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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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The Short Circuit (Sci) Phenotype Results from a Mutation in the Transcription Factor Zfhx3(A) Representative double-plotted actograms of wheel-running activity in Zfhx3Sci/+ and Zfhx3+/+ mice (7 days on a 12-hr light:dark (LD) schedule, followed by 2 weeks in constant darkness). Yellow shading represents periods when lights are on. Vertical black bars represent wheel running activity.(B) Zfhx3Sci/+ mice have a shorter free-running period than littermate controls in constant darkness (n = 6). ∗p = 0.0009.(C) The Zfhx3Sci mutation mapped to the zinc-finger homeobox 3 (Zfhx3) locus and results in a G → T transversion at position 6620.(D) Multiple protein sequence alignment of ZFHX3 protein and its paralogues. The Zfhx3Sci mutation, a V1963F substitution, is in a highly conserved region.(E) A schematic of the functional domains of ZFHX3; the Zfhx3Sci mutation lies upstream of a zinc-finger domain.(F) Representative plots showing circadian activation of PER2::LUC expression in ex vivo SCN organotypic slices from Zfhx3Sci/+ (gray line) or Zfhx3+/+ (black line) animals.(G and H) The mean (G) period and (H) amplitude of PER2::LUC expression were decreased in Zfhx3Sci/+ (gray bars, n = 29) compared to Zfhx3+/+ (black bars, n = 21). ∗p < 0.05, t test.Error bars indicate SEM.See also Figures S1 and S2.
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fig1: The Short Circuit (Sci) Phenotype Results from a Mutation in the Transcription Factor Zfhx3(A) Representative double-plotted actograms of wheel-running activity in Zfhx3Sci/+ and Zfhx3+/+ mice (7 days on a 12-hr light:dark (LD) schedule, followed by 2 weeks in constant darkness). Yellow shading represents periods when lights are on. Vertical black bars represent wheel running activity.(B) Zfhx3Sci/+ mice have a shorter free-running period than littermate controls in constant darkness (n = 6). ∗p = 0.0009.(C) The Zfhx3Sci mutation mapped to the zinc-finger homeobox 3 (Zfhx3) locus and results in a G → T transversion at position 6620.(D) Multiple protein sequence alignment of ZFHX3 protein and its paralogues. The Zfhx3Sci mutation, a V1963F substitution, is in a highly conserved region.(E) A schematic of the functional domains of ZFHX3; the Zfhx3Sci mutation lies upstream of a zinc-finger domain.(F) Representative plots showing circadian activation of PER2::LUC expression in ex vivo SCN organotypic slices from Zfhx3Sci/+ (gray line) or Zfhx3+/+ (black line) animals.(G and H) The mean (G) period and (H) amplitude of PER2::LUC expression were decreased in Zfhx3Sci/+ (gray bars, n = 29) compared to Zfhx3+/+ (black bars, n = 21). ∗p < 0.05, t test.Error bars indicate SEM.See also Figures S1 and S2.

Mentions: We conducted an ENU screen to uncover genetic factors affecting mammalian circadian behavior (Bacon et al., 2004). Among G1 animals in a dominant screen, we identified a mouse with a circadian period (τDD; behavioral circadian period of the animals in constant darkness) shorter than the population mean (23.6 ± 0.08 hr; mean ± SEM). This phenotype was inherited in a dominant fashion, with a τDD (23.0 ± 0.05 hr) ranging from 21.4 to 23 hr (Figures 1A and 1B). We named the mutation short circuit (Sci) and mapped the dominant phenotype to mouse chromosome 8 between D8Mit138 (107.67 Mb) and D8Mit213 (110.57 Mb), containing 25 annotated genes. Among the candidates, zinc-finger homeobox 3 (Zhfx3) was highly and almost exclusively expressed in adult SCN (Lein et al., 2007). We identified a point mutation in exon 9, resulting in a G → T transversion at position 6620 (Zfhx3 transcript, ENSMUST00000043896) (Figure 1C). The Sci mutation substitutes a phenylalanine for a valine at residue 1963 (V1963F) in a highly conserved region just upstream of the 17th zinc-finger motif (Figures 1D and 1E). Following the identification of the causative mutation, genotype and phenotype correlation demonstrated that the mutation causes homozygous lethality during embryonic development; therefore, only Zfhx3Sci/+ adult animals could be assessed phenotypically.


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)

The Short Circuit (Sci) Phenotype Results from a Mutation in the Transcription Factor Zfhx3(A) Representative double-plotted actograms of wheel-running activity in Zfhx3Sci/+ and Zfhx3+/+ mice (7 days on a 12-hr light:dark (LD) schedule, followed by 2 weeks in constant darkness). Yellow shading represents periods when lights are on. Vertical black bars represent wheel running activity.(B) Zfhx3Sci/+ mice have a shorter free-running period than littermate controls in constant darkness (n = 6). ∗p = 0.0009.(C) The Zfhx3Sci mutation mapped to the zinc-finger homeobox 3 (Zfhx3) locus and results in a G → T transversion at position 6620.(D) Multiple protein sequence alignment of ZFHX3 protein and its paralogues. The Zfhx3Sci mutation, a V1963F substitution, is in a highly conserved region.(E) A schematic of the functional domains of ZFHX3; the Zfhx3Sci mutation lies upstream of a zinc-finger domain.(F) Representative plots showing circadian activation of PER2::LUC expression in ex vivo SCN organotypic slices from Zfhx3Sci/+ (gray line) or Zfhx3+/+ (black line) animals.(G and H) The mean (G) period and (H) amplitude of PER2::LUC expression were decreased in Zfhx3Sci/+ (gray bars, n = 29) compared to Zfhx3+/+ (black bars, n = 21). ∗p < 0.05, t test.Error bars indicate SEM.See also Figures S1 and S2.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4537516&req=5

fig1: The Short Circuit (Sci) Phenotype Results from a Mutation in the Transcription Factor Zfhx3(A) Representative double-plotted actograms of wheel-running activity in Zfhx3Sci/+ and Zfhx3+/+ mice (7 days on a 12-hr light:dark (LD) schedule, followed by 2 weeks in constant darkness). Yellow shading represents periods when lights are on. Vertical black bars represent wheel running activity.(B) Zfhx3Sci/+ mice have a shorter free-running period than littermate controls in constant darkness (n = 6). ∗p = 0.0009.(C) The Zfhx3Sci mutation mapped to the zinc-finger homeobox 3 (Zfhx3) locus and results in a G → T transversion at position 6620.(D) Multiple protein sequence alignment of ZFHX3 protein and its paralogues. The Zfhx3Sci mutation, a V1963F substitution, is in a highly conserved region.(E) A schematic of the functional domains of ZFHX3; the Zfhx3Sci mutation lies upstream of a zinc-finger domain.(F) Representative plots showing circadian activation of PER2::LUC expression in ex vivo SCN organotypic slices from Zfhx3Sci/+ (gray line) or Zfhx3+/+ (black line) animals.(G and H) The mean (G) period and (H) amplitude of PER2::LUC expression were decreased in Zfhx3Sci/+ (gray bars, n = 29) compared to Zfhx3+/+ (black bars, n = 21). ∗p < 0.05, t test.Error bars indicate SEM.See also Figures S1 and S2.
Mentions: We conducted an ENU screen to uncover genetic factors affecting mammalian circadian behavior (Bacon et al., 2004). Among G1 animals in a dominant screen, we identified a mouse with a circadian period (τDD; behavioral circadian period of the animals in constant darkness) shorter than the population mean (23.6 ± 0.08 hr; mean ± SEM). This phenotype was inherited in a dominant fashion, with a τDD (23.0 ± 0.05 hr) ranging from 21.4 to 23 hr (Figures 1A and 1B). We named the mutation short circuit (Sci) and mapped the dominant phenotype to mouse chromosome 8 between D8Mit138 (107.67 Mb) and D8Mit213 (110.57 Mb), containing 25 annotated genes. Among the candidates, zinc-finger homeobox 3 (Zhfx3) was highly and almost exclusively expressed in adult SCN (Lein et al., 2007). We identified a point mutation in exon 9, resulting in a G → T transversion at position 6620 (Zfhx3 transcript, ENSMUST00000043896) (Figure 1C). The Sci mutation substitutes a phenylalanine for a valine at residue 1963 (V1963F) in a highly conserved region just upstream of the 17th zinc-finger motif (Figures 1D and 1E). Following the identification of the causative mutation, genotype and phenotype correlation demonstrated that the mutation causes homozygous lethality during embryonic development; therefore, only Zfhx3Sci/+ adult animals could be assessed phenotypically.

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