The Regulatory Factor ZFHX3 Modifies Circadian Function in SCN via an AT Motif-Driven Axis.
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.Moreover, mutant ZFHX3 had a decreased ability to activate AT motifs in the promoters of these neuropeptide genes.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.
Affiliation: MRC Harwell, Harwell Science and Innovation Campus, Oxfordshire OX11 0RD, UK.Show MeSH
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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).
Affiliation: MRC Harwell, Harwell Science and Innovation Campus, Oxfordshire OX11 0RD, UK.