2005 — 2007 |
Tittle, Rachel K. |
F31Activity Code Description: To provide predoctoral individuals with supervised research training in specified health and health-related areas leading toward the research degree (e.g., Ph.D.). |
Mechanism of Zinc Potentiation of P2x Receptors @ University of Michigan At Ann Arbor
DESCRIPTION (provided by applicant): P2X receptors are cation channels gated by extra cellular ATP. Defects in signaling by these channels result in deficits in pain perception, male fertility, and sound transduction. Homomeric channels made up of the P2X2 subunit show potentiation of ATP-induced current when extra cellular zinc is applied. In P2X2, extra cellular histidines 120 and 213 are independently required for zinc potentiation. It is hypothesized that these residues directly participate in a zinc binding site, and that when zinc is bound, the distance between these two residues changes such that the open state of the ion channel is stabilized. To test this hypothesis, a search will be made for other residues potentially involved in binding zinc. Additionally, residues 120 and 213 will be blocked by the binding of a small molecule, and the amount of zinc potentiation will be measured. Based on the hypothesis, it is expected that zinc potentiation will decrease. Finally, residues 120 and 213 will be cross-linked with various molecules. It is expected that rigid cross linkers of certain size will yield channels that are permanently either in the potentiated or non-potentiated state, regardless of the presence of zinc.
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1 |
2010 — 2012 |
Tittle, Rachel K. |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
The Role of Dna Methylation in Lens Fiber Cell Terminal Differentiation @ University of Texas, Austin
DESCRIPTION (provided by applicant): Methylation of DNA cytosine residues results in epigenetic transcriptional repression, and aberrant methylation plays a role in human cancers. During development, DNA methylation is a mechanism whereby cell-type specific gene expression patterns are set during terminal differentiation. However, the specific role of DNA methylation in terminal differentiation and organogenesis has so far been studied in very few cell types. This is due in part to the early embryonic lethality of knockout mice lacking genes required for DNA methylation. Unlike mouse models, zebrafish with mutations in two key epigenetic regulators, DNA Methyltransferase 1 (dnmt1) and Ubiquitin-like, Containing PHD and RING Finger Domains 1 (uhrf1), survive to late embryonic stages, at which time many complex organs (including the eye) have formed. I have taken advantage of these mutant zebrafish lines to study the role of DNA methylation in development of the vertebrate lens. Consisting of only two cell types: proliferative epithelial cells and terminally differentiated lens fibers, the lens is an ideal tissue in which to study gene regulation. Despite this, little is currently known about the role of DNA methylation in lens development. My preliminary data show that loss of Dnmt1 and Uhrf1 function leads to cataracts and morphologically abnormal lenses that contain disorganized and apoptotic lens fibers. Methylation of genomic DNA in dnmt1 and uhrf1 mutants is reduced to 25% of wild type levels, supporting a model in which DNA methylation is required for normal lens development. The goal of the proposed research is to determine how DNA methylation functions to silence genes during lens fiber terminal differentiation. This will be determined with the following specific aims: Specific Aim 1: To determine whether lens epithelial cell genes, which are downregulated during lens fiber terminal differentiation, are silenced by DNA methylation. Specific Aim 2: To determine whether the lens defects in dnmt1 and uhrf1 mutant zebrafish are caused by deficient histone H3K9 tri-methylation. The experiments proposed are novel in that they will begin to elucidate the role and mechanism of DNA methylation-mediated gene silencing in the process of lens formation. This topic has relevance as a mechanism for understanding cataract formation, and it will increase our understanding of how deregulated DNA methylation can contribute to cancer. PUBLIC HEALTH RELEVANCE: When the normal process of lens development goes awry in humans due to genetic mutations or other factors, cataracts result. The zebrafish models utilized in this study link cataracts to the epigenetic regulatory mechanism of DNA methylation. Very little is currently known about the role of DNA methylation in lens development, and the results of this study will improve medical understanding of cataract formation, and may additionally lead to improved therapeutic options. Beyond lens development and cataractogenesis, aberrant DNA methylation plays a role in human cancers, and the results of this study will provide an additional in vivo system through which tissue- specific roles for methylation during differentiation can be studied.
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0.951 |