Abstract
Purpose: :
FIZ1 (Flt-3 Interacting Zinc-finger) interacts with NRL, an essential activator of rod-specific genes. Increasing FIZ1 concentration coincides with photoreceptor maturation, when NRL and CRX become detectable on the promoters of photoreceptor-specific genes in vivo (Rho, Pde6b and Rbp3). We pursued genome wide analysis of 26,000 promoters to find gene targets of FIZ1 in vivo.
Methods: :
Using the mouse neural retina as a model: Transmission electron microscopy (immunogold), protein interaction tests (Y2H, GST-Pulldown, Co-IP), electrophoretic mobility shifts assays (EMSA), ChIP-on-ChIP using Affymetrix tiling arrays, Quantitative-ChIP (Q-ChIP), and Taqman gene expression assays.
Results: :
TEM revealed that FIZ1 is a nuclear protein in mature retinal neurons, including photoreceptors. Native FIZ1 and CRX were co-precipitated from retinal nuclear extracts and FIZ1 can bind directly to CRX, in addition to NRL. EMSA of NRL- and CRX-binding elements, with retinal nuclear protein extracts, were altered by antibody to FIZ1. ChIP-on-ChiP, Q-ChIP, and Taqman gene expression assays revealed that FIZ1 targets include numerous photoreceptor and neuronal genes whose expression rises during maturation of the neural retina, including: Rho, Pde6b, Arrestin, Rs1.
Conclusions: :
This first use of ChIP-on-Chip tiling arrays, with neural retinal tissue, revealed that FIZ1 is part of the protein complex on the promoters of many photoreceptor and neuronal genes that are expressed during maturation of retinal neurons. Focusing on specific genes as a maturation markers, such as Rho, Q-ChIP analysis found that increased binding of FIZ1 corresponds to increased binding of actively transcribing RNA Polymerase-II.
Keywords: retinal development • gene/expression • gene microarray