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W. Xiao, X. Wang, A. Giddabasappa, D. A. Fox; Bioinformatic Analysis Identified Upstream Transcription Factors and Indirect Regulators of Differentially Expressed Mouse Retinal Genes During Gestational Lead Exposure (GLE). Invest. Ophthalmol. Vis. Sci. 2009;50(13):3995.
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© ARVO (1962-2015); The Authors (2016-present)
GLE increases and prolongs the proliferation of retinal progenitor cells, which resulted in an increased number of late-born rods and bipolar cells in adult C57BL/6 mouse retinas [ARVO 2007]. Using Affymetrix arrays and Gene Ontology analysis, we identified 730 differentially expressed genes in GLE retinas during the late embryonic (E16.5-18.5) and early postnatal (PN2-10) periods that were mainly involved in cell cycle control and neuronal differentiation [ARVO 2008]. Our current goal was to identify the common upstream transcription factors (TFs) and other expression regulators of these differentially expressed genes.
To accomplish this goal, two different bioinformatic software platforms were utilized: sub-network enrichment analysis (SNEA) and TF binding site over-representation analysis (TFBSORA). SNEA used Ariadne’s Pathway Studio 6.0 software, which is based on protein interaction networks described in the literature and curated databases. Promoter binding TFs and other expression regulators of the differentially expressed genes in GLE retinas with statistically significant enrichment were extracted. TFBSORA used Promoter Analysis and Interaction Network Toolset (PAINT). The 4,000 bp promoter sequence of each differentially expressed gene in GLE was retrieved from the Ensembl annotated mouse genome database and TFBSs on these sequences were located with the Match program (Transfac Public). Based on coincidence of regulatory sites, TFs with statistically significant over-representation were identified.
SNEA revealed several major hubs for the differentially expressed genes in developing GLE retinas. The major TFs were E2f1, Fos, Fosb, Junb, Junc, Jund, Nfyb, Notch and Rb. The major indirect expression regulators were Cdk2, Cdkn2a[p16] and Pten. TFBSORA revealed several major TF hubs for the differentially expressed genes in developing GLE retinas such as Cebpa, Creb1, E2f, Elk1, Junc, Myc and Pou2f1.
SNEA and TFBSORA identified mostly non-overlapping regulators. The combination of SNEA and TFBSORA identified major groups of TFs and signaling molecules that directly and indirectly regulate essential cell cycle and neuronal differentiation genes in the developing GLE retina. Ongoing ChIP-qPCR, Westerns and Co-IP studies are examining the binding and expression level of these regulators to determine the molecular mechanisms underlying the unique GLE retinal phenotype.
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