HOXA13 AMINO-TERMINAL FUNCTIONAL DOMAINS

HOXA13 氨基末端功能域

• 批准号: 6745148
• 负责人: JEFFREY W INNIS
• 金额: $ 23.78万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6745148
• 关键词: developmental genetics; embryonic stem cell; gene targeting; genetically modified animals; histogenesis; laboratory mouse; limbs; nucleic acid sequence; protein binding; protein protein interaction; protein structure function; reproductive development; site directed mutagenesis; transcription factor; transfection; yeast two hybrid system

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.

HOX基因编码调节动物身体计划模式的转录因子。 鼠Hoxa13基因对于数字和生殖道形态发生至关重要。 脊椎动物HOXA13蛋白直系同源物的N末端，非整形域段（NTD）是高度保守的。 旁程第13个HOX蛋白中共享了直系同源特异性的保守NTD残基的子集，这意味着HOX蛋白NTD的古代和独特的功能活性存在。 我们假设HOXA13 NTD内的保守残基对于转录活性和与Hoxa13的正常蛋白质伴侣的相互作用至关重要。在目的中，我们将使用带有细胞启动子的体外瞬时转染测定系统，该系统已证明HOXA13激活了20倍，以测试NTD突变的Hoxa13蛋白的功能。 体外系统将直接解释保守序列改变对正常转录因子功能的影响。 这些实验将使我们能够有效地定义保守的氨基酸和肽结构域的作用，并确定修饰对正常HOXA13活性的特定功能效应。在AIM II中，我们将使用酵母两杂交系统分离和研究与Hoxa13的三个不同域特异性相互作用的蛋白质。 结合的特异性将通过体内和体外方法和结合必不可少的氨基酸进行验证。 将确定目标I中描述的蛋白质改变对候选辅助因子结合的影响。 分离蛋白的功能评估将在体外转染测定中进行测试。在AIM III中，我们将使用ES细胞中的同源重组来修饰从AIM I工作中知道的进化保存的HOXA13氨基酸基序，对于体外特定的转录活性至关重要。 我们将把这些修饰的ES细胞注入胚泡中，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。

项目成果

Group 13 HOX proteins interact with the MH2 domain of R-Smads and modulate Smad transcriptional activation functions independent of HOX DNA-binding capability.

• DOI: 10.1093/nar/gki761
• 发表时间: 2005
• 期刊: NUCLEIC ACIDS RESEARCH
• 影响因子: 14.9
• 作者: Williams, TM;Williams, ME;Heaton, JH;Gelehrter, TD;Innis, JW
• 通讯作者: Innis, JW

A group 13 homeodomain is neither necessary nor sufficient for posterior prevalence in the mouse limb.

• DOI: 10.1016/j.ydbio.2006.05.027
• 发表时间: 2006-09
• 期刊: Developmental biology
• 影响因子: 2.7
• 作者: Melissa E. Williams;J. Lehoczky;J. Innis

Candidate downstream regulated genes of HOX group 13 transcription factors with and without monomeric DNA binding capability.

• DOI: 10.1016/j.ydbio.2004.12.015
• 发表时间: 2005-03
• 期刊: Developmental biology
• 影响因子: 2.7
• 作者: Thomas M. Williams;Melissa E. Williams;R. Kuick;David E. Misek;K. McDonagh;S. Hanash;J. Innis