S., Swaroop A. retina, we show that a 0.9-kb sequence upstream of the transcription initiation site is sufficient to drive reporter gene expression in photoreceptors. We further determine a 0.3-kb sequence including a proximal promoter (cluster A1) and an enhancer (cluster B) that can direct rod-specific expression as a direct transcriptional target of ROR and suggest that combinatorial action of multiple regulatory factors modulates the expression of in developing and mature retina. convenience and a well defined cell repertoire, serves as an excellent model for investigating the origin and maintenance of cellular diversity. The retina consists of six types of neurons and one type of glia. Rod and cone photoreceptors function as specialized sensory neurons that are responsible for scotopic and photopic vision, respectively. Rod photoreceptors are highly vulnerable to genetic defects and environmental abuse (8) and are needed for cone cell viability (9). Hence, elucidation of genesis and functional maintenance of rod photoreceptors would permit better design of strategies for treatment of retinal and macular degenerative diseases. Distinct retinal cell types originate in a conserved temporal order from multipotent retinal progenitor cells that undergo progressive changes in transcriptional says (10). Both extrinsic cues and intrinsic factors play critical functions in retinal development; however, intrinsic mechanisms largely dictate the acquisition of Rabbit Polyclonal to AL2S7 cell type specificity (11, 12). MASH1, NEUROD1, MATH5, and other basic helix-loop-helix transcription factors bias cells toward specific neuronal fates (13, 14). One of the important regulatory proteins that guides photoreceptor lineage from retinal progenitor cells is the homeodomain transcription factor orthodenticle homolog 2 (OTX2)3; its loss results in amacrine-like cells instead of photoreceptors (15). However, OTX2 is not sufficient to induce specific photoreceptor cell fate and requires conversation with other specific regulators (16, 17). BLIMP1, a zinc finger protein, appears to control the choice between photoreceptor and bipolar cell fate (18, 19). Downstream from OTX2 (and probably BLIMP1) in photoreceptor transcriptional hierarchy, retinoid-related orphan nuclear receptor (ROR) controls appropriate differentiation of both rod and cone photoreceptors (20, 21). The retina of cone photoreceptors are dependent on the expression and activity of four transcription factors: cone rod homeobox (CRX), thyroid hormone receptor 2 (TR2), neural retina leucine zipper (NRL), and nuclear receptor subfamily 2, group E, member 3 (NR2E3) (17). The studies using knock-out mice suggest that the homeodomain protein CRX does not specify photoreceptor cell fate, yet it critically contributes to photoreceptor-specific gene activation and homeostasis FICZ (22, 23). TR2, together with ROR and retinoid X receptor , modulates cone differentiation and patterning (24, 25). The key transcriptional regulator of photoreceptor cell fate choice is usually NRL (26), a basic motif leucine zipper (bZIP) protein that induces postmitotic precursors to become rods instead of cones (27). Ablation of in mouse prospects photoreceptor precursors to acquire a default short wavelength-sensitive opsin-expressing cone (S-cone) state (28, 29). NR2E3 is usually a direct transcriptional target of NRL (30). The primary role of NR2E3 is usually to repress the expression of cone genes. Loss of NR2E3 results in a retina with enhanced S-cone function and many hybrid photoreceptors expressing both S-opsin and rhodopsin (17, 31C33). Together with CRX, NR2E3, and other transcription factors, NRL activates the rod differentiation pathway by inducing the expression of rod-specific genes, including rhodopsin and cGMP-phosphodiesterase (22, 34C36). Not surprisingly, mutations in are associated with retinal degenerative diseases (37C40). Previously we showed that a 2.5-kb genomic sequence, upstream of the transcription initiation site, contains four conserved regions (cluster ICIV) that might control expression (41, 42). Transgenic mice expressing GFP under the control of this sequence selectively express the reporter gene in developing and mature rod photoreceptors (41). Here, we statement the identification of specific expression and implicate CRX, OTX2, and cyclic AMP response element-binding protein (CREB) in modulating expression. EXPERIMENTAL PROCEDURES Bioinformatic FICZ Analysis Genomic sequences were analyzed using the July 2007 (mm9) mouse genome assembly (University or college of California Santa Cruz Genome Browser FICZ Project, Santa Cruz, CA) (43). The conserved regions upstream of transcription start site were aligned with CLUSTALW (44). The TFsearch program (45), MultiTF tool, and Mulan program (46) were used to find predicted transcription factor binding sites annotated in the TRANSFAC database (version 4.0) (47). Plasmid DNA Constructs and Mutagenesis Genomic sequences upstream of the mouse transcription start site were PCR-amplified and cloned into the pEGFP-N1 vector (Clontech). The SV40 basal promoter driving mCherry-IRES-alkaline phosphatase was generated by replacing GFP with mCherry sequence in SV40-GFP-IRES-alkaline phosphatase plasmid (48). Conserved sequence clusters were cloned into pEGFP-N1 and SV40-mCherry-IRES-alkaline phosphatase. Sequences contained in each cluster were as follows: cluster A (?304 to +119; relative to the transcriptional start site), A1 (?34 to +16), B (?938 to ?657), B.