Elucidating the transcriptional network in p63 expressing mammary myoepithelium

描述（由申请人提供）：转录因子Trp 63是一种谱系特异性转录因子，在富含上皮的组织中高度表达并发挥重要作用。p63基因敲除小鼠的显著表型集中体现了这一点，这些小鼠缺乏成熟的分层表皮，并表现出多个外胚层附属物（如乳腺、毛发和牙齿）发育受阻。值得注意的是，在小鼠模型的完美表型中，Trp 63基因的显性突变在具有外胚层发育不良、口面裂和肢体畸形的各种组合的人类疾病中被发现。破译p63转录调节发育过程的潜在分子机制已被证明是相当具有挑战性的，部分原因是由于存在多种p63亚型。然而，最近的研究强烈表明，？Np 63蛋白是在上皮细胞中表达的主要同种型，在上皮细胞中它们可以作为有效的转录调节因子起作用，决定细胞的分化。 干/祖细胞更新和协调增殖和分化之间的平衡。因此，有一个新的需要确定特定的分子目标？Np 63，特别是其他发育调节转录因子，与？Np 63介导p63的某些生物学功能。但我们的知识如何？Np 63适合乳腺的整体转录回路没有得到很好的表征。 我们创造了一个新的等位基因？Np 63-GFP小鼠，其中GFP的表达是由？Np 63特异性调控元件，从而允许前瞻性隔离？富含Np 63的肌上皮/基底细胞群。此外，我们已经开发并成功地利用ChIP级抗体专门针对？Np 63同种型。这些宝贵的资源加上下一代测序的强大技术将使我们能够a）使用抗？Np 63特异性抗体和深度测序（ChIP-seq）来识别相关性？Np 63以高分辨率和深覆盖率靶向乳腺中的位点。B）分析从全局？Np 63结合位点，通过比较它们对现有的p63靶数据库，以确定关键成员的？肌上皮细胞中Np 63驱动的转录通路可能与乳腺的生物学功能相关。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？Np 63调控程序是理解其功能的分子机制和整合其功能的关键步骤。Np 63与现有的监管网络。我们的研究是在体内探测？Np 63调控的转录组在一个特定的和全球的方式将提供新的见解乳腺发育和分化的机制，这是不可能的。

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.