Determinants of X-chromosome transcriptional regulation in C. elegans

Ercan, Sevinc

Abstract

Chromosome-based mechanisms of sex determination cause an imbalance in the transcript dosage of the X chromosomes between the sexes. In C. elegans, X chromosome dose is compensated by at least two distinct mechanisms. First males (XO) and females (XX) equalize X-linked transcript levels between the sexes. This regulation is mediated by the Dosage Compensation Complex (DCC) that binds specifically to both X chromosomes of XX hermaphrodites to down-regulate expression by a factor of two. A second uncharacterized mechanism equalizes average transcript levels between X and autosomes within each sex. This second mechanism is present in other metazoans including humans. This project aims to test models that pertain to both mechanisms. I propose to identify the DNA sequence and chromatin determinants required for X-specific localization of the DCC. DNA sequence motifs will be derived from initial DCC recruitment sites identified by ChlP-chip (Chromatin ImmunoPrecipitation followed by microarray analysis) of the DCC subunit that initially confers X specificity to the complex. In addition, nucleosome occupancy will be measured by FAIRE (Formaldehyde- Assisted Isolation of Regulatory Elements) and nucleosome positions will be determined by microarray analysis of mononucleosomal DNA generated by micrococcal nuclease digestion. Factors that block or assist local spreading will be identified by ChlP-chip of DCC in X-to-autosome fusion strains. I propose determine the molecular basis of DPY-30 requirement for DCC localization by identifying its role in transcription related methylation of histone H3 lysine 4. Finally, I aim to determine whether the second mechanism of dosage compensation that occurs between X and autosomes occurs at the level of transcription or translation by determining RNA Polymerase II levels on X and autosomal-linked genes. Epigenetic regulation of X-chromosome transcription occurs both in humans and C elegans, yet many of the molecular mechanisms remain uncharacterized. It is known that X-linked transcripts are particularly highly expressed in brain tissues and that the epigenetics of X-linked genes maybe linked to mental diseases. We plan to use the genetic and molecular tools available in C. elegans to understand how the X chromosome evolved to regulate its transcription to compensate for both sex-specific dosage differences and transcriptional haploinsufficiency. Additionally, determining the molecular mechanism of DCC activity and condensin binding will contribute to our understanding of how a complex of proteins with evolutionary roots in chromatin compaction have been co-opted to fine-tune transcription chromosome-wide. ? ? ?