Figure 5.

ZFHX3^SCI Differentially Activates a Circadian Motif in SCN

(A) In vitro activation of the AT motif by overexpressing Zfhx3 without and with the Sci mutation (Zfhx3⁺ and Zfhx3^Sci respectively) using a luciferase reporter construct driven by the AT motif (×7) in HEK293 cells. Zfhx3⁺ transcriptionally activated the AT motif, while Zfhx3^Sci did not (p < 0.05, t test).

(B and C) As shown, (B) raw data and (C) de-trended data show circadian activation of AT sequences in SCN slices transduced by LVs coding for the luciferase reporter driven by the AT motif. A.U., arbitrary units.

(D) AT-motif-driven luciferase expression in SCN slices from Zfhx3^Sci/+ (gray lines) and Zfhx3^+/+ (black lines) mice.

(E and F) There was a substantial decrease in (E) the amplitude of AT motif activation in Zfhx3^Sci/+ and a small, but significant decrease in (F) period compared to Zfhx3^+/+ (p < 0.05, t test).

(G and H) Representative double-plotted actograms of wheel-running activity in C57Bl/6 mice injected with (G) control siRNA (siNT) or (H) siZfhx3 (arrow denotes time of injection). Blue shading represents periods when lights are on. Vertical black bars represent wheel running activity. LD, light:dark cycle.

(I) Animals injected with siZfhx3 had a significantly lengthened τ_DD (23.74 ± 0.05 hr, mean ± SEM) compared to control siRNA (23.46 ± 0.05 hr). p < 0.05, t test.

(J) Injection of siZfhx3 led to a 43% downregulation of Zfhx3 mRNA levels compared to control siRNA (p < 0.05, t test).

(K and L) In (K), representative plots are shown of AT activation before and after transduction with the Syn-CRE vector in ex vivo SCN of Zfhx3^+/+ (black lines) and Zfhx3^Flox/+ mice (gray lines). (L) Zfhx3 deletion in Zfhx3^Flox/+ SCN significantly lengthened the period of AT activation relative to Zfhx3^+/+.

Error bars indicate SEM. ^∗p < 0.05, t test.

See also Figure S4.