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