The long-term objective of this research is to understand how chromatin remodeling controls the development of the vertebrate eye and central nervous system (CNS). As in all developmental pathways, eye development depends upon precise regulation of spatial and temporal patterns of gene expression. Transcription activation and repression occurs in the context of chromatin, a complex of DNA and histone proteins that compacts DNA into the eukaryotic nucleus. Chromatin remodeling enzymes positively and negatively regulate the accessibility of DNA in chromatin, and are thereby essential regulators of gene expression. The Imitation Switch (ISWI) chromatin remodeling enzyme is required for normal development of the eye and CNS in Xenopus laevis, and embryos lacking ISWI develop congenital subcapsular cataracts. However, ISWI is the catalytic subunit of multiple remodeling complexes in vivo. We are specifically interested in how different ISWI-dependent chromatin remodeling enzymes regulate eye development during embryogenesis. To accomplish this, we will assess the roles of individual ISWI complexes by inhibiting subunits unique to each complex. We will begin by using morpholino injection to inhibit WSTF, a subunit of the WICH complex, and CHRAC-17, a subunit of the CHRAC complex. We will assess the effects of these knockdowns in the developing Xenopus embryo, specifically within the developing eye, and analyze the resulting eye malformations by microscopic examination, sectioning and fine structure analysis, and gene expression analysis. These studies will provide critical insights into the role of chromatin remodeling in eye development. This proposal has three specific aims.
Aim 1 is to characterize the developmental phenotypes of Xenopus embryos lacking functional WICH or CHRAC complexes, using morpholino inhibition, addressing both morphological and transcriptional defects.
Aim 2 is to identify the contributions of the conserved domains within WSTF (bromodomain, PHD finger, and DDT domains) to its developmental functions.
This aim will include injection of deletion mutants of WSTF.
Aim 3 is to determine the stage(s) during which ISWI activity is required for eye development, by generating transgenic Xenopus that express a dominant-negative ISWI mutant under control of inducible and tissue-specific promoters. Congenital cataracts are the leading cause of blindness in children. Lack of ISWI remodeling activity in developing frog embryos results in numerous defects in brain and eye development, including the formation of subcapsular cataracts. Our work will contribute to the understanding of the mechanisms of normal eye development and provides a new model for studying congenital cataract formation. ? ? ?
| Barnett, Chris; Krebs, Jocelyn E (2011) WSTF does it all: a multifunctional protein in transcription, repair, and replication. Biochem Cell Biol 89:12-23 |
