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POLR3G Dependent PolyA+ and smallRNA Transcriptomes in Human Pluripotent Stem Cells

ABSTRACT: POLR3G is expressed at high levels in human pluripotent stem cells (hPSCs) and is required for maintenance of stem cell state through mechanisms, which are not known in detail. In order to explore how POLR3G regulates stem cell state, we have carried out deep sequencing analysis of polyA+ and smallRNA transcriptomes present in hPSCs and regulated in POLR3G dependent manner. Our data reveals that POLR3G regulates a specific subset of the hPSC transcriptome, including multiple transcripts types, such as protein coding genes, lincRNAs, miRNAs and snoRNAs. The primary function of POLR3G is in the maintenance rather than repression of transcription. The majority of POLR3G polyA+ transcriptome is regulated during differentiation and the key pluripotency factors bind to the promoters of at least 30 % of the POLR3G regulated transcripts. The POLR3G transcriptome is functionally linked to embryonic development and important tasks in cellular maintenance and function. #!/bin/bash

ORGANISM(S): Homo sapiens

PROVIDER: GSE94696 | GEO | 2017/05/10

SECONDARY ACCESSION(S): PRJNA371846

REPOSITORIES: GEO

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Homo sapiens

Project description:POLR3G Dependent PolyA+ and smallRNA Transcriptomes in Human Pluripotent Stem Cells

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RNA-seq from ENCODE/Caltech (Mouse)

Project description:This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (Georgi K. Marinov mailto:georgi@caltech.edu (data coordination/informatics/experimental), Diane Trout mailto:diane@caltech.edu (informatics) Brian Williams mailto:bawilli_91125@yahoo.com (experimental)). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:genome@soe.ucsc.edu). RNA-seq is a method for mapping and quantifying the transcriptome of any organism that has a genomic DNA sequence assembly (Mortazavi et al., 2008). RNA-seq is performed by reverse-transcribing an RNA sample into cDNA, followed by high-throughput DNA sequencing, which was done here on the Illumina HiSeq sequencer. The transcriptome measurements shown on these tracks were performed on polyA selected RNA (http://hgwdev.cse.ucsc.edu/cgi-bin/hgEncodeVocab?term=longPolyA&type=rnaExtract) from total cellular RNA (http://hgwdev.cse.ucsc.edu/cgi-bin/hgEncodeVocab?term=cell&type=localization). PolyA-selected RNA was fragmented by magnesium-catalyzed hydrolysis and then converted into cDNA by random priming and amplified. Paired-end 2x100 bp reads were obtained from each end of a cDNA fragment. Reads were aligned to the mm9 human reference genome using TopHat (Trapnell et al., 2009), a program specifically designed to align RNA-seq reads and discover splice junctions de novo. All sequence and alignments files are available at http://hgwdev.cse.ucsc.edu/cgi-bin/hgFileUi?db=mm9&g=wgEncodeCaltechRnaSeq.

2012-05-10 | GSE37909 | GEO

Genome-wide maps of RNA Pol III subunits POLR3C, POLR3G, and PLOR3GL in ES-E14TG2a and primary MEF cells

Project description:Mammalian cells contain two isoforms of RNA Polymerase III (Pol III) that differ in only a single subunit, with POLR3G in one form and the related POLR3GL in the other form. Previous research indicates that POLR3G and POLR3GL are differentially expressed, with POLR3G expression being largely restricted to embryonic stem cells (ESCs) and tumor cells in contrast to the ubiquitously expressed POLR3GL. To date, the functional differences between these two subunits remain largely unexplored, especially in vivo. Here, we show that POLR3G and POLR3GL containing Pol III complexes bind the same target genes and assume the same functions both in vitro and in vivo and, to a certain degree, can compensate for each other in vivo. Moreover, an observed defect in the differentiation ability of POLR3G knockout ESCs can be rescued by exogenous expression of POLR3GL. Interestingly, POLR3G knockout mice die at a very early embryonic stage, whereas POLR3GL knockout mice complete embryonic development without noticeable defects but die at about 3 weeks after birth with signs of both general growth defects and potential cerebellum-related neuronal defects. The different phenotypes of the knockout mice likely reflect differential expression levels of POLR3G and POLR3GL across developmental stages and between tissues and insufficient amounts of total Pol III in vivo.

2020-01-22 | GSE143969 | GEO

ESRG, a novel target of OCT4, maintains self-renewal and pluripotency of human pluripotent stem cells in collaboration with MCM2

Project description:Long non-coding RNA ESRG was first identified in our previous study, but its physiological function, regulatory and action mechanisms in human pluripotent stem cells (hPSCs) remain largely unexplored. Here, we found that ESRG is specifically and highly expressed in hPSCs, and its transcription is directly regulated by OCT4, suggesting that ESRG may be an integral component of the core regulatory circuit regulating the pluripotent state of hPSCs. Knockdown of ESRG induces hPSC differentiation, cell cycle arrest, and apoptosis. Mechanistically, ESRG binds to minichromosome maintenance protein 2 (MCM2), a replication-licensing factor, to sustain its steady-state level and nuclear translocation, safeguarding error-free DNA replication. Further study showed that inhibition of the interaction bewteen ESRG and MCM2 results in DNA damage and activation of p53 signaling pathway, ultimately deregulates deregulates pluripotency and self-renewal of hPSCs. In sum, our observations suggest that ESRG, as a novel target of OCT4, plays an essential role in maintaining the pluripotency and self-renewal of hPSCs in collaboration with MCM2 to suppress p53 signaling. These findings provide critical insights into the mechanisms underlying the maintenance of self-renewal and pluripotency in hPSCs.

2023-03-02 | GSE190957 | GEO

Midgut transcriptome of Culex quinquefasciatus larvae treated with LC90 doses of Bin toxin from Lysinibacillus sphaericus

Project description:Lysinibacillus sphaericus produces the mosquito larvicidal binary toxin consisting of BinA and BinB, which are both required for toxicity against Culex and Anopheles larvae. The molecular mechanisms behind Bin toxin-induced damage remain unexplored. We used whole-genome microarray-based transcriptome analysis to better understand how Culex larvae respond to Bin toxin treatment at the molecular level. Our analyses of Culex quinquefasciatus larvae transcriptome changes at 6, 12, and 18 h after Bin toxin treatment revealed a wide range of transcript signatures, including genes linked to the cytoskeleton, metabolism, immunity, and cellular stress, with a greater number of down-regulated genes than up-regulated genes. Bin toxin appears to mainly repress the expression of genes involved in metabolism, the mitochondrial electron transport chain, and the protein transporter of the outer/inner mitochondrial membrane. The induced genes encode proteins linked to mitochondrial-mediated apoptosis and cellular detoxification including autophagic processes and lysosomal compartments.

2017-03-22 | GSE96838 | GEO

Stirred suspension bioreactors maintain naïve pluripotency of human pluripotent stem cells (hPSCs)

Project description:Although cell therapies require large numbers of quality-controlled hPSCs, existing technologies are limited in their ability to efficiently grow and scale stem cells. We report here that cell-state conversion from primed-to-naïve pluripotency enhances the biomanufacturing of hPSCs. Naïve hPSCs exhibit superior growth kinetics and aggregate formation characteristics in stirred suspension bioreactors compared to their primed counterparts. Moreover, we demonstrate the role of the bioreactor mechanical environment in the maintenance of naïve pluripotency, through transcriptomic enrichment of mechano-sensing signaling for cells in the bioreactor along with a decrease in expression of lineage-specific and primed pluripotency hallmarks. Bioreactor-cultured, naïve hPSCs express epigenetic regulatory transcripts associated with naïve pluripotency, and display hallmarks of X-chromosome reactivation. They exhibit robust production of naïve pluripotency metabolites and display reduced expression of primed pluripotency cell surface markers. We also show that these cells retain the ability to undergo targeted differentiation into beating cardiomyocytes, hepatocytes, and neural rosettes. They additionally display faster kinetics of teratoma formation compared to their primed counterparts. Naïve bioreactor hPSCs also retain structurally stable chromosomes. Our research corroborates that converting hPSCs to the naïve state enhances hPSC manufacturing and indicates a potentially important role for the bioreactor’s mechanical environment in maintaining naïve pluripotency.

2020-05-08 | ST001377 | MetabolomicsWorkbench

Comparison of hPSCs-derived cholangiocyte-like cells with primary biliary tissue.

Project description:We established a step-wise protocol for the generation of human induced pluripotent stem cells (hPSCs) derived cholangiocyte-like cells (CLCs). The aim of this experiment was to provide in depth characterisation of the transcriptome of CLCs. These cells were compared with two of their in vitro progenitors, hPSCs and hepatoblasts (HBs), and to freshly isolated primary biliary tissue.

2015-05-26 | E-MTAB-2965 | biostudies-arrayexpress

POLR3G and POLR3GL-RNA polymerase III target genes

2013-10-01 | GSE47849 | GEO

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. 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.

2013-10-01 | E-GEOD-47849 | biostudies-arrayexpress

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