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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.

The cone-rod homeobox protein Crx

Figure 3: Immunostaining of Crx (purple) with nucleus labeled in blue and cone outer segments labeled in yellow.

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. Human CRX mutations (Figure 4) are associated with dominant photoreceptor degenerative diseases including autosomal dominant cone rod dystrophy (adCRD), retinitis pigmentosa (adRP) and Leber congenital amaurosis (LCA) (Figure 4). These show a wide range of onset ages. Homozygous Crx knockout mice (Crx-/-) develop a phenotype similar to LCA; failure to developouter 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 have created and are characterizing 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 are being carried out in cultured cells and transgenic mice are being carried out.

Figure 4: Crx protein and mutations associated with human retinal degeneration diseases. Ad, autosomal dominant; CRD, cone-rod dystrophy; LCA, Leber congenital amaurosis; RP, retinitis pigmentosa.

The photoreceptor nuclear receptor Nr2e3 (PNR)

Figure 5: Nr2e3 domain structure (A) and immunostaining on monkey retinal sections (B), demonstrating that Nr2e3 is predominantly expressed in rods. PNA, peanut agglutinin (which binds a carbohydrate on the surface of cone photo receptor).

Nr2e3 (also called Photoreceptor Nuclear Receptor, or PNR) is an orphan nuclear receptor specifically expressed by rod photoreceptor cells (Figure 5). Nr2e3 acts as 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 within the same cell.

Epigenetic regulatory role of network photoreceptor transcription factors

The opsin genes are not expressed immediately after the birth of photoreceptors. Instead, the chromatin of each opsin gene undergoes sequential “epigenetic changes” during photoreceptor terminal differentiation as shown schematically in Figure 6, leading to the expression of a specific opsin in its respective photoreceptor subtype at the appropriate time, and maintaining such expression at appropriate levels. We hypothesize that a network of photoreceptor transcription factors, such as Otx2/Crx, Nrl and Nr2e3, plays a role in regulating chromatin configurations of the opsin genes during photoreceptor development and maintenance. We have begun to test this hypothesis using various biochemical tools developed for epigenetic studies. Chromatin immunoprecipitation assays are used to profile histone modifications on each opsin gene and investigate recruitment of histone modification enzymes by a specific transcription factor; Chromosomal conformation capture (3C) assays to reveal local chromosomal organization of each opsin gene in a given photoreceptor subtype. Changes of epigenetic signatures in diseased retinas lacking either a key photoreceptor transcription factor or an epigenetic co-regulator are also being studied. Our goal is to understand how coordinated actions of specific transcription factors and general epigenetic regulators precisely regulate photoreceptor gene expression and how mutations in either type of factor contribute to retinal diseases.

Figure 6: Model for the role of Crx in chromatin modification and transcription activation (adapted from Hum. Mol. Genet. 16:2433-2452, 2007). In retinal progenitor cells that have not committed to the photoreceptor lineage, chromatin associated with the opsin genes exists in a compacted state that excludes the transcription machinery and keeps these genes silenced. As these cells become committed to the photoreceptor lineage, they express Crx, which binds to opsin promoters. Bound Crx then recruits co-activators including histone acetyltransferases (HATs), which then modify lysine residues in the tails of histone H3 and H4, derepressing the chromatin associated with these genes and allowing basal levels of transcription. As additional transcription factors are expressed and recruited to these promoters, and as chromatin associated with enhancer elements becomes derepressed, transcription reaches high levels, allowing photoreceptors to build and maintain their outer segments and perform phototransduction.
 
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