Figure 3. ChIP analysis demonstrating that network transcription factors bind to their own and each other's promoters.

Antibodies against Crx, Nrl, Nr2e3, Trβ2, and NeuroD1 were used to immunoprecipitate the bound chromatin fragments from wild-type (WT), Crx^−/−, Nrl^−/−, or Nr2e3^rd7/rd7 (“Nr2e3^−/−“) retinae. Primers specific to the promoter regions of the genes listed on the left [(Peng and Chen, 2005); Table 3] were used to detect the presence of the candidate promoter regions in the immunoprecipitates by PCR. A band indicates that the transcription factor recognized by the immunoprecipitating antibody is bound to the promoter region of the indicated regulator or target gene. Target genes examined include S-opsin (Sop), M-opsin (Mop), rhodopsin (Rho), and interphotoreceptor binding protein (Rbp3), all of which are expressed in photoreceptors. GluR6, which is expressed in bipolar cells but not photoreceptors, serves as a control for photoreceptor specificity. In addition, PCR reactions using primers against DNA sequences immediately 3’ of each gene gave no bands (data not shown), confirming regulatory region-specific binding. Control immunoprecipitates using purified non-specific rabbit or goat IgG yielded no specific promoter sequences from WT (second lanes from the right) or knockout (data not shown) mice. Samples of retina homogenates (“input”) from WT (far right lanes) and knockout (data not shown) mice serve as positive controls for PCR.