Identification Of Transcriptional Factor MEOX2 Binding Protein

Background: The proliferation of vascular smooth muscle cells (VSMCs) plays a crucial role in both the development of atherosclerosis and the restenosis that occurs following surgical procedures such as either balloon angioplasty or coronary bypass grafts. In contrast to VSMCs, cardiomyocytes do not proliferate sufficiently to completely ameliorate the damage following insults such as a myocardial infarction. Therefore, reducing excessive proliferation of VSMCs in atherosclerotic vascular disease and promoting cardiomyocyte proliferation in ischemic heart disease represent novel approaches to the treatment of cardiovascular diseases.Homeobox genes encode transcription factors that contain an evolutionarily conserved 60 amino acid helix-turn-helix DNA binding region called the homeodomain. Homoeobox genes have been shown to regulate cellular growth and differentiation in developing embryos and during disease progression . Previous studies have shown that the overexpression of Mesenchyme Homeobox 2 (Meox2) gene induced cell cycle arrest of VSMCs and cardiomyocytes. As well ectopic Meox2 expression reduced the amount of restenosis that occurred following vascular injury in rodent models of balloon angioplasty by inducing both cell cycle arrest and apoptosis. These findings support a role for Meox2 in regulating the pathological growth of VSMCs. The molecular mechanisms by which Meox2 regulates the proliferation, differentiation and migration of vascular smooth muscle cells and cardiomyocytes are not known. Discovery of the partners with which the MEOX2 protein associates, is essential to gain insight into the molecular mechanism of how MEOX2 regulates the growth of both cardiomyocytes and VSMCs.Objective: The aim of this experiment is to identify the ME0X2 bingding protein, and the binding region, furthermore, to investigate the binding proteins have the function on regulating the transcriptional activity of ME0X2 or not.Methods: (1) Construction of the "bait" plasmids. A cassette encoding the Meox2 protein with its histidine/glutamine (H/Q) rich domain deleted (Meox2AHQ) was PCR amplified and then cloned into pGBKT7 to gain the pGBKT7-Meox2AHQ bait plasmid. Similarly, the pGBKT7-Meox2, pGBKT7-Meox2AHD, pGBKT7-Meox2-Nterm, pGBKT7-Meox2-Cterm, pGBKT7-Meox2-mid, and pGBKT7-Meoxl bait plasmid were cloned by using the different primer. (2) Texting the auto-activation of reporter genes by the bait. AH 109 yeast cells transformed with the different bait plasmids were plated on different synthetic dropout (SD) plates, SD/-Trp/X-a-GAL, SD/-Trp/-His and SD/-Trp/-Ade/X-a-GAL, to determine whether the baits themselves auto-activated the following reporter genes Ade2, His3, and Mell respectively. 3-Amino-l, 2, 4-triazole (3-AT) was added to the dropout media to inhibit low endogenous levels of His3 expression. To optimize the 3-AT concentration, cells transformed with the different bait plasmids were plated on the SD/-His/-Trp plates containing 0, 2.5, 5, 7.5, 10, 12.5, 15, and 40 mM concentrations of 3-AT, then incubated at 30 °C for two week. P-Galactosidase filter lift assays were also performed to determine the level of activation of the LacZ gene by the different bait plasmids. (3) Screening a human heart cDNA library. The AH 109 transformed with the pGBKT7-Meox2AHQ were combined with the human heart cDNA library and incubated at 30°C with shaking at 30 rpm for 24 hours. Cells were pelleted and resuspended. Onto each 150-mm SD/-Trp/-Leu/-His/-Ade (Quadruple Dropout, QDO) plate, 200 ul of the mating mixture was spread and the plates were incubated at 30°C for up to 14 days. The plasmids isolated from the positive clone were sequenced. (4) Pull-down assay. Meox2 was cloned into the pMAL-c2x vector inframe with the maltose-binding protein (MBP). Recombinant MBP and MBP-MEOX2-FLAG proteins were purified from the TBl transformed with either the pMAL-c2x or pMAL-c2-Meox2-FLAG plasmids respectively. The full length cDNA encoding RING Finger protein 10 (RNF10) was cloned into pCMVTag4Ainframe with the FLAG epitope. MBP and MBP-ME0X2-FLAG proteins were mixed with 50 \x\ of amylose resin, then the lysate from 3T3 cells transfected with pCMVTag4A or pCMVTag4A-RNF10-FLAG was added and the slurry was mixed for a further two hours. The resin was pelleted and the bound proteins were eluted with D-Maltose. The western blot was performed by using the anti-FLAG M2 monoclonal antibody. (5) Immunoprecipitation. NIH 3T3 cells were transfected with different combinations of pCMVTag4A, pCMVTag5A, pCMVTag4A-RNF10-FLAG, pCMVTag5A-Meox2 by using Lipofectamine 2000. The cells were homogenized in lysis buffer. 3 mg of cellular proteins were incubated with 40ul of anti-FLAG M2 agarose affinity gel overnight. The beads were pelleted, then the 3 * FLAG peptide was added to elute the bound proteins from the beads. Western blotting was performed by using an anti-Myc monoclonal antibody 9B11. (6) Luciferase assays. NIH 3T3 cells were transfected with 2 ug pGL3-p21wafl reporter plasmid, 1 ug pCMV-p-gal and 3 ug pCMV-Tag4A, or pC-RNF10-FLAG, or pC-Meox2-FLAG, or co-transfected with Meox2 and RNF10 using Lipofectamine 2000. Cells were harvested 24 h after transfection and luciferase and P-Gal assays were performed on the cell lysates. The luciferase levels were divided by the P-Gal levels to normalize for transfection efficiency. The data was analyzed using the Student's t-test.Results: (1) Transactivation potential of the different MEOX bait proteins. On SD/-His/-Trp plates containing 5 mM 3-AT, only pGBKT7-Meoxl and pGBKT7-Meox2 transformed yeast grew whereas the yeast transformed with pGBKT7, pGBKT7-Meox2AHQ, pGBKT7-Meox2AHD did not grow at all. pGBKT7-Meox2 yeast grew very poorly at the 10 mM concentration of 3-AT. pGBKT7-Meoxl transformed yeast grew well at 40 mM 3-AT. All of the transformed AH 109 grew on SD/-Trp/X-a-GAL plates however, only the positive control and the pGBKT7-Meoxl transformed yeast turned blue. On the SD/-Trp/-Ade/X-a-GAL plates, only the positive control and the AH 109 yeast transformed with pGBKT7-Meoxl grew and both of them turned blue. p-Galactosidase assays demonstrated that the MEOXl bait protein, but not MEOX2, activated expression of the LacZ gene as well. (2) Isolation of MEOX2 binding proteins. After screening...