reflects our work on three different specific aims: 1. TRANSCRIPTIONAL ACTIVATION AND SWI/SNF-DEPENDENT NUCLEOSOME MOBILIZATION. In collaboration with Dr. Bruce H. Howard (NICHD). We chose budding yeast as a model organism because biochemical studies of chromatin structure could be combined with molecular genetics. Current models for the role of the SWI/SNF ATP-dependent chromatin remodeling complex in gene regulation are focused on promoters, where the most obvious changes in chromatin structure occur. However, using our plasmid model system with HIS3, a SWI/SNF-regulated gene, we discovered that transcriptional activation creates a domain of remodeled chromatin structure that extends far beyond the promoter, to include the entire gene. We addressed the effects of transcriptional activation on the chromatin structure of HIS3 by mapping the precise positions of nucleosomes in non-induced and transcriptionally activated chromatin. In the absence of the Gcn4 activator, the HIS3 gene is organized into a dominant nucleosomal array. In wild type chromatin, this array is disrupted, and several alternative, overlapping, nucleosomal arrays are formed. Disruption of the dominant array also requires the SWI/SNF remodeling machine, indicating that the SWI/SNF complex plays an important role in nucleosome mobilization. We suggest that the net effect of interplay among remodeling machines at HIS3 is to create a highly dynamic chromatin structure (Clark, 2010). Our work on HIS3 and our earlier work on CUP1 indicate that, at least for these two genes, the target of remodeling complexes is a domain rather than just the promoter. This is an important finding, because it suggests that remodeling complexes act on chromatin domains. We speculate that remodeling entire genes might facilitate elongation through nucleosomes by RNA polymerase II. The advent of high throughput sequencing has allowed us to analyse nucleosome positioning on a genome-wide scale, confirming and extending our previous observations. We are using paired-end sequencing, which increases the accuracy of the position measurements and facilitates the bioinformatics analysis. We have shown that major disruptions occur in the chromatin structure of genes when they are activated for transcription, involving a dramatic loss of canonical nucleosomes and a re-positioning of the remaining nucleosomes (Cole et al., 2011a). We also discovered that the specialized nucleosome at the centromere is essentially perfectly positioned, unlike canonical nucleosomes, which occupy one of several alternative overlapping positions (Cole et al., 2011b). We attribute the perfect positioning of the centromeric nucleosome to the presence of sequence-specific factors built into the nucleosome. Currently, we are exploring the roles of remodeling complexes capable of nucleosome mobilization in disruption of chromatin structure. Clark DJ (2010). Nucleosome positioning, nucleosome spacing and the nucleosome code. J Biomol Struc Dyn 27, 781793. Cole HA, Howard BH, Clark DJ (2011a). Activation-induced disruption of nucleosome position clusters on the coding regions of Gcn4-dependent genes extends into neighbouring genes. Nucl Acids Res PubMed ID: 21880600 Cole HA, Howard BH, Clark DJ (2011b). The centromeric nucleosome of budding yeast is perfectly positioned and covers the entire centromere. Proc Natl Acad Sci USA 108, 12687-12692. 2. A NUCLEOSOMAL BARRIER TO TRANSCRIPTION BY RNA POLYMERASE II IN VITRO AND IN VIVO. In collaboration with the Studitsky Lab (UMDNJ) and the Bartholomew Lab (USI). Many eukaryotic genes are regulated at the level of transcript elongation. Nucleosomes are likely targets for this regulation. Previously, the Studitsky Lab has shown that nucleosomes formed on very strong positioning sequences (601 and 603), present a high, orientation-dependent barrier to transcription by RNA polymerase II in vitro. The existence of this polar barrier correlates with the interaction of a 16-bp polar barrier signal (PBS) with the promoter-distal histone H3-H4 dimer. We find that the polar barrier is relieved by ISW2, an ATP-dependent chromatin remodeler, which translocates the nucleosome over a short distance, such that the PBS no longer interacts with the distal H3-H4 dimer, although it remains within the nucleosome. In vivo, insertion of the 603 positioning sequence into the yeast CUP1 gene results in a modest reduction in transcription, but this reduction is orientation-independent, indicating that the polar barrier can be circumvented. Surprisingly, the 603-nucleosome is present at the expected position in only a small fraction of cells. Thus, the polar barrier is probably non-functional in vivo because the nucleosome is not positioned appropriately, presumably due to nucleosome sliding activities. Interactions between polar barriers and chromatin remodelers might have significant regulatory potential (Gaykalova et al., 2011). Gaykalova DA, Nagarajavel V, Bondarenko VA, Bartholomew B, Clark DJ, Studitsky VM (2011). Nucl Acids Res 39, 3520-3528. 3. SPT10 AND SWI4 CONTROL THE TIMING OF HISTONE H2A/H2B GENE ACTIVATION IN BUDDING YEAST. We have shown previously that induction of the CUP1 gene results in acetylation of nucleosomes at the CUP1 promoter. This acetylation is dependent on SPT10, which encodes a putative histone acetylase (HAT) and has been implicated as a global regulator. We confirmed that Spt10 has global effects on transcription using expression microarrays. Surprisingly, the global effects of Spt10 are the indirect result of defects in chromatin structure, reflecting its role as activator of the core histone genes. We demonstrated that Spt10 binds specifically to pairs of upstream activating sequences (UAS elements) in the core histone promoters (consensus: (G/A)TTCCN6TTCNC), consistent with a direct role in histone gene regulation. The DNA-binding domain of Spt10 contains an unusual zinc finger with homology to foamy retrovirus integrase, which we propose to be a sequence-specific DNA-binding protein. Thus, 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. 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 show that Spt10 binds to two pairs of UAS elements in the HTA1-HTB1 promoter: UAS1/UAS2 drive HTA1 expression and UAS3/UAS4 drive HTB1. UAS3 and UAS4 also contain binding sites for the cell cycle regulator SBF (a Swi4-Swi6 heterodimer), which overlap the Spt10 binding sites. Spt10 and SBF binding to UAS3 and UAS4 is mutually exclusive in vitro. 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 (Eriksson et al., 2011). Our current work is focused on the identification of negative regulatory proteins that bind at the histone promoters, which might counteract activation by Spt10. Eriksson PR, Ganguli D, Clark DJ (2011). Spt10 and Swi4 control the timing of histone H2A/H2B gene activation in budding yeast. Mol Cell Biol 31, 557-572.

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Rawal, Yashpal; Chereji, R?zvan V; Qiu, Hongfang et al. (2018) SWI/SNF and RSC cooperate to reposition and evict promoter nucleosomes at highly expressed genes in yeast. Genes Dev 32:695-710
Johnson, Thomas A; Chereji, Razvan V; Stavreva, Diana A et al. (2018) Conventional and pioneer modes of glucocorticoid receptor interaction with enhancer chromatin in vivo. Nucleic Acids Res 46:203-214
Ouda, Ryota; Sarai, Naoyuki; Nehru, Vishal et al. (2018) SPT6 interacts with NSD2 and facilitates interferon-induced transcription. FEBS Lett 592:1681-1692
Rawal, Yashpal; Chereji, R?zvan V; Valabhoju, Vishalini et al. (2018) Gcn4 Binding in Coding Regions Can Activate Internal and Canonical 5' Promoters in Yeast. Mol Cell 70:297-311.e4
Chereji, R?zvan V; Clark, David J (2018) Major Determinants of Nucleosome Positioning. Biophys J 114:2279-2289
Maree, Johannes Petrus; Povelones, Megan Lindsay; Clark, David Johannes et al. (2017) Well-positioned nucleosomes punctuate polycistronic pol II transcription units and flank silent VSG gene arrays in Trypanosoma brucei. Epigenetics Chromatin 10:14
Chereji, R?zvan V; Ocampo, Josefina; Clark, David J (2017) MNase-Sensitive Complexes in Yeast: Nucleosomes and Non-histone Barriers. Mol Cell 65:565-577.e3
Gerasimova, N S; Pestov, N A; Kulaeva, O I et al. (2016) Transcription-induced DNA supercoiling: New roles of intranucleosomal DNA loops in DNA repair and transcription. Transcription 7:91-5
Ocampo, Josefina; Chereji, R?zvan V; Eriksson, Peter R et al. (2016) The ISW1 and CHD1 ATP-dependent chromatin remodelers compete to set nucleosome spacing in vivo. Nucleic Acids Res 44:4625-35
Qiu, Hongfang; Chereji, R?zvan V; Hu, Cuihua et al. (2016) Genome-wide cooperation by HAT Gcn5, remodeler SWI/SNF, and chaperone Ydj1 in promoter nucleosome eviction and transcriptional activation. Genome Res 26:211-25

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