Lysine Acetyltransferases CBP and EP300 are Essential for Histone H3 K18 and K27 Acetylation in the Lens Placode and to Determine the Fate of Lens Progenitor Cells

Abstract

Purpose: CBP and p300 are chromatin remodeling enzymes that catalyze acetylation of specific lysine residues in core histone subunits and other nuclear and cytoplasmic substrates. Mouse models have shown CBP and p300 are essential for early embryonic development. Previous studies have shown that CBP/p300 have distinct temporal/spatial expression patterns in the mouse lens, and are required for expression of lens crystallins.

Methods: We inactivated CBP and p300 in ectodermal cells that give rise to the lens and corneal epithelium using Le-cre and Pax6-cre and generated double CBP and p300 mutant embryos. Expression of CBP/p300, crystallins and the lens-specific regulatory proteins Pax6, Six3, Sox2, Foxe3 and Prox1, and the levels of core histone modifications was evaluated by immunofluorescence in mouse embryos. RNAs were isolated from lens placodes by laser microdissection followed by RNA expression profiling.

Results: Deletion of either CBP or p300 alone caused minimal changes in lens and corneal development. However, compound mutants indicate proper lens morphology requires the presence of at least one allele of either CBP or p300. In CBP-/- ;p300+/- mutants, defects in lens fiber cells were accompanied by disrupted corneal morphogenesis. In contrast, CBP+/-;p300-/- mutants showed relatively normal cornea formation, suggesting CBP has distinct functions from p300 in cornea and eyelid formation. Homozygous deletion of CBP/p300 blocked lens placode formation, and, consequently, completely disrupted eye formation. Expression of Six3 and Sox2 was reduced in the mutated ectoderm compared to the wild type lens placodes. The mutated ectoderms exhibited markedly reduced levels of H3 K18 and K27 acetylation and no expression of αA- or αB-crystallins. Microarray analysis revealed that 204 of the top 1000 lens-preferred transcripts identified in the iSyte database were decreased in CBP/p300 knockout lens placodes.

Conclusions: CBP and p300 are required to establish lens cell-type identity during the transition from specified into determined lens cells. Our studies suggest that lens placode formation requires the coordinated action of different chromatin remodeling enzymes and the establishment of “stable” patterns of histone posttranslational modifications at key genes/loci to control lens cell identity/cell-type memory.

Keywords: 738 transcription • 497 development • 539 genetics