The eukaryotic genome is packaged as chromatin with nucleosomes comprising its basic structural unit, but the detailed structure of chromatin and its dynamic remodeling in terms of individual nucleosome positions has not been completely defined experimentally for any genome. We used ultra-high–throughput sequencing to map the remodeling of individual nucleosomes throughout the yeast genome before and after a physiological perturbation that causes genome-wide transcriptional changes. Nearly 80% of the genome is covered by positioned nucleosomes occurring in a limited number of stereotypical patterns in relation to transcribed regions and transcription factor binding sites. Chromatin remodeling in response to physiological perturbation was typically associated with the eviction, appearance, or repositioning of one or two nucleosomes in the promoter, rather than broader region-wide changes. Dynamic nucleosome remodeling tends to increase the accessibility of binding sites for transcription factors that mediate transcriptional changes. However, specific nucleosomal rearrangements were also evident at promoters even when there was no apparent transcriptional change, indicating that there is no simple, globally applicable relationship between chromatin remodeling and transcriptional activity. Our study provides a detailed, high-resolution, dynamic map of single-nucleosome remodeling across the yeast genome and its relation to global transcriptional changes.

Although chromatin structure is known to affect transcriptional activity, it is not clear how broadly patterns of changes in histone modifications and nucleosome occupancy affect the dynamic regulation of transcription in response to perturbations. The identity and role of chromatin remodelers that mediate some of these changes are also unclear. Here, we performed temporal genome-wide analyses of gene expression, nucleosome occupancy, and histone H4 acetylation during the response of yeast (Saccharomyces cerevisiae) to different stresses and report several findings. First, a large class of predominantly ribosomal protein genes, whose transcription was repressed during both heat shock and stationary phase, showed strikingly contrasting histone acetylation patterns. Second, the SWI/SNF complex was required for normal activation as well as repression of genes during heat shock, and loss of SWI/SNF delayed chromatin remodeling at the promoters of activated genes. Third, Snf2 was recruited to ribosomal protein genes and Hsf1 target genes, and its occupancy of this large set of genes was altered during heat shock. Our results suggest a broad and direct dual role for SWI/SNF in chromatin remodeling, during heat shock activation as well as repression, at promoters and coding regions.

Transcription of the U6 snRNA gene (SNR6) in Saccharomyces cerevisiae by RNA polymerase III (pol III) requires TFIIIC and its box A and B binding sites. In contrast, TFIIIC has little or no effect on SNR6 transcription with purified components in vitro due to direct recognition of the SNR6 TATA box by TFIIIB. When SNR6 was assembled into chromatin in vitro by use of the Drosophila melanogaster S-190 extract, transcription of these templates with highly purified yeast pol III, TFIIIC, and TFIIIB displayed a nearabsolute requirement for TFIIIC but yielded a 5-to 15-fold-higher level of transcription relative to naked DNA (>100-fold activation over repressed chromatin). Analysis of chromatin structure demonstrated that TFIIIC binding leads to remodeling of U6 gene chromatin, resulting in positioning of a nucleosome between boxes A and B. The resulting folding of the intervening DNA into the nucleosome could bring the suboptimally spaced SNR6 box A and B elements into greater proximity and thus facilitate activation of transcription. In the absence of ATP, however, the binding of TFIIIC to box B in chromatin was not accompanied by remodeling and the transcription activation was ϳ35% of that seen in its presence, implying that both TFIIIC binding and ATP-dependent chromatin remodeling were required for the full activation of the gene. Our results suggest that TFIIIC, which is a basal transcription factor of pol III, also plays a direct role in remodeling chromatin on the SNR6 gene.Packing of DNA as chromatin generates gene-specific architectures that are instrumental in poising genes for or against immediate or eventual expression (23,32,43,45,61,70). These gene-specific chromatin structures are generated in vivo by defined positioning of histone octamers (72). Chromatin is generally repressive for transcription (50, 71), but this repression can be overcome with help from chromatin structure modulators (41). The first step in returning an inactive gene to activity frequently involves the binding of an activator to its cognate binding site over the surface of a nucleosome; binding sites that are not exposed on the nucleosome surface can be unmasked by ubiquitous ATP-dependent chromatin remodeling factors working in conjunction with histone acetylation (1,5,11,24,28,39,(63)(64)(65). Chromatin remodeling and histone modification have been largely analyzed in the context of RNA polymerase II (pol II) genes, but chromatin structure has also been shown to play an important role in pol III gene transcription (44,67,70).pol III genes are characteristically short, with intragenic promoter elements (boxes A, B, and C) to which the core transcription factors TFIIIC and TFIIIA bind (15,54,68). TFIIIC is very large, and the nine Zn fingers of TFIIIA also cover an extended DNA site. This ability of pol III to nearly cover entire transcription units with its "own" transcription factors in competition with occupancy by nucleosomes suggests a relationship of pol III genes in vivo to their chromatin background that is quite different ...

Transcription from the yeast SNR6 (U6 small nuclear RNA) chromatin, a gene transcribed by the enzyme RNA polymerase III, depends on its transcription factor IIIC (TFIIIC) and the promoter elements (the intragenic box A and box B located downstream to its terminator) to which TFIIIC binds. The genes transcribed by polymerase III generally lack the upstream promoter elements where TFIIIC is known to recruit the transcription initiation factor TFIIIB. The TFIIIC-dependent chromatin remodeling of the gene in vitro that involves translational positioning of a nucleosome between boxes A and B is found to be essential for its transcriptional activation. We show here that the role of TFIIIC is not limited to the recruitment of TFIIIB on chromatin templates. The pre-binding of TFIIIB to the SNR6 TATA box in the upstream gene region does not alleviate TFIIIC requirement for transcriptional activation of the chromatin. Binding of TFIIIC to an array of pre-positioned nucleosomes results in an upward shift of the single nucleosome between boxes A and B. The ϳ40-bp shift of this nucleosome in the 3 to 5 direction leads to increased nuclease sensitivity of the ϳ40-bp DNA 3 to the upstream TATA box. Further chromatin remodeling accompanies the binding of TFIIIB in the next step. This two-step remodeling mechanism using the basal factors of the gene yields high transcription levels and generates a chromatin structure similar to that reported for the gene in vivo.Organization of the eukaryotic genome into chromatin links the process of transcription intimately with the structure of its template. Gene expression in eukaryotes is regulated by several mechanisms that alter the chromatin structure either to allow or disallow an activity using it as the substrate or template. These mechanisms include the covalent histone modifications, exchange with histone variants, and ATP-dependent chromatin remodeling, demonstrated in a large number of studies with the genes transcribed by the enzyme RNA polymerase II (pol II) 3 (1-4). Fewer examples of chromatin transcription by the RNA pol III are available that may have a different relationship with its template structure due to the short gene units and characteristically intragenic promoter elements.Pol III transcribes genes encoding structural RNAs required for translation (tRNA, 5 S rRNA), tRNA processing (RPR1), and splicing (U6 snRNA). The pol III transcription machinery in yeast is made up of the 17-subunit polymerase and its two complex factors (TFIIIB and TFIIIC) that are involved in promoter recognition and/or initiation of all the three classes of the pol III genes (5-8). Transcription factor IIIA, which binds to the intragenic box C, is required only for the 5 S ribosomal DNA transcription (9). TFIIIC binds to the intragenic promoter elements, boxes A and B (9), and the role attributed to it in pol III transcription has been to recruit the true transcription initiation factor TFIIIB in the upstream region of the pol III-transcribed genes (10). The TFIIIB-DNA complex is extremely stab...

The eukaryotic genome is packaged into chromatin, and chromatin modification and remodeling play an important role in transcriptional regulation, DNA replication, recombination and repair. Recent findings have shown that various post-translational histone modifications cooperate to recruit different effector proteins that bring about mobilization of the nucleosomes and cause distinct downstream consequences. The combination of chromatin immunoprecipitation (ChIP) using antibodies directed against the core histones or specific histone modifications, with high-resolution tiling microarray analysis allows the examination of nucleosome occupancy and histone modification status genome-wide. Comparing genome-wide chromatin status with global gene expression patterns can reveal causal connections between specific patterns of histone modifications and the resulting gene expression. Here, we describe current methods based on recent advances in microarray technology to conduct such studies.