Chromatin Regulation of Epithelial Progenitor Cell Self-Renewal by Pygo2

许多上皮组织，如乳腺，可以持续地自我更新 成年人的生活。在发育和再生过程中，上皮干/祖细胞池 细胞增殖和分化以产生多种细胞类型。的长期目标是 我们的研究是使用多学科方法来研究遗传学和表观遗传学。 控制这些细胞的形成、维持和分化的机制 干细胞/祖细胞。这项建议的重点是研究老鼠侏儒的作用。 2(Pygo2)基因，编码表观遗传激活剂，使用乳房干/祖细胞 细胞作为模型系统。第一个具体目标是确定关键的下游目标 乳腺干/祖细胞中的PYGO-2。干细胞/祖细胞丰富的群体将 从正常和PYGO_2缺陷动物的乳腺上皮细胞中制备 转录图谱和RT-PCR分析以鉴定PYGO-2反应基因。就地 然后进行表达、染色质免疫沉淀和报告分析 测试Pygo2是否直接激活假定的干/祖细胞特异性基因和 在这些靶基因座上，PYGO2是否增加组蛋白H3K4的三甲基化。第二 其具体目的是验证PYGO2在乳腺干/祖细胞中发挥作用的假说 通过与K4-二甲基化和三甲基化组蛋白H3(H3K4me2/3，活性)结合实现细胞自我更新 染色质标志)和组蛋白组蛋白H3 K4甲基转移酶(HMT) 复合体。H3K4me2/3和/或HMT缺失的野生型和突变型Pygo2蛋白 结合将在祖细胞扩增试验中进行测试。串联亲和纯化 并将进行质谱学蛋白质鉴定以鉴定特定的 乳腺前体细胞中的HMT及其相关因子。这一事件的意义 拟议的研究有几个方面：1)它们将有助于我们理解正常程度 上皮干/祖细胞的发育和动态平衡在基因上受到调控 和表观遗传水平；2)这些知识将为我们思考 上皮癌，因为已知癌细胞可以重新激活对发育具有重要意义的细胞 成人环境中的基因；3)关于涉及PYGO2的新的分子相互作用的知识 可能涉及上皮性疾病的治疗干预的可能靶点或 干细胞/祖细胞起源的癌症。

Many epithelial tissues such as mammary glands can self-renew continuously in adult life. During development and regeneration, a pool of epithelial stem/progenitor cells proliferate and differentiate to generate multiple cell types. The long-term goal of our research is to use a multidisciplinary approach to study both genetic and epigenetic mechanisms that control the formation, maintenance, and differentiation of these stem/progenitor cells. This proposal focuses on investigating the role of mouse pygopus 2 (Pygo2) gene, which encodes an epigenetic activator, using mammary stem/progenitor cells as a model system. The first specific aim is to identify critical downstream targets of Pygo2 in mammary stem/progenitor cells. Stem/progenitor cell-enriched populations will be prepared from mammary epithelia of normal and Pygo2-deficient animals for transcriptional profiling and RT-PCR analysis to identify Pygo2-responsive genes. In situ expression, chromatin immunoprecipitation and reporter assays will then be performed to test whether Pygo2 directly activates putative stem/progenitor cell-specific genes and whether Pygo2 increases histone H3 K4 trimetylation at these target loci. The second specific aim is to test the hypothesis that Pygo2 functions in mammary stem/progenitor cell self-renewal by binding to K4-di- and tri-methylated histone H3 (H3K4me2/3, active marks of chromatin) and SET domain histone H3 K4 methyltransferase (HMT) complexes. Wild-type and mutant Pygo2 proteins deficient in H3K4me2/3 and/or HMT binding will be tested in progenitor cell expansion assays. Tandem affinity purification and mass spectrometric protein identification will be performed to identify the specific HMT and its associating factors in mammary progenitor cells. The significance of the proposed studies are several-fold: 1) they will aid our understanding of how normal development and homeostasis of epithelial stem/progenitor cells is regulated at genetic and epigenetic levels; 2) such knowledge will provide a framework for our thinking on epithelial cancers, as cancer cells are known to re-activate developmentally important genes in an adult setting; 3) knowledge on novel molecular interactions involving Pygo2 may implicate possible targets for therapeutic intervention of epithelial diseases or cancers of a stem/progenitor cell origin.