B.S. Stanford University
Ph.D. Massachusetts Institute of Technology
Bio 25 Investigations in Molecular Biology
Bio 110 Genetics
Bio 171 Ethical Issues in Biotechnology & Genetics
Bio 189 Topics in Cell and Molecular Biology
The long-term objective of my research is to contribute to our understanding of how cell fates are specified during development. The free-living soil nematode, Caenorhabditis elegans, is an excellent model system in which to study cell fate. Its amenability both to genetics and molecular biology, in addition to the fact that its complete cell lineage and genome sequence are known, make it an ideal choice for genetic, molecular, and cellular approaches to the analysis of development.
Proteins belonging to the winged-helix family of transcription factors appear to play important roles in many developmental processes, including establishment of body axes, cell fate specification, differentiation of tissues, maintenance of cellular differentiation, and tumorigenesis. The LIN-31 protein, a member of this winged-helix family, is required for the proper specification of vulval cell fates during C. elegans development. This protein is believed to play two roles during development: 1) to promote vulval cell fates (when phosphorylated by the signal transduction protein MAP kinase) and 2) to promote non-vulval cell fates (when heterodimerized with another transcription factor, LIN-1). My research aims to further investigate LIN-31's role in cell fate specification during vulval development by extending a functional analysis of this multifaceted protein.
1. Functional Analysis of the winged helix transcription factor LIN-31.
Preliminary experiments identified the DNA-binding domain as critical for both functions of LIN-31 described above. Subsequent experiments revealed that both the acidic domain and one of the MAP kinase consensus phosphorylation sites are required for non-vulval cell fates. Ongoing site-directed mutagenesis experiments will identify functions of the other three MAP kinase phosphorylation sites, a region of homology at the C-terminus of LIN-31, and other sites. In addition, in vitro phosphorylation experiments will determine which of the consensus sites are actually phosphorylated.
2. Identification of LIN- 31::LIN-1 heterodimerization elements.
The LIN-31 protein is known to heterodimerize with another transcription factor, LIN-1, in order to promote non-vulval cell fates (and/or repress vulval cell fates). Preliminary site-directed mutagenesis experiments suggest that the acidic domain may be a candidate for this interaction. This hypothesis is currently being tested using co-immunoprecipitation experiments.
3. Identification of High-Affinity LIN-31 Binding Sequences.
The short-term goal of these experiments is to identify high-affinity LIN-31 binding sequences. The long-term goal is to understand the nature of LIN-31's interaction with DNA and to identify transcriptional targets of LIN-31. Using a sequential selection technique, we will identify oligonucleotides that bind the LIN-31 DNA binding domain with high affinity. We will then use those sequences to look for genomic clones of genes that may be direct targets of LIN-31. We can also use the sequences to determine the optimal LIN-31 binding sequence and show how the phosphorylation and heterodimerization state of LIN-31 might alter that sequence. Identification of the genes that LIN-31 activates and/or represses will greatly enhance the understanding of how cell fates are specified in response to extracellular signals.
Wagmaister, J.A., G.R. Miley, C.A. Morris, J.G. Gleason, L.M. Miller, K. Kornfeld, and D.M. Eisenmann (2006). "Identification of sites in the C. elegans Hox gene lin-39 required for early embryonic expression and for direct regulation by the transcription factors LIN-1 and LIN-31" Developmental Biology 297:550-65).
L. M. Miller, H. A. Hess, D. B. Doroquez, and N. Andrews (2000). Null mutations in the lin-31 gene indicate two functions during C. elegans vulval development. Genetics 156: 1595-1602.
Miller, L.M., D. A. Waring, and S. K. Kim (1996). Mosaic analysis using a ncl-1(+) extrachromosomal array reveals that lin-31 acts in the Pn.p cells during Caenorhabditis elegans vulval development. Genetics 143:1181-1191.
Rhind, N., L.M. Miller, J. Kopczynski, B. J. Meyer (1995). xol-1 acts as an early switch in the C. elegans male/hermaphrodite decision. Cell 80: 71-82.
Lackner, M.R., K. Kornfeld, L.M. Miller, H.R. Horvitz, and S. K. Kim (1994). A MAP kinase homolog, mpk-1, is involved in ras-mediated induction of vulval cell fates in C. elegans. Genes and Development 8:160-173.
Miller, L.M., M.E. Gallegos, B.A. Morisseau, and S.K. Kim (1993). lin-31, a Caenorhabditis elegans HNF-3/fork head transcription factor homolog, specifies three alternative cell fates in vulval development. Genes and Development 7: 933- 947.
Miller, L.M., J.D. Plenefisch, L.P. Casson, and B.J. Meyer. (1988). xol-1: A gene that controls the male modes of both sex determination and X chromosome dosage compensation in C. elegans. Cell 55: 167-183.