Chen Lab / Projects
Photoreceptor transcriptome and transcription factor network
Rod and cone photoreceptors are special neurons carrying out phototransduction that converts the light signal into neuronal signal. Rods are responsible for night vision, while cones are responsible for color vision and visual acuity. Both rods and cones have a unique structure called outer segment (OS) where opsins, the visual pigments, are localized (Figure 1).
Figure 1: Rod and Cone photoreceptors in mammalian retina. A) A human retinal section showing three neuronal cell layers: outer nuclear layer (ONL) containing the nucleus of rods and cones; inner nuclear layer (INL) containing the nucleus of bipolar, horizontal and amacrine and Muller glial cells; gonglion cell layer (GCL). B) Diagram of rod and cone structure. C) Scan EM showing the outer segments
Figure 2: Photoreceptor transcription factor network induces differentiation of photoreceptor subtypes.
Rods and cones preferentially express a set of genes coding for specific proteins essential for photoreceptor function, including opsins. These genes are so called photoreceptor transcriptome. The expression of these genes is coordinately and precisely regulated. Over-expression and under-expression of some genes in the photoreceptor transcriptome can lead to developmental defects or photoreceptor degeneration. This regulation is mediated by a network of photoreceptor transcription factors (Figure 2), the regulatory proteins that bind to the promoter and/or enhancer region of each photoreceptor gene to activate or repress transcription. They are intrinsic factors determining the development and maintenance of photoreceptor subtypes.
Figure 3: Immunostaining of Crx (purple) with nucleus labeled in blue and cone outer segments labeled in yellow.
The cone-rod homeobox protein Crx
Crx is an otd/Otx-like homeodomain transcription factor required for development and maintenance of photoreceptor function. It is expressed in both rods and cones (Figure 3), as well as their precursors. Crx serves as the first molecular switch that directs progenitor cells to differentiate into photoreceptors. Crx activates transcription of many photoreceptor genes by recruiting co-activator/chromatin remodeling complexes and interacting with other transcription factors. Crx mutations are associated with dominant photoreceptor degenerative diseases with a wide-age of onset, including autosomal dominant cone rod dystrophy (adCRD), retinitis pigmentosa (adRP) and Leber congenital amaurosis (LCA) (Figure 4). Homozygous Crx knockout mice (Crx-/-) develop a phenotype similar to LCA; lack of the outer segments, undetectable photoreceptor function and progressive degeneration of rods and cones. However, heterozygous Crx knockout mice (Crx+/-) are essentially normal.
Our research on Crx has been focusing on 1) how Crx mutations cause dominant disease in humans, whether by haploid-insufficiency or dominant-negative effects. We will create and characterize knock-in mouse models carrying selected human Crx mutations; 2) the mechanism by which Crx activates transcription. We are using protein-protein interaction assays to identify Crx interacting proteins and co-factors required for Crx trans-activating activity; 3) regulation of Crx expression. We are analyzing the promoter region of the mouse and human Crx gene to identify regulatory DNA elements and transcription factors controlling Crx expression in photoreceptor cells. Reporter assays in cultured cells and transgenic mice are being carried out.
Figure 4: Crx protein and mutations associated with human retinal degeneration
Figure 5: Nr2e3 domain structure (A) and immunostaining on monkey retinal sections (B), demonstrating that Nr2e3 is predominantly expressed in rods.
The photoreceptor nuclear receptor Nr2e3 (PNR)
Nr2e3 is an orphan nuclear receptor specifically expressed by rod photoreceptor cells (Figure 5). Nr2e3 is a dual transcription regulator. It interacts with Crx to activate the transcription of rod genes, but repress cone genes. Nr2e3 mutations cause enhanced S-cone syndrome (ESCS) in humans and rd7 in mice, featuring excess blue cones and degeneration of rods.
Our research on Nr2e3 has been focusing on identifying proteins that interact with Nr2e3 and modify its activity, such as co-activators and co-repressors. Our goal is to determine how Nr2e3 activates and represses transcription of different genes.