Molecular analysis of TEF-1 family transcription factors involved in cardiovascular development and function

TEF-1转录因子家族包括Tead 1/TEF-1、Tead 2/ETF、Tead 3/ETFR-1和Tead 4/ETFR-2四个成员，含有高度保守的TEA DNA结合结构域，并与心血管发育和功能有关。最近有报道说，扇贝（Sd）蛋白，TEF-1的果蝇同源物，通过蛋白质-蛋白质相互作用与Vestigial（Vg）控制参与翅膀发育的基因表达。我们通过RTPCR和RACE方法分离了一个小鼠编码与Vg的Sd结合结构域（SdBD）相似的氨基酸序列的cDNA。从cDNA推导的蛋白质，称为小鼠Vg相关因子（mVRF），由307个氨基酸残基组成。mVRF的氨基酸序列与最近鉴定的人类Vg对应物Tondu的氨基酸序列具有49%的同源性，但除SdBD外，与果蝇Vg的氨基酸序列存在差异。北方杂交结果显示mVRF mRNA在胎盘、胚胎和睾丸中均有表达。使用荧光素酶报告基因和mVRF表达载体的瞬时转染试验表明，mVRF的过表达抑制TEF-1依赖的转录活性。另一方面，我们已经鉴定并表征了在表达ETF/Tead 2基因的细胞（例如P19细胞）中转录激活所需的小鼠ETF/Tead 2基因的第一内含子中的细胞特异性117-bp增强子序列。我们还确定了一个GC丰富的序列（GC盒）和两个串联重复的GA丰富的序列（近端和远端GA元件）的增强子，并表征其可能的结合蛋白，这可能在细胞特异性增强子功能中发挥作用。

The TEF-1 family of transcription factors consists of at least four members, Tead1/TEF-1, Tead2/ETF, Tead3/ETFR-1 and Tead4/ETFR-2, containing a highly conserved TEA DNA-binding domain, and have been implicated in cardiovascular development and function. It has recently been reported that the scalloped (Sd) protein, a Drosophila homologue of TEF-1, controls the gene expression involved in the wing development through protein-protein interaction with Vestigial (Vg). We have isolated a mouse cDNA encoding amino acid sequences similar to those of the Sd-binding domain (SdBD) of Vg by RTPCR and RACE methods. The protein deduced from the cDNA, termed mouse Vg-related Factor (mVRF), consists of 307 amino acid residues. The amino acid sequence of mVRF showed 49 % identity to that of Tondu, a recently identified human counterpart of Vg, but diverged from that of Drosophila Vg except for that of SdBD. Northern blot analysis revealed that mVRF mRNA expressed in placenta, embryo and testis. Transient transfection assay using the luciferase reporter gene and mVRF expression vector suggested that overexpression of mVRF represses the TEF-1-dependent transcriptional activity. On the other hand, we have identified and characterized the cell-specific 117-bp enhancer sequence in the first intron of the mouse ETF/Tead2 gene required for transcriptional activation in ETF/Tead2 gene expressing cells, such as P19 cells. We have also identified one GC-rich sequence (the GC box) and two tandemly repeated GA-rich sequences (the proximal and distal GA elements) in the enhancer and characterized their possible binding proteins, which may play a role in the cell-specific enhancer function.