The RPB2 flap loop of human RNA polymerase II is dispensable for transcription initiation and elongation.
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE29734 | GEO | 2011/06/24
SECONDARY ACCESSION(S): PRJNA141155
REPOSITORIES: GEO
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Project description:Deletion of the flap loop in the Rp2 subunit of human RNAPII did not alter the ability of human RNAPII to initiate transcription or elongate the initiated transcripts in vivo and in vitro, and was also dispensable for elongation factor-mediated enhancement and inhibition of transcript elongation in vitro. ChIP:chip of wild-type and flap deletion human RNAPII revealed that the mutant RNAPII was not defective for promoter binding, initiation and elongation in vivo. Eight ChIP datasets were generated as log2(IP/input) fluorescence intensities using anti-FLAG IP of wild-type RPB2-FLAG cells in biological triplicate, anti-FLAG IP of delta-FL RPB2 FLAG cells in biological triplicate, and anti-RPB1 CTD, 8WG16 of both wild-type and delta-FL cells in biological duplicate. The two 8WG16- IP datasets were combined to measure the distribution of RPB1 signal.
2011-06-24 | E-GEOD-29734 | biostudies-arrayexpress
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Project description:Here we characterized a genetic regulatory system named as the hpDNA-assisted structure-guided nuclease mediating interference (HpSGNi) system, which was composed of flap endonuclease 1 (FEN1) and mis-hairpin DNA probe (mis-hpDNA). To investigate gene regulation effective of the HpSGNi system, we co-transformed HEK293A (human embryonic kidney epithelial cells) with plasmids encoding FEN1 with NES and two groups of mis-hpDNAs targeting the EGFP gene and negative control, respectively. RNA-seq was used to examine the regulation effective. We proved the HpSGNi system can knock down specific gene in human cells. At the same time, it indicated that a degree of off-targets occur in HpSGNi-treated cells.
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Project description:As a central component during Okazaki fragment maturation, flap endonuclease 1 (FEN1) removes the 5â flap and maintains genomic stability. Here, FEN1 was cloned as a suppressor of transcriptional gene silencing (TGS) from a forward genetic screen. FEN1 is abundant in the root and shoot apical meristems and FEN1-GFP shows a nucleolus-localized signal in tobacco cells. Arabidopsis fen1-1 mutant is hypersensitive to MMS and shows reduced telomere length. Interestingly, genome-wide ChIP-seq and RNA-seq results demonstrate that FEN1 mutation leads to a decrease in the H3K27me3 level and an increase in the expression of a subset of genes marked with H3K27me3. Overall, these results uncover a role for FEN1 in mediating TGS besides maintaining genome stability in Arabidopsis. To characterized the role of FEN1 in epigenetic silencing, we examine histone modification and RNA expression changes by whole-genome RNA sequencing; H3K27me3-, H3K4me3-, H3K9me2-, H3-ChIP-seq in A. thaliana transgenic wild type (TWT) and fen1 mutant
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Project description:As a central component during Okazaki fragment maturation, flap endonuclease 1 (FEN1) removes the 5’ flap and maintains genomic stability. Here, FEN1 was cloned as a suppressor of transcriptional gene silencing (TGS) from a forward genetic screen. FEN1 is abundant in the root and shoot apical meristems and FEN1-GFP shows a nucleolus-localized signal in tobacco cells. Arabidopsis fen1-1 mutant is hypersensitive to MMS and shows reduced telomere length. Interestingly, genome-wide ChIP-seq and RNA-seq results demonstrate that FEN1 mutation leads to a decrease in the H3K27me3 level and an increase in the expression of a subset of genes marked with H3K27me3. Overall, these results uncover a role for FEN1 in mediating TGS besides maintaining genome stability in Arabidopsis.
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Project description:Here we characterized a flap endonuclease 1 (FEN1) plus hairpin DNA probe (hpDNA) system, called the HpSGN system, for both DNA and RNA editing without sequence limitation.To investigate the mRNA on/off target cleavage effective of the HpSGN system,we co-transformed HepG2 (liver hepatocellular cells) with plasmids encoding FEN1 with NES and two groups of hpDNAs targeting the mRNA of AFP (Alpha Fetoprotein) gene and CDK9 (Cyclin-dependent kinase 9) gene, respectively. RNA-seq was used to examine the on/off taget cleavage effective.We proved the HpSGN system can knock down specific mRNAs in human cells at a level of ~25%.At the same time,it indicated that a degree of off-targets occur in HpSGN-treated cells.
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Project description:Single stranded DNA binding proteins play many roles in nucleic acid metabolism, but their importance during transcription remains unclear. Quantitative proteomic analysis of RNA polymerase II (RNApII) pre-initiation complexes (PICs) identified Sub1 and the Replication Protein A complex (RPA), both of which bind single-stranded DNA (ssDNA). Sub1, homolog of mammalian coactivator PC4, exhibits strong genetic interactions with factors necessary for promoter melting. Sub1 localizes near the transcription bubble in vitro and binds to promoters in vivo dependent upon PIC assembly. In contrast, RPA localizes to transcribed regions of active genes, strongly correlated with transcribing RNApII but independently of replication. RFA1 interacts genetically with transcription elongation factor genes. Interestingly, RPA levels increase at active promoters in cells carrying a Sub1 deletion or ssDNA binding mutant, suggesting competition for a common binding site. We propose that Sub1 and RPA interact with the non-template strand of RNApII complexes during initiation and elongation, respectively. Chip-chip from wt and sub1D cells with Rfa1 Chromatin immunoprecipitation (ChIP) of Rfa1 in wt and sub1D yeast demonstrated that Rfa1 localization correlates with RNA Polymerase II and is increased at some transcription start sites when Sub1 has been deleted. Comparison of Rfa1 localization in wt vs sub1D yeast
2011-09-26 | E-GEOD-32416 | biostudies-arrayexpress
Project description:Dynamic control of gene expression is crucial for most aspects of cell physiology and at its core is RNA polymerase II (RNAPII). Using 53 RNAPII point mutants, we generated a point mutant epistatic miniarray profile (pE-MAP) comprising ~60,000 quantitative genetic interactions in Saccharomyces cerevisiae. This enabled functional assignment of RNAPII sub-domains, including connections to protein complexes. Using splicing microarrays and point mutants altering elongation rates in vitro, we found an inverse relationship between RNAPII speed and in vivo splicing efficiency. Furthermore, the pE-MAP classified groups of fast and slow mutants that favor upstream and downstream start site selection, respectively. Finally, the pE-MAP identified Sub1 as a positive transcription factor regulating start site selection and splicing. These data reveal striking coordination of polymerization rate with transcription initiation and splicing, suggesting transcription rate is tuned to coordinate multiple gene expression steps. The pE-MAP approach provides a powerful strategy to understand other multi-functional machines at amino acid resolution. Two channel microarrays were used. RNA isolated from a large amount of wt yeast from a single culture was used as a common reference. This common reference was used in one of the channels for each hybridization and used in the statistical analysis to obtain an average expression-profile for each deletion mutant relative to the wt. Two independent cultures were hybridized on two separate microarrays. For the first hybridization the Cy5 (red) labeled cRNA from the deletion mutant is hybridized together with the Cy3 (green) labeled cRNA from the common reference. For the replicate hybridization, the labels are swapped. Each gene is represented twice on the microarray, resulting in four measurements per mutant. Using the Erlenmeyer growth protocol up to five deletion strains were grown on a single day. In the tecan platereader, up to eleven deletion strains could be grown on a single day. Wt cultures were grown parallel to the deletion mutants to assess day-to-day variance. Strains with point mutations in either rpb1, rpb2 or rpb7 subunits of RNA polymerase II (RNAPII) examined under normal growth conditions. The mutated RNAPII subunit is present on URA3 or LEU2 marked CEN plasmid, and the corresponding genomic RNAPII subunit deleted. Strains labeled as wildtype also have the relevant genomic RNAPII subunit deleted, but complemented with wildtype RNAPII subunit on CEN plasmid.
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