Polycomb/Trithorax group proteins collaborate with Heterochromatin protein 1 to regulate Drosophila sex determination

摘要

BackgroundA combination of histone modifications, a ‘histone epigeneticcode’, is created and altered by specific enzymes, andrecognized by proteins that bind to these modificationsthrough specific domains [1]. These proteins modify thechromatin environment for precise transcriptional regulation.Some of these well studied chromatin remodelingcomplexes include the Polycomb and the Trithorax GroupProteins (PcG and Trx-G). The two groups are primarilyantagonistic and control patterning of the body duringembryogenesis, through the regulation of gene expression.The Polycomb Repressive Complexes mediate the initiationof gene repression and its maintenance. The Trx-Gproteins are involved in maintaining the activation of geneexpression [1]. In Drosophila, one of the earliest developmentaldecisions made in the embryo is that of determiningits sex. This process involves regulating the expressionof the X chromosome sensing promoter of Sex-lethal (Sxl)at its establishment promoter, Sxlpe [2]. Using this sensitivesystem which differentiates one versus two X chromosomes,our lab has shown that heterochromatin proteinsare required for proper Sxlpe regulation. We found thatHeterochromatin Protein 1 a (HP1a) plays both a repressiveand activating role in regulating Sxlpe [3].Materials and methodsDrosophila Stocks: ash1[MB03235]/TM6C, Sb; E(z)[EY21318]; E(z)32A40/TM6C, Sb, Tb; Su(z)12[4]/TM6C,Sb, Tb; wild-type w[1118] and Ore R. In situ hybridizationin 0-4 hour embryos was used to analyze Sxlpeexpression levels using a Digoxygenin labeled RNA probespecific to Sxlpe transcripts; Chromatin Immunoprecipitationswere performed with antibodies specific toH3K4me3, H3K27me3 and HP1a on chromatin preparedfrom 1-3 and 2-4 hour embryos, also from the abovementioned stocks. Quantitative real-time PCR quantifiedSxl sequences in the DNA from each ChIP.ResultsWe find that the PcG/Trx-G proteins genetically interactwith mutations in the sex determination pathway andinfluence the ability of females to determine their sex. Insitu analysis of ash1, Su(z)12 and E(z) mutant embryosshow they affect both the timing and strength of transcriptionof the sex establishment promoter, Sxlpe. qRT-PCRanalyses support the in situs, showing a change in SxlPemRNA levels. We also observe that these proteins arenecessary for proper histone 3 lysine 4 (H3K4) and histone3 lysine 27 (H3K27) methylation at the promoter. Surprisingly,we find that embryos deficient in E(z), Su(z)12 orASH1 protein also affect the binding of HP1a at SxlPesequences.ConclusionsOur data support the idea that PcG proteins function on alarger set of target genes with a broader role in gene regulationand development. Additionally, we find a novel heterochromatinrole for PcG/Trx-G proteins which influencesHP1a in the regulation of gene expression.