1. ROLES OF THE SWI/SNF AND RSC CHROMATIN REMODELING COMPLEXES IN NUCLEOSOME PHASING. In collaboration with Dr James Iben (NICHD). Paired-end sequencing performed by Dr Jun Zhu's Lab (NHLBI). RSC and SWI/SNF are related ATP-dependent chromatin remodeling machines that move nucleosomes, regulating access to DNA. These complexes are conserved from yeast to man, where they play critical roles in gene regulation and other essential processes. Furthermore, it has become clear that mutations in SWI/SNF and related complexes are strongly associated with various cancers, particularly pediatric cancers. In this project, we address the roles of SWI/SNF and RSC in nucleosome phasing relative to transcription start sites in budding yeast, in which it is relatively easy to eliminate their activities, either using a null mutation for SWI/SNF (snf2), or by depleting cells of an essential subunit (the Rsc8 subunit of RSC). In vivo, gene promoters are typically depleted of nucleosomes (nucleosome-depleted regions or """"""""NDRs""""""""). Thus, nucleosomes on genes are organized into regularly spaced arrays, where the first (+1) nucleosome is located either directly over the transcription start site (TSS) or very close to it. The ordering of nucleosomes with respect to the TSS is referred to as nucleosome phasing. We have obtained genome-wide nucleosome maps from wild type and mutant cells by isolating DNA protected from micrococcal nuclease (MNase) digestion and subjecting it to paired-end sequencing. Rather surprisingly, SWI/SNF has no effect on nucleosome phasing at the global level. To account for this observation, we propose that SWI/SNF regulates relatively few genes. In contrast, RSC depletion results in global nucleosome re-positioning: both upstream and downstream nucleosomal arrays shift toward the NDR, with no change in spacing, resulting in a narrower and partly filled NDR. The global picture of RSC-depleted chromatin represents the average of a range of chromatin structures, with most genes showing a shift of the +1 or the -1 nucleosome into the NDR. Using RSC ChIP data reported by others, we show that RSC occupancy is highest on the coding regions of heavily transcribed genes, though not at their NDRs. We propose that RSC has a role in restoring chromatin structure after transcription. 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. Thus, RSC plays a critical role in organizing yeast chromatin. Currently, we are extending these studies to examine the roles of additional remodelers in organizing yeast chromatin (ISW1, ISW2 and CHD1). Do they have specific roles, or are they functionally redundant? Ganguli D, Chereji RV, Iben JR, Cole HA, Clark DJ (2014). RSC-dependent constructive and destructive interference between opposing arrays of phased nucleosomes in yeast. Genome Res., epub (PMID 25015381). 2. INTERACTION BETWEEN RNA POLYMERASE II LARGE SUBUNIT AND CORE HISTONES DURING TRASCRIPTION THROUGH THE NUCLEOSOME. In collaboration with the Studitsky Lab (Fox Chase Cancer Center). The problem of how RNA polymerase II transcribes through a nucleosome has been studied for many years: the nucleosome is very compact and yet RNA polymerase II can transcribe through it, displacing only a histone H2A-H2B dimer. How does the bulky polymerase achieve this feat? We collaborated with the Studitsky Lab to compare the kinetics of transit through a single nucleosome by purified wild type and mutated RNA polymerase II (Chang et al., 2014). The mutations are located in a negatively charged region of the polymerase identified by modeling, which was postulated to interact with the positively charged histones during transit, such that the histones are more likely to be retained after transcription. We found that although the mutated polymerase transcribes at a similar rate to wild type, the histone octamer is more likely to be displaced from the DNA, consistent with a histone-polymerase interaction during transcription. Chang HW, Kulaeva OI, Shaytan AK, Kibanov M, Kuznedelov K, Severinov KV, Kirpichnikov MP, Clark DJ, Studitsky VM (2014). Analysis of the mechanism of nucleosome survival during transcription. Nucl. Acids Res. 42, 1619-1627. 3. SPT10 AND SBF CONTROL THE TIMING OF HISTONE H2A/H2B GENE ACTIVATION IN BUDDING YEAST. We have shown that Spt10 is a very 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. More recently, we have addressed the role of Spt10 in the cell cycle-dependent regulation of the histone genes, which is necessary to provide histones for nucleosome assembly during DNA replication. Histones H2A and H2B are expressed from divergent promoters at the HTA1-HTB1 and HTA2-HTB2 loci. We showed that Spt10 and the cell cycle regulator SBF (a Swi4-Swi6 heterodimer) have overlapping binding sites in the HTA1-HTB1 promoter. Both SBF and Spt10 are bound in cells arrested with alpha-factor, apparently awaiting a signal to activate transcription. Soon after removal of alpha-factor, SBF initiates a small, early peak of HTA1 and HTB1 transcription, which is followed by a much larger peak due to Spt10. Both activators dissociate from the HTA1-HTB1 promoter after expression has been activated. Thus, SBF and Spt10 cooperate to control the timing of HTA1-HTB1 expression. Our current work has two goals: (1) Understanding the dynamics of chromatin structure during the cell cycle at the histone genes and genome-wide. (2) Testing our proposed model for histone gene regulation (Eriksson et al., 2012), which posits that the extent to which histone chaperones are saturated with histones is the critical signal for negative feedback control of transcription. Eriksson PR, Ganguli D, Nagarajavel V, Clark DJ (2012). Regulation of histone gene expression in budding yeast. Genetics 191, 7-20.
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