Abstract
BackgroundThe Myc oncoprotein is a transcriptional regulator whose function is essential for normal development. Myc is capable of binding to 10% of the mammalian genome, and it is unclear how a developing embryo controls the DNA binding of its abundant Myc proteins in order to avoid Myc's potential for inducing tumorigenesis.ResultsTo identify chromatin binding proteins with a potential role in controlling Myc activity, we established a genetic assay for dMyc activity in Drosophila. We conducted a genome-wide screen using this assay, and identified the Trithorax Group protein Ash1 as a modifier of dMyc activity. Ash1 is a histone methyltransferase known for its role in opposing repression by Polycomb. Using RNAi in the embryo and Affymetrix microarrays, we show that ash1 RNAi causes the increased expression of many genes, suggesting that it is directly or indirectly required for repression in the embryo, in contrast to its known role in maintenance of activation. Many of these genes also respond similarly upon depletion of Pc and pho transcripts, as determined by concurrent microarray analysis of Pc and pho RNAi embryos, suggesting that the three are required for low levels of expression of a common set of targets. Further, many of these overlapping targets are also activated by Myc overexpression. We identify a second group of genes whose expression in the embryo requires Ash1, consistent with its previously established role in maintenance of activation. We find that this second group of Ash1 targets overlaps those activated by Myc and that ectopic Myc overcomes their requirement for Ash1.ConclusionGenetic, genomic and chromatin immunoprecipitation data suggest a model in which Pc, Ash1 and Pho are required to maintain a low level of expression of embryonic targets of activation by Myc, and that this occurs, directly or indirectly, by a combination of disparate chromatin modifications.
Highlights
The Myc oncoprotein is a transcriptional regulator whose function is essential for normal development
Pho and Posterior sex combs (Psc) are involved in repression of dmyc Pc binds to methylated histone H3 at lysine 27 (H3K27), mediating repression of many genetic loci by Myc [18]
In an investigation of other Polycomb Group (PcG) gene products potentially involved in this repression by Myc and Pc, we chose three candidates to test: Posterior sex combs (Psc), which is in a core repressive complex with Pc [34]; E(z), which is a histone methyltransferase that methylates histone H3 lysine 27 and recruits Pc repression [35,36,37]; and Pho, which is one of two DNA-binding proteins of the group and recruits E(z) [28,30,31,32]
Summary
The Myc oncoprotein is a transcriptional regulator whose function is essential for normal development. Myc is capable of binding to 10% of the mammalian genome, and it is unclear how a developing embryo controls the DNA binding of its abundant Myc proteins in order to avoid Myc's potential for inducing tumorigenesis. Myc is a transcription factor, and its C-terminal domain is required for dimerization with its partner Max and its subsequent binding to DNA. It possesses an N-terminal transactivation domain that is required for both Myc's biological activities of transcriptional activation and repression. Activation by Myc involves binding to the nuclear protein TRRAP, which recruits histone acetyltransferase complexes to Myc activation targets. Myc binding to DNA is dependent on chromatin context, such that Myc sites having certain chromatin modifications are almost always bound, and Myc sites lacking those certain modifications are rarely bound by Myc [6]
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