In heterogametic species there is an imbalance in the number of sex-linked genes between males and females. Therefore, it is essential that transcription is equalized between the sexes and between the sex chromosomes and autosomes, a process called dosage compensation. Though different dosage compensation mechanisms have evolved, they all share a common initial step of distinguishing the X-chromosome from the autosomes. The model organism Drosophila melanogaster has evolved a single-step mechanism for dosage compensation, providing a simple model for studying how a single chromosome is discriminated from the rest of the genome. In Drosophila, selective two-fold upregulation of the single male X-chromosome equalizes transcription between the sexes. The selective identification of the X-chromosome involves the recruitment of a ribonucleoprotein complex, Male-Specific Lethal (MSL) that is expressed only in males. The MSL complex initially localizes to GA-rich sequences within 'seed sites' on the X-chromosome, termed MSL Recognition Elements (MREs). However, MSL complex cannot bind directly to MREs and these sequences are distributed throughout the genome. The Larschan laboratory has recently identified a previously unstudied zinc finger protein, Chromatin-Linked Adapter for MSL Proteins (CLAMP), that links the MSL complex to MRE sequences. Furthermore, CLAMP is highly enriched on the X-chromosome independent of MSL complex and therefore is likely to be involved in the earliest step of X-identification. Therefore, the identification of CLAMP provides the first opportunity to define the X-chromosome features that promote its identification for dosage compensation. Based on strong preliminary data, I hypothesize that identification of the X-chromosome requires contributions from primary genome sequence and higher-order nuclear organization mediated by the CLAMP protein. I will test my hypothesis by defining the role of linear and three-dimensional genomic organization in enrichment of CLAMP on the X-chromosome. First, using Chromatin Immunoprecipitation- qPCR (ChIP-qPCR), I will determine whether enrichment of CLAMP on the X-chromosome occurs cooperatively or additively as the number of tandem MRE sequences increases. Second, I will determine if putative initial seed sites cluster together in three-dimensional nuclear space using the Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-pet) method. The proposed experiments will use state-of-the- art approaches to provide key insight into how initial identification of the X-chromosome is achieved.
Dosage compensation is a conserved process that corrects for the imbalance in sex chromosome number in heterogametic species. A key obstacle in all species that perform dosage compensation is the selective identification of the X-chromosome from the autosomes. Using a combination of genetic and genomic techniques; this project will provide insight into understanding how this evolutionarily conserved mechanism is initiated.