Elucidating the transcriptional network in p63 expressing mammary myoepithelium

描述（由申请人提供）：转录因子Trp63是一种高表达的谱系特异性转录因子，在富上皮组织中起重要作用。p63基因敲除小鼠的显著表型体现了这一点，它们缺乏成熟的分层表皮，并且在多个外胚层附属物（如乳腺、毛发和牙齿）的发育中表现出阻滞。值得注意的是，在小鼠模型的完美表型中，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、口面裂和肢体畸形的各种组合。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，部分原因是存在多种p63亚型。然而，最近的研究强烈表明？Np63蛋白是上皮细胞中表达的主要亚型，在上皮细胞中它们可以作为有效的转录调节因子发挥作用

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.