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Oliver J Rando

Publications and source records attributed to Oliver J Rando.

12 recordsLinked to original sources
PubMed2006

Chromatin structure in the genomics era.

The packaging of eukaryotic genomes into chromatin has a large influence on DNA-templated processes, such as transcription. The availability of genome sequences and 'genomics' technologies such as DNA microarrays and high-throughput sequencing had an immediate effect on the study of transcriptional regulation, by enabling researchers to identify the coregulation patterns of thousands of genes. These same resources are now being used successfully to study the structure of chromatin. Here, I review some of these new genomics approaches to understanding chromatin structure in eukaryotes.

Animals↗
PubMed2006

Distinct pathways for snoRNA and mRNA termination.

Transcription termination at mRNA genes is linked to polyadenylation. Cleavage at the poly(A) site generates an entry point for the Rat1/Xrn2 exonuclease, which degrades the downstream transcript to promote termination. Small nucleolar RNAs (snoRNAs) are also transcribed by RNA polymerase II but are not polyadenylated. Chromatin immunoprecipitation experiments show that polyadenylation factors and Rat1 localize to snoRNA genes, but mutations that disrupt poly(A) site cleavage or Rat1 activity do not lead to termination defects at these genes. Conversely, mutations of Nrd1, Sen1, and Ssu72 affect termination at snoRNAs but not at several mRNA genes. The exosome complex was required for 3' trimming, but not termination, of snoRNAs. Both the mRNA and snoRNA pathways require Pcf11 but show differential effects of individual mutant alleles. These results suggest that in yeast the transcribing RNA polymerase II can choose between two distinct termination mechanisms but keeps both options available during elongation.

Blotting, Northern↗
PubMed2006

Noisy silencing of chromatin.

Chromatin-based repression is a major mechanism for epigenetically heritable variation. Work in the July 21 issue of Molecular Cell quantitatively examines transcriptional silencing in individual yeast cells, demonstrating locus-specific effects and finding that different silencing mutants exhibit qualitatively distinct single-cell defects.

Chromatin↗
PubMed2005

Histone variant H2A.Z marks the 5' ends of both active and inactive genes in euchromatin.

In S. cerevisiae, histone variant H2A.Z is deposited in euchromatin at the flanks of silent heterochromatin to prevent its ectopic spread. We show that H2A.Z nucleosomes are found at promoter regions of nearly all genes in euchromatin. They generally occur as two positioned nucleosomes that flank a nucleosome-free region (NFR) that contains the transcription start site. Astonishingly, enrichment at 5' ends is observed not only at actively transcribed genes but also at inactive loci. Mutagenesis of a typical promoter revealed a 22 bp segment of DNA sufficient to program formation of a NFR flanked by two H2A.Z nucleosomes. This segment contains a binding site of the Myb-related protein Reb1 and an adjacent dT:dA tract. Efficient deposition of H2A.Z is further promoted by a specific pattern of histone H3 and H4 tail acetylation and the bromodomain protein Bdf1, a component of the Swr1 remodeling complex that deposits H2A.Z.

Acetylation↗
PubMed2005

Single-nucleosome mapping of histone modifications in S. cerevisiae.

Covalent modification of histone proteins plays a role in virtually every process on eukaryotic DNA, from transcription to DNA repair. Many different residues can be covalently modified, and it has been suggested that these modifications occur in a great number of independent, meaningful combinations. Published low-resolution microarray studies on the combinatorial complexity of histone modification patterns suffer from confounding effects caused by the averaging of modification levels over multiple nucleosomes. To overcome this problem, we used a high-resolution tiled microarray with single-nucleosome resolution to investigate the occurrence of combinations of 12 histone modifications on thousands of nucleosomes in actively growing S. cerevisiae. We found that histone modifications do not occur independently; there are roughly two groups of co-occurring modifications. One group of lysine acetylations shows a sharply defined domain of two hypo-acetylated nucleosomes, adjacent to the transcriptional start site, whose occurrence does not correlate with transcription levels. The other group consists of modifications occurring in gradients through the coding regions of genes in a pattern associated with transcription. We found no evidence for a deterministic code of many discrete states, but instead we saw blended, continuous patterns that distinguish nucleosomes at one location (e.g., promoter nucleosomes) from those at another location (e.g., over the 3' ends of coding regions). These results are consistent with the idea of a simple, redundant histone code, in which multiple modifications share the same role.

Chromatin Immunoprecipitation↗
PubMed2005

Genome-scale identification of nucleosome positions in S. cerevisiae.

The positioning of nucleosomes along chromatin has been implicated in the regulation of gene expression in eukaryotic cells, because packaging DNA into nucleosomes affects sequence accessibility. We developed a tiled microarray approach to identify at high resolution the translational positions of 2278 nucleosomes over 482 kilobases of Saccharomyces cerevisiae DNA, including almost all of chromosome III and 223 additional regulatory regions. The majority of the nucleosomes identified were well-positioned. We found a stereotyped chromatin organization at Pol II promoters consisting of a nucleosome-free region approximately 200 base pairs upstream of the start codon flanked on both sides by positioned nucleosomes. The nucleosome-free sequences were evolutionarily conserved and were enriched in poly-deoxyadenosine or poly-deoxythymidine sequences. Most occupied transcription factor binding motifs were devoid of nucleosomes, strongly suggesting that nucleosome positioning is a global determinant of transcription factor access.

Binding Sites↗
PubMed2005

Developmentally induced changes in transcriptional program alter spatial organization across chromosomes.

Although the spatial location of genes within the nucleus has been implicated in their transcriptional status, little is known about the dynamics of gene location that accompany large-scale changes in gene expression. The mating of haploid yeast Saccharomyces cerevisiae is accompanied by a large-scale change in transcription and developmental program. We examined changes in nuclear organization that accompany stimulus by the mating pheromone alpha factor and found that most alpha-factor-induced genes become associated with components of the nuclear envelope. The myosin-like protein Mlp1, which has been implicated in mRNA export, was further shown to exhibit RNA dependence in its association with alpha-factor-induced genes. High-resolution mapping of association of chromosome III with Mlp1 revealed alpha-factor-dependent determinants of nuclear pore association, including origins of replication, specific intergenic regions, and the 3' ends of transcriptionally activated genes. Taken together, these results reveal RNA- and DNA-dependent determinants of nuclear organization as well as a detailed picture of how an entire chromosome alters its spatial conformation in response to a developmental cue.

Chromosomes, Fungal↗
PubMed2005

Genomic characterization reveals a simple histone H4 acetylation code.

The histone code hypothesis holds that covalent posttranslational modifications of histone tails are interpreted by the cell to yield a rich combinatorial transcriptional output. This hypothesis has been the subject of active debate in the literature. Here, we investigated the combinatorial complexity of the acetylation code at the four lysine residues of the histone H4 tail in budding yeast. We constructed yeast strains carrying all 15 possible combinations of mutations among lysines 5, 8, 12, and 16 to arginine in the histone H4 tail, mimicking positively charged, unacetylated lysine states, and characterized the resulting genome-wide changes in gene expression by using DNA microarrays. Only the lysine 16 mutation had specific transcriptional consequences independent of the mutational state of the other lysines (affecting approximately 100 genes). In contrast, for lysines 5, 8, and 12, expression changes were due to nonspecific, cumulative effects seen as increased transcription correlating with an increase in the total number of mutations (affecting approximately 1,200 genes). Thus, acetylation of histone H4 is interpreted by two mechanisms: a specific mechanism for lysine 16 and a nonspecific, cumulative mechanism for lysines 5, 8, and 12.

Acetylation↗
PubMed2004

The yeast Rat1 exonuclease promotes transcription termination by RNA polymerase II.

The carboxy-terminal domain (CTD) of the RNA polymerase II (RNApII) largest subunit consists of multiple heptapeptide repeats with the consensus sequence YSPTSPS. Different CTD phosphorylation patterns act as recognition sites for the binding of various messenger RNA processing factors, thereby coupling transcription and mRNA processing. Polyadenylation factors are co-transcriptionally recruited by phosphorylation of CTD serine 2 (ref. 2) and these factors are also required for transcription termination. RNApII transcribes past the poly(A) site, the RNA is cleaved by the polyadenylation machinery, and the RNA downstream of the cleavage site is degraded. Here we show that Rtt103 and the Rat1/Rai1 5' --> 3' exonuclease are localized at 3' ends of protein coding genes. In rat1-1 or rai1Delta cells, RNA 3' to polyadenylation sites is greatly stabilized and termination defects are seen at many genes. These findings support a model in which poly(A) site cleavage and subsequent degradation of the 3'-downstream RNA by Rat1 trigger transcription termination.

Chromatin Immunoprecipitation↗
PubMed2002

Genomic expression programs and the integration of the CD28 costimulatory signal in T cell activation.

Optimal activation of T cells requires effective occupancy of both the antigen-specific T cell receptor and a second coreceptor such as CD28. We used cDNA microarrays to characterize the genomic expression program in human peripheral T cells responding to stimulation of these receptors. We found that CD28 agonists alone elicited few, but reproducible, changes in gene expression, whereas CD3 agonists elicited a multifaceted temporally choreographed gene expression program. The principal effect of simultaneous engagement of CD28 was to increase the amplitude of the CD3 transcriptional response. The induced genes whose expression was most enhanced by costimulation were significantly enriched for known targets of nuclear factor of activated T cells (NFAT) transcription factors. This enhancement was nearly abolished by blocking the nuclear translocation of NFATc by using the calcineurin inhibitor FK506. CD28 signaling promoted phosphorylation, and thus inactivation, of the NFAT nuclear export kinase glycogen synthase kinase-3 (GSK3), coincident with enhanced dephosphorylation of NFATc proteins. These results provide a detailed picture of the transcriptional program of T cell activation and suggest that enhancement of transcriptional activation by NFAT, through inhibition of its nuclear export, plays a key role in mediating the CD28 costimulatory signal.

CD28 Antigens↗
PubMed2002

Phosphatidylinositol-dependent actin filament binding by the SWI/SNF-like BAF chromatin remodeling complex.

Recently, several chromatin remodeling complexes in yeast, Drosophila, and mammals have been shown to contain actin and actin-related proteins (arps). However, the function of actin in these complexes is unclear. Here, we show that the mammalian SWI/SNF-like BAF complex binds to phosphatidylinositol 4,5-bisphosphate (PIP2) micelles and PIP2-containing mixed lipid vesicles, and that PIP2 binding allows the complex to associate with actin pointed ends and branch points. Actin binds to at least two distinct domains in the C terminus of the Brg1 protein, and interaction with only one of these domains is sensitive to PIP2. Based on these findings, we propose a model for PIP2 activation of actin binding by relief of intramolecular capping of actin by Brg1.

Actin Cytoskeleton↗