Restriction of histone gene transcription to S phase by phosphorylation of a chromatin boundary protein.

The cell cycle-regulated expression of core histone genes is required for DNA replication and proper cell cycle progression in eukaryotic cells. Although some factors involved in histone gene transcription are known, the molecular mechanisms that ensure proper induction of histone gene expression during S phase remain enigmatic. Here we demonstrate that S-phase transcription of the model histone gene HTA1 in yeast is regulated by a novel attach-release mechanism involving phosphorylation of the conserved chromatin boundary protein Yta7 by both cyclin-dependent kinase 1 (Cdk1) and casein kinase 2 (CK2). Outside S phase, integrity of the AAA-ATPase domain is required for Yta7 boundary function, as defined by correct positioning of the histone chaperone Rtt106 and the chromatin remodeling complex RSC. Conversely, in S phase, Yta7 is hyperphosphorylated, causing its release from HTA1 chromatin and productive transcription. Most importantly, abrogation of Yta7 phosphorylation results in constitutive attachment of Yta7 to HTA1 chromatin, preventing efficient transcription post-recruitment of RNA polymerase II (RNAPII). Our study identified the chromatin boundary protein Yta7 as a key regulator that links S-phase kinases with RNAPII function at cell cycle-regulated histone gene promoters.

Two-color cell array screen reveals interdependent roles for histone chaperones and a chromatin boundary regulator in histone gene repression.

We describe a fluorescent reporter system that exploits the functional genomic tools available in budding yeast to systematically assess consequences of genetic perturbations on gene expression. We used our Reporter-Synthetic Genetic Array (R-SGA) method to screen for regulators of core histone gene expression. We discovered that the histone chaperone Rtt106 functions in a pathway with two other chaperones, Asf1 and the HIR complex, to create a repressive chromatin structure at core histone promoters. We found that activation of histone (HTA1) gene expression involves both relief of Rtt106-mediated repression by the activity of the histone acetyltransferase Rtt109 and restriction of Rtt106 to the promoter region by the bromodomain-containing protein Yta7. We propose that the maintenance of Asf1/HIR/Rtt106-mediated repressive chromatin domains is the primary mechanism of cell-cycle regulation of histone promoters. Our data suggest that this pathway may represent a chromatin regulatory mechanism that is broadly used across the genome.

Comprehensive genetic analysis of transcription factor pathways using a dual reporter gene system in budding yeast.

The development and application of genomic reagents and techniques has fuelled progress in our understanding of regulatory networks that control gene expression in eukaryotic cells. However, a full description of the network of regulator-gene interactions that determine global gene expression programs remains elusive and will require systematic genetic as well as biochemical assays. Here, we describe a functional genomics approach that combines reporter technology, genome-wide array-based reagents and high-throughput imaging to discover new regulators controlling gene expression patterns in Saccharomyces cerevisiae. Our strategy utilizes the synthetic genetic array (SGA) method to systematically introduce promoter-GFP (green fluorescent protein) reporter constructs along with a control promoter-RFP (red fluorescent protein) gene into the array of approximately 4500 viable yeast deletion mutants. Fluorescence intensities from each reporter are assayed from individual colonies arrayed on solid agar plates using a scanning fluorimager and the ratio of GFP to RFP intensity reveals deletion mutants that cause differential GFP expression. We are exploiting this screening approach to construct a detailed map describing the interplay of regulators controlling the eukaryotic cell cycle. The method is extensible to any transcription factor or signalling pathway for which an appropriate reporter gene can be devised.

Reporter-based synthetic genetic array analysis: a functional genomics approach for investigating the cell cycle in Saccharomyces cerevisiae.

Temporal control of gene expression is a widespread feature of cell cycles, with clear transcriptional programs in bacteria, yeast, and metazoans. In budding yeast, approximately 1,000 genes are transcribed during a specific interval of the cell cycle. Although a number of factors that contribute to this periodic pattern of gene expression have been studied in Saccharomyces cerevisiae, pathways of cell cycle-regulated transcription remain largely undefined. To identify regulators of genes exhibiting cell cycle periodicity, we have developed a functional genomics approach termed reporter-based synthetic genetic array (R-SGA) analysis. Based on synthetic genetic array (SGA) analysis, R-SGA allows rapid and easily automated incorporation of a cell cycle reporter gene into the array of viable haploid yeast gene-deletion mutants. Scoring of reporter activity in mutant strains compared to wild type identifies candidate regulators of the cell cycle gene of interest. In contrast to microarrays, which generally provide information about the expression of all genes under a particular condition (for example, a single gene deletion), R-SGA analysis facilitates the study of the expression of a single gene in all deletion mutants. Our system can be adapted to examine the expression of any gene not only in the context of haploid deletion mutants but also using other array-based strain collections available to the yeast community.

Signature proteins that are distinctive of alpha proteobacteria.

BACKGROUND: The alpha (alpha) proteobacteria, a very large and diverse group, are presently characterized solely on the basis of 16S rRNA trees, with no known molecular characteristic that is unique to this group. The genomes of three alpha-proteobacteria, Rickettsia prowazekii (RP), Caulobacter crescentus (CC) and Bartonella quintana (BQ), were analyzed in order to search for proteins that are unique to this group. RESULTS: Blast analyses of protein sequences from the above genomes have led to the identification of 61 proteins which are distinctive characteristics of alpha-proteobacteria and are generally not found in any other bacteria. These alpha-proteobacterial signature proteins are generally of hypothetical functions and they can be classified as follows: (i) Six proteins (CC2102, CC3292, CC3319, CC1887, CC1725 and CC1365) which are uniquely present in most sequenced alpha-proteobacterial genomes; (ii) Ten proteins (CC1211, CC1886, CC2245, CC3470, CC0520, CC0365, CC0366, CC1977, CC3010 and CC0100) which are present in all alpha-proteobacteria except the Rickettsiales; (iii) Five proteins (CC2345, CC3115, CC3401, CC3467 and CC1021) not found in the intracellular bacteria belonging to the order Rickettsiales and the Bartonellaceae family; (iv) Four proteins (CC1652, CC2247, CC3295 and CC1035) that are absent from various Rickettsiales as well as Rhodobacterales; (v) Three proteins (RP104, RP105 and RP106) that are unique to the order Rickettsiales and four proteins (RP766, RP192, RP030 and RP187) which are specific for the Rickettsiaceae family; (vi) Six proteins (BQ00140, BQ00720, BQ03880, BQ12030, BQ07670 and BQ11900) which are specific to the order Rhizobiales; (vii) Four proteins (BQ01660, BQ02450, BQ03770 and BQ13470) which are specific for the order Rhizobiales excluding the family Bradyrhizobiaceae; (viii) Nine proteins (BQ12190, BQ11460, BQ11450, BQ11430, BQ11380, BQ11160, BQ11120, BQ11100 and BQ11030 which are distinctive of the Bartonellaceae family;(ix) Six proteins (CC0189, CC0569, CC0331, CC0349, CC2323 and CC2637) which show sporadic distribution in alpha-proteobacteria, (x) Four proteins (CC2585, CC0226, CC2790 and RP382) in which lateral gene transfers are indicated to have occurred between alpha-proteobacteria and a limited number of other bacteria. CONCLUSION: The identified proteins provide novel means for defining and identifying the alpha-proteobacteria and many of its subgroups in clear molecular terms and in understanding the evolution of this group of species. These signature proteins, together with the large number of alpha-proteobacteria specific indels that have recently been identified http://www.bacterialphylogeny.com, provide evidence that all species from this diverse group share many unifying and distinctive characteristics. Functional studies on these proteins should prove very helpful in the identification of such characteristics.

Illuminating transcription pathways using fluorescent reporter genes and yeast functional genomics.

Technological advances have enabled researchers to probe gene regulatory pathways on an unprecedented scale. Here, we summarize our recent work that exploits a systematic screening approach in the budding yeast to discover regulators of a promoter of interest. We discuss future applications of our approach based on emerging themes in the literature.

Quantitative cell array screening to identify regulators of gene expression.

In the last decade or so, advances in genome-scale technologies have allowed systematic and detailed analysis of gene function. The experimental accessibility of budding yeast makes it a test-bed for technology development and application of new functional genomic tools and resources that pave the way for comparable efforts in higher eukaryotes. In this article, we review advances in reporter screening technology to discover trans-acting regulators of promoters (or cis-elements) of interest in the context of a novel functional genomics approach called Reporter Synthetic Genetic Array (R-SGA) analysis. We anticipate that this methodology will enable researchers to collect quantitative data on hundreds of gene expression pathways in an effort to better understand transcriptional regulatory networks.

Functional dissection of IME1 transcription using quantitative promoter-reporter screening.

Transcriptional regulation is a key mechanism that controls the fate and response of cells to diverse signals. Therefore, the identification of the DNA-binding proteins, which mediate these signals, is a crucial step in elucidating how cell fate is regulated. In this report, we applied both bioinformatics and functional genomic approaches to scrutinize the unusually large promoter of the IME1 gene in budding yeast. Using a recently described fluorescent protein-based reporter screen, reporter-synthetic genetic array (R-SGA), we assessed the effect of viable deletion mutants on transcription of various IME1 promoter-reporter genes. We discovered potential transcription factors, many of which have no perfect consensus site within the IME1 promoter. Moreover, most of the cis-regulatory sequences with perfect homology to known transcription factor (TF) consensus were found to be nonfunctional in the R-SGA analysis. In addition, our results suggest that lack of conservation may not discriminate against a TF regulatory role at a specific promoter. We demonstrate that Sum1 and Sok2, which regulate IME1, bind to nonperfect consensuses within nonconserved regions in the sensu stricto Saccharomyces strains. Our analysis supports the view that although comparative analysis can provide a useful guide, functional assays are required for accurate identification of TF-binding site interactions in complex promoters.