Elucidating the transcriptional network in p63 expressing mammary myoepithelium

描述(申请人提供)：转录因子Trp63是一种谱系特异的转录因子，在富含上皮的组织中高度表达并发挥重要作用。这一点以p63基因敲除小鼠的显著表型为代表，这些小鼠缺乏成熟的复层表皮，并阻碍了乳腺、毛发和牙齿等多个外胚层附件的发育。值得注意的是，在完美的小鼠模型表型中，Trp63基因的显性突变在人类疾病中被发现，这些疾病包括外胚层发育不良、口裂和肢体畸形的各种组合。破译p63转录调控发育过程的潜在分子机制已被证明是相当具有挑战性的，部分原因是存在多种p63亚型。然而，最近的研究强烈表明，np63蛋白是在上皮细胞中表达的主要亚型，在那里它们可以起到强大的转录调节作用。 干细胞/祖细胞更新，协调增殖和分化之间的平衡。因此，迫切需要确定Np63的特定分子靶点，特别是其他发育调控的转录因子，这些转录因子与Np63共同作用于P63的某些生物学功能。然而，我们对Np63如何与乳腺的整体转录回路相匹配的知识还没有得到很好的描述。我们已经产生了一个新的Np63-GFP小鼠等位基因，其中GFP的表达是由Np63特异的调控元件驱动的，因此可以预期分离出富含Np63的肌上皮/基础种群。此外，我们已经开发并成功利用了针对？Np63亚型的芯片级抗体。这些宝贵的资源与强大的下一代测序技术相结合，将使我们能够a)使用抗Np63特异性抗体和深度测序(CHIP-SEQ)进行芯片操作，以高分辨率和深度覆盖的方式识别乳腺中相关的Np63靶点；b)通过与现有的p63靶点数据库进行比较，分析从全球？Np63结合位点获得的结果，以确定可能与乳腺生物功能相关的肌上皮细胞中？Np63驱动的转录电路的关键成员。鉴于p63在乳腺发育中的重要作用及其与乳腺癌的密切关系，确定一个明确的？Np63调控程序是理解其功能的分子机制以及将？Np63与现有的调控网络整合的关键一步。我们以特定和全局的方式探索体内Np63调节的转录组的研究将为乳腺发育和分化的机制提供新的见解，这是以前不可能的。

DESCRIPTION (provided by applicant): The transcription factor Trp63 is a lineage-specific transcription factor that is highly expressed and plays an important role in epithelial-rich tissue. This is epitomized by the striking phenotype of the p63 knockout mice, which lack a mature stratified epidermis and exhibit a block in development of multiple ectodermal appendages such as mammary glands, hair and teeth. Remarkably, in perfect phenocopy of the mouse model, dominant mutations of the Trp63 gene are found in human disorders with various combinations of ectodermal dysplasia, orofacial clefting and limb malformations. Deciphering the underlying molecular mechanism by which p63 transcriptionally regulates developmental processes has proven to be quite challenging, in part, due to the existence of multiple p63 isoforms. However, recent studies have strongly suggested that ?Np63 proteins are the dominant isoforms expressed in epithelial cells where they can function as potent transcriptional regulators, dictate stem/progenitor cell renewal and orchestrate the balance between proliferation and differentiation. Hence, there is an emerging need for identifying specific molecular targets of ?Np63, particularly other developmentally regulated transcription factors, which act together with ?Np63 in mediating some of the biological functions of p63. However our knowledge of how ?Np63 fits into the global transcriptional circuitry of mammary glands is not well characterized. We have generated a novel allele of ?Np63-GFP mice, where the expression of GFP is driven by ?Np63-specific regulatory elements thus allowing prospective isolation of ?Np63-enriched myoepithelial/basal populations. Furthermore, we have developed and successfully utilized ChIP-grade antibodies directed specifically to the ?Np63 isoform. These valuable resources coupled with the powerful technology of next-generation sequencing will allow us to a) Perform ChIP using anti-?Np63 specific antibodies and deep sequencing (ChIP-seq) to identify relevant ?Np63 targets sites in mammary glands at high resolution and deep coverage, b) Analyze the results obtained from the global ?Np63-binding sites by comparing them against the existing p63-target database to identify key members of the ?Np63-driven transcriptional circuitry in myoepithelial cells that are likely to be relevant for the biological function of mammary glands. Given the essential role of p63 in mammary gland development and its close association with breast cancer, the identification of a well-defined ?Np63 regulatory program is a critical step in understanding the molecular mechanism of its function and in integrating ?Np63 with existing regulatory networks. Our studies to probe the in vivo ?Np63-regulated transcriptome in a specific and global fashion will provide novel insights into mechanisms of mammary gland development and differentiation that has not been possible before.