Abstract
The importance of histone H1 heterogeneity and total H1 stoichiometry in chromatin has been enigmatic. Here we report a detailed characterization of the chromatin structure of cells overexpressing either H1(0) or H1c. Nucleosome spacing was found to change during cell cycle progression, and overexpression of either variant in exponentially growing cells results in a 15-base pair increase in nucleosome repeat length. H1 histones can also assemble on chromatin and influence nucleosome spacing in the absence of DNA replication. Overexpression of H1(0) and, to a lesser extent, H1c results in a decreased rate of digestion of chromatin by micrococcal nuclease. Using green fluorescent protein-tagged H1 variants, we show that micrococcal nuclease-resistant chromatin is specifically enriched in the H1(0) variant. Overexpression of H1(0) results in the appearance of a unique mononucleosome species of higher mobility on nucleoprotein gels. Domain switch mutagenesis revealed that either the N-terminal tail or the central globular domain of the H1(0) protein could independently give rise to this unique mononucleosome species. These results in part explain the differential effects of H1(0) and H1c in regulating chromatin structure and function.
Highlights
The fundamental repeating unit of the eukaryotic chromatin is the nucleosome, which consists of an octamer of two molecules each of the core histones H2A, H2B, H3, and H4, and in higher eukaryotes, at least a single molecule of H1 histone [1, 2]
Overexpression of H1 Histone Variants Increases Nucleosome Spacing—micrococcal nuclease (MN) digestion of chromatin from H1-overexpressing cells or control cells reveals that the vast majority of material is organized in a regularly repeating nucleosomal ladder (Fig. 1A)
The total amount of H1 is estimated to be 1.2–1.4 times that of control cells [32]. These results indicate that overexpression of H1 histones to high levels does not lead to the formation of aberrant chromatin structures
Summary
Generation of Expression Vectors and Cell Lines—The H10- and H1coverexpressing cell lines, MTH10 and MTH1c, were described previously [26, 27]. For the overexpression of H1 histone variants under density arrest, cells were grown to confluence prior to induction with ZnCl2 as described above. Quantitation of GFP-tagged H1 Variants in the Micrococcal Nuclease-soluble and -insoluble Chromatin Fractions by Fluorometry—Nuclei from density-arrested cells expressing either H10GFP or H1cGFP were mixed with a 20-fold excess of control nuclei, digested with MN and fractionated as described above. The MN-insoluble chromatin pellet and the combined S1 and S2 fractions were extracted with high salt buffer (0.5 M KCl, 10 mM Tris-HCl, pH 7.2, 5 mM MgCl2, 0.5 mM phenylmethylsulfonyl fluoride, 2 mM EDTA, and 1 mM EGTA) for 30 min to extract all chromatin-bound H1 histones. For the NP patterns of chromatin from cells expressing H1GFP fusion proteins, the gels were first scanned in the blue fluorescence mode on a Storm fluorimager (Molecular Dynamics) to visualize GFP fluorescence from nucleosome species carrying bound H1GFP, and stained with EtBr and scanned on the fluorimager to visualize all nucleosome species
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