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Y. Yang, L. V. Wolf, J. Zavadil, A. Cvekl; Chromatin Structure and Epigenetic Regulation of Pax6 Target Genes in Mouse Lens and Pancreas. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5028. doi: https://doi.org/.
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Cell-lineage formation and differentiation requires a coordinated action of specific DNA-binding transcription factors, chromatin remodeling and signaling molecules. Pax6 is a lineage-specific DNA-binding transcription factor that acts as a "master" gene for eye development while also functions in the brain and developing pancreas. Our study is aimed to identify the chromatin structure and epigenetic regulation of Pax6 downstream targets in mouse lens and pancreas.
Expression levels of molecular markers of the mouse lens (e.g. crystallins) and pancreas (e.g. insulin and glucagon) were determined by quantitative RT-PCR, respectively. Differential gene expression analysis of wild type mouse newborn (P1) lenses and cultured βTC3 pancreatic cells in conjunction with analysis of Pax6 heterozygous lenses by Affymetrix mouse 430A 2.0 expression microarrays. To identify genes directly regulated by Pax6, ChIP-on-chip experiments were performed using chromatin obtained from mouse P1 lenses and pancreas βTC3 cells. Both immunoprecipitated and control genomic fragments were amplified and hybridized with the Affymetrix Mouse GeneChip 1.0R promoter tiling arrays.
As expected, lens and pancreas cells are characterized through their own "fingerprint" expression profiles. We found no expression of crystallins in the pancreas. In contrast, low levels of insulin and glucagon expression were identified in the lens. ChIP-on-chip studies identified a few hundreds of promoters occupied by Pax6 in vivo in lens and β-cell chromatin. A group of 12 direct targets of Pax6 were confirmed by quantitative chromatin immunoprecipitations (qChIPs) in both systems. In addition, the precise location of Pax6-binding sites within regions identified by ChIP-on-chip was mapped by using known Pax6-binding site "consensus" sequences. Interestingly, Pax6 binding to the regulatory regions is not necessarily associated with active gene transcription. For example, Pax6 binds to the αA-crystallin promoter in pancreas βTC3 cells although no αA-crystallin expression was detected. To determine which core histone tail modifications are associated with Pax6-bound in transcriptionally permissive or repressive environment, we are currently mapping modifications such as H3K9Ac, H3K4Me3 and H3K27Me surrounding those Pax6-binding sites.
Collectively, our studies identify a large number of novel direct targets of Pax6 during lens and pancreas development and form the foundation of understanding specific epigenetic regulatory mechanisms in cellular differentiation.
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