Otx2 and other transcription factors are required for <i>Blimp1</i> spatial and temporal enhancer activity.

<p><b>(A)</b> Enhancer protein binding assays. 5’-biotinylated (BIO) oligonucleotides were made double stranded and bound to streptavidin (SA) coated dynabeads. P0 retinal nuclear protein lysate was incubated with the beads and the bound proteins purified and subjected to polyacrylamide gel electrophoresis (PAGE). (Left) A Coomassie gel containing unmodified nuclear lysate (input) and pulldowns with wild-type F3.1d oligos, <i>Otx2</i> binding site mutant oligos, and with unbound beads (no- oligo). The ladder band sizes are in kDa. Only a subset of the input proteins bind to the F3.1d and Otx2 mutant oligos. The no-oligo condition showed modest non-specific bead binding background. The intense band at 12 kDa represents the SA boiled from the dynabeads. (Right) Western blot of these samples shows a robust Otx2 signal in the input lane at 32–35 kDa (expected size of 31-32kDa). The F3.1d wild-type sequence pulls down Otx2 more strongly than the mutant oligo. Otx2 is not pulled down in the no-oligo condition. This shows that Otx2 is preferentially binding to its consensus site and not elsewhere in the sequence. <b>(B)</b> Otx2 chromatin immunoprecipitation (ChIP) on P0 retinal cells as a percentage of the input signal. Error bars represent S. D. The <i>Id3</i> promoter region lacks <i>Otx2</i> binding sites and Otx2 (red) does not pulldown the <i>Id3</i> promoter more than goat IgG control (black). <i>Rbp3</i> is a known Otx2 target and is immunoprecipitated strongly by Otx2 versus goat IgG (*, unpaired t-test, P < 0.03). Otx2 also pulls down the <i>Blimp1</i> F3.1d enhancer region significantly (*, unpaired t-test, P < 0.02) compared to IgG control. <b>(C)</b> F3.1d sequence showing required (red) sequence regions. JASPAR was used to predict transcription factor binding (underlined). Factors with a 90% relative score threshold that are expressed in the P2 retina are shown. The 5’ most required sequence strongly matches a ROR response element. Rod-specific Nr2e3 is predicted to bind in non-essential sequence, while several homeodomain transcription factors are predicted to bind in subsections of the X and Y regions. Additional candidate transcription factors are seen when the score threshold is lowered to 80% (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176905#pone.0176905.s004" target="_blank">S2 Table</a>). <b>(D)</b> Multi-component regulation of <i>Blimp1</i> gene expression. Other work has implicated RORβ as the ROR family member bound to F3.1d [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176905#pone.0176905.ref043" target="_blank">43</a>]. Otx2, RORβ, and factors bound to the X and Y sites are required for expression. At early stages of eye development and within the RPE, Otx2 is present while RORβ is absent, preventing <i>Blimp1</i> expression. Later, Otx2, RORβ, and activators that bind X and Y must be present to achieve robust expression in photoreceptors. In contrast to <i>Blimp1</i> and its enhancer, Otx2 and RORβ remain expressed in mature photoreceptors. This suggests that temporal regulation of the enhancer occurs through the X and Y sites. This could be due to the loss of activating factors and/or the accumulation of silencing factors that are able to supersede or compete with the activators.