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的调控等级的调节，导致转录网络的阶段和组织特异性表达。理解这样的网络构成了发育生物学的一个重大挑战。通过对果蝇性二型性的调控，为果蝇的性分化提供了一个模型。M.Weiss(CWRU)和B.Baker(Stanford)实验室的这一合作R01应用程序专注于Doublesex(DSX)，它是新发现的DM转录因子家族的成员，广泛参与后生动物的发育，包括在人类中。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导致性别之间形态、生化和行为差异的基因的。双性(Dsx)基因控制着黑腹隐翅虫性别决定层级的一个主要分支。双性(Dsx)基因编码性别特异的蛋白质亚型(命名为dsxf(雌性)和dsxm(雄性))，这是性别特异的RNA剪接的结果。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，因此可能识别目标基因中相同的顺式作用调控元件。Dsxf(但不是dsxm)与转录辅活化子(IX)相互作用，推测导致在靶基因上组装女性特有的多蛋白-DNA复合体。这种性别特有的调节特性可能是作为神经系统中典型的“行为基因”的DSX功能的基础：DSXM调节男性求爱歌曲中的正弦歌声，而DSXF被假设为调节女性求爱行为的主要特征，包括拒绝行为。我们试图通过在行为遗传学中的应用来研究dsxf和dsxm的性别特异性功能的分子基础。为此，提出了一种创新的跨学科战略，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。研究将集中在含有性别特异性尾巴的DSXF/M各自的C-末端结构域。此外，新描述的体内基因打靶方法将被用来研究作为行为基因的dsx与离散的和可量化的行为元素库之间的关系。由于人类9号染色体上的dsx相关基因的缺失与新生儿的两性畸形有关，我们的结果也可能为我们提供对人类一大类出生缺陷的洞察。 公共卫生相关性：雄性和雌性动物之间的差异是如何由基因决定的，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，以深入了解人类的出生缺陷、不育综合征和性腺癌症。

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.