Figure 3.

Pias3 is a transcriptional target of Trβ2 and Rxrγ. (a) Identification of an evolutionarily conserved thyroid hormone response element (TRE) in the mammalian Pias3 promoter. (b) Trβ2 and Rxrγ cooperate to activate expression of a Pias3 promoter luciferase reporter construct, and enhanced it in the presence of 9-cis retinoic acid (9CRA). Pink and green triangles represent increasing doses treatment of 9CRA (5 μM and 10 μM) and 3,5,3′-triiodothyronine (T₃, 1 nM and 5 nM), respectively. ‘+’ and ‘++’ represent 15 ng and 30 ng of transfected plasmid, respectively. All data are represented as mean ± s.d. (n = 3). (c) Trβ2 and Rxrγ selectively co-occupy the Pias3 promoter. Chromatin immunoprecipitation (ChIP) assays were performed using P14 mouse retinas. Results are shown as gel images of PCR products representing promoter sequences of the Pias3 promoter. (d) Quantitative real-time PCR ChIP analysis of the Pias3 promoter for the antibodies used in Fig. 3b. Relative IP versus Input ratio is presented as percent of Input, which is calculated based on a formula: (2^−ΔCt) x 100%, where ΔCt=Ct [IP] – Ct [Input]. All data are represented as mean ± s.d. (n = 3). (e) Flat mount IHC of the dorsal region of an 8wk WT and Trβ2^−/− mouse retinas labeled with Sop, Mop and Pias3 antibodies. Immunostaining with Pias3 (red channel) is visualized separately on right panels. Scale bar: 20 μm. (f) Quantification of Pias3 immunofluorescence by cone photoreceptor subtype. All data are represented as mean ± s.e.m. (n = 100 cells).