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: Using ex vivo organotypic slices from Zfhx3Sci/+ or Zfhx3+/+ animals on a PER2::LUC background, we found that Zfhx3Sci/+ SCN had a shorter circadian period and a decreased amplitude of fusion protein expression relative to wild-type (p < 0.05, t test) (Figures 1F–1H). Similar decreases were found in individual neurons from Zfhx3Sci/+ SCN slices compared to wild-type neurons (Figures S1A and S1B). Furthermore, the period distribution was broader, and RAE increased in individual Zfhx3Sci/+ neurons imaged across the SCN circuit (Figures S1C and S1D) (p < 0.05, t test). There were no period differences in organotypic lung slices (Figure S1E), suggesting a central oscillator specific effect. These ex vivo SCN findings mirror the differences seen in locomotor behavior and suggest that photic inputs are not necessary for the expression of the short-period phenotype. Conversely, there were no significant differences in mRNA expression patterns of core circadian genes in the SCN of Zfhx3Sci/+ and Zfhx3+/+ animals sampled at six time points across the light:dark cycle (Figure S2), suggesting that any differences in clock gene expression are masked by the light:dark cycle. Collectively, these data predict that the mutation disrupts a Zfhx3-dependent and canonical TTFL-independent effect on SCN circadian period.
Affiliation: MRC Harwell, Harwell Science and Innovation Campus, Oxfordshire OX11 0RD, UK.