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

ABSTRACT: 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

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE205987 | GEO | 2022/12/31

REPOSITORIES: GEO

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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

DNA binding activities of the C-clamp

Project description:Investigated the DNA binding specificity of the C-clamp of dnTCF1E Used a doxycycline inducible system to investigate the role of the C-clamp in the genome-wide binding of dnTCF1E. Induced dnTCF1EWT and dnTCF1Emut (C-clamp mutant that is null for C-clamp DNA binding) over 2 time points (2 hours and 9 hours). A 0 hour (no doxycycline, uninduced) control was also included.

2014-12-04 | E-GEOD-53536 | biostudies-arrayexpress

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

CLAMP Protein Binding Microarray (PBM) experiment

Project description:To investigate the DNA binding specificity of the CLAMP protein, we have designed a custom PBM to interrogate the binding of CLAMP to DNA sequences extracted from the Drosophila melanogaster genome. Specific regions were extracted based on ChIP-seq data, motif occurence and proximity to gene transcription start sites. N-terminal GST-tagged protein samples were made for the C-terminal four and six zinc finger portions of the protein CLAMP; samples were made by in vitro transcription translation (IVT). IVT reaction mixtures for the two CLAMP constructs were applied directly to the PBM microarray and incubated for 1hour. Microarray-bound protein was fluorescently labeled using Alexa488-conjugated antibodies targeting GST, and the microarray was scanned in using a standard microarray scanner. Median fluorescence intensity over eight replicate probes was reported for each unique DNA sequence on the microarray.

2016-07-17 | E-GEOD-83442 | biostudies-arrayexpress

Synergistic interactions between CLAMP and MSL complex facilitate Drosophila dosage compensation

Project description:ChIP-seq and mRNA-seq experiments were performed to understand the role of the CLAMP protein in dosage compensation ChIP-seq experiments compared the binding profiles of CLAMP in male and female cells and mRNA-seq data to define the role of CLAMP in regulating genes on the X-chromosome

2013-07-15 | E-GEOD-39271 | biostudies-arrayexpress

The zinc finger protein CLAMP promotes long-range chromatin interactions that mediate dosage compensation of the Drosophila male X-chromosome [4C-seq]

Project description:Hi-C and 4C-seq anlaysis of Drosophila S2 cells Drosophila dosage compensation is an important model system for defining how active chromatin domains are formed. The Male-specific lethal dosage compensation complex (MSLc) increases transcript levels of genes along the length of the single male X-chromosome to equalize with that on the two female X-chromosomes. The strongest binding sites for MSLc cluster together in three-dimensional space independent of MSLc because clustering occurs in both sexes. CLAMP, a non-sex specific, ubiquitous zinc finger protein, binds synergistically with MSLc to enrich the occupancy of both factors on the male X-chromosome. Here, we demonstrate that CLAMP promotes the observed clustering of MSLc bindings sites. Genome-wide, CLAMP promotes interactions between active chromatin regions and represses interactions between inactive chromatin regions. Moreover, the X-enriched CLAMP protein promotes longer-range interactions on the active X-chromosome than autosomes. Overall, we define how long-range interactions, mediated by a locally-enriched ubiquitous transcription factor, generate a three-dimensional active chromatin domain.

2019-12-01 | GSE130544 | GEO

The zinc finger protein CLAMP promotes long-range chromatin interactions that mediate dosage compensation of the Drosophila male X-chromosome [Hi-C seq]

2019-12-01 | GSE130545 | GEO

CLAMP Protein Binding Microarray (PBM) experiment

2016-07-17 | GSE83442 | GEO

The zinc-finger protein CLAMP promotes gypsy chromatin insulator function in Drosophila: differential ChIP-seq of CP190 and CLAMP under CP190 knockdown

Project description:Chromatin insulators are DNA-protein complexes that establish higher order independent DNA domains to influence transcriptional regulation. Insulators are defined by two different functions: they can block communication between an enhancer and a promoter and also act as a barrier between heterochromatin and euchromatin. In Drosophila, the gypsy-insulator complex contains three core components: Su(Hw), CP190 and Mod(mdg4)67.2. Here we identify a novel role for Chromatin-linked adaptor for MSL proteins (CLAMP) in promoting gypsy chromatin insulator function. When Clamp is depleted by RNAi, gypsy-dependent enhancer blocking activity decreases and barrier activity is reduced in all tissues. Furthermore, Clamp RNAi knockdowns and mutation result in disorganized insulator complex localization in the nucleus. Co-immunoprecipitation experiments showed that CLAMP physically associates with core gypsy-insulator proteins. Co-localization of CLAMP with gypsy components on polytene chromosomes and ChIP-seq analysis demonstrates co-localization of CLAMP with a subset of insulator sites across the genome. Thus, our findings suggest a ubiquitous, genome-wide role for CLAMP in promoting gypsy-dependent chromatin insulator activity.

2019-01-28 | GSE118699 | GEO

Skeletal muscle biopsies before and after hyperinsulinemic-euglycemic clamp

Project description:6 lean humans were submitted to a 3 hours hyperinsulinemic-euglycemic clamp. Skeletal muscle biopsies were taken before and after the clamp. Set of arrays that are part of repeated experiments

2010-06-04 | GSE11868 | GEO

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