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

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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

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Ab

un

da

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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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80

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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

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0

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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

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Ab

un

da

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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

80

60

40

20

0

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Ab

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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

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Ab

un

da

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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

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Ab

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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

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Ab

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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

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Ab

un

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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

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Ab

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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

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Ab

un

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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

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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).

Page 9 of 9

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)