Abstract
Hox genes contain a homeobox encoding a 60βamino acid DNA binding sequence. The Hoxa1 gene (Hox1.6, ERA1) encodes two alternatively spliced mRNAs that encode distinct proteins, one with the homeodomain (Hoxa1β993), and another protein lacking this domain (Hoxa1β399). The functions of Hoxa1β399 are unknown. We detected Hoxa1β993 and Hoxa1β399 by immunoprecipitation using Hoxa1 antibodies. To assess whether Hoxa1β399 functions in cellular differentiation, we analyzed Hoxb1, a Hoxa1 target gene. Hoxa1β993 and its cofactor, Pbx1, bind to the Hoxb1 SOctβR3 promoter to transcriptionally activate a luciferase reporter. Results from F9 stem cells that stably express ectopic Hoxa1β399 (the F9β399 line) show that Hoxa1β399 reduces this transcriptional activation. Gel shift assays demonstrate that Hoxa1β399 reduces Hoxa1β993/Pbx1 binding to the Hoxb1 SOctβR3 region. GST pullβdown experiments suggest that Hoxa1β399, Hoxa1β993, and Pbx1 form a trimer. However, the F9β399 line exhibits no differences in RAβinduced proliferation arrest or endogenous Hoxb1, Pbx1, Hoxa5, Cyp26a1, GATA4, or Meis mRNA levels when compared to F9 wildβtype. J. Cell. Biochem. 106: 427β443, 2009. Β© 2008 WileyβLiss, Inc.