Epithelial Stem Cell Programs

描述（由申请人提供）：成体干细胞具有巨大的内在增殖能力，但避免了与肿瘤生长相关的长生不老。在干细胞中控制这种“自我更新”的遗传途径尚不清楚，但这种程序的关键“主调节器”已被描述为造血和精原干细胞。层状上皮（包括表皮、乳腺和前列腺）的相应主调控因子仍然难以捉摸，这暴露了我们对这些高度再生和易患癌症的组织的理解存在重大差距。然而，最近的数据确定这种缺失的上皮干细胞调节因子为p63，它是p53转录因子家族的一员。令人不安的是，全基因组chip - in -chip数据表明，p63直接调节超过800个基因，这暗示着干细胞自我更新的遗传程序可能是复杂的，并且对分析具有抗性。在这一建议中，我们提出了一个假设，即自我更新是由不同谱系的干细胞的共同机制控制的，尽管已知的自我更新的主要调节因子是组织特异性的这一矛盾正在逼近。我们假设的关键是，这些主要的调节因子必须协调三个遗传程序来维持干细胞，其中两个决定了它们的组织特异性。因此，除了控制自我更新程序外，这些主调节器还需要驱动与干细胞生态位的相互作用，以及阻止组织特异性分化程序。为了确定潜在的自我更新的共同遗传程序，我们在四个目标中建立了利用上皮干细胞独特的克隆特性的实验策略。在Aim 1中，我们通过“掩盖”p63的组织特异性作用来识别参与自我更新的基因。为此，我们比较了表型相似的同一谱系上皮干细胞的基因表达谱，这些细胞仅在增殖史上有所不同。在Aim 2中，我们鉴定了造血细胞中由Bmi-1直接调节的基因，并将这些基因与上皮干细胞中由p63调节的基因进行比较，以寻找相互调节的基因亚群，这些基因亚群包含一个共同的自我更新程序。在Aim 3中，我们通过筛选针对p63调节的转录因子子集的shrna来解决相同的问题，以了解它们在复制时间内的代表性。我们的目标是使用目标1、2和3中的所有三个数据集来生成候选基因的有限列表，以深入分析自我更新途径中的功能。在Aim 4中，我们研究了控制p63稳定性的分子机制，因为这一特征最终控制了自我更新。我们预计这些策略将为p63和其他组织特异性主调控因子赋予再生细胞干性的假定“共同”途径提供基本见解。

DESCRIPTION (provided by applicant): Adult stem cells possess an immense, intrinsic capacity for proliferation and yet avoid the immortality associated with neoplastic growth. The genetic pathways that control this "self-renewal" in stem cells are unknown, but key, "master regulators" of such programs have been described for hematopoietic and spermatogonial stem cells. A corresponding master regulator of stratified epithelia, including epidermis, breast, and prostate, had remained elusive, exposing a major gap in our understanding of these highly regenerative and cancer-prone tissues. However, recent data identify this missing epithelial stem cell regulator as p63, a member of the p53 family of transcription factors. Disconcertingly, whole-genome ChIp-on-chip data indicate that p63 directly regulates more than 800 genes, a hint that genetic programs underlying self-renewal in stem cells are likely complex and resistant to analysis. In this proposal, we develop the hypothesis that self- renewal is controlled by common mechanisms in stem cells of different lineages, despite the looming contradiction that the known master regulators of self-renewal are tissue-specific. Key to our hypothesis is that these master regulators must coordinate three genetic programs to maintain stem cells, two of which dictate their tissue specificity. Thus in addition to controlling a self-renewal program, these master regulators also need to drive interactions with the stem cell niche as well as forestall tissue-specific differentiation programs. To identify the putative common genetic program underlying self-renewal, we establish experimental strategies in four aims that take advantage of the unique clonogenic properties of epithelial stem cells. In Aim 1, we identify genes involved in self-renewal by "masking" the tissue-specific roles of p63. To do this, we compare the gene expression profiles of phenotypically similar, epithelial stem cells of the same lineage that differ only in their proliferation history. In Aim 2, we identify the genes directly regulated by Bmi-1 in hematopoietic cells and compare these with those regulated by p63 in epithelial stem cells to search for mutually regulated subsets of genes that comprise a common program of self-renewal. In Aim 3, we approach this same question by screening shRNAs against a subset of transcription factors regulated by p63 for their representation over replicative time. Our goal here is to use all three datasets from Aims 1, 2, and 3 to generate a restricted list of candidate genes for individual, in-depth analysis of function in self-renewal pathways. In Aim 4, we examine the molecular mechanisms that control p63 stability as this feature ultimately controls self-renewal. We anticipate that these strategies will provide fundamental insights into the putative "common" pathways by which p63 and other, tissue-specific master regulators impart stemness to regenerative cells. PUBLIC HEALTH RELEVANCE: Answers to the questions posed here will unveil information critical to the therapeutic use of adult stem cells in general and of epithelial stem cells in particular. Given that epithelial tissues contribute to the majority of human cancers, this work should also help to unravel how p63-dependent regenerative properties contribute to the process of tumorigenesis.