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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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POLR3G and POLR3GL-RNA polymerase III target genes

ABSTRACT: Purpose: Characterization of the target genes of two versions of the Polymerase III (pol III), one containing POLR3G subunit or POLR3GL subunit, in different tissues. Methods: We performed ChIP_seq of POLR3D and the two variants subunits, POLR3G and POLR3GL in Human IMR90 cells, Mouse liver and Mouse Hepatocarcinoma Hepa 1-6 cells in order to locate and analyse total pol III, POLR3G and POLR3GL-containing pol III in various tissues. Results: We show that POLR3G- as well as POLR3GL-containing pol III are present in cultured cell lines and in normal mouse liver, although the relative amounts of the two forms vary, with the POLR3G-containing pol III relatively more abundant in dividing cells. Both forms of pol III occupy the same target genes, in very constant proportions within one cell line, suggesting that the two forms of pol III have similar function with regard to specificity for target genes. Conclusions: POLR3G and POLR3GL occupy the same target genes, but they are differentially expressed under different conditions and in different cell types. Pol III target genes in human IMR cells, Mouse liver, and Mouse Hepatocarcinoma cells were identified by chromatin Immuno-precipitation followed by deep sequencing using Illumina HiSeq. For Human IMR90 cells, we used antibodies against POLR3D, POLR3G, POLR3GL and BDP1. For Mouse data, we targeted POLR3D, POLR3G and POLR3GL and did ChIP_seq replicates. We also sequenced the corresponding inputs (crosslinked DNA of IMR90 cells, Mouse liver, and Mouse Hepatocarcinoma cells). This represents a total of 20 samples.

ORGANISM(S): Mus musculus

SUBMITTER: Viviane Praz

PROVIDER: E-GEOD-47849 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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POLR3G and POLR3GL-RNA polymerase III target genes

Project description:Purpose: Characterization of the target genes of two versions of the Polymerase III (pol III), one containing POLR3G subunit or POLR3GL subunit, in different tissues. Methods: We performed ChIP_seq of POLR3D and the two variants subunits, POLR3G and POLR3GL in Human IMR90 cells, Mouse liver and Mouse Hepatocarcinoma Hepa 1-6 cells in order to locate and analyse total pol III, POLR3G and POLR3GL-containing pol III in various tissues. Results: We show that POLR3G- as well as POLR3GL-containing pol III are present in cultured cell lines and in normal mouse liver, although the relative amounts of the two forms vary, with the POLR3G-containing pol III relatively more abundant in dividing cells. Both forms of pol III occupy the same target genes, in very constant proportions within one cell line, suggesting that the two forms of pol III have similar function with regard to specificity for target genes. Conclusions: POLR3G and POLR3GL occupy the same target genes, but they are differentially expressed under different conditions and in different cell types.

2013-10-01 | GSE47849 | GEO

Quantifying ChIP-seq data: a spiking method providing an internal reference for sample-to-sample normalization.

Project description:Purpose: Description of a spike-adjusting-method (SAM) to normalize ChIP-seq data . Methods: We performed ChIP-seq of POLR3D and POLR2B with mouse liver supplemented with 2.5% of human DNA. Human DNA will be used as an internal control for ChIP-seq quantification. Results: We show that using the SAM for ChIP-seq quantification improve similarity of POLR3D and POLR2B ChIP-seq replicates samples and improve difference between samples originate from different conditions. Conclusions: The SAM improves comparison of ChIP-seq samples, either by increasing similarity between replicates or by emphasise differences between conditions. Chromatin Immuno-precipitations were performed with antibodies directed against POLR3D (Pol III) and POLR2B (Pol II) using mouse liver material supplemented with human DNA. Immuno-precipitated DNA was next sequenced using Illumina HiSeq. Three different concentrations of human spiked DNA were tested for the Pol III ChIP (2.5%, 5% and 10%). We also sequenced the corresponding inputs (crosslinked DNA from mouse liver). Two concentrations of human spiked DNA (5% and 10%) were tested for the Pol 2 ChIP. We also sequenced the corresponding inputs (crosslinked DNA from mouse liver).

2014-04-02 | E-GEOD-52049 | biostudies-arrayexpress

Genome-wide MAF1-dependent regulation of RNA polymerase III transcription [ChIP-Seq]

Project description:In higher eukaryotes, an important mechanism to tune translation in different tissues and conditions is mTORC1-dependent regulation of tRNAs transcription by RNA polymerase III (Pol III), as the relative amount of tRNAs tightly coordinates with the translational needs of the cell. mTORC1 contributes to regulate protein synthesis through its direct substrate MAF1, which functions as a negative regulator of Pol III transcription in response to stimuli such as serum starvation or rapamycin treatment. Here, we applied ChIP-seq to examine the Pol III occupancy profile in human fibroblasts and report evidence of a genome wide, MAF1-dependent coordinated response to favorable or stress growth conditions. Strikingly, while a set of genes is extremely responsive in terms of Pol III binding, other genes are mostly unperturbed, yet associated with transcriptionally engaged polymerases as revealed by nascent EU-labeled RNA-seq (neuRNA-seq). As shown by DamIP-seq, the responsiveness of a subset of genes is tightly connected to the rapid and transient interaction of MAF1 with DNA-bound Pol III. We performed duplicate ChIP-seq experiments for the Rpc4 (POLR3D) subunit of RNA polymerase III in IMR90hTert cells grown in the presence of fetal bovine serum (FBS), serum starved (SS), serum starved and treated with insulin (SS+I), serum starved and treated with insulin and rapamycin (SS+R+I). Additional ChIP-seq profiles were generated in cells treated with MAF1 siRNAs and serum starved. MAF1 binding was addressed by DamIP-seq, using two replicates per clone of IMR90hTert cells expressing HA-tagged MAF1-DamK9A (2 different clones) or EGFP-DamK9A (2 different clones). To monitor dynamic transcription profiles we did neusRNA-seq in IMR90hTert cells EU-labeled or mock (DMSO)-labeled. For both DamIP-seq and neusRNA-seq, cells were either unperturbed or serum starved.

2016-02-15 | E-GEOD-73926 | biostudies-arrayexpress

Genome-wide MAF1-dependent regulation of RNA polymerase III transcription [RNA-Seq]

2016-02-15 | E-GEOD-73929 | biostudies-arrayexpress

Genome-wide MAF1-dependent regulation of RNA polymerase III transcription [DamIP-Seq]

2016-02-15 | E-GEOD-73928 | biostudies-arrayexpress

A Multiplicity of Factors Contributes to Selective RNA polymerase III occupancy of a Subset of RNA polymerase III Genes in Mouse Liver

Project description:The genomic loci occupied by RNA polymerase (pol) III have been characterized in human culture cells by genome-wide chromatin immunoprecipitation experiments followed by deep sequencing (ChIP-Seq). These studies have in particular shown that only about 40 % of the annotated 622 human tRNA genes and pseudogenes are occupied by pol III, and that these genes are often in regions of open chromatin rich in active pol II transcription units. Here we have used ChIP-Seq to characterize pol III-occupied loci in a differentiated tissue, the mouse liver. Our studies define the mouse liver pol III-occupied loci and point to a conserved pol III-occupied mammalian interspersed repeat (MIR) as a potential regulator of a pol III subunit-encoding gene. They reveal that synteny relationships can be established between a number of human and mouse pol III genes, and that the expression levels of these genes are significantly linked. They establish that variations within the A and B promoter boxes, as well as the strength of the terminator sequence can strongly affect pol III occupancy of tRNA genes. They reveal correlations with various genomic features that together describe the pol III occupancy scores over some 50% of tRNA genes. In mouse liver, pol III-occupied loci represented in the NCBI37/mm9 genome assembly comprise fifty 5S genes, fourteen known non-tRNA genes, nine 4.5S genes, and some twenty nine SINEs. In addition, out of the 433 annotated tRNA genes, half are occupied by pol III. Transfer RNA gene expression levels reflect both an underlying genomic organization that is conserved in dividing human culture cells and resting mouse liver cells, and the particular promoter and terminator strengths of individual genes. 12 samples examinded, 4 on pol III, 2 on pol II, 2 on H3K4me3, 2 on H3k36me3, 2 input samples.

2012-01-19 | E-GEOD-33421 | biostudies-arrayexpress

A constant level of Pol III is maintained in the nucleus by nuclear retention and degradation-supplementery

Project description:Pol III functions in the nucleus for transcription and cytoplasm as a viral-DNA sensor, but how cells maintain the cytoplasmic-to-nuclear partitioning of Pol III activity is unknown. Here, we found that Pol III forms transcription-dependent protein clusters in the nucleus and preferentially associates with assembly factors in the cytoplasm. Acute protein degradation of Pol III subunits followed by transient protein labeling showed that nuclear and cytoplasmic Pol III response differently after the loss of their subunits.

2023-05-29 | PXD042522 | iProX

Subcellular Localization Shapes the Fate of RNA Polymerase III

Project description:RNA Pol III plays a vital role in transcription and as a viral-DNA sensor, but how it is assembled and distributed within cells remain poorly understood. Here, we show that Pol III is assembled with chaperones in the cytoplasm and forms transcription-dependent protein clusters upon transport into the nucleus. The largest subunit (RPC1) depletion through an auxin-inducible degron leads to rapid degradation and disassembly of Pol III complex in the nucleus and cytoplasm, respectively. This generates a pool of partially assembled Pol III intermediates, which can be rapidly mobilized into the nucleus upon the restoration of RPC1. Our study highlights the critical role of subcellular localization in determining Pol III's fate and provide insight into the dynamic regulation of nuclear Pol III levels and the origin of cytoplasmic Pol III complexes involved in mediating viral immunity.

2022-11-06 | PXD037971 | iProX

Transcriptome analysis of immortalized and transformed human IMR90 fibroblasts

Project description:RNA polymerase (Pol) III transcribes small untranslated RNAs that are essential for cellular homeostasis and growth. Its activity is regulated by inactivation of tumor suppressor proteins and overexpression of the oncogene c-MYC, but the concerted action of these tumor-promoting factors on Pol III transcription has not yet been assessed. In order to comprehensively analyse the regulation of Pol III transcription during tumorigenesis we employ a model system that relies on the expression of five genetic elements to achieve cellular transformation. Expression of these elements in six distinct transformation intermediate cell lines leads to the inactivation of TP53, RB1, and protein phosphatase 2A, as well as the activation of RAS and the protection of telomeres by TERT, thereby conducting to full tumoral transformation of IMR90 fibroblasts. Transformation is accompanied by moderately enhanced levels of a subset of Pol III-transcribed RNAs (7SK; MRP; H1). In addition, mRNA and/or protein levels of several Pol III subunits and transcription factors are upregulated, including increased protein levels of TFIIIB and TFIIIC subunits, of SNAPC1 and of Pol III subunits. Strikingly, the expression of POLR3G and of SNAPC1 is strongly enhanced during transformation in this cellular transformation model. Collectively, our data indicate that increased expression of several components of the Pol III transcription system accompanied by a 2-fold increase in steady state levels of a subset of Pol III RNAs is sufficient for sustaining tumor formation.

2018-04-23 | GSE99212 | GEO

Genome-wide localization of the RNA polymerase III transcription machinery in human cells

Project description:To characterize the pol III transcriptome in actively growing human cells, we used MR90hTeIrt lung fibroblasts, which are immortal but not transformed, with an intact Rb pathway. After formaldehyde cross-linking, chromatin was extracted and submitted to immunoprecipitation. To identify actively transcribed RNAP-III transcription units, we used antibodies directed against the RPC4 subunit of RNAP-III. To identify promoter regions, we used antibodies directed against Bdp1, which is part of both Brf1-TFIIIB and Brf2-TFIIIB, and thus should mark all types of active RNAP-III promoters. Antibodies directed against Brf1 were used to mark type 1 and 2 promoters. For type 3 promoters, we engineered an IMR90hTert cell line expressing the SNAPc subunit SNAP45 tagged at its C-terminal end with the biotin acceptor domain as well as the biotin ligase BirA and performed M-bM-^@M-^\chromatin affinity purificationM-bM-^@M-^] (ChAP) with streptavidin beads. In each case, the precipitated material was then processed for deep sequencing. ChIP-enriched DNA or ChAP-enriched DNA from MR90hTeIrt cells were analyzed by Illumina high-throughput sequencing. This analysis includes ChIP data done with antibodies against : RPC4, Bfr1, Bdp1 and SNAP45. Two lanes were run for RPC4, Brf1 and SNAP45, and three for Bdp1. One lane was run with Input DNA for control.

2010-02-11 | E-GEOD-18184 | biostudies-arrayexpress

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