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Lineage-specific DNA-binding transcription factors regulate development by activating and repressing particular set of genes required for the acquisition of a specific cell type. Pax6 is a paired domain and homeodomain-containing transcription factor essential for development of central nervous, olfactory and visual systems, as well as endocrine pancreas. Haploinsufficiency of Pax6 results in perturbed lens development and homeostasis. Loss-of-function of Pax6 is incompatible with lens lineage formation and results in abnormal telencephalic development. Using DNA microarrays, we have identified 559 genes expressed differentially between 1-day old mouse Pax6 heterozygous and wild type lenses. Of these, 178 (31.8%) were similarly increased and decreased in Pax6 homozygous embryonic telencephalon [Holm PC, Mader MT, Haubst N, Wizenmann A, Sigvardsson M, Götz M (2007) Loss- and gain-of-function analyses reveals targets of Pax6 in the developing mouse telencephalon. Mol Cell Neurosci 34: 99-119]. In contrast, 381 (68.2%) genes were differently regulated between the lens and embryonic telencephalon. Differential expression of nine genes implicated in lens development and homeostasis: Cspg2, Igfbp5, Mab21l2, Nrf2f, Olfm3, Spag5, Spock1, Spon1 and Tgfb2, was confirmed by quantitative RT-PCR, with five of these genes: Cspg2, Mab21l2, Olfm3, Spag5 and Tgfb2, identified as candidate direct Pax6 target genes by quantitative chromatin immunoprecipitation (qChIP). In Mab21l2 and Tgfb2 promoter regions, twelve putative individual Pax6-binding sites were tested by electrophoretic mobility shift assays (EMSAs) with recombinant Pax6 proteins. This led to the identification of two and three sites in the respective Mab21l2 and Tgfb2 promoter regions identified by qChIPs. Collectively, the present studies represent an integrative genome-wide approach to identify downstream networks controlled by Pax6 that control mouse lens and forebrain development.

Gene regulation by enhancers is important for precise temporal and spatial gene expression. Enhancers can drive gene expression regardless of their location, orientation or distance from the promoter. Changes in chromatin conformation and chromatin looping occur to bring the promoter and enhancers into close proximity. αA-crystallin ranks among one of the most abundantly expressed genes and proteins in the mammalian lens. The αA-crystallin locus is characterized by a 16 kb chromatin domain marked by two distal enhancers, 5′ DCR1 and 3′ DCR3. Here we used chromatin conformation capture (3C) analysis and transgenic approaches to analyze temporal control of the mouse αA-crystallin gene. We find that DCR1 is necessary, but not sufficient alone to drive expression at E10.5 in the mouse lens pit. Chromatin looping revealed interaction between the promoter and the region 3′ to DCR1, identifying a novel enhancer region in the αA-crystallin locus. We determined that this novel enhancer region, DCR1S, recapitulates the temporal control by DCR1. Acting as shadow enhancers, DCR1 and DCR1S are able to control expression in the lens vesicle at E11.5. It remains to be elucidated however, which region of the αA-crystallin locus is responsible for expression in the lens pit at E10.5.