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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 Interacts with and Differentially Activates the AT Motif in Avp and Vip PromotersQuantitative ChIP of Zfhx3+/+ SCN tissue samples using ZFHX3 antiserum.(A) Primer pairs were designed to span the AT motif (Avp-AT/Vip-AT) and an adjacent region (Avp-1/Vip-1) upstream of the TSS (primers in the coding region of each gene were used to normalize each reaction).(B) Zfhx3 binds to the promoter around the AT motif of both Avp and Vip compared to the control gene, Gapdh. Data are shown as fold change normalized for input relative to the corresponding coding region of each gene and are taken at ZT3 (n = 3).(C and D) Overexpression of ZFHX3 with the Sci mutation (ZFHX3Sci) was ineffective in activation of luciferase driven by AT-motif-containing promoters of (C) Avp and (D) Vip in HEK293 cells (p < 0.05, t test). R.U., relative units.(E and F) Zfhx3+ activation (black bars) of the (E) Avp and (F) Vip promoters was more than three times that seen for either DBP (white bars) or CLOCK/BMAL (gray bars) (p < 0.05, t test).(G) Overexpression ZFHX3Sci was also ineffective in activation of the Prokr2 promoter Prokr2 in HEK293 cells (p < 0.05, t test).(H and I) Mutation of the three most conserved residues (positions 6, 8, and 10) in the AT motifs of both the (H) Avp and (I) Vip promoter constructs (gray bars) resulted in significantly decreased levels of ZFHX3+ activation compared to those with AT motifs intact (black bars) (p < 0.05, t test). White bars represent the activation of the mutated promoter constructs alone.Error bars indicate SEM. ∗p < 0.05, t test.See also Figure S5.
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fig6: ZFHX3SCI Interacts with and Differentially Activates the AT Motif in Avp and Vip PromotersQuantitative ChIP of Zfhx3+/+ SCN tissue samples using ZFHX3 antiserum.(A) Primer pairs were designed to span the AT motif (Avp-AT/Vip-AT) and an adjacent region (Avp-1/Vip-1) upstream of the TSS (primers in the coding region of each gene were used to normalize each reaction).(B) Zfhx3 binds to the promoter around the AT motif of both Avp and Vip compared to the control gene, Gapdh. Data are shown as fold change normalized for input relative to the corresponding coding region of each gene and are taken at ZT3 (n = 3).(C and D) Overexpression of ZFHX3 with the Sci mutation (ZFHX3Sci) was ineffective in activation of luciferase driven by AT-motif-containing promoters of (C) Avp and (D) Vip in HEK293 cells (p < 0.05, t test). R.U., relative units.(E and F) Zfhx3+ activation (black bars) of the (E) Avp and (F) Vip promoters was more than three times that seen for either DBP (white bars) or CLOCK/BMAL (gray bars) (p < 0.05, t test).(G) Overexpression ZFHX3Sci was also ineffective in activation of the Prokr2 promoter Prokr2 in HEK293 cells (p < 0.05, t test).(H and I) Mutation of the three most conserved residues (positions 6, 8, and 10) in the AT motifs of both the (H) Avp and (I) Vip promoter constructs (gray bars) resulted in significantly decreased levels of ZFHX3+ activation compared to those with AT motifs intact (black bars) (p < 0.05, t test). White bars represent the activation of the mutated promoter constructs alone.Error bars indicate SEM. ∗p < 0.05, t test.See also Figure S5.

Mentions: To confirm that ZFHX3 binds to the promoter of the key predicted target genes Avp and Vip in vivo, we performed quantitative ChIP on SCN tissue from Zfhx3+/+ animals (ZT3) using ZFHX3 antiserum. We found a significant increase in immunoprecipitated DNA upstream of the transcriptional start site (TSS) for both Avp and Vip compared to the Gapdh control promoter region, suggesting that ZFHX3 binds to both target gene promoters (Figures 6A and 6B). At the Vip promoter, this binding occurred in the vicinity of the AT motif to a far greater extent than in an adjacent upstream region. These data strongly suggest that ZFHX3 binds to Avp and Vip promoters in close proximity to AT consensus motifs.


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 Interacts with and Differentially Activates the AT Motif in Avp and Vip PromotersQuantitative ChIP of Zfhx3+/+ SCN tissue samples using ZFHX3 antiserum.(A) Primer pairs were designed to span the AT motif (Avp-AT/Vip-AT) and an adjacent region (Avp-1/Vip-1) upstream of the TSS (primers in the coding region of each gene were used to normalize each reaction).(B) Zfhx3 binds to the promoter around the AT motif of both Avp and Vip compared to the control gene, Gapdh. Data are shown as fold change normalized for input relative to the corresponding coding region of each gene and are taken at ZT3 (n = 3).(C and D) Overexpression of ZFHX3 with the Sci mutation (ZFHX3Sci) was ineffective in activation of luciferase driven by AT-motif-containing promoters of (C) Avp and (D) Vip in HEK293 cells (p < 0.05, t test). R.U., relative units.(E and F) Zfhx3+ activation (black bars) of the (E) Avp and (F) Vip promoters was more than three times that seen for either DBP (white bars) or CLOCK/BMAL (gray bars) (p < 0.05, t test).(G) Overexpression ZFHX3Sci was also ineffective in activation of the Prokr2 promoter Prokr2 in HEK293 cells (p < 0.05, t test).(H and I) Mutation of the three most conserved residues (positions 6, 8, and 10) in the AT motifs of both the (H) Avp and (I) Vip promoter constructs (gray bars) resulted in significantly decreased levels of ZFHX3+ activation compared to those with AT motifs intact (black bars) (p < 0.05, t test). White bars represent the activation of the mutated promoter constructs alone.Error bars indicate SEM. ∗p < 0.05, t test.See also Figure S5.
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fig6: ZFHX3SCI Interacts with and Differentially Activates the AT Motif in Avp and Vip PromotersQuantitative ChIP of Zfhx3+/+ SCN tissue samples using ZFHX3 antiserum.(A) Primer pairs were designed to span the AT motif (Avp-AT/Vip-AT) and an adjacent region (Avp-1/Vip-1) upstream of the TSS (primers in the coding region of each gene were used to normalize each reaction).(B) Zfhx3 binds to the promoter around the AT motif of both Avp and Vip compared to the control gene, Gapdh. Data are shown as fold change normalized for input relative to the corresponding coding region of each gene and are taken at ZT3 (n = 3).(C and D) Overexpression of ZFHX3 with the Sci mutation (ZFHX3Sci) was ineffective in activation of luciferase driven by AT-motif-containing promoters of (C) Avp and (D) Vip in HEK293 cells (p < 0.05, t test). R.U., relative units.(E and F) Zfhx3+ activation (black bars) of the (E) Avp and (F) Vip promoters was more than three times that seen for either DBP (white bars) or CLOCK/BMAL (gray bars) (p < 0.05, t test).(G) Overexpression ZFHX3Sci was also ineffective in activation of the Prokr2 promoter Prokr2 in HEK293 cells (p < 0.05, t test).(H and I) Mutation of the three most conserved residues (positions 6, 8, and 10) in the AT motifs of both the (H) Avp and (I) Vip promoter constructs (gray bars) resulted in significantly decreased levels of ZFHX3+ activation compared to those with AT motifs intact (black bars) (p < 0.05, t test). White bars represent the activation of the mutated promoter constructs alone.Error bars indicate SEM. ∗p < 0.05, t test.See also Figure S5.
Mentions: To confirm that ZFHX3 binds to the promoter of the key predicted target genes Avp and Vip in vivo, we performed quantitative ChIP on SCN tissue from Zfhx3+/+ animals (ZT3) using ZFHX3 antiserum. We found a significant increase in immunoprecipitated DNA upstream of the transcriptional start site (TSS) for both Avp and Vip compared to the Gapdh control promoter region, suggesting that ZFHX3 binds to both target gene promoters (Figures 6A and 6B). At the Vip promoter, this binding occurred in the vicinity of the AT motif to a far greater extent than in an adjacent upstream region. These data strongly suggest that ZFHX3 binds to Avp and Vip promoters in close proximity to AT consensus motifs.

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