Abstract
The effects of inhibiting histone deacetylation on the maturation of newly replicated chromatin have been examined. HeLa cells were labeled with [3H]thymidine in the presence or absence of sodium butyrate; control experiments demonstrated that butyrate did not significantly inhibit DNA replication for at least 70 min. Like normal nascent chromatin, chromatin labeled for brief periods (0.5-1 min) in the presence of butyrate was more sensitive to digestion with DNase I and micrococcal nuclease than control bulk chromatin. However, chromatin replicated in butyrate did not mature as in normal replication, but instead retained approximately 50% of its heightened sensitivity to DNase I. Incubation of mature chromatin in butyrate for 1 h did not induce DNase I sensitivity: therefore, the presence of sodium butyrate was required during replication to preserve the increased digestibility of nascent chromatin DNA. In contrast, sodium butyrate did not inhibit or retard the maturation of newly replicated chromatin when assayed by micrococcal nuclease digestion, as determined by the following criteria: 1) digestion to acid solubility, 2) rate of conversion to mononucleosomes, 3) repeat length, and 4) presence of non-nucleosomal DNA. Consistent with the properties of chromatin replicated in butyrate, micrococcal nuclease also did not preferentially attack the internucleosomal linkers of chromatin regions acetylated in vivo. The observation of a novel chromatin replication intermediate, which is highly sensitive to DNase I but possesses normal resistance to micrococcal nuclease, suggests that nucleosome assembly and histone deacetylation are not obligatorily coordinated. Thus, while deacetylation is required for chromatin maturation, histone acetylation apparently affects chromatin organization at a level distinct from that of core particle or linker, possibly by altering higher order structure.
Highlights
Experiments demonstrated that buryrate did not sig- One meansof distinguishing among theabove possibilities nificantly inhibit DNA replication forat least 70 min. is touse specific inhibitors of cellular metabolism, in order to
Chromatin labeled foruncouple a selected process from the manyrequired for chrobrief periods (0.5-1 min) in the presence of butyrate was more sensitive to digestion withDNase I and micrococcal nuclease than control bulk chromatinH. owever, chromatin replicated in butyratedid not mature as in normal replication, but instead retaine-d50% of its heightenedsensitivitytoDNase I
Incubation of mature chromatin in butyrate for 1 h did not induce DNase I sensitivity: the presenceof sodium butyrate was required during replication to preserve the increased digestibilityof nascent chromatinDNA
Summary
Cell Culture and Labeling-HeLa cells were maintained in spinner alteration innuclease accessibility are poorly understood; the culture at 37 “C in Eagle’s minimal essential medium supplemented complex processes involved in chromatin biosynthesis prob- with 5% calf serum. Ably involve several structural modifications that contribute Long term labeling of cells with [14C]thymidine Cells ( “ 4 X 105/ml) were harvested by centrifugation (250 X g; 1.5 min) and concentrated 20-fold (0.5-1-min pulse) or 5-. This article must be pCi/ml; labeling in the presence of cycloheximide was performed a t hereby marked “aduertisement” in accordance with 18U.S.C. Section 4 pCi/ml. Deacetylation Is Required for ChrMomaatutirnation performed at 0.3-0.8 mCi/ml, after concentrating cells 20-30-fold in chromatinsamples were adjustedto mM magnesium acetate, prewarmed whole medium.
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