Genome analysis in several eukaryotes shows a surprising number of transcripts which do not encode conventional messenger RNAs. Once considered noise, these non-coding RNAs (ncRNAs) appear capable of controlling gene expression by various means. We find Drosophila sex determination, specifically the master-switch gene Sex-lethal (Sxl), is regulated by long ncRNAs (>200 nt). The lncRNAs influence the dose sensitive establishment promoter of Sxl, SxlPe, which must be activated to specify female sex. They are primarily from two regions, R1 and R2, upstream of SxlPeand show a dynamic developmental profile. Of the four lncRNA strands only one, R2 antisense, has its peak coincident with SxlPe transcription, suggesting it may promote activation. Indeed, its expression is regulated by the X chromosome counting genes, whose dose determines whether SxlPe is transcribed. Transgenic lines which ectopically express each of the lncRNAs show they can act in trans, impacting the process of sex determination but also altering the levels of the other lncRNAs. Generally, expression of R1 is negative whereas R2 is positive to females. This ectopic expression also results in a change in the local chromatin marks, affecting the timing and strength of SxlPe transcription. The chromatin marks are those deposited by the Polycomb and Trithorax groups of chromatin modifying proteins, which we find bind to the lncRNAs. We suggest the increasing numbers of non-coding transcripts being identified are a harbinger of interacting networks similar to the one we describe.

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Polycomb group (PcG) mutants were first identified in Drosophila on the basis of their failure to maintain proper Hox gene repression during development. The proteins encoded by the corresponding fly genes mainly assemble into one of two discrete Polycomb repressive complexes: PRC1 or PRC2. However, biochemical analyses in mammals have revealed alternative forms of PRC2 and multiple distinct types of noncanonical or variant PRC1. Through a series of proteomic analyses, we identify analogous PRC2 and variant PRC1 complexes in Drosophila , as well as a broader repertoire of interactions implicated in early development. Our data provide strong support for the ancient diversity of PcG complexes and a framework for future analysis in a longstanding and versatile genetic system.