Cintia Hongay, Ph.D.
143 Science Center
Suffolk University, Boston, MA: BS in Biology, Summa Cum Laude, 1996
Harvard University, GSAS, Cambridge MA: PhD in Genetics, the BBS Program at Harvard Medical School, DMS. Dr. Fred Winston, mentor, 2003.
The Whitehead Institute for Biomedical Research, Cambridge, MA: Postdoctoral Fellow, Dr. Gerald R. Fink, mentor, 2004-2007. Postdoctoral Fellow, Dr. Terry L. Orr-Weaver, mentor, 2008-2011.
BY412 Molecular Biology Lab (Fall)
BY160 Biology II (Spring)
Soma-germline interactions are required for proper gamete formation in all multicellular organisms. In S. cerevisiae, a unicellular eukaryote, each individual cell interacts with the environment, sensing available nutrients, to determine when to switch from mitotic growth (vegetative asexual state) to meiosis (gametogenesis). IME4 (Inducer of Meiosis 4) is an N6 adenosine mRNA methyltransferase exclusively expressed in a/a diploid cells upon meiotic entry and I found that it regulates the mitotic:meiotic switch in yeastIME4 has highly conserved homologs (60% overall homology) in D. melanogaster, A. thaliana, M. musculus, H. sapiens, and other metazoans, but its function in multicellular organisms is presently unknown.
The Drosophila IME4 homolog, Dm ime4 (CG5933), is expressed in adult ovaries and testes, suggesting an evolutionarily conserved function in gametogenesis. Deletion of this gene or its ablation via RNAi produced the unexpected result that, in contrast to yeast but similar to Arabidopsis, Dm ime4 is essential for viability. Hypomorphic mutations in Dm ime4 were recovered that yield viable adults whose phenotypes reveal critical functions for this gene in gametogenesis. In addition, Dm ime4 mutants (male and female) display phenotypes that indicate developmental defects (cleft thorax and cuticle aberrations or etched tergites). These phenotypes resemble those displayed in defective Notch/Decapentaplegic and Hedgehog signaling. I have generated inducible transgenic lines carrying a wild-type copy or one with point mutations in the conserved catalytic domain of Dm IME4 and found that only the wild-type Dm ime4 transgene rescues these mutant phenotypes, indicating that the conserved catalytic domain, which is responsible for carrying out N6 Adenosine methylation, is essential.
The Drosophila melanogaster ovary is a powerful yet simple system with only a few cell types in which to study the molecular requirements for proper soma-germline communications leading to the successful production of the mature egg. Each ovary pair consists of strings of ovarioles, a conveyor belt of progressively maturing egg chambers that start at the germarium, where primary sources of soma and germline-derived cells, upon receiving the appropriate signals, specify cell fates to initiate gametogenesis. Dm ime4 is required for this developmental process. Dm IME4 is mostly nuclear and it is localized primarily in the soma-derived follicle cells, starting with strong signal in prefollicle cell niches in the germarium, the source of follicle cells that will surround the emerging 16-cyst of sister germline cells and, in later stages, in the polar cells, specialized follicle cells necessary for establishing the anterior-posterior axial organization of the egg. Ovarioles from Dm ime4 mutants have high incidence of fused egg-chambers that contain several germline cysts. Polar cells are absent or mislocalized, and there are no stalk cells, another specialized follicle cell type required to keep each egg chamber separate from one another. Although this phenotype is complex, the fusion is consistent with defects in cyst individualization at the posterior end of the germarium, a process regulated by Delta-Notch signaling. Indeed, Notch signaling is defective in Dm ime4 mutants.
The question I am interested in addressing through my current and future research is how this mRNA methyltransferase affects several aspects of development. Preliminary data obtained by immunoprecipitation of Dm IME4 from ovarian protein extracts, followed by mass-spectrometry, show co-immunoprecipitation of several splicing factors in an RNA-mediated fashion. High throughput yeast two-hybrid screens (BioGrid) also show splicing factors as interactors of Dm IME4 (CG5933). In addition, the A. thaliana homolog of IME4 interacts with a putative splicing factor by yeast two-hybrid. Alternative splicing of pre-mRNAs, with its best studied example in the Drosophila sex-determination pathway, can be a powerful and versatile regulatory mechanism for control of gene expression and functional diversification of proteins. Nothing is known about the role of N6mA, a non-editing RNA modification, in this process, nor its involvement in gametogenesis or in any other developmental process. Taken together, my findings suggest it contributes to major developmental decisions. Due to the high degree of conservation of this gene, my work will provide a paradigm to investigate the role of Dm ime4 homologs in other organisms, including vertebrates. To this end, my lab is currently collaborating with Dr. Kenneth Wallace’s lab here at Clarkson University to study the developmental role of IME4 in zebrafish. Our preliminary results indicate that the zebrafish IME4 homolog is expressed in Notch-regulated regions of the zebrafish embryo, suggesting a functional conservation of this gene in vertebrates. The ultimate goal is to extrapolate our findings in higher vertebrates, particularly mice and ultimately humans, where the homolog of IME4 exists but remains unstudied, and Notch signaling is known to be crucial in development as well as in diseased states such as cancer.
Hongay, C., Jia, N., Bard, M., and Winston, F. (2002). Mot3 is a transcriptional repressor of ergosterol biosynthetic genes and is required for normal vacuolar function in Saccharomyces cerevisiae. Embo J 21, 4114-4124.
Hongay, C., Grisafi, P., Galitski, T., and Fink, G. (2006) Antisense transcription controls cell fate in Saccharomyces cerevisiae. Cell 127, 735-745.
Hongay, C. and Orr-Weaver, T. (2011). Drosophila Inducer of MEiosis 4 (IME4) is required for Notch signaling during oogenesis. Proc Natl Acad Sci U S A. 2011 Aug 22. [Epub ahead of print]
Roach, G., Wallace, R.H., Cameron, A., Ozel, R.E., Hongay, C. , Andreescu, S., and Wallace, K.N. (2012). Loss of ascl 1a prevents secretory cell differentiation within the zebrafish intestinal epithelium resulting in a loss of posterior intestinal motility.