SWI-SNF-mediated nucleosome remodeling: role of histone octamer mobility in the persistence of the remodeled state.

SWI-SNF is an ATP-dependent chromatin remodeling complex that disrupts DNA-histone interactions. Several studies of SWI-SNF activity on mononucleosome substrates have suggested that remodeling leads to novel, accessible nucleosomes which persist in the absence of continuous ATP hydrolysis. In contrast, we have reported that SWI-SNF-dependent remodeling of nucleosomal arrays is rapidly reversed after removal of ATP. One possibility is that these contrasting results are due to the different assays used; alternatively, the lability of the SWI-SNF-remodeled state might be different on mononucleosomes versus nucleosomal arrays. To investigate these possibilities, we use a coupled SWI-SNF remodeling-restriction enzyme assay to directly compare the remodeling of mononucleosome and nucleosomal array substrates. We find that SWI-SNF action causes a mobilization of histone octamers for both the mononucleosome and nucleosomal array substrates, and these changes in nucleosome positioning persist in the absence of continued ATP hydrolysis or SWI-SNF binding. In the case of mononucleosomes, the histone octamers accumulate at the DNA ends even in the presence of continued ATP hydrolysis. On nucleosomal arrays, SWI-SNF and ATP lead to a more dynamic state where nucleosomes appear to be constantly redistributed and restriction enzyme sites throughout the array have increased accessibility. This random positioning of nucleosomes within the array persists after removal of ATP, but inactivation of SWI-SNF is accompanied by an increased occlusion of many restriction enzyme sites. Our results also indicate that remodeling of mononucleosomes or nucleosomal arrays does not lead to an accumulation of novel nucleosomes that maintain an accessible state in the absence of continuous ATP hydrolysis.

Interplay of yeast global transcriptional regulators Ssn6p-Tup1p and Swi-Snf and their effect on chromatin structure.

Transcriptional regulation in yeast involves a number of general trans-acting factors affecting chromatin structure. The Swi-Snf complex is required for expression of a large number of genes and has the ability to remodel chromatin in vitro. The Ssn6p-Tup1p repressor complex may be involved in chromatin organization through the interaction with pathway-specific DNA-binding proteins. To study the interplay of these factors and their effect on chromatin we have analyzed SUC2 chromatin structure in wild-type cells and in strains bearing combinations of ssn6/tup1 and swi1 mutations. We have mapped nucleosome positioning of the repressed gene in wild-type cells using primer extension methodology, allowing base pair resolution, and have analyzed details of chromatin remodeling in the derepressed state. In ssn6 or tup1 mutants under repressing conditions the observed changes in SUC2 chromatin structure may be suppressed by the swi1 mutation, suggesting that Ssn6p-Tup1p is not required for the establishment of nucleosome positioning at the SUC2 promoter. Our data indicate the involvement of chromatin remodeling factors distinct from the Swi-Snf complex in SUC2 transcriptional regulation and suggest that Swi-Snf may antagonize Ssn6p-Tup1p by controlling remodeling activity. We also show that a relatively high level of SUC2 transcription can coexist with positioned nucleosomes.

Nucleosome structural transition during chromatin unfolding is caused by conformational changes in nucleosomal DNA.

We have recently reported that certain core histone-DNA contacts are altered in nucleosomes during chromatin unfolding (Usachenko, S. I., Gavin I. M., and Bavykin, S. G. (1996) J. Biol. Chem. 271, 3831-3836). In this work, we demonstrate that these alterations are caused by a conformational change in the nucleosomal DNA. Using zero-length protein-DNA cross-linking, we have mapped histone-DNA contacts in isolated core particles at ionic conditions affecting DNA stiffness, which may change the nucleosomal DNA conformation. We found that the alterations in histone-DNA contacts induced by an increase in DNA stiffness in isolated core particles are identical to those observed in nucleosomes during chromatin unfolding. The change in the pattern of micrococcal nuclease digestion of linker histone-depleted chromatin at ionic conditions affecting chromatin compaction also suggests that the stretching of the linker DNA may alter the nucleosomal DNA conformation, resulting in a structural transition in the nucleosome which may play a role in rendering the nucleosome competent for transcription and/or replication.

Alterations in nucleosome core structure in linker histone-depleted chromatin.

We have previously shown that the sequential arrangement of histone-DNA contacts is essentially the same in the nucleosomal core of sea urchin sperm nuclei, where chromatin is highly condensed and repressed, and in nuclei from lily bud sepals or yeast, where chromatin is highly active in transcription and replication and is significantly or completely depleted of histone H1. However, the difference in the strength of some histone-DNA contacts has not been understood or discussed. In this work, we demonstrate that some of these differences are due to a conformational change in the nucleosomal core. We show that the nucleosomal core in linker histone-depleted chromatin is in a different conformational state compared with the nucleosomal core in folded chromatin or in isolated core nucleosomes. This conformational state is characterized by altered strengths in the histone H4 and H2A/H2B contacts with the regions of sharply bent nucleosomal DNA around sites +/-1 and +/-4 and site +/-5, respectively. We demonstrate that this conformation, which we call the "stretched nucleosome," is a general feature of unfolded linker histone-depleted chromatin and may occur during chromatin activation. Our results suggest that this nucleosome structural alteration does not depend on chromatin sources and histone variants studied in this work. In addition, we show that this alteration is reversible and is caused by the stretching of linker DNA during chromatin unfolding.

Tup1p represses Mcm1p transcriptional activation and chromatin remodeling of an a-cell-specific gene.

In yeast, a number of regulatory proteins expressed only in specific cell types interact with general transcription factors in a combinatorial manner to control expression of cell-type-specific genes. We report a detailed analysis of activation and repression events that occur at the promoter of the a-cell-specific STE6 gene fused to a beta-galactosidase gene in a yeast minichromosome, as well as factors that control the chromatin structure of this promoter both in the minichromosome and in the genomic STE6 locus. Mcm1p results in chromatin remodeling and is responsible for all transcriptional activity from the STE6 promoter in both wild-type a and alpha cells. Matalpha2p cooperates with Tup1p to block both chromatin remodeling and Mcm1p-associated activation. While Matalpha2p represses only Mcm1p, the Tup1p-mediated repression involves both Mcm1p-dependent and -independent mechanisms. Swi/Snf and Gcn5p, required for full induction of the STE6 gene, do not contribute to chromatin remodeling. We suggest that Tup1p can contribute to repression by blocking transcriptional activators, in addition to interacting with transcription machinery and stabilizing chromatin.

Rearrangement of the histone H2A C-terminal domain in the nucleosome.

Using zero-length covalent protein-DNA crosslinking, we have mapped the histone-DNA contacts in nucleosome core particles from which the C- and N-terminal domains of histone H2A were selectively trimmed by trypsin or clostripain. We found that the flexible trypsin-sensitive C-terminal domain of histone H2A contacts the dyad axis, whereas its globular domain contacts the end of DNA in the nucleosome core particle. The appearance of the histone H2A contact at the dyad axis occurs only in the absence of linker DNA and does not depend on the absence of linker histones. Our results show the ability of the histone H2A C-terminal domain to rearrange. This rearrangement might play a biological role in nucleosome disassembly and reassembly and the retention of the H2A-H2B dimer (or the whole octamer) during the passing of polymerases through the nucleosome.

Zero-length protein-nucleic acid crosslinking by radical-generating coordination complexes as a probe for analysis of protein-DNA interactions in vitro and in vivo.

Redox-active coordination complexes such as 1,10-phenanthroline-Cu(II) (OP-Cu) and bleomycin-Fe(III) are commonly used as "chemical nucleases" to introduce single-strand breaks in nucleic acids. Here we report that under certain conditions these complexes may crosslink proteins to nucleic acids. In vitro experiments suggest that proteins are crosslinked to DNA by a mechanism similar to dimethyl sulfate-induced crosslinking. Furthermore, we demonstrate that the OP-Cu complex can generate protein-DNA crosslinks in mammalian cells in vivo. By combining the OP-Cu crosslinking and a "protein shadow" hybridization assay we identify proteins interacting with DNA in isolated pea chloroplasts and show that this methodology can be applied to detect DNA-binding proteins on specific DNA sequences either in vitro or in vivo.