Chromatin remodeling ATPases Brg1 and Snf2h in ocular lens development

Abstract

Temporal and spatial regulation of gene transcription is fundamental to the development of multicellular organisms. ATP-dependent chromatin remodeling employs ATP energy to translocate nucleosomes and coordinate gene transcription. Brg1 (Smarca4) and Snf2h (Smarca5) are catalytic subunits of the chromatin remodeling complexes SWI/SNF and ISWI, respectively. These complexes mediate transcriptional regulation, DNA replication, and DNA repair. Disrupted functions of either Brg1 or Snf2h lead to severe ocular abnormalities. My hypothesis is: Chromatin remodeling enzymes Brg1 and Snf2h independently regulate mammalian lens development through their control of lens fiber cell differentiation, regulation of gene transcription, and execution of lens fiber cell denucleation.;My specific aims are: Aim 1: To determine the roles of Brg1 in mammalian lens development. Aim 2: To determine the roles of Snf2h in mammalian lens development. Aim 3: To identify novel molecular mechanisms that govern differentiation-coupled lens fiber cell denucleation.;We found that Brg1 is required for lens terminal differentiation (Aim 1). Brg1 mutants exhibited abnormal lens morphology, reduced expression of alphaA-crystallin, retention of nuclei, and cataract. RNA expression profiling using Brg1 mutant lenses followed by comparative studies with Pax6 +/- and Hsf4-/- lens microarrays identified hundreds of commonly regulated targets including DNase IIbeta, the key endonuclease responsible for lens DNA degradation. Lens-specific deletion of Snf2h led to reduced lens cell growth, perturbed lens cell cycle regulation, denucleation defects, and lens opacity (Aim 2). In addition, Snf2h regulated lens epithelium integrity and Notch signaling. Snf2h-/- lenses exhibited disturbed expression of autophagy regulatory proteins LC3B (microtubule-associated protein 1 light chain 3B) and FYCO1 (FYVE and coiled-coil domain-containing 1), suggesting a potential role of autophagy in the degradation of lens nuclei proteins (Aim 3). RNA expression studies found reduced expression of Dnase2b and Hsf4 in both Brg1 and Snf2h knockout lenses. Chromatin immunoprecipitation detected Brg1 and Snf2h proteins at the Dnase2b and Hsf4 loci in lens chromatin. Collectively, these studies demonstrate that Brg1 and Snf2h regulate unique and joint aspects of lens formation, and provide novel insights into the molecular mechanisms of lens fiber cell differentiation, denucleation and cataract formation.