Doctor of Philosophy, Stanford University, GENE-PHD (2009)
Michael Snyder, Postdoctoral Faculty Sponsor
Progenitor cells maintain self-renewing tissues throughout life by sustaining their capacity for proliferation while suppressing cell cycle exit and terminal differentiation. DNA methylation provides a potential epigenetic mechanism for the cellular memory needed to preserve the somatic progenitor state through repeated cell divisions. DNA methyltransferase 1 (DNMT1) maintains DNA methylation patterns after cellular replication. Although dispensable for embryonic stem cell maintenance, the role for DNMT1 in maintaining the progenitor state in constantly replenished somatic tissues, such as mammalian epidermis, is unclear. Here we show that DNMT1 is essential for epidermal progenitor cell function. DNMT1 protein was found enriched in undifferentiated cells, where it was required to retain proliferative stamina and suppress differentiation. In tissue, DNMT1 depletion led to exit from the progenitor cell compartment, premature differentiation and eventual tissue loss. Genome-wide analysis showed that a significant portion of epidermal differentiation gene promoters were methylated in self-renewing conditions but were subsequently demethylated during differentiation. Furthermore, UHRF1 (refs 9, 10), a component of the DNA methylation machinery that targets DNMT1 to hemi-methylated DNA, is also necessary to suppress premature differentiation and sustain proliferation. In contrast, Gadd45A and B, which promote active DNA demethylation, are required for full epidermal differentiation gene induction. These data demonstrate that proteins involved in the dynamic regulation of DNA methylation patterns are required for progenitor maintenance and self-renewal in mammalian somatic tissue.
View details for DOI 10.1038/nature08683
View details for Web of Science ID 000273981100056
View details for PubMedID 20081831
To elucidate mechanisms of cancer progression, we generated inducible human neoplasia in three-dimensionally intact epithelial tissue. Gene expression profiling of both epithelia and stroma at specific time points during tumor progression revealed sequential enrichment of genes mediating discrete biologic functions in each tissue compartment. A core cancer progression signature was distilled using the increased signaling specificity of downstream oncogene effectors and subjected to network modeling. Network topology predicted that tumor development depends on specific extracellular matrix-interacting network hubs. Blockade of one such hub, the beta1 integrin subunit, disrupted network gene expression and attenuated tumorigenesis in vivo. Thus, integrating network modeling and temporal gene expression analysis of inducible human neoplasia provides an approach to prioritize and characterize genes functioning in cancer progression.
View details for DOI 10.1016/j.ccr.2009.04.002
View details for Web of Science ID 000266686500006
View details for PubMedID 19477427
Ras proteins are membrane-bound GTPases that play a central role in transmitting signals from the cell surface to the nucleus and affect a wide array of biological processes. The overall cellular response to Ras activation varies with cell type, experimental conditions, signal strength, and signal duration. Most current studies, however, rely on expression of constitutively active protein to study Ras function and thus ignore temporal variables, as well as signal strength. These experiments may provide contradictory results, as seen in the case of epidermal keratinocytes. In this setting, Ras has been shown to both promote and oppose proliferation and differentiation. By providing control over timing, duration, and signal magnitude, conditional systems allow for more precise investigation of the role of Ras in carcinogenesis, as well as normal cellular physiology. This chapter focuses on use of a ligand-responsive steroid hormone receptor fusion of Ras, ER-Ras, to study aspects of cellular transformation in epidermal keratinocytes.
View details for DOI 10.1016/S0076-6879(05)07054-0
View details for Web of Science ID 000237082800054
View details for PubMedID 16757362
Despite numerous attractive intracellular targets, protein therapeutics have been principally confined to the extracellular space due to the lack of a straightforward way to deliver functional polypeptides to the cell interior. Peptide sequences facilitating intracellular protein delivery have been identified; however, current strategies to apply them require problematic steps, such as generation of new in-frame fusion proteins, covalent chemical conjugation, and denaturation. We have developed a new approach to protein transfer into cells and tissues that relies on single-step decoration by cysteine-flanked, arginine-rich transporter peptides. This approach facilitated cell and tissue delivery of a variety of functional proteins, including antibodies and enzymes. Decoration with transporter peptides thus provides an attractive general means of intracellular delivery of functional proteins in vitro and in tissue.
View details for DOI 10.1016/j.ymthe.2004.02.004
View details for Web of Science ID 000221288900011
View details for PubMedID 15120333