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R.K. Hyde, A.E. Griep; E2F Target Gene Promoters Are Preferentially Regulated by Different E2F Family Members in the Mouse Ocular Lens . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1072.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Cell cycle regulation, controlled in part by the activity of the retinoblastoma protein (pRB), is essential for maintaining the normal patterns of growth and differentiation of lens. It is thought that the expression of many genes involved in cell cycle progression are regulated by complexes containing a member of the pRB family and a member of the E2F family of transcription factors. Evidence from studies in the lens support this model. However, to date it is not known if individual E2F target genes are preferentially regulated by specific pRB:E2F combinations. Methods: To characterize the profile of E2F family members associated with the promoters of various E2F target genes in the context of the normal mouse lens, we performed chromatin immunoprecipitation (ChIP) experiments. Chromatin was isolated from the lenses of neonatal mice after using formaldehyde to crosslink protein and DNA. The chromatin was immunoprecipitation with antibodies specific for E2F1,2,3,4,or 5. Crosslinking was reversed, and promoter fragments were PCR amplified. Products were analyzed by Southern blot and quantified by phosphoimager. Results: By ChIP analysis, E2F2, E2F3a and E2F4 are all associated with the bmyb promoter at nearly equal levels. In contrast, E2F3a is the predominant E2F associated with the p19ARF and cdc2 promoters. Interestingly, E2F family members are associated with nearly 100% of the p19ARF and bmyb promoters but associated with only approximately 25% of cdc2 promoters. E2Fs were not associated with the αA crystallin promoter, indicating the specificity of the immunoprecipitations. We are presently determining if specific pRB proteins are preferentially associated with these same promoters. Conclusions: In the lenses of neonatal mice, multiple E2Fs are associated with the p19ARF, bmyb and cdc2 promoters. However, the profile of E2Fs associated with each of these promoters differs. Thus, specific subsets of E2Fs may play unique roles in the lens by preferentially regulating the expression of individual E2F target genes. These results also indicate that in the lens, cdc2 expression is not solely regulated by pRB:E2F complexes.
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