The histone chaperones Nap1 and Vps75 bind histones H3 and H4 in a tetrameric conformation

Histone chaperones physically interact with histones to direct proper assembly and disassembly of nucleosomes regulating diverse nuclear processes such as DNA replication, promoter remodeling, transcription elongation, DNA damage, and histone variant exchange. Currently, the best-characterized chaperone-histone interaction is that between the ubiquitous chaperone Asf1 and a dimer of H3 and H4. Nucleosome assembly proteins (Nap proteins) represent a distinct class of histone chaperone. Using pulsed electron double resonance (PELDOR) measurements and protein crosslinking, we show that two members of this class, Nap1 and Vps75, bind histones in the tetrameric conformation also observed when they are sequestered within the nucleosome. Furthermore, H3 and H4 trapped in their tetrameric state can be used as substrates in nucleosome assembly and chaperone-mediated lysine acetylation. This alternate mode of histone interaction provides a potential means of maintaining the integrity of the histone tetramer during cycles of nucleosome reassembly

The chromatin remodelling enzymes SNF2H and SNF2L position nucleosomes adjacent to CTCF and other transcription

Within the genomes of metazoans, nucleosomes are highly organised adjacent to the binding sites for a subset of transcription factors. Here we have sought to investigate which chromatin remodelling enzymes are responsible for this. We find that the ATP-dependent chromatin remodelling enzyme SNF2H plays a major role organising arrays of nucleosomes adjacent to the binding sites for the architectural transcription factor CTCF sites and acts to promote CTCF binding. At many other factor binding sites SNF2H and the related enzyme SNF2L contribute to nucleosome organisation. The action of SNF2H at CTCF sites is functionally important as depletion of CTCF or SNF2H affects transcription of a common group of genes. This suggests that chromatin remodelling ATPase's most closely related to the Drosophila ISWI protein contribute to the function of many human gene regulatory elements

Sin mutations alter inherent nucleosome mobility

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 dimer–tetramer 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

Depletion of SNF2H affects the transcription of many genes regulated by CTCF.

<p>(A) RNA seq was performed for cells depleted of CTCF and SNF2H. Expression of 3294 genes could be significantly (p<0.05 and q<0.05) measured across four repeats in both cell types. The total number of affected genes following depletion of CTCF and SNF2H is shown in brackets. There is significant overlap between genes with altered expression (>1.5 fold) following SNF2H and CTCF depletion. Amongst these co-regulated genes there is a high correlation in the extent to which expression is affected in each depletion (Spearman’s correlation 0.83, p = 2.19e-94). This indicates that a significant subset of CTCF regulated genes are co-regulated by SNF2H. (B) Nuc seq of CTCF sites that retain CTCF occupancy (top 10%) following SNF2H depletion (blue) in comparison to control RNA depletion (red). Despite the retention of CTCF nucleosome organisation is reduced at these locations in the absence of SNF2H. (C) Nuc seq of genes where CTCF occupancy is reduced following SNF2H depletion (blue) in comparison to scrambled RNA depletion (red). At these sites a more dramatic loss of nucleosome positioning is observed following loss of both CTCF and SNF2H. (D) CTCF ChIP following depletion of SNF2H and control scrambled RNA is shown for all CTCF sites (blue) or CTCF sites within 10kb of 368 genes identified as being co-regulated by CTCF and SNF2H (as described in A). Least squares fits and Spearman correlation for each data series are also shown. The changes in CTCF occupancy at sites within the vicinity of co-regulated genes is very similar to that observed for all other sites. This indicates that changes in CTCF occupancy following SNF2H depletion do not provide a simple explanation for co-regulation by SNF2H and CTCF.</p

Cohesin does not organize nucleosomes at CTCF sites.

<p>(A, B) ChIP seq for RAD21 was plotted with respect to CTCF sites following depletion of CTCF (A) or SNF2H (B). In each case enrichment for RAD21 is reduced following depletion (blue) in comparison to control depletions (red). (C) ChIP seq showing SNF2H enrichment at CTCF sites in RAD21 (blue) depleted and control (red) cells. SNF2H enrichment is not affected by depletion of RAD21. (D) Depletion of RAD21 (blue) has little effect on nucleosome organization at CTCF sites in comparison to a control depletion.</p

Interdependence of SNF2H and CTCF binding.

<p>(A) Chromatin of control cells (red) and CTCF depleted (blue) HeLa cells was immuno-precipitated using an anti-SNF2H antibody and the resulting DNA fragments sequenced. SNF2H is enriched at CTCF sites in comparison to the surrounding DNA, and this enrichment is reduced following depletion of CTCF. (B) ChIP seq experiment for CTCF showing enrichment at CTCF sites (red). Following depletion of SNF2H CTCF occupancy is reduced (blue). (C) Protein levels of SNF2H (green bands) and CTCF (top red bands) in cells depleted for SNF2H, control cells or CTCF depleted cells. Two different quantities (lane 1 and 2) of whole cell extracts of the respective cells were immuno-blotted for SNF2H, CTCF and beta-actin as a loading control. Depletion of SNF2H does not result in any global change in CTCF protein levels.</p

siRNA Oligos Used for RNA Interference.

<p>siRNA Oligos Used for RNA Interference.</p