The Function of the CLAMP Zinc Finger Protein in Targeting MSL Complex to the X-chromosome during Dosage Compensation in Drosophila Melanogaster Chery , Jessica creator 2014 28, 308 p. born digital Thesis (Ph.D. -- Brown University (2014) Larschan, Erica Director Freiman, Richard Reader Delong, Alison Reader Bender, Judith Reader Buratowski, Stephen Reader Brown University. BIOMED: Molecular Biology, Cell Biology, and Biochemistry sponsor theses Coordinate gene regulation is critical to promote normal development and prevent disease. How domains of co-regulation are established in vivo remains poorly understood. X-chromosome dosage compensation system provides an excellent model for studying coordinate gene regulation because all of the active genes along the length of a whole chromosome are precisely co-regulated. Both Drosophila and mammals coordinately increase expression of genes on the male X-chromosome to equalize expression with autosomes and the female two X-chromosomes. Drosophila dosage compensation is mediated by the Male-Specific Lethal (MSL) ribonucleoprotein complex, which is expressed only in males and specifically identifies the X-chromosome. MSL complex is recruited to the X-chromosome via 21-bp MSL Recognition Elements (MRE) that contain a highly conserved 8-bp core. MREs are two-fold enriched on the X-chromosome but this enrichment alone cannot explain the complete X-specificity of MSL complex. Furthermore, it was unknown how MRE motifs are identified by MSL complex, because none of the known components of MSL specifically recognize MREs. Using a genetic screen, we identified a previously unstudied protein called CLAMP (Chromatin Linked Adaptor for MSL Proteins) that provides the previously unknown link between MSL complex and X-chromosome. CLAMP is a seven C2H2 zinc finger protein with a glutamine-rich domain, similar to other highly conserved transcriptional activators. Using chromatin immunoprecipitation (ChIP), custom protein binding microarrays (PBMs), structure-function analysis, Electrophorectic Mobility Shift Assays (EMSAs), and RNA immunoprecipitation (RIP), we determined the following: 1) CLAMP directly binds MREs; 2) CLAMP has an in vivo association with the non-coding roX (RNA on X) of MSL complex, and 3) CLAMP’s glutamine-rich domain is critical for MSL recruitment. It was previously known that MSL complex requires the entire GA-repeat containing 21-bp MRE for recruitment although only the 8-bp core is highly conserved. We determined that increasing the number of GA repeats increases CLAMP binding and the X-chromosome has higher number of GA-repeats than autosomes. Together, our data supports a model in which increasing GA-repeats facilitates increased CLAMP binding, thereby promoting X-chromosome identification. CLAMP zinc finger protein GA-repeats gene regulation dosage compensation Zinc-finger proteinsGenetic regulation RPB 20141006 engEnglish10.7301/Z0TX3CQXIn CopyrightCollection is open for research.dissertations