Role Of Sirna Pathway In Epigenetic Modifications Of The Drosophila Melanogaster X Chromosome

Eukaryotic genomes are organized into large domains of coordinated regulation. The role of small RNAs in formation of these domains is largely unexplored. An extraordinary example of domain-wide regulation is X chromosome compensation in Drosophila melanogaster males. This process occurs by hypertranscription of genes on the single male X chromosome. Extensive research in this field has shown that the Male Specific Lethal (MSL) complex binds X-linked genes and modifies chromatin to increase expression. The components of this complex, and their actions on chromatin, are well studied. In contrast, the mechanism that results in exclusive recruitment to the X chromosome is not understood. Our research focuses on the process by which male flies selectively modulate expression from their single X chromosome. Prior studies in the lab have found that the siRNAs produced from repetitive sequences on the X chromosome and the repeat DNA itself, participates in dosage compensation in flies. Interestingly, the siRNA pathway contributes to X-localization of the MSL complex. The basis of enhanced localization is unknown, and no RNAi components have been found to interact directly with the MSL complex. This suggests that siRNA influences X-recognition by an indirect and novel mechanism. I found evidence that chromatin around these repeats is modulated by the siRNA pathway. I demonstrated that FLAG-tagged Argonaute2 protein localizes at these repeats. I show that numerous Agonaute2-interacting proteins show evidence of participation in compensation. One of these, Su(var)3-9, deposits H3K9me2 in and near the repeats. When a repeat-containing transgene is inserted on an autosome, H3K9me2 is enriched in surrounding chromatin, an effect that is enhanced by ectopic production of cognate siRNA. In accord with the idea that these repeats contribute to recruitment of dosage compensation, genes as much as 100 kb from the autosomal insertion increase in expression upon expression of ectopic siRNA. My studies demonstrate that chromatin around a group of X-enriched sequences is modulated by siRNA, and supports the idea that siRNA contributes to the elevated expression that characterizes the compensated male X chromosome. This study advances our understanding of the mechanism of X recognition by showing a direct relationship between siRNA-directed chromatin modification and a class of repetitive elements that helps mark the X chromosome