Abstract
Gaining new insights into gene regulation involves an in-depth understanding of protein-protein interactions on chromatin. A powerful model for studying mechanisms of gene regulation is dosage compensation, a process that targets the X-chromosome to equalize gene expression between XY males and XX females. We previously identified a zinc finger protein in Drosophila melanogaster that plays a sex-specific role in targeting the Male-specific lethal (MSL) dosage compensation complex to the male X-chromosome, called the Chromatin-Linked Adapter for MSL Proteins (CLAMP). More recently, we established that CLAMP has non-sex-specific roles as an essential protein that regulates chromatin accessibility at promoters genome-wide. To identify associations between CLAMP and other factors in both male and female cells, we used two complementary mass spectrometry approaches. We demonstrate that CLAMP associates with the transcriptional regulator complex Negative Elongation Factor (NELF) in both sexes and determine that CLAMP reduces NELF recruitment to several target genes. In sum, we have identified many new CLAMP-associated factors and provide a resource for further study of this little understood essential protein.
Highlights
Identification of functional chromatin-associated protein-protein interactions has been important in understanding the establishment of dosage compensation in Drosophila melanogaster [1]
While it is known that the Male-specific lethal (MSL) complex facilitates the process of D. melanogaster dosage compensation [3], the MSL complex does not include any sequence-specific DNA binding proteins with high affinity for the GA-rich cis-elements that target it to the male X-chromosome [4]
To identify factors that associate with Chromatin-linked adapter for MSL Proteins (CLAMP) in vivo, we performed two biological replicates of CLAMP immunoprecipitation under non-crosslinked conditions followed by mass spectrometry analysis from whole cell extracts of male (S2) and female (Kc) cells
Summary
Identification of functional chromatin-associated protein-protein interactions has been important in understanding the establishment of dosage compensation in Drosophila melanogaster [1]. Dosage compensation is a conserved process, which in D. melanogaster occurs by increasing transcript levels expressed from the single male X-chromosome to equal those expressed from the two female X-chromosomes [2]. While it is known that the Male-specific lethal (MSL) complex facilitates the process of D. melanogaster dosage compensation [3], the MSL complex does not include any sequence-specific DNA binding proteins with high affinity for the GA-rich cis-elements that target it to the male X-chromosome [4]. Using a cell-based RNA interference approach, we recently demonstrated that a zinc finger protein is a key regulator of MSL complex recruitment to the X-chromosome [5,6], which we named Chromatin-linked adapter for MSL Proteins (CLAMP).
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