Both human and Drosophila males must dosage compensate to equalize X-chromosome dosage between XX females and XY males. Although these species use diverse approaches for dosage compensation, the common first step by which the X chromosome is specifically distinguished from the autosomes for subsequent regulation remains poorly understood. Drosophila provides an ideal system in which to study how the X- chromosome is specifically identified because all of the active genes along the X-chromosome are targeted and a large number of genetic, biochemical, and genomic approaches are available. In Drosophila males, transcription of X-linked genes is increased two-fold, a process mediated by the MSL (Male Specific Lethal) complex, which specifically identifies and spreads along the male X-chromosome. However, the MSL complex, comprised of protein and RNA components, does not directly interact with DNA, and the mechanism by which it is specifically targeted to the male X-chromosome remains unknown. A recently identified zinc-finger protein, CLAMP (Coupling Lethal Adapter for MSL Proteins), recruits MSL complex to the X-chromosome by associating with both the complex and short DNA sequences called MREs (MSL Recognition Elements). Therefore, the identification of CLAMP provides a key opportunity to understand the initial stages of X identification and subsequent dynamic complex spreading. The first goal of this project is to determine how MSL complex specifically recognizes genes on the Drosophila male X-chromosome.
We aim to capture the dynamics of MSL complex spreading along the X by inducing MSL expression in female Drosophila cells, where it is normally not present, creating a pseudo-male system. We will then assay MSL complex spreading along the X-chromosome at various time points, representing a process too rapid to capture in male cells in real-time. Via these experiments we hope to answer the key question: What is the mechanism by which CLAMP functionally links the MSL complex to the X-chromosome? Our second goal is to determine the non sex-specific function of the CLAMP protein, which colocalizes with known components of the Histone Locus Body (HLB). We will use microscopy in a variety of in vivo systems as well as a cell-based reporter to determine the function of this specific CLAMP chromatin localization in both males and females. We will then use a candidate RNAi screen to identify other factors involved in CLAMP recruitment. We hope to address the key question: What is the function of non sex-specific CLAMP localization on the chromatin?
Both human and Drosophila males must dosage compensate to equalize X-chromosome dosage between XX females and XY males. Drosophila dosage compensation is mediated by the Male Specific Lethal (MSL) complex, which specifically identifies and spreads along the male X-chromosome, yet the mechanism by which MSL is specifically targeted and spreads remains unknown. CLAMP, a newly identified protein that recruits MSL complex to the X-chromosome, provides a key opportunity to understand the initial mechanism of X identification and subsequent dynamic complex spreading.
|Rieder, Leila E; Koreski, Kaitlin P; Boltz, Kara A et al. (2017) Histone locus regulation by the Drosophila dosage compensation adaptor protein CLAMP. Genes Dev 31:1494-1508|