Analysis of factor interactions with RNA polymerase II elongation complexes using a new electrophoretic mobility shift assay

The elongation phase of transcription by RNA polymerase II (RNAP II) is controlled by a carefully orchestrated series of interactions with both negative and positive factors. However, due to the limitations of current methods and techniques, not much is known about whether and how these proteins physically associate with the engaged polymerases. To gain insight into the detailed mechanisms involved, we established an experimental system for analyzing direct factor interactions to RNAP II elongation complexes on native gels, namely elongation complex electrophoretic mobility shift assay (EC-EMSA). This new assay effectively allowed detection of interactions of TFIIF, TTF2, TFIIS, DSIF and P-TEFb with elongation complexes generated from a natural promoter using an immobilized template. As an application of this assay system, we characterized the association of transcription elongation factor DSIF with RNAP II elongation complexes and discovered that the nascent transcript facilitated recruitment of DSIF. Examples of how the system can be manipulated to address different questions are provided. EC-EMSA should be useful for further investigation of factor interactions with RNAP II elongation complexes

Interlocking Transcriptional Feedback Loops Control White-Opaque Switching in Candida albicans

The human pathogen Candida albicans can assume either of two distinct cell types, designated “white” and “opaque.” Each cell type is maintained for many generations; switching between them is rare and stochastic, and occurs without any known changes in the nucleotide sequence of the genome. The two cell types differ dramatically in cell shape, colony appearance, mating competence, and virulence properties. In this work, we investigate the transcriptional circuitry that specifies the two cell types and controls the switching between them. First, we identify two new transcriptional regulators of white-opaque switching, Czf1 and white-opaque regulator 2 (Wor2). Analysis of a large set of double mutants and ectopic expression strains revealed genetic relationships between CZF1, WOR2, and two previously identified regulators of white-opaque switching, WOR1 and EFG1. Using chromatin immunoprecipitation, we show that Wor1 binds the intergenic regions upstream of the genes encoding three additional transcriptional regulators of white-opaque switching (CZF1, EFG1, and WOR2), and also occupies the promoters of numerous white- and opaque-enriched genes. Based on these interactions, we have placed these four genes in a circuit controlling white-opaque switching whose topology is a network of positive feedback loops, with the master regulator gene WOR1 occupying a central position. Our observations indicate that a key role of the interlocking feedback loop network is to stably maintain each epigenetic state through many cell divisions

The loss of histone H3 lysine 9 acetylation due to dSAGA-specific dAda2b mutation influences the expression of only a small subset of genes

In Drosophila, the dADA2b-containing dSAGA complex is involved in histone H3 lysine 9 and 14 acetylation. Curiously, although the lysine 9- and 14-acetylated histone H3 levels are drastically reduced in dAda2b mutants, these animals survive until a late developmental stage. To study the molecular consequences of the loss of histone H3 lysine 9 and 14 acetylation, we compared the total messenger ribonucleic acid (mRNA) profiles of wild type and dAda2b mutant animals at two developmental stages. Global gene expression profiling indicates that the loss of dSAGA-specific H3 lysine 9 and 14 acetylation results in the expression change (up- or down-regulation) of a rather small subset of genes and does not cause a general transcription de-regulation. Among the genes up-regulated in dAda2b mutants, particularly high numbers are those which play roles in antimicrobial defense mechanisms. Results of chromatin immunoprecipitation experiments indicate that in dAda2b mutants, the lysine 9-acetylated histone H3 levels are decreased both at dSAGA up- and down-regulated genes. In contrast to that, in the promoters of dSAGA-independent ribosomal protein genes a high level of histone H3K9ac is maintained in dAda2b mutants. Our data suggest that by acetylating H3 at lysine 9, dSAGA modifies Pol II accessibility to specific promoters differently

Genic and Global Functions for Paf1C in Chromatin Modification and Gene Expression in Arabidopsis

In budding yeast, intragenic histone modification is linked with transcriptional elongation through the conserved regulator Paf1C. To investigate Paf1C-related function in higher eukaryotes, we analyzed the effects of loss of Paf1C on histone H3 density and patterns of H3 methylated at K4, K27, and K36 in Arabidopsis genes, and integrated this with existing gene expression data. Loss of Paf1C did not change global abundance of H3K4me3 or H3K36me2 within chromatin, but instead led to a 3′ shift in the distribution of H3K4me3 and a 5′ shift in the distribution of H3K36me2 within genes. We found that genes regulated by plant Paf1C showed strong enrichment for both H3K4me3 and H3K27me3 and also showed a high degree of tissue-specific expression. At the Paf1C- and PcG-regulated gene FLC, transcriptional silencing and loss of H3K4me3 and H3K36me2 were accompanied by expansion of H3K27me3 into the promoter and transcriptional start regions and further enrichment of H3K27me3 within the transcribed region. These results highlight both genic and global functions for plant Paf1C in histone modification and gene expression, and link transcriptional activity with cellular memory

ChromaSig: A Probabilistic Approach to Finding Common Chromatin Signatures in the Human Genome

Computational methods to identify functional genomic elements using genetic information have been very successful in determining gene structure and in identifying a handful of cis-regulatory elements. But the vast majority of regulatory elements have yet to be discovered, and it has become increasingly apparent that their discovery will not come from using genetic information alone. Recently, high-throughput technologies have enabled the creation of information-rich epigenetic maps, most notably for histone modifications. However, tools that search for functional elements using this epigenetic information have been lacking. Here, we describe an unsupervised learning method called ChromaSig to find, in an unbiased fashion, commonly occurring chromatin signatures in both tiling microarray and sequencing data. Applying this algorithm to nine chromatin marks across a 1% sampling of the human genome in HeLa cells, we recover eight clusters of distinct chromatin signatures, five of which correspond to known patterns associated with transcriptional promoters and enhancers. Interestingly, we observe that the distinct chromatin signatures found at enhancers mark distinct functional classes of enhancers in terms of transcription factor and coactivator binding. In addition, we identify three clusters of novel chromatin signatures that contain evolutionarily conserved sequences and potential cis-regulatory elements. Applying ChromaSig to a panel of 21 chromatin marks mapped genomewide by ChIP-Seq reveals 16 classes of genomic elements marked by distinct chromatin signatures. Interestingly, four classes containing enrichment for repressive histone modifications appear to be locally heterochromatic sites and are enriched in quickly evolving regions of the genome. The utility of this approach in uncovering novel, functionally significant genomic elements will aid future efforts of genome annotation via chromatin modifications

The Set2/Rpd3S Pathway Suppresses Cryptic Transcription without Regard to Gene Length or Transcription Frequency

In cells lacking the histone methyltransferase Set2, initiation of RNA polymerase II transcription occurs inappropriately within the protein-coding regions of genes, rather than being restricted to the proximal promoter. It was previously reported that this “cryptic” transcription occurs preferentially in long genes, and in genes that are infrequently transcribed. Here, we mapped the transcripts produced in an S. cerevisiae strain lacking Set2, and applied rigorous statistical methods to identify sites of cryptic transcription at high resolution. We find that suppression of cryptic transcription occurs independent of gene length or transcriptional frequency. Our conclusions differ with those reported previously because we obtained a higher-resolution dataset, we accounted for the fact that gene length and transcriptional frequency are not independent variables, and we accounted for several ascertainment biases that make cryptic transcription easier to detect in long, infrequently transcribed genes. These new results and conclusions have implications for many commonly used genomic analysis approaches, and for the evolution of high-fidelity RNA polymerase II transcriptional initiation in eukaryotes

Protein Rates of Evolution Are Predicted by Double-Strand Break Events, Independent of Crossing-over Rates

Theory predicts that, owing to reduced Hill–Robertson interference, genomic regions with high crossing-over rates should experience more efficient selection. In Saccharomyces cerevisiae a negative correlation between the local recombination rate, assayed as meiotic double-strand breaks (DSBs), and the local rate of protein evolution has been considered consistent with such a model. Although DSBs are a prerequisite for crossing-over, they need not result in crossing-over. With recent high-resolution crossover data, we now return to this issue comparing two species of yeast. Strikingly, even allowing for crossover rates, both the rate of premeiotic DSBs and of noncrossover recombination events predict a gene's rate of evolution. This both questions the validity of prior analyses and strongly suggests that any correlation between crossover rates and rates of protein evolution could be owing to slow-evolving genes being prone to DSBs or a direct effect of DSBs on sequence evolution. To ask if classical theory of recombination has any relevance, we determine whether crossover rates predict rates of protein evolution, controlling for noncrossover DSB events, gene ontology (GO) class, gene expression, protein abundance, nucleotide content, and dispensability. We find that genes with high crossing-over rates have low rates of protein evolution after such control, although any correlation is weaker than that previously reported considering meiotic DSBs as a proxy. The data are consistent both with recombination enhancing the efficiency of purifying selection and, independently, with DSBs being associated with low rates of evolution

Computational study of associations between histone modification and protein-DNA binding in yeast genome by integrating diverse information

<p>Abstract</p> <p>Background</p> <p>In parallel with the quick development of high-throughput technologies, <it>in vivo (vitro) </it>experiments for genome-wide identification of protein-DNA interactions have been developed. Nevertheless, a few questions remain in the field, such as how to distinguish true protein-DNA binding (functional binding) from non-specific protein-DNA binding (non-functional binding). Previous researches tackled the problem by integrated analysis of multiple available sources. However, few systematic studies have been carried out to examine the possible relationships between histone modification and protein-DNA binding. Here this issue was investigated by using publicly available histone modification data in yeast.</p> <p>Results</p> <p>Two separate histone modification datasets were studied, at both the open reading frame (ORF) and the promoter region of binding targets for 37 yeast transcription factors. Both results revealed a distinct histone modification pattern between the functional protein-DNA binding sites and non-functional ones for almost half of all TFs tested. Such difference is much stronger at the ORF than at the promoter region. In addition, a protein-histone modification interaction pathway can only be inferred from the functional protein binding targets.</p> <p>Conclusions</p> <p>Overall, the results suggest that histone modification information can be used to distinguish the functional protein-DNA binding from the non-functional, and that the regulation of various proteins is controlled by the modification of different histone lysines such as the protein-specific histone modification levels.</p

Replication and Active Demethylation Represent Partially Overlapping Mechanisms for Erasure of H3K4me3 in Budding Yeast

Histone modifications affect DNA–templated processes ranging from transcription to genomic replication. In this study, we examine the cell cycle dynamics of the trimethylated form of histone H3 lysine 4 (H3K4me3), a mark of active chromatin that is viewed as “long-lived” and that is involved in memory during cell state inheritance in metazoans. We synchronized yeast using two different protocols, then followed H3K4me3 patterns as yeast passed through subsequent cell cycles. While most H3K4me3 patterns were conserved from one generation to the next, we found that methylation patterns induced by alpha factor or high temperature were erased within one cell cycle, during S phase. Early-replicating regions were erased before late-replicating regions, implicating replication in H3K4me3 loss. However, nearly complete H3K4me3 erasure occurred at the majority of loci even when replication was prevented, suggesting that most erasure results from an active process. Indeed, deletion of the demethylase Jhd2 slowed erasure at most loci. Together, these results indicate overlapping roles for passive dilution and active enzymatic demethylation in erasing ancestral histone methylation states in yeast