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.
Reproductive Biology and Endocrinology 2008, 6:28
Statistical analysis Data were compared by analysis of variance followed by Duncan's test for multi-group comparison and Student t test for between-group comparison. All data were expressed as mean ± standard deviations. The level of significance was taken at P < 0.01 and P < 0.05.
J. Clin. Invest. 118(1): 51-63 (2007)
Tumor escape in a Wnt1-dependent mouse breast cancer model is enabled by p19Arf/p53 pathway lesions but not p16Ink4a loss J. Clin. Invest. Michael T. Debies, et al. 118:51 doi:10.1172/JCI33320 [Go to this article.] Figure 1Mechanisms of mammary tumor relapse in MTB/TWNT mice. (A) Mammary expression of putative Wnt pathway target genes. Northern hybridization analyses are shown. Mammary gland RNA came from 5-week-old virgin female MTB/TWNT mice that were either Dox naive (Off Dox) or treated with Dox for 96 hours (On Dox). Tumor RNA came from clonally related outgrowths derived from a Dox-dependent MTB/TWNT tumor that was explanted onto the flanks of Dox-treated host mice. Paired flank explants were harvested during ongoing Dox treatment or after timed Dox withdrawal. (B) Tumor gene expression patterns. Northern hybridization analysis was performed on RNA samples from primary and relapsed MTB/TWNT mammary tumors. Three relapsed DITs expressed Wnt1 transgene in an inducer-independent manner (lanes marked T), and 3 expressed aberrant transcripts encoding activated β-catenin variants (β). (C) Molecular genetic analysis of the Ctnnb1 (β-catenin) gene. Segments of transcripts encoding the regulatory domain of β-catenin were amplified from tumor-derived RNA via RT-PCR and subjected to DNA sequencing. The coding region of mouse β-catenin is shown schematically, with arrows indicating the primers used for RT-PCR placed relative to their approximate annealing sites along the open reading frame. The blowup depicts critical aa residues encoded within exon 3; known hot spots for cancer-associated aa substitutions are in b. Asterisks denote the residue affected by the S33Y mutation identified in 2 DITs. The upper chromatograms show detection of only the wild-type β-catenin allele in an antecedent primary tumor but additional detection of the S33Y allele in a descendant recurrent tumor. The lower panels depict RT-PCR–based detection of an aberrantly spliced β-catenin transcript lacking exon 3 within a relapsed tumor (R) and not within the antecedent primary tumor (P). (D) Tumor histology. Photomicrographs of H&E-stained sections derived from representative primary-relapse tumor pairs. The mode of tumor escape identified for each relapse is indicated. Scale bar: 50 μm.
