Sin mutations alter inherent nucleosome mobility

Abstract

Previous studies have identified sin mutations that alleviate the requirement for the yeast SWI/SNF chromatin remodelling complex, which include point changes in the yeast genes encoding core histones. Here we characterise the biochemical properties of nucleosomes bearing these mutations. We find that sin mutant nucleosomes have a high inherent thermal mobility. As the SWI/SNF complex can alter nucleosome positioning, the higher mobility of sin mutant nucleosomes provides a means by which sin mutations may substitute for SWI/SNF function. The location of sin mutations also provides a new opportunity for insights into the mechanism for nucleosome mobilisation. We find that both mutations altering histone DNA contacts at the nucleosome dyad and mutations in the dimertetramer interface influence nucleosome mobility. Furthermore, incorporation of H2A.Z into nucleosomes, which also alters dimer-tetramer interactions, affects nucleosome mobility. Thus, variation of histone sequence or subtype provides a means by which eukaryotes may regulate access to chromatin through alterations to nucleosome mobility. The EMBO Journal (2004) IntroductionNucleosomes are the universal molecular packaging state of DNA in nuclei. They are responsible for compacting eukaryotic genomes to allow them to fit into the limited volume of the cell nucleus. The nucleosome core particle and an additional variable length of unbound linker DNA together comprise the fundamental repeating unit of chromatin (Kornberg, 1974). The nucleosome core particle consists of a core of eight polypeptides, two copies each of the four highly conserved histone proteins H2A, H2B, H3 and H4, around which 147 bp of DNA are wrapped in 1.7 superhelical turns (Luger et al, 1997). H3 and H4 associate to form histone fold dimers as do histones H2A and H2B. Each histone fold dimer associates to form an octameric spiral with dyad symmetry.The H2AÀH2B histone fold dimer units are less stably associated within the octamer than the two H3ÀH4 histone fold dimers (Eickbush and Moudrianakis, 1978). A number of extra protein elements decorate the regular spiral of histone fold dimers, including unstructured 'tails' that extend outside the DNA superhelix, the additional H3 aN helix that organises the most exterior turn of bound DNA, a structured H2A C-terminal extension that passes over the top face of the histone octamer, and an H2B aC helix lying above the histone dimer (Luger and Richmond, 1998).A consequence of the organisation of DNA into nucleosomes is that all genetic processes must contend with a chromatin substrate. In the case of gene regulation, wrapping into nucleosomes makes DNA sequences differentially accessible to transcription factors (Owen-Hughes and Workman, 1994). Accessibility of a particular site depends on the absolute 'position' of the nucleosome (Polach and Widom, 1995), and many cases have been recognised in which nucleosome positioning affects genomic accessibility (Simpson, 1990;Lomvardas and Thanos, 2002;Miller and Widom, 2003). In this way, nucleoso...

Reconciling the positive and negative roles of histone H2A.Z in gene transcription

“…35 In yeast, it has been demonstrated that transcription factor binding sites tend to be rotationally exposed on the H2A.Z nucleosome surface near its border, while transcriptional start sites tend to reside about one helical turn inside the nucleosome border. 35 In vitro, H2A.Z-containing nucleosomes have also been shown to be more mobile, even in the absence of chromatin remodelers, 76 which may explain the high dependence of cells on SWI/SNF remodelling complex when H2A.Z is absent. 23 This is in contrast with other in vitro and in vivo experiments, where H2A.Z seems to reduce translational mobility of nucleosomes.…”

Insight Into the Mechanism of Nucleosome Reorganization From Histone Mutants That Suppress Defects in the FACT Histone Chaperone

“…Three independent samples for each condition were analyzed as in Figure 5, A and B, with the average and standard deviation of the three measurements presented. (C and D) Nucleosomes were constructed using recombinant histones from X. laevis (with normal histones or the mutation indicated) and a 255-bp MMTV DNA fragment (Flaus et al 2004) labeled with Cy5. Samples were treated with FACT for the amount of time indicated, excess unlabeled genomic DNA was added to disrupt the FACT-nucleosome complexes, and then products were separated by native PAGE (Rhoades et al 2004).…”

“…These conditions increase the total dimer loss caused by FACT (Xin et al 2009) and increase the electrophoretic separation of octameric and tetrameric nucleosomes. These nucleosomes migrate as one major band in native gels (Flaus et al 2004) (Figure 6C, "Nuc") along with several minor translational variants, while reconstructions show that tetrasomes migrate to four main bands ( Figure 6C, "Tet"). FACT does not appear to promote translocation of nucleosomes (Rhoades et al 2004), so we infer that the multiple forms observed with this larger MMTV fragment are corresponding pairs of octasomal and tetrasomal species occupying the same preferred translational positions.…”

“…Instead of the 181-bp 5S rDNA nucleosomes containing recombinant yeast histones used above, we used a 255-bp MMTV (Mouse Mammary Tumor Virus) sequence (Flaus et al 2004), recombinant Xenopus laevis histones, and low Mg 2+ ion concentrations. These conditions increase the total dimer loss caused by FACT (Xin et al 2009) and increase the electrophoretic separation of octameric and tetrameric nucleosomes.…”

Human SWI/SNF Drives Sequence-Directed Repositioning of Nucleosomes on C-myc Promoter DNA Minicircles

“…(24,25,45,(47)(48)(49)(50)), suggesting that mammalian body temperature might promote some level of nucleosome redistribution in vivo. Thermal repositioning is most rapid at high temperatures (up to 65°C) and in the absence of divalent cations or linker histones (45,48,51,52)). In general, thermal repositioning appears to result in redistribution between sites that are all at least moderately-favored during nucleosome assembly (causing increases in some moderately favored positions and decreases in some strongly favored positions).…”