner surprisingly similar to deposition/replicationrelated acetylation.Although having both of these biologically important acetylations car...

Dissecting the Molecular Roles of Histone Chaperones in Histone Acetylation by Type B Histone Acetyltransferases (HAT-B)

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Recruitment of the Type B Histone Acetyltransferase Hat1p to Chromatin Is Linked to DNA Double-Strand Breaks

“…Consistent with its classification as a type B histone acetyltransferase, Hat1p is highly specific for histones that are free in solution with no demonstrable activity when chromatin-associated histones are used as a substrate. In addition, the enzyme is specific for histone H4 lysine residues 5 and 12, which are the sites of acetylation found on the newly synthesized pool of histone H4 in a wide variety of eukaryotic organisms (11,13,20,22,30,34,42,50). Hence, Hat1p has the requisite biochemical activities to function in the acetylation of newly synthesized histone H4.…”

Hat1: the emerging cellular roles of a type B histone acetyltransferase

“…Type A histone acetyltransferases possess the properties normally associated with chromatin-modifying enzymes as they are localized to the nucleus and are capable of modifying histones that are incorporated into chromatin. An extensive series of biochemical fractionation studies identified type B histone acetyltransferases as a distinct class of enzymes that can be found in the cytoplasm and, most importantly, have the ability to acetylate free, but not nucleosomal, histones (Garcea and Alberts, 1980;Sures and Gallwitz, 1980;Wiegand and Brutlag, 1981;Richman et al, 1988;Lopez-Rodas et al, 1991a, b;Mingarro et al, 1993;Brownell and Allis, 1996). Therefore, these enzymes likely modify histones before their assembly into chromatin rather than directly acting on preexiting chromatin structures.…”