HMG-D and histone H1 alter the local accessibility of nucleosomal DNA

Abstract

There is evidence that HMGB proteins facilitate, while linker histones inhibit chromatin remodelling, respectively. We have examined the effects of HMG-D and histone H1/H5 on accessibility of nucleosomal DNA. Using the 601.2 nucleosome positioning sequence designed by Widom and colleagues we assembled nucleosomes in vitro and probed DNA accessibility with restriction enzymes in the presence or absence of HMG-D and histone H1/H5. For HMG-D our results show increased digestion at two spatially adjacent sites, the dyad and one terminus of nucleosomal DNA. Elsewhere varying degrees of protection from digestion were observed. The C-terminal acidic tail of HMG-D is essential for this pattern of accessibility. Neither the HMG domain by itself nor in combination with the adjacent basic region is sufficient. Histone H1/H5 binding produces two sites of increased digestion on opposite faces of the nucleosome and decreased digestion at all other sites. Our results provide the first evidence of local changes in the accessibility of nucleosomal DNA upon separate interaction with two linker binding proteins.

Figure 1

HMG-D and histone H1/H5 binding to nucleosomes induces changes in nucleosome structure. (A) Schematic of the restriction enzyme sites on nucleosomes reconstituted on the 601.2 sequence [adapted with permission from fig. 1A in Anderson et al. (3)]. (B) Means and standard deviations for the ratio of the rate of digestion of protein-bound nucleosomes to unbound nucleosomes are plotted as a function of the location on the nucleosome. Means are results of a least three independent assays. Full-length HMG-D, black bars; histone H1/H5, grey bars; HMG-D100, open bars and hatched bars. The hatched bars indicate sites at which HMG-D100 inhibits restriction enzyme digestion to the extent that no significant rate could be calculated. HMG-D74 (data not shown) has little or no effect on digestion. (C) Means and standard deviations as in (B) for H1/H5 bound versus unbound nucleosomes.

Figure 2

(A) Nucleosomes reconstituted on the 601.2 positioning sequence do not have intrinsic mobility. Aliquots of 50 fmol of nucleosomes, reconstituted on the 601.2 sequence positioning sequence in the presence of 0.5 µM HMG-D, 40 nM histone H1/H5 or 0.4 µM HMGB1, were resolved on native 8% polyacrylamide gels at 4°C before incubation of half gel slices at 4 or 37°C for 1 h as indicated, followed by second dimension electrophoresis. Arrows indicate the directions of electrophoresis. Nucleosome mobility would be observed as nucleoprotein complexes migrating away from the diagonal, illustrated by the arrowheads. The asterisk indicates complexes of HMG-D with naked DNA. (B) The influence of HMG-D, histone H1/H5 and HMGB1 on nucleosome mobility. The assay was conducted as described above, with 50 fmol of nucleosomes reconstituted on the sea urchin 5S positioning sequence. Arrowheads indicate nucleosomes that have moved away from the diagonal and hence are mobile. Unlike H1/H5 and HMGB1, HMG-D does not restrict the intrinsic mobility of nucleosomes.

Figure 2

(A) Nucleosomes reconstituted on the 601.2 positioning sequence do not have intrinsic mobility. Aliquots of 50 fmol of nucleosomes, reconstituted on the 601.2 sequence positioning sequence in the presence of 0.5 µM HMG-D, 40 nM histone H1/H5 or 0.4 µM HMGB1, were resolved on native 8% polyacrylamide gels at 4°C before incubation of half gel slices at 4 or 37°C for 1 h as indicated, followed by second dimension electrophoresis. Arrows indicate the directions of electrophoresis. Nucleosome mobility would be observed as nucleoprotein complexes migrating away from the diagonal, illustrated by the arrowheads. The asterisk indicates complexes of HMG-D with naked DNA. (B) The influence of HMG-D, histone H1/H5 and HMGB1 on nucleosome mobility. The assay was conducted as described above, with 50 fmol of nucleosomes reconstituted on the sea urchin 5S positioning sequence. Arrowheads indicate nucleosomes that have moved away from the diagonal and hence are mobile. Unlike H1/H5 and HMGB1, HMG-D does not restrict the intrinsic mobility of nucleosomes.