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. 2019 Jul 11;8:e43235. doi: 10.7554/eLife.43235

Figure 4. TIP60-deficiency evokes a circadian phenotype in mice and disrupts rhythmic clock gene expression in the SCN and in MEFs.

(A) Double-plotted actograms of control (Syt10Cre/+Tip60+/+) and mutant (Syt10Cre/+Tip60 fl/-, Syt10Cre/Cre Tip60fl/-) mice under 12 hr:12 hr light-dark and constant darkness conditions. Gray shadings indicate dark phases. (B) Free-running periods determined by χ2 periodogram analysis. AR: arrhythmic (***p<0.001, one-way ANOVA with Bonferroni post-test). (C) TIP60-immunoreactivity (ir) in the SCN of control (Syt10Cre/+Tip60+/+) and arrhythmic mutant (Syt10Cre/+Tip60 fl/-) mice. (D) Densitometric quantification of clock gene mRNA expression from radioactive in situ hybridization analysis of control (Syt10Cre/+Tip60+/+; black) and mutant (Syt10Cre/+Tip60 fl/-; gray) SCN sections (n = 3; two-way ANOVA, see Supplementary file 1). (E) Bioluminescence recordings of synchronized Tip60fl/-; Bmal1-LUC MEFs transduced with AdGFP (black) or AdCre (gray) (n = 4). (F) mRNA expression analysis of Dex synchronized Tip60fl/- MEFs transduced with either AdGFP (black) or AdCre (gray) (n = 3; two-way ANOVA, see Supplementary file 1). All data are shown as mean ± SD.

Figure 4.

Figure 4—figure supplement 1. Generation and validation of the Tip60-deficient mice and MEFs derived from such animals.

Figure 4—figure supplement 1.

(A) Targeting strategy for the Tip60 locus. Tip60 wildtype allele, targeting vector, targeted allele, floxed allele after removal of the PGK-Neo cassette, and the mutant Tip60 allele created by Cre-mediated recombination are shown. LoxP (black triangle) and FRT (open triangle) sites are marked. The arrows indicate the position of diagnostic primers P1-P3 (see Supplementary file 1). (B) PCR genotyping of Tip60 alleles. The wildtype allele produces a 331 bp amplicon (primers P1, (P2) while the floxed allele produces a 429 bp amplicon (P1, P2). Deletion of the LoxP-flanked region of the Tip60 locus leads to a 487 bp fragment (P1, P3). (C) TUNEL immunoreactivity on brain sections of Syt10Cre/+Tip60+/+ (control), arrhythmic Syt10Cre/+Tip60 fl/- animals, and Syt10Cre/+Tip60 fl/- sections treated in situ with DNase I. (D) mRNA expression of housekeeping genes in Tip60fl/- MEFs transduced with either AdGFP or AdCre, 24 hr after Dex synchronization. Data are mean ± SD (n = 3; ns > 0.05 and ***p<0.001, Student’s t-test). (E) Flow cytometric analysis of AdGFP, AdCre transduced, and heat-treated (3 min, 95°C) confluent Tip60fl/- MEFs. (F) Bioluminescence tracing of Dex-synchronized Bmal1-LUC MEFs transduced with AdGFP (black) or AdCre (red) (n = 4).