Am J Pathol. 2008 July; 173(1): 68–76.

Figure 5 Egr-1 binds and transactivates the human phosphacan promoter. A: EMSAs with biotin-labeled OGNs carrying the putative phosphacan promoter Egr-1 site (Phos-1) were incubated with: 0, no nuclear extract; 1, nuclear extract from astrocytes; 2, nuclear extract from astrocytes plus unlabeled OGN carrying the fibronectin promoter Egr-1 site; and 3, nuclear extract from astrocytes plus the same fibronectin promoter OGN, but with a mutated Egr-1 site. The OGN with the fibronectin promoter Egr-1 site (lane 2) competes effectively with binding to Phos-1. B: EMSA using the Phos-1 OGN and nuclear extracts from astrocytes in the presence of antibodies against different Egr family members. The numbered lanes represent the following conditions: 0, no antibody; 1, anti-Egr-1; 2, anti-Egr-2; 3, anti-Egr-3; and 4, anti-Egr-4. Only the anti-Egr-1 antibody in lane 2 hinders the nuclear protein/Phos-1 DNA binding complex. C: EMSA using Phos-1 and OCT-1 OGN and nuclear extracts isolated from wild-type (WT) or Egr-1-deficient mouse brain tissue. Numbered lanes represent: 1, Phos-1 OGN incubated with nuclear extract from WT brain tissue; 2, Phos-1 OGN incubated with nuclear extract from Egr-1−/− brain tissue; 3,: OCT-1 OGN incubated with nuclear extract from WT brain; and 4, OCT-1 OGN incubated with nuclear extract from Egr-1−/− brain tissue. Nuclear extracts from Egr-1−/− brain tissue (lane 2) produce less Phos-1 shift. D: Schematic drawing of the four constructs used to study the activity of the phosphacan promoter Egr-1 binding site. Construct 1: pGL3 basic, empty vector; construct 2: contains the 402-bp promoter fragment of the phosphacan gene cloned in pGL3; construct 3: same as 2 except point mutations were introduced to destroy the Egr-1 site; and construct 4: same as 2 except for a short nucleotide deletion that removes the Egr-1 site. E: The constructs outlined in D were transfected in primary human astrocytes and cell extracts were assayed for luciferase activity 24 hours later. Bars labeled 1 to 4 represent the activities of constructs 1 to 4, respectively. Mann-Whitney rank sum test shows a statistically significant difference between construct 2 and constructs 3 and 4, P < 0.001. F: Luciferase assay in HeLa cells after transfection of: a, construct 1, empty vector; b, construct 4 lacking the Egr-1 site; c, construct 4 together with the Egr-1 expression vector; d, construct 2 including the Egr-1 site; e, construct 2 together with the Egr-1 expression vector. Mann-Whitney rank sum test shows a statistical significant difference between d and e, P < 0.001. RLU, relative luciferase units. The activity of construct 2 (bars 2 in E and d in F) in one assay was set arbitrarily as 100. Am J Pathol. 2008 July; 173(1): 77–92.

Reproductive Biology and Endocrinology 2008, 6:49

In this study, the expression of HOX cofactors, PBX1, PBX2, and MEIS1/2, were examined by using RT-PCR, immunofluorescence in cultured immortalized human granulosa (SVOG) cells. The distribution of these HOX cofactors in human ovaries was examined by immunohistochemistry. The effects of growth differentiation factor-9 (GDF-9) and follicle-stimulating hormone (FSH) on PBX2 in SVOG cells were investigated by western blot analysis. Binding activities of HOXA7 and PBX2 to the specific sequences in granulosa cells were determined by electrophoretic mobility shift assay (EMSA).

Reproductive Biology and Endocrinology 2008, 6:49

Electrophoretic Mobility-Shift Assay (EMSA) Nuclear extracts were prepared with a nuclear extract kit (Panomics, Inc., Redwood City, CA) according to the manufacturer's instructions. DNA-binding reactions were performed with equal amounts of nuclear proteins (10 μg) and 32P-labeled probes at 25°C for 10 min. The following synthetic double-strand oligonucleotide probes were used: Pbx, 5'-CGAATTGATTGATGCACTAATTGGAG-3', and EMX2, 5'-AGGAAGCTGTTTATGTGATCCCCG-3', which have been previously shown to contain the consensus recognition sequences for the HOX-Pbx complexes [23,24]. For cold competition assays, a 100-fold excess of unradiolabeled oligonucleotides was added to the reactions prior to the 32P-labeled probes. Anti-HOXA7 and anti-PBX2 antibodies used in the supershift experiments were added to the nuclear extract at 25°C for 30 min before the addition of labeled probe. Protein-DNA complexes were resolved in 5% polyacrylamide gel containing 1× TBE (Tris-borate-EDTA: 0.09 M Tris-borate and 2 mM EDTA, pH 8.0). Before loading of the samples, the gel was pre-run for 90 min at 100 V at 4°C. Electrophoresis was carried out at 120 V at 4°C. The gel was then dried under vacuum and exposed to Kodak X-OMAT AR film (Kodak, Rochester, NY).

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.

Mol Cell Biol. 2003 February; 23(3): 777–790.

Electrophoretic mobility shift assays. Nuclear and cytoplasmic extracts of primary keratinocytes were prepared according to Schreiber et al. (63) with slight modifications as previously described (53). The oligonucleotide probe used was a consensus NF-κB binding site derived from the κB element of the murineinterleukin-2 promoter (TCEdA>C) (6): 3′-GACCAAGAGGGATTTCACCCCTAAATC-5′. The following antibodies were used for the supershift electrophoretic mobility shift assay: anti-p65-NF-κB (Santa Cruz Biotechnology Inc., Santa Cruz, Calif.; A, sc-109), anti-c-Rel (Santa Cruz; N, sc-070), anti-RelB (Santa Cruz; C-19, sc-226), anti-p50-NF-κB (Santa Cruz; NLS, sc-114), and anti-p52-NF-κB (Upstate Biotechnology Inc., Lake Placid, N.Y.; 06-413).