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永生性。控制干细胞“自我更新”的遗传途径尚不清楚，但造血干细胞和精原干细胞的关键“主调节因子”已被描述。复层上皮（包括表皮、乳腺和前列腺）的相应主调节器仍然难以捉摸，暴露了我们对这些高度再生和易患癌症的组织的理解中的一个重大空白。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，p53家族转录因子的一员。令人不安的是，全基因组ChIP芯片数据表明，p63直接调节800多个基因，这暗示干细胞自我更新的遗传程序可能很复杂，难以分析。在这个提议中，我们提出了这样的假设，即自我更新是由不同谱系的干细胞中的共同机制控制的，尽管已知的自我更新的主调节因子是组织特异性的这一矛盾迫在眉睫。我们假设的关键是，这些主调节器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。为了确定公认的共同的遗传程序自我更新的基础上，我们建立了实验策略，在四个目标，利用上皮干细胞的独特的克隆形成特性。在目标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。为了做到这一点，我们比较了表型相似的基因表达谱，相同谱系的上皮干细胞，只是在他们的增殖历史不同。在目标2中，我们确定了Bmi-1在造血细胞中直接调控的基因，并将其与上皮干细胞中p63调控的基因进行比较，以寻找相互调控的基因子集，这些基因包括一个共同的自我更新程序。在目标3中，我们通过筛选针对由p63调节的转录因子子集的shRNA来解决相同的问题，以确定其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，用于自我更新途径中功能的个体深入分析。在目标4中，我们研究了控制p63稳定性的分子机制，因为这个功能最终控制自我更新。我们预计，这些策略将提供基本的见解推定的“共同”途径，p63和其他组织特异性的主调节器赋予干细胞再生细胞。 公共卫生相关性：对这里提出的问题的回答将揭示一般的成体干细胞和特别是上皮干细胞的治疗用途的关键信息。鉴于上皮组织导致了大多数人类癌症，这项工作也应该有助于揭示p63依赖的再生特性如何促进肿瘤发生的过程。

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.