Compositional and structural analysis of selected chromosomal domains from Saccharomyces cerevisiae

Hamperl, Stephan; Brown, Christopher R.; Villar-Garea, Ana; Pérez-Fernández, Jorge; Bruckmann, Astrid; Huber, Katharina; Wittner, Manuel; Babl, Virginia; Stoeckl, Ulrike; Deutzmann, Rainer; Boeger, Hinrich; Tschochner, Herbert; Milkereit, Philipp; Griesenbeck, Joachim

doi:10.1093/nar/gkt891

Cited by 26 publications

(32 citation statements)

References 89 publications

(111 reference statements)

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“…4 these genes is transcribed at any given time [8][9][10][11][12]. Modulation of rRNA synthesis rates in response to growth factors, stress, and cell cycle cues appears to be controlled almost exclusively by changing the activity of a fixed number of "transcriptionally competent genes" rather than epigenetically silencing or reactivating rRNA genes [13,14].…”

Section: Accepted Manuscriptmentioning

confidence: 99%

Cell cycle and growth stimuli regulate different steps of RNA polymerase I transcription

Hung

Lesmana

Peck

et al. 2017

Gene

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, Cell cycle and growth stimuli regulate different steps of RNA polymerase I transcription. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Gene(2016Gene( ), doi: 10.1016Gene( /j.gene.2016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T 3 AbstractTranscription of the ribosomal RNA genes (rDNA) by RNA Polymerase I (Pol I) is a major control step for ribosome synthesis and is tightly linked to cellular growth.However, the question of whether this process is modulated primarily at the level of transcription initiation or elongation is controversial. Studies in markedly different cell types have identified either initiation or elongation as the major control point. In this study, we have re-examined this question in NIH3T3 fibroblasts using a combination of metabolic labelling of the 47S rRNA, chromatin immunoprecipitation analysis of Pol I and overexpression of the transcription initiation factor Rrn3. Acute manipulation of growth factor levels altered rRNA synthesis rates over 8-fold without changing Pol I loading onto the rDNA. In fact, robust changes in Pol I loading were only observed under conditions where inhibition of rDNA transcription was associated with chronic serum starvation or cell cycle arrest. Overexpression of the transcription initiation factor Rrn3 increased loading of Pol I on the rDNA but failed to enhance rRNA synthesis in either serum starved, serum treated or G0/G1 arrested cells. Together these data suggest that transcription elongation is rate limiting for rRNA synthesis. We propose that transcription initiation is required for rDNA transcription in response to cell cycle cues, whereas elongation controls the dynamic range of rRNA synthesis output in response to acute growth factor modulation.

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Section: Accepted Manuscriptmentioning

confidence: 99%

Cell cycle and growth stimuli regulate different steps of RNA polymerase I transcription

Hung

Lesmana

Peck

et al. 2017

Gene

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“…Although many chromatin factors have already been identified by genetics and protein-protein interaction studies, direct, unbiased and comprehensive analyses of chromatin interactions at specific genomic loci has remained a major challenge [5][6] . A commonly considered strategy towards solving this problem is introducing an affinity handle at a locus of interest, purifying the locus (capture) and analyzing the co-purified proteins by mass spectrometry (MS) [7][8] . However, a major challenge with capture-MS is the need for very high levels of enrichment of the locus of interest versus the rest of the large genome while at the same time obtaining sufficient amounts of material for comprehensive and quantitative MS analysis [5][6] .…”

Section: Introductionmentioning

confidence: 99%

Decoding the chromatin proteome of a single genomic locus by DNA sequencing

Korthout

Poramba‐Liyanage

Verzijlbergen

et al. 2017

Preprint

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Transcription, replication and repair involve interactions of specific genomic loci with many different proteins. How these interactions are orchestrated at any given location and under changing cellular conditions is largely unknown because systematically measuring protein-DNA interactions at a specific locus in the genome is challenging. To address this problem, we developed Epi-Decoder, a Tag-ChIPBarcode-Seq technology in budding yeast to identify and quantify in an unbiased and systematic manner the proteome of an individual genomic locus. Epi-Decoder is orthogonal to proteomics approaches because it does not rely on mass spectrometry but instead takes advantage of DNA sequencing. Analysis of the proteome of a transcribed locus proximal to an origin of replication revealed more than 400 proteins. Moreover, replication stress induced changes in local chromatin-proteome composition prior to local origin firing, affecting replication proteins as well as transcription proteins. Epi-Decoder will enable the delineation of complex and dynamic protein-DNA interactions across many regions of the genome.

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“…S1 A and B) (10,11). PHO5 chromatin isolated in this way was indistinguishable from chromatin at the chromosomal locus on the basis of digestion with specific and nonspecific endonucleases ( Fig.…”

Section: Resultsmentioning

confidence: 93%

“…It remains to be determined whether nonhistone components of promoter chromatin contribute to the potentiation of transcription. MS of purified PHO5 chromatin circles has thus far revealed only histones (11), but proteins present in one copy or a small number of copies on the circles may have escaped detection by this analysis.…”

Section: Discussionmentioning

confidence: 97%

Chromatin potentiates transcription

Nagai

Davis

Mattei

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Chromatin isolated from the chromosomal locus of the PHO5 gene of yeast in a transcriptionally repressed state was transcribed with 12 pure proteins (80 polypeptides): RNA polymerase II, six general transcription factors, TFIIS, the Pho4 gene activator protein, and the SAGA, SWI/SNF, and Mediator complexes. Contrary to expectation, a nucleosome occluding the TATA box and transcription start sites did not impede transcription but rather, enhanced it: the level of chromatin transcription was at least sevenfold greater than that of naked DNA, and chromatin gave patterns of transcription start sites closely similar to those occurring in vivo, whereas naked DNA gave many aberrant transcripts. Both histone acetylation and trimethylation of H3K4 (H3K4me3) were important for chromatin transcription. The nucleosome, long known to serve as a general gene repressor, thus also performs an important positive role in transcription.RNA polymerase II | PHO5 | Saccharomyces cerevisiae A ssembly of purified histones on promoter DNA interferes with the initiation of transcription by RNA polymerase II and general transcription factors (GTFs) in vitro (1) and in vivo (2). Factors that relieve inhibition by histones have been identified by transcribing chromatin reconstituted with purified histones and genetic analysis. These factors include ATP-dependent chromatin remodelers (3, 4), histone-modifying enzymes (5-7), FACT (8), and TFIIS (9). Although informative, these studies are incomplete, because chromatin reconstituted with purified histones differs from chromatin assembled in vivo. Reconstituted chromatin lacks the patterns of histone modification, histone variants, and nonhistone proteins shown to play important roles in transcription in vivo. Nucleosome positioning, also important for transcription in vivo, cannot be accurately reconstituted in vitro. We have, therefore, investigated chromatin assembled in vivo as a template for transcription in vitro. ResultsPHO5 chromatin in the transcriptionally repressed state was excised from yeast chromosomes in circular form by recombination and purified by affinity chromatography as described ( Fig. S1 A and B) (10, 11). PHO5 chromatin isolated in this way was indistinguishable from chromatin at the chromosomal locus on the basis of digestion with specific and nonspecific endonucleases ( Fig. S1 C and D) (10,12). Because the chromatin was derived from a single copy gene, very small quantities were obtained: on the order of 10 fmol/L cell culture. At this low level, transcription cannot be detected directly by radioisotope incorporation or fluorescence, and therefore, we turned to RT-PCR. Two sets of primers were used: one to amplify the region downstream of the transcription start sites (TSSs) and detect all transcripts ("downstream" primer pair) and one to amplify any signal from cryptic transcripts originating upstream and reading through the promoter ("upstream" primer pair) (Fig. 1A and Fig. S2A). Subtraction of the upstream signal from the downstream signal revealed the level of p...

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Compositional and structural analysis of selected chromosomal domains from Saccharomyces cerevisiae

Cited by 26 publications

References 89 publications

Cell cycle and growth stimuli regulate different steps of RNA polymerase I transcription

Cell cycle and growth stimuli regulate different steps of RNA polymerase I transcription

Decoding the chromatin proteome of a single genomic locus by DNA sequencing

Chromatin potentiates transcription

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