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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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RNA Sequencing Reveals Transcripts Differentially Expressed in Zfhx3Sci/+ SCN(A) Venn diagrams depicting the number of differentially regulated transcripts, at ZT3 and ZT15, with a log2 fold change of >1 between Zfhx3Sci/+ and Zfhx3Sci/+ SCN. Genes were considered differentially expressed if they passed significance (left, p < 0.05; right, q < 0.05) for at least one of the three analysis methods (EdgeR, DESeq, or Cufflinks).(B) Transcripts with significant q values at each time point were used to populate heatmaps. For each time point, a heatmap shows the average expression in reads per kilobase per million (RPKM) for each transcript. wt, wild-type; mut, mutant.(C–E) Representative box plots of gene expression for (C) Vip, (D) Prokr2, and (E) Vipr2 (q < 0.05).See also Tables S1 and S2.
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fig2: RNA Sequencing Reveals Transcripts Differentially Expressed in Zfhx3Sci/+ SCN(A) Venn diagrams depicting the number of differentially regulated transcripts, at ZT3 and ZT15, with a log2 fold change of >1 between Zfhx3Sci/+ and Zfhx3Sci/+ SCN. Genes were considered differentially expressed if they passed significance (left, p < 0.05; right, q < 0.05) for at least one of the three analysis methods (EdgeR, DESeq, or Cufflinks).(B) Transcripts with significant q values at each time point were used to populate heatmaps. For each time point, a heatmap shows the average expression in reads per kilobase per million (RPKM) for each transcript. wt, wild-type; mut, mutant.(C–E) Representative box plots of gene expression for (C) Vip, (D) Prokr2, and (E) Vipr2 (q < 0.05).See also Tables S1 and S2.

Mentions: We used RNA sequencing to identify transcriptional targets of ZFHX3. RNA was extracted from SCN tissue punches from Zfhx3Sci/+ and Zfhx3+/+ animals at zeitgeber time (ZT)3 and ZT15 (n = 3 for each time by genotype combination). RNA sequencing revealed that 242 genes were differentially expressed at either one or both time points (log2 fold change > 1, p < 0.05) (Table S1), with 28 of those surviving multiple testing correction (q < 0.05 in at least two quantification methods). At this more stringent level, 19 genes were differentially expressed at ZT3 and 13 at ZT15, while 4 genes were affected at both time points (Figure 2A). The majority of genes (17 of 28) showed a decrease in expression in Zfhx3Sci/+ (Figure 2B). Interestingly, the expression of a number of circadian-related neuropeptides was decreased in Zfhx3Sci/+ SCN, including Vip, its receptor (Vipr2), and prokineticin receptor 2 (Prokr2) (Figures 2C–2E). We conducted gene ontology (GO) enrichment analysis using all of the differentially expressed genes (q < 0.05 in at least one quantification method, n = 169) and found significant enrichment for a number of GO terms including neuron differentiation, regulation of cellular metabolic process, and regulation of cell proliferation (Table S2).


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)

RNA Sequencing Reveals Transcripts Differentially Expressed in Zfhx3Sci/+ SCN(A) Venn diagrams depicting the number of differentially regulated transcripts, at ZT3 and ZT15, with a log2 fold change of >1 between Zfhx3Sci/+ and Zfhx3Sci/+ SCN. Genes were considered differentially expressed if they passed significance (left, p < 0.05; right, q < 0.05) for at least one of the three analysis methods (EdgeR, DESeq, or Cufflinks).(B) Transcripts with significant q values at each time point were used to populate heatmaps. For each time point, a heatmap shows the average expression in reads per kilobase per million (RPKM) for each transcript. wt, wild-type; mut, mutant.(C–E) Representative box plots of gene expression for (C) Vip, (D) Prokr2, and (E) Vipr2 (q < 0.05).See also Tables S1 and S2.
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Related In: Results  -  Collection

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fig2: RNA Sequencing Reveals Transcripts Differentially Expressed in Zfhx3Sci/+ SCN(A) Venn diagrams depicting the number of differentially regulated transcripts, at ZT3 and ZT15, with a log2 fold change of >1 between Zfhx3Sci/+ and Zfhx3Sci/+ SCN. Genes were considered differentially expressed if they passed significance (left, p < 0.05; right, q < 0.05) for at least one of the three analysis methods (EdgeR, DESeq, or Cufflinks).(B) Transcripts with significant q values at each time point were used to populate heatmaps. For each time point, a heatmap shows the average expression in reads per kilobase per million (RPKM) for each transcript. wt, wild-type; mut, mutant.(C–E) Representative box plots of gene expression for (C) Vip, (D) Prokr2, and (E) Vipr2 (q < 0.05).See also Tables S1 and S2.
Mentions: We used RNA sequencing to identify transcriptional targets of ZFHX3. RNA was extracted from SCN tissue punches from Zfhx3Sci/+ and Zfhx3+/+ animals at zeitgeber time (ZT)3 and ZT15 (n = 3 for each time by genotype combination). RNA sequencing revealed that 242 genes were differentially expressed at either one or both time points (log2 fold change > 1, p < 0.05) (Table S1), with 28 of those surviving multiple testing correction (q < 0.05 in at least two quantification methods). At this more stringent level, 19 genes were differentially expressed at ZT3 and 13 at ZT15, while 4 genes were affected at both time points (Figure 2A). The majority of genes (17 of 28) showed a decrease in expression in Zfhx3Sci/+ (Figure 2B). Interestingly, the expression of a number of circadian-related neuropeptides was decreased in Zfhx3Sci/+ SCN, including Vip, its receptor (Vipr2), and prokineticin receptor 2 (Prokr2) (Figures 2C–2E). We conducted gene ontology (GO) enrichment analysis using all of the differentially expressed genes (q < 0.05 in at least one quantification method, n = 169) and found significant enrichment for a number of GO terms including neuron differentiation, regulation of cellular metabolic process, and regulation of cell proliferation (Table S2).

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