Identification of a Keratinocyte Stem Cell Regulatory Gene in the KSC2 Locus

描述（由申请人提供）：角化细胞干细胞在维持表皮和毛囊的正常结构和功能方面具有毋庸置疑的作用，并且在遗传性和获得性皮肤病中发挥重要作用。因此，鉴定调节其数量和增殖潜力的基因是皮肤生物学中的一个关键问题。利用我们实验室最近取得的几项进展，我们采取了一种新的基因鉴定策略，包括1)对角质细胞干细胞进行特别敏感和定量的体外检测，2)发现这些集落的数量和大小是定量的多基因特征，以及3)基因定位与形成最大集落的大量干细胞相关的位点。本研究的目的是在小鼠4号染色体上我们命名为角质细胞干细胞2号位点（Ksc2）的位点上鉴定一个基因或基因簇。我们的假设是，这个位点上的一个基因（或多个基因）调节形成最大角质细胞集落的干细胞的数量。具体目标1的重点是使用额外的遗传和统计工具来缩小我们之前确定的Ksc2位点的连锁间隔，从大约10厘米到大约0.5厘米。在Specific Aim 2中，我们将使用候选基因方法，使用数据库挖掘和基于实验室的方法进行干细胞基因鉴定。在目标3中，我们将使用互补基因表达谱来识别差异过表达或过表达的基因以及相关的分子途径，并评估导致基因表达差异的调控多态性。利用这三个目标的组合，相互作用的方法，我们期望识别和表征角质形成细胞干细胞形成最大菌落的新调控基因，因此，最高的增殖潜力。这个项目的意义在于，Ksc2位点上的基因或基因簇，因为它显示出与大量最大菌落的连锁，可能是一个令人惊讶和意想不到的皮肤癌治疗和预防的靶点。我们提出的方法代表了一种强大的遗传学方法的新应用，以一种全新的方式应用于一种全新的表型：角质形成细胞菌落的数量和大小。我们之前发现，集落数量反映了角质形成细胞干细胞增殖潜能的数量。这项拟议的研究将影响我们对上皮干细胞调控的理解。该项目不仅对了解毛囊和表皮稳态具有基础科学意义，而且可能对皮肤癌、增生性皮肤病以及干细胞功能下降的潜在状况的诊断、预防和治疗具有临床意义。

DESCRIPTION (provided by applicant): Keratinocyte stem cells have an unquestioned role in maintaining the normal structure and function of the epidermis and hair follicles and are important players in inherited and acquired skin diseases. Therefore, identification of genes regulating their numbers and proliferative potential is a critical problem in cutaneous biology. We have taken a novel strategy for gene identification taking advantage of several recent advances made by our laboratory including 1) a particularly sensitive and quantitative in vitro assay for keratinocyte stem cells, 2) discovering that the number and size of those colonies are quantitative multigenic traits, and 3) genetically mapping a locus linked to a high number of stem cells forming the largest colonies. The objective of this proposed research is to identify a gene or gene cluster in a locus we have named Keratinocyte stem cell locus 2 (Ksc2) on mouse chromosome 4. Our hypothesis is that a gene (or genes) in this locus regulates the number of stem cells forming the largest keratinocyte colonies. The focus of Specific Aim 1 is to use additional genetic and statistical tools to narrow the linkage interval of the Ksc2 locus that we have previously identified from approximately 10 cM to approximately 0.5 cM. In Specific Aim 2, we will use a candidate gene approach for stem cell gene identification using database mining and lab-based methods. In Aim 3, we will use complementary gene expression profiling to identify differentially over- or under-expressed genes as well as associated molecular pathways, and to assess regulatory polymorphisms causing differences in gene expression. Using the combined, interactive approaches of these three aims, we expect to identify and characterize a new regulatory gene for the keratinocyte stem cells forming the largest colonies, and hence, the highest proliferative potential. The significance of this project is that the gene or gene cluster in the Ksc2 locus, because it shows linkage with a high number of the largest colonies, may be a surprising and unanticipated target for skin cancer therapy and prevention. The approach we present represents a novel application of powerful genetics methods applied in a completely new way to a completely new phenotype: the number and size of keratinocyte colonies. We previously found that colony number reflected the number of proliferative potential of keratinocyte stem cells. This proposed research will impact our understanding of epithelial stem cell regulation. This project not only has basic science ramifications for understanding hair follicle and epidermal homeostasis, it may have clinical impact for the diagnosis, prevention, and treatment of skin cancer, hyperplastic skin disease, as well as potential conditions of declining stem cell function.