Science 14Feb2020

( 30 – 32 ), but not inBmal1−/−tissues. After syn-
chronization with DEX, as described above,
samples were harvested over two circadian
cycles, labeled with 10-plex tandem mass tags
(TMTs), and then analyzed by mass spectrom-
etry to quantify the abundance of rhythmic
proteins (Fig. 4A). A 24-hour oscillation in
abundance of proteins and phosphosites was
detected in both wild-type andBmal1−/−MSFs
and liver tissues (Fig. 4, B to D; fig. S9, A to D,
and fig. S10, A to E). We detected 585 rhythmic
proteins in wild-type and 364 inBmal1−/−
MSFs, with comparable peak phases of protein
rhythms in both genotypes (Fig. 4C). Akin to
the rhythmic transcriptome, we saw minimal
overlap between the cyclic proteins and phospho-
proteins identified in wild-type andBmal1−/−
cells (fig. S10, B to D). Asexpected, amplitudes
for rhythmic phosphoproteins (phosphosites)
were higher compared to proteins in both geno-
types (fig. S10, F to H). The global rhythms of
protein phosphorylation seen inBmal1−/−im-
plicates posttranslational processes as critical
regulators of tissue rhythmicity, even in the
absence of BMAL1.
The functions of rhythmic transcripts and
proteins inBmal1knockouts were diverse
(fig. S11). Biological processes that appeared
to be enriched significantly inBmal1−/−com-
pared to wild-type included metabolism, in-
tracellular transport, and oxidation-reduction
(Fig. 4E and figs. S11 and S12). Furthermore,
specific cellular components were overrep-
resented, particularly the mitochondrion and
its different subcompartments (Fig. 4F and
fig. S13). In agreement with these observa-
tions,Bmal1function has been reported to
be important for mitochondrial fission and
fusion dynamics ( 33 ), and for respiratory
function in the liver ( 33 , 34 ). We have thus

demonstrated that althoughBmal1is neces-

sary for the expression of 24-hour behavioral

cyclicity, it is not required for 24-hour mo-

lecular rhythms at the transcriptional, trans-

lational, and posttranslational levels.

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    ACKNOWLEDGMENTS
    We thank the Advanced Sequencing and Bioinformatics scientific
    technology platforms at the Francis Crick Institute for their support
    with next-generation sequencing.Funding:A.B.R. acknowledges
    funding from the Perelman School of Medicine, University of
    Pennsylvania, and the Institute for Translational Medicine and
    Therapeutics (ITMAT), Perelman School of Medicine, University of
    Pennsylvania. A.B.R. also acknowledges funding from the European
    Research Council (ERC Starting Grant no. 281348, MetaCLOCK),
    the EMBO Young Investigators Programme, and the Lister Institute
    of Preventive Medicine. A.B.R. was supported in part by a
    Wellcome Trust Senior Fellowship in Clinical Science (100333/Z/
    12/Z) at the University of Cambridge, and also in part by the
    Francis Crick Institute, which receives its core funding from Cancer
    Research UK (FC001534), the UK Medical Research Council
    (FC001534), and the Wellcome Trust (FC001534).Author
    contributions: S.R., U.K.V., and A.B.R. conceived and designed the
    experiments. S.R., U.K.V., A.S., and G.D. performed the MSFs
    and liver tissue time-course experiments. U.K.V. performed the
    RNA-Seq and quantitative real-time reverse-transcriptase
    polymerase chain reaction experiments and analyzed the data. S.R.
    performed the quantitative proteomics and phosphoproteomics
    and analyzed the data with support from A.P.S. and S.A.H. A.B.R.
    supervised the whole study. The manuscript was written by S.R.,
    U.K.V., and A.B.R. All authors agreed on the interpretation of data
    and approved the final version of the manuscript.Competing
    interests:The authors declare no competing interests.Data
    and materials availability:The RNA-seq data have been deposited
    in the Gene Expression Omnibus (accession nos. GSE111696
    and GSE134333). The mass spectrometry proteomics and
    phosphoproteomics data have been deposited to the
    ProteomeXchange Consortium via the PRIDE ( 35 ) partner
    repository with the dataset identifier PXD009243.

SUPPLEMENTARY MATERIALS

science.sciencemag.org/content/367/6479/800/suppl/DC1

Materials and Methods

Figs. S1 to S13

Tables S1 to S4

References ( 36 – 57 )

View/request a protocol for this paper fromBio-protocol.

20 January 2019; resubmitted 18 July 2019

Accepted 14 January 2020

10.1126/science.aaw7365

Rayet al.,Science 367 , 800–806 (2020) 14 February 2020 6of6

RESEARCH | REPORT