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Identifying differential binding of transcription factor CLAMP on chromatin in S2 (male) and Kc (female) emmbryonic cell lines

ABSTRACT: Identifying differential binding of transcription factor CLAMP on chromatin in S2 (male) and Kc (female) emmbryonic cell lines

PROVIDER: PRJNA908252 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR22523151_1.fastq.gz	Fastqsanger.gz
	SRR22523151_2.fastq.gz	Fastqsanger.gz
	SRR22523152_1.fastq.gz	Fastqsanger.gz
	SRR22523152_2.fastq.gz	Fastqsanger.gz
	SRR22523153_1.fastq.gz	Fastqsanger.gz

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Identifying splicing targets of CLAMP by mRNA-sequencing

Project description:CLAMP is a GA-repeat motif binding transcription factor that regulates gene expression. We hypothesized that CLAMP also regulates RNA processing, specifically alternative splicing that occurs co-transcriptionally because CLAMP is bound to intronic regions that are rich in polypyrimidine tracts which contain GA-rich sequences. Furthermore, GA-rich repeat sequences are thought to have evolved from polypyrimidine tracts that regulate splicing. Also, MALDI-mass spectrometry data identifying putative CLAMP interactors found association with 33 RNA binding proteins, including 6 that regulate alternative splicing. Thus, to test our hypothesis that CLAMP shapes transcriptome diversity by regulating RNA transcript splicing, we performed mRNA-sequencing in the presence and absence of CLAMP in Kc (female) and S2 (male) cell lines and used a SUPPA based pipeline called time2splice (https://github.com/ashleymaeconard/time2splice) to analyze the sequencing data. Using both cell lines helped us to identify female (Kc) and male (S2) specific splicing events. We identified 452 genes differentially spliced beetween Kc and S2 cells. There are 46 and 113 CLAMP-dependent splicing events in Kc and S2 cells, respectively. Interestingly, 45 CLAMP-dependent events (belonging to 42 genes) are specific to Kc cells, i.e female-specific, whereas 112 CLAMP dependent events (belonging to 100 genes) in S2 cells are specific to S2 cells, i.e male-specific. Therefore, we identified a new role for the transcription factor CLAMP in regulated sex-specific splicing events in Kc and S2 cells.

2023-08-02 | GSE220439 | GEO

Identifying differential binding of transcription factor CLAMP on chromatin in S2 (male) and Kc (female) emmbryonic cell lines

Project description:CLAMP is a maternally deposited transcription factor binding to GA-rich genomic region. It recruits male-specific MSL (Male sex-lethal) complex on male X-chromosome that regulates dosage compensation in males, and competes with other GA-binding proteins like GAF, acting both synergistically as well as antagonistically in a context dependent manner. Considering CLAMP's male specific role in dosage compensation but ubiquitous expression in both males and females, what regulates differential binding of CLAMP is still unknown. Furthermore, ChiP-seq analysis has being long used to study protein-DNA interactions, however, since it uses physical force to shear DNA after protein capture, it is prone to both false negative and false positive peaks being called. To minimise these discrepancies, a recent CutnRun MNase based technique have being developed (Heinkoff Lab) which captures the DNA fragments bound to protein of interest. Due to higher accuracy in identifying the size and sequence of the protein bound DNA region, one can also identify monomeric or multimeric protein binding on the chromatin. We have used this technique to identify differential and shared CLAMP binding sites in male and female cells. We also compared CLAMP DNA binding peaks with CLAMP RNA binding peaks (iCLIP data) in male and female cells to understand sex-biased co-transcriptional RNA processing mechanism.

2023-12-31 | GSE220053 | GEO

RNAseq S2 Clamp RNAi knockdown

Project description:RNAseq S2 Clamp RNAi knockdown

| PRJEB112555 | ENA

SMF-ONT S2 clamp RNAi knockdown

Project description:SMF-ONT S2 clamp RNAi knockdown

| PRJEB112557 | ENA

Determining sex-specific RNA interactors of maternal Transcription factor CLAMP in Drosophila cell lines

Project description:We performed RNA-protein interaction study iCLIP, i.e single nucleotide resolution uv crosslinking and immunoprecipitation for DNA binding transcription factor CLAMP in different cellular fractions (Chromatin fraction, Nucleoplasmic fraction, Cytoplasmic Fraction) to explore sex-specific RNA binding properties of CLAMP in female (Kc) and Male (S2) cells

2022-12-31 | GSE205987 | GEO

RNAseq S2 Clamp RNAi knockdown

Project description:Three biological replicates of RNA-seq were performed using S2 cells incubated for 72 hours with RNAi targeting Clamp or LacZ (control) transcripts. For each target, 10 µg of each purified dsRNA was combined to a total of 30 µg and directly added to 1.5 × 10⁶ S2 cells seeded in a 6-well plate. Cells were incubated for 72 hours at 25 °C. Total RNA was extracted from treated cells using the RNeasy Mini Kit (Qiagen, 74104). RNA-seq libraries were prepared from the isolated RNA using the NEBNext UltraExpress RNA Library Prep Kit (NEB, E3330S), following the manufacturer’s protocol. Libraries were sequenced on an Illumina NextSeq 2000 platform, generating 122-base single-end reads.

2026-08-04 | E-MTAB-17016 | biostudies-arrayexpress

Identifying splicing targets of CLAMP by mRNA-sequencing

Project description:Identifying splicing targets of CLAMP by mRNA-sequencing

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SMF-ONT S2 clamp RNAi knockdown

Project description:Collection of 2 biological replicates of SMF-ONT performed in S2 cell incubated for 72hrs with RNAi targeting Clamp or LacZ (ctrl) transcripts. The footprinting protocol was adapted from Kleinendorst and Barzaghi et al., 2021 and optimised for long-read sequencing and high weight DNA extraction. In summary, 2.5 million intact nuclei per SMF reaction have been treated with successive incubation with GpC (M.CviPI) and CpG (M.SssI) methyltransferase enzymes. Footprinted DNA was extracted using the Quick-DNA HMW MagBead kit following the manufacturer protocol and using wide-bore tips. Nanopore ligation library (LSK114) preparation has been performed using 1µg of purified footprinted HWM DNA. Sequencing has been performed using promethION flow cells for the R10 chemistry (LSK114). The data have been analysed using the SMF-ONT nextflow pipeline (https://git.embl.de/grp-krebs/nf-smfont) using custom methylation call models.

2026-08-04 | E-MTAB-17018 | biostudies-arrayexpress

Identifying RNA cargo of Drosophila S2 exosomes

Project description:Identifying RNA cargo of Drosophila S2 exosomes

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Transcriptional profiling of Drosophila melanogaster cell lines S2 and Kc with Nup153 RNAi-mediated depletion.

Project description:Expression profiling of Nup153 RNAi-mediated depletion in Drosophila S2 and Kc cells. This experiment is related to experiment E-MEXP-2525.

2010-02-12 | E-MEXP-2523 | biostudies-arrayexpress

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