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Gene
2002 Jun 12;2921-2:173-81. doi: 10.1016/s0378-1119(02)00675-3.
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Histone H1 variants differentially inhibit DNA replication through an affinity for chromatin mediated by their carboxyl-terminal domains.
De S
,
Brown DT
,
Lu ZH
,
Leno GH
,
Wellman SE
,
Sittman DB
.
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Multiple forms of histone H1 are found in most mammalian tissues, and diversity in their temporal and spatial expression likely corresponds to diversity in function. Here, using Xenopus egg extracts, we show that while the somatic H1s significantly inhibit DNA replication in Xenopus sperm nuclei, little or no inhibition is seen in the case of the testes-specific variant, H1t. We suggest that differences in H1-chromatin interactions might explain some of the diversity in H1 function. To demonstrate this, we show that the somatic H1 variants preferentially assemble into chromatin relative to H1t. Differences in chromatin structure are seen depending on whether chromatin assembly occurs in the presence of somatic H1s or H1t. These data suggest that the mechanistic basis for some of the functional differences of H1 variants lies in their relative affinity for chromatin. Using a series of domain-switch mutants of H1(0) and H1t we identify the H1 carboxyl-terminal domains as the domains responsible for the differential affinity for chromatin and, concurrently, for the differential effects of H1 variants upon DNA replication.
Fig. 1. Effect of H10 and H1t upon DNA replication. Xenopus sperm nuclei (3 ng DNA/μl extract) were incubated in the absence or presence of increasing concentrations of either H10 or H1t. Error bars represent replication assays performed in a minimum of three different egg extracts to account for extract-to-extract variations.
Fig. 2. Association of H1 variants with Xenopus sperm chromatin. Xenopus sperm nuclei were incubated in egg extract containing H10 (A) or H1t (B). Bound histones were acid extracted and analysed by SDS–PAGE. The relative amounts of H10 and H1t bound are shown (C). In competitive binding assays, Xenopus sperm nuclei were incubated in extract containing either pairs of somatic H1 variants (D), or one somatic variant and H1t (E). Bound histones were acid extracted and analysed by SDS–PAGE. The amounts of different H1 variants bound to Xenopus sperm nuclei are shown (F). The x-axis represents the input concentration of each of the two variants used in an assay. Error bars represent binding assays of different sets of variants performed in at least three different extracts.
Fig. 3. Effect of H10 and H1t on nucleosome spacing. Xenopus sperm nuclei were incubated in egg extract with or without H10 or H1t. Remodeled chromatin was subjected to a limited MN digest, resolved on a 1.8% Metaphor agarose gel, and stained with Vistra Green. DNA bands corresponding to mono-, di-, tri-, tetra-, and pentanucleosomes are shown (black arrows).
Fig. 4. Effect of H10 and H1t on decondensation of Xenopus sperm chromatin. Xenopus sperm nuclei were incubated for 3, 15 and 30 min either in buffer (XSN) or egg extract (XSN+ELSS) with or without H10 or H1t. All incubations were carried out in the same extract. Remodeled chromatin was visualized using Hoechst 33258.
Fig. 5. Domain-switch hybrids of H10 and H1t. A schematic of the structural domains of H10 and H1t showing the number of amino acids in each domain is shown (A). Expressed hybrids were analysed by SDS–PAGE to show purity (B).
Fig. 6. Effect of H1 structural domains upon DNA replication. Xenopus sperm nuclei were incubated at 3 ng DNA/μl extract in the absence or presence of increasing concentrations of H10, H1t, as well as domain-switch hybrids of these variants. Results are sorted according to replications performed in the presence of H10 and hybrids containing H10 C-termini (A) and H1t and hybrids containing H1t C-termini (B). Error bars represent replication assays performed in a minimum of three different egg extracts to account for extract-to-extract variations.
Fig. 7. Effect of H10 and H1t domain-switch hybrids on decondensation of Xenopus sperm chromatin. Xenopus sperm nuclei were incubated for 3, 15 and 30 min either in buffer (XSN) or egg extract (XSN+ELSS), in the absence or presence of H10, H1t and their domain-switch hybrids. All incubations were carried out in the same egg extract. Remodeled chromatin was visualized using Hoechst 33258.
