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.
The proliferation and differentiation of a stem/progenitor cell is important for building a tissue with the correct final size and cell compositions. Defective proliferation during development and regeneration leads to undergrowth, reduced tissue size, and possibly birth defects. Excessive proliferation of stem cells depletes a stem cell reserve and may cause premature aging. Unstoppable stem/progenitor cell proliferation results in an inability to initiate terminal differentiation and consequently tissue dysfunction, as well as abnormal growth and consequently cancer. The proposed studies will help us understand the genetic and epigenetic determinants of the expansive self-renewal of epithelial stem/progenitor cells. They may provide fundamental knowledge necessary to understand human birth defects and/or to design successful stem cell therapy for tissue regeneration. Moreover, they will likely implicate novel molecular targets for treating cancers, particularly those of a stem/progenitor cell origin. Current cancer treatment is directed towards the tumor mass, while the putative cancer stem cells are thought, and known in some cases, to evade available therapeutic interventions. A thorough understanding of the genetic and epigenetic control of normal epithelial stem/progenitor cells will shed light on the regulation of cancerous stem cells and provide clues for us to find ways to efficiently target the latter. The work in the end will generate two deliverables that will ultimately benefit cancer patients: 1) new putative biomarkers of cancer stem cells, 2) critical molecular interactions that may serve as targets for developing new therapeutics of stem/progenitor cell-derived cancers that involve activated Wnt target gene expression.
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