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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.

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

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

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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.

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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.

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

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