John J. Wyrick

Dissecting the Regulatory Circuitry of a Eukaryotic Genome

Genome-wide expression analysis was used to identify genes whose expression depends on the functions of key components of the transcription initiation machinery in yeast. Components of the RNA polymerase II holoenzyme, the general transcription factor TFIID, and the SAGA chromatin modification complex were found to have roles in expression of distinct sets of genes. The results reveal an unanticipated level of regulation which is superimposed on that due to gene-specific transcription factors, a novel mechanism for coordinate regulation of specific sets of genes when cells encounter limiting nutrients, and evidence that the ultimate targets of signal transduction pathways can be identified within the initiation apparatus.

Serial Regulation of Transcriptional Regulators in the Yeast Cell Cycle

Genome-wide location analysis was used to determine how the yeast cell cycle gene expression program is regulated by each of the nine known cell cycle transcriptional activators. We found that cell cycle transcriptional activators that function during one stage of the cell cycle regulate transcriptional activators that function during the next stage. This serial regulation of transcriptional activators forms a connected regulatory network that is itself a cycle. Our results also reveal how the nine transcriptional regulators coordinately regulate global gene expression and diverse stage-specific functions to produce a continuous cycle of cellular events. This information forms the foundation for a complete map of the transcriptional regulatory network that controls the cell cycle.

Deciphering gene expression regulatory networks

In the past year, great strides have been made in our understanding of the regulatory networks that control gene expression in the model eukaryote Saccharomyces cerevisiae. The development and use of a number of genomic tools, including genome-wide location and expression analysis, has fueled this progress. In addition, a variety of computational algorithms have been devised to mine genomic sequence for conserved regulatory motifs in co-regulated genes. The recent description of the genetic network controlling the cell cycle illustrates the tremendous potential of these approaches for deciphering gene expression regulatory networks in eukaryotic cells.

Interplay of Positive and Negative Regulators in Transcription Initiation by RNA Polymerase II Holoenzyme

ABSTRACT Activation of protein-encoding genes involves recruitment of an RNA polymerase II holoenzyme to promoters. Since the Srb4 subunit of the holoenzyme is essential for expression of most class II genes and is a target of at least one transcriptional activator, we reasoned that suppressors of a temperature-sensitive mutation in Srb4 would identify other factors generally involved in regulation of gene expression. We report here that MED6 and SRB6, both of which encode essential components of the holoenzyme, are among the dominant suppressors and that the products of these genes interact physically with Srb4. The recessive suppressors include NCB1 (BUR6),NCB2, NOT1, NOT3, NOT5, and CAF1, which encode subunits of NC2 and the Not complex. NC2 and Not proteins are general negative regulators which interact with TATA box binding protein (TBP). Taken together, these results suggest that transcription initiation involves a dynamic balance between activation mediated by specific components of the holoenzyme and repression by multiple TBP-associated regulators.

Deciphering the Roles of the Histone H2B N-Terminal Domain in Genome-Wide Transcription†

ABSTRACT Histone N-terminal domains are frequent targets of posttranslational modifications. Multiple acetylated lysine residues have been identified in the N-terminal domain of H2B (K6, K11, K16, K17, K21, and K22), but little is known about how these modifications regulate transcription. We systematically mutated the N-terminal domain of histone H2B, both at known sites of lysine acetylation and elsewhere, and characterized the resulting changes in genome-wide expression in each mutant strain. Our results indicate that known sites of lysine acetylation in this domain are required for gene-specific transcriptional activation. However, the entire H2B N-terminal domain is principally required for the transcriptional repression of a large subset of the yeast genome. We find that the histone H2B repression (HBR) domain, comprised of residues 30 to 37, is necessary and sufficient for this repression. Many of the genes repressed by the HBR domain are located adjacent to telomeres or function in vitamin and carbohydrate metabolism. Deletion of the HBR domain also confers an increased sensitivity to DNA damage by UV irradiation. We mapped the critical residues in the HBR domain required for its repression function. Finally, comparisons of these data with previous studies reveal that a surprising number of genes are coregulated by the N-terminal domains of histone H2B, H3, and H4.

Ctk Complex-Mediated Regulation of Histone Methylation by COMPASS

ABSTRACT A comparative global proteomic screen identified factors required for COMPASS (complex of proteins associated with Set1)-mediated mono-, di-, and trimethylation of the fourth lysine of histone H3 (H3K4), which included components of a cyclin-dependent protein kinase (Ctk complex) that phosphorylates the C-terminal domain of the largest subunit of RNA polymerase II (Pol II). Our results indicate that histone H3K4 methylation levels are regulated by the Ctk1, Ctk2, and Ctk3 components of the Ctk complex. We show that loss of Ctk1 kinase activity results in reduced histone H3K4 monomethylation levels, followed by a global increase in histone H3K4 trimethylation levels on chromatin. Ctk1 loss does not appear to have a substantial effect on histone H2B monoubiquitination levels or COMPASS and Paf1 complex phosphorylation. Our chromatin immunoprecipitation studies demonstrate that histone H3 eviction during active transcription is decelerated in a CTK1 deletion strain in response to reduced levels of Pol II recruitment. Our in vitro studies show that the onset of monomethylation on an unmethylated histone H3 by COMPASS is virtually immediate, while the onset of trimethylation occurs upon extended time of association between the histone tail and COMPASS. Our study suggests a role for the Ctk complex in the regulation of the pattern of H3K4 mono-, di-, and trimethylation via COMPASS.

The role of histone H2A and H2B post-translational modifications in transcription : A genomic perspective

In eukaryotic cells, the genome is packaged with histones H2A, H2B, H3, and H4 to form nucleosomes. Each of the histone proteins is extensively post-translationally modified, particularly in the flexible N-terminal histone tail domains. Curiously, while post-translational modifications in histone H3 and H4 have been extensively studied, relatively little is known about post-translational modifications in the N-terminal domains of histone H2A and H2B. In this review, we will summarize current knowledge of post-translational modifications in the N-terminal domains of histone H2A and H2B, and the histone variant H2AZ. We will examine the distribution of these modifications in genomic chromatin, and the function of these modifications in transcription.

Osiris: an integrated promoter database for Oryza sativa L.

SUMMARY Rice (Oryza sativa L.) is an important model monocot and cereal crop. While the rice genome sequence has been published and annotated, relatively little is known about the transcriptional networks that regulate rice gene expression. For this reason, we have developed Osiris, a database containing promoter sequences, predicted transcription factor (TF) binding sites, gene ontology annotation and microarray expression data for 24 209 genes in the rice genome. These tools are seamlessly integrated in the Osiris web site, allowing the user to visualize TF binding sites in multiple promoters; analyze the statistical significance of enriched TF binding sites; query for genes containing similar promoter regulatory logic or gene function and visualize the microarray expression patterns of queried or selected gene sets. AVAILABILITY http://www.bioinformatics2.wsu.edu/Osiris

Histone H3 Lys79 methylation is required for efficient nucleotide excision repair in a silenced locus of Saccharomyces cerevisiae

Methylation of specific histone lysine residues regulates gene expression and heterochromatin function, but little is known about its role in DNA repair. To examine how changes in conserved methylated residues of histone H3 affect nucleotide excision repair (NER), viable H3K4R and H3K79R mutants were generated in Saccharomyces cerevisiae. These mutants show decreased UV survival and impaired NER at the transcriptionally silent HML locus, while maintaining normal NER in the constitutively expressed RPB2 gene and transcriptionally repressed, nucleosome loaded GAL10 gene. Moreover, the HML chromatin in these mutants has reduced accessibility to Micrococcal nuclease (MNase). Importantly, chromatin immunoprecipitation analysis demonstrates there is enhanced recruitment of the Sir complex at the HML locus of these mutants, and deletion of the SIR2 or SIR3 genes restores the MNase accessibility and DNA repair efficiency at this locus. Furthermore, following UV irradiation expression of NER genes in these mutants remains at wild type levels, with the exception of RAD16 which decreases by more than 2-fold. These results indicate that impaired NER occurs in the silenced chromatin of H3K79R and H3K4,79R mutants as a result of increased binding of Sir complexes, which may reduce DNA lesion accessibility to repair enzymes.

Nucleosomes Inhibit Cas9 Endonuclease Activity in Vitro

During Cas9 genome editing in eukaryotic cells, the bacterial Cas9 enzyme cleaves DNA targets within chromatin. To understand how chromatin affects Cas9 targeting, we characterized Cas9 activity on nucleosome substrates in vitro. We find that Cas9 endonuclease activity is strongly inhibited when its target site is located within the nucleosome core. In contrast, the nucleosome structure does not affect Cas9 activity at a target site within the adjacent linker DNA. Analysis of target sites that partially overlap with the nucleosome edge indicates that the accessibility of the protospacer-adjacent motif (PAM) is the critical determinant of Cas9 activity on a nucleosome.