Biochemical Studies of a Transcription Factor

描述（由申请人提供）：后生动物的发育受到特定调控层次的调节，导致转录网络的阶段和组织特异性表达。理解此类网络对发育生物学提出了重大挑战。果蝇性二态性的调节提供了一个模型。 M. Weiss (CWRU) 和 B. Baker (斯坦福) 实验室的 R01 合作应用重点关注 Doublesex (DSX)，它是新近认识的 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角度理解信息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。双性（dsx）基因控制黑腹果蝇性别决定等级的一个主要分支。 doublesex (dsx) 基因编码性别特异性蛋白质亚型（称为 DSXF（女性）和 DSXM（男性）），作为性别特异性 RNA 剪接的结果。尽管 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，因此可能识别靶基因中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，可能导致在靶基因处组装女性特异性多蛋白-DNA 复合物。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 调节正弦歌唱，这是雄性求爱歌曲的组成部分，而 DSXF 被认为调节雌性求爱行为的主要特征，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。研究将集中于包含性别特异性尾部的 DSXF/M 各自的 C 末端结构域。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且可量化的行为元素库之间的关系。由于人类第 9 号染色体中 DSX 相关基因的缺失与新生儿的两性异常有关，因此我们的结果也可能有助于深入了解一类主要的人类出生缺陷。 公共健康相关性：雄性和雌性动物之间的差异如何由基因决定，这是一个重大的科学问题。我们正在研究果蝇的性别特异性基因，以深入了解人类出生缺陷、不孕综合症和性腺癌症。

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.