HOXA13 AMINO-TERMINAL FUNCTIONAL DOMAINS

Hox基因编码的转录因子调节动物的身体计划的模式。 小鼠Hoxa13基因对指和生殖道形态发生至关重要。 脊椎动物HOXA 13蛋白同源物的N-末端非同源结构域片段（NTD）是高度保守的。 同源物特异性保守NTD残基的一个子集是共享的parabolic组13 HOX蛋白，并意味着存在古老的和独特的功能活动的NTD的HOX蛋白。 我们推测，保守的残基内的NTD的HOXA 13的转录活性和与正常蛋白质的HOXA 13的合作伙伴的相互作用是至关重要的。在目的I中，我们将使用具有细胞启动子的体外瞬时转染测定系统来测试NTD突变HOXA 13蛋白的功能，我们已经证明该细胞启动子被HOXA 13激活20倍。 体外系统将提供一个保守的序列改变对正常转录因子功能的影响的直接解释。 这些实验将使我们能够有效地定义保守氨基酸和肽结构域的作用，并确定修饰对正常HOXA 13活性的特定功能效应。在目的II中，我们将使用酵母双杂交系统分离和研究蛋白质特异性地与HOXA 13的三个不同结构域相互作用。 将采用体内和体外方法验证结合特异性，并测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。 将在体外转染试验中检测分离蛋白的功能评估。在目标III中，我们将在ES细胞中使用同源重组来修饰目标I中已知的进化保守的HOXA 13氨基酸基序，这些基序对体外特异性转录活性至关重要。 我们将这些修饰的ES细胞注射到胚泡中以产生嵌合小鼠，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.