Reproductive Biology and Endocrinology 2008, 6:49
HOXA7 and PBX2 bind as a heterodimer to the Pbx sequence, but not to EMX2. EMSA analysis was performed with 32P-labeled oligonucleotides containing the (A) Pbx consensus sequence and (B) EMX2 consensus sequence. Supershift assays were also performed with antibodies to HOXA7 and PBX2. Lanes are designated as follows: lane 1, negative control using excess unlabeled cold probe (100×) with nuclear extract; lane 2, 32P-labeled probe without nuclear extract, showing the migration of free probe in absence of nuclear extract; lane 3, 32P-labeled probe with nuclear extract; lane 4, supershift reaction with 32P-labeled probe, nuclear extract and anti-PBX2 antibody; lane 5, supershift reaction with 32P-labeled probe, nuclear extract and anti-HOXA7 antibody; lane 6, supershift reaction with 32P-labeled probe, nuclear extract, anti-Pbx2 and anti-HOXA7 antibodies; lane 7, 32P-labeled probe without nuclear extract but with anti-PBX2 and anti-HOXA7 antibodies. Abbreviation: n.s., non-specific complex. Ota et al. Reproductive Biology and Endocrinology 2008 6:49 doi:10.1186/1477-7827-6-49
Reproductive Biology and Endocrinology 2008, 6:49
To examine whether HOXA7 and PBX2 were present in the complexes, supershift analysis was performed using antibodies directed against the two proteins. Strikingly, the addition of anti-HOXA7 and/or anti-PBX2 antibodies caused supershifts of the complexes bound to the Pbx sequence, resulting in two bands with apparently higher molecular weights (Lanes 4, 5 and 6, Figure 4A). In contrast, whereas addition of the anti-HOXA7 antibody produced a characteristic shift in the complexes formed with the EMX2 probe, the PBX2 antibody alone did not result in a marked supershift (Lanes 4, 5, Figure 4B). These data revealed a differential recruitment of PBX2 to the Pbx and EMX2 consensus sequences. HOXA7 may bind to Pbx as a heterodimer with PBX2 and to EMX2 with other cofactor(s).
Reproductive Biology and Endocrinology 2008, 6:49
To validate the PBX2 and HOXA7 protein-protein interaction in SVOG cells, we examined their binding to two target sequences: PBX and EMX2. Previous studies showed that complexes of Pbx1 and HOX1–4 display optimal binding to the target sequence 5'-CGAATTGATTGATGCACTAATTGGAG-3' [23] and Pbx2 is also known to bind to this sequence [26]. TGAT is a Pbx binding site and TNAT is a HOX site. The TAAT, the HOX binding site which was used in this study, is accepted to bind the middle paralog groups 3–8 [29]. In addition, PBX2 and HOXA10 interactions with EMX2 were demonstrated in endometrial cancer cell lines [24]. EMX2 is expressed in the epithelial components of the urogenital system during development, and, as shown by its knockout studies, this gene is essential for the development of the female reproductive system [30]. In this study, HOXA7 and PBX2 complexes bound to the Pbx sequence, but not to the EMX2 sequence. The results indicate that a HOXA7 and PBX2 interaction occurs in granulosa cells. EMX2 can be a target of HOXA7, but it did not bind to PBX2 in granulosa cells. These results suggest that HOXA7 and PBX2 can make dimers in granulosa cells. However, when HOXA7 binds to the EMX2 promoter in granulosa cells, different cofactors might be used to enhance the HOXA7 binding specificity and strength.
