1. TRANSCRIPTIONAL ACTIVATION AND ATP-DEPENDENT CHROMATIN REMODELING MACHINES. In collaboration with Dr. Feng Cui and Dr Victor Zhurkin (NCI). The new high-throughput sequencing technology has made it possible to map nucleosomes genome-wide in a relatively simple experiment involving digestion of nuclei with micrococcal nuclease (MNase). Recently, we described a genome-wide paired-end sequencing study of nucleosomes derived from cells treated with 3-aminotriazole (3AT), which induces Gcn4-dependent genes, and control cells (Cole et al., 2011). We concluded that each nucleosome may adopt one of several alternative positions within a cluster and that this cluster organization is genome-wide. The implication is that different cells within a population have different chromatin structures. Thus, binding sites for regulatory factors might be nucleosomal in some cells and in the linker in other cells. Induction with 3AT results in a major disruption of nucleosome positioning, sometimes with altered nucleosome spacing (Cole et al., 2011). The most affected genes (50 in all) exhibit a dramatic loss of occupancy over the transcribed region, sometimes extending into neighboring genes. In contrast, nucleosome-depleted promoters are generally unaffected by induction. A small number of genes are repressed by 3AT; these show similar changes in their chromatin structure, but in reverse: loss of nucleosome occupancy is observed in control cells rather than in 3AT-treated cells. Thus, loss of nucleosome occupancy correlates with gene activation. We have now examined the extensive nucleosomal rearrangements that occur when genes are activated for transcription by 3AT using a global, bioinformatic, approach (Cui et al., 2012). At the genomic level, nucleosomes are regularly phased relative to the transcription start site. However, for a subset of strongly induced genes, this phasing is much more irregular after induction, consistent with the loss of some nucleosomes and the re-positioning of the remaining nucleosomes. To address the nature of this rearrangement, we developed the inter-nucleosome distance auto-correlation (DAC) function. At long range, DAC analysis indicates that nucleosomes have an average spacing of 162 bp, as expected. At short range, DAC reveals a 10.25-bp periodicity, implying that nucleosomes in overlapping positions are rotationally related. That is, nucleosome positions overlap by multiples of 10 bp, the helical period of B-DNA, such that the energy required to bend the DNA around the central core histone octamer is minimally affected. DAC analysis of the 3AT-induced genes suggests that transcription activation coincides with rearrangement of nucleosomes into irregular arrays with longer spacing. Sequence analysis of the +1 nucleosomes belonging to the 45 most strongly activated genes reveals a distinctive periodic oscillation in the A/T-dinucleotide occurrence that is present throughout the nucleosome and extends into the linker. This unusual pattern suggests that the +1 nucleosomes might be prone to sliding, thereby facilitating transcription. Currently, we are exploring the roles of remodeling complexes that are capable of nucleosome mobilization, such as SWI/SNF and RSC, in disruption of chromatin structure. We have written a review of our work, placing it in the context of the chromatin field (Cole et al., 2012a) and we have described our methodology in detail (Cole et al., 2012b). Cole HA, Howard BH, Clark DJ (2011). Activation-induced disruption of nucleosome position clusters on the coding regions of Gcn4-dependent genes extends into neighbouring genes. Nucl Acids Res 39, 9521-9535. Cole HA, Nagarajavel V, Clark DJ (2012a). Perfect and imperfect nucleosome positioning in yeast. Biochim Biophys Acta 1819, 639-643. Cole HA, Nagarajavel V, Clark DJ (2012b). Genome-wide mapping of nucleosomes in yeast using paired-end sequencing. Methods in Enzymology 513, 145-168. Cui F, Cole HA, Clark DJ, Zhurkin VB (2012). Transcriptional activation of yeast genes disrupts intragenic nucleosome phasing. Nucl Acids Res 40, 10753-10764. 2. STABLE RNA POLYMERASE III TRANSCRIPTION COMPLEXES IN VIVO In collaboration with James R. Iben, Bruce H. Howard and Richard J. Maraia (NICHD). TFIIIB and TFIIIC are multi-subunit factors required for transcription by RNA polymerase III. During the course of our nucleosome mapping studies, we discovered that the tRNA genes are occupied by non-nucleosomal complexes, which are in fact stable transcription complexes composed of two multi-subunit factors: TFIIIB and TFIIIC. Accordingly, we have described a high-resolution footprint map of TFIIIBTFIIIC complexes in Saccharomyces cerevisiae, obtained by paired-end sequencing of MNase-resistant DNA (Nagarajavel et al., 2013). On tRNA genes, TFIIIB and TFIIIC form stable complexes with the same distinctive occupancy pattern but in mirror image, termed bootprints. Global analysis reveals that the TFIIIBTFIIIC transcription complex exhibits remarkable structural elasticity: tRNA genes vary significantly in length but remain protected by TFIIIC. Introns, when present, are markedly less protected. The RNA polymerase III transcription terminator is flexibly accommodated within the transcription complex and, unexpectedly, plays a major structural role by delimiting its 3'-boundary. The ETC sites, where TFIIIC binds without TFIIIB, exhibit different bootprints, suggesting that TFIIIC forms complexes involving other factors. We confirm six ETC sites and report a new site (ETC10). Surprisingly, TFIIIC, but not TFIIIB, interacts with some centromeric nucleosomes, suggesting that interactions between TFIIIC and the centromere may be important in the 3-dimensional organization of the nucleus. Currently, we are pursuing this connection between centromeres and transcription complexes. Nagarajavel V, Iben JR, Howard BH, Maraia RJ, Clark DJ (2013). Global 'bootprinting' reveals the elastic architecture of the yeast TFIIIB-TFIIIC transcription complex in vivo. Nucl Acids Res PMID 23856458. 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.