HOXA13 AMINO-TERMINAL FUNCTIONAL DOMAINS

HOX基因编码转录因子，这些转录因子调节动物身体平面的模式。小鼠Hoxa13基因对数字和生殖道的形态发生至关重要。脊椎动物HOXA13蛋白的N端非同源结构域片段(NTD)高度保守。部分同源基因保守的NTD残基在13类HOX蛋白中共享，暗示HOX蛋白的NTDS存在古老而独特的功能活性。我们假设HOXA13的NTD中的保守残基对于转录活性和与HOXA13的正常蛋白质伙伴的相互作用至关重要。在目标I中，我们将使用一个带有细胞启动子的体外瞬时转染实验系统，我们已经证明HOXA13可以激活20倍，以测试NTD突变的HOXA13蛋白的功能。体外系统将为保守序列改变对正常转录因子功能的影响提供直接的解释。这些实验将使我们能够有效地确定保守氨基酸和多肽结构域的作用，并确定修饰对正常HOXA13活性的特定功能影响。在AIM II中，我们将使用酵母双杂交系统来分离和研究与HOXA13的三个不同结构域特异相互作用的蛋白质。结合的特异性将通过体内和体外方法进行验证，并将确定结合所必需的氨基酸。将确定目标I中描述的蛋白质改变对候选辅因子结合的影响。分离蛋白的功能评估将在体外转染实验中进行测试。在Aim III中，我们将使用ES细胞中的同源重组来修改在Aim I中已知的进化保守的HOXA13氨基酸基序，这些基序对体外特定转录活动至关重要。我们将把这些修饰的ES细胞注射到囊胚中，创造嵌合小鼠，获得突变等位基因的种系传递，并表征与这些突变相关的肢体表型。

Hox genes encode transcription factors that regulate patterning of the body plan of animals. The murine Hoxa13 gene is critical for digital and reproductive tract morphogenesis. The N-terminal, non-homeodomain segment (NTD) of vertebrate HOXA13 protein orthologues is highly conserved. A subset of the orthologue-specific conserved NTD residues are shared among paralogue group 13 HOX proteins and implies the existence of ancient and distinct functional activities for the NTDs of HOX proteins. We hypothesize that conserved residues within the NTD of HOXA13 are critical for transcriptional activity and for interactions with normal protein partners of HOXA13. In Aim I we will use an in vitro transient transfection assay system with a cellular promoter, that we have shown to be activated 20-fold by HOXA13, to test the function of NTD-mutant HOXA13 proteins. The in vitro system will provide a direct interpretation of the effect of a conserved sequence alteration on normal transcription factor function. These experiments will allow us to define the role of conserved amino acids and peptide domains efficiently, and to determine the specific functional effect of a modification on normal HOXA13 activity. In Aim II we will use the yeast two-hybrid system to isolate and study proteins specifically interacting with three different domains of HOXA13. Specificity of binding will be verified with in vivo and in vitro methods and amino acids essential for binding will be determined. The effect of the protein alterations described in Aim I on binding by the candidate cofactors will be determined. Functional assessment of isolated proteins will be tested in the in vitro transfection assay. In Aim III we will use homologous recombination in ES cells to modify evolutionarily-conserved HOXA13 amino acid motifs known from work in Aim I to be critical for specific transcriptional activities in vitro. We will inject these modified ES cells into blastocysts to create chimeric mice, obtain germline transmission of the mutant alleles, and characterize the limb phenotypes associated with these mutations.