Our basic goal is to understand how chromatin structure influences gene regulation. Chromatin is generally repressive in nature but its structure is manipulated by cells in a regulated way to determine which genes are potentially transcriptionally active and which genes remain repressed in a given cell type. This regulation depends on interactions between DNA sequence-specific transcription factors, chromatin enzymes and chromatin. The structural subunit of chromatin is the nucleosome core, which contains 147 bp of DNA wrapped 1.7 times around a central histone octamer composed of two molecules each of the four core histones (H2A, H2B, H3 and H4). Generally, nucleosomes are regularly spaced along the DNA, like beads on a string. At physiological salt concentrations, the beads-on-a-string structure folds spontaneously to form a fiber 30 nm wide, assisted by the linker histone (H1), which binds to the nucleosome core and to the linker DNA. Thus, collectively, the histones control DNA accessibility. Gene activation involves the recruitment of a set of factors to a promoter in response to appropriate signals, ultimately resulting in the formation of an initiation complex by RNA polymerase II (Pol II) and transcription. These events occur in the presence of nucleosomes, which are compact structures capable of blocking transcription at every step. To circumvent and regulate this chromatin block, eukaryotic cells possess dedicated enzymes, including ATP-dependent chromatin remodeling machines, histone modifying complexes and histone chaperones. The remodeling machines use ATP to move nucleosomes along or off DNA (e.g. the SWI/SNF, RSC, CHD and ISWI complexes), or to exchange histone variants between nucleosomes (e.g. the SWR complex). The histone modifying complexes contain enzymes which modify the histones post-translationally to alter their DNA-binding properties and to mark them for recognition by other complexes, which have activating or repressive roles (the histone code hypothesis). Histone-modifying enzymes include histone acetylases (HATs), deacetylases (HDACs), methylases and kinases. Histone chaperones mediate histone transfer reactions that occur during transcription and DNA replication (e.g. Asf1 and the CAF-1 complex). These enzymes, together with DNA methylating and de-methylating enzymes, are central to epigenetics. Many human diseases have been linked to chromatin remodeling enzymes and epigenetic modifications. For example, mutations in the hSNF5 subunit of the SWI/SNF complex are strongly linked to pediatric rhabdoid tumors. The CHD class of ATP-dependent remodelers has also been linked to cancer and to autism. Cancer therapies and drugs aimed at epigenetic targets are being tested. Recent studies have revealed a correlation between a linker histone variant and tumor heterogeneity. A full understanding of the functions of chromatin structure, enzymes and modifications is therefore vital.
Our aim i s to dissect chromatin remodeling mechanisms in vivo and elucidate their contributions to gene regulation. Our current efforts are focused on elucidating the contributions of the various ATP-dependent chromatin remodeling complexes to chromatin organization in vivo. Most of our work involves the use of budding yeast, Saccharomyces cerevisiae, as a model organism, but we are also involved in some mouse chromatin studies. During this Fiscal Year, we have made significant progress towards understanding the nature of promoter chromatin; more specifically, on the question of what occupies the nucleosome-depleted region that is characteristic of most yeast promoters and many mouse and human promoters. Our study was published in Molecular Cell and is summarized below. 1. Non-histone barrier complexes occupy nucleosome-depleted promoters. Most genes in yeast and in higher organisms have a characteristic chromatin organization, in which the promoter, just upstream of the transcription start site (TSS), is depleted of nucleosomes (the nucleosome-depleted region or NDR). The NDR is flanked on both sides by well-positioned nucleosomes in phased arrays with a characteristic spacing. These observations are based primarily on experiments using micrococcal nuclease (MNase), which digests linker DNA rapidly and nucleosome core DNA much more slowly. A central question in our work is the nature of the NDR - is it protein-free or is it occupied by a large non-histone complex? An important factor is the DNA sequence itself: poly(dA) sequences are commonly found in promoters (though they are not universal) and exclude nucleosomes to some extent in vitro, but this effect is relatively weak. Recently, several labs have reported that many promoter NDRs are not actually nucleosome-free but occupied by easily digested, unstable fragile nucleosomes. However, other labs have reported high-resolution mapping by ChIP and tiling microarray that provide little evidence for histones at yeast promoters. Micrococcal nuclease (MNase)-sensitive nucleosomes have also been reported in higher eukaryotes. In this report, we addressed the important issue of the nature of the NDR in yeast. We confirm that an MNase-sensitive complex is present at yeast promoters. The critical question is whether histones are present at NDRs, as predicted if fragile nucleosomes are formed at NDRs. We used two different approaches to detect histones H4 and H2B: MNase-ChIP-seq, which involves immuno-precipitation (IP) of nucleosomes from MNase digests; and standard ChIP-seq using sonication, which does not depend on the use of MNase. Both sets of data show that NDRs are strongly depleted of histones. Although we find no evidence for fragile nucleosomes at promoters, we do detect MNase-sensitive nucleosomes elsewhere in the genome. However, they have high A/T-content, suggesting that MNase sensitivity does not indicate structural instability, but the preference of MNase for A/T-rich DNA - simply that A/T-rich nucleosomes are digested faster than G/C-rich nucleosomes. We confirm our conclusions by analyzing ChIP-exo, chemical mapping and ATAC-seq data from other labs. By analogy with stable RNA polymerase III transcription complexes at tRNA genes, which are composed of TFIIIB and TFIIIC, we propose that RNA polymerase II promoters are occupied by similar stable transcription complexes. Currently, we are endeavoring to identify the MNase-sensitive complexes which occupy promoter NDRs. Chereji RV, Ocampo J, Clark DJ (2017). MNase-Sensitive Complexes in Yeast: Nucleosomes and Non-histone Barriers. Mol Cell 65:565-577. 2. Maree JP, Povelones ML, Clark DJ, Rudenko G, Patterton HG (2017). Well-positioned nucleosomes punctuate polycistronic Pol II transcription units and flank silent VSG gene arrays in Trypanosoma brucei. Epigenetics & Chromatin 10:14. 3. Invited Review: Ocampo J, Cui F, Zhurkin VB, Clark DJ (2016). The proto-chromatosome: A fundamental subunit of chromatin? Nucleus 7:382-7.
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