Fig. 1.

Identification of Caprin-1 as a Pou4f3 target gene. (A) Four replicate clone arrays obtained from the reverse subtraction experiment. Replicate dot blots were hybridised with radiolabelled cDNA from forward (increased Pou4f3 minus decreased Pou4f3) and reverse (decreased Pou4f3 minus increased Pou4f3) experiments, with both subtracted and unsubtracted cDNA. fu, forward unsubtracted; fs, forward subtracted; ru, reverse unsubtracted; rs, reverse subtracted. The Caprin-1 clone at position A10 on each filter (arrows) is highly enriched in the reverse subtracted cDNA, suggesting that it is downregulated by Pou4f3. (B) Putative Pou4f3-binding sites used as EMSA probe sequences (A1, A2, B, C1 and C2) are shown. Underlined bases correspond to sequences identified by MatInspector, matches to the core sequence are shown in capital letters, and the asterisk indicates nucleotides with a matrix position conservation (Ci) value of >60/100. (C) EMSA analysis. OC-2 cell nuclear extract was incubated with the radiolabelled probes shown in B. These were incubated alone (lane 1), or with a 250-fold excess of the indicated unlabelled competitor sequences (lanes 2–4). Bound protein is indicated (arrowheads). (D) ChIP analysis. Binding of Halo-tagged Pou4f3 to sites A1 and C1, and to a control sequence (NS) within the Caprin-1 5′ flanking sequence, in native OC-2 cell chromatin as determined by qPCR. Levels are plotted relative to the levels bound by a control vector after 40 cycles; however, the control sequence was not detected in all three replicates, therefore the minimum possible fold differences are shown on the basis of an assigned cycle threshold of 40, rather than the absolute threshold. Other sites were not precipitated above background levels. Error bars represent 95% confidence intervals (n=3).