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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 Interaction with the AT Motif in Circadian Gene Promoters, Related to Figure 6(A–C) Overexpression of recombinant ZFHX3Sci failed to activate the (A) Grp, (B) Vipr2, and (C) Drd1a promoter containing vectors (p > 0.05, t test, in HEK293 cells).(D) Cry2, Per1, and Per2 have a predicted AT motif in their promoters, whereas Cry1 does not (Pscan score and sequences shown; position is relative to start of first exon).(E) Co-transfection of ZFHX3+ failed to activate the Cry1 promoter, while it did activate the (F) Cry2, (G) Per1, and (H) Per2 promoters (p < 0.05, t test, in HEK293 cells). We found a small decrease in ZFHX3Sci activation compared to ZFHX3+ for the Per2 promoter (p < 0.05, t test, in HEK293 cells).
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figs5: ZFHX3SCI Interaction with the AT Motif in Circadian Gene Promoters, Related to Figure 6(A–C) Overexpression of recombinant ZFHX3Sci failed to activate the (A) Grp, (B) Vipr2, and (C) Drd1a promoter containing vectors (p > 0.05, t test, in HEK293 cells).(D) Cry2, Per1, and Per2 have a predicted AT motif in their promoters, whereas Cry1 does not (Pscan score and sequences shown; position is relative to start of first exon).(E) Co-transfection of ZFHX3+ failed to activate the Cry1 promoter, while it did activate the (F) Cry2, (G) Per1, and (H) Per2 promoters (p < 0.05, t test, in HEK293 cells). We found a small decrease in ZFHX3Sci activation compared to ZFHX3+ for the Per2 promoter (p < 0.05, t test, in HEK293 cells).

Mentions: As several genes downregulated in module 1 had predicted AT motifs upstream of their TSSs (Figure 3B), we hypothesized that their diminished expression may be due to a decreased ability of ZFHX3Sci to activate transcription via the AT motif. To test this, we cloned the module 1 gene putative promoters with strong predicted AT motifs (Avp, Vip; Pscan threshold > 0.85), with moderate predicted motifs (Grp, Prokr2; Pscan threshold > 0.80), or without one (Drd1a, Vipr2; Pscan threshold ≤0.8) into the pGL3-Enhancer Luciferase Reporter Vector. We co-transfected these reporters together with the Zfhx3Sci or Zfhx3+ expression vectors into HEK293 cells. In both cases, there was increased transcriptional activation of reporters containing strong predicted AT motifs when we overexpressed ZFHX3+, while this activation was dramatically less when we overexpressed ZFHX3Sci (Figures 6C and 6D). Furthermore, ZFHX3+-driven expression was significantly higher than that driven by DBP or by CLOCK/BMAL (Figures 6E and 6F). Co-transfection of reporters containing moderate predicted AT motifs with ZFHX3+ led to increased activation of the Prokr2 reporter (Figure 6G; Figure S5A) but not the Grp reporter. Finally, we found no significant activation of those reporters without predicted AT motifs (Figures S5B and S5C).


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 Interaction with the AT Motif in Circadian Gene Promoters, Related to Figure 6(A–C) Overexpression of recombinant ZFHX3Sci failed to activate the (A) Grp, (B) Vipr2, and (C) Drd1a promoter containing vectors (p > 0.05, t test, in HEK293 cells).(D) Cry2, Per1, and Per2 have a predicted AT motif in their promoters, whereas Cry1 does not (Pscan score and sequences shown; position is relative to start of first exon).(E) Co-transfection of ZFHX3+ failed to activate the Cry1 promoter, while it did activate the (F) Cry2, (G) Per1, and (H) Per2 promoters (p < 0.05, t test, in HEK293 cells). We found a small decrease in ZFHX3Sci activation compared to ZFHX3+ for the Per2 promoter (p < 0.05, t test, in HEK293 cells).
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figs5: ZFHX3SCI Interaction with the AT Motif in Circadian Gene Promoters, Related to Figure 6(A–C) Overexpression of recombinant ZFHX3Sci failed to activate the (A) Grp, (B) Vipr2, and (C) Drd1a promoter containing vectors (p > 0.05, t test, in HEK293 cells).(D) Cry2, Per1, and Per2 have a predicted AT motif in their promoters, whereas Cry1 does not (Pscan score and sequences shown; position is relative to start of first exon).(E) Co-transfection of ZFHX3+ failed to activate the Cry1 promoter, while it did activate the (F) Cry2, (G) Per1, and (H) Per2 promoters (p < 0.05, t test, in HEK293 cells). We found a small decrease in ZFHX3Sci activation compared to ZFHX3+ for the Per2 promoter (p < 0.05, t test, in HEK293 cells).
Mentions: As several genes downregulated in module 1 had predicted AT motifs upstream of their TSSs (Figure 3B), we hypothesized that their diminished expression may be due to a decreased ability of ZFHX3Sci to activate transcription via the AT motif. To test this, we cloned the module 1 gene putative promoters with strong predicted AT motifs (Avp, Vip; Pscan threshold > 0.85), with moderate predicted motifs (Grp, Prokr2; Pscan threshold > 0.80), or without one (Drd1a, Vipr2; Pscan threshold ≤0.8) into the pGL3-Enhancer Luciferase Reporter Vector. We co-transfected these reporters together with the Zfhx3Sci or Zfhx3+ expression vectors into HEK293 cells. In both cases, there was increased transcriptional activation of reporters containing strong predicted AT motifs when we overexpressed ZFHX3+, while this activation was dramatically less when we overexpressed ZFHX3Sci (Figures 6C and 6D). Furthermore, ZFHX3+-driven expression was significantly higher than that driven by DBP or by CLOCK/BMAL (Figures 6E and 6F). Co-transfection of reporters containing moderate predicted AT motifs with ZFHX3+ led to increased activation of the Prokr2 reporter (Figure 6G; Figure S5A) but not the Grp reporter. Finally, we found no significant activation of those reporters without predicted AT motifs (Figures S5B and S5C).

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