Pax6 Interactions with Chromatin and Identification of Its Novel Direct Target Genes in Lens and Forebrain

Abstract

Pax6 encodes a specific DNA-binding transcription factor that regulates the development of multiple organs, including the eye, brain and pancreas. Previous studies have shown that Pax6 regulates the entire process of ocular lens development. In the developing forebrain, Pax6 is expressed in ventricular zone precursor cells and in specific populations of neurons; absence of Pax6 results in disrupted cell proliferation and cell fate specification in telencephalon. In the pancreas, Pax6 is essential for the differentiation of α-, β- and δ-islet cells. To elucidate molecular roles of Pax6, chromatin immunoprecipitation experiments combined with high-density oligonucleotide array hybridizations (ChIP-chip) were performed using three distinct sources of chromatin (lens, forebrain and β-cells). ChIP-chip studies, performed as biological triplicates, identified a total of 5,260 promoters occupied by Pax6. 1,001 (133) of these promoter regions were shared between at least two (three) distinct chromatin sources, respectively. In lens chromatin, 2,335 promoters were bound by Pax6. RNA expression profiling from Pax6+/− lenses combined with in vivo Pax6-binding data yielded 76 putative Pax6-direct targets, including the Gaa, Isl1, Kif1b, Mtmr2, Pcsk1n, and Snca genes. RNA and ChIP data were validated for all these genes. In lens cells, reporter assays established Kib1b and Snca as Pax6 activated and repressed genes, respectively. In situ hybridization revealed reduced expression of these genes in E14 cerebral cortex. Moreover, we examined differentially expressed transcripts between E9.5 wild type and Pax6−/− lens placodes that suggested Efnb2, Fat4, Has2, Nav1, and Trpm3 as novel Pax6-direct targets. Collectively, the present studies, through the identification of Pax6-direct target genes, provide novel insights into the molecular mechanisms of Pax6 gene control during mouse embryonic development. In addition, the present data demonstrate that Pax6 interacts preferentially with promoter regions in a tissue-specific fashion. Nevertheless, nearly 20% of the regions identified are accessible to Pax6 in multiple tissues.

PAX6, a novel target of miR-335, inhibits cell proliferation and invasion in glioma cells

“…As a key transcription factor, PAX6 has been demonstrated to be involved in the development of the eye, central nervous system and pancreas (3,24). Previously, PAX6 was revealed to be involved in the regulation of malignant glioma (4,25).…”

“…Accumulating evidence has demonstrated that PAX6 is crucial in the development of the central nervous system (3). Previously, several studies have suggested that PAX6 acts as a tumor suppressor in malignant glioma (4,5).…”

A schizophrenia-associated HLA locus affects thalamus volume and asymmetry

“…While ZNRD1-AS has no known biological role, several lines of evidence suggest that one or both of HLA-A and ZNRD1 have neuronal functions (Boulanger, 2009;Elmer and McAllister, 2012;Xie et al, 2013) and possible roles in schizophrenia (Bergen et al, 2012; Cross-Disorder Group of the Psychiatric Genomics Consortium, 2013b; McAllister, 2014) (in addition to the evidence for neuronal function of HLA class I proteins outlined in the Introduction). Both of these genes are expressed in the thalamus (Allen Brain Atlas: http://human.brain-map.org; Braineac: http://www.braineac.org/ (Ramasamy et al, 2014)), and Chip-seq data in mouse brain has suggested that their homologues (H2-K1 and Znrd1) are regulated by the transcriptional factor Pax6 (Xie et al, 2013), a major actor in the developing central nervous system. Notably Pax6 interacts with Baf150 and Baf177 to regulate cerebral size and thickness (Tuoc et al, 2013;Xie et al, 2013).…”

“…Both of these genes are expressed in the thalamus (Allen Brain Atlas: http://human.brain-map.org; Braineac: http://www.braineac.org/ (Ramasamy et al, 2014)), and Chip-seq data in mouse brain has suggested that their homologues (H2-K1 and Znrd1) are regulated by the transcriptional factor Pax6 (Xie et al, 2013), a major actor in the developing central nervous system. Notably Pax6 interacts with Baf150 and Baf177 to regulate cerebral size and thickness (Tuoc et al, 2013;Xie et al, 2013). Furthermore, both HLA-A and ZNRD1 contain SNPs associated with schizophrenia by GWAS; rs2524005 is intronic in HLA-A (Bergen et al, 2012) and rs8321 is in the 3 0 untranslated region of ZNRD1 (Cross-Disorder Group of the Psychiatric Genomics Consortium, 2013b).…”

Functional dissection of the paired domain of Pax6 reveals molecular mechanisms of coordinating neurogenesis and proliferation

“…Development 140 (5) with Pax6 ChIP data obtained with E15 WT cerebral cortex, lens and pancreas cells (Xie and Cvekl, 2011;Xie et al, 2013). Interestingly, Pax6 was bound to the promoters of ~20% of the genes regulated in either Pax6…”