“PKC-mediated phosphorylation of BCL11B at Serine 2 negatively

1

“PKC-mediated phosphorylation of BCL11B at Serine 2 negatively regulates its 1

interaction with NuRD complexes during CD4+ T cell activation” 2

3

Marion Dubuissez1, £, Ingrid Loison1, Sonia Paget1, Han Vorng2, Saliha Ait-Yahia2, 4

Olivier Rohr3,4, Anne Tsicopoulos2 and Dominique Leprince1,* 5

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(1) Univ. Lille, CNRS, Institut Pasteur de Lille, UMR 8161 - M3T - Mechanisms of 7

Tumorigenesis and Targeted Therapies, F-59000 Lille 8

(2) Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - UMR8204 - 9

CIIL - Center for Infection and Immunity of Lille, F-59000 Lille, 10

(3) University of Strasbourg, IUT Louis Pasteur, EA7292, Dynamic of Host Pathogen 11

interactions, Institut of Parasitology and Tropical pathology, F-67000 Strasbourg France (4) 12

Institut Universitaire de France, Paris, France 13

* Corresponding author. Dominique Leprince, Institut de Biologie de Lille, CNRS UMR8161 14

1, rue du Pr. Calmette, CS 50447, 59021 Lille Cedex, France. 15

E mail : [email protected]; Phone : +33 3 20 87 11 19. Fax : +33 3 20 87 11 11 16

£ Present address: Maisonneuve-Rosemont Hospital Research Center, Maisonneuve-17

Rosemont Hospital, 5415 boulevard l'Assomption Montreal, QC H1T 3W5, Canada; 18

19

Key Words: BCL11B, NuRD chromatin remodeling complexes, PKC, CD4+ T cell 20

activation, Phosphorylation, SUMOylation, IL2, Id2 21

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Running title: PKC switches BCL11B to an activator in CD4+ T cells 23

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Number of characters (excluding spaces): 38878. 25

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MCB Accepted Manuscript Posted Online 9 May 2016Mol. Cell. Biol. doi:10.1128/MCB.00062-16Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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Abstract 27

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The transcription factor BCL11B/CTIP2 is a major regulatory protein implicated in various 29

aspects of development, function and survival of T cells. MAPK-mediated phosphorylation 30

and SUMOylation modulate BCL11B transcriptional activity, switching it from a repressor in 31

naïve murine thymocytes to a transcriptional activator in activated thymocytes. Here, we 32

show that BCL11B interacts via its conserved N-terminal MSRRKQ motif with endogenous 33

MTA1 and MTA3 proteins to recruit various NuRD complexes. Furthermore, we demonstrate 34

that PKC-mediated phosphorylation of BCL11B Ser2 does not significantly impact on 35

BCL11B SUMOylation but negatively regulates NuRD recruitment by dampening the 36

interaction with MTA1/3 and RbAp46 proteins. We detected increased phosphorylation of 37

BCL11B Ser2 upon in vivo activation of transformed and primary human CD4+ T cells. We 38

show that following activation of CD4+ T cells, BCL11B still binds to IL2 and Id2 promoters 39

but activates their transcription by recruiting P300 instead of MTA1. Prolonged stimulation 40

results in the direct transcriptional repression of BCL11B by KLF4. 41

Our results unveil Ser2 phosphorylation as a new BCL11B post-translational modification 42

linking PKC signalling pathway to TCR activation and define a simple model for the 43

functional switch of BCL11B from a transcriptional repressor to an activator during TCR 44

activation of human CD4+ T cells. 45

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INTRODUCTION 47

48

Post translational modifications (PTMs) of transcription regulatory proteins allow integrating 49

various signalling and environmental cues into highly dynamic and controlled responses, 50

thereby achieving coordinated gene expression programs essential for cell proliferation or 51

differentiation. 52

The transcription factor BCL11B/CTIP2 was independently isolated as an interacting partner 53

of COUP-TF in neurons and as a tumour suppressor gene in mouse models of γ-ray induced 54

thymic lymphomas (1-3). Besides its expression in the CNS, BCL11B was shown to be widely 55

expressed in all T-cell subsets, starting from the DN2 stage and to be involved in various 56

aspects of development, function and survival of T-cells (4). Indeed, BCL11B is a focal point 57

essential for several checkpoints involved in T-cell commitment in early progenitors, 58

selection at the DN2 stage and differentiation of peripheral T cells (5-9). Furthermore, 59

monoallelic BCL11B deletions or missense mutations have been identified in the major 60

molecular subtypes of T-cell acute lymphoblastic leukaemia (10). Therefore, these 61

observations together with the occurrence of deletions and mutations in γ-ray induced 62

thymomas in mice identify BCL11B as a haploinsufficient tumor suppressor gene (11). 63

BCL11B is essential for T-cell development and is considered as a “guardian of T cell fate” 64

(12). Its closely related paralog BCL11A is essential for normal lymphopoiesis and 65

haemoglobin switching during erythroid differentiation (13-15). These two transcription 66

factors appear thus as key regulators of fundamental differentiation programs during normal 67

hematopoiesis. 68

BCL11B represses transcription of its targets genes through interaction with several 69

chromatin remodelling complexes and notably recruits NuRD complexes via interaction with 70

MTA1 and MTA2 (4, 11, 16-18). Albeit, originally characterized as a sequence-specific 71


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transcriptional repressor, BCL11B also behaves as a context-dependent transcriptional 72

activator of the IL2 and Cot kinase genes in CD4+ T-cell activation (19, 20). 73

This dual behaviour of BCL11B as a transcriptional repressor and activator is not fully 74

understood but clearly rely on a dynamic cross-talk between BCL11B PTMs. Indeed, mass 75

spectrometry analyses of thymocytes isolated from 4-8 week-old mice and stimulated with a 76

mixture of phorbol ester and calcium ionophore used as an in vitro model mimicking TCR 77

activation identified several MAPK phosphorylation sites of Bcl11b and confirmed its 78

SUMOylation on Lysine 679 (21). These phosphorylation events then initiate a rapid and 79

complex cycle of Bcl11b PTMs including deSUMOylation, rephosphorylation and 80

reSUMOylation allowing recruitment of the transcriptional coactivator P300 to activate Id2 81

transcription (21, 22). 82

Here, we found that BCL11B interacts with the three MTA family members through its 83

conserved N-terminal MSRRKQ motif which is embedded in a potential PKC 84

phosphorylation consensus site. We demonstrated that a S2D phosphomimetic point mutation 85

is sufficient to abolish the interaction of BCL11B with all MTA corepressors and hence with a 86

wide range of NuRD complexes. Through generation of phosphospecific antibodies, we 87

identified in vivo Serine 2 phosphorylation of endogenous BCL11B proteins. We found that 88

activation of transformed Jurkat or primary human CD4+ T-cells results in a rapid and 89

transient PKC-induced phosphorylation of this BCL11B Ser2 culminating at 30 minutes of 90

treatment. In contrast with the MAPK-induced phosphorylations in late T-cell development, 91

this PKC phosphorylation peak precedes and does not impact on the SUMOylation peak 92

during activation of CD4+ T-cells. After prolonged activation (5 hours), the decrease of 93

BCL11B protein levels observed is due to the direct transcriptional repression of BCL11B by 94

KLF4. As shown by co-immunoprecipitation of endogenous proteins and chromatin 95

immunoprecipitation experiments, this BCL11B Ser2 phosphorylation through a decreased 96


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interaction with MTA1 and a concomitant increased interaction with P300 participates to a 97

strong transcriptional up-regulation of Id2 and IL2, two BCL11B direct target genes, during 98

CD4+ T cell activation. A PKC inhibitor, Gö6983 abolishes this co-repressor/co-activator 99

switch in Jurkat cells. Thus, our findings identify BCL11B Ser2 phosphorylation as a new 100

mandatory step in the interconnected post-translational modifications converting BCL11B 101

from a transcriptional repressor to an activator and provide compelling evidence that together 102

with SUMOylation this PKC-mediated phosphorylation is essential for transcriptional 103

activation of IL2 during human CD4+ T-cell activation. 104

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MATERIALS AND METHODS 106

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Cell culture 108

HEK293T cells were maintained in DMEM (Gibco) supplemented with 10% FCS, non 109

essential amino acids and gentamycin. Jurkat and MOLT4 cells were maintained in RPMI 110

1640 (Gibco) supplemented with 10% FCS, non essential amino acids and 111

penicillin/streptomycin. 112

113

Isolation and activation of human T CD4+ lymphocytes. 114

Donor blood was provided by Etablissement Français du Sang - Nord de France (EFS), in 115

agreement with the official ethics statement between EFS and the Centre National de la 116

Recherche Scientifique (CNRS) - Délégation Nord Pas-de-Calais et Picardie. The study was 117

approved by the Institut de Biologie de Lille (CNRS) and EFS Institutional Review Board, 118

and informed consent was obtained in writing for each donor. Isolation of human CD4+ T 119

cells was performed as previously described (23). Briefly, human peripheral blood 120

mononuclear cells (PBMC) were isolated by Ficoll Hypaque density gradient centrifugation 121

(Amersham Pharmacia Biotech, Uppsala, Sweden) and CD4+ T cells were obtained by 122

negative selection using CD4+ T Cell Isolation Kit II according to the manufacturer’s 123

instructions (Miltenyi Biotech). The purity was determined by flow cytometry, and was 124

always ≥ 95%. CD4+ cells (106 cells/ml) were cultured in complete RPMI medium (RPMI 125

1640 (Gibco, Carlsbad, CA, USA), 10% heat-inactivated fetal calf serum (FCS) (Gibco), 126

2mM L-glutamine) and stimulated or not in 96-well flat-bottomed microculture plates coated 127

with anti-CD3 antibodies (10µg/ml, OKT3 clone) and soluble anti-CD28 (2µg/ml; BD 128

Biosciences) for 24 hr or with Phorbol 12-myristate 13-acetate (PMA 50ng/ml) and 129


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Ionomycin (1μg/ml) for 5 hours. Cells were then recovered by centrifugation for further 130

analyses. 131

132

Plasmids and chemicals 133

The MTA1, MTA3, SENP2 and BCL11A expression vectors were kind gifts of Drs R. 134

Kumar, P. Wade, R. Hay and P. Tucker, respectively. The various BCL11B-Gal4 expression 135

constructs used in this study were generated as follows. Using as a template the wild-type 136

pcDNA3-FLAG-BCL11B plasmid (17, 24), we PCR amplified using relevant 137

oligonucleotides fragments corresponding to amino acids 1-20 and 1-58 of wild-type BCL11B 138

as well as 1-20 fragments with point mutation of Serine 2 (S2A, S2D, S2T). Next, these 139

fragments were cloned in frame with a C-terminal Gal-4 DNA-binding domain followed by a 140

Nuclear localization signal (NLS) and an HA epitope into the pSG5-Gal4-NLS-HA 141

eukaryotic vector to mimic their location in the full-length protein (25). The point (S2A, S2D) 142

and deletion (ΔMSRRKQ) mutants of BCL11B were PCR amplified from the wt BCL11B 143

plasmid with relevant oligonucleotides and subcloned in pcDNA3-FLAG-BCL11B using 144

standard procedures. All constructs were verified by sequencing. PMA (Phorbol-12-145

Myristate-13-Acetate) and Ionomycin both from Sigma-Aldrich were used at a final 146

concentration of 50 ng/ml and 1 μg/ml, respectively. Bisindolylmaleimide II (BIM II) (used at 147

a final concentration of 10μM for 30 minutes) and Gö6983 (used at a final concentration of 148

1μM for 15 minutes) which are general inhibitors of protein kinase C (PKC) subtypes were 149

purchased from Sigma-Aldrich and Selleckchem, respectively (26, 27, 28). The MEK 150

inhibitor U0126 was purchased from Merck Millipore and used at a final concentration of 10 151

μM for 30 minutes. Okadaic Acid (OA) and Calyculin A (Cal. A) which are potent inhibitors 152

of all three type 2A Ser/Thr phosphatases (PP1, PP2A and PP6) (29) were both obtained from 153

Santa Cruz. They were dissolved in DMSO and used at a final concentration of 1 μM and 154


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50 nM, respectively. Calyculin A also inhibits arsenic-induced PML SUMOylation (30) and 155

BCL11B SUMOylation (21, 31). The SUMOylation inhibitors 2-DO8 which is specific and 156

blocks SUMO transfer from the Ubc9-thioester complex to the substrates (32) and Anacardic 157

Acid which blocks SUMOylation without affecting ubiquitination (33) were both obtained 158

from Sigma and used at a final concentration of 50 μM (34). Anarcadic acid is also a potent 159

inhibitor of several Histone Acetyl Transferase (HATs) such as PCAF, P300 and TIP60 (35). 160

161

Transfection, immunoprecipitation and co-immunoprecipitation 162

Cells were transfected in Opti-MEM (Invitrogen) by the PEI method using ExGen 500 163

(Euromedex), as previously described with 2.5 µg of DNA corresponding to the relevant 164

expression vectors or the empty vector used as control. Cells were transfected for 6h and then 165

incubated in fresh complete medium. After 48h transfection, cells were rinsed with ice PBS 166

and lysed in cold IPH buffer (50mM Tris, 150 mM NaCl, 5 mM EDTA, 0.5% NP40, protease 167

inhibitor cocktail (Roche)) for co-immunoprecipitation. Cell lysates were sonicated briefly 168

and cleared by centrifugation (14 000 rpm, 4°C, 15 min). The supernatants were pre-cleared 169

with 15 µl of protein A/G sepharose beads (Amersham Bioscience) incubating during 1 hour 170

on a rotator at 4°C. Then, lysates were incubated with 2 µg of antibody on a rotator at 4°C 171

overnight. Later, 20 µl of protein A/G beads were added and incubated 30 min at 4°C. Finally, 172

the beads were washed three times with IPH buffer. Bound proteins were eluted by boiling in 173

Laemmli buffer (36). 174

To detect SUMOylation of endogenous BCL11B proteins by immunoprecipitation studies, 175

Jurkat cells were lysed in a lysis buffer containing 1% SDS, 20 mM Tris-HCl pH 8, 10% 176

glycerol, 1 mM DTT and 15 mM NEM. These cells extracts were immediately boiled for 10 177

minutes and diluted in 9 volumes of RIPA buffer (20 mM Tris-HCl pH8, 0.5 mM EDTA, 150 178


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mM NaCl, 0.5% NP40, 10% glycerol) supplemented with 10mM N-ethylmaleimide (NEM) 179

and protease inhibitor cocktail for immunoprecipitation with anti BCL11B antibodies (37). 180

To detect endogenous interactions between BCL11 (A or B) and MTA (1 or 3) proteins, we 181

prepared the combined cytoplasmic and nucleoplasmic fractions and the micrococcal 182

nuclease-solubilised chromatin fractions as previously described (36, 38, 39). Two chromatin 183

fractions prepared in parallel from the same number of cells were analyzed in co-184

immunoprecipitation assays as described above, with control rabbit IgG or MTA1 and MTA3 185

specific antibodies. 186

187

Transfection and luciferase transactivation test 188

HEK293T cells were plated in 12-well plates Cell Bind (Corning) and transfected with 500 ng 189

of DNA for 6h in triplicate with the luciferase reporter plasmid pGL3-5xGal4 RE-tk luc 190

(luciferase expression under the control of Gal4 responsive elements) or the pGL3-empty 191

vector in combination with various BCL11B-Gal4 constructs and then incubated in fresh 192

complete medium. 48 hours after transfection, cells were rinsed with ice PBS and lysed in the 193

Luc assay buffer. After normalization to the β-galactosidase activity, the data were expressed 194

as the Luc activity relative to the activity of pGl3-Luc with pSG5-Gal4-NLS-HA which 195

represents the basal condition and was given an arbitrary value of 1. The results represent the 196

mean values from three independent transfections in triplicate. Luciferase and β-galactosidase 197

activities were measured using respectively beetle luciferin (Promega) and the Galacto-Light 198

kit (Tropix) with a Berthold chemiluminometer (36). 199

200

Antibodies and Western Blot analyses 201

To generate polyclonal antibodies against Phosphorylated-Serine 2 of BCL11B, control and 202

phosphorylated peptides corresponding to AA 1-19 of human BCL11B (H2N-203


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MS(PO3H2)RRKQGNPQHLC-CONH2) peptide were synthesized, coupled to KLH and used 204

to immunize rabbit (Eurogentec, Belgium). Control and phosphospecific antibodies were 205

purified by affinity chromatography using standard protocols. Commercial antibodies of the 206

following specificity were used: FLAG M2 from Sigma; HA from BABCO; BCL11B 207

(ab18465 for Western Blot, ab28448 for IP and ChIP and A300-383A for IP) from Abcam 208

and Bethyl Laboratories; Phospho-Erk1/2 (9106) from Cell Signaling; Actin (sc-1616R); 209

MTA1 (sc-9445 for WB, sc-10813 for IP and sc-10813 or ab50263 for ChIP) from Santa Cruz 210

and Abcam; MTA3 (ab87275) from Abcam; Erk2 (sc-154) from Santa Cruz; HDAC2 (sc-211

7899 H-54) from Santa Cruz; CHD4 (ab70469) from Abcam; RbAP46 (sc-8272) from Santa 212

Cruz; RbAP48 (C15200206 for ChIP) from Diagenode; Anti phospho-PKC substrate (2261) 213

from Cell Signaling; KLF4 (ab106629) and RanGAP1 (ab92360) from Abcam. 214

Western blots were performed as previously described (40). The secondary antibodies were 215

horseradish peroxidase-linked antibodies against rabbit, rat and mouse immunoglobulins 216

(Amersham Biosciences); goat immunoglobulins (Southern Biotech). 217

To analyze the SUMOylation of BCL11B proteins by Western blotting analyses, transfected 218

HEK293 cells or Jurkat and human CD4+ T cells pelleted by centrifugation were directly 219

lysed in Laemmli loading buffer, boiled for 10 minutes and processed for Western blotting as 220

described above. 221

222

Chromatin Immunoprecipitation 223

Control or activated Jurkat and primary human CD4+ T-cells were washed with PBS and 224

resuspended in 0.5 ml PBS for 5x106 cells. Then, cells were fixed by adding formaldehyde to 225

a final concentration of 1% for 8 min at room temperature. To stop fixation, glycine was 226

added to a final concentration of 0.125M. After 5 min at room temperature, cells were 227

collected by centrifugation (1500 rpm, at 4°C, 5 min). The supernatants were removed and 228


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cells were lysed by resuspension in chilled cell lysis buffer for 10 min on a rotator at 4°C. 229

Then, the samples were pelleted, resuspended in 200 µl nuclei lysis buffer and sonicated to 230

chromatin with an average size of 250 bp using a cooling BioRuptor (Diagenode, Belgium). 231

20 µg of chromatin was immunoprecipitated with indicated antibodies and real-time PCR 232

analyses were performed as described (36). The primers used are summarized in 233

Supplementary Table 1. For the IL2 promoter, we designed primers around the BCL11B US1 234

binding sites (19). For the Id2 promoter, the Transcription Start Site (TSS) is derived from the 235

Id2 human locus found in the NCBI Nucleotide database (gi: 568815596). The region just 236

upstream of the TSS contains several BCL11B binding sites as shown by ChIP-Seq analyses 237

of BCL11B binding in double positive human thymocytes (9). Clustal alignments of the 238

corresponding human and murine regions highlighted a conserved potential BCL11B direct 239

binding site, TGGGC, which has been analyzed in ChIP-qPCR experiments using relevant 240

oligonucleotides. Similar analyses identified a potential KLF4 binding site in the BCL11B 241

promoter. For the ChIP in presence of the PKC inhibitors, Jurkat cells were first incubated 242

with Gö6983 (1μM for 15 minutes) before the relevant activation treatment. 243

244

RT and Quantitative PCR 245

Total RNAs were reverse transcribed using random primers and MultiScribeTM reverse 246

transcriptase (Applied Biosystems). Real-time PCR analyses were performed by Power SYBR 247

green (Applied Biosystems) in a MX3005P fluorescence temperature cycler (Stratagene) 248

according to the manufacturer’s instructions. Results were normalized to 18S RNA used as 249

internal control. The primers used are summarized in Supplementary Table 2. 250

251


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Statistics 252

Experiments were realised at least twice independently in duplicates or triplicates. Statistical 253

analyses were performed by Student’s test. The asterisk (*) indicates p < 0.05, (**) indicates p 254

< 0.01 and (***) indicates p < 0.001. 255

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RESULTS 257

258

BCL11B interacts with the three MTA proteins 259

MTA1, MTA2 or MTA3 are found in a mutually exclusive manner in different specialized 260

NuRD complexes (41, 42). BCL11B interacts with the closely related MTA1 and MTA2 261

proteins (18). We thus investigated if BCL11B also interacts with the functionally distinct 262

MTA3 protein. Co-immunoprecipitation experiments (Co-IPs) in transiently transfected 263

HEK293T (Fig. 1A-B) or between endogenous proteins in the human acute lymphoblastic 264

leukemia cell line, MOLT4 (Fig. 1C-D) and in the human CD4+ T-cell line Jurkat (Supp. Fig. 265

1A-B) (19), demonstrated that BCL11B interacts with MTA1 and MTA3. Contrary to Jurkat, 266

MOLT4 cells express both BCL11B and BCL11A but with lower levels of MTA1 and MTA3 267

(Supplemental Fig. 1C-F) (43, 44). Similarly, we unravelled an interaction between BCL11A 268

and MTA1 or MTA3 proteins either ectopically expressed in HEK293T (Supp. Fig. 1G-H) or 269

endogenous in MOLT4 cells (Fig. 1E-F). Taken together, these results demonstrate that 270

BCL11B and BCL11A can interact with the MTA1/MTA2 and MTA3 proteins and hence 271

with a wide variety of NuRD complexes. 272

273

Mimicked phosphorylation of the BCL11B N-terminal domain (S2D) disrupts its 274

interaction with MTA proteins and relieves its transcriptional repression activity 275

The 1-45 amino acids of BCL11B containing the MSRRKQ motif shared with FOG-1 and 276

SALL1 are sufficient for the interaction with MTA1 in GST pull-down experiments (18, 26, 277

45, 46). To assess this interaction in vivo, we fused the 1-20 or 1-58 amino acids of BCL11B, 278

in frame with a C-terminal Gal4 DNA-binding domain (Fig. 2A). Transient transfection 279

assays in HEK293T demonstrated that these 1-20- and 1-58-BCL11B-Gal4 chimeras are 280

nuclear, interact with MTA1 and MTA3 and mediate strong transcriptional repression in 281


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luciferase reporter assays (Fig. 2B-C-D) (Supp. Fig. 2A-C). Serine 2 (Ser2) of the conserved 282

MSRRKQ motif is embedded in a potential consensus site (S/T-X2-0-R/K1-3) for 283

phosphorylation by protein kinase C (PKC), a family of Serine/Threonine kinases involved in 284

many cellular functions, including T-cell activation (47-48). To study the importance of Ser2 285

and the effect of its potential phosphorylation, we constructed several point mutants of Ser2 286

(S2X) in the 1-20 BCL11B-Gal4 chimera (Fig. 2A). First, the phosphomimetic S2D mutant is 287

unable to co-immunoprecipitate MTA1 or MTA3 proteins and in luciferase transactivation 288

assays, its repression activity is significantly relieved (Fig. 2B-C-E-F) (Supp. Fig. 2). In 289

addition, the repression activity of the phosphodeficient S2A mutant is also inhibited (49) 290

whereas a Serine to Threonine substitution (S2T) has no significant impact on the repression 291

potential of the BCL11B-Gal4 chimera (Fig. 2F-G) (Supp. Fig. 2). 292

Thus, the first 20 amino acids of BCL11B, including the conserved MSRRKQ motif are 293

sufficient to interact with MTA1 or MTA3 and have autonomous repression activity. Ser2 is 294

important for these two properties which are severely impaired by a phosphomimetic point 295

mutation, S2D. 296

297

A phophosmimetic mutation of BCL11B Ser2, S2D, impedes NuRD recruitment 298

We engineered a S2D and a S2A full-length BCL11B point mutant and a deletion mutant, 299

BCL11B-ΔMSRRKQ (Fig. 3A). They all display a nuclear localization (Supp Fig. 2G). In 300

Co-IPs, the phosphomimetic S2D mutant weakly interacts with MTA1 and MTA3 301

overexpressed in HEK293T (Fig. 3B-C). Similarly, co-IPs assays demonstrated that the S2A 302

and S2D point mutants ectopically expressed in HEK293T still weakly interact with 303

endogenous MTA1 proteins whereas the ΔMSRRKQ deletion mutant is unable to do so (Fig. 304

3D). The MSRRKQ motif of FOG1 also interacts with RbAp48 (49). We thus investigated the 305

interaction of these mutants with other endogenous components of NuRD. We demonstrated 306


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that the S2A mutant still significantly interacts with MTA1, MTA3 and RbAp46 as compared 307

to wt BCL11B (Fig. 3E). By contrast, the S2D mutant is totally unable to interact with 308

endogenous RbAp46 whereas it weakly interacts with MTA1, MTA3, CHD4 and HDAC2 309

(Fig. 3E-F). These results are in close agreement with the crystallographic structure of the 310

FOG 1-15 peptide bound to RbAp48 since Ser2 is engaged in hydrogen bond interaction 311

whereas the adjacent Arg3, Arg4 and Lys5 participate to ion pair contacts with Glu residues 312

in RbAp48 (49). Thus, a S2D phosphomimetic mutation in the N-terminal MSRRKQ motif of 313

BCL11B negatively regulates the interactions with several endogenous NuRD components. 314

315

BCL11B is phosphorylated by PKC on Ser2 upon PMA activation of HEK293T cells 316

and is SUMOylated. 317

We next raised antibodies against Phospho-Ser2 BCL11B which were able to detect 318

phosphorylation of BCL11B on Ser2 residue in transfected HEK293T cells treated with PMA, 319

a potent PKC activator (48) (Fig. 4A) (Supp. Fig. 3). These antibodies detected a strong 320

induction of Ser2 phosphorylation after overexpression in PMA-treated HEK293T of wt 321

BCL11B but not of the S2A mutant, thereby demonstrating their specificity (Fig. 4B). Finally, 322

addition of BIMII, a PKC inhibitor decreased BCL11B phosphorylation on Ser2 whereas 323

Okadaic acid, a Ser/Thr phosphatase inhibitor, increased it (Fig. 4C-D). 324

In P/A-stimulated murine thymocytes, Bcl11b is SUMOylated (21). In PMA-activated-325

HEK293T cells transfected with BCL11B, we also observed a slowly migrating band (above 326

the 150KDa Marker) which corresponds to the SUMOylation of BCL11B since it totally 327

disappeared upon cotransfection of the deSUMOylase SENP2 (Fig. 5A) (Supp. Fig. 3). In 328

addition, the PKC inhibitor BIMII seems to have no significant effects on BCL11B 329

SUMOylation level while the ERK inhibitor U0126 seemed to slightly reduce it (Fig. 5B). 330

The PKC inhibitor also partially inhibits Erk activation as previously shown (51). Bcl11b 331


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SUMOylation is important for its interaction with p300 (21). Interestingly, whereas the S2A 332

and S2D point mutants are severely affected in their interaction with MTA1 as compared with 333

wt BCL11B (Fig. 3), they display similar levels of SUMOylation and hence of interaction 334

with endogenous p300 proteins (Fig. 5C-D), correlating the lack of significant effects of the 335

PKC inhibitor on BCL11B SUMOylation. 336

Thus, the PKC-mediated phosphorylation of BCL11B in HEK293T impinges on its 337

interaction with MTA1 but not on its SUMOylation and interaction with P300. 338

339

PKC-mediated Ser2 phoshorylation of endogenous BCL11B proteins upon PMA-340

Ionomycin (P/I) activation of Jurkat cells has no impact on its SUMOylation. 341

We next addressed this PKC-mediated Ser2 phosphorylation on endogenous BCL11B 342

proteins during human CD4+ T-cell activation. Western blot analyses of Jurkat cell extracts 343

with the anti pSer2 BCL11B serum confirmed a peak of transient phosphorylation of BCL11B 344

Ser2 after 30 minutes of activation with PMA-Ionomycin (P/I) (Fig. 6A). 345

Immunoprecipitation of Jurkat cell extracts by a C-terminal BCL11B antibody followed by 346

Western blot analyses with the anti pSer2 BCL11B serum identified BCL11B proteins 347

phosphorylated on Serine 2 upon T cell activation, notably in presence of the phosphatase 348

inhibitor, Okadaic acid (Fig. 6B). Treatment with Okadaic acid induced a supershift of 349

BCL11B both in basal and activated conditions due to its phosphorylation by ERK and PKC 350

pathways as detected by the anti pSer2 (Fig. 6B, lane 3 and Fig. 6C, lanes 2 and 4). 351

Furthermore, Ser2 phosphorylation observed in presence of Okadaic acid is lost if, before 352

activation, cells are treated with the PKC inhibitor, Gö6983 whereas the supershift of 353

BCL11B is still observed (Fig. 6D, lanes 4 and 5). 354

In activated Jurkat cells, BCL11B phosphorylation is transient and precedes its SUMOylation 355

(Fig. 6A). As observed in HEK293T cells, the PKC inhibitor seems to have no significant 356


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effects on BCL11B SUMOylation after 60 minutes of activation, whereas ERK inhibition by 357

U0126 seems to slightly decrease it (Fig. 6E). Another Ser/Thr phosphatase inhibitor, 358

Calyculin A, is also a SUMOylation inhibitor as initially shown for PML (30) and later for 359

BCL11B (21, 31). Upon activation and Calyculin A treatment of Jurkat cells, we observed an 360

electromobility shift for the major BCL11B proteins whereas the slowest migrating BCL11B-361

SUMO band totally disappeared (Fig. 6F), as shown in murine thymocytes (21). Similar 362

results were obtained with the specific SUMOylation inhibitor 2-D08 (32, 34) (Fig. 6G) and 363

with Anacardic Acid (AA), an inhibitor of SUMOylation (33) and of various Histone Acetyl 364

transferase (HATs) (35). This might explain through transcriptional inhibition, the decrease of 365

BCL11B protein levels as well as of several SUMOylated proteins observed with AA (Supp. 366

Fig. 5). Finally, Western blot analyses with anti-SUMO-2 after immunoprecipitation of 367

endogenous BCL11B proteins under denaturing conditions, allowed the detection of 368

SUMOylated BCL11B proteins in activated Jurkat cells (Fig. 6H) (37). As a whole, these data 369

identify phosphorylation of BCL11B Ser2 by PKC as a new post-translational modification of 370

BCL11B during human CD4+ T-cell activation, preceding and acting independently of its 371

SUMOylation. 372

373

KLF4 directly represses BCL11B transcription upon PMA-Ionomycin (P/I) activation of 374

Jurkat cells. 375

SUMOylation of Bcl11b in P/A-treated murine thymocytes ultimately results in its 376

ubiquitination and degradation (21). However, whereas prolonged stimulation of Jurkat cells 377

with P/I also resulted in a decrease of BCL11B protein levels after 5 hours (Fig. 6A), this 378

correlated with transcriptional repression of BCL11B rather than degradation after 379

ubiquitination (Supp. Fig. 5). Indeed, qRT-PCR analyses demonstrated a strong decrease of 380

BCL11B mRNAs starting at 2 hours of stimulation (Fig. 7A). Furthermore, BCL11B proteins 381


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levels decreased after 5 hours of P/I stimulation, even in presence of three different 382

proteasome inhibitors, MG132, ALLN or Lactacystin (Fig. 7B). Inducible overexpression of 383

the transcriptional repressor KLF4 in Jurkat cells inhibits the expression of T-cell associated 384

factors including BCL11B and induces its degradation after 48 hours (31). These results 385

prompted us to further investigate the relationship between KLF4 and BCL11B. In Jurkat 386

cells, qRT-PCR analyses detected a strong induction of KLF4 expression peaking 30-60 387

minutes after activation (Fig. 7C) and thus preceding the repression of BCL11B expression 388

(Fig. 7A). Furthermore, in silico analyses of the BCL11B promoter identified a conserved 389

KLF4 binding site upstream of the TSS (Fig. 7D). ChIP analyses demonstrated the direct 390

binding of KLF4 on the BCL11B promoter and on CXCR4, a bona fide KLF4 direct target-391

gene (31) (Fig. 7E) after 5 hours of activation when BCL11B and CXCR4 expression are 392

severely repressed (Fig. 7A) (Supp. Fig. 6A-B). 393

Thus, BCL11B is a new direct target gene of the transcriptional repressor KLF4 during human 394

CD4+ T cell activation. 395

396

Endogenous BCL11B binds the IL2 and Id2 promoters but with the MTA1 co-repressor 397

or the P300 co-activator in resting versus PMA-Ionomycin (P/I) activated Jurkat T cells. 398

BCL11B participates through interaction with the P300 co-activator in the transcriptional 399

activation of IL2 and of Id2 (19, 21). Activation of Jurkat cells with P/I induced a huge up-400

regulation of IL2 gene expression starting as early as 30 minutes of treatment and a 401

significant, albeit slightly delayed up-regulation of Id2 around 120 minutes (Fig. 8A-B and 402

Supp. Fig. 6C), whereas BCL11B mRNA levels decreased after 2 hours of treatment (Fig. 403

7A). Co-immunoprecipitation of BCL11B with MTA1 or P300 endogenous proteins in Jurkat 404

cells treated for 30 minutes with P/I demonstrated that the peak of BCL11B Ser2 405


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phosphorylation, we reproducibly observed (Fig. 6A), is concomitant with a slight decrease of 406

BCL11B-MTA1 interaction and a clear increase of BCL11B-P300 interaction (Fig. 8C). 407

To correlate these results with promoter occupancy, we first performed pilot ChIP 408

experiments with chromatin prepared from Jurkat cells and showed that BCL11B and MTA1 409

are bound on the Upstream Site 1 (US1) of the IL2 promoter (19) and on the Id2 promoter on 410

a newly identified and conserved BCL11B binding site located just upstream of the 411

transcription start site (Fig. 8D and Supp. Fig. 7). ChIP experiments conducted with Jurkat 412

cells treated with DMSO or with P/I for 30 minutes failed to detect any significant differences 413

in BCL11B binding to the IL2 and Id2 promoters (Fig. 8E-F). By contrast, in Jurkat cells 414

activated for 30 minutes to induce the peak of BCL11B Ser2 phosphorylation (Fig. 6), IL2 415

and Id2 promoter occupancy by MTA1 and MTA3 was markedly decreased as compared to 416

steady-state (Fig. 8E-H). This effect was not due to a down-regulation of MTA1 expression 417

since the levels of MTA1 RNAs (Supp. Fig. 6) are slightly increased by the treatment. 418

Conversely and in agreement with the Co-IP experiments, recruitment of the P300 coactivator 419

at the IL2 and Id2 promoters is significantly increased in activated Jurkat cells (Fig. 8I). As 420

control, none of these proteins were detected on the GAPDH promoter (Fig. 8G-I). 421

Ectopic expression of S2D or S2A BCL11B mutants in Jurkat cells to directly address the 422

impact of Ser2 phosphorylation on this MTA1/P300 switch is not feasible. Indeed, the prior 423

inactivation of the highly expressed endogenous BCL11B proteins will rapidly inhibit the 424

proliferation of Jurkat cells and induce apoptosis (51, 52). To obtain further evidence of the 425

specific involvement of PKC in regulating BCL11B interactions with MTA1 and P300, Jurkat 426

cells were activated in presence of a pan-PKC inhibitor, Gö6983. Pretreatment of Jurkat cells 427

with Gö6983 has no effects on BCL11B or MTA1 and RbAp48 expression levels but blocked 428

the P/I-induced activation of IL2 expression (Fig. 9A-B) (Supp. Fig. 6G-H). Furthermore, in 429

presence of the PKC inhibitor similar levels of BCL11B are bound to the IL2 promoter but 430


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MTA1 and RbAp48 are not released from the promoter (Fig. 9C-D). Conversely, Gö6983 431

inhibits the P/I-induced increase of P300 binding on the IL2 US1 site (Fig. 9D). 432

Together, these data show that P/I treatment does not affect direct BCL11B binding to IL2 433

and Id2 promoters but induces its phosphorylation on Ser2 by PKC, to disrupt interaction with 434

NuRD repressive complexes and to mediate interaction with P300, thereby participating to its 435

transcriptional activation. 436

Activation of primary human CD4+ T cells switches BCL11B from a transcriptional 437

repressor to a transcriptional activator of IL2 438

Primary human CD4+ T cells were activated with P/I for 5 hours or with antiCD3/CD28 for 439

24 hours to induce TCR stimulation (23). In both conditions, we observed a 2-fold repression 440

of BCL11B mRNA levels whereas BCL11B protein levels were roughly identical suggesting 441

some post-translational stabilization mechanisms (Fig 10A-B). Interestingly, a concomitant 442

increase of BCL11B Ser2 phosphorylation and SUMOylation nicely correlating with 443

transcriptional activation of IL2 was observed, especially with the anti CD3/CD28 antibodies 444

(Fig. 10B). ChIP analyses were thus performed after 24h stimulation with anti CD3/CD28 445

antibodies, a treatment mimicking the canonical activation of T-cells through antigen 446

presenting cells, since IL2 induction was particularly efficient (ca. 4000-fold) in these 447

conditions (Fig. 10C). This activation had no effect on the binding of BCL11B on the IL2 448

promoter but led to a strongly increased recruitment of P300 to this region, concomitant with 449

the transcriptional activation of IL2 (Fig. 10D-E). 450

These results therefore show that upon physiological activation of primary human CD4+ T 451

cells, BCL11B through phosphorylation of Ser2 in its N-terminal repression domain is 452

switched from a transcriptional repressor to an activator of IL2 expression. 453

454


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DISCUSSION 455

456

Multiple post-translational modifications (PTMs) of a given protein substrate can occur 457

simultaneously or sequentially to modulate its function, localization and stability through 458

cooperative or antagonizing effects on interacting partners. BCL11B is essential for T-cell 459

development. Recently, Bcl11b function was shown to be finely regulated during TCR 460

activation of resting murine thymocytes by a MAPK-initiated dynamic sequence of PTMs. 461

Indeed, treatment with phorbol ester and calcium ionophore resulted in rapid phosphorylation 462

on 23 S/TP sites, deSUMOylation, dephosphorylation, reSUMOylation, ubiquitination and 463

ultimately degradation of Bcl11b (21, 22, 53). Importantly, this complex pathway facilitates 464

derepression of repressed direct target-genes of Bcl11b, as shown for Id2, when immature 465

thymocytes need to initiate differentiation programs (21). 466

Here, we describe a different and much simpler PKC-mediated pathway which through 467

phosphorylation of Ser2 in the BCL11B conserved MSRKKQ motif participates to the up-468

regulation of IL2, another BCL11B direct target gene, during activation of human CD4+ T 469

cells by PMA/Ionomycin (P/I) or by anti CD3/CD28 antibodies as a surrogate for canonical 470

activation of T-cells through antigen-presenting cells (Fig. 11). Notably, this is the first 471

description at the endogenous level of a PTM targeting this important MSRRKQ repression 472

motif shared by several distinct nuclear proteins. The phosphorylation of Bcl11b Ser2 has not 473

been detected during differentiation of resting murine thymocytes (90% being DP cells) (21), 474

a population clearly distinct of the human CD4+ T cells analyzed here. However, the N-475

terminal region was not present in the coverage map of Bcl11b (21) since trypsin digests 476

which cut after Arg and Lys residues are predicted to generate hardly detectable MSR 477

peptides. 478


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The Ser2 phosphorylation peak in Jurkat cells precedes the SUMOylation peak but, a specific 479

PKC inhibitor has no effect on the SUMOylation of BCL11B (Fig. 6). By contrast, the 480

multiple phosphorylation events induced by the MAPK pathway in murine thymocytes favor 481

the interaction of Bcl11b with the deSUMOylase SENP1 (21). Another major difference is the 482

lack of ubiquitination of human BCL11B after prolonged P/I treatment of Jurkat cells. In fact, 483

the decrease of BCL11B proteins levels observed after 5 hours of activation seems to mainly 484

rely on direct transcriptional repression of BCL11B by KLF4 rather than on BCL11B protein 485

degradation by the proteasome (Fig. 7). 486

These different MAPK- or PKC-mediated pathways however both orchestrate the 487

transcriptional activity of BCL11B to allow derepression of direct target genes, albeit through 488

strikingly different mechanisms. Indeed, through quantitative ChIP experiments in activated 489

Jurkat cells or in purified normal human CD4+ T cells, we observed similar binding of 490

BCL11B to the IL2 promoter as compared to basal conditions. However, in these activating 491

conditions, which result in BCL11B Ser2 phosphorylation, the levels of MTA1 are severely 492

reduced at the IL2 promoter whereas P300 levels reciprocally increase on line with the 493

induction of IL2 expression (Fig. 8-10). In the Co-IP experiments (Fig. 8C) whereas a net 494

increase of BCL11B-P300 interaction is clearly seen, the impact of T-cell activation on 495

BCL11B-MTA1 interaction is relatively low. However, in contrast with Co-IPs, the ChIP 496

experiments were performed on chromatin and are thus more likely to reflect the function of a 497

transcription factor and of its different partners in their natural context (54, 55). In developing 498

murine thymocytes, Bcl11b and MTA1 remained associated at the Id2 promoter both in basal 499

conditions and activating conditions, as shown by ChIP experiments (21). However, as stated 500

by these authors, the possibility that MTA1 and/or other NuRD component proteins became 501

post-translationally modified as a consequence of this treatment has not been investigated in 502

detail. This is an important issue since the C-terminal end of MTA proteins involved in the 503


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interaction with MSRRKQ-proteins is subject to extensive post-translational modifications. 504

Notably, the methylation-demethylation of MTA1 Lysine 532 regulates its cyclical- and 505

signaling- dependent association with NuRD corepressor or NuRF co-activator complexes, 506

respectively (56). The transcriptional co-factor FOG-1 (Friend of GATA) also interacts with 507

NuRD through a N-terminal MSRRKQ motif and this interaction plays a key role for lineage 508

commitment during erythropoiesis and megakaryopoiesis (45, 57-58). Strikingly, ChIP 509

experiments revealed that NuRD components are present at both repressed and active 510

GATA1/FOG1 target genes (59). These results suggest two possible mechanisms for 511

interaction of MTA1 with different transcription factors depending on the cellular context: (i) 512

binding of MTA1 to the target genes both in active or repressive conditions but with PTMs 513

fine-tuning its interaction with repressive or activating complexes as reported for 514

GATA1/FOG1 or (ii) presence only in repressive conditions and loss of interaction in 515

activating conditions due to specific PTMs in short MTA1-interacting motifs, as shown here 516

for PKC-mediated phosphorylation of BCL11B in CD4+ T cells. 517

Here, we demonstrated that this short motif is necessary and sufficient to mediate binding of 518

BCL11A and BCL11B to the three members of MTA co-repressors family, thus enabling 519

BCL11A, BCL11B and more generally MSRRKQ-containing proteins to interact with the 520

complete range of NuRD complexes. This highlights their flexibility to participate to various 521

differentiation programs involved in the development of the immune and central nervous 522

(CNS) systems and to regulate tissue-specific gene expression in many distinct cellular types 523

including T and B cells, ameloblasts or keratinocytes for BCL11B (16) or lymphopoiesis and 524

erythroid precursors for BCL11A (15). Interestingly, the interaction of the BTB/POZ 525

transcriptional repressors HIC1 and BCL6 with MTA1 and MTA3, respectively, are inhibited 526

by acetylation of a lysine residue in different conserved peptidic interaction motifs. HIC1 527

acetylation on K314 in the MKHEP SUMOylation/acetylation motif negatively regulates its 528


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interaction with MTA1 whereas its SUMOylation favors it, notably in the DNA-damage 529

response (40, 60). BCL6 acetylation on K379 in the KKYK motif abrogates its ability to 530

interact with MTA3 which is a major repression mechanism controlling Germinal Center B 531

cells differentiation (61-63) as well as CD4+ T-cell fate and function (64). 532

Although our studies have been focused on BCL11B, it is tempting to speculate that similar 533

PTMs could be identified in other MSRRKQ-containing nuclear proteins. Indeed, the 534

developmental regulator SALL1 is certainly regulated by PKC-phosphorylation on Ser2, as 535

suggested by in vitro studies (27). For FOG1, the role of Ser2 has been investigated only 536

through Alanine scanning experiments which demonstrated that this S2A mutation did not 537

affect the repression potential of a FOG1 1-12 Gal4 chimera (57). However, these in vitro 538

studies or in vivo Knock-in models in mice and the crystal structure of RbAp48 (another 539

NuRD component) bound to the 15 N-terminal amino-acids of FOG-1 have provided 540

compelling evidences for the key role played by the RKK residues in the conserved 541

MSRRKQ motif (45, 50, 57-59). Whereas Ser2 is only involved in Hydrogen bond 542

interaction, these RKK residues are engaged both in hydrogen bond interaction and in ion pair 543

contacts with acidic Glu residues in RbAp48 (49). These strong interactions could be 544

impaired by the negative charge brought by PKC-mediated phosphorylation of the adjacent 545

Ser2 residue as shown for the interaction between the phosphomimetic S2D BCL11B mutant 546

and the related RbAp46 protein (Fig. 3E). Recently, ZNF827 was shown to recruit NuRD to 547

telomeres in cells using ALT (Alternative Lengthening of Telomeres) to enable telomere 548

extension. This recruitment relies on RRK residues found in an N-terminal MPRRKQ motif 549

highly homologous to the motif found in FOG, SALL1 and BCL11A/B except for the 550

replacement of Ser2 by a Pro residue (65). Thus, the PKC-mediated phosphorylation of Ser2 551

that we have demonstrated here for BCL11B would provide to these MSRRKQ-containing 552


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proteins a rapid and efficient mechanism to fine tune their interaction via a RRK motif with 553

NuRD. 554

In conclusion, we have identified a novel mechanism for how the essential regulator of T cell 555

development BCL11B is regulated by PKC-mediated phosphorylation of Ser2 in its conserved 556

N-terminal repression domain during activation of CD4+ T cells. In addition, this BCL11B N-557

terminal domain is essential for the transcriptional repression of HIV-1 LTR sequences which 558

thus affects both HIV-1 replication and virus production in CD4+ T lymphocytes (66). In 559

future studies, it would be interesting to explore if this switch between transcriptional 560

repression and activation by phosphorylation in their MSRRKQ motifs could be generalized 561

to other NuRD-interacting proteins, in particular to BCL11A or FOG-1, two important 562

regulators of differentiation programs during normal hematopoiesis. 563

564

ACKNOWLEDGMENTS 565

Marion Dubuissez and Sonia Paget are supported by predoctoral fellowships from the 566

University of Lille. We thank Dr Capucine Van Rechem for critical reading of the manuscript. 567

568

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Pei XY, Smits AH, Kloet SL, Wang H, Shepherd NE, Stokes PH, Blobel GA, 760 Vermeulen M, Glover DM, Mackay JP, Laue ED. 2014. Insight into the 761 architecture of the NuRD complex: structure of the RbAp48-MTA1 subcomplex. J 762 Biol Chem 289:21844-21855. 763

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determinants for protein kinases and protein phosphatases. J Biol Chem 266:15555-766 15558. 767

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immunological synapse. Trends Immunol 34:234-242. 770 771 49] Lejon S, Thong SY, Murthy A, AlQarni S, Murzina NV, Blobel GA, Laue ED, 772

Mackay JP. 2011. Insights into association of the NuRD complex with FOG-1 from 773 the crystal structure of an RbAp48.FOG-1 complex. J Biol Chem 286:1196-1203. 774

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Schmidt CA, Li Y. 2011. Down regulation of BCL11B expression inhibits 777 proliferation and induces apoptosis in malignant T cells by BCL11B-935-siRNA. 778 Hematology:16236-242. 779

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Przybylski GK, Schmidt CA, Li. Y. 2011. Gene expression profiles in BCL11B-787 siRNA treated malignant T cells. J Hematol Oncol 4:23. 788

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1-mediated recruitment of NuRD is required for cell lineage re-enforcement during 810 haematopoiesis. Embo J 29:457-468. 811

812 [59] Miccio A, Wang Y, Hong W, Gregory GD, Wang H, Yu X, Choi JK, Shelat S, Tong 813

W, Poncz M, Blobel GA. 2010. NuRD mediates activating and repressive functions 814 of GATA-1 and FOG-1 during blood development. Embo J 29:442-456. 815

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2010. Differential regulation of HIC1 target genes by CtBP and NuRD, via an 818 acetylation/SUMOylation switch, in quiescent versus proliferating cells. Mol Cell Biol 819 30:4045-4059. 820

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2004. MTA3 and the Mi-2/NuRD complex regulate cell fate during B lymphocyte 823 differentiation. Cell 119:75-86. 824

825 [62] Huang C, Gonzalez DG, Cote CM, Jiang Y, Hatzi K, Teater M, Dai K, Hla T, 826

Haberman AM, Melnick A. 2014. The BCL6 RD2 domain governs commitment of 827 activated B cells to form germinal centers. Cell Rep 8:1497-1508. 828

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middle domain repressor function is required for T follicular helper cell differentiation 836 and utilizes the corepressor MTA3. Proc Natl Acad Sci U S A 112:13324-13329. 837

838 [65] Conomos D, Reddel RR, Pickett HA. 2014. NuRD-ZNF827 recruitment to telomeres 839

creates a molecular scaffold for homologous recombination. Nat Struct Mol Biol 840 21:760-770. 841

842 [66] Cismasiu VB, Paskaleva E, Suman Daya S, Canki M, Duus K, Avram D. 2008 843

BCL11B is a general transcriptional repressor of the HIV-1 long terminal repeat in T 844 lymphocytes through recruitment of the NuRD complex. Virology 380:173-181. 845

846 TABLES 847

Two Tables listing the various oligonucleotides used in the RT-qPCR and ChIP-qPCR 848

experiments can be found as Supplemental Information on line. 849

850

FIGURE LEGENDS 851

852

Figure 1. BCL11A and BCL11B interact with MTA1 and MTA3. (A) MTA1 interacts with 853

BCL11B. HEK293T were transfected with MTA1 and BCL11B expression vectors. Whole 854

cells extracts incubated with anti-FLAG antibodies (IP FLAG) and 1% of each lysate (Input) 855

were immunoblotted with the indicated antibodies. FLAG-BCL11B co-immunoprecipitate 856

endogenous MTA1 proteins (lane 3) visualized as a doublet (*). (B) BCL11B interacts with 857

MTA3. A similar experiment was conducted in HEK293T cells with BCL11B and MTA3 858

expression vectors. (C) Endogenous BCL11B and MTA1 proteins interact. Solubilized 859

chromatin fractions were prepared in duplicate from the same number of MOLT4 cells and 860

1% was retained for direct analyses by Western blot (Input). The interaction between 861

BCL11B and MTA1 was evaluated by co-immunoprecipitation assays. (D) Endogenous 862

BCL11B and MTA3; (E) BCL11A and MTA1 and (F) BCL11B and MTA3 proteins interact 863

in MOLT4 cells. The same procedure was used for panels C to E. 864


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865

Figure 2. Ser 2 in the conserved N-terminal motif of BCL11B is essential for its interaction 866

with MTA1 and MTA3 and for its transcriptional repression activity (A) Schematic drawing 867

of the BCL11B-Gal4 (DNA-binding domain)-NLS (Nuclear localization signal)-HA fusion 868

proteins. (B) A phosphomimetic point mutation S2D in the BCL11B N-terminal domain 869

inhibits its interaction with MTA1. After transfection with the indicated expression vectors, 870

HEK293T cells lysates were immunoprecipitated with anti-c-myc antibodies. 871

Immunoprecipitated samples [IP c-myc (MTA1)] and 1% of whole cell extracts (Input) were 872

analyzed by immunoblotting with the indicated antibodies. (C) The BCL11B-MTA3 873

interaction is also negatively regulated by the S2D mutation. A similar experiment was 874

performed in HEK293T with a FLAG-MTA3 plasmid and the BCL11B-Gal4 chimeras. (D) 875

The conserved BCL11B N-terminal domain represses transcription. The transcriptional 876

activity of BCL11B-Gal4 constructs was tested by transient luciferase reporter assays in 877

HEK293T cells. The results represent the mean values from three independent transfections in 878

triplicate. (E) The S2D phosphomimetic mutation in the BCL11B N-terminal domain partially 879

inhibits its transcriptional repression potential. A similar luciferase reporter assay was 880

conducted in HEK293T with the 1-20 BCL11B wild-type (WT) and the S2D point mutant. (F-881

G) Repression potential of the various S2X point mutants BCL11B N-terminal domain. 882

Luciferase reporter assays were conducted with the S2A, S2D, S2T and wt BCL11B-Gal4 883

constructs as described above. 884

885

Figure 3. The phosphomimetic S2D mutation in BCL11B inhibits NuRD recruitment. (A) 886

Schematic drawing of the full-length BCL11B proteins tested. (B) The interaction with MTA1 887

is impaired by the S2D phosphomimetic mutation of BCL11B. HEK293T were transfected 888

with the indicated combination of expression vectors and Co-IP assays followed by 889


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immunoblotting with the indicated antibodies were performed. (C) The BCL11B-MTA3 890

interaction is also strongly reduced by the S2D phosphomimetic mutation. A similar Co-IP 891

experiment was realized in HEK293T but with the FLAG-MTA3 expression vector. Relevant 892

pieces of the membranes were cut (around the 80KDa marker) to separate the MTA3 and 893

BCL11B proteins and probed with anti-FLAG antibodies. (D) Interaction of wt, S2A, S2D 894

and ΔMSRKKQ BCL11B with endogenous MTA1 proteins. Total extracts of HEK293T 895

transfected with the indicated plasmids were analysed by Co-IP with anti-FLAG antibodies 896

and imunoblotted with MTA1 and FLAG antibodies. (E-F) Interaction of the S2A and S2D 897

BCL11B point mutants with endogenous NuRD components. Co-IP experiments followed by 898

immunoblotting with the indicated antibodies were performed as in Panel D with the S2A and 899

S2D (Panel E) or S2D (Panel F) BCL11B mutants. 900

901

Figure 4. Phosphorylation of BCL11B Ser2 in HEK293T cells upon PKC activation. (A) 902

PMA-induced in vitro phosphorylation of BCL11B Ser2. HEK293T cells transfected with 903

FLAG-BCL11B were mock-treated (-), incubated with vehicle (DMSO) or with PMA (1μM) 904

for 20 minutes. Immunoprecipitated samples IP (FLAG) and Input lysates were analyzed by 905

immunoblotting with antibodies specific for phosphorylated Ser2 (pSer2 BCL11B) and FLAG 906

as control. (B) Specificity of the anti-pSer2 BCL11B antibodies. A similar experiment was 907

performed to compare in the three conditions the BCL11B S2A mutant and wt BCL11B. * 908

refers to a non-specific band. (C) PKC is implicated in BCL11B Ser2 phosphorylation. 909

HEK293T cells were transfected with BCL11B and activated exactly as in panel A. One plate 910

was pre-incubated with the pan-PKC inhibitor BIMII before PMA activation. The PKC 911

inhibitor also partially inhibits Erk activation as previously shown (51). (D) The phosphatase 912

inhibitor Okadaic Acid (OA) allows detection of BCL11B Ser2 phosphorylation in basal 913

conditions. HEK293T cells transfected with the indicated expression vectors were incubated 914


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with DMSO or treated with OA. Total cell lysates were analyzed by immunoblotting with the 915

indicated antibodies. 916

917

Figure 5. BCL11B SUMOylation in HEK293T cells is independent of Ser2 Phosphorylation. 918

(A) BCL11B is SUMOylated. Total cells extracts of HEK293T transfected with the indicated 919

combinations of BCL11B, SUMO2 and the deSUMOylase SENP2 were prepared in 920

denaturing conditions and immunoblotted with indicated antibodies. The arrowhead 921

corresponds to SUMOylated forms of BCL11B (B) The PKC pathway does not impinge on 922

BCL11B SUMOylation in contrast with the ERK pathway. HEK293T cells were transfected 923

with BCL11B and pre-incubated with the PKC inhibitor BIMII or with the Erk1/2 inhibitor, 924

U0126 before PMA activation. The PKC inhibitor also partially inhibits Erk activation as 925

previously shown (51). Total cells extracts prepared in denaturing conditions were 926

immunoblotted as indicated. (C) The S2D and S2A point mutations have no significant 927

impact on BCL11B SUMOylation. HEK293T were transfected as indicated, lysed in 928

denaturing conditions and immunoblotted with anti FLAG antibodies to detect BCL11B and 929

its SUMOylated forms. Hsp60 was used to quantify the ratio of SUMOylated BCL11B to 930

total BCL11B using the Fujifilm MultiGauge software. The value obtained for BCL11B wt in 931

absence of SUMO2 (lane 5) was set up as 1. (D) The S2D and S2A point mutations do not 932

affect BCL11B interaction with endogenous P300 proteins. HEK293T transfected with the 933

indicated plasmids were subjected to Co-IPs analyses with anti-FLAG antibodies followed by 934

immunoblotting with P300 or FLAG antibodies. The ratio of interacting P300 relative to 935

BCL11B was measured as in panel C. 936

937

Figure 6. Endogenous BCL11B proteins are phosphorylated on Ser2 and SUMOylated in 938

PMA/Ionomycin-activated Jurkat cells. (A) Activation of Jurkat cells with P/I induces Ser2 939


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Phosphorylation of endogenous BCL11B proteins. Jurkat cells were treated with P/I in a time-940

dependent manner and extracts prepared in denaturing conditions were analysed by 941

immunoblotting of two different SDS/PAGE gels. In gel A1, pSer2 BCL11B is shown as a 942

green arrow and a non-specific band as an *. In gel A2, slowly migrating BCL11B-943

SUMOylated species are shown as red arrows. Quantification of total BCL11B proteins in 944

DMSO versus P/I conditions (bottom panel) and of Ser2-phosphorylated and SUMO-945

BCL11B to BCL11B (right panel) were performed with the Fujifilm MultiGauge software. 946

(B) Detection of pSer2 BCL11B in activated Jurkat cells by IP/WB. Jurkat cells were treated 947

with DMSO or activated for 30 minutes and pre-treated or not with the phosphatase inhibitor 948

Okadaic acid. Total cell extracts were immunoprecipitated by BCL11B and immunoblotted 949

with the pSer2 BCL11B specific antibodies. The membrane was striped and reprobed with 950

anti-BCL11B antibodies. The green arrow corresponds to pSer2-BCL11B and the black 951

arrows to the supershift of phosphorylated BCL11B observed with OA. (C) The phosphatase 952

inhibitor Okadaic Acid (OA) favors detection of pSer2 BCL11B and induces a supershift of 953

BCL11B upon P/I activation of Jurkat cells. Cells were treated with DMSO (-) or activated 954

with P/I for 30 minutes and pre-treated or not with OA. Total cell extracts were prepared and 955

immunoblotted as indicated. Green arrows correspond to BCL11B and the black arrows to the 956

supershift of phosphorylated BCL11B observed with OA. (D) The PKC inhibitor Gö6983 957

abolishes Ser2 phosphorylation but not the supershift of BCL11B upon P/I activation. An 958

experiment was conducted essentially as described in panel C) but with pre-treatment with 959

Gö6983. (E) SUMOylation of endogenous BCL11B proteins is not affected by PKC 960

inhibition. Jurkat cells were treated with DMSO or activated for 60 minutes to favor 961

SUMOylation and treated or not with Gö6983 and U0126. Total cell extracts prepared in 962

denaturing conditions were immunoblotted as indicated. 963


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(F) The SUMOylation inhibitor Calyculin A inhibits BCL11B SUMOylation. Jurkat cells 964

were treated with DMSO or activated for 60 minutes and pretreated or not with the 965

SUMOylation inhibitor Calyculin A (Cal A). Cal. A is also a phosphatase inhibitor and thus 966

induces a supershift for BCL11B. Red arrows correspond to SUMOylated forms of BCL11B. 967

(G) The specific SUMOylation inhibitor 2D-08 slightly inhibits BCL11B SUMOylation. A 968

similar experiment was performed with Jurkat cells pretreated or not with 2D-08 for the 969

indicated times. (H) Detection of endogenous BCL11B SUMOylation in Jurkat cells by 970

immunoprecipitation analyses. Jurkat cells were treated with DMSO or P/I for 1 hour to 971

favour SUMOylation (panel A). To detect SUMOylated proteins, the cells were lysed in 1 972

volume 1% SDS. The lysates were immediately boiled for 10 minutes, then diluted with 9 973

volumes of RIPA buffer without SDS and immunoprecipitated with anti-BCL11B antibodies. 974

The immunoprecipitates were then analysed by Western blotting with anti-SUMO2 (upper 975

panel). The SUMOylated BCL11B proteins are indicated by an arrowhead. The membranes 976

were striped and reprobed with anti-BCL11B antibodies. Please note that in contrast with the 977

other gels in this Figure, this gel is a 6% SDS/PAGE which might explain the “ladder-like” 978

aspect of the BCL11B bands. 979

980

Figure 7. Activation of Jurkat cells induces BCL11B down regulation by direct transcriptional 981

repression mediated by KLF4 (A) Prolonged activation of Jurkat cells induces down-982

regulation of BCL11B expression. Jurkat cells were treated with DMSO or activated with P/I 983

for the indicated times. BCL11B mRNAs levels were determined by RT-qPCR analyses. (B) 984

BCL11B proteins are not targeted for degradation by ubiquitination. Jurkat cells were 985

pretreated with different proteasome inhibitors for 1h before activation and activated for 5h as 986

indicated. Total cells extracts were immunoblotted with anti-BCL11B antibodies. (C) KLF4 987

expression is induced in activated Jurkat cells. KLF4 mRNA levels were analyzed by qRT-988


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PCR in the samples used in panel A for BCL11B (D) Schematic drawing of the human 989

BCL11B proximal promoter region. The transcription Start Site (TSS), the conserved potential 990

KLF4 binding site (underlined) and the oligonucleotides used in ChIP-qPCR experiments are 991

shown. (E) KLF4 binds the BCL11B promoter. Chromatin was prepared from DMSO-treated 992

and P/I-activated Jurkat and ChIP analyses performed for KLF4 or IgG at KLF4 binding sites 993

in BCL11B and CXCR4 promoters. GAPDH was used as a nonbinding control. 994

995

Figure 8. BCL11B-mediated repression of IL2 expression through MTA1 or MTA3 996

recruitment in un-stimulated cells is lost during Jurkat cells activation by P/I treatment. (A, 997

B) Activation of human Jurkat CD4+ T cells by P/I induced an increase of IL2 and Id2 998

expression. Jurkat cells were treated with DMSO or activated with P/I for the indicated times. 999

Total RNAs were extracted and RT-qPCR experiments were performed to analyse the 1000

increase of IL2 (A) and Id2 (B) expression levels after activation of Jurkat cells. Due to the 1001

huge induction of IL2 expression after prolonged stimulation, the IL2 expression levels 1002

measured after 2 and 5 hours of treatment are shown with a different scale as Supplementary 1003

Figure 6C (C) Endogenous P300 preferentially interacts with BCL11B in activated Jurkat T 1004

cells. Cells were activated or not with P/I, lysed in IPH buffer and immunoprecipitated with 1005

anti BCL11B. As control, P/I-treated cells were immunoprecipitated with rabbit IgG. The 1006

immunoprecipitates were then analyzed by Western blot with the indicated antibodies. (D) 1007

Schematic drawing of the human Id2 proximal promoter region with the Transcription Start 1008

Site (TSS) and the conserved potential BCL11B direct binding site, TGGGC. The 1009

oligonucleotides used in ChIP-qPCR experiments are shown as arrowheads. (E- I) ChIP 1010

experiments were performed on Jurkat cells chromatin with antibodies against BCL11B, 1011

MTA1, MTA3 and rabbit IgG (panels E-H) or against P300 and mouse IgG (panel I). The 1012

bound material was eluted and analysed by quantitative PCR using primers flanking the US1 1013


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BCL11B binding site in IL2 (19) and the newly identified BCL11B binding in Id2. For each 1014

promoter and antibody, GAPDH was used as an internal nonbinding control. 1015

1016

Figure 9. The PKC inhibitor Gö6983 abolishes the co-repressor-coactivator switch of 1017

BCL11B on the IL2 promoter. (A-B) Jurkat cells were treated with DMSO or activated with 1018

P/I for 30 minutes without or with pre-incubation with the PKC inhibitor Gö6983. RT-qPCR 1019

experiments were performed to analyse the expression levels of BCL11B and IL2. (C-D) 1020

Relative occupancy of BCL11B, MTA1, RbAp48 and P300 on IL2 promoter during Jurkat 1021

activation with or without treatment with the PKC inhibitor Gö6983. These bindings were 1022

assessed by ChIP-qPCR experiments in triplicate before and after activation and inhibitor 1023

treatment, as described in panels A-B. Error bars indicate standard deviation. The ChIP results 1024

for the US1 site in the IL2 promoter are shown on the left whereas those for the GAPDH 1025

promoter used as a nonbinding control are shown on the right. 1026

Figure10. Activation of primary human CD4+ T cells induces phosphorylation of BCL11B 1027

Ser2 and the corepressor-coactivator switch on the IL2 promoter (A) Activation of primary 1028

human CD4+ T cells by P/I or by anti CD3/CD28 antibodies induced down-regulation of 1029

BCL11B expression. RT-qPCR experiments were performed to analyse BCL11B expression. 1030

(B) Analyses of endogenous BCL11B phosphorylation on Ser2 and SUMOylation in 1031

activated human CD4+ T cells. CD4+ T cells were directly lysed in Laemmli buffer, boiled 1032

and analysed by Western blotting with the indicated antibodies, 5h or 24h after P/I- or anti-1033

CD3/CD28-treatment respectively. The red arrowhead corresponds to SUMOylated BCL11B 1034

proteins. (C) Increase of IL2 expression in activated human CD4+ T cells. RT-qPCR 1035

experiments were performed to analyse IL2 expression upon activation with P/I (left panel) or 1036

anti-CD3/CD28 antibodies (right panel). (D-E) Relative occupancy of BCL11B and P300 on 1037

the IL2 promoter during human CD4+ T-cell activation. BCL11B (D) and P300 (E) occupancy 1038


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on the US1 site in the IL2 promoter (19) was analyzed by ChIP-qPCR experiments in human 1039

CD4+ T cells with and without activation. Error bars indicate standard deviation. Similar ChIP 1040

experiments were performed with control IgG. For each antibody, the ChIP results for the 1041

GAPDH promoter used as a nonbinding control are shown. 1042

1043

Figure 11. Working model on the co-repressor/activator switch on BCL11B target genes 1044

during activation of Jurkat or primary human CD4+ T cells. 1: In un-stimulated Jurkat or 1045

primary human CD4+ T cells isolated from PBMCs of healthy donors, BCL11B bound 1046

together with MTA1 and NuRD complex represses transcription of direct target genes such as 1047

IL2 and Id2. 2: Upon activation, phosphorylation of Ser2 in the conserved N-terminal 1048

repression domain allows disruption of the interaction with MTA1 and hence NuRD 1049

repressive complexes while BCL11B remains bound to the IL2 and Id2 promoters. 3: Then, 1050

SUMOylation on Lys679 in the C-terminal end of BCL11B allows the recruitment of P300 to 1051

activate transcription. 4: After 2-5 hours of treatment, a transcriptional down-regulation of 1052

BCL11B by KLF4 would stop and/or limit the IL2 and Id2 activation. 1053

1054


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C D

Dubuissez et al., Figure 1

E F

A

Input 1%

IP MTA3 FLAG-BCL11B

FLAG-MTA3

FLAG-BCL11B

WB :

1 2 3 4

FLAG-MTA3

HEK293T

FLAG-BCL11B

MTA1

FLAG-BCL11B Input 1%

IP FLAG

(BCL11B)

MTA1

WB :

1 2 3 4

Short exp.

Long exp.

HEK293T

* *

* *

* *

IP

WB :

MTA1

BCL11A

Input 1%

1 2

MOLT4

1’ 2’

1 2

MOLT4

1’ 2’

Input 1%

IP

WB :

BCL11B

MTA3

1 2 1’ 2’

WB :

IP

Input 1%

MTA1

BCL11B

MOLT4

MOLT4

IP

WB :

1 2

Input 1%

1’ 2’

BCL11A

MTA3


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Figure 1. BCL11A and BCL11B interact with MTA1 and MTA3. (A) MTA1 interacts with BCL11B.

HEK293T were transfected with MTA1 and BCL11B expression vectors. Whole cells extracts

incubated with anti-FLAG antibodies (IP FLAG) and 1% of each lysate (Input) were

immunoblotted with the indicated antibodies. FLAG-BCL11B co-immunoprecipitate endogenous

MTA1 proteins (lane 3) visualized as a doublet (*). (B) BCL11B interacts with MTA3. A similar

experiment was conducted in HEK293T cells with BCL11B and MTA3 expression vectors. (C)

Endogenous BCL11B and MTA1 proteins interact. Solubilized chromatin fractions were prepared

in duplicate from the same number of MOLT4 cells and 1% was retained for direct analyses by

Western blot (Input). The interaction between BCL11B and MTA1 was evaluated by co-

immunoprecipitation assays. (D) Endogenous BCL11B and MTA3; (E) BCL11A and MTA1 and

(F) BCL11B and MTA3 proteins interact in MOLT4 cells. The same procedure was used for

panels C to E.


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D

A

B C

WB :

MTA1

MTA1

HA

Input 1%

IP c-myc

(MTA1)

+ MTA1

HA

1 2 3 4 5 6 7 8 9 10

FLAG-MTA3

FLAG-MTA3

Input 1%

HA

HA

WB :

+ MTA3

1 2 3 4 5 6 7 8

0

0,2

0,4

0,6

0,8

1

1,2

1 2 3

*** ***

Gal4-NLS-HA 1-20 BCL11B

Gal4-NLS-HA

1-58 BCL11B

Gal4-NLS-HA

Fold

activation

0

0,2

0,4

0,6

0,8

1

1,2

1 2 3

**

**

Gal4-NLS-HA 1-20 S2D

BCL11B

Gal4-NLS-HA

1-20 WT

BCL11B

Gal4-NLS-HA

**

Fold

activation

***

BCL11B 1 894 ZF MSRRKQ

Gal4 1 58 1-58 BCL11B

NLS-HA

1 20 1-20 WT BCL11B

GAL4-NLS-HA 1 20 1-20 S2X BCL11B

MDRRKQ

Gal4

NLS-HA

Gal4

NLS-HA

Gal4

NLS-HA

IP FLAG

(MTA3)

MARRKQ MTRRKQ

S2D S2A S2T

E


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Dubuissez et al., Figure 2 continued

**

0

0,2

0,4

0,6

0,8

1

1,2

gal4 1-20 S2T 1-20 WT

Fold

activation

Gal4-NLS-HA 1-20 WT

BCL11B

Gal4-NLS-HA

1-20 S2T

BCL11B

Gal4-NLS-HA

NS **

G

0

0,2

0,4

0,6

0,8

1

1,2

gal4 1-20 S2A 1-20 S2D 1-20 WT

F F

old

activation

Gal4-NLS-HA 1-20 S2A

BCL11B

Gal4-NLS-HA

1-20 WT

BCL11B

Gal4-NLS-HA

***

** **

1-20 S2D

BCL11B

Gal4-NLS-HA

*** *** **

Figure 2. Ser 2 in the conserved N-terminal motif of BCL11B is essential for its interaction with

MTA1 and MTA3 and for its transcriptional repression activity (A) Schematic drawing of the

BCL11B-Gal4 (DNA-binding domain)-NLS (Nuclear localization signal)-HA fusion proteins. (B)

A phosphomimetic point mutation S2D in the BCL11B N-terminal domain inhibits its interaction

with MTA1. After transfection with the indicated expression vectors, HEK293T cells lysates

were immunoprecipitated with anti-c-myc antibodies. Immunoprecipitated samples [IP c-myc

(MTA1)] and 1% of whole cell extracts (Input) were analyzed by immunoblotting with the

indicated antibodies. (C) The BCL11B-MTA3 interaction is also negatively regulated by the

S2D mutation. A similar experiment was performed in HEK293T with a FLAG-MTA3 plasmid

and the BCL11B-Gal4 chimeras. (D) The conserved BCL11B N-terminal domain represses

transcription. The transcriptional activity of BCL11B-Gal4 constructs was tested by transient

luciferase reporter assays in HEK293T cells. The results represent the mean values from three

independent transfections in triplicate. (E) The S2D phosphomimetic mutation in the BCL11B

N-terminal domain partially inhibits its transcriptional repression potential. A similar luciferase

reporter assay was conducted in HEK293T with the 1-20 BCL11B wild-type (WT) and the S2D

point mutant. (F-G) Repression potential of the various S2X point mutants BCL11B N-terminal

domain. Luciferase reporter assays were conducted with the S2A, S2D, S2T and wt BCL11B-

Gal4 constructs as described above.


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BCL11B WT

MDRRKQ

BCL11B S2D

MARRKQ

BCL11B S2A

DMSRRKQ

BCL11B DMSRRKQ

1

MSRRKQ

894 ZF


B

Input 1%

IP c-myc

(MTA1) MTA1

FLAG-

BCL11B

MTA1

WB :

1 2 3 4 5 6

FLAG-

BCL11B

HEK293T

D

Input 1%

IP FLAG (BCL11B)

WB :

MTA1

MTA1

FLAG-

BCL11B

1 2 3 4 5 6

FLAG-

BCL11B

HEK293T

FLAG-BCL11B

FLAG-BCL11B

FLAG-MTA3

FLAG-MTA3

WB :

Input 1%

1 2 3 4 5 6

IP MTA3

C HEK293T

A

MTA1

RbAp46

MTA3

FLAG-

BCL11B

Input 1% IP FLAG (BCL11B

1 2 3 4

WB :

1’ 2’ 3’ 4’

HEK293T E

Input 1%

1 2 3

CHD4

HDAC2

RbAp46

MTA1

FLAG-

BCL11B

(BCL11B)

WB :

1’ 2’ 3’

IP FLAG

F HEK293T


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Figure 3. The phosphomimetic S2D mutation in BCL11B inhibits NuRD recruitment. (A) Schematic

drawing of the full-length BCL11B proteins tested. (B) The interaction with MTA1 is impaired by the

S2D phosphomimetic mutation of BCL11B. HEK293T were transfected with the indicated

combination of expression vectors and Co-IP assays followed by immunoblotting with the indicated

antibodies were performed. (C) The BCL11B-MTA3 interaction is also strongly reduced by the S2D

phosphomimetic mutation. A similar Co-IP experiment was realized in HEK293T but with the FLAG-

MTA3 expression vector. Relevant pieces of the membranes were cut (around the 80KDa marker)

to separate the MTA3 and BCL11B proteins and probed with anti-FLAG antibodies. (D) Interaction

of wt, S2A, S2D and DMSRKKQ BCL11B with endogenous MTA1 proteins. Total extracts of

HEK293T transfected with the indicated plasmids were analysed by Co-IP with anti-FLAG

antibodies and imunoblotted with MTA1 and FLAG antibodies. (E-F) Interaction of the S2A and

S2D BCL11B point mutants with endogenous NuRD components. Co-IP experiments followed by

immunoblotting with the indicated antibodies were performed as in Panel D with the S2A and S2D

(Panel E) or S2D (Panel F) BCL11B mutants.


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C HEK293T + FLAG-BCL11B WT

IP FLAG

(BCL11B)

Input 1%

FLAG-BCL11B

pSer2 BCL11B

WB:

1 2 3

pErk1/2

A

IP FLAG

(BCL11B)

Input 1%

pSer2

BCL11B

FLAG-BCL11B

FLAG-BCL11B

pErk1/2

Short exp.

Long exp.

HEK293T + FLAG-BCL11B WT

1 2 3

WB:

B HEK293T +

-

IP FLAG

(BCL11B)

Input 1%

pSer2 BCL11B

FLAG-BCL11B

*

BCL11B S2A

*

BCL11B WT

- WB:

1 2 3 4 5 6

D

100

OA

DMSO - - -

- - -

+ + +

+ + +

HEK293T +

pSer2 BCL11B

FLAG-BCL11B

hsp60

100

1 2 3 4 5 6

Figure 4. Phosphorylation of BCL11B Ser2 in HEK293T cells upon PKC activation. (A) PMA-

induced in vitro phosphorylation of BCL11B Ser2. HEK293T cells transfected with FLAG-BCL11B

were mock-treated (-), incubated with vehicle (DMSO) or with PMA (1mM) for 20 minutes.

Immunoprecipitated samples IP (FLAG) and Input lysates were analyzed by immunoblotting with

antibodies specific for phosphorylated Ser2 (pSer2 BCL11B) and FLAG as control. (B) Specificity of

the anti-pSer2 BCL11B antibodies. A similar experiment was performed to compare in the three

conditions the BCL11B S2A mutant and wt BCL11B. * refers to a non-specific band. (C) PKC is

implicated in BCL11B Ser2 phosphorylation. HEK293T cells were transfected with BCL11B and

activated exactly as in panel A. One plate was pre-incubated with the pan-PKC inhibitor BIMII

before PMA activation. The PKC inhibitor also partially inhibits Erk activation as previously shown

(51). (D) The phosphatase inhibitor Okadaic Acid (OA) allows detection of BCL11B Ser2

phosphorylation in basal conditions. HEK293T cells transfected with the indicated expression

vectors were incubated with DMSO or treated with OA. Total cell lysates were analyzed by

immunoblotting with the indicated antibodies.


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A

C D


0

12

34

5

S2A S2D WT

- SUMO2

+ SUMO2

SU

MO

-BC

L11B

re

lative

to B

CL

11B

/hsp60

BCL11B

FLAG-BCL11B

FLAG-

BCL11B

0

0,5

1

1,5

2

1 2 3

HEK293T

Inte

racting P

300

rela

tive

to

IP

BC

L11B

2 3 4

Lanes

P300 Input 1%

P300

IP FLAG

(BCL11B)

WB :

1 2 3 4

Short exp.

Short exp.

Long exp.

Long exp.

FLAG-BCL11B

B

HEK293T +

150

100

150

100

SUMO2 - + - + - +

FLAG-

BCL11B

Short exp.

Long exp.

Hsp60

WB :

1 2 3 4 5 6

SUMO

100

150

HEK293T +

WB :

Actin

SUMO 2

SENP 2

FLAG-BCL11B WT

SENP2

- - +

1 2 3

SUMO

pErk 1/2

BCL11B SUMO BCL11B

FLAG

HEK293T +

FLAG-BCL11B WT

100

150

100

150

Erk 2 total

1 2 3 4

WB :


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Figure 5. BCL11B SUMOylation in HEK293T cells is independent of Ser2 Phosphorylation. (A)

BCL11B is SUMOylated. Total cells extracts of HEK293T transfected with the indicated

combinations of BCL11B, SUMO2 and the deSUMOylase SENP2 were prepared in denaturing

conditions and immunoblotted with indicated antibodies. The arrowhead corresponds to

SUMOylated forms of BCL11B (B) The PKC pathway does not impinge on BCL11B SUMOylation in

contrast with the ERK pathway. HEK293T cells were transfected with BCL11B and pre-incubated

with the PKC inhibitor BIMII or with the Erk1/2 inhibitor, U0126 before PMA activation. The PKC

inhibitor also partially inhibits Erk activation as previously shown (51). Total cells extracts prepared

in denaturing conditions were immunoblotted as indicated. (C) The S2D and S2A point mutations

have no significant impact on BCL11B SUMOylation. HEK293T were transfected as indicated,

lysed in denaturing conditions and immunoblotted with anti FLAG antibodies to detect BCL11B and

its SUMOylated forms. Hsp60 was used to quantify the ratio of SUMOylated BCL11B to total

BCL11B using the Fujifilm MultiGauge software. The value obtained for BCL11B wt in absence of

SUMO2 (lane 5) was set up as 1. (D) The S2D and S2A point mutations do not affect BCL11B

interaction with endogenous P300 proteins. HEK293T transfected with the indicated plasmids were

subjected to Co-IPs analyses with anti-FLAG antibodies followed by immunoblotting with P300 or

FLAG antibodies. The ratio of interacting P300 relative to BCL11B was measured as in panel C.


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0

0,5

1

1,5

2

0 5' 30' 60' 120' 300'


pSer2 BCL11B

BCL11B

BCL11B

Short

exp.

Long

exp.

pErk 1/2

Erk 2 total

DMSO P/I

Jurkat cells

(min) WB: 0 5 30 60 120 300 0 5 30 60 120 300

1 2 3 4 5 6 7 8 9 10 11 12

A

100

150

A1

A2 2 1

*

0

1

2

3

4

0 5' 30' 60' 120' 300'

1

2SUMO-BCL11B

0

50

100

150

0 5' 30' 60' 120' 300'

Rela

tive t

o B

CL11B

/Erk

2 a

nd

norm

alis

ed t

o D

MS

O

pSer2 BCL11B

Treatment (min)

BCL11B proteins level

Treatment (min)

BC

L1

1B

(S

ho

rt

exp

.) / E

rk2

to

tal

DMSO

P/I

D

B

C Pre-treatment 30’

Activation 30’

Jurkat cells

P/I

OA -

WB:

1 2 3 4

100

150

100

150

BCL11B

pErk 1/2

Short

exp.

Long

exp.

pSer2

BCL11B

Erk 2 total

+ + -

- - + +

100

100 Long exp.

Short exp.

100

100

150

100

1 2 3 4 5

100

150

pSer2 BCL11B

pErk 1/2

Erk 2 total

BCL11B

WB:

Short exp. Long exp.

Pre-treatment 30’

Activation 30’

Pre-treatment 15’

P / I

OA

Gö6983

-

-

- - -

- -

+ +

+ +

+ + + +

Jurkat cells

100

150 100

150

pSer2

BCL11B

Erk 2 total

BCL11B

pErk 1/2

WB:

*

Input 1%

100

100

100

1 2 3 4

Pre-treatment 30’

Activation 30’

Jurkat cells

P/I

OA - + + -

- - + +

BCL11B

IP:

BCL11B


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E

pErk 1/2

BCL11B 100

150

1 2 3 4

Erk2 total

Jurkat cells

P/I

U0126

Gö6983

-

Pre-treatment 15’

Activation 60’ + + +

- - - +

+ - - -

WB: 2 1

F

H

WB :

Input 1.5%

P/I 60’ + - + + - +

BCL11B

SUMO2

Long

exp.

Short

exp.

pErk 1/2

Actin

100

150

100

150

100

150

Jurkat cells

IP

1 2 3 1’ 2’ 3’

G

Pre-treatment 30’

Activation 60’

100

150 BCL11B

pErk 1/2

Jurkat cells

P/I

Cal. A -

WB:

Erk 2 total

1 2 3

- +

- + +

2 1


pErk 1/2

Erk 2 total

1 2 3 4 5 6 7 8 9

100

150 2 1 BCL11B

WB:

1h 2h 4h

Jurkat cells

DMSO

1h 2h 4h

DMSO

1h 2h 4h

2D-08

DMSO P/I P/I Activation 60’

Pre-treatment


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Figure 6. Endogenous BCL11B proteins are phosphorylated on Ser2 and SUMOylated in

PMA/Ionomycin-activated Jurkat cells. (A) Activation of Jurkat cells with P/I induces Ser2

Phosphorylation of endogenous BCL11B proteins. Jurkat cells were treated with P/I in a time-

dependent manner and extracts prepared in denaturing conditions were analysed by

immunoblotting of two different SDS/PAGE gels. In gel A1, pSer2 BCL11B is shown as a green

arrow and a non-specific band as an *. In gel A2, slowly migrating BCL11B-SUMOylated species

are shown as red arrows. Quantification of total BCL11B proteins in DMSO versus P/I conditions

(bottom panel) and of Ser2-phosphorylated and SUMO-BCL11B to BCL11B (right panel) were

performed with the Fujifilm MultiGauge software. (B) Detection of pSer2 BCL11B in activated

Jurkat cells by IP/WB. Jurkat cells were treated with DMSO or activated for 30 minutes and pre-

treated or not with the phosphatase inhibitor Okadaic acid. Total cell extracts were

immunoprecipitated by BCL11B and immunoblotted with the pSer2 BCL11B specific antibodies.

The membrane was striped and reprobed with anti-BCL11B antibodies. The green arrow

corresponds to pSer2-BCL11B and the black arrows to the supershift of phosphorylated BCL11B

observed with OA. C) The phosphatase inhibitor Okadaic Acid (OA) favors detection of pSer2

BCL11B and induces a supershift of BCL11B upon P/I activation of Jurkat cells. Cells were treated

with DMSO (-) or activated with P/I for 30 minutes and pre-treated or not with OA. Total cell

extracts were prepared and immunoblotted as indicated. Green arrows correspond to BCL11B and

the black arrows to the supershift of phosphorylated BCL11B observed with OA. (D) The PKC

inhibitor Gö6983 abolishes Ser2 phosphorylation but not the supershift of BCL11B upon P/I

activation. An experiment was conducted essentially as described in panel C) but with pre-

treatment with Gö6983. (E) SUMOylation of endogenous BCL11B proteins is not affected by PKC

inhibition. Jurkat cells were treated with DMSO or activated for 60 minutes to favor SUMOylation

and treated or not with Gö6983 and U0126. Total cell extracts prepared in denaturing conditions

were immunoblotted as indicated.

(F) The SUMOylation inhibitor Calyculin A inhibits BCL11B SUMOylation. Jurkat cells were treated

with DMSO or activated for 60 minutes and pretreated or not with the SUMOylation inhibitor

Calyculin A (Cal A). Cal. A is also a phosphatase inhibitor and thus induces a supershift for

BCL11B. Red arrows correspond to SUMOylated forms of BCL11B. (G) The specific SUMOylation

inhibitor 2D-08 slightly inhibits BCL11B SUMOylation. A similar experiment was performed with

Jurkat cells pretreated or not with 2D-08 for the indicated times. (H) Detection of endogenous

BCL11B SUMOylation in Jurkat cells by immunoprecipitation analyses. Jurkat cells were treated

with DMSO or P/I for 1 hour to favour SUMOylation (panel A). To detect SUMOylated proteins, the

cells were lysed in 1 volume 1% SDS. The lysates were immediately boiled for 10 minutes, then

diluted with 9 volumes of RIPA buffer without SDS and immunoprecipitated with anti-BCL11B

antibodies. The immunoprecipitates were then analysed by Western blotting with anti-SUMO2

(upper panel). The SUMOylated BCL11B proteins are indicated by an arrowhead. The membranes

were striped and reprobed with anti-BCL11B antibodies. Please note that in contrast with the other

gels in this Figure, this gel is a 6% SDS/PAGE which might explain the “ladder-like” aspect of the

BCL11B bands.


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B

0

0,2

0,4

0,6

0,8

1

1,2

0 5 30 60 120 300

A BCL11B mRNA levels in Jurkat cells

Re

lative

exp

ressio

n

Treatment (min)

NS NS

NS NS ** ***

DMSO

P/I

0

10

20

30

40

50

60

70

80

0 5 30 60 120 300

C

Treatment (min)

KLF4 mRNA levels in Jurkat cells

Re

lative

exp

ressio

n

NS NS

**

**

** ***

D

100 bp

ATG 5’ 3’ BCL11B

TSS

Mu:TTTGGGTTTTTGAGGGGGCGGGGTGAGGGGGTAC

Hu:TTTGGGTTTTTTGAGGGGGCGGGGTGAGGGGGTA

KLF4 binding site

Actin

P/I

BCL11B 100

150

WB :

Jurkat cells

MG132

ALLN

Lactacystin - - - + - - - - - +

+ - - - - -

Pre-treatment 60’

Activation 300’ - + + + +

DMSO

P/I

E CXCR4 promoter GAPDH promoter BCL11B promoter

0

0,2

0,4

0,6

0,8

1

1,2

KLF4 IgG

0

0,2

0,4

0,6

0,8

1

1,2

KLF4 IgG

0

0,2

0,4

0,6

0,8

1

1,2

KLF4 IgG

% In

pu

t

*

NS NS NS NS

% In

pu

t

% In

pu

t

ChIP KLF4 ChIP IgG ChIP KLF4 ChIP IgG ChIP KLF4 ChIP IgG

* DMSO 300’

P/I 300’


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Figure 7. Activation of Jurkat cells induces BCL11B down regulation by direct

transcriptional repression mediated by KLF4 (A) Prolonged activation of Jurkat cells

induces down-regulation of BCL11B expression. Jurkat cells were treated with DMSO or

activated with P/I for the indicated times. BCL11B mRNAs levels were determined by RT-

qPCR analyses. (B) BCL11B proteins are not targeted for degradation by ubiquitination.

Jurkat cells were pretreated with different proteasome inhibitors for 1h before activation

and activated for 5h as indicated. Total cells extracts were immunoblotted with anti-

BCL11B antibodies. (C) KLF4 expression is induced in activated Jurkat cells. KLF4 mRNA

levels were analyzed by qRT-PCR in the samples used in panel A for BCL11B (D)

Schematic drawing of the human BCL11B proximal promoter region. The transcription Start

Site (TSS), the conserved potential KLF4 binding site (underlined) and the oligonucleotides

used in ChIP-qPCR experiments are shown. (E) KLF4 binds the BCL11B promoter.

Chromatin was prepared from DMSO-treated and P/I-activated Jurkat and ChIP analyses

performed for KLF4 or IgG at KLF4 binding sites in BCL11B and CXCR4 promoters.

GAPDH was used as a nonbinding control.


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0

5

10

15

0 5 30 60 120 300

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

ChIP BCL11B ChIP MTA1 ChIP IgG

D

100 bp

ATG 5’ 3’ Id2

TSS

Mu: ATTTGCCACCCCAGCTGGGCTCTCGCCCGCTCC

Hu: ATTTACCACCCCAGCTGGGCTCGCGCCC----C

BCL11 B binding site IL2 promoter

GAPDH promoter G

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7


Id2 promoter F


% In

pu

t

NS NS

NS

D 30’

P/I 30’

ChIP IgG ChIP BCL11B ChIP MTA1

NS

NS

**

% In

pu

t

D 30’

P/I 30’

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7


% In

pu

t


NS

NS

*** D 30’

P/I 30’

E


**

P300

BCL11B

MTA1

P300

MTA1

BCL11B

IP

Input 2%

IgG

P/I 30’

BCL11B

WB : + - +

Jurkat cells

1 2 3

0

2

4

6

0 5 30

A IL2 mRNA levels in Jurkat cells R

ela

tive

exp

ressio

n

0 30 5

NS NS

Treatment (min)

DMSO

P/I

B Id2 mRNA levels in Jurkat cells

Re

lative

exp

ressio

n

Treatment (min)

DMSO

P/I ***

NS NS NS NS

0 30 60 5 120 300

**

C

0

0,5

1

1,5

2

1 2

0

0,5

1

1,5

2

1 2

D 30’

P/I 30’

H

% In

pu

t

ChIP MTA3 ChIP IgG

IL2 promoter

NS

*

ChIP MTA3 ChIP IgG

**

NS

Id2 promoter

ChIP MTA3 ChIP IgG

GAPDH promoter

NS % In

pu

t

% In

pu

t

NS

0

0,5

1

1,5

2

1 2


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D 30’

P/I 30’

I

0

1

2

3

4

5

6

7

P300 308 IgG 206

% In

pu

t

ChIP P300 ChIP IgG

IL2 promoter

NS

*

0

1

2

3

4

5

6

7

1 2ChIP P300 ChIP IgG

*

NS

Id2 promoter

0

1

2

3

4

5

6

7

1 2ChIP P300 ChIP IgG

GAPDH promoter

NS

*

% In

pu

t

% In

pu

t

Figure 8. BCL11B-mediated repression of IL2 expression through MTA1 or MTA3

recruitment in un-stimulated cells is lost during Jurkat cells activation by P/I treatment. (A,

B) Activation of human Jurkat CD4+ T cells by P/I induced an increase of IL2 and Id2

expression. Jurkat cells were treated with DMSO or activated with P/I for the indicated

times. Total RNAs were extracted and RT-qPCR experiments were performed to analyse

the increase of IL2 (A) and Id2 (B) expression levels after activation of Jurkat cells. Due to

the huge induction of IL2 expression after prolonged stimulation, the IL2 expression levels

measured after 2 and 5 hours of treatment are shown with a different scale as

Supplementary Figure 6C (C) Endogenous P300 preferentially interacts with BCL11B in

activated Jurkat T cells. Cells were activated or not with P/I, lysed in IPH buffer and

immunoprecipitated with anti BCL11B. As control, P/I-treated cells were

immunoprecipitated with rabbit IgG. The immunoprecipitates were then analyzed by

Western blot with the indicated antibodies. (D) Schematic drawing of the human Id2

proximal promoter region with the Transcription Start Site (TSS) and the conserved

potential BCL11B direct binding site, TGGGC. The oligonucleotides used in ChIP-qPCR

experiments are shown as arrowheads. (E- I) ChIP experiments were performed on Jurkat

cells chromatin with antibodies against BCL11B, MTA1, MTA3 and rabbit IgG (panels E-H)

or against P300 and mouse IgG (panel I). The bound material was eluted and analysed by

quantitative PCR using primers flanking the US1 BCL11B binding site in IL2 (19) and the

newly identified BCL11B binding in Id2. For each promoter and antibody, GAPDH was used

as an internal nonbinding control.


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0

1

2

3

4

1 2


0

0,5

1

1,5

2

DMSO PMA/Iono Gö6983+PMA/Iono

IL2 mRNA levels in Jurkat cells

Re

lative

exp

ressio

n

* NS

**

A B

0

0,2

0,4

0,6

0,8

1

1,2

DMSO PMA/Iono Gö6983+PMA/Iono

BCL11B mRNA levels in Jurkat cells R

ela

tive

exp

ressio

n NS NS

NS

IL2 promoter

% In

pu

t

NS NS

NS NS

DMSO 30’

P/I 30’

Gö6983 15’+ P/I 30’

GAPDH promoter C

ChIP BCL11B ChIP IgG

D

0

1

2

3

4

5

6

MTA1 RbAp48 P300 IgG

% In

pu

t

IL2 promoter

*

**

NS NS

** *

* **

0

1

2

3

4

5

6

MTA1 RbAp48 P300 IgG

NS NS

NS NS

NS NS

NS NS

GAPDH promoter

% In

pu

t

DMSO 30’

P/I 30’

Gö6983 15’+ P/I 30’

ChIP MTA1 ChIP IgG ChIP RbAp48 ChIP P300 ChIP MTA1 ChIP IgG ChIP RbAp48 ChIP P300

DMSO 30’ P/I 30’ Gö6983+P/I 30’ DMSO 30’ P/I 30’ Gö6983+P/I 30’

0

1

2

3

4

1 2 ChIP BCL11B ChIP IgG

% In

pu

t NS NS

NS NS

Figure 9. The PKC inhibitor Gö6983 abolishes the co-repressor-coactivator switch of

BCL11B on the IL2 promoter. (A-B) Jurkat cells were treated with DMSO or activated with

P/I for 30 minutes without or with pre-incubation with the PKC inhibitor Gö6983. RT-qPCR

experiments were performed to analyse the expression levels of BCL11B and IL2. (C-D)

Relative occupancy of BCL11B, MTA1, RbAp48 and P300 on IL2 promoter during Jurkat

activation with or without treatment with the PKC inhibitor Gö6983. These bindings were

assessed by ChIP-qPCR experiments in triplicate before and after activation and inhibitor

treatment, as described in panels A-B. Error bars indicate standard deviation. The ChIP

results for the US1 site in the IL2 promoter are shown on the left whereas those for the

GAPDH promoter used as a nonbinding control are shown on the right.


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0

2

4

6

8

10

12

1 2

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

1 2

24h medium

24h CD3/CD28

ChIP BCL11B ChIP IgG 0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

1 2

% In

put

IL2 promoter

NS

NS

ChIP BCL11B ChIP IgG

NS

NS

GAPDH promoter


D

C IL2 mRNA levels in LT CD4+ cells

0

1000

2000

3000

4000

5000

CD4 5h CD4 5h

PMA/iono

0

10

20

30

40

50

CD4 24h CD4 24h

CD3/CD28

Re

lative

exp

ressio

n

24h

medium

24h

CD3/CD28

5h

medium

5h P/I

** **

0

0,2

0,4

0,6

0,8

1

1,2

1,4

CD4 5h CD4 5h

PMA/iono

CD4 24h CD4 24h

CD3/CD28

Re

lative

exp

ressio

n

5h

medium

5h P/I 24h

medium

24h

CD3/CD28

A BCL11B mRNA levels in LT CD4+ cells

** *

B

24h medium

24h CD3/CD28

% In

put

0

2

4

6

8

10

12

1 2

GAPDH promoter IL2 promoter

ChIP P300 ChIP IgG

*

NS

ChIP P300 ChIP IgG

* NS

E

LT CD4+ cells

pSer2 BCL11B

WB:

P/I 5h

CD3/CD28 24h - - - +

Short exp.

Long exp.

- + - -

actin

BCL11B

0

1

2

1 2 3 4BC

L11B

/actin

80

80

80


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Figure 10. Activation of primary human CD4+ T cells induces phosphorylation of BCL11B

Ser2 and the corepressor-coactivator switch on the IL2 promoter (A) Activation of primary

human CD4+ T cells by P/I or by anti CD3/CD28 antibodies induced down-regulation of

BCL11B expression. RT-qPCR experiments were performed to analyse BCL11B expression.

(B) Analyses of endogenous BCL11B phosphorylation on Ser2 and SUMOylation in activated

human CD4+ T cells. CD4+ T cells were directly lysed in Laemmli buffer, boiled and analysed

by Western blotting with the indicated antibodies, 5h or 24h after P/I- or anti-CD3/CD28-

treatment respectively. The red arrowhead corresponds to SUMOylated BCL11B proteins.

(C) Increase of IL2 expression in activated human CD4+ T cells. RT-qPCR experiments were

performed to analyse IL2 expression upon activation with P/I (left panel) or anti-CD3/CD28

antibodies (right panel). (D-E) Relative occupancy of BCL11B and P300 on the IL2 promoter

during human CD4+ T-cell activation. BCL11B (D) and P300 (E) occupancy on the US1 site in

the IL2 promoter (19) was analyzed by ChIP-qPCR experiments in human CD4+ T cells with

and without activation. Error bars indicate standard deviation. Similar ChIP experiments were

performed with control IgG. For each antibody, the ChIP results for the GAPDH promoter

used as a nonbinding control are shown.


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IL2 repression

NuRD

BCL11B

MTA 1

X

1

IL2, Id2

IL2, Id2 repression BCL11B IL2, Id2

NuRD

MTA 1 X

X

2 PKC

Stop IL2, Id2 repression

P

Ser2

30’

Time scale Inactivated LT CD4+

TCR

Activation

Activated LT CD4+

BCL11B

Downregulation BCL11B

by KLF4

Stop IL2, Id2 activation

4 2H

Gène cible IL2, Id2

IL2, Id2 activation

3 P

Ser2

30’ p300

BCL11B


Figure 11. Working model on the co-repressor/activator switch on BCL11B target

genes during activation of Jurkat or primary human CD4+ T cells. 1: In un-stimulated

Jurkat or primary human CD4+ T cells isolated from PBMCs of healthy donors,

BCL11B bound together with MTA1 and NuRD complex represses transcription of

direct target genes such as IL2 and Id2. 2: Upon activation, phosphorylation of Ser2 in

the conserved N-terminal repression domain allows disruption of the interaction with

MTA1 and hence NuRD repressive complexes while BCL11B remains bound to the IL2

and Id2 promoters. 3: Then, SUMOylation on Lys679 in the C-terminal end of BCL11B

allows the recruitment of P300 to activate transcription. 4: After 2-5 hours of treatment,

a transcriptional down-regulation of BCL11B by KLF4 would stop and/or limit the IL2

and Id2 activation.


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Documents

“PKC-mediated phosphorylation of BCL11B at Serine 2 negatively