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Nuclear regulatory landscape of the circadian clock in mouse liver
Jingkui Wang1,, Daniel Mauvoisin2, Eva Martin2, Florian Atger2, Antonio Nunes Galindo2, Federico Sizzano2, Loïc Dayon2, Martin Kussmann2, Patrice Waridel3, Manfredo Quadroni3, Felix Naef1 and Frédéric Gachon2
1Institute of Bioengineering, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne
and Swiss Institute of Bioinformatics, Lausanne, Switzerland 2Nestlé Institute of Health Sciences, Lausanne, Switzerland.
3Protein Analysis Facility, University of Lausanne, Lausanne, Switzerland.
Abstract Diurnal oscillations of gene expression dictated by the circadian clock enable living organisms to coordinate their physiological processes with daily environmental changes. Although such rhythms have been extensively studied at the level of transcription and mRNA accumulation, comparably little is known at the proteins level, though recent proteomics studies indicated that total protein rhythms generally appeared damped compared to their cognate mRNAs. In order to further dissect how diurnal rhythms affect key functions such as transcription or chromatin remodeling, we quantified the temporal nuclear accumulation of proteins and phosphoproteins from mouse liver by SILAC-based MS. Our analysis identified ~5000 nuclear proteins, including all core-clock and clock-related proteins, over 500 of which are found to be rhythmic under a stringent statistical threshold (FDR <5%). These rhythmic nuclear proteins are mainly controlled at the post-transcriptional level and are often parts of complexes showing robust diurnal nuclear accumulation. These rhythmic complexes are notably involved in transcriptional regulation, rRNA synthesis, ribosome assembly, as well as DNA damage repair. From the parallel analysis of the nuclear phospho-proteome, we could infer the temporal activity o f k i n a s e s c o n t r i b u t i n g t o t h e s e r h y t h m i c phosphorylations. A large fraction of the kinase activities were implicated in cell signaling and cell cycle regulation. In addition, 80 transcription factors and about 100 transcriptional coregulators showed clear diurnal oscillations in the nucleus, enlarging the extent of transcriptional and epigenetic regulations by the circadian clock and/or systemic cues. Finally, a number of proteins with functions in the cytoplasm are detected in the nucleus at a common and sharp time near the night-day transition. This phenomenon is probably linked to the rhythmic endoreplication occurring in hepatic cells and associated to leakage of the nuclear membrane. Taken together, these findings provide unprecedented insights into the regulatory landscape of the diurnal liver nucleus.
0 12 24 36 45
Every 3 hours 2 WT mice
0 12 24
Every 12 hours 8 SILAC mice
SILAC mix (1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1) WT mix (1:1)
mix (1:1)
Trypsin Digest Fractionation
Unlabeled mice
F2
13C6-Lysine
MS analysis
0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45
Methods & Results
Nuclei purification and proteins extraction
Phospho-enrichment
MS analysis
1) 5-10% of total proteins show diurnal accumulations. 2) ~50% of these rhythmic proteins do not have corresponding
rhythmic mRNAs and are highly enriched in secretory proteins. Question: How about the rhythmicity of proteins in different subcompartments such as nucleus ?
CoveragePercent.rhythmic
0.0
0.2
0.4
0.6
0.8
1.0
Fig. 1 ~ 5000 nuclear proteins were quantified by SILAC-based MS including
core-clock and clock-related proteins
TF
Cor
egul
ator
RN
A pr
oces
sing
Kin
ase
Pho
spha
tase
4820 proteins quantified
Nucleus Both Cytoplasm
mRNA protein mRNA protein mRNA protein
rhyt
hmic
no
n-rh
ythm
ic
FASN
ALB
ACACA
LC
NE
LC
NE
LC
NE
αβGSK3
LC
NE SIRT7 LC
NE
NR3C1 LC
NE
Fig. 3 Subunits of nuclear protein complexes showed highly similar and
diurnal accumulations
Fig. 5 Transcription factors and coregulators (FDR<5%) involved in the
circadian transcription
FASN ZT0 confocal
Lamin A/C IF
P-CDC2
LC
NE
T-MCM2
LC
NE
P-‐CDC2
MCM2
LC
NE
LC
NE
3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 ZT 0
A) Distribution of samples numbers quantified for detected nuclear proteins. B) Coverage of nuclear proteins and percentages of rhythmic ones
(FDR<5%) for different functional categories. C) Phases and peak-trough amplitudes of core-clock and clock-related
proteins with examples in D).
A B
C
A) Phase distribution for the rhythmic nuclear proteins (FDR<0.05) grouped by their annotated localizations (UNIPROT) B) Heat maps of the rhythmic proteins and their corresponding mRNAs. Data is standardized by rows and gray blocks indicate missing data. C) Western blot of individual rhythmic proteins performed on nuclear extract. The graphs represents the quantification of the blots and the corresponding mass spec data.
A
B
C
Fig. 2 Rhythmic nuclear proteins are mainly post-transcriptionally regulated
Take-home messages
Fig. 6 Rhythmic endoreplication probably causes leakage of cytoplasmic proteins by weakening nuclear envelope
A
B
A
B
C LC
NE CDC6
P27 Kip1 LC
NE
LC
NE
A) FACS analyses of nuclei isolated from mouse liver around the clock and separated into ploidy populations by (n = 4 light dark cycles) B) Localization of FASN at ZT0 (confocal fluorescence) and immunofluorescence of Lamin A/C in purified liver nuclei. C) Western blot of individual protein involved in cell cycle regulation and DNA replication.
A) Peak phases of rhythmic nuclear protein complexes and individual examples are found in B).
1. SILAC nuclear proteomics in mouse liver - High resolution (~5000 proteins), - ~12% highly rhythmic (FDR<5%). - Almost all core clock and clock
related genes identified and quantified.
2. Within these rhythmic proteins - 81 transcription factors and more than
100 coregulators : most of them are new.
- Many nuclear protein complexes also display diurnal expression.
3. Annotated cytoplasmic proteins are also
- detected in the nucleus with a sharp day night transition phase.
è may be due to a weakening of the nuclear envelope resulting from rhythmic endoreplication/replication.
Previous work
SILAC mass spectrometry analysis of mouse liver nuclei
nb of quantified samples
nb o
f pro
tein
s
0 5 10 150
500
1000
1500
2000
2500
3000
●NFIL3
●ARNTL,CLOCK
●
●NR1D1
●NR1D2
●DBP
●HLF
●TEF
●PER1●PER2
●CRY2
●RORC
● RORA
●CRY1
●
●
●
core clockstablizing loopoutput regulators
ZT0
ZT6
ZT12
ZT18
D ARNTL
●
●●
●
●
●
●●
●●
●●
0 6 12 18 24
−0.6
−0.3
0.0
0.3
CLOCK
●
●
●
●
●
● ●
●
●●
● ●
0 6 12 18 24
−0.5
−0.2
0.1
0.4
PER1
●
●
●●
●
●
● ●
●
●
●
●
0 6 12 18 24−2
−1
0
1
PER2
●
●
●
●
●
● ●
●
0 6 12 18 24−2
−1
0
1
Fig.4 Rhythmic activities of kinases predicted by nuclear phospho-proteome
nucleus phospho C A B
C
A) and B) Examples and heat maps of rhythmic nuclear phospho-proteins with non-rhythmic nuclear proteins. C) Peak phases of rhythmic kinase activities predicted by the nuclear phospho-proteins, some of which are confirmed by their nuclear protein accumulations in D)
ZT6 ZT18
ZT0
ZT12
NFIA, S280
●
●
●
●
●
●
●
● ● ●
●●
●
● ●●
●
●
●
●●
●
● ●
●
●
●
●●
●
●
●
0 12 24 36 48−0.7
−0.4
−0.1
0.2
0.5
0.8CIC, S1080
●
●
●
●
● ●
●●
●●
●
●
●
● ●
●●
●
●●
●
●
●
●
●
●
●
●
●
●● ●
0 12 24 36 48−0.9
−0.6
−0.3
0.0
0.3
0.6
NCBP1, S22
● ●●
● ● ●●
●● ●
● ●
●
● ●●
●
● ●
●
●
●
●
●
●
●
●
●
● ●●
●
0 12 24 36 48−0.7
−0.4
−0.1
0.2
0.5
0.8
CHD4, S1517
●● ●
●● ●
● ●● ● ●
●
●●
●●
●
●
●
●
●
●
●
●
●
●●
● ●
●
●●
0 12 24 36 48
−0.5−0.3−0.10.10.30.50.7
D ●
●
● ●
●
●
● ●●
●
●●
●
●
●
●
CSNK1D
0 12 24 36 48
−1.0−0.40.20.81.4
●
●
●●
●
● ●
●
●
●
●
●
●
●
●
●
GSK3A
0 12 24 36 48
−2.0−1.2−0.40.41.2
● ●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
MAPK14
0 12 24 36 48
−1.2−0.40.41.2
● ●
●
●
●
●
●
●
●
●●
●
●
●
●
●
PRKAA2
0 12 24 36 48
−1.0−0.40.20.81.42.0
REP
IN1
DIDO
1NF
IL3
AATF
CIZ1
MEF
2DHNF1
BPHC2
PPARD
ILF2NR2F6
ZKSCAN3
IRF9HNF1A
TBX3ZMYND8GTF2IRD1CLOCKARNTLTFEB
ELF1CICATF1IKZF1NFIATCF7L2FLI1JUNBZFHX3FOXP1RFX1HBP1ELF2NR1D1
NR2C1
ZFHX4
MGAE4F1
SP2NFIBHM
BOX1
SCMH1
CCNT2
FOXP2
USF2TERF1N
R1D
2ZN
F187PPAR
AD
BPAR
ID5BR
XRA
PATZ
1H
LFN
R3C
2KLF1
3TE
F
NR3C
1
BHLH
E40
MAFB
ERF
FOXA3
NR1H4NFIX
HCFC1PCBP1
HSF1FOXK1FOXA1
KLF3
SREBF1
ESRRAETV3
HNF4A
RORC
TCF12
RORA
CEBPGESR1
TP53
●
IRF2
BP1
●
ZFPM
1
●
CAC
YBP
●
PAF1
●
ZFP1
28
●
CXXC
5
●
SETD
8
●
BCOR
●
EDF1
●
NRIP1
●
ZBTB
33
●
MAML1
●
IRF2
BP2
●
RB1
●DDX17
● WIZ
● CBX2
● ZMYND8
● CBFA2T2
● TBL1X
● PHF21A● EHMT2● SETDB1
● KAT5● JMJD1C●ATF7IP
●MTA2●NCOR1●TBL1XR1●HDAC3
●CABIN1●RBBP7●EMSY
●N4BP2L2●MLLT10
●ZBTB1
●BPTF
●CHD6
●EP400
●KDM3A
●HIRA
●MED12
●MTA3
●NACC1
●MRGBP
●KDM5A
●DAXX
●TINF2
●MED28
●EPC2
●TRRAP
●PPARGC1B
●SIN3A
●MED
23
●CIPC
●SAP130
●CR
EBBP
●CEC
R2
●BAZ2A
●MED
13
●
WBP
7
●
PHF1
7
●
MED
16
●
ASH
1L
●
EP30
0
●
MLL
3
●
VPS7
2
●
MED
20
●
DDX5
●
LIN5
4
●
PSIP
1
●
ARID
2
●
BAZ2
B
●
LIN3
7
●
THRA
P3
●
BAP1
●
ATRX
●
NIPB
L
●
RAD5
4L2
●
TAF2
●
TRIM
24
●
AFF4
●
CRTC2
●PER3
●PER1 ●PER2
●
CRY2 ●
NONO
●
BRD2
●
BAZ1B
●
ABT1 ●
CRY1 ●
YAF2
●
BCCIP ●
SKI ●
KDM
1B
●
SUPT6H
●
HDAC
5
TF.nuclearTF.bothTF.phospho
●●●●●●●
CoregulatorAcetylDeacetylMethylDemethylUbiqDeubiq
M NMA
Arp2/3Arpc2Actr2Arpc1bActr3Arpc3Arpc4Arpc5
0 12 24 36 48−1.8
−0.8
0.2
1.2
2.2CSN/CSA/DDB2
Polr2aCops3Cops5Cops8Cops4Cops2Gps1Cops6Cops7aDdb1Rbx1Cops7bGferCul4aDdb2
0 12 24 36 48−3
−2
−1
0
1
2
NuRDRbbp7Mta2Mta3Zfpm1Gatad2bChd4Smarca4Mbd3Hdac2Mta1Smarcb1Arid1aMbd2Smarcd2Hdac1;Gm10093Dpf2Actl6aSmarcc2Smarcc1Mbd3Rbbp4HnrnpcSmarce1
0 12 24 36 48−2.8
−1.8
−0.8
0.2
1.2
2.2
TRAP/SMCC/DRIPMed16Thrap3Med13TrrapMed12Esr1Med23Cdk8CcncMed21Med17Med1Med24RxrbCcnt1Med14Med10Med6Kat2aMed27Med26Med4Brd4Med7Cdk9Med31
0 12 24 36 48−3.5
−2.5
−1.5
−0.5
0.5
1.5
2.5Mediator/asscociated
Med16Thrap3Med13Med12Med28Med20;Gm20517Med23Cdk8CcncMed21Med17Med1Med24Med25Med29Med13lMed15Med14Med10Med6Acad8Med27Med26Med22Med4Brd4Opa1Med19Med11Med18Med8Med7Cdk19Med31
0 12 24 36 48−3.5−2.5−1.5−0.50.51.52.5
Rev−ErbA−Ncor1−Hdac3Nr1d1Ncor1Hdac3Ncor1
0 12 24 36 48−2.1
−1.1
−0.1
0.9
1.9NCOR/SMRT
Ncor1Tbl1xr1Tbl1xHdac3Zbtb33Ncor2Ncor2Trim33Ncor1Gps2
0 12 24 36 48−2.3
−1.3
−0.3
0.7
1.7
2.7PER
Cry2Per1Cry1Per2Per3NonoWdr5
0 8 16 24 32 40 48
−2.1
−1.1
−0.1
0.9
1.9
●
AP2
●
Arp2/3
●
Profilin 1
●
Endo
cytic
coa
t
●
RICH
1/AM
OT p
olar
ity
●
20S
met
hyltr
ansfe
rase
●
KCNQ
1
●
CLIC
4
●
AQP2−f
orce−g
ener
ator
●
Neph
rin
●
WIP−W
ASp−
actin−m
yosin−I
ia
●
Emer
in
● DNMT1−RB1−HDAC1−E2F1
● IMP3−IMP4−MPP
● Exosome
● RNA PolI
● Bmal1−Clock
●CoREST−HDAC
●NCOR/SMRT●TFC4−CTNNB1●SETDB1−HMTase●HDAC1/2−associated●DNA ligase III−XRCC1
●NuRD●Ikaros●Rev−ErbA−Ncor1−Hdac3
●NCOR−SIN3
●HIRA●NURF●SIN3−associated
●APC/C
●NuA4/Tip60 HAT
●ZNF198−PML
●MRE11−RAD50−NBN
●TRF1
●p300−CBP
●SWI/SNF
●TRAP/SMCC/DRIP
●CDK8−CCNC−RNA PolII
●Mediator/asscociated
●
Bmal1−C
lock−
Pers
●
PA28
●
PER
●CPSF ●
PSF−P54 ●
Parvulin pre−rRNP ●
WICH ●CKII ●
p130Cas−ER−alpha−cSrc ●
RNA PolII/associated ●
Core Cohensin ●
Retromer ●
HSP90−associated ●
AMPK
●
Alpha−GDI−Hsp9●
MNK1−eIF4F●
Mt3−Hsp84−Ck●
AP1●
ESCRT−II●
APPBP1−UBA3●
SURF●
CSN/CSA/DDB2●
CCT
●
●
●
●
●
●
●
●
transcriptioncytoskeletonchaperonecell cycleDNA repairproteolisisprotein transprotothers
PER
ZT6
ZT12
ZT18
ZT0
B ●E−Box
●
RRE
ClockArntl
Nr1d1
Nr1d2
Rorc
Rora
ActivatorRepressor
●D−Box
●KLFE
●E−Box
Nfil3
DbpH
lf
Klf13Tef
Bhlhe40
Klf3 ●HNF4A_NR2Fs
●IR3
Nr2f6
Nr3c2Nr3c1
Esrra
Esr1
●
HNF1s● TFE
B.m
● CRE● FOXE
●RXRE
●
NR1H4.
m●
HSE
●HNF4A_NR2Fs
●E−Box
Hnf1b
Hnf1a
Tfeb
Atf1Foxp1
Foxp2
Rxra
Foxa3
Nr1h4
Hsf1Foxk1Foxa1
Hnf4a
Tcf12
●
ETS
Elf1Fli1
Elf2Erf
Etv3
core-clock output-regulator hormone cues Ets family metabolic cues
0 1 2345678
91011121314
1516171819202122 23
0 1 2345678
91011121314
1516171819202122 23 0 1 2
345678
91011121314
1516171819202122 23
●
Map4k2_ST
●
Mapk14_ST
●
Mapk15_ST
●
Plk2_ST
●
Mapk9_ST
●Stk16_
ST
● Gsk3a_
ST
● Mapk1_ST● Sik1_ST● Dstyk_ST●Ripk3_ST●Camkk2_ST●Nek9_ST●Tesk2_ST●Cdk5_ST●Mapk3_ST●Tgfbr2_ST
●Cdk6_ST●Mapk12_ST●Mapkapk5_ST
●Acvr2b_ST●Prkcb_ST●Gsk3b_ST
●Mapk7_ST
●Prkx_ST●Raf1_ST
●Wee1_ST
●Mapk8_ST
●Nuak1_ST
●Map3k13_ST
●Rps6ka3_ST
●Cdk4_ST
●
Uhmk1_ST
●
Camk2d_ST
●
Cdk7_ST
●Clk1_
ST●
Stk24_ST
●Map3k8_ST●Dyrk1b_ST●
Csnk2a2_ST ●Akt1_ST ●Cdk16_ST ●Srpk2_ST ●Scyl2_ST ●Srpk1_ST ●
Pim3_ST ●
Plk3_ST ●
Pim2_ST ●
Camk1d_ST ●
Snrk_ST ●
Chek2_ST ●
Vrk3_ST ●
Nek2_ST ●
Cdk18_ST ●
Limk2_ST ●
Csnk1d_ST ●
Vrk1_ST ●
Araf_ST ●
Clk3_ST●
Prkaa2_ST
●
Csnk1a1_ST
●
Cdk9_ST
●
Nek4_ST
●
Mapkapk3_ST
●
Nek8_ST
●
Fastk_ST
●
Mknk2_ST
●
Dyrk2_ST
●
Mapk6_ST
●
Cdk1_ST
Peak phases of TF accumulations vs. those of predicted motifs
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