Differential association of HMG1 and linker histones B4 and H1 with dinucleosomal DNA: structural transitions and transcriptional repression.

Abstract

Health and Human Development. NIH. Bldg 6. Room Bethesda. MD 2t)892-2710. USA We examined the structural and functional consequences of incorporating either histone HI, histone B4 or HMG1 into a synthetic dinucleosome containing two 5S rRNA genes. We found that all three proteins bind to linker DNA, stabilizing an additional 20 bp from micrococcal nuclease digestion and restrict nucleosome mobility. Histone HI has the highest-affinity interaction with the dinucleosome; histone B4 and HMG1 associate with significantly reduced affinities. We found that histone Hi binds to the dinucleosome template with a dissociation constant (KD) of 7.4 nM, whereas the KD is 45 nM for histone B4 and 300 nM for HMG1. The KDS for the interaction of these proteins with naked DNA are 18 nM for Hi, 80 nM for B4 and 300 nM for HMG1. The differences in association of these proteins with the dinucleosome are reflected in the efficiency with which the different proteins repress transcription from the 5S rRNA genes. Thus, although all three proteins can contribute to the organization of chromatin, the stability of the structures they assemble will vary. Our results provide a molecular explanation for the transcriptional promiscuity of Xenopus early embryonic chromatin, which is enriched in HMG1 and linker histone B4, but deficient in histone Hi.

Linker histone variants control chromatin dynamics during early embryogenesis

“…This system reveals, that in contrast to the effects of somatic H1, the association of oocyte͞ embryonic linker histone B4 with dinucleosomes has little effect on the accessibility of the linker DNA and allows ATPdependent chromatin remodeling, even though it reduces accessibility around the nucleosomal dyad axis, as does somatic H1. These distinct properties of linker histone variants may explain why replacement of histone B4 with somatic H1 directly leads to gene repression and the loss of mesodermal competence observed in previous studies (10,11).…”

“…Microinjection of targeted ribozymes into early embryos revealed that replacement of B4 with somatic H1 is required for selective gene repression and proper development (10). The previous study using model chromatin templates also suggested that the repressive effect of histone B4 on transcription is weaker than that of somatic H1 (11). It may be tempting to speculate that histone B4 contributes directly to dynamic chromatin in early embryos.…”

Nucleosome assembly protein-1 is a linker histone chaperone in Xenopus eggs

“…In some experiments, recombinant B4 from which the tags for purification have been removed was prepared as described (25), and dinucleosomes were incubated with B4 or His-xNAP-1͞B4 complexes in buffer (50 mM KCl͞0.57 mM EDTA͞0.5% glycerol͞1.0 mg͞ml BSA͞10 mM Tris⅐HCl, pH 8.0) at room temperature for 30 min. Each dinucleosome sample (55 fmol) was digested with 0.075, 0.15, 0.30, or 0.60 units of nucleococcal nuclease and analyzed as described (26).…”

“…Thus, in the in vitro reconstitution system, the formation of a complex between B4 and xNAP-1 is an essential requirement for successful remodeling of sperm chromatin into nucleosomes containing the appropriate deposition of linker histones. To investigate directly a role of xNAP-1 in the deposition of B4 onto the nucleosomal linker DNA, we used synthetic 5S dinucleosomes that contain two nucleosome cores spaced by intact linker DNA; the physiological binding of one or two molecules of linker histones to dinucleosomes results in a slight decrease in mobility on nucleoprotein agarose gel electrophoresis and can be distinguished from nonspecific histone binding (26). Dinucleosomes, whose DNA had been radiolabeled, were incubated with various concentrations of free His-B4 or the His-xNAP-1͞ His-B4 complex at physiological ionic strength, and incorporation of B4 into these dinucleosomes was analyzed by gel electrophoresis.…”

Activators and repressors: making use of chromatin to regulate transcription

“…However, mutations in SIN1 decrease expression of some genes, suggesting a more complex structural role for this protein in chromatin. In vertebrates, HMG1/2 have been shown to replace linker histones within the nucleosome and to directly influence the transcription process in a chromatin context Ura et al 1996).…”