Maeda et al.
Supplementary Figure Legends
Supplementary Fig. 1 a, The protein expression levels of introduced oncogenes were
comparable in WT and Zbtb7-/- MEFs (described in Fig 1a and 1b). Western blot analyses
were carried out with the indicated antibodies. b, The protein expression levels of
introduced oncogenes in MEFs (described in Fig 1c and 1d) were shown. Exogenous
Pokemon protein showed higher molecular weight due to FLAG tag. c, Pokemon
antagonizes Myc-induced cellular apoptosis. Rat1 MycErTM cells were infected with either
pWZL empty vector or pWZL-FLAG Pokemon vector. After 24h serum starvation, cells
were treated with 4-Hydroxytamoxifen (4-OHT) in 0.1% serum containing medium.
Apoptotic cells were counted by trypan blue exclusion method on the indicated days. The
Pokemon expression levels are also shown (right).
Supplementary Fig. 2 a, Schematic representation of CASTing procedure. In vitro
translated FLAG-tagged Pokemon was incubated with a degenerate oligonucleotide pool
(binding reaction). Pokemon/oligonucleotide complexes were precipitated with anti-FLAG
antibody-conjugated beads. After extensive washing, binding oligonucleotides were
eluted from beads, amplified by PCR, sub-cloned into plasmids and subsequently
sequenced. b, Alignment of individual Pokemon oligonucleotide binding sequences
identified from the CAST analysis. The color code represents the percentage of
conserved sequence among pulled-down selected oligos.
c, EMSA performed with a
radio-labeled Pokemon binding sequence containing oligo-probe. Wheat extracts
containing in vitro translated Pokemon (Pok+), or wheat extracts without Pokemon (Pok-),
were incubated with radio-labeled POK probes (see also e in this figure). Non-labeled
POK oligos were used as cold competitors. d, Transrepression assays performed in 293T
cells transfected with increasing amounts of Pokemon expression vector and a luciferase
reporter plasmid (3POKBB-Luc) that contains three artificial Pokemon binding sites. The
y-axis represents fold activation of reporter normalized by a dual-luciferase system.
Pokemon expression level is shown in lower panel. e, Schematic representation of
mutated oligos used as probes or cold competitors in EMSA assay. Two to five bases
were replaced with thymine residues (shown in blue) and utilized in EMSA to identify the
core sequence for Pokemon binding. f, EMSA was performed with radio-labeled POK,
POK m1 and POK m2 probes with or without in vitro translated Pokemon. POK m1 probe
did not bind with in vitro translated Pokemon, indicating the core sequence for Pokemon
binding is located within the mutated sequence in POK m1 oligo. g, EMSA was further
performed to identify the core sequence of Pokemon binding site utilizing the mutated
oligos as cold competitors (POK, m3, m4, m5 and m6). The oligo m4 (and m6) failed to
compete with radio-labeled POK probe, which indicates that the two cytosine residues
mutated in the m4 oligo are indispensable for Pokemon binding.
Supplementary Fig. 3 a, Schematic representation of mouse p19Arf and human p14ARF
promoters. Mouse (left) and human (right) ARF promoter sequences [500 bp upstream
the transcription start site (+1)] are shown. Putative binding sites for DMP1 (orange),
E2F1 [blue; (+) and (–) indicates sense and antisense, respectively], Sp-1 (purple) and
TBX2/3 (green) are indicated. Pokemon putative binding sites are highlighted in red
characters. Promoter sequences included in the luciferase reporter constructs utilized in
our studies are indicated in purple (mouse) and orange (human) circles. Arrows
represents the primer sets that were utilized for the ChIP assays. b, Pokemon
transcriptional repression depends on both POZ and Zinc finger DNA binding domains.
Transrepression assays in NIH3T3 cells transfected with vectors encoding Pokemon
deletion mutants along with a mouse p19Arf luciferase based reporter (top left).
Expression levels of each Pokemon mutant were examined by Western blot (top right).
Schematic representation of Pokemon mutants is also shown (bottom). c, Schematic
representation of the mutated mouse p19Arf luciferase reporters. d, Pokemon antagonizes
E2F1-dependent ARF transactivation/de-repression. E2F1 expression vector and the
p19Arf luciferase reporter were transfected in NIH3T3 cells along with increasing amounts
of Pokemon expression vector. e, Real-Time PCR analysis for quantification of p19Arf
mRNA in serially passaged MEFs. MEFs were prepared from three independent
littermates. PCR reactions were performed in duplicate and the relative amount of p19Arf
mRNA is shown together with standard deviation. f, Pokemon is not capable of repressing
mouse p16Ink4a promoter activity. Transrepression assays were performed in NIH3T3 cells.
Increasing amounts of Pokemon expression vector were transfected along with mouse
p16Ink4a luciferase reporters. Expression control is also shown (bottom). g, p53
up-regulation in Zbtb7-/- (Null) MEFs. Western blot analysis of p53 expression in various
preparations of WT and Zbtb7-/- MEFs. Normalization of p53 protein levels over Hsp90 is
also shown (bottom).
Supplementary Fig. 4 a, Detection of transgenic and total Pokemon expression by
RT-PCR in splenocytes from two wild type controls (Cont.1, Cont. 2) and from F1 animals
of three independent lckE-Pokemon founder lines (numbers 16, 21 and 26). Primers
used were either specific for the transgenic FLAG-Pokemon or for both transgenic and
endogenous (total) Pokemon. Control reactions incubated without Reverse Transcriptase
(RT) were included. RT-PCR for a housekeeping gene, hprt, is also shown. b,
Monoclonal anti-POKEMON 13E9 antibody produces specific staining for Pokemon.
Immunohistochemical Pokemon staining of 13.5 d.p.c fetal liver (FL) cells are shown.
Pokemon shows strong and specific nuclear staining only in Zbtb7+/+ FL cells but not
Zbtb7-/- FL cells. c, Sections of an lckE-Pokemon thymic lymphoma immunostained with
antibodies to Pokemon and TdT (x400 magnification). d, Western blot for total Pokemon
on lysates from thymocytes of 4 wild type control mice and cells from 4 independent
lckE-Pokemon thymic lymphomas. Specificity of the Pokemon antibody is demonstrated
by the lack of a signal from lysate of Zbtb7-/- MEFs. Hsp90 is shown as a loading control.
Supplementary Fig. 5 Pokemon expression in normal human and mouse lymphoid
organs. a, Immunohistochemical staining of normal human thymus for POKEMON, CD3
and AE1:AE3 (at low power x40 (inset) and at high power x400 magnifications). The
arrows indicate medullary epithelial cells and Hassle’s corpuscles, which are highly
positive for POKEMON and AE1:AE3 but negative for CD3. b, Immunohistochemical
staining of normal/reactive human tonsil for POKEMON at x40, x100 and x400
magnifications. Pokemon is expressed in squamous epithelium and germinal center
lymphocytes. The arrows indicate germinal centers (GCs). c, Immunohistochemical
staining of normal mouse spleen for Pokemon and CD3 (at x40, x100 and x400
magnifications). Pokemon is mainly expressed in the white pulp germinal centers, which
are negative for CD3. d, Immunohistochemical staining of normal mouse thymus for
Pokemon and CD3 (at x40, x100 and x400 magnifications).
Supplementary Fig. 6 a, POKEMON expression in human T cell lymphomas. The table
shows a summary of T cell lymphoma TMA. Pictures of two POKEMON positive
lymphoblastic lymphoma cases are shown. Abbreviations: ALCL; Anaplastic large cell
lymphoma, AIL; Angioimmunoblastic lymphoma, LBL; Lymphoblastic lymphoma, PTCL;
Peripheral T cell lymphoma. b, POKEMON expression in human B cell lymphomas. The
table shows a summary of B cell lymphoma TMA. Pictures of two POKEMON positive
CLL cases are also shown. Abbreviations: DLBCL; Diffuse large B cell lymphoma, FL;
Follicular Lymphoma, MZL; Marginal zone lymphoma, SLL/CLL; Small cell lymphocytic
lymphoma/Chronic lymphocytic lymphoma, MCL; Mantle cell lymphoma. c, Kaplan-Meier
curves of overall survival (OS) in DLBCL cases stratified according to the indicated
markers. Case numbers (n) are also indicated. A log-rank test was used to calculate
P-values. The Age (>60), IPI (3 and 4), AA stage (3 and 4) and BCL2 positivity were
predictive of worse prognosis. Abbreviations (see also Supplementary tables): IPI;
International Prognostic Index, AA stage; Ann Arbor Stage. d, POKEMON is highly
expressed in BCL6 positive lymphomas. Bar graphs show the percentage of POKEMON
positivity among BCL6 positive lymphoma samples. Colors indicate levels of POKEMON
expression. e, The protein expression levels of introduced oncogenes in MEFs
(described in Fig. 5d) were comparable in WT and Zbtb7-/- MEFs. Western blot analyses
were carried out with the indicated antibodies. f, The expression levels of p14ARF mRNA
in 37 DLBCL cases. Cases were stratified according to relative POKEMON mRNA levels
(POKEMONmRNA/PBGDmRNA). Relative POKEMON mRNA levels ranged from 0.45 to
13.5 in this cohort and the cases with POKEMON mRNA levels higher than 5.5 were
classified into High group. P-value was generated by a Welch two sample t-test.