Identification of Pax6-Dependent Gene Regulatory Networks in the Mouse Lens

Abstract

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.

Olfactomedin III expression contributes to anoikis-resistance in clonal variants of a human lung squamous carcinoma cell line

“…OLFM3 is often localised to the Golgi and is a secreted protein. In mouse lens, the Pax6 transcription factor directly targets OLFM3 expression [25]. However, while Pax6 was detected in our microarray studies, the mRNA levels were slightly lower in DLKP-SQ than in DLKP-I or DLKP-M.…”

“…OLFM1 and 3 are expressed mainly in the brain, OLFM2 in the pancreas and prostate, and OLFM4 in the bone marrow, small intestine, colon and prostate [15]. Although originally identified in neural tissue, OLFM3 expression has been reported in the lung and spinal cord [23][24][25] and commercially available OLFM3 antibodies have been shown to stain lung cancer tissue. Lung cancers have also been observed to express OLFM1 and OLFM4 [19,22].…”

Primate-specific RFPL1 gene controls cell-cycle progression through cyclin B1/Cdc2 degradation

“…31,32 Thus, cross talk between Pax6 signaling and p53 activity appears to regulate specific genes in a given environment as the identification of either Pax6 or p53 target genes greatly varies depending on the cell system used. 4,13,[33][34][35] Although we observed that hRFPL1 impact on cell number and on cyclin B1 and Cdc2 levels was shared by hRFPL2, hRFPL3 and mRFPL, indicating that this activity was conserved during the evolution of this gene family, it is noteworthy that such an activity involved the RDM domain. This RDM, present in all RFPL proteins but restricted to them, is the result of a neofunctionalization event that occurred after the emergence of the RFPL gene ancestor.…”

The role of Pax6 in regulating the orientation and mode of cell division of progenitors in the mouse cerebral cortex

“…We searched the available transcriptome and ChIP data of Pax6 in cerebral cortex and lens (Holm et al, 2007;Sansom et al, 2009;Wolf et al, 2009) to identify possible target genes of Pax6 that might affect cell division. One candidate was the sperm associated antigen 5 (Spag5 or mAstrin), a microtubule-associated protein (Thein et al, 2007; Cheng et al, 2007) localizing to spindle poles and kinetochores in mammalian cells (Mack and Compton, 2001).…”