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The Regulatory Factor ZFHX3 Modifies Circadian Function in SCN via an AT Motif-Driven Axis.

Parsons MJ, Brancaccio M, Sethi S, Maywood ES, Satija R, Edwards JK, Jagannath A, Couch Y, Finelli MJ, Smyllie NJ, Esapa C, Butler R, Barnard AR, Chesham JE, Saito S, Joynson G, Wells S, Foster RG, Oliver PL, Simon MM, Mallon AM, Hastings MH, Nolan PM - Cell (2015)

Bottom Line: Using RNA sequencing, we found minimal effects on core clock genes in Zfhx3(Sci/+) SCN, whereas the expression of neuropeptides critical for SCN intercellular signaling was significantly disturbed.Lentiviral transduction of SCN slices showed that the ZFHX3-mediated activation of AT motifs is circadian, with decreased amplitude and robustness of these oscillations in Zfhx3(Sci/+) SCN slices.In conclusion, by cloning Zfhx3(Sci), we have uncovered a circadian transcriptional axis that determines the period and robustness of behavioral and SCN molecular rhythms.

View Article: PubMed Central - PubMed

Affiliation: MRC Harwell, Harwell Science and Innovation Campus, Oxfordshire OX11 0RD, UK.

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ZFHX3SCI 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 Zfhx3Sci respectively) using a luciferase reporter construct driven by the AT motif (×7) in HEK293 cells. Zfhx3+ transcriptionally activated the AT motif, while Zfhx3Sci 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 Zfhx3Sci/+ (gray lines) and Zfhx3+/+ (black lines) mice.(E and F) There was a substantial decrease in (E) the amplitude of AT motif activation in Zfhx3Sci/+ 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 τDD (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 Zfhx3Flox/+ mice (gray lines). (L) Zfhx3 deletion in Zfhx3Flox/+ SCN significantly lengthened the period of AT activation relative to Zfhx3+/+.Error bars indicate SEM. ∗p < 0.05, t test.See also Figure S4.
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fig5: ZFHX3SCI 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 Zfhx3Sci respectively) using a luciferase reporter construct driven by the AT motif (×7) in HEK293 cells. Zfhx3+ transcriptionally activated the AT motif, while Zfhx3Sci 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 Zfhx3Sci/+ (gray lines) and Zfhx3+/+ (black lines) mice.(E and F) There was a substantial decrease in (E) the amplitude of AT motif activation in Zfhx3Sci/+ 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 τDD (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 Zfhx3Flox/+ mice (gray lines). (L) Zfhx3 deletion in Zfhx3Flox/+ SCN significantly lengthened the period of AT activation relative to Zfhx3+/+.Error bars indicate SEM. ∗p < 0.05, t test.See also Figure S4.

Mentions: To test whether the Sci mutation affects the ability of ZFHX3 to regulate transcription via the AT consensus motif, we cloned this motif (×7) into the pGL3-Enhancer Luciferase Reporter Vector. This reporter was then co-transfected with an expression vector containing recombinant Zfhx3, with or without the Sci mutation (Zfhx3Sci and Zfhx3+, respectively), into HEK293 cells. We assayed transcriptional activity in cell lysates using the dual luciferase assay and found that Zfhx3+ showed an increased activation relative to the empty vector. In contrast, activation by Zfhx3Sci was no different from that of the empty vector, supporting our hypothesis that the Sci mutation results in a decreased ability of ZFHX3 to activate transcription via the AT motif (p < 0.05, t test) (Figure 5A). We confirmed that the AT reporter response was specific to ZFHX3, as activation was at least three times higher than that of DBP or CLOCK/BMAL (p < 0.05, t test) (Figure S4A).


The Regulatory Factor ZFHX3 Modifies Circadian Function in SCN via an AT Motif-Driven Axis.

Parsons MJ, Brancaccio M, Sethi S, Maywood ES, Satija R, Edwards JK, Jagannath A, Couch Y, Finelli MJ, Smyllie NJ, Esapa C, Butler R, Barnard AR, Chesham JE, Saito S, Joynson G, Wells S, Foster RG, Oliver PL, Simon MM, Mallon AM, Hastings MH, Nolan PM - Cell (2015)

ZFHX3SCI 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 Zfhx3Sci respectively) using a luciferase reporter construct driven by the AT motif (×7) in HEK293 cells. Zfhx3+ transcriptionally activated the AT motif, while Zfhx3Sci 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 Zfhx3Sci/+ (gray lines) and Zfhx3+/+ (black lines) mice.(E and F) There was a substantial decrease in (E) the amplitude of AT motif activation in Zfhx3Sci/+ 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 τDD (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 Zfhx3Flox/+ mice (gray lines). (L) Zfhx3 deletion in Zfhx3Flox/+ SCN significantly lengthened the period of AT activation relative to Zfhx3+/+.Error bars indicate SEM. ∗p < 0.05, t test.See also Figure S4.
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fig5: ZFHX3SCI 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 Zfhx3Sci respectively) using a luciferase reporter construct driven by the AT motif (×7) in HEK293 cells. Zfhx3+ transcriptionally activated the AT motif, while Zfhx3Sci 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 Zfhx3Sci/+ (gray lines) and Zfhx3+/+ (black lines) mice.(E and F) There was a substantial decrease in (E) the amplitude of AT motif activation in Zfhx3Sci/+ 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 τDD (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 Zfhx3Flox/+ mice (gray lines). (L) Zfhx3 deletion in Zfhx3Flox/+ SCN significantly lengthened the period of AT activation relative to Zfhx3+/+.Error bars indicate SEM. ∗p < 0.05, t test.See also Figure S4.
Mentions: To test whether the Sci mutation affects the ability of ZFHX3 to regulate transcription via the AT consensus motif, we cloned this motif (×7) into the pGL3-Enhancer Luciferase Reporter Vector. This reporter was then co-transfected with an expression vector containing recombinant Zfhx3, with or without the Sci mutation (Zfhx3Sci and Zfhx3+, respectively), into HEK293 cells. We assayed transcriptional activity in cell lysates using the dual luciferase assay and found that Zfhx3+ showed an increased activation relative to the empty vector. In contrast, activation by Zfhx3Sci was no different from that of the empty vector, supporting our hypothesis that the Sci mutation results in a decreased ability of ZFHX3 to activate transcription via the AT motif (p < 0.05, t test) (Figure 5A). We confirmed that the AT reporter response was specific to ZFHX3, as activation was at least three times higher than that of DBP or CLOCK/BMAL (p < 0.05, t test) (Figure S4A).

Bottom Line: Using RNA sequencing, we found minimal effects on core clock genes in Zfhx3(Sci/+) SCN, whereas the expression of neuropeptides critical for SCN intercellular signaling was significantly disturbed.Lentiviral transduction of SCN slices showed that the ZFHX3-mediated activation of AT motifs is circadian, with decreased amplitude and robustness of these oscillations in Zfhx3(Sci/+) SCN slices.In conclusion, by cloning Zfhx3(Sci), we have uncovered a circadian transcriptional axis that determines the period and robustness of behavioral and SCN molecular rhythms.

View Article: PubMed Central - PubMed

Affiliation: MRC Harwell, Harwell Science and Innovation Campus, Oxfordshire OX11 0RD, UK.

Show MeSH
Related in: MedlinePlus