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Investigating the role of Hmo1 in transcription by Pol I in yeast.

ABSTRACT: The purpose of this study was to investigate the role of the yeast protein, high mobility group protein (Hmo1), in rRNA synthesis. For over twenty years, Hmo1 has been proposed to be an activator of transcription by Pol I, but its regulatory mechanism remains largely undefined. Therefore, we used native elongating transcript sequencing (NET-seq) to explore the role of this factor in transcription by Pol I. Our NET-seq results demonstrated that in cells lacking Hmo1 (hmo1D), Pol I occupancy was significantly increased across the rDNA template, indicating an increased pause propensity in these cells. These data suggest that Hmo1 is an important transcription elongation factor for Pol I.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE247981 | GEO | 2023/11/20

REPOSITORIES: GEO

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Determining the effect of RPA49 on RNA polymerase I occupancy in vivo

Project description:The central goal of this project was to determine the role of the RNA polymerase I (Pol I) subunits RPA34 and RPA49 in rRNA synthesis. Previous studies by various groups have demonstrated that these two subunits may play a role in transcription initiation and elongation by on Pol I occupancy in vivo, but this is still not clearly defined. Here, we deployed a high-resolution technique, native elongating transcript sequencing (NET-seq), to test the effects of RPA34 and RPA49 on Pol I occupancy in vivo. We found that when RPA34 was deleted, there was a significant reduction in Pol I occupancy across the rDNA template. Additionally, when RPA49 was deleted, Pol I occupancy was even further reduced across the template. Collectively, our findings suggest that perhaps RPA34 acts to stabilize RPA49 and these subunits may act primarily as transcription initiation factors for Pol I.

2023-05-22 | GSE232731 | GEO

BMH-21 inhibits RNA polymerase I transcription cycle and rDNA occupancy

Project description:The objective of this study was to determine the effect of a small-molecule Pol I inhibitor, BMH-21, on rRNA synthesis in vivo. NET-seq was performed to determine the Pol I occupancy after BMH-21 treatment, as compared to vehicle-treatment (phosphate buffer control). Our findings suggest that BMH-21 treatment reduces Pol I occupancy on the rDNA template. Additionally, BMH-21 induces repositioning of Pol I in AT-rich rDNA regions that are directly upstream from GC-rich regions. This study suggests that BMH-21 is a powerful inhibitor of transcription by Pol I, and gives a potential mechanism of action for this inhibitor in vivo.

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Hmo1 ChIP-chip

Project description:Yeast Hmo1 ChIP-chip in triplicate with single untagged control Yeast Hmo1 ChIP-chip was performed in yDH419 (Hmo1-3myc) in triplicate using anti-myc (9E10 monoclonal antibody). A control experiment was performed using the untagged isogenic strain, BY4742

2010-07-01 | E-GEOD-4046 | biostudies-arrayexpress

Defining the influence of the A12 subunit on transcription elongation and termination by rna polymerase I

Project description:we performed Native Elongating Transcript Sequencing (NET-seq) on an rpa12Δ strain of S. cerevisiae and evaluated the resultant change in Pol I occupancy throughout the 35S gene and the IGS. Compared to WT, we observed template sequence-specific changes in Pol I occupancy throughout the 35S gene. We also observed rpa12Δ Pol I occupancy downstream of both termination sites and throughout most of the IGS, including the 5S gene. Occupancy of rpa12Δ Pol I increased just upstream of the promoter proximal Reb1 binding site and dropped significantly after, implicating this site as a third terminator for Pol I transcription.

2019-12-21 | GSE142457 | GEO

Spt4 promotes Pol I processivity and transcription elongation

Project description:The goal of this study was to determine the effect of Spt4 on transcription by Pol I in vivo. Therefore, Native Elongating Transcript Sequencing (NET-seq) was used to evaluate the occupancy of Pol I in wild-type and spt4△ strains. We determined that in spt4△ yeast, there was a processivity defect and Pol I accumulated at the 5' end of the 35S gene. Additionally, we observed that there was an enrichment for G-rich sequences just downstream of a high A content in the spt4△ strain, indicating that perhaps Spt4 plays a role in helping to propel Pol I through these regions of the rDNA. Altogether, this study suggests that Spt4 is an important transcription factor for Pol I by promoting polymerase processivity.

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Hmo1 ChIP-chip

Project description:Yeast Hmo1 ChIP-chip in triplicate with single untagged control Keywords: ChIP-chip

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A Top2 and Hmo1-mediated genome architectural pathway preventing chromosome fragility.

Project description:Specialized topoisomerases solve the topological constraints arising when replication forks encounter transcription. We have investigated Top2 contribution in S phase transcription. Specifically in S phase, Top2 binds intergenic regions close to transcribed genes without influencing their transcription. The Top2-bound loci exhibit low nucleosome density and accumulate yH2A when Top2 is defective. These intergenic loci associate with the HMG-protein Hmo1 throughout the cell cycle and are refractory to the histone variant Htz1. In top2 mutants, Hmo1 is deleterious and accumulates at pericentromeric regions in G2/M. Our data indicate that Top2 is dispensable for transcription and that Hmo1 and Top2 bind in the proximity of genes transcribed in S phase suppressing chromosome fragility at the M-G1 transition. We propose that an Hmo1-dependent epigenetic signature together with Top2 mediate a S-phasen architectural pathway controlling replicon dynamics when forks encounter transcriptionto preserve genome integrity. Signal tracks in BED format suitable for visualization on the UCSC genome browser can be found at http://bio.ifom-ieo-campus.it/supplementary/Bermejo_et_al_CELL_2009

2009-09-05 | GSE16258 | GEO

NET-prism enables RNA polymerase-dedicated transcriptional interrogation at nucleotide resolution

Project description:The advent of quantitative approaches that enable interrogation of transcription at single nucleotide resolution has allowed a novel understanding of transcriptional regulation previously undefined. To better map transcription genome-wide at base pair resolution and with transcription/elongation factor dependency we developed an adapted NET-seq protocol called NET-prism (Native Elongating Transcription by Polymerase-Regulated Immunoprecipitants in the Mammalian genome). NET-prism introduces an immunoprecipitation to capture RNA Pol II – associated proteins, which reveals the interaction of these proteins with active RNA Pol II. Application of NET-prism on different Pol II variants (Pol II S2ph, Pol II S5ph), elongation factors (Spt6, Ssrp1), splicing factors (Sf1), and components of the pre-initiation complex (PIC) (TFIID, and Mediator) reveals diverse Pol II signals, at a single nucleotide resolution, with regards to directionality and intensity over promoters, splice sites, and enhancers/super-enhancers. NET-prism will be broadly applicable as it exposes transcription factor/Pol II dependent topographic specificity and thus, a new degree of regulatory complexity.

2019-07-02 | GSE107257 | GEO

Native Elongating Transcript Sequencing Reveals Human Transcriptional Activity at Nucleotide Resolution

Project description:Major features of transcription by human RNA polymerase II (Pol II) remain poorly defined due to a lack of quantitative approaches for visualizing Pol II progress at nucleotide resolution. We developed a simple and powerful approach for performing native elongating transcript sequencing (NET-seq) in human cells that globally maps strand-specific Pol II density at nucleotide resolution. NET-seq exposes a mode of antisense transcription that originates downstream and converges on transcription from the canonical promoter. Convergent transcription is associated with a distinctive chromatin configuration and is characteristic of lower-expressed genes. Integration of NET-seq with genomic footprinting data reveals stereotypic Pol II pausing coincident with transcription factor occupancy. Finally, exons retained in mature transcripts display Pol II pausing signatures that differ markedly from skipped exons, indicating an intrinsic capacity for Pol II to recognize exons with different processing fates.

2015-04-23 | GSE61332 | GEO

Native elongating transcript sequencing (NET-seq) of wild type Saccharomyces cerevisiae and of DST1, RCO1, SET1, SET2, EAF3 deletion strains

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2011-01-19 | E-GEOD-25107 | biostudies-arrayexpress

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