Academic journal article Genetics

The Relationship between Long-Range Chromatin Occupancy and Polymerization of the Drosophila ETS Family Transcriptional Repressor Yan

Academic journal article Genetics

The Relationship between Long-Range Chromatin Occupancy and Polymerization of the Drosophila ETS Family Transcriptional Repressor Yan

Article excerpt

ABSTRACT ETS family transcription factors are evolutionarily conserved downstream effectors of Ras/MAPK signaling with critical roles in development and cancer. In Drosophila, the ETS repressor Yan regulates cell proliferation and differentiation in a variety of tissues; however, the mechanisms of Yan-mediated repression are not well understood and only a few direct target genes have been identified. Yan, like its human ortholog TEL1, self-associates through an N-terminal sterile a-motif (SAM), leading to speculation that Yan/TEL1 polymers may spread along chromatin to form large repressive domains. To test this hypothesis, we created a monomeric form of Yan by recombineering a point mutation that blocks SAM-mediated self-association into the yan genomic locus and compared its genome-wide chromatin occupancy profile to that of endogenous wild-type Yan. Consistent with the spreading model predictions, wild-type Yan-bound regions span multiple kilobases. Extended occupancy patterns appear most prominent at genes encoding crucial developmental regulators and signaling molecules and are highly conserved between Drosophila melanogaster and D. virilis, suggesting functional relevance. Surprisingly, although occupancy is reduced, the Yan monomer still makes extensive multikilobase contacts with chromatin, with an overall pattern similar to that of wild-type Yan. Despite its near-normal chromatin recruitment, the repressive function of the Yan monomer is significantly impaired, as evidenced by elevated target gene expression and failure to rescue a yan null mutation. Together our data argue that SAM-mediated polymerization contributes to the functional output of the active Yan repressive complexes that assemble across extended stretches of chromatin, but does not directly mediate recruitment to DNA or chromatin spreading.

DYNAMIC regulation of gene expression during development requires the combined and coordinated action of transcriptional activators and repressors across multiple cisregulatory modules (CRMs). Research over the last decade has led to a growing appreciation of the existence and importance of both short-range linear and long-range threedimensional chromatin interactions to overall regulation of gene expression (Hong et al. 2008; Frankel et al. 2010; Perry et al. 2010, 2011; Bulger and Groudine 2011; Dunipace et al. 2011; He et al. 2011). However, the molecular determinants underlying long-range transcriptional regulation remain poorly understood.

One long-standing hypothesis of transcriptional repression is that the biochemical ability of a factor to polymerize might drive spreading of repressive complexes along the chromatin, thereby providing a mechanism of long-range repression (Courey and Jia 2001; Roseman et al. 2001). Well-studied examples include multiple Polycomb Group (PcG) corepressors and the ETS family transcriptional repressors TEL1 (ETV6) and Yan, all of which carry a strong oligomerization domain termed the sterile a-motif (SAM) (Kim et al. 2001, 2002, 2005; Tran et al. 2002; Qiao et al. 2004; Qiao and Bowie 2005). In vitro, the isolated SAM domains from these proteins form helical, head-to-tail polymers whose overall structural homology suggests a common mode of function. In vivo, mutations that disrupt SAMmediated self-association have been shown to reduce or ablate repression activity of both the PcG and the ETS proteins in a variety of cultured cell and transgenic overexpression assays (Roseman et al. 2001; Song et al. 2005; Zhang et al. 2010; Robinson et al. 2012). Genome-wide occupancy analyses of two polymerization-competent PcG proteins in Drosophila, Polycomb (Pc) and Polyhomeotic (Ph), have shown that chromatin occupancy "spreads" over regions ranging from several to hundreds of kilobases (Negre et al. 2006; Schwartz et al. 2006; Tolhuis et al. 2006). Comparable studies have not been performed yet for either human TEL1 or Drosophila Yan, and although it is widely inferred, it has not been demonstrated that SAM-mediated oligomerization drives the long-range PcG chromatin occupancy patterns. …

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