Abstract 1060: Identification of a Novel Downstream Target for TBX5 Regulation of Cardiac Development
A prominent member of the T-box transcription factor family, TBX5, plays an important role in cardiogenesis. It is expressed in the myocardium of the fetal and adult human heart. Previously, we used a retroviral strategy to overexpress wildtype (wt) and mutant isoforms of TBX5 in vitro in a myc-transformed embryonic quail cardiomyocyte-like cell line (MEQC) and in vivo in the embryonic chick heart. Overexpression of wt-TBX5 leads to non-cell autonomous inhibition of cell proliferation in vitro and in vivo. The molecular mechanism by which TBX5 controls myocardial development through regulation of cell proliferation is being elucidated. We now propose a new TBX5 downstream target gene that may play a role in this process. We observed increased expression of a histone deacetylase (HDAC) family member, HDAC2, in vitro in wt-TBX5-MEQCs. Expression of HDAC2 was also increased in developing chick hearts expressing human wt-TBX5 in vivo. These chromatin-modifying enzymes have been implicated in a number of cellular processes, including regulation of cardiogenesis and cell proliferation. Bioinformatic analyses of human genomic DNA upstream of the HDAC2 translation initiation site revealed several putative T-box binding elements (TBEs) required for recruiting TBX5 to this promoter. We now demonstrate that a minimal region of the putative HDAC2 promoter is required for transcriptional activation by TBX5. TBX5 binds directly to at least 6 TBEs within the minimal HDAC2 promoter. In addition, TBX5 synergistically activates transcription of HDAC2 in concert with its partner proteins, GATA4 and NKX2.5. Electrophoretic mobility shift assays show that these factors transcriptionally activate HDAC2 by binding directly to at least 1 GATA and 2 NK binding elements within the minimal HDAC2 promoter. Chromatin immunoprecipitation was performed on lysates of cells overexpressing wt-TBX5 with a specific antibody recognizing TBX5. We now demonstrate recruitment of TBX5 to several T-box binding sites within the HDAC2 promoter. Specificity of this interaction was confirmed by the lack of detection of TBX5 recruitment to the HDAC2 promoter by a non-immune IgG. Our study provides evidence for a novel mechanism by which TBX5 enlists HDAC2 to participate in cardiac development.