Defense Date


Document Type


Degree Name

Doctor of Philosophy


Human Genetics

First Advisor

Dr. Rita Shiang


Treacher Collins syndrome (TCS) is an autosomal dominant craniofacial development disorder and is caused by mutations in the TCOF1 gene. The TCOFl protein treacle is a nucleolar protein and may function in ribosome biogenesis.Previously, we identified downstream candidate genes using microarray analysis after manipulating Tcofl levels in a murine neuroblastoma (NB) cell line. The list of genes includes cell cycle genes as well as the transcription factors Cnbp and Tbx2, which are known to affect the cell cycle through the c-myc and p19-Mdm2-p53-p21 pathways respectively. To further characterize the cellular effects of Tcofl, stably transfected NB cell lines with overexpression or knockdown of Tcofl were generated. Growth curves were generated by counting cell numbers. BrdU incorporation and TUNEL assays were used to determine proliferation and apoptosis levels. Western blot analysis was used to detect protein level changes of candidate downstream pathway genes. Bothoverexpression and knockdown of Tcofl are detrimental to cell growth. Overexpression of Tcofl causes increased apoptosis and knockdown of Tcofl causes reduced cell proliferation and increased apoptosis. Western blot analysis shows that Cnbp and Tbx2 protein levels change with Tcofl, c-myc level is decreased in Tcofl knockdown cells and p19 (Cdkn2d), p53 and p21 (Cdkn1a) levels are increased in Tcofl overexpressing cells. Our results suggest that an optimal Tcofl level is required for cell proliferation and survival, and that overexpression and knockdown of Tcofl may affect cell proliferation and apoptosis through the p19-Mdm2-p53-p21 and Cnbp-c-myc pathways respectively.Heterozygous Tcofl knock out mice are neonatal lethal, which circumvents further analysis of the heterozygous and homozygous mice. In this study, we generated Tcofl conditional allele mice with loxP sites flanking exon 1. These mice were crossed with Wntl-Cre transgenic mice to generate a conditional knockout of Tcofl specifically in neural crest (NC) cells. Homozygous conditional knockout mice show craniofacial abnormalities resembling TCS patients. Heterozygous conditional knockout mice are phenotypically normal, which suggests that Tcofl functions in tissues other than NC cells during development. Cnbp expression is decreased in a proportion of the homozygous conditional knockout mouse embryos. Our results suggest that Tcofl may affect craniofacial development through Cnbp by maintaining cell growth.


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Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

June 2008