Trithorax and ASH1 interact directly and associate with the trithorax group-responsive bxd region of the Ultrabithorax promoter

Trithorax (TRX) and ASH1 belong to the trithorax group (trxG) of transcript ional activator proteins, which maintains homeotic gene expression during D rosophila development. TRX and ASH1 are localized on chromosomes and share several homologous domains with other chromatin-associated proteins, includ ing a highly conserved SET domain and PHD fingers. Based on genetic interac tions between trx and ash1 and our previous observation that association of the TRX protein with polytene chromosomes is ash1 dependent, we investigat ed the possibility of a physical linkage between the two proteins. We found that the endogenous TRX and ASH1 proteins coimmunoprecipitate from embryon ic extracts and colocalize on salivary gland polytene chromosomes. Furtherm ore, we demonstrated that TRX and ASH1 bind in vivo to a relatively small ( 4 kb) bxd subregion of the homeotic gene Ultrabithorax (Ubx), which contain s several trx response elements. Analysis of the effects of ash1 mutations on the activity of this regulatory region indicates that it also contains a sh1 response element(s). This suggests that ASH1 and TRX act on Ubx in rela tively close proximity to each other. Finally, TRX and ASH1 appear to inter act directly through their conserved SET domains, based on binding assays i n vitro and in yeast and on coimmunoprecipitation assays with embryo extrac ts. Collectively, these results suggest that TRX and ASH1 are components th at interact either within trxG protein complexes or between complexes that act in close proximity on regulatory DNA to maintain Ubx transcription.