Extended Data Fig. 1: Splice mutations of Hdac4 and Hdac5 genes increased NREMS.

a. Haplotype analysis of the Slp2 pedigree for wake time based on C57B6J/C57B6N SNPs (N₂, n = 31). b. Exome sequencing identified single nucleotide changes within the LOD score peak (upper) and total wake time (lower) of each mouse. Blue and red bars indicate normal and short wake time, respectively. c. RT-PCR showing short Hdac4 transcripts specific to Hdac4^Slp2/+ mice. Hrt, heart; Ctx, cerebral cortex; Hyp, hypothalamus. Representative images from three biological replicates are shown. d. Immunoblot of brain homogenates of wild-type and Hdac4^Slp2/+ mice using anti-HDAC4 antibody. Anti-β-actin antibody was used as loading control. Quantitation of HDAC4 band intensity against β-actin (Right). Representative blots from three biological replicates are shown. e. Structures of HDAC4,5,7 and 9 with phosphorylation sites and deacetylase (DAC) domain. Numbers on the DAC domain indicate the region encoded by the skipped exon. f. Genomic DNA and transcripts of class IIa Hdacs. Red triangles indicate the splice acceptor site mutations (SA) of Hdac4, 5, 7, and 9 genes. Black triangles indicate stop codon produced due to the frameshift. g, h. RT-PCR of Hdac4^SA (g) and Hdac5^SA (h) mouse cortical tissues (3 mice per group). Shorter bands (asterisk) corresponding to exon skipping variants. i–k. Total wake time (i), NREMS episode duration (j), and total time spent in REMS (k) of Hdac4 mice (+/+, n = 26; SA/+, n = 26). l. NREMS delta power under the basal condition and after 6-hour sleep deprivation (SD) of Hdac4^SA mice. m. Change in NREMS delta power after sleep deprivation relative to each ZT under the basal condition of Hdac4 mice (+/+, n = 10; SA/+, n = 13). n, o. Relative NREMS delta power to the basal condition (n) and time spent in NREMS (o) for 3 h from ZT13. HDAC4 inhibitor, LMK235, was intracerebroventricularly administered at ZT11.5. n = 12. p–r. Total time spent in wake (p), NREMS episode duration (q), and total time spent in REMS (r) of Hdac5 mice (+/+, n = 17; SA/+, n = 21; SA/SA, n = 7). s, t. NREMS delta power under the basal condition and after 6-hour sleep deprivation (s) and change in NREMS delta power after sleep deprivation relative to each ZT under the basal condition (t) of Hdac5 mice (+/+, n = 14; SA/+, n = 21; SA/SA, n = 7). u, v. Diurnal NREMS delta density (u) and EEG power spectra during NREMS (v) of Hdac4^SA; Hdac5^SA mice. Hdac4^+/+; Hdac5^SA/+ mice (n = 16) and Hdac4^SA/+; Hdac5^SA/+ mice (n = 10). Data shown as mean ± s.e.m.; two-sided P values. Unpaired t-test (f, v); two-way ANOVA with Tukey’s test (i–k, p–r); mixed-effects model (m, t, u), with Dunnett’s test (n, o); two-way repeated measures ANOVA with Sidak’s (l) or Tukey’s test (s).

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