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Mammalian PERIOD2 regulates H2A.Z incorporation in chromatin to orchestrate circadian negative feedback

Abstract

Mammalian circadian oscillators are built on a feedback loop in which the activity of the transcription factor CLOCK–BMAL1 is repressed by the PER–CRY complex. Here, we show that murine Per/ fibroblasts display aberrant nucleosome occupancy around transcription start sites (TSSs) and at promoter-proximal and distal CTCF sites due to impaired histone H2A.Z deposition. Knocking out H2A.Z mimicked the Per null chromatin state and disrupted cellular rhythms. We found that endogenous mPER2 complexes retained CTCF as well as the specific H2A.Z-deposition chaperone YL1—a component of the ATP-dependent remodeler SRCAP and p400–TIP60 complex. While depleting YL1 or mutating chaperone-binding sites on H2A.Z lengthened the circadian period, H2A.Z deletion abrogated BMAL1 chromatin recruitment and promoted its proteasomal degradation. We propose that a PER2-mediated H2A.Z deposition pathway (1) compacts CLOCK–BMAL1 binding sites to establish negative feedback, (2) organizes circadian chromatin landscapes using CTCF and (3) bookmarks genomic loci for BMAL1 binding to impinge on the positive arm of the subsequent cycle.

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Fig. 1: PER-TKO cells have disrupted patterns of cell growth.
Fig. 2: Altered nucleosome occupancy around PER2- and BMAL1-binding sites and TSS in PER-TKO cells.
Fig. 3: A PER complex mediates H2A.Z deposition on chromatin.
Fig. 4: PER2 interacts with YL1 to determine circadian period.
Fig. 5: H2A.Z deletion mimics nucleosome occupancy changes at TSS and CTCF sites as in Per−/− cells.
Fig. 6: H2A.Z nucleosome occupancy at CLOCK–BMAL1 sites peaks during the early repressive phase of circadian feedback.
Fig. 7: H2A.Z modulates BMAL1 chromatin recruitment and stability to regulate circadian period and amplitude.
Fig. 8: PER-dependent H2A.Z incorporation regulates BMAL1 DNA binding and its stability and establishes NORs to repress transcription.

Data availability

Data sets generated by the lab are available in the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo) under the accession number GSE153939.

H2A.Z, H3K27me3 and H3K9me2 positions were obtained from GSE51505. H2A.Z coverage over several ZT points was obtained from GSE47145. PER1, PER2 and BMAL1 positions were obtained from GSE39860. CTCF sites were taken from GSE102997.

The reference mouse genome used in the study was mm10, gencode vM22 from UCSC (https://hgdownload.soe.ucsc.edu/goldenPath/mm10/bigZips/). Source data are provided with this paper.

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Acknowledgements

The work was supported by an ATIP Avenir installation grant, an ENS emergence award, Projét Fondation ARC (PJA 20191209722) and a La Ligue contre le Cancer, Comité de l’Isère mono-équipe grant (R15026CC) to K. P. E. G. F. was funded by the ATIP Avenir postdoctoral fellowship, F. A. was supported by a grant from CLARA Oncostarter (CVPPRCAN000174) and K. T. was funded by an INSERM Plan Cancer in Physics (CP17067-00) grant to C. A. and K. P.. We would like to thank S. Yamazaki for providing us with PER-TKO cells, to D. Weaver for reaching out to S. Yamazaki; S. Dimitrov for access to the H2A.Z conditional mouse model; M.-P. Felder Schmittbuhl and U. Albrecht for Per1/Per2 Brdm/ mice; S. Brown for Bmal1–luciferase lentiviral plasmid; A. Kramer for U2OS Bmal1–luc cells; C. Weitz for BMAL1 antibody; S. Romand for generating Per constructs; A. Ors for primer design and comments; M. Fackeure, S. Arenales and O. Bartle for technical help; B. Gillet and S. Hughes at the IGFL sequencing platform (PSI) for Illumina sequencing; and team members, A. Garces, Y. Ghavi-Helm and F. Leulier for discussions pertaining to the manuscript. We gratefully acknowledge support from C. Kabir at IGFL and the Pôle Scientifique de Modélisation Numérique at ENS de Lyon for computing resources. We particularly would like to extend our sincere gratitude to K. Koronowski and J. Smith, members of the laboratory of P. Sassone-Corsi, a pioneer in the field, for their help with shipping liver specimens from the Liver specific Bmal1-/- KO mice.

Author information

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Contributions

K. P. conceived and directed the project with inputs from K. T., F. A. and E. G. F. K. T., F. A., E. G. F., R. S., I. S., D. L. and K. P. conducted the experiments. K. T. and R. S. performed all the bioinformatics analyses. T. S. and P.-S.W. generated and isolated tissues from liver-specific Bmal1−/− mice. K. P. wrote the manuscript with input from C. A., K. T., F. A., E. G. F., D. L., S. T., P.-S.W. and S. A. B.

Corresponding author

Correspondence to Kiran Padmanabhan.

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The authors declare no competing interests.

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Nature Structural and Molecular Biology thanks Paul Wade, and Ueli Schibler for their contribution to the peer review of this work. Beth Moorefield was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Characterization of cell lines by Sir-DNA dye incorporation.

A) Left panel, wildtype, Per DKO, Per TKO cells and mPer2 rescue fibroblasts were treated with DMSO or 1mM of siR-DNA live cell Fluorogenic Labelling Probe (SpirochromeSiR-DNA) and analyzed by flow cytometry (representative of N=3 experiments). Right panel, Results of three independent experiments are represented as mean±SD of the geometric mean. A one-tailed t-test was performed. B) Total genomic DNA from Per TKO cells, mPer2 rescue cell lines, wildtype fibroblasts and Per DKO cells was extracted and quantified. Results are expressed as mean±SD of N=3 independent experiments. A one-tailed t-test was performed. C) Sequential salt extraction (80-600mM NaCl) of wildtype and Per TKO nuclei were performed. Extracts as well as the final pellet were blotted for H2A.Z and H3. Results are representative of 4 experiments.

Source data

Extended Data Fig. 2 Nucleosome occupancy analysis (low MNase-seq) in H2A.Z DKO and Per TKO cells.

A) Tapestation profile of S0 nucleosomal fraction for wildtype, Per TKO and H2A.Z DKO cells prior to library preparation and sequencing. B) Average profile of low MNase-seq signal enrichment in wildtype Per TKO and H2AZ DKO cells at PER1 binding site, and at some repetitive elements (LTR, SINE and simple repeats), at TSS of expressed genes (solid lines) or non-expressed genes (dashed lines) (RNAseq analysis, this study, Extended Data Fig. 6), and around H2A.Z nucleosome peaks in heterochromatin at H3K9me2 or H3K27me3 loci. H2A.Z, H3K27me3 and H3K9me2. N= 2 biological replicates were performed. C) Average profiles of signal enrichment for low and high MNase-seq (top and bottom, respectively) in wildtype fibroblasts and Per TKO cells for genes classified in GO term gene cluster ‘Estrogen pathway’.

Extended Data Fig. 3 Nucleosome occupancy analysis (high MNase-seq) in H2A.Z DKO and Per TKO cells.

Average profile of high MNase-seq signal enrichment in wildtype, Per TKO and H2AZ DKO cells at BMAL1, PER1 and PER2 binding site, at TSS of all genes, at H3K9me2 and H3K27me3 marked heterochromatin and at the border of repetitive elements (LINE, LTR, Simple repeats and SINE).

Extended Data Fig. 4 Interdependence of PER2 and H2A.Z loading in chromatin.

A) ChIP-seq profiles for H2A.Z, PER2 and BMAL1 in mouse liver at BMAL1-PER2 binding sites during the repressive phase. B) Western blot analysis showing the levels of H2A.Z in chromatin of different cell lines. H2A.Z DKO cells, two different cultures of Per TKO, mPer2-rescue or Bmal1−/− cell lines are represented. H3 was used as loading control. Results are representative of 3 experiments. C) Nuclear fractionation of cells into nucleoplasm (Np), chromatin fraction using MNase digestion (Ch1), chromatin fraction using MNase digestion with sonication (Ch2) and insoluble pellets post centrifugation (P1, P2). Fractions were analyzed for PER2, BMAL1, H2A.Z and H3. Results are representative of n>4 experiments. D) Bioluminescence profiles from U2OS cells stably expressing different shRNA constructs targeting YL1 (shYL1a (green), shYL1b (red) and shYL1c (purple)) compared to a scrambled control (shSCR). E) Images of cells at the end of lumicycle analysis (5X and 10X mag), Scale bar is 100 µm. Results are representative of 3 experiments. F) Immunoblot for H2A.Z in cells from the lumicycle at the end of the bioluminescence analysis, βActin was used as a loading control. Results are representative of 2 experiments. G) Western blot analysis for total cell extracts or for chromatin fraction for PER2, BMAL1 and H2A.Z on wildtype cells or following 3, 5 or 7 days of tamoxifen treatment. βActin or Histone H3 were used as loading controls. Results are representative of 4 experiments. H) Western blot analysis for total cell extracts or for the chromatin fraction for BMAL1, H2A.Z and H3 on wildtype cells (WT) and H2A.Z DKO treated with doxycycline to induce H2A.Z1. Non doxycycline treated cells were used as control. Results are representative of 2 experiments.

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Extended Data Fig. 5 H2A.Z regulation of circadian period and amplitude.

A) Period determination in wildtype and H2A.Z DKO fibroblasts N=6 independent experiments (for for example, Fig. 5a), mean±SEM and p-values (t-test, paired, two-tailed) are indicated. B) Upper panel. Tamoxifen treated H2A.Z1 KO cells or H2A.Z2 KO cells (day 4+TAM) total RNA were subjected to RT-qPCR analysis for the expression of H2afz, H2afz, Bmal1 and Per2. The expression levels are normalized with Rps9 gene and compared to the wildtype cells. Results are expressed as fold change mean±SD in N=3 independent experiments. A two tailed paired t-test was performed (p-values<0.05 are indicated). Lower panel. Untreated wildtype cells (black traces), H2A.Z1 KO or H2A.Z2 KO cells (day 4+TAM, red traces) expressing a Bmal1:luciferase reporter were synchronized and bioluminescence was recorded for 5 days. C) Left panel. Bioluminescence recordings from wildtype fibroblasts, or rescue lines (following doxycycline mediated induction of H2A.Z1 expression in Tamoxifen treated cells). Right panel. Immunoblots for H2A.Z and H3 (loading control) from wildtype or H2A.Z DKO cells expressing H2A.Z1 rescue constructs as indicated above the blot. Results are representative of N=3 experiments. D) Phase contrast images of cells prior to and after real-time luminescence recordings for wildtype, H2A.Z DKO, and H2A.Z DKO rescued with H2A.Z1-Nter K5R mutant. Results are representative of N=3 experiments.

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Extended Data Fig. 6 Transcriptomic analysis of H2A.Z DKO and Per TKO cells.

A) Illumina total RNASeq Pearson correlation plots of gene read counts for quadruplicate experiments for wildtype, H2A.Z DKO (+tamoxifen, day 4) and Per TKO cells. B) Volcano plot summarizing RNA-Seq data for Per TKO vs wildtype expression. DESeq analysis identified 5275 differentially expressed genes (>2 fold difference in expression; p-value <0.01 (DE test)). C) Volcano plot showing deregulated genes in Per TKO cells and focused on genes belonging to the GO term: P400-TIP60 SRCAP complex. In red, genes with >1.5 fold difference and p-value <0.01 (DE test). D) Log2 fold change and p-values (DE test) for some specific core clock genes from RNAseq (wildtype vs H2A.Z DKO) data are highlighted. E) Log fold change in expression between wildtype and H2A.Z DKO RNA for GO terms ‘ATP generation pathway’ (light grey) and ‘oxidoreductase activity on NAD(P)H’ (dark grey) pathways. Center line denotes the median value (50th percentile), while the box contains the 25th and 75th percentiles of the dataset. Mean is indicated by the cross. The whiskers mark the 5th and the 95th percentiles and values beyond these bounds are consider outliers in circles F) Volcano plot showing deregulated genes in H2A.Z DKO fibroblasts, for subset belonging to the GO term ‘regulation of circadian rhythms’. In red are highlighted genes with >2 fold difference and p-value <0.01(DE test).

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Supplementary information

Reporting Summary

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Supplementary Table 1

Number of sequences and statistics for the different sequencing datasets

Supplementary Table 2

ChIP–qPCR primers list

Supplementary Table 3

RT–qPCR primers list

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Tartour, K., Andriani, F., Folco, E.G. et al. Mammalian PERIOD2 regulates H2A.Z incorporation in chromatin to orchestrate circadian negative feedback. Nat Struct Mol Biol 29, 549–562 (2022). https://doi.org/10.1038/s41594-022-00777-9

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