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Page 1 of 9
SUPPLEMENTAL DATA
Coordinate Regulation of Bcl11b Activity in Thymocytes by the MAPK Pathways and Protein Sumoylation
Ling-juan Zhang, Walter K. Vogel, Xiao Liu, Acharawan Topark-Ngarm, Brian L. Arbogast, Claudia S. Maier, Theresa M. Filtz, and Mark Leid
Supplemental Figure S1 Bcl11b phosphorylation sites identified by tandem mass spectrometry (spectra)
Supplemental Figure S2 Coverage map of Bcl11b post-translational modifications identified by tandem mass spectrometry
Supplemental Table 1 QPCR primers used in this study
Supplemental Table 2 Bcl11b co-immunoprecipitating NuRD complex proteins indentified in primary mouse thymocytes
Page 2 of 9
100
80
60
40
20
0
Re
lati
ve
Ab
un
da
nc
e
12001000800600400200m/z
A 91-VLDKpSpSPPPSSR-102 m/z (exp) = 675.32629 (2+)m/z (th) = 675.32667Error = 0.00038 (0.56 ppm)
y6640.4
ºDKb2
ºy2y2
262.1
b3328.2 y3
349.2ºb4
y4446.3
b4456.3
y9++ –P
ºy9++
y9++
511.8y10
++
–P
b5pS96543.3
y10++
b6–P
[ºMH2–P]++
[MH2–P]++
b5pS95632.3
[ºMH2]++
b5–PpS95
y7pS96807.3
y8–P
ºy7y7
pS95727.5
b6710.3
y8894.3 y9
1022.5
y101137.42b10
1088.5b11
1175.4
ºb6–PDKp(SS)
–P ºy8–P
20
15
10
5
0
Re
lati
ve
Ab
un
da
nc
e
1600140012001000800600400m/z
100
! !B [MH3–P]3+
946.3
[ºMH3–P]3+
107-RVpSEPVEIGIQVTPDEDDHLLpSPTK-131m/z (exp) = 978.7909 (3+)m/z (th) = 978.7893Error = 0.0016 (1.7 ppm)
y3345.2
[MH3–2P]3+
913.6
y7++
438.2b9++–Py4
b4–Pb12++
b12++–P
645.3 y12++–P
674.6
y12++
723.5y121446.4
y111349.3
b121387.5
b131488.4
y131547.5
b7877.1 y14
1646.6 b151700.5
y14–P1548.5
y13–P
1449.5
ºb13y12–Pb11
1288.4
b22++–P
1246.8b21++
1212.6
y21++
1192.3
y20++
b10
b20++–P
1106.6
y20++–P
b91047.1
y8,b8
y14++
y13++
774.3
b13++
b13++–P
695.7
b4552.2
30
25
20
15
10
5
0
Re
lati
ve
Ab
un
da
nc
e
140012001000800600400m/z
100
! ! ! !C[MH3–P]
3+
867.6
[ºMH3-P]3+
108-VSEPVEIGIQVTPDEDDHLLpSPTK-131m/z (exp) = 900.0986 (3+)m/z (th) = 900.1002Error = 0.0015 (1.7 ppm)
y3345.3
ºy4,ºb5
ºb3298.1
ºb7
y131547.4
y121446.4
y12++
724.0
y223+
838.3
b7
b8
y143+
550.1b4b3
ºb4395.2
y4,b5512.2 ºb6
623.2
b6ºy223+
y14++
824.0
y213+
ºy213+
y13++
774.3
y203+
y11++
675.1 y23++
1300.0
y22++ºy22
++
y101234.4
y21++
1192.0
y21++
–P1143.3
y20++
ºy20++
y20++
–P1094.6
y18++
1029.7
ºy18++
y17++
972.9
y16++
944.3b9924.2
y15++
ºy15++
879.3
Page 3 of 9
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40
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0
Re
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ve
Ab
un
da
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e
12001000800600400m/z
D
b4353.2
b5424.3
y3385.2
y4552.2
b12++
559.2
y5–P552.2
b13++
y11++
–P575.3
y11++
624.3
[MH3–P]3+
658.0[ºMH3–P]
3+
652.1
y13++
736.6
y6766.2
b15++
709.1
y16++
859.2y15++
823.6
y16++
–P810.1
y14++
780.1
b9y7
y17++
894.7
y18++
951.2
y8
ºy18++
942.2y18++–P
902.2
ºy17++
885.6
y17++
–P845.7
ºy16++
y91053.5
b121117.6
y101150.5
y111247.6 b14
1303.5
b16++
759.5
153-APIAASSSHPPTSVITpSPLR-172 m/z (exp) = 690.35519 (3+)m/z (th) = 690.3524Error = 0.0028 (4.0 ppm)
100
80
60
40
20
0
Re
lati
ve
Ab
un
da
nc
e
12001000800600400m/z
E 248-IYLEPGPASTSLpTPR-262m/z (exp) = 841.41426 (2+)m/z (th) = 841.41348Error = 0.00078 (0.93 ppm)
y111163.5
y2272.2 b2
277.1
y3453.2
b3390.1 a4
b4519.1
y11++
582.3
y12++
646.9y5653.4
y13++
703.3 y7–P743.4
[MH2–P]++
792.3
ºy7,ºb8823.3
y91009.4
y101066.4
y11–P1065.5
b13–P1312.5
b121229.6
y121292.5
ºy12ºy11y8
y9–Py++11–P
a2 YLE
100
80
60
40
20
0
Re
lati
ve
Ab
un
da
nc
e
18001600140012001000800m/z
F 263-LTIPPPLGPETVAQpSPLMNFLGDSNPFNLLR-293m/z (exp) = 1143.58322 (3+)m/z (th) = 1143.58231Error = 0.00091 (0.80 ppm)
y28++
1551.2
y12++
697.0b7732.6
y6759.5
y7873.4
y16++
929.0 y17++
–P
y8960.6
y18++
1072.0
y17++
1008.0
y91075.3
y19++
1107.5
ºy10
y101132.5
ºb12
b121215.6
y111245.6
y23++
–Pb13
y23++
y24++
1349.0
y121392.7
ºb14
b141414.8
y28++–P
1502.4 y28++–P
1542.4
b171791.7
b181922.9
b20++
1092.4
b24++
b29++
1571.2
b30++
1627.6
ºb30++
y161848.0
ºb20++–P
ºy28++
–P
[MH3–P]3+
1110.9
Page 4 of 9
100
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60
40
20
0
Re
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ve
Ab
un
da
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e
140012001000800600400m/z
G
y3329.3
y4–P329.3
y4496.1
b5583.4 y6–P
y6692.4 b7
754.5
y7789.5
y8–P788.4
b8841.4
y8886.4
b9
[MH2–P]++
949.2
[ºMH2–P]++
[MH2–2P]++
900.3
b101109.5
ºb10
b10–P1011.5
b15–P1568.6b14
1499.7y131412.5
b111206.6
y111241.4
367-LRELAGNSSpTPPPVpSPGR-384m/z (exp) = 997.95888 (2+)m/z (th) = 997.95846Error = 0.00043 (0.43 ppm)
100
80
60
40
20
0
Re
lati
ve
Ab
un
da
nc
e
1000800600400200m/z
H
y8994.4
y7880.3
b6713.3[MH2–P]++
561.8
y91107.5
b9–P976.3
y8–P896.4
y6783.3
ªb6696.2
y9++
b5
y4508.2
y8++
497.7
ªy8++
y8++ –P
448.7
y7++ –P
b3341.1
y2
b2227.1
y5y3 b7a2
391-LLNPFQPpSPK-400m/z (exp) = 610.8087 (2+)m/z (th) = 610.8074Error = 0.0013 (2.2 ppm)
b8PFQ
y4–P410.2
y7++
100
80
60
40
20
0
Re
lati
ve
Ab
un
da
nc
e
18001600140012001000800600400m/z
I 391-LLNPFQPSPKpSPFLSTPPLPPMPAGpTPPPQPPAK-424m/z (exp) = 1235.2869 (3+)m/z (th) = 1235.2848Error = 0.0020 (1.6 ppm)
y4412.3
PPMPSGpT–P634.2
y15++
782.0
PPMPSGpT,y15++–P
782.0
b6713.3
y8831.5
y17++
886.7 b8897.5
y18++
935.6
b19++–P
1022.7
y19++
1071.7
y313+
1122.3
y22++
1160.1
[MH3–P]3+
1202.8
y23++
1208.8
b26++–P
1388.0
PPMPSGpTPPPQ–P
1053.6
y181869.9
b161816.8
ºb16ººb16
b141646.8
y31++
1682.3
y151562.7
y28++
1496.4
b30++
–P1597.9
b16–Pºb15
ºy28++
y15–P1464.7
y28++–P
1447.4
ºb26++
–Pb26++–2P
1339.4
y26++
1404.2
Page 5 of 9
40
30
20
10
0
Re
lati
ve
Ab
un
da
nc
e
1600140012001000800600400m/z
100
! !J
b161767.5
b16–P1669.6
b121361.5
y121237.5
y8++
416.2
y4412.2
y7++
367.8
b5532.3
b4445.3
b6713.0
b12++
681.3
401-SPFLSpTPPLPPMoxPAGpTPPPQPPAK-424m/z (exp) = 866.74231 (3+)m/z (th) = 866.74201Error = 0.00030 (0.34 ppm)
y12++
619.3 PPMoxPAGpTPPPQ
y21++b20
++
y20++
1077.7
y19++
1034.2
y18++
943.7
b91020.3
ºb9
b6–P615.2
y14++
741.4
y15++
790.0
[MH3–P]3+
834.2
b9–P992.5
PPMoxPAGpT–P
y8831.4
100
80
60
40
20
0
Re
lati
ve
Ab
un
da
nc
e
1000800600400m/z
K 413-PAGpTPPPQPPAK-424m/z (exp) = 619.30286 (2+)m/z (th) = 619.30247Error = 0.00039 (0.63 ppm)
y101069.4
b111091.3
y9–P1069.4
y8831.4
b8826.2
y7734.4
b8–P728.3y6
637.3b6
601.5
[MH2–P]++
570.4[ºMH2–P]
++
ªy10++
526.8
y11++
–P521.5
y10++
–P486.2
y9++
–P457.8
y4412.2
b4407.1
QPPA394.2
b4–P309.1
ºy8813.4
100
80
60
40
20
0
Re
lati
ve
Ab
un
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nc
e
18001600140012001000800600m/z
L 487-SDDGLSAASpSPEPGTSELPGDLK-509m/z (exp) = 1155.5069 (2+)m/z (th) = 1155.5047Error = 0.0022 (1.9 ppm)
y5529.3
ºy5ºb8
b8717.2
y7ºb6
y8858.4
y++18
911.9
y++20
996.9
y++21
[MH2–P]++
1106.5
[ºMH2–P]++
y111113.5
ºb12
b121197.3
y121242.5
y141506.6y14
–P ºb15
y151593.6
y161664.6 b18
–P
y17 y181822.8
b181781.5
ºb18b10–P
b6ºb7 b7
y6642.4
b9ºb9
ºb10–P855.3
ººb10–P
y9
y++22–P1063.4
ºb12–P1081.2 [ººMH2]
++
y131339.6
y13–P
y15–P
ºy15–P
ªºy15–P
ºy15
ººb18
b17ºb17ºy16
Page 6 of 9
15
10
5
0
Re
lati
ve
Ab
un
da
nc
e
800700600500400300m/z
100
! !M [MH3–P]3+
587.8
[ºMH3–P]3+
y2218.1
b2226.1
y3289.2 b3
297.2
y7++
354.2 y8++
382.8
y9++
411.3
b9++
–P
y4426.1
y153+
–P411.3 y10
++
b10++
–P470.8
b11++
–P499.3
b5507.2 b12
++
–P527.8
y183+
–P544.7
y173+
545.4
y14++
–P627.9
y14++
676.7
b7–P673.2
y7707.2 y8
764.3
y16++
781.9
y18++
–P817.1
y9, b17++
821.3
b18++
857.0
y18++
866.1
b10++
519.5
658-KPAPLPpSPGLGGPALHAAK-676m/z (exp) = 620.33665 (3+)m/z (th) = 620.33837Error = 0.00055 (0.89 ppm)
y12++
543.8
b11++y5
14
12
10
8
6
4
2
0
Re
lati
ve
Ab
un
da
nc
e
18001600140012001000800600m/z
100
! ! !N [ªMH2–P]++
943.4
[ªºMH2]++
983.3[ªºMH2–P]
++
y5546.4
[ªMH2–P]++
951.8
[ªººMH2]++
974.4
y4432.2
ªb10pS7341055.3 ªb10
pS7311135.2
y6675.3
y7762.4
y8pS731849.5
y8pS734929.3
y9pS731986.4 y9
pS7341066.3 y11
1282.5y121369.6
y131470.4
y151688.9
ªb171809.4
ªºb171791.7
ªºb161678.8
ªb141552.5 ªb15
1609.7
ªb131438.4
ªºb13ªºb13–P
ªb111222.5
y16++
893.5
y15++
844.9
y14++
771.3
y15++
736.0
y12++
ªºb7684.1ªb6
615.4
ªºb6ªb5514.2
724-QSPFATSpSEHpSSENGSLR-741m/z (exp) = 1000.9226 (2+)m/z (th) = 1000.9209Error = 0.0017 (1.7 ppm)
100
80
60
40
20
0
Re
lati
ve
Ab
un
da
nc
e
12001000800600400m/z
O 742-FSpTPPGDLLDGGLSGR-757m/z (exp) = 834.88543 (2+)m/z (th) = 834.88508Error = 0.00035 (0.42 ppm)
ºy3301.2
b3–P318.2 ºb3
398.2
b3416.2
y4432.2
y5489.3
y6546.3
b6–P569.1
y12++
579.0
ºy13++
618.4
y13++
627.4
y7661.3
y14++–P
668.9
ºy14++
708.5
y14++
717.8
ºb7764.4
y8774.4
b7782.2
[MH2–P]++
785.9
y9887.4
b9–P910.5
y101002.6 b9
1008.4
y111059.4
b101123.3
b12–P1139.5
y121156.5
ºy131235.7
y131253.6
b131350.4
Page 7 of 9
SUPPLEMENTAL FIGURE S1. Bcl11b phosphorylation sites identified by tandem mass spectrometry. Ion trap tandem mass spectra were recorded by a Thermo LTQ-FTMS Ultra. Exact mass determinations of the parent ion were derived from full-scan spectra recorded in the ICR cell at a resolution of 100,000 (m/z = 400). Ions resulting from the neutral lose of H2O and NH3 are identified by prefixing º- and ª-symbols while the neutral lose of H3PO4 is identified by a –P suffix in the spectral annotations. Panels A, N, P, and Q are singly phosphorylated peptides where the site of phosphorylation is assigned to multiple sites. Diagnostic phosphosite specific tandem masses are noted in the annotations.
20
15
10
5
0
Re
lati
ve
Ab
du
na
cn
e
1000900800700600500400300m/z
100
! !P[MH3–P]
3+
678.0[ºMH3–P]
3+
672.0
[ººMH3]3+
698.6
[ºººMH3]3+
[ººMH3–P]3+
758-SGTApSGGp(ST)PHLGGPGPGRPSSK-780m/z (exp) = 710.33083 (3+)m/z (th) = 710.33056Error = 0.00027 (0.37 ppm)
ºb3228.1
b3
ºb4299.1
ººb4 b4
b5–P386.2 y4
ºb5–P368.1
ºb6–P425.2
b6–P
y++9
y++10
ºb7–P
y++11 y
++12
555.5ºb8–P
y++13
624.0
y++16p(765/6)807.3
y++16
p762766.7
y++18p(765/6)863.7
y++18
p762823.7
y++22
1021.5y++21
992.4
y11996.5
y++21–P943.1y
++20
942.4
y++20–P893.8
y++19
906.9
ºy++19
y++19–P
ºy++19–P849.7
y++17
p762795.1
y++17p(765/6)835.2
ºy++18
y++15
p766763.1
y3+20–P596.3
y3+19
y3+21–P629.9
y3+21
662.6
y3+22–P
ºy3+21
656.5
b6p(765/6)
b7p(765/6)
y3+20
15
10
5
0
Rel
ativ
e A
bund
ance
120011001000900800700600500400300m/z
100
≈ ≈Q
b3246.1
ºb4299.2
b4
b5–PpS762
b6pS762
b6–PpS762
758-SGTApSGGp(ST)PHLGGPGPGRPp(SS)KEGR-783 m/z (exp) = 824.38609 (3+)m/z (th) = 824.38562Error = 0.00047 (0.56 ppm)
b7p(765–79)
b7–PpS762
b5p(765–79)
y++7
ºb5–PpS762
ºb6–P
ºb7–PpS762
y4b14
p(778/9)1167.6
b8–Pp(762/5)
ºb8–P
b9–P
ºb9–P
[MH3–P]3+
792.1[ºMH3–P]
3+
y3+22
719.2
y3+23
742.8 y3+24
776.5
b14–Pp(762–66)1149.5
b14p(762–66)1247.5
y++17
p(778/9)883.6
y++17p(762–66)843.7
y++16
p(778/9)834.7
[ººMH3–P]3+
y5p(762–78)576.4
y5p779
b++24
y++23
ºb++24
y++22
b++24–P
y++23–P
y++22–P
y++21
p(765–79)1034.7
y++20
p(765–79)1006.2
y9p(778/9)
y++21
p762
y++21–P
p(765–79)985.8
y++20–Pp(765–79)956.8
y++19–P
p(765–79)
y++18p(766–79)
b21p779
y++15
p(778/9)
y3+23–P709.9
y++11
p(778/9)
y++12p(762–66)
y6p778/9
b11p778/9
y++19
p(765–79)977.4
Page 8 of 9
1 msrRKQGNPQ HLSQRelitp eadhveatil eedegleiee psslglmVGG 51 PDPDLLTCGQ CQMNFPLGDI LVFIEHKKKQ CGGLGPCYDK VLDKSSPPPS 101 SRSELRRVSE PVEIGIQVTP DEDDHLLSPT KGICPKQENI AGPCRPAQLP 151 SMAPIAASSS HPPTSVITSP LRALGVLPPC FPLPCCGARP ISGDGTQGEG 201 QMeapfgcqc elsGKDEPSS YICTTCKQPF NSAWFLLQHA QNTHGFRIYL 251 EPGPASTSLT PRLTIPPPLG PETVAQSPLM NFLGDSNPFN LLRMTGPILR 301 DHPGFGEGRL PGTPPLFSPP PRHHLDPHRL SAEEMGLVAQ HPSAFDRVMr 351 LNPMAIDSPA MDFSRrLREL AGNSSTPPPV SPGRGNPMHR LLNPFQPSPK 401 SPFLSTPPLP PMPAGTPPPQ PPAKskSCEF CGKTFKFQSN LIVHRRSHTG 451 EKPYKCQLCD HACSQASKlk rhmkTHMHKa gslagrSDDG LSAASSPEPG 501 TSELPGDLKA ADGDFRhhes dpslgpeped dedeeeeeee lllenesrpe 551 aafsmdselg rGRENGGGVP PGVAGAGAAA AALADEKALA LGKVMEDAGL 601 GALPQYGEKR GAFLKRAGDT GDAGAVGCGD AGAPGAVNGR GGAFAPGAEP 651 FPALFPRKPA PLPSPGLGGP ALHAAKRIKV EKDLELPPAA LIPSENVYSQ 701 WLVGYAASRH FMKDPFLGFT DARQSPFATS SEHSSENGSL RFSTPPGDLL 751 DGGLSGRSGT ASGGSTPHLG GPGPGRPSSK EGRRSDTCEY CGKvfkNCSN 801 LTVHRRSHTG ERPYKCELCN YACAQSSKlt rhmkTHGQIG KEVYRCDICQ 851 MPFSVYSTLE KhmkKWHGEH LLTNDVKIEQ AERS
SUPPLEMENTAL FIGURE S2. Coverage map of Bcl11b post-translational modifications identified by tandem mass spectrometry. Combined trypsin and cyanogen bromide/trypsin digests of natively expressed mouse thymus Bcl11b sequenced by tandem spectrometry. Phosphorylated residues are shown in red and the underlined pairs indicate sites to which at least a single phosphorylation is ambiguously assigned. The SUMO1 and SUMO2/3 adduction site, lysine 679, is shown in green. Sequences covered by these experiments are shown in uppercase bold characters. Sequence presented is full-length mouse Bcl11b (Swiss-Prot: Q99PV8) but includes coverage derived from the shortened splice variant missing residues 142–213, isoform 2 (Swiss-Prot: Q99PV8-2).
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SUPPLEMENTAL TABLE 1 Primers used for RT-qPCR amplifications
Gene ID Forward Primer Reverse Primer
Id2 5’-GTCCTTGCAGGCATCTGAAT-3’ 5’-CTCCTGGTGAAATGGCTGAT-3’ Gapdh 5’-AGGTCGGTGTGAACGGATTTG-3’ 5’-GTAGACCATGTAGTTGAGGTCA-3’
ChIP primer sets for mouse Id2 promoter Locus Forward Primer Reverse Primer
–3 kb 5’-TGCCAATTCATCACCTGTC-3’ 5’-CCTCGGTTTTGTTCTCACAG-3’ TSS 5’-GGCGAGTGCACATAAAAGAC-3’ 5’-CCTCTGGTTGAGCAGTCAGT-3’
3´ IGR 5’-GGCTCGGTTCAGAATGAAG-3’ 5’-TCAAAATTCAGGCTGGAGAG-3’
SUPPLEMENTAL TABLE 2 Bcl11b co-immunoprecipitating NuRD complex proteins identified in primary mouse thymocytes NuRD complex proteins in Bcl11b immune complex were identified by mass spectrometry. Primary mouse thymo-cytes were stimulated with PMA and A23187 for the indicated times and immunoprecipitated with anti-Bcl11b antibody as described the Experimental Procedures. Immune complexes were prepared for mass spectrometry analysis after SDS-PAGE size-fractionation (10 fractions/condition). Each in-gel tryptic digest was separately analyzed on an LTQ-FTMS Ultra mass spectrometer and the resulting data sets combined for protein identity analysis; see Experimental Procedures. NuRD complex members identified are shown with their percent sequence coverage and the number of peptides unique to the indicated protein identification shown in parentheses for each experimental determination.
MW P/A-Stimulated Protein Identification (kDa) Gene ID Basal 5 min 30 min 120 min Bcl11b immune-target 95 Bcl11b 68% (53) 73% (61) 74% (66) 68% (52) Metastasis-associated protein 1 79 Mta1 45% (22) 43% (22) 48% (26) 46% (23) Metastasis-associated protein 2 75 Mta2 57% (31) 53% (30) 55% (33) 58% (33) Metastasis-associated protein 3 67 Mta3 19% (3) 19% (3) 23% (6) 31% (9) Histone deacetylase 1 55 Hdac1 37% (14) 40% (14) 46% (15) 39% (14) Histone deacetylase 2 55 Hdac2 36% (7) 34% (7) 38% (8) 42% (9) Transcriptional repressor p66-α 67 Gatad2a 45% (19) 49% (22) 56% (24) 54% (23) Transcriptional repressor p66-β 65 Gatad2b 54% (22) 58% (24) 52% (21) 46% (21) Histone-binding protein (RbAp46) 48 Rbbp7 30% (4) 32% (4) 33% (5) 32% (5) Histone-binding protein (RbAp48) 48 Rbbp4 34% (13) 36% (13) 36% (15) 35% (14) Methyl-CpG-binding domain protein 2 44 Mbd2 12% (4) 21% (6) 24% (7) 13% (4) Methyl-CpG-binding domain protein 3 32 Mdb3 21% (6) 58% (14) 42% (11) 43% (12) Chromodomain-helicase-DNA-binding protein 4 (Mi2-β)
218 Chd4
25% (37)
38% (53)
34% (48)
32% (50)