student research day - Case Western Reserve University School of ...

David Reznick
Altered Gene Expression in RFX4-hml/hml
Homozygous Mutant Mice
David Reznick, Cong Tian, Hui Chen, Heping Yu, Fenchan Han, Cindy Benedict-Alderfer and Dr. Qing Zheng
Department of Otolaryngology – Head and Neck Surgery
Case Western Reserve University
Regulatory factor X 4 (RFX4) is a DNA binding protein. A mutation of this gene, hypoplasia of the membranous
labyrinth (hml), was analyzed in this lab using two-dimensional differential in-gel electrophoresis (2D-DIGE) and
mass spectrometry. Cochleae from Rfx4hml/hml mutant showed down regulation of two proteins, aldolase C and
prohibitin, when compared to the cochleae of their wild type littermates. Aldolase is a glycolytic enzyme, where
aldolase C is the isoform found in the brain. Prohibitin (Phb) is a regulatory protein within cells whose most
predominant function is the inhibition of DNA synthesis. Because Rfx4hml/hml mice exhibit hearing impairment and
hypoplasia of the membranous labyrinth, we hypothesized that the mutant phenotype can be correlated with a
decrease in these proteins Phb and Aldolase C. To test our hypothesis and to further confirm the 2D-DIGE results,
we investigated expression of these two genes at the mRNA and protein level in Rfx4hml/hml mutant and wild type
littermates. Semi-quantitative RT-PCR was used to determine the level of mRNA expression of these genes in
cochleae from Rfx4hml/hml mutant and wild type mice. Cochleae cryosections from the same mice were analyzed
by immunofluorescence to further elucidate any alteration in prohibitin protein expression. The semi-quantitative
RT-PCR assay of Rfx4hml/hml and wild type littermates showed a decreased expression of aldolase C in mutants,
while prohibitin had no statistically significant change in expression. Immunofluorescence in these mouse tissues
showed the presence of prohibitin in the organ of Corti with no significant alterations in expression quantity and
location. In conclusion, aldolase C was found to have altered expression in the Rfx4hml/hml mice compared to wild
type littermates, whereas prohibitin was not altered to statistically significant levels in the Rfx4hml/hml mice.
These results regarding prohibitin are still significant as they are the first demonstration of prohibitin expression in
the inner ear.
Supported by NIH/NIDCD R01DC007392 to Dr. Qing Yin Zheng and the Crile Fellowship to David Reznick
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