Human SWI/SNF nucleosome remodeling activity is partially inhibited by linker histone H1

The physical structure and the compact nature of the eukaryotic genome present a functional barrier for any cellular process that requires access to the DNA. The linker histone H1 is intrinsically involved in both the determination of and the stability of higher order chromatin structure. Because histone H1 plays a pivotal role in the structure of chromatin, we investigated the effect of histone H1 on the nucleosome remodeling activity of human SWI/SNF, an ATP-dependent chromatin remodeling complex. The results from both DNase I digestion and restriction endonuclease accessibility assays indicate that the presence of H1 partially inhibits the nucleosome remodeling activity of hSWI/SNF. Neither H1 bound to the nucleosome nor free H1 affected the ATPase activity of hSWI/SNF, suggesting that the observed inhibition of hSWI/SNF nucleosome remodeling activity depends on the structure formed by the addition of H1 to nucleosomes.     

Composition and functional specificity of SWI2/SNF2 class chromatin remodeling complexes

By regulating the structure of chromatin, ATP-dependent chromatin remodeling complexes (remodelers) perform critical functions in the maintenance, transmission and expression of the eukaryotic genome. Although all known chromatin-remodeling complexes contain an ATPase as a central motor subunit, a number of distinct classes have been recognized. Recent studies have emphasized a more extensive functional diversification among closely related chromatin remodeling complexes than previously anticipated. Here, we discuss recent insights in the functional differences between two evolutionary conserved subclasses of SWI/SNF-related chromatin remodeling factors. One subfamily comprises yeast SWI/SNF, fly BAP and mammalian BAF, whereas the other subfamily includes yeast RSC, fly PBAP and mammalian PBAF. We review the subunit composition, conserved protein modules and biological functions of each of these subclasses of SWI/SNF remodelers. In particular, we will focus on the roles of specific subunits in developmental gene control and human diseases. Recent findings suggest that functional diversification among SWI/SNF complexes allows the eukaryotic cell to fine-tune and integrate the execution of diverse biological programs involving the expression, maintenance and duplication of its genome.     

The SWI/SNF complex acts to constrain distribution of the centromeric histone variant Cse4

In order to gain insight into the function of the Saccharomyces cerevisiae SWI/SNF complex, we have identified DNA sequences to which it is bound genomewide. One surprising observation is that the complex is enriched at the centromeres of each chromosome. Deletion of the gene encoding the Snf2 subunit of the complex was found to cause partial redistribution of the centromeric histone variant Cse4 to sites on chromosome arms. Cultures of snf2Δ yeast were found to progress through mitosis slowly. This was dependent on the mitotic checkpoint protein Mad2. In the absence of Mad2, defects in chromosome segregation were observed. In the absence of Snf2, chromatin organisation at centromeres is less distinct. In particular, hypersensitive sites flanking the Cse4 containing nucleosomes are less pronounced. Furthermore, SWI/SNF complex was found to be especially effective in the dissociation of Cse4 containing chromatin in vitro. This suggests a role for Snf2 in the maintenance of point centromeres involving the removal of Cse4 from ectopic sites.     

Structure-function relationships in nucleosomal arrays containing linker histone H5

To study the structural and functional changes accompanying the integration of histone H5 into the nucleosome structure, linear DNA species have been employed with a terminal promoter for bacteriophage T7 RNA polymerase followed by tandem repeats of a 207-bp nucleosome positioning sequence. The oligonucleosomes assembled from 12-repeat DNA and saturating amounts of core histone octamer plus histone H5 are compacted, in the presence of 1 mM free magnesium ions, to the level of the 30-nm fiber. Under these ionic conditions the efficiency in RNA synthesis and the size distribution of RNA chains obtained with this template are the same as those corresponding to the template without H5, indicating that the 30-nm fiber stabilized by H5 does not impair RNA elongation. Therefore, under our experimental conditions, incorporation of one molecule of histone H5 per nucleosome does not affect elongation of RNA even when a folded structure is produced. However, elongation is inhibited by binding of an excess of H5.     

SWI/SNF chromatin remodeling requires changes in DNA topology

ySWI/SNF complex belongs to a family of enzymes that use the energy of ATP hydrolysis to remodel chromatin structure. Here we examine the role of DNA topology in the mechanism of ySWI/SNF remodeling. We find that the ability of ySWI/SNF to enhance accessibility of nucleosomal DNA is nearly eliminated when DNA topology is constrained in small circular nucleosomal arrays and that this inhibition can be alleviated by topoisomerases. Furthermore, we demonstrate that remodeling of these substrates does not require dramatic histone octamer movements or displacement. Our results suggest a model in which ySWI/SNF remodels nucleosomes by using the energy of ATP hydrolysis to drive local changes in DNA twist.     

Structural analysis of the yeast SWI/SNF chromatin remodeling complex

Elucidating the mechanism of ATP-dependent chromatin remodeling is one of the largest challenges in the field of gene regulation. One of the missing pieces in understanding this process is detailed structural information on the enzymes that catalyze the remodeling reactions. Here we use a combination of subunit radio-iodination and scanning transmission electron microscopy to determine the subunit stoichiometry and native molecular weight of the yeast SWI/SNF complex. We also report a three-dimensional reconstruction of yeast SWI/SNF derived from electron micrographs.     

Chromatin remodeling by ISW2 and SWI/SNF requires DNA translocation inside the nucleosome

Chromatin-remodeling complexes regulate access to nucleosomal DNA by mobilizing nucleosomes in an ATP-dependent manner. In this study, we find that chromatin remodeling by SWI/SNF and ISW2 involves DNA translocation inside nucleosomes two helical turns from the dyad axis at superhelical location-2. DNA translocation at this internal position does not require the propagation of a DNA twist from the site of translocation to the entry/exit sites for nucleosome movement. Nucleosomes are moved in 9- to 11- or approximately 50-base-pair increments by ISW2 or SWI/SNF, respectively, presumably through the formation of DNA loops on the nucleosome surface. Remodeling by ISW2 but not SWI/SNF requires DNA torsional strain near the site of translocation, which may work in conjunction with conformational changes of ISW2 to promote nucleosome movement on DNA. The difference in step size of nucleosome movement by SWI/SNF and ISW2 demonstrates how SWI/SNF may be more disruptive to nucleosome structure than ISW2.