RNA polymerase II | EPFL Graph Search

RNA polymerase II (RNAP II and Pol II) is a multiprotein complex that transcribes DNA into precursors of messenger RNA (mRNA) and most small nuclear RNA (snRNA) and microRNA. It is one of the three RNAP enzymes found in the nucleus of eukaryotic cells. A 550 kDa complex of 12 subunits, RNAP II is the most studied type of RNA polymerase. A wide range of transcription factors are required for it to bind to upstream gene promoters and begin transcription. Discovery Early studies suggested a minimum of two RNAPs: one which synthesized rRNA in the nucleolus, and one which synthesized other RNA in the nucleoplasm, part of the nucleus but outside the nucleolus. In 1969, science experimentalists Robert Roeder and William Rutter definitively discovered an additional RNAP that was responsible for transcription of some kind of RNA in the nucleoplasm. The finding was obtained by the use of ion-exchange chromatography via DEAE coated Sephadex beads. T

Phase specific transcriptional regulation of circadian clock and metabolism in mouse liver

Jonathan Aryeh Sobel

The molecular clock has been conserved from cyanobacteria to mammals and is believed to align behavioral and biochemical processes with the diurnal cycle. This cellular mechanism has been an advantage to increase the fitness of organisms through the ability to anticipate food availability or predator presence. Accumulating evidence has revealed that the circadian clock is intimately interconnected with the metabolic cycle. But, the nature of these interconnections is yet not clear at the transcriptional level, and the contribution of cis-regulatory modules has not been elucidated. Accessible chromatin regions of the genome are implicated in diverse processes, such as gene regulation through enhancers and promoters, insulation of genomic domains or alternative splicing. They allow cell-type specific programs that are controlled by tissue-specific transcription factors and chromatin modifiers, although tissue-specific regulation of the circadian clock remains unclear. Therefore, we compared genome-wide BMAL1 binding and chromatin accessibility in the liver and in NIH3T3 fibroblasts. Moreover, for the first time, our study explores the dynamics of accessible regions, histone 3 lysine 27 acetylation (H3K27ac) and RNA polymerase II (Pol II) every 4 hours over one day in mouse liver. In this study, we show that a substantial fraction of these accessible sites are oscillating during a diurnal cycle with a circadian period. We observed that these accessible regions are enriched in the proximity of actively transcribed genes, and that they are dynamically affected by the binding of transcription factors such as BMAL1. We investigated wild-type (WT) and Bmal1-/- genotypes, in night restricted feeding regimen and in Light-Dark (LD) cycle, to study the circadian clock regulatory network underlying diurnal transcription. Therefore, we applied a penalized generalized linear model to infer the activity of transcription factor binding motifs in oscillating accessible sites using Pol II loadings at the transcription start sites of nearby genes. We were able to recapitulate the known regulatory elements of the circadian clock, notably E-box, D-Box, and ROR-responsive elements (RRE) in the WT genotype. On the other hand, we found that Forkhead box (FOX), glucocorticoids responsive elements (GRE), and C-AMP Response Element (CRE) were the main contributors of the regulation of oscillating genes in Bmal1-/-. Finally, using a mixture model to detect footprints with a base pair resolution, we studied the dynamics of the accessibility overlapping E-box. Our last analysis suggested that BMAL1/CLOCK is binding on double E-boxes with a spacer of 6 or 7 bp in a hetero-tetramer configuration. A 3D structure model further supported this binding mode. In Summary, we used DNase I-seq and ChIP-seq of Pol II and H3K27ac to study the circadian chromatin landscape in a 4h time-resolved experimental design. We uncovered the underlying circadian transcriptional regulatory network and, we dissected the chromatin accessibility around BMAL1 binding sites at a base pair resolution, which led to an unappreciated mode of binding of BMAL1/CLOCK in a hetero-tetramer conformation on double E-boxes. Lastly, we found tissue-specific factors that might contribute to tissue-specific binding of BMAL1/CLOCK.

EPFL2016

Telomeric Repeat Containing RNAs and the Response to Telomere Deprotection in Saccharomyces Cerevisiae

Andrea Bernardo Panza

Telomeres are the DNA/RNA/protein structures at the end of linear eukaryotic chromosomes, which protect them against the DNA damage repair machinery, preventing chromosome end-to-end fusions and aberrant recombination. Moreover, telomeres compensate for the erosion that occurs at every cycle of DNA replication maintaining telomere length through the action of the telomerase reverse transcriptase. Even if telomeres have many features in common with heterochromatic DNA, they have been shown to be transcribed into telomeric repeat-containing RNA (TERRA) both in vertebrates and plants. In the first part of this work we demonstrate that TERRA expression is conserved in the yeast Saccharomyces cerevisiae. Moreover, TERRA is transcribed by RNA polymerase II, it is polyadenylated and its levels in wild type cells are maintained low through the action of the 5’ to 3’ RNA exonuclease Rati. Indeed cells harboring the thermosensitive mutant allele ratl-1 accumulate high levels of TERRA even at semipermissive temperature. Moreover, ratl-1 mutant cells display a telomere shortening phenotype that is rescued by RNase H overexpression, indicating that DNA/RNA hybrids, which can accumulate in this mutant, inhibit the normal telomere maintenance. In the second part of this work we tried to define TERRA function(s) in response to telomere alterations in yeast cells. We observed that TERRA increases during senescence and survivor formation in est2A mutant cells, which undergo through telomere shortening due to telomerase deletion. The observed increase in TERRA does not require the recombination machinery. Moreover, though TERRA levels are not altered by global DNA damage, they are raised in cdcl3-l, stnl-13 and ku70A mutants, which display telomere resection and DNA damage checkpoint activation at restrictive temperature, suggesting a specific role for TERRA in response to telomere alterations. Since the TERRA increase in telomere deprotection-mutants is suppressed by the deletion of either the 5’ to 3’ exonuclease EXOl or one of the checkpoint factors MEC1, TEL1 and RAD9, we suggest that TERRA is part of a specific checkpoint response, which is induced upon telomere resection. Finally, since est2A cells harboring the ratl-1 mutation display a prolonged senescence phenotype, we suspect a role for TERRA in telomere protection and in particular in preventing telomeric recombination.

EPFL2012

A large-scale, in vivo transcription factor screen defines bivalent chromatin as a key property of regulatory factors mediating Drosophila wing development

Monica Albarca Aguilera, Bart Deplancke

Transcription factors (TFs) are key regulators of cell fate. The estimated 755 genes that encode DNA binding domain-containing proteins comprise similar to 5% of all Drosophila genes. However, the majority has remained uncharacterized so far due to the lack of proper genetic tools. We generated 594 site-directed transgenic Drosophila lines that contain integrations of individual UAS-TF constructs to facilitate spatiotemporally controlled misexpression in vivo. All transgenes were expressed in the developing wing, and two-thirds induced specific phenotypic defects. In vivo knockdown of the same genes yielded a phenotype for 50%, with both methods indicating a great potential for misexpression to characterize novel functions in wing growth, patterning, and development. Thus, our UAS-TF library provides an important addition to the genetic toolbox of Drosophila research, enabling the identification of several novel wing development-related TFs. In parallel, we established the chromatin landscape of wing imaginal discs by ChIP-seq analyses of five chromatin marks and RNA Pol II. Subsequent clustering revealed six distinct chromatin states, with two clusters showing enrichment for both active and repressive marks. TFs that carry such "bivalent"chromatin are highly enriched for causing misexpression phenotypes in the wing, and analysis of existing expression data shows that these TFs tend to be differentially expressed across the wing disc. Thus, bivalently marked chromatin can be used as a marker for spatially regulated TFs that are functionally relevant in a developing tissue.

Cold Spring Harbor Laboratory Press2015