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

描述（由申请人提供）：角质形成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无可争议的作用，并且是遗传性和获得性皮肤病的重要参与者。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。我们采用了一种新的基因鉴定策略，利用了我们实验室最近取得的几项进展，包括 1) 对角质形成细胞干细胞进行特别敏感和定量的体外测定，2) 发现这些集落的数量和大小是定量的多基因性状，3) 对与形成最大集落的大量干细胞相关的基因座进行了基因定位。这项拟议研究的目的是鉴定小鼠 4 号染色体上我们命名为角质形成细胞干细胞基因座 2 (Ksc2) 的基因座中的基因或基因簇。我们的假设是，该基因座中的一个或多个基因调节形成最大角质形成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将我们之前确定的 Ksc2 基因座的连锁间隔从约 10 cM 缩小到约 0.5 cM。在具体目标 2 中，我们将使用数据库挖掘和基于实验室的方法，使用候选基因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性过度表达或表达不足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、交互方法，我们期望识别和表征角质形成细胞干细胞形成最大集落的新调控基因，从而形成最高的增殖潜力。该项目的意义在于，Ksc2 基因座中的基因或基因簇，因为它显示出与大量最大菌落的连锁，可能是皮肤癌治疗和预防的令人惊讶和意想不到的靶标。我们提出的方法代表了强大的遗传学方法的新颖应用，以全新的方式应用于全新的表型：角质形成细胞集落的数量和大小。我们之前发现集落数反映了角质形成细胞干细胞增殖潜力的多少。这项拟议的研究将影响我们对上皮干细胞调节的理解。该项目不仅对了解毛囊和表皮稳态具有基础科学影响，还可能对皮肤癌、增生性皮肤病以及干细胞功能下降的潜在病症的诊断、预防和治疗产生临床影响。

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.