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A signalling pathway for transcriptional regulation of sleep amount in mice

Nature volume 612, pages 519–527 (2022)Cite this article

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Abstract

In mice and humans, sleep quantity is governed by genetic factors and exhibits age-dependent variation1,2,3. However, the core molecular pathways and effector mechanisms that regulate sleep duration in mammals remain unclear. Here, we characterize a major signalling pathway for the transcriptional regulation of sleep in mice using adeno-associated virus-mediated somatic genetics analysis4. Chimeric knockout of LKB1 kinase—an activator of AMPK-related protein kinase SIK35,6,7—in adult mouse brain markedly reduces the amount and delta power—a measure of sleep depth—of non-rapid eye movement sleep (NREMS). Downstream of the LKB1–SIK3 pathway, gain or loss-of-function of the histone deacetylases HDAC4 and HDAC5 in adult brain neurons causes bidirectional changes of NREMS amount and delta power. Moreover, phosphorylation of HDAC4 and HDAC5 is associated with increased sleep need, and HDAC4 specifically regulates NREMS amount in posterior hypothalamus. Genetic and transcriptomic studies reveal that HDAC4 cooperates with CREB in both transcriptional and sleep regulation. These findings introduce the concept of signalling pathways targeting transcription modulators to regulate daily sleep amount and demonstrate the power of somatic genetics in mouse sleep research.

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Fig. 1: LKB1–SIK3 signalling inhibits HDAC4/5 to regulate NREMS.
Fig. 2: ABC knockout of HDAC4 and/or HDAC5 increases NREMS amount and delta power.
Fig. 3: ABC expression HDAC4/5(CN) reduces NREMS amount and delta power.
Fig. 4: HDAC4 regulates NREMS amount in posterior hypothalamus.
Fig. 5: HDAC4 and CREB coordinate to regulate sleep downstream of SIK3 kinase.
Fig. 6: HDAC4 and CREB function cooperatively in transcriptional regulation.

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Data availability

All reagents and raw data generated in this study are available from the corresponding author upon reasonable request. The raw and processed RNA-seq data have been deposited at NCBI Gene Expression Omnibus under accession GSE182907. The raw and processed ChIP–seq data have been deposited at NCBI Gene Expression Omnibus under accession GSE182805. Because of their large size, the source data for mouse sleep phenotype analysis will be provided upon reasonable request. Source data are provided with this paper.

Code availability

The automated SleepV software for video-based analysis of sleep–wake behaviours4 has been deposited at GitHub (https://github.com/wochiguodong/SleepV.git.)4. The custom-designed C++ language-based automated sleep staging software for EEG/EMG data has been deposited at Zenodo (https://doi.org/10.5281/zenodo.7102778).

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Acknowledgements

We thank M. Luo and F. Shao for sharing reagents; Y. Zhou, M. Zhao, Z. Li, W. Min, Y. Zhuang, Y. Sun, H. Huang, J. Wang and Y. Yin for technical assistance; and X. Wang, F. Shao, T. Chen, T. Han and M. Xu for discussion and comments on the manuscript. This work was supported by the National Major Project of China Science and Technology Innovation 2030 for Brain Science and Brain-Inspired Technology (2021ZD0203400 to Q.Liu), the innovation grant (Z181100001318004 to Q.Liu), and institution grants from the Beijing Municipal Commission of Science and Technology Commission and Chinese Ministry of Science and Technology.

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Author notes

  1. These authors contributed equally: Rui Zhou, Guodong Wang

Authors and Affiliations

  1. College of Biological Sciences, China Agriculture University, Beijing, China

    Rui Zhou & Meimei Liao

  2. National Institute of Biological Sciences, Beijing (NIBS), Beijing, China

    Rui Zhou, Guodong Wang, Qi Li, Fanxi Meng, Can Liu, Rui Gan, Meimei Liao, Junjie Xu, Di Sang, Xue Gao, Shuang Zhou, Kejia Wu, Quanzhi Sun, Ying Guo, Chongyang Wu, Zhiyu Chen, Lin Chen, Bihan Shi, Haiyan Wang, Xia Wang, Huaiye Li, Tao Cai, Bin Li, Fengchao Wang, Eric Erquan Zhang & Qinghua Liu

  3. Graduate School of Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China

    Guodong Wang, Fanxi Meng & Di Sang

  4. Tsinghua Institute of Multidisciplinary Biomedical Research (TIMBR), Tsinghua University, Beijing, China

    Qi Li, Tao Cai, Fengchao Wang, Eric Erquan Zhang & Qinghua Liu

  5. Peking University–Tsinghua University–NIBS Joint Graduate Program, School of Life Sciences, Tsinghua University, Beijing, China

    Can Liu

  6. Department of Anesthesiology, The Second Affiliated Hospital, Chongqing Medical University, Chongqing, China

    Dapeng Ju

  7. College of Life Sciences, Beijing Normal University, Beijing, China

    Junjie Xu & Shuang Zhou

  8. Department of Anatomy, Faculty of Medicine, Toho University, Tokyo, Japan

    Hiromasa Funato

  9. International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, Tsukuba, Japan

    Hiromasa Funato, Masashi Yanagisawa & Qinghua Liu

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  1. Rui Zhou
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Contributions

R.Z., G.W., Q.Li and J.X. executed most of the experiments with input from Q.Liu, E.E.Z., H.F. and M.Y. F.W. helped to generate multiple mouse strains. X.G., S.Z., Z.C., L.C. and B.S. performed EEG/EMG recording and analysis. R.Z. and D.J. performed RNA-seq and ChIP–seq analyses with help from B.L., R.G. and T.C. D.S. performed ex vivo recording of SCN of Per2::Luc mice. Q.S., Y.G. and C.W. performed AAV packaging and purification. F.M. and C.L. performed stereotactic injection of AAVs in the hypothalamus and thalamus. H.W., X.W. and H.L. helped with mouse husbandry. R.Z., G.W. and Q.Liu prepared the figures and Q.Liu wrote the manuscript.

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Correspondence to Qinghua Liu.

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Extended data figures and tables

Extended Data Fig. 1 LKB1-SIK3 signalling regulates NREMS by suppressing HDAC4/5.

a, Representative images showing co-immunostaining of LKB1 (green) and mCherry or Cre (HA, red) neurons in the cortex, hippocampus, thalamus, midbrain and medulla of AAV-hSyn-mCherry or AAV-hSyn-cre injected Lkb1flox/flox mice. Scale bars, 50 μm. b and c, REMS (b) or Wake (c) amount of AAV-hSyn-mCherry (control (Ctrl), n = 11) or AAV-hSyn-cre (Lkb1KO, n = 12) injected Lkb1flox/flox mice. d-g, Hourly NREMS time (d), EEG power spectra analysis (e), and REMS (f) or Wake (g) amount of Lkb1flox/flox mice co-injected with AAV-hSyn-cre and AAV-hSyn-mCherry (Lkb1KO/mCherry, n = 7), AAV-hSyn-SIK3-ST221E (Lkb1KO/SIK3-S(T221E), n = 7), or AAV-hSyn-SLP-ST221E (Lkb1KO/SLP-S(T221E), n = 8). h, Schematic of H4N/H5N-VP16 interfering with transcriptional activities of endogenous HDAC4/5 proteins. i, Representative images (two independent experiments with similar results) showing the effects of H4N-VP16 and H5N-VP16 on expression of co-transfected 3x MEF2 response element (MRE)-copGFP reporter gene in AAVpro 293T cells. Scale bars, 200 μm. j-m, Hourly NREMS time (j), EEG power spectra analysis (k), and REMS (l) or Wake (m) amount of Lkb1flox/flox mice injected with AAV-hSyn-cre and AAV-hSyn-mCherry (Lkb1KO/mCherry, n = 7) or AAV-hSyn-H4N/H5N-VP16 (Lkb1KO/H4N/H5N-VP16, n = 6). Data are mean ± s.e.m. Two-way ANOVA with Sidak’s test (b, c, l and m); mixed-effects model (d and j); one-way ANOVA with Dunnett’s test (e); two-way ANOVA with Dunnett’s test (f and g); and two-tailed unpaired t-test (k). n refers to the number of biological replicates.

Source data

Extended Data Fig. 2 ABC knockout of HDAC4/5 increases NREMS amount and delta power.

a-d, Hourly NREMS time (a), EEG power spectra analysis (b), and REMS (c) or Wake (d) amount of C57BL/6J mice injected with AAV-hSyn-mCherry (n = 15), AAV-hSyn-H4N-VP16 (n = 7) or AAV-hSyn-H4N/H5N-VP16 (n = 7). e, Schematic of ABC-KO of Hdac4 or Hdac5 by triple-target CRISPR. f and g, Immunoblotting (f) and quantification (g) of HDAC4 and HDAC5 proteins in whole brain lysates of Cas9 mice (n = 3 per group) injected with AAV-sgRNANT (ABC-NT), AAV-sgRNAHD4 (ABC-Hdac4KO), AAV-sgRNAHD5 (ABC-Hdac5KO), or AAV-sgRNAHD4 and AAV-sgRNAHD5 (ABC-Hdac4/5DKO). h-m, NREMS (h), REMS (i) or Wake (j) amount, hourly NREMS time (k), delta power density (l) and EEG power spectra analysis (m) of ABC-NT, ABC-Hdac4KO, ABC-Hdac5KO, or ABC- Hdac4/5DKO mice (n = 8 per group). n-q, Hourly NREMS time (n), EEG power spectra analysis (o), and REMS (p) or Wake (q) amount of AAV-hSyn-mCherry (ABC-Hdac4WT, n = 10) or AAV-hSyn-cre (ABC-Hdac4KO, n = 9) injected Hdac4flox/flox mice and AAV-hSyn-mCherry (ABC-Hdac5WT, n = 11) or AAV-hSyn-cre (ABC-Hdac5KO, n = 8) injected Hdac5flox/flox mice. Data are mean ± s.e.m. Mixed-effects model (a, k, l and n); one-way ANOVA with Dunnett’s test (b, g and m); two-way ANOVA with Sidak’s test (c-d, h—j and p-q); and two-tailed unpaired t-test (o). See Supplementary Fig. 1 for gel source data. n refers to the number of biological replicates.

Source data

Extended Data Fig. 3 ABC-Hdac4KO mice display normal vision and behaviors.

a-d, Hourly NREMS time (a), EEG power spectra analysis (b), and REMS (c) or Wake (d) amount of AAV-hSyn-rTTA and AAV-TRE-cre co-injected Hdac4flox/flox (iHdac4KO) mice without (-Dox, n = 6) or with (+Dox, n = 8) doxycycline treatment. e, Representative images (two independent experiments with similar results) showing co-immunostaining of S-Opsin (green) and Rhodopsin (red) in the retina of AAV-hSyn-mCherry (ABC-Ctrl) or AAV-hSyn-cre (ABC-Hdac4KO) injected Hdac4flox/flox mice. Scale bars, 20 μm. f, Representative images showing immunostaining of c-Fos (green) in the SCN of ABC-Ctrl or ABC-Hdac4KO mice after 15 min of light exposure during the dark phase. Scale bars, 50 μm. g, Quantification of light-induced c-Fos expression in (f) (n = 3). h-l, Fear conditioning (h), open field (i, j), sucrose preference (k), and rotarod (l) behavioral tests of ABC-Ctrl (n = 7) and ABC-Hdac4KO (n = 8) mice. Data are mean ± s.e.m. Mixed-effects model (a); two-way ANOVA with Sidak’s test (c-d, i, and k-l); and two-tailed unpaired t-test, two tailed (b, g-h and j). n refers to the number of biological replicates.

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Extended Data Fig. 4 ABC expression HDAC4(CN) or HDAC5(CN) reduces NREMS amount and delta power.

a-d, Hourly NREMS time (a), EEG power spectra analysis (b), and REMS (c) or Wake (d) amount of AAV-hSyn-mCherry (n = 9), AAV-hSyn-HDAC4WT (n = 10) or AAV-hSyn-HDAC4CN (n = 8) injected C57BL/6J mice. e-h, Hourly NREMS time (e), EEG power spectra analysis (f), and REMS (g) or Wake (h) amount of AAV-hSyn-mCherry (n = 18), AAV-hSyn-HDAC5WT (n = 10) or AAV-hSyn-HDAC5CN (n = 9) injected C57BL/6J mice. i-l, Hourly NREMS time (i), EEG power spectra analysis (j), and REMS (k) or Wake (l) amount of AAV-hSyn-mCherry (n = 8), AAV-hSyn-HDAC7WT (n = 8) or AAV-hSyn-HDAC7CN (n = 9) injected C57BL/6J mice. m-p, Hourly NREMS time (m), EEG power spectra analysis (n), and REMS (o) or Wake (p) amount of AAV-hSyn-mCherry (n = 8), AAV-hSyn-HDAC4CN (n = 9), AAV-hSyn-HDAC4CN/D934N (n = 9) or AAV-hSyn-HDAC4CN/D840N (n = 6) injected C57BL/6J mice. Data are mean ± s.e.m. Mixed-effects model (a, e and m); one-way ANOVA with Dunnett’s test (b, f, j and n); and two-way ANOVA with Dunnett’s test (c-d, g-h, k-l and o-p). n refers to the number of biological replicates.

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Extended Data Fig. 5 Regulation of HDAC4/5 by sleep-wake cycle and homeostatic sleep response of ABC-Hdac4KO and ABC-Hdac4CN mice.

a and b, Immunoblotting (a) and quantification (b) of S245 phosphorylation of HDAC4/5 in total lysates of cortex from C57BL/6J mice at ZT2 and ZT14 (n = 3). c and d, Immunoblotting (c) and quantification (d) of nuclear:cytoplasmic ratio of HDAC4/5 in cytoplasmic and nuclear extracts of cortex from C57BL/6J mice at ZT2 and ZT14 (n = 3). e and i, Immunoblotting of total lysates of cortex (e) or hypothalamus (i) from 6-h slept (S6) and sleep-deprived (SD6) C57BL/6J mice. f and j, Quantification of S245 phosphorylation of HDAC4/5 in (e) and (i) (n = 3). g and k, Immunoblotting of cytoplasmic and nuclear extracts of cortex (g) or hypothalamus (k) from S6 and SD6 mice. h and l, Quantification of nuclear:cytoplasmic ratio of HDAC4/5 in (g) and (k) (n = 3). m-p, Hourly NREMS time (m) or delta power density (o) and accumulated NREMS time (n) or delta power (p) change of AAV-hSyn-GFP (Ctrl, n = 8) or AAV-hSyn-cre (Hdac4KO, n = 7) injected Hdac4flox/flox mice before (BS) and after sleep deprivation (SD). q-t, Hourly NREMS time (q) or delta power density (s) and accumulated NREMS time (r) or delta power change (t) of AAV-hSyn-mCherry (n = 9), AAV-hSyn-HDAC4WT (n = 10) or AAV-hSyn-HDAC4CN (n = 7) injected C57BL/6J mice before (BS) and after sleep deprivation (SD). Data are mean ± s.e.m. Two-tailed unpaired t-test (b, f, j and p); two-way ANOVA with Sidak’s test (d, h and l); mixed-effects model (m and q); and one-way ANOVA with Dunnett’s test (t). See Supplementary Fig. 1 for gel source data. n refers to the number of biological replicates.

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Extended Data Fig. 6 HDAC4/5 regulates NREMS amount in posterior hypothalamus without altering circadian clock.

a—d, Hourly NREMS time (a), EEG power spectra analysis (b), and REMS (c) or Wake (d) amount of C57BL/6J mice injected with AAV-hSyn-mCherry (n = 6), AAV-hSyn-HDAC4CN (n = 7) or AAV-hSyn-H4N/H5N-VP16 (n = 7) in posterior hypothalamus. e and f, Schematic and representative images showing HDAC4(CN) expression in SCN following stereotactic injection of AAV-hSyn-HDAC4CN in POA (n = 6) or posterior hypothalamus (n = 7). Scale bars, 1 mm. g, Representative ex vivo SCN slice bioluminescence recording of AAV-hSyn-mCherry, AAV-hSyn-HDAC4CN, or AAV-hSyn-H4N/H5N-VP16VP16 injected Per2::Luc mice (n = 3), and the short period control Cry1−/−;Per2::Luc mice (n = 1). Circadian time 0 was set as 24 h post medium change. h, Quantitative analysis of circadian periods in (g). Solid line represents the mean of SCN bioluminescence recording of AAV-hSyn-mCherry injected Per2::Luc mice, whereas dashed lines represent cut-offs of abnormality (≥ three standard deviations). i, RNAi knockdown of all four genes caused no clock phenotype in Bmal1:dLuc U2OS cells. Data were re-analyzed from a previous study30. siGL2 serves as the negative and siCRY2 as the positive controls for period changes. Circadian time 0 was set as 24 h post-medium change. j, Quantitative analyses of each knockdown result in (i) using Multicycle software. The solid line represents the mean of negative control, and dash lines indicate the cut-offs of abnormality (≥ three standard deviations). Each dot represents one experiment and duplicate experiments are applied for each gene. Data are mean ± s.e.m. Mixed-effects model (a); two-way ANOVA with Dunnett’s test (c and d); and one-way ANOVA with Dunnett’s test (h and j). n refers to the number of biological replicates.

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Extended Data Fig. 7 HDAC4(CN) regulates NREMS amount in a CREB-dependent manner.

a, Schematics of wild-type and mutant MEF2A, MEF2C,MEF2D, FOXO1 and FOXO3. b-e, Hourly NREMS time (b), EEG power spectra analysis (c), and REMS (d) or Wake (e) amount of AAV-hSyn-eGFP (n = 21), AAV-hSyn-CREB (n = 14), or AAV-hSyn-A-CREB (n = 21) injected C57BL/6J mice. f, Representative images showing co-immunostaining of A-CREB-T7 (purple), HDAC4(CN)-HA (red) and NeuN (green) of cortical and thalamic sections from C57BL/6J mice co-injected with AAV-hSyn-HDAC4CN and AAV-hSyn-A-CREB. Scale bars, 20 μm. g, Quantification of the percentage of HDAC4(CN)+ neurons that co-express A-CREB and vice versa in the cortex and thalamus in (f) (n = 6). h-k, Hourly NREMS time (h), EEG power spectra analysis (i), and REMS (j) or Wake (k) amount of C57BL/6J mice injected with AAV-hSyn-mCherry (n = 6), AAV-hSyn-HDAC4CN and AAV-hSyn-mCherry (HD4(CN)+mCherry, n = 7), or AAV-hSyn-HDAC4CN and AAV-hSyn-A-CREB (HD4(CN)+A-CREB, n = 6). Data are mean ± s.e.m. Mixed-effects model (b and h); and two-way ANOVA with Dunnett’s test (d-e and j-k). n refers to the number of biological replicates.

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Extended Data Fig. 8 HDAC4 and CREB coordinately regulate sleep downstream of SIK3 kinase.

a, Immunoblotting of brain lysates from AAV-hSyn-eGFP injected wild-type (WT+eGFP) mice and AAV-hSyn-eGFP (SLP+eGFP), AAV-hSyn-CREB (SLP+CREB), or AAV-hSyn-HDAC4CN (SLP+HD4(CN)) injected Sik3E13∆/+ mice. b, Daily sleep time in WT+eGFP (n = 10), SLP+eGFP (n = 9), SLP+CREB (n = 9), or SLP+HD4(CN) (n = 8) mice. c-f, Hourly NREMS time (c), EEG power spectra analysis (d), and REMS (e) or Wake (f) amount of WT+eGFP (n = 10), SLP+eGFP (n = 9), SLP+CREB (n = 6), or SLP+HD4(CN) (n = 8) mice. g, Immunoblotting of brain lysates from AAV-hSyn-eGFP (SLP+eGFP), AAV-hSyn-HDAC4WT (SLP+HD4(WT)), or AAV-hSyn-HDAC4S245A (SLP+HD4(S245A)) injected Sik3E13∆/+ mice (two independent experiments with similar results). h-m, Hourly NREMS time (h) and delta power density (i), EEG power spectra analysis (j), and NREMS (k), REMS (l), or Wake (m) amount of SLP+eGFP (n = 12), SLP+HD4(WT) (n = 8), or SLP+HD4(S245A) (n = 8) mice. n, Immunoblotting of brain lysates from AAV-hSyn-eGFP (SLP+eGFP), AAV-hSyn-HDAC5WT (SLP+HD5(WT)), or AAV-hSyn-HDAC5CN (SLP+HD5(CN)) injected Sik3E13∆/+ mice (two independent experiments with similar results). o-t, Hourly NREMS time (o) and delta power density (p), EEG power spectra analysis (q), and NREMS (r), REMS (s) or Wake (t) amount of SLP+eGFP (n = 8), SLP+HD5(WT) (n = 8), or SLP+HD5(CN) (n = 9) mice. Data are mean ± s.e.m. One-way ANOVA with Dunnett’s test (b, d and j); mixed-effects model (c, h, i and o); and two-way ANOVA with Dunnett’s test (e-f, k-m and r-t). See Supplementary Fig. 1 for gel source data. n refers to the number of biological replicates.

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Extended Data Fig. 9 HDAC4 and CREB specifically rescue transcriptomic changes in the cortex and hypothalamus of Sik3E13∆/+ mice.

a, Venn diagram showing the common and unique sets of DEGs among the cortex (CTX), thalamus (THA) and hypothalamus (HPT) of SLP+eGFP mice. b, Venn diagrams showing the numbers of unique and overlapped DEGs among the SLP+eGFP, SLP+CREB and SLP+HDAC4(CN) mouse cortex, thalamus and hypothalamus, respectively. To identify DEGs, we compare the transcriptomes of WT+eGFP vs SLP+eGFP samples as well as the transcriptomes of SLP+eGFP vs SLP+CREB or SLP+HDAC4(CN) samples (n = 4 per group). c, A table listing overlapped DEGs in the cortex, thalamus and hypothalamus, respectively, among the SLP+eGFP (normalized to WT+eGFP) and SLP+CREB and SLP+HDAC4(CN) (normalized to SLP+eGFP) brain samples.

Extended Data Fig. 10 HDAC4 and CREB function cooperatively in transcriptional regulation.

a, Volcano plots of RNA-seq data from ABC-HDAC4(CN) or ABC-CREB mouse brain tissues (n = 4 for thalamus or hypothalamus, n = 3 for cortex) by binomial test via edgeR. The horizontal dashed line shows the two-sided unadjusted P = 0.05 as cutoff. The vertical dashed lines show +/-0.38-log2 fold changes as cutoff. Orange and blue dots indicate up- or down-regulated DEGs. b, RNA-seq heatmaps showing DEGs in HDAC4(CN) brain samples that are documented as target genes of MEF2, RUNX2, FOXO, or SRF, respectively. c, Percentage of CREB and/or HDAC4(CN)-bound genes (by ChIP-seq) in the DEGs of ABC-HDAC4(CN), ABC-CREB or both brain samples (by RNA-seq). d, Venn diagrams showing the numbers of unique and overlapped DEGs by comparing the RNA-seq and ChIP-seq results. e, Circadian oscillation of the overlapped DEGs (by RNA-seq) or HDAC4(CN)/CREB-bound genes (by ChIP-seq) based on previous circadian transcriptome analysis of the synaptosome or forebrain47. f, A model for transcriptional regulation of sleep amount in mice.

Supplementary information

Supplementary Fig. 1

The unprocessed Immunoblots associated with the data presented in main figures and extended data figures.

Reporting Summary

Supplementary Table 1

The DEGs of ABC-HDAC4CN or ABC-CREB mouse brain tissues (relative to the corresponding ABC-mCherry mouse brain tissues). Linked to Fig. 6.

Supplementary Table 2

The overlapping DEGs in the cortex (CTX), hypothalamus (HPT) and thalamus (THA) of ABC-HDAC4CN and ABC-CREB mouse brains (relative to the corresponding ABC-mCherry mouse brain tissues). Linked to Fig. 6.

Source data

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Zhou, R., Wang, G., Li, Q. et al. A signalling pathway for transcriptional regulation of sleep amount in mice. Nature 612, 519–527 (2022). https://doi.org/10.1038/s41586-022-05510-6

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