Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

New Chromatin Run-On Reaction Enables Global Mapping of Active RNA Polymerase Locations in an Enrichment-free Manner

ABSTRACT: The status of cellular RNA polymerases’ locations mirrors global transcriptional activity. A number of methods have been developed to map the distribution of RNA polymerase II (RNPII) genomewide, most of which took advantages of im-munoprecipitations of RNPII complexes or nucleotide analog-labeled nascent RNAs followed by high-throughput sequenc-ing. Given the tedious procedures involved in enrichment and sequencing library construction experiments, development of a simple method that conquers these issues is highly beneficial to the field. Here we report a mutation-based and enrich-ment-free Global chromatin Run-On sequencing (mGRO-seq) technique to map strand-specific RNPII locations ge-nomewide at near base resolution. An ATP analog, named N6-allyladenosine triphosphate (a6ATP), was designed and incor-porated into nascent RNAs at RNPII-located positions during a chromatin run-on assay. The chromatin RNAs were purified and subjected to a mild iodination reaction, which quantitatively transformed a6A into 1,N6-cyclized adenosine (cyc-A). During reverse transcription of RNA to complementary DNA (cDNA), base misincorporation at opposite site of cyc-A oc-curred and mutation reads were generated in the cDNA high-throughput sequencing. Bioinformatics analysis identified the genomewide RNPII location distributions from the characteristic A-mutated sites. mGRO-seq yields both the profile of RNPII sites and the chromatin RNA abundance from one experiment. To sum up, our work demonstrated a new simple and cost-effective method for mapping RNA polymerase locations genomewide, which holds great promise for single-cell char-acterization of RNPII distributions.

ORGANISM(S): Homo sapiens

PROVIDER: GSE160073 | GEO | 2023/10/25

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Similar Datasets

Identification and Characterization of the Mammalian Nuclear RNA N6-Adenosine Methyltransferase Core Complex

Project description:N6-methyladenosine (m6A) is the most prevalent internal modification found in mammalian messenger and non-coding RNAs. The discoveries of functionally significant demethylases that reverse this methylation as well as the recently revealed m6A distributions in mammalian transcriptomes strongly indicate regulatory functions of this modification. Here we report the identification and characterization of the mammalian nuclear RNA N6-adenosine methyltransferase core (RNMTC) complex. Besides METTL3, a methyltransferase which was the only known component of RNMTC in the past, we discovered that a previously uncharacterized methyltransferase, METTL14, exhibits a N6-adenosine methyltransferase activity higher than METTL3. Together with WTAP, the third component that dramatically affects the cellular m6A level, these three proteins form the core complex that orchestrates m6A deposition on mammalian nuclear RNA. Biochemistry assays, imaging experiments, as well as transcriptome-wide analyses of the binding sites and their effects on m6A methylation support methylation function and reveal new insights of RNMTC. PAR-CLIP and m6A-seq in HeLa cells

2013-11-29 | E-GEOD-46705 | biostudies-arrayexpress

m6A-Label-seq directly reports m6A methylome at base resolution

Project description:Here we report a metabolic labeling method to map mRNA N6-methyladenosine (m6A) modification transcriptome-wide at base resolution, termed m6A-label-seq. The cells were fed with Se-allyl-L-selenohomocysteine, an analog of methoine, which serves as the precursor of methylation enzyme cofactor, so that cellular RNAs were continuously deposited with N6-allyladenosine (a6A) at supposed m6A sites. We enriched a6A-containing mRNAs and sequenced their a6A sites which are identical to m6A sites, based on iodination-induced misincorporation during reverse transcription.

2020-04-21 | GSE131316 | GEO

Mapping polycomb complexes in human and mouse embryonic stem cells

Project description:In embryonic stem (ES) cells, bivalent chromatin domains with overlapping repressive (H3 lysine 27 tri-methylation) and activating (H3 lysine 4 tri-methylation) histone modifications mark the promoters of more than 2000 genes. To gain insight into the structure and function of bivalent domains, we mapped key histone modifications and subunits of Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) genomewide in human and mouse ES cells by chromatin immunoprecipitation followed by ultra high-throughput sequencing. We find that bivalent domains can be segregated into two classes: the first occupied by both PRC2 and PRC1 (PRC1-positive) and the second specifically bound by PRC2 (PRC2-only). PRC1-positive bivalent domains appear functionally distinct as they more efficiently retain lysine 27 tri-methylation upon differentiation, show stringent conservation of chromatin state, and associate with an overwhelming number of developmental regulator gene promoters. We also used computational genomics to search for sequence determinants of Polycomb binding. This analysis revealed that the genomewide locations of PRC2 and PRC1 can be largely predicted from the locations, sizes and underlying motif contents of CpG islands. We propose that large CpG islands depleted of activating motifs confer epigenetic memory by recruiting the full repertoire of Polycomb complexes. Keywords: cell type comparison

2008-11-01 | GSE13084 | GEO

Identification and Characterization of the Mammalian Nuclear RNA N6-Adenosine Methyltransferase Core Complex

2013-11-29 | GSE46705 | GEO

Mapping polycomb complexes in human and mouse embryonic stem cells

2008-11-01 | E-GEOD-13084 | biostudies-arrayexpress

Drosophila CLOCK Target Gene Characterization: Implications for Circadian Tissue-Specific Gene Expression

Project description:CLOCK (CLK) is a master transcriptional regulator of the circadian clock in Drosophila. To identify CLK direct target genes and address circadian transcriptional regulation in Drosophila, we performed chromatin immunoprecipitation-tiling array assays (ChIP-chip) with a number of circadian proteins. CLK binding cycles on at least 800 sites with maximal binding in the early night. The CLK partner protein CYCLE (CYC) is on most of these sites. The CLK/CYC heterodimer is joined 4-6 hrs later by the transcriptional repressor PER, indicating that the majority of CLK targets are regulated similarly to core circadian genes (Menet et al. 2010). About 30% of target genes also show cycling Pol II binding. Many of these generate cycling RNAs despite not being documented in prior RNA cycling studies. This is due in part to different RNA isoforms and to fly head tissue heterogeneity. CLK has specific targets in different tissues, implying that important CLK partner proteins and/or mechanisms contribute to gene-specific and tissue-specific regulation.

2011-11-15 | GSE32613 | GEO

CLOCKWORK ORANGE promotes CLOCK-CYCLE activation via the Drosophila ortholog of CLOCK INTERACING PROTEIN, CIRCADIAN (ChIP-Seq)

Project description:CWO reinforces PER-TIM repression of core clock gene transcription by antagonizing CLK-CYC binding to E-boxes, but also functions to promote CLK-CYC transcription of core clock genes. To determine the relationship between CWO and CLK-CYC binding across the genome, we identified CWO and CLK binding sites via ChIP-seq.

2021-07-23 | GSE165042 | GEO

N6-adenosine Methylation of Enhancer RNAs and YTHDC1 Facilitate Transcriptional Condensate Formation and 3D Chromatin Organization

Project description:Eukaryotic chromatin is pervasively transcribed into nascent RNAs. However, a mechanistic understanding of their general roles remains lacking. Here, we characterized the landscapes of N6-adenosine methylation (i.e. m6A) on nascent RNAs using a new method that we referred to as MINT-Seq. We found widespread m6A deposition on nascent RNAs produced from regulatory elements including enhancer RNAs (eRNAs) and promoter upstream antisense RNAs (uaRNAs).

2021-08-08 | GSE143441 | GEO

CLOCKWORK ORANGE promotes CLOCK-CYCLE activation via the Drosophila ortholog of CLOCK INTERACING PROTEIN, CIRCADIAN (RNA-Seq)

Project description:The Drosophila circadian clock is driven by a transcriptional feedback loop in which the bHLH transcription factor CLOCK-CYCLE (CLK-CYC) binds E-boxes to transcribe genes encoding the PERIOD-TIMELESS (PER-TIM) repressor, which releases CLK-CYC from E-boxes to inhibit transcription. The bHLH-Orange transcription factor CLOCKWORK ORANGE (CWO) reinforces repression by binding E-boxes to displace CLK-CYC, but also acts through an unknown mechanism to promote CLK-CYC transcription. To determine how CWO activates CLK-CYC transcription, we identified CWO DNA binding targets that are upregulated in the absence of CWO repression, conserved in mammals and preferentially expressed in brain pacemaker neurons. Among the genes identified was the Drosophila ortholog of mammalian Clock Interacting Protein Circadian (Cipc) that acts to repress CLOCK-BMAL1 transcription. Reducing or eliminating Drosophila Cipc expression shortens circadian period while overexpressing Cipc lengthens circadian period in flies, consistent with previous analysis showing that Drosophila Cipc represses CLK-CYC transcription in S2 cell culture. Long period rhythms of cwo mutant flies are largely rescued when Cipc expression is reduced or eliminated, indicating that increased Cipc expression mediates period lengthening of cwo mutants. These results suggest a mechanism for CWO-dependent CLK-CYC activation: CWO inhibition of CIPC repression promotes CLK-CYC transcription. Such a mechanism may be conserved given that orthologs of cwo and Cipc carry out analogous roles in the mammalian circadian clock.

2021-07-23 | GSE165043 | GEO

Methyltransferase METTL5 interactome

Project description:Cellular RNAs are covalently modified and these modifications can impact on all biological processes and hence are implicated in different types of diseases. Amongst RNA modifications, N6-methyladenosine (m6A) is one of the most widespread and has been found on messenger (mRNA), ribosomal (rRNA), non-coding and spliceosomal RNAs. We undertook a systematic screen to uncover new RNA-methyltransferases. We demonstrate that the methyltransferase-like 5 protein (METTL5) is an 18S rRNA specific methyltransferase and interacts specifically with Trmt122.

2020-09-09 | PXD015582 | Pride

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data