Regulatory architecture of the Androgen Receptor locus in development & evolution

描述(申请人提供)：雄激素受体(AR)在许多不同的哺乳动物组织中，包括软骨、骨、肌肉、脂肪、皮肤和分泌腺，对性二型表型的发育至关重要。尽管这种基因在正常发育和人类疾病中都很重要，但目前人们对AR在何时何地正常表达的机制知之甚少。AR编码区被超过1MB的基因间DNA包围。这些侧翼区域包含许多在不同哺乳动物中高度保守的短非编码序列。我用比较基因组学证明，人类AR基因侧翼区域有一个不寻常的缺失，它移除了在黑猩猩、恒河猴、狗和小鼠中高度保守的非编码序列。我在转基因小鼠中使用了功能增强子分析，以表明同源黑猩猩和小鼠的序列驱动着发育中的面部触须和生殖器结节的可复制表达，这两个解剖位置也是人类与大多数其他哺乳动物在形态上不同的地方。我的研究目的是确定这种缺失对人类生物学的意义，并确定在哺乳动物发育过程中指定AR表达的解剖分布的顺式调控机制。我将利用可诱导的Cre重组酶报告分析来进一步表征由人类缺失的AR控制区指定的表达模式和细胞谱系。为了严格测试这种保守序列丢失的功能后果，我还将使用一种靶向基因敲入策略来删除小鼠中的这一元件，并将其替换为黑猩猩的元件，从而在这个增强子区域复制人类和黑猩猩的遗传条件。最后，我还将使用细菌人工染色体(BAC)克隆和转基因小鼠的功能测试，扫描AR基因周围一个大的650kb的基因间隔区，以寻找其他组织特异性和发育增强剂。这些实验将极大地提高我们对AR在哺乳动物发育和性别分化过程中表达的机制，以及它在指定人类独特表型中所起的作用的理解。这项研究与公共健康相关，因为AR的错误表达与多种人类疾病有关，如雄激素不敏感综合征、脊柱球肌萎缩和前列腺癌。确定AR表达调控的机制可能有助于这些疾病和其他人类疾病的长期诊断和治疗。

DESCRIPTION (provided by applicant): Androgen Receptor (AR) is critical for the development of the sexually dimorphic phenotypes in many different mammalian tissues, including cartilage, bone, muscle, fat, skin, and secretory glands. Despite the importance of this gene in both normal development and human disease, little is currently known about the mechanisms that control where and when AR is normally expressed. The AR coding region is surrounded by over 1 MB of intergenic DNA. These flanking regions contain numerous short non-coding sequences that are highly conserved in different mammals. I have used comparative genomics to show that humans have an unusual deletion in the flanking regions of the AR gene, which removes non-coding sequences that are otherwise highly conserved in the chimpanzee, rhesus, dog, and mouse. I have used functional enhancer assays in transgenic mice to show that the homologous chimpanzee and mouse sequences drive reproducible expression in the developing facial vibrissae and genital tubercle, two anatomical locations, where humans are also morphologically distinct from most other mammals. The objectives of my research are to determine the significance of this deletion for human biology and to identify cis-regulatory mechanisms that specify the anatomical distribution of AR expression during mammalian development. I will utilize an inducible Cre-recombinase reporter assay to further characterize the expression pattern and cell lineages specified by the AR control region that is missing in humans. To rigorously test the functional consequence of the loss of this conserved sequence, I will also use a targeted gene knock-in strategy to delete this element in the mouse and replace it with that of the chimpanzee, thus replicating both the human and chimpanzee genetic conditions in this enhancer region. Finally, I will also scan a large 650 kb intergenic region surrounding the AR gene for other tissue specific and developmental enhancers, using bacterial artificial chromosome (BAC) clones and functional assays in transgenic mice. These experiments will greatly improve our understanding of the mechanisms by which AR expression is established during mammalian development and sexual differentiation, and the role it plays in specifying the unique human phenotype. This research is relevant to public health because the misexpression of AR is associated with multiple human diseases such as androgen insensitivity syndrome, spinal bulbar muscular atrophy, and prostate cancer. Identifying the mechanisms by which AR expression is regulated may aid in the long-term diagnosis and treatment of these and other human diseases.