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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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ZFHX3+ Preferentially Activates a Novel, Circadian Motif in SCN, Related to Figure 5(A) In vitro activation of the AT motif by recombinant ZFHX3, DBP, or combined CLOCK and BMAL using a luciferase reporter construct driven by the AT motif (×7) in HEK293 cells. Zfhx3+ activation of the AT motif was more than 3 times that seen for either DBP or CLOCK/BMAL (p < 0.05, t test). Activation of AT sequences in SCN slices transduced by LVs showed cyclic activation in SCN slices from Zfhx3+/+ animals (Figures 5B and 5C).(B–D) Mutations of the three most conserved residues (positions 6, 8, 10) in the AT motifs strongly reduced AT activation rates (gray bars) when compared to the AT consensus (black bars). This effect was quantified by measuring both the (B) amplitude and robustness of these oscillations, (C) relative amplitude error, and (D) period error (p < 0.05, t test).(E) SCN slices expressing the AT-luciferase reporters were treated with pzf 670462 1 μM (blue lines) or vehicle (black lines), respectively.(F) Period of AT-mediated oscillations was significantly lengthened by pzf 670462 (p < 0.001, 2-way ANOVA repeated-measures), whereas it was unaffected by vehicle treatment. This effect was reversible upon removal of the drug.
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figs4: ZFHX3+ Preferentially Activates a Novel, Circadian Motif in SCN, Related to Figure 5(A) In vitro activation of the AT motif by recombinant ZFHX3, DBP, or combined CLOCK and BMAL using a luciferase reporter construct driven by the AT motif (×7) in HEK293 cells. Zfhx3+ activation of the AT motif was more than 3 times that seen for either DBP or CLOCK/BMAL (p < 0.05, t test). Activation of AT sequences in SCN slices transduced by LVs showed cyclic activation in SCN slices from Zfhx3+/+ animals (Figures 5B and 5C).(B–D) Mutations of the three most conserved residues (positions 6, 8, 10) in the AT motifs strongly reduced AT activation rates (gray bars) when compared to the AT consensus (black bars). This effect was quantified by measuring both the (B) amplitude and robustness of these oscillations, (C) relative amplitude error, and (D) period error (p < 0.05, t test).(E) SCN slices expressing the AT-luciferase reporters were treated with pzf 670462 1 μM (blue lines) or vehicle (black lines), respectively.(F) Period of AT-mediated oscillations was significantly lengthened by pzf 670462 (p < 0.001, 2-way ANOVA repeated-measures), whereas it was unaffected by vehicle treatment. This effect was reversible upon removal of the drug.

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)

ZFHX3+ Preferentially Activates a Novel, Circadian Motif in SCN, Related to Figure 5(A) In vitro activation of the AT motif by recombinant ZFHX3, DBP, or combined CLOCK and BMAL using a luciferase reporter construct driven by the AT motif (×7) in HEK293 cells. Zfhx3+ activation of the AT motif was more than 3 times that seen for either DBP or CLOCK/BMAL (p < 0.05, t test). Activation of AT sequences in SCN slices transduced by LVs showed cyclic activation in SCN slices from Zfhx3+/+ animals (Figures 5B and 5C).(B–D) Mutations of the three most conserved residues (positions 6, 8, 10) in the AT motifs strongly reduced AT activation rates (gray bars) when compared to the AT consensus (black bars). This effect was quantified by measuring both the (B) amplitude and robustness of these oscillations, (C) relative amplitude error, and (D) period error (p < 0.05, t test).(E) SCN slices expressing the AT-luciferase reporters were treated with pzf 670462 1 μM (blue lines) or vehicle (black lines), respectively.(F) Period of AT-mediated oscillations was significantly lengthened by pzf 670462 (p < 0.001, 2-way ANOVA repeated-measures), whereas it was unaffected by vehicle treatment. This effect was reversible upon removal of the drug.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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figs4: ZFHX3+ Preferentially Activates a Novel, Circadian Motif in SCN, Related to Figure 5(A) In vitro activation of the AT motif by recombinant ZFHX3, DBP, or combined CLOCK and BMAL using a luciferase reporter construct driven by the AT motif (×7) in HEK293 cells. Zfhx3+ activation of the AT motif was more than 3 times that seen for either DBP or CLOCK/BMAL (p < 0.05, t test). Activation of AT sequences in SCN slices transduced by LVs showed cyclic activation in SCN slices from Zfhx3+/+ animals (Figures 5B and 5C).(B–D) Mutations of the three most conserved residues (positions 6, 8, 10) in the AT motifs strongly reduced AT activation rates (gray bars) when compared to the AT consensus (black bars). This effect was quantified by measuring both the (B) amplitude and robustness of these oscillations, (C) relative amplitude error, and (D) period error (p < 0.05, t test).(E) SCN slices expressing the AT-luciferase reporters were treated with pzf 670462 1 μM (blue lines) or vehicle (black lines), respectively.(F) Period of AT-mediated oscillations was significantly lengthened by pzf 670462 (p < 0.001, 2-way ANOVA repeated-measures), whereas it was unaffected by vehicle treatment. This effect was reversible upon removal of the drug.
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