Pax6 interactions with chromatin and identification of its novel direct target genes in lens and forebrain.

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.