David J. Clark PhD

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We study the role of chromatin remodeling complexes in gene regulation. Gene activation must occur in the presence of nucleosomes, which are compact structures capable of blocking transcription at every step. The nucleosome is composed of ~147 bp of DNA wrapped ~1.7 times around a central octamer composed of two molecules each of the four core histones (H2A, H2B, H3 and H4). To circumvent the chromatin block, eukaryotic cells possess ATP-dependent chromatin remodeling machines, which slide or dismantle nucleosomes, and histone-modifying complexes, which mark nucleosomes for interaction with other chromatin-binding proteins. The central roles of these complexes in gene regulation, epigenetics and disease have generated huge interest in their functions and mechanisms of action.

            We are exploiting the new massively parallel sequencing technologies to create genome-wide maps of nucleosomes (MNase-seq) and other chromatin components (ChIP-seq and PESCI) in budding yeast and, in collaboration with other labs, in mouse cells. In vivo, active gene promoters and enhancers are occupied by sequence-specific transcription factors and associated proteins. Such nucleosome-depleted regions (NDRs) are flanked by arrays of regularly spaced nucleosomes that are phased relative to the NDR. In yeast, the first (+1) nucleosome is usually located directly over the transcription start site (TSS). What happens to nucleosomes on genes when they are activated? We find that the chromatin structure of the most transcriptionally active yeast genes is heavily disrupted: some nucleosomes are lost, others are stripped of one or both of their H2A-H2B dimers, and those that remain are out of position. Furthermore, RNA polymerase II complexes queue up on these genes, apparently waiting to terminate. We are actively exploring the implications of this fascinating observation.

            Our current focus is on the functions of RSC and SWI/SNF in gene activation. These complexes are related ATP-dependent chromatin remodeling machines capable of moving nucleosomes, thereby regulating access to DNA. They are conserved from yeast to man. Mutations in SWI/SNF subunits are strongly associated with various cancers, particularly pediatric cancers. We are addressing the roles of SWI/SNF and RSC in nucleosome phasing, either using a null mutation for SWI/SNF (snf2Δ), or by depleting cells of an essential RSC subunit. We find that SWI/SNF has no effect on global nucleosome phasing, probably because it regulates relatively few genes. In contrast, depletion of RSC results in global re-positioning of nucleosomes: both upstream and downstream nucleosomal arrays shift toward the NDR, with no change in spacing, resulting in a narrower NDR. Analysis of gene pairs in different orientations demonstrates that phasing patterns reflect competition between phasing signals emanating from neighboring NDRs. These signals may be in phase, resulting in constructive interference and a regular array, or out of phase, resulting in destructive interference and fuzzy positioning. We propose a modified barrier model, in which a stable complex located at the NDR acts as a bidirectional phasing barrier. In RSC-depleted cells, this barrier has a smaller footprint, resulting in narrower NDRs. We conclude that RSC has a critical role in organizing yeast chromatin, perhaps involving restoration of chromatin structure following transcription. Currently, we are examining the roles of additional remodelers in organizing yeast chromatin (including ISW1, ISW2 and CHD1). Do they have specific roles, or are they functionally redundant?

            In a related project, we are addressing the cell cycle-dependent regulation of the yeast histone genes. We have shown that they are activated by both Spt10 and SBF. Spt10 is an unusual trans-activator, in which a HAT domain, normally recruited as a co-activator to promoters through an activation domain, is attached directly to a sequence-specific DNA-binding domain. Currently, we are following chromatin structure dynamics during the cell cycle. We aim to test our model for histone gene regulation, which posits that the extent to which histone chaperones are saturated with histones is the critical signal for negative feedback control of transcription.

Selected Publications

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