Biochemical Studies of a Transcription Factor

描述（由申请人提供）：后生动物的发育受特定的调控等级调节，导致转录网络的阶段和组织特异性表达。理解这样的网络是发育生物学的一个重大挑战。黑腹果蝇性别二态性的调节提供了一个模型。这项来自M. Weiss （CWRU）和B. Baker （Stanford）实验室的合作R01申请专注于double - esex (DSX)，这是一个新发现的DM转录因子家族的成员，广泛参与后生动物的发育，包括人类。我们的长期目标是从分子上理解信息是如何从性别的主要决定因素传递到负责两性之间形态、生化和行为差异的基因的。双性（dsx）基因控制着d.m anogaster性别决定等级的一个主要分支。作为性别特异性RNA剪接的结果，双性（dsx）基因编码性别特异性蛋白质亚型（指定为DSXF（女性）和DSXM（男性））。虽然DSXF和DSXM具有相反的生物学功能，但它们表现出相似的dna结合特性，因此可能在靶基因中识别相同的顺式作用控制元件。DSXF（但不是DSXM）与转录共激活因子Intersex （IX）相互作用，可能导致靶基因上女性特异性多蛋白- dna复合物的组装。这种性别特异性调节特性可能是dsx作为神经系统中典型“行为基因”功能的基础：DSXM调节雄性求偶歌曲的一个组成部分——正弦鸣叫，而DSXF被假设调节雌性求偶行为的主要特征，包括拒绝行为。我们试图研究DSXF和DSXM性别特异性功能的分子基础，并将其应用于行为遗传学。为此，提出了一种创新的跨学科策略，将分子遗传学与生物化学、蛋白质组学和结构生物学相结合。研究将集中在DSXF/M含有性别特异性尾巴的c端结构域。此外，新描述的体内基因靶向方法将用于研究dsx作为行为基因与离散和可量化的行为元素之间的关系。由于人类9号染色体上dsx相关基因的缺失与新生儿的性间性异常有关，我们的研究结果也可能为人类出生缺陷的主要类别提供见解。

DESCRIPTION (provided by applicant): Development in metazoans is regulated by specific regulatory hierarchies leading to the stage- and tissue-specific expression of transcriptional networks. Understanding such networks poses a major challenge in developmental biology. A model is provided by the regulation of sexual dimorphism in Drosophila melanogaster. This collaborative R01 application from the laboratories of M. Weiss (CWRU) and B. Baker (Stanford) focuses on Doublesex (DSX), a member of a newly recognized family of DM transcription factors broadly involved in metazoan development, including in humans. Our long-term goal is a molecular understanding of how information is transmitted from the primary determinants of sex to the genes responsible for the morphological, biochemical, and behavioral differences between the sexes. The doublesex (dsx) gene controls a major branch of the sex-determining hierarchy of D. melanogaster. The doublesex (dsx) gene encodes sex-specific protein isoforms (designated DSXF (female) and DSXM (male)) as a consequence of sex-specific RNA splicing. Although DSXF and DSXM have opposing biological functions, they exhibit similar DNA-binding properties and so presumably recognize the same cis- acting control elements in target genes. DSXF (but not DSXM) interacts with transcriptional coactivator Intersex (IX), presumably leading to assembly of a female-specific multiprotein-DNA complex at target genes. Such sex-specific regulatory properties presumably underlie the function of dsx as a prototypical "behavioral gene" in the nervous system: DSXM regulates sine singing, a component of the male courtship song, whereas DSXF is hypothesized to regulate major features of female courtship behavior, including rejection behavior. We seek to investigate the molecular bases of the sex-specific functions of DSXF and DSXM with application to behavioral genetics. To these ends, an innovative interdisciplinary strategy is proposed that integrates molecular genetics with biochemistry, proteomics, and structural biology. Studies will focus on the respective C-terminal domains of DSXF/M containing sex-specific tails. In addition, newly described methods for gene targeting in vivo will be employed to investigate the relationship between dsx as a behavioral gene and a discrete and quantifiable repertoire of behavioral elements. Because deletions of DSX-related genes in human chromosome 9 are associated with intersexual abnormalities of the newborn, our results may also provide insight into a major class of human birth defects. Public Health Relevance: How the differences between male and female animals are specified by genes defines a major scientific problem. We are studying sex-specific genes in fruit flies to gain insight into human birth defects, infertility syndromes, and cancers of the gonads.