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LYMPHOID NEOPLASIA
Deregulation of Aiolos expression in chronic lymphocytic leukemia is associated
with epigenetic modifications
Katy Billot,1 Jérémie Soeur,2 Fanny Chereau,3,4 Issam Arrouss,1 Hélène Merle-Béral,5,6 Meng-Er Huang,2
Dominique Mazier,1 Véronique Baud,3,4 and Angelita Rebollo1
1Hôpital
Pitié-Salpêtrière, Université Pierre et Marie Curie (UPMC) Paris VI, Inserm, Paris, France; 2CNRS UMR 3348, Institut Curie, Université Paris Sud-11,
Orsay, France; 3Institut Cochin, Université Paris Descartes, CNRS, Paris, France, 4Inserm, Paris, France; 5Hôpital Pitié-Salpêtrière, Service d’Hématologie
Biologique, Paris, France; and 6CDR des Cordeliers, UPMC Paris VI, Inserm, Paris, France
Chronic lymphocytic leukemia (CLL) is
characterized by a clonal accumulation of
mature neoplastic B cells that are resistant to apoptosis. Aiolos, a member of the
Ikaros family of zinc-finger transcription
factors, plays an important role in the
control of mature B lymphocyte differentiation and maturation. In this study, we
showed that Aiolos expression is upregulated in B-CLL cells. This overexpression does not implicate isoform imbal-
ance or disturb Aiolos subcellular
localization. The chromatin status at the
Aiolos promoter in CLL is defined by the
demethylation of DNA and an enrichment
of euchromatin associated histone markers, such as the dimethylation of the
lysine 4 on histone H3. These epigenetic
modifications should allow its upstream
effectors, such as nuclear factor-␬B, constitutively activated in CLL, to gain access to promoter, resulting up-regulation
of Aiolos. To determine the consequences
of Aiolos deregulation in CLL, we analyzed the effects of Aiolos overexpression or down-regulation on apoptosis.
Aiolos is involved in cell survival by regulating the expression of some Bcl-2 family members. Our results strongly suggest that Aiolos deregulation by
epigenetic modifications may be a hallmark of CLL. (Blood. 2011;117(6):
1917-1927)
Introduction
The lymphocyte developmental is tightly controlled by a large
number of transcription factors.1 Among these proteins, Aiolos has
been identified as a homologue of the largest Ikaros isoform,2 with
strong similarities in the DNA binding, activation, and dimerization
domains.3 At least 16 isoforms of human Aiolos, generated by
alternative splicing, have been described.4 These isoforms can be
divided into 2 groups. One possesses at least 3 zinc fingers at the
N-terminus and binds DNA, and the other possesses fewer than
3 N-terminal zinc fingers and is unable to bind DNA. Isoforms that
cannot bind DNA but retain the capacity to dimerize are considered
to act in a dominant-negative fashion.5,6 Aiolos is not expressed in
mouse hematopoietic stem cells but is detectable in pro-B and
double-negative CD4/CD8 thymocyte precursors. Expression of
Aiolos is further up-regulated as these cells progress to the pre-B
and double-positive CD4/CD8 stages of differentiation, respectively. Aiolos is highly expressed in mature murine peripheral
B cells but not significantly in splenic T cells.7 In humans, B cells
express the highest level of total Aiolos compared with T cells,
natural killer cells, monocytes, and CD34⫹ hematopoietic progenitors. T and natural killer cells express comparable levels of Aiolos,
and monocytes express almost undetectable level of Aiolos whereas
CD34⫹ progenitors are negative for Aiolos expression.8 Aiolos
plays an essential role during B-cell maturation and its inactivation
in knockout mice results in an increase in B-cell precursors,
breakdown in B-cell tolerance, and the development of B-cell
lymphomas. In contrast, peritoneal, marginal, and recirculating
B cells are severely depleted.7 Aiolos functions and the parameters
involved in its transcriptional regulation are largely unknown in
humans and need be better defined. Recently, we reported for the
first time that Aiolos expression is deregulated in CLL,9 which was
subsequently confirmed by the authors of other studies.10,11
Chronic lymphocytic leukemia (CLL)12 is characterized by the
accumulation of monoclonal B lymphocytes in blood, bone marrow, and peripheral lymphoid tissues. CLL can be divided in
2 subsets on the basis of immunoglobulin heavy chain variable
region (IgVH) gene mutation status. Patients whose CLL cells
exhibit IgVH gene mutation have better a clinical prognosis than the
patients whose CLL cells do not exhibit IgVH gene mutation.13
ZAP-70, which is involved in T-cell receptor signaling, is aberrantly expressed in some patients14 and shows partial overlap with
overexpression of other risk factors such as the presence of CD38
or unmutated IgVH genes. CLL cells do not display a unique
recurrent genomic alteration but have several chromosomal alterations, including 11q22-q23 (ATM, or ataxia telangiectasia mutated), 13q14, 17p13 (TP53), 6q21 deletions, and 12 trisomy. 11q
and 17p deletions are associated with a poor outcome, whereas
isolated 13q deletion is associated with a favorable prognosis.15
Aberrant gene function and altered gene expression are key
features of cancer. Growing evidence shows that both acquired
epigenetic abnormalities and genetic alterations contribute to cause
deregulation of gene expression.16 Epigenetic is defined as heritable changes in gene expression that are not accompanied by
changes in DNA sequence. Covalent modifications of histones but
also of CpG sites on DNA participate in the establishment of local
chromatin structures that influence permissivity of gene promoter
for the subsequent assembly and/or activation of the transcriptional
Submitted September 13, 2010; accepted November 21, 2010. Prepublished
online as Blood First Edition paper, December 7, 2010; DOI 10.1182/blood2010-09-307140.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2011 by The American Society of Hematology
BLOOD, 10 FEBRUARY 2011 䡠 VOLUME 117, NUMBER 6
1917
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1918
BLOOD, 10 FEBRUARY 2011 䡠 VOLUME 117, NUMBER 6
BILLOT et al
machinery.17 Among histones marks, covalent modifications of Lys
4, Lys 9, and Lys 27 of the histone H3 tail are known to play crucial
roles. Histone H3 di/trimethylation on K4 and/or acetylation on K9
have been extensively associated with active regions of the genome
(euchromatin) whereas histone H3 trimethylation on K9 and/or
K27 and DNA methylation participate in the establishment and
maintenance of silent domains (heterochromatin).18 In this study,
we showed that Aiolos expression is up-regulated in a panel of
32 CLL patients. The overexpression does not implicate isoforms
imbalance or disturbing Aiolos subcellular localization. To investigate the causes of this Aiolos deregulation, we analyzed the profiles
of DNA methylation and histones modifications on Aiolos promoter. We show that enriched euchromatin-associated markers
together with upstream Aiolos regulators such as nuclear factor ␬B
(NF-␬B) may generate Aiolos overexpression. Furthermore, we
demonstrate that ectopic Aiolos overexpression decreases phorbol
myristate acetate (PMA)/ionomycin-mediated apoptosis in CLL
cells. These data suggest that Aiolos plays a role in apoptosismediated events and may be a hallmark of CLL.
turer’s instructions, followed by flow cytometry analysis. B cells CD19⫹
were scanned in FL1-H (fluorescein isothiocyanate) versus FL2-H (PI)
channels with a FACSCalibur instrument equipped with CellQuest software
(Becton Dickinson). Data analysis was performed with the quadrant
statistics of WEASEL software.
Aiolos siRNA knockdown
The siRNA against human Aiolos, nontargeting negative control siRNA,
and siRNA delivery media were from Thermo Scientific Dharmacon. The
siRNA used were specially modified by Dharmacon’s proprietary Accell
technology. Cells were cultured at a 1 ⫻ 106 cells/mL density with siRNAs
at a final concentration of 1␮M in Accell siRNA delivery media for at least
72 hours. Modifications in gene expression were assessed by real-time
polymerase chain reaction (PCR).
Nucleofection
The pcDNA3.1 vector (Invitrogen) was used to clone the cDNA encoding
wild-type Aiolos (Aio-1) in the BamHI/HindIII cloning sites. Transient
transfection of B cells was achieved by the Human B cell Nucleofactor Kit,
following the Amaxa guidelines for B-cell transfection.
RNA preparation and reverse-transcriptase PCR
Methods
Patients and B-cell isolation
In accordance with a protocol approved by the institutional ethics committee, peripheral blood mononuclear cell (PBMC) samples were collected
upon informed consent obtained from patients with CLL at the Hematological Department of Pitié-Salpêtrière Hospital. Diagnosis was assessed
according to the recent World Health Organization classification. In all
cases, blood sample collection was performed at the time of diagnosis.
Fresh blood from healthy donors (HDs) was collected by the Etablissement
Français du Sang. Mononuclear cells isolated from peripheral blood were
prepared by Ficoll gradient centrifugation, and B cells were isolated by
negative selection with the use of Dynabeads untouched human B cells
kit (Dynal).
Total RNA extraction was performed with the QIAGEN RNeasy kit
according to the manufacturer’s instructions. RNA (500 ng) was reverse
transcribed (RT) and amplified by the Titanium one-step RT-PCR (Clontech; for primers sequences, see supplemental Table 1, available on the
Blood Web site; see the Supplemental Materials link at the top of the online
article). The RT-PCR products were resolved by agarose gel electrophoresis
and visualized by ethidium bromide staining.
Quantitative RT-PCR
RNA was reverse transcribed by Superscript VILO cDNA synthesis kit
(Invitrogen). Quantitative PCR (qPCR) was performed by use of the
TaqMan gene expression assay (Applied Biosystems), and SYBR Green
assays were performed with the SYBR Green PCR Master Mix (Applied
Biosystems; for primers sequences, see supplemental Table 1).
Cell culture and inhibitors treatment
Daudi (human Burkitt lymphoma) and U937 (human histiocytic lymphoma) cell lines, purified normal and leukemic B cells, or PBMCs were
cultured in RPMI 1640 medium supplemented with 10% heat-inactivated
fetal bovine serum, 2mM L-glutamine, 1mM sodium pyruvate, 25mM
HEPES (N-2-hydroxyethylpiperazine-N⬘-2-ethanesulfonic acid), 0.1mM
nonessential amino-acids, and 100 U/mL penicillin/streptomycin. BAY
11-7082 was supplied by Calbiochem (Merck) in dimethyl sulfoxide
(DMSO) and was used at 5␮M final concentration. An equivalent volume of
DMSO was used in all experiments as a control. The cell-permeable NEMO
binding domain (NBD) peptide and its mutated control, mNBD, were a gift
from Dr Fabrice Agou (Institut Pasteur) and used at 20␮M final concentration. PMA and ionomycin (Sigma-Aldrich) was used for 8 hours at
10 ng/mL and 1 ␮g/mL, respectively.
Immunofluorescence and confocal analysis
The subcellular localization of Aiolos protein was examined as previously
described.19 In brief, B cells were fixed (4% paraformaldehyde for 20 minutes at room temperature), permeabilized (0.1% Triton X-100 for 5 minutes
at room temperature), and then incubated with “in-house” rabbit polyclonal
antiAiolos antibody overnight in phosphate-buffered saline/bovine serum
albumin at 4°C. After several washing steps, samples were mounted and
visualized with a Leica Leitz DMRB microscope fitted with a Leica
DFC300FX camera.
Annexin V/propidium iodide staining
Annexin V (AV)/propidium iodide (PI) labeling was performed by use of
the Vybrant Apoptosis Assay Kit #3 (Invitrogen) according to the manufac-
Chromatin immunoprecipitation
Chromatin immunoprecipitation (ChIP) assays were performed as described.20 The primers sequences match the CpG island, as well as upstream
and downstream of the CpG island (for primers sequences, see supplemental Table 1).
Methylated DNA immunoprecipitation
The methylated DNA immunoprecipitation (MeDIP) assay was performed
as described.20 Commercial glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) and testis/sperm-specific histone 2B (TSH2B) were used as a
negative and positive control (Diagenode) for MeDIP, respectively.
Protein extraction and Western blotting
Cytosolic extracts were prepared with buffer A (10mM HEPES, pH
7.8; 2mM MgCl2; 10 mM KCl; 0.1mM ethylenediaminetetraacetic acid
[EDTA]) and nuclear extracts with buffer B (500mM NaCl; 20mM HEPES,
pH 7.8; 1.5mM MgCl2; 0.5mM EDTA; 25% glycerol), with both buffers
containing 1mM phenylmethylsulfonyl fluoride, 5mM Na2VO4, 2mM
dithiothreitol, and the Complete Protease Inhibitor Cocktail (Roche Applied
Science). Total cellular lysates were prepared with the use of a high-salt
lysis buffer (400mM NaCl; 25mM HEPES, pH 7.8; 1.5mM MgCl2; 0.2mM
EDTA; 1% NP-40; 1mM phenylmethylsulfonyl fluoride; 5mM Na2VO4;
2mM dithiothreitol; and the Complete Protease Inhibitor Cocktail). Cell
lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis, transferred to nitrocellulose membrane (Bio-Rad), blocked
in Odyssey Blocking Buffer (LI-COR), and incubated with a primary
antibody. Blotted proteins were detected and quantified with the Odyssey
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BLOOD, 10 FEBRUARY 2011 䡠 VOLUME 117, NUMBER 6
infrared imaging system LI-COR. The following primary antibodies were
used: Aiolos (“in-house” rabbit Ab), actin (rabbit Ab; Cell signaling),
NF-␬B RelA (rabbit Ab; Santa Cruz), and fibrillarin (mouse Ab; Abcam).
Electrophoretic mobility shift assays
Nuclear extracts were prepared and analyzed for DNA binding activity by
use of the HIV-LTR tandem ␬B oligonucleotide as ␬B probe as previously described.21 For supershift assays, nuclear extracts were preincubated with antibodies specific for 30 minutes on ice before the addition
of the labeled probe.
Statistical analysis
We used Prism 5.0c (GraphPad Software) for statistical analysis. Unpaired
t test and Mann-Whitney U tests were realized, and statistical significance
was set at P ⬍ .05 with the following degrees: *P ⬍ .05; **P ⬍ .01; and
***P ⬍ .001.
Results
Expression of Aiolos but not Ikaros is up-regulated in B cells
from CLL patients
The level of Aiolos mRNA expression was determined by real-time
PCR analysis. Aiolos transcripts were amplified with primers
specific for the second and third exons, which amplify all Aiolos
isoforms. We analyzed a total of 32 CLL samples and 28 HDs and
normalized the amount of mRNA in each sample by using Abelson
(Abl) RNA as internal control. Clinical and pathologic characteristics of patients used in this study and their Aiolos expression data
are shown in Table 1. Total Aiolos mRNA was overexpressed in
B cells from CLL patients compared with HDs (Table 1). A 1.5-fold
increase of Aiolos transcripts in CLL samples compared with
normal B cells was observed, which is statistically significant
(P ⬍ .0001; Figure 1A). Given that unmutated IgVH gene status,
high ZAP-70 expression, or high CD38 expression are associated
with worse clinical outcome, we analyzed by qPCR the level of
Aiolos expression in B cells from these CLL patient subgroups. No
correlation of these clinical markers with Aiolos expression levels
was observed (Figure 1B). We further analyzed Aiolos protein
expression in PBMC from 2 HDs and 7 CLL patients. Aiolos
protein was overexpressed in PBMCs isolated from all CLL
patients except for P20, which shows a very weak expression as the
HDs (Figure 1C). This new data set confirmed our previous
observation that Aiolos expression is up-regulated in B cells from
CLL patients.9 To investigate whether Ikaros, an homologue of
Aiolos, is deregulated in B-CLL cells, we analyzed by RT-PCR the
expression pattern of different Ikaros isoforms in PBMC from
5 CLL patients and 2 HDs. An analysis of HDs revealed the
presence of 4 Ikaros variants: Ik-1, Ik-2, Ik-3, and Ik-4 but not Ik-6
(Figure 1D). No major differences in the distribution of Ikaros
isoforms were detected between HDs and CLL patients.
It is interesting to notice that, different from acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and chronic
myelogenous leukemia (CML), CLL cells analyzed in this study do
not express the dominant-negative isoform Ik-6. Nevertheless, this
result should be verified on a larger group of CLL patients. In all
cases, Ik-2/3 was the most abundant mRNA species, and the shorter
variant Ik-4 was equally expressed in cells from HD and CLL
patients. To investigate whether there was a change of total
amounts of Ikaros transcripts, we performed quantitative analysis
of total Ikaros expression on CD19-positive peripheral blood cells
in a group of 14 CLL patients and 13 HDs. In contrast to Aiolos, the
DEREGULATION OF AIOLOS IN CLL
1919
total Ikaros expression is similar between CLL patients and HDs
(Figure 1E). Thus, in contrast to Aiolos, Ikaros expression is not
deregulated in B cells from CLL. Therefore, B-CLL cells seem
to exhibit a specific up-regulation of the Aiolos member of the
Ikaros family.
B cells from CLL patients show a normal Aiolos subcellular
distribution
The authors of previous studies revealed an isoform-specific cell
distribution in the Aiolos isoforms. Aio-1, the full-length isoform,
and Aio-⌬5, the isoform lacking one zinc finger, are both present in
nuclei. By contrast, the Aio-⌬3,4,5,6 isoform, which lacks the
DNA-binding domain, is predominantly localized in the cytoplasm,4 similar to the distribution described for the Ikaros dominantnegative isoforms. To investigate putative modifications in the
subcellular localization of Aiolos in CLL cells, we analyzed by
confocal microscopy the Aiolos cellular distribution by using the
specific anti-Aiolos antibody. Daudi (B cells) and U937 (monocytes) cell lines were used as a positive and negative control for
Aiolos expression, respectively. We observed that Aiolos protein
has same localization in B cells from HD and from CLL patients,
with intense punctuate green fluorescent staining in their nuclei
(Figure 2).
Aiolos promoter is associated with enriched active chromatin
marks in B-CLL cells
We have recently shown that Aiolos promoter activity is regulated
by epigenetic mechanisms.22 To assess the involvement of this type
of regulation in the Aiolos up-regulation, we first analyzed histone
posttranslational modifications. We performed ChIP assays to
investigate the chromatin status at the Aiolos promoter of B cells
from CLL patients. Cross-linked chromatin from B cells of 8 HDs
and 14 CLL patients was immunoprecipitated by the use of specific
antibodies for histone H3 dimethylated on lysine 4 (H3K4me2),
histone H3 trimethylated on lysine 4 (H3K4me3), 9 (H3K9me3)
and 27 (H3K27me3), or histone H3 acetylated on lysine 9 (H3K9ac).
PCR was performed by the use of primers spanning a 2-kb region
upstream and downstream of the transcriptional initiation site
(from ⫺1 kb to ⫹ 1.5 kb; primers a-f, Figure 3A). The Aiolos
promoter exhibited active chromatin marks (euchromatin), such as
H3K4me2 (Figure 3B), H3K4me3 (Figure 3C), and H3K9ac
(Figure 3D) from ⫺1 to ⫹ 1.5-kb region spanning the Aiolos
promoter in B cells from both CLL patients and HDs. However, the
Aiolos promoter in CLL cells displays statistically significant
enrichment for H3K4me2 and H3K4me3 (P ⬍ .05), but not for
H3K9ac, compared with normal B cells. Of note we were not able
to detect heterochromatin (inactive chromatin) markers such as
H3K27me3 (Figure 3E) or H3K9me3 (Figure 3F) on Aiolos
promoter, independently of the promoter region analyzed. This
result suggests a more marked open chromatin structure of the
Aiolos promoter in B-CLL cells compared with HDs, which
highlights an increased transcriptional activity and correlates with
an up-regulation of Aiolos expression in B-CLL cells.
We then determined whether modifications in chromatin structure at the Aiolos promoter were associated with changes in
histone-modifying enzymes expression. Because H3K4me2 and
H3K4me3 were enriched significantly in CLL patients, we analyzed the SMYD3 (SET and MYND domain-containing protein 3)
expression, which has a histone H3-lysine 4–specific methyltransferase activity and is frequently up-regulated in some human
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BLOOD, 10 FEBRUARY 2011 䡠 VOLUME 117, NUMBER 6
BILLOT et al
Table 1. Clinicopathological characteristics of CLL patients used in this study
Patient no.
Age, y
Sex
Stage*
VH gene status†
CD38%
Cytogenetic
Ratio total Aiolos/Abl (mRNA)
1
67
M
A
M
0
13q⫺ (mono), 11q⫺
1.99
2
74
F
B
M
1
ND
3.72
3
60
M
A
M
0
N
4.06
4
42
M
A
M
2
N
3.79
5
64
F
A
M
1
ND
4.66
6
70
F
A
M
1
13p⫺, 13q⫺ (bi)
4.07
7
51
F
C
M
3
N
3.66
8
60
M
A
M
2
N
3.84
9
59
M
B
M
0
6q⫺, 13q⫺ (bi), ⫹ 8
5.4
10
75
M
C
M
93
ND
3.37
11
70
F
A
U
72
⫹ 12
4.28
12
54
M
C
U
2
⫹ 12
3.63
13
51
M
A
M
84
⫹ 12
3.87
14
69
M
B
M
2
ND
3.9
15
49
M
B
U
0
13q⫺ (mono), 11q⫺
4.58
16
60
F
B
U
91
13q⫺, t(8;8)
5.36
4.14
17
59
F
C
U
88
Normal FISH
18
58
M
A
M
20
13q⫺ mono
2.73
19
60
M
C
U
70
ND
ND
20
54
M
B
ND
41
13q⫺ (mono)
4.87
21
45
M
B
M
37
Normal FISH
2.84
22
56
M
A
ND
30
ND
3.45
23
80
M
B
U
71
del 13q
2.93
24
65
F
A
M
0
ND
4.2
25
65
M
ND
ND
97
ND
4.41
26
80
M
A
ND
91
⫹ 12
3.06
27
60
F
C
U
90
⫹ 12
3.98
28
77
F
A
U
1
13q⫺, t(2;18), t(6;13)
2.53
29
62
F
A
ND
1
N
3.87
30
87
F
A
ND
0
13q⫺ (mono)
3.22
31
79
M
A
M
56
13q⫺ (mono)
2.32
32
62
M
A
M
0
13q⫺ (mono)
3.42
33
78
M
A
M
0
13q⫺ (mono), t(9; 13; 14)
3.37
34
67
M
B
M
ND
N
ND
35
75
F
A
M
ND
ND
ND
36
63
M
A
U
ND
13q⫺, 11q⫺
ND
37
78
M
A
M
1
ND
ND
38
74
M
B
U
28
13q⫺
ND
39
77
M
B
U
1
13q⫺, 11q⫺
ND
40
50
M
A
M
0
13q⫺
ND
41
79
F
A
ND
ND
ND
ND
ND
42
80
M
A
M
56
ND
43
66
M
A
M
2
N
ND
44
64
M
A
M
1
ND
ND
Abl indicates Abelson; bi, biallelic; CLL, chronic lymphocytic leukemia; FISH, fluorescence in situ hybridization; M, mutated; mono, monoallelic; N, normal; ND, not
determined; t, translocation; U, unmutated; ⫹ 12, trisomy 12; 13q⫺, deletion 13q; 13p⫺, deletion 13p; 11q⫺, deletion 11q; and 6q⫺, deletion 6q.
*Stage is according to Binet scale.
†Greater than 2% deviation from germline sequence was considered as mutated. In our study, ZAP-70 negative cases are IgVH mutated. The average age for HD is
⬃ 54 and ⬃ 64 for CLL patients. The average of ratio total Aiolos/Abl for HD is ⬃ 2.4.
carcinomas. As shown in Figure 3G, SMYD3 mRNA was significantly overexpressed in B cells from CLL patients, compared with
HDs (P ⫽ .0031). This result shows for the first time a SMYD3
up-regulation in CLL.
Demethylated Aiolos promoter CpG island in malignant and
normal B cells
In addition to modification of the N-terminal tail of histones, DNA
methylation of CpG islands plays a crucial role in chromatin
organization and function. The main CpG island on Aiolos
promoter was detected between ⫺344 bp and the transcription
initiation site (Figure 3A) by Methprimer software analysis. We
have previously shown that this CpG island exhibits differential
methylation between Aiolos expressing and nonexpressing cell
lines. We analyzed by the use of MeDIP assays the methylation
patterns of a 2-kb region spanning the upstream and downstream
transcriptional initiation site that contains the CpG-rich region of
the Aiolos promoter. Sonicated genomic DNA from 3 HDs and 3 CLL
patients’ B cells was immunoprecipited with antibody specific for
5-methylcytidine. PCR was performed by use of the previously
described primers for ChIP assays (Figure 3A). The primer pairs
c and d amplified the CpG-rich region on Aiolos promoter.
Promoters of actively transcribed GAPDH gene and silent TSH2B
gene in B cells were used as negative and positive controls for
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BLOOD, 10 FEBRUARY 2011 䡠 VOLUME 117, NUMBER 6
A
1921
B
P < .0001
5.5
***
Total Aiolos / Abl
5.5
Total Aiolos / Abl
4.5
3.5
2.5
1.5
HD
4.5
3.5
2.5
1.5
CLL
P = n.s.
p = n.s.
p = n.s.
U
M
VH gene
pos neg
ZAP-70
pos neg
CD38
C
P13
WB
P34
P36
P35
P20
P32
P33
HD
HD
AIOLOS
58 kDa
ACTIN
45 kDa
AIOLOS / ACTIN
4
(Densitometric analysis)
Figure 1. Analysis of Aiolos and Ikaros expression in CLL.
(A) Quantification of total Aiolos transcripts in B cells from 32 CLL patients
(P1-P18 and P20-P33) and 28 HDs obtained by RT-qPCR of B-cell RNA
and normalized to the Abl gene. Absolute quantification of Aiolos and Abl
was performed with the use of known quantities of pcDNA3.1-hAio1
construct and Daudi total RNA, respectively. Respective data are summarized and presented as the mean ⫾ SEM. (B) Comparison of Aiolos levels
in CLL subsets defined by VH gene mutation status, ZAP-70, and CD38
expression. Respective data are summarized and presented as the
mean ⫾ SEM. Differences between groups were tested using unpaired
t test (Prism5.0c software; ns indicates non significant; *P ⬍ .05; **P ⬍ .01;
***P ⬍ .001). (C) Aiolos protein levels in PBMCs from 7 CLL patients and
2 HDs were detected by Western blot with a specific anti-Aiolos antibody.
Actin was used as internal loading control (top). The histogram shows the
densitometric analysis of 2 independent Western blot experiments with
same protein samples. The error bars represent standard deviation
(bottom). (D) Expression of Ikaros transcripts in PBMC from HD and CLL
patients. Representative results of Ikaros isoforms expression in 2 HD and
5 CLL patients (P2, P25, P31, P32, and P34) after one-step RT-PCR
amplification of PBMC RNA. ␤-actin was used as internal control.
(E) Quantitative analysis of total Ikaros mRNA levels in B cells from 13 HD
and 14 CLL patients (P17, P18, P20-P23, P25, P26, and P28-P33). Data
are expressed as normalized expression by use of the 2⫺⌬Ct calculation
method (Ct indicates cycle threshold) and ␤-Actin as reference gene.
n.s. indicates nonsignificant; *P ⬍ .05; **P ⬍ .01; ***P ⬍ .001.
DEREGULATION OF AIOLOS IN CLL
3
2
1
0
P13
P34
P36
D
CLL
Ik6
β-Actin
MeDIP, respectively. Figure 4A shows that the Daudi cell line has
demethylated promoter, which correlates with Aiolos expression.
In contrast, Aiolos promoter is densely methylated on the CpG-rich
region in U937 cell line, which correlates with Aiolos silencing.
These results are in agreement with our previous results obtained
by bisulfite sequencing. The Aiolos promoter is densely demethylated in B cells from CLL and HD patients (Figure 4B). This result
strongly suggests that Aiolos up-regulation in B cells from CLL
patients is not caused by an abnormal promoter demethylation but
rather by an increase of euchromatin markers, notably H3K4.
Inhibition of NF-␬B activation induces down-regulation of
Aiolos expression
CLL cells show increased NF-␬B–binding activity compared with
normal B cells23 and we have previously shown functional NF-␬B
binding sites in the Aiolos promoter.24 The NF-␬B proteins are key
regulators of differentiation and survival in B cells. In mammals,
this protein family includes NF-␬B1 (p50 and its precursor p105),
NF-␬B2 (p52 and its precursor p100), RelA (p65), Rel (cRel), and
RelB. In the inactive state, NF-␬B proteins occur as homodimeric or
heterodimeric complexes in the cytoplasm bound to I␬B proteins. After
P20
P32
P33
HD
HD
E
B
n.s.
Total Ikaros / β-Actin
P32 P2 P34 P31 P25 A
Ikaros
Ik1
Ik2/3
Ik4
HD
P35
0.100
0.075
0.050
0.025
0.000
HD
CLL
appropriate stimulation, I␬B are phosphorylated, ubiquinated, and
degraded, allowing the translocation of NF-␬B dimers to the
nucleus and subsequent transcription of NF-␬B target genes.
We decided to analyze the contribution of NF-␬B to the control
of Aiolos expression in CLL cells. We first analyzed NF-␬B
DNA-binding activity in whole cell extracts from the PBMCs of
7 CLL patients and 3 HDs. All CLL patients, except P20, presented
a strong constitutive NF-␬B DNA binding activity that was not
observed in the HDs (Figure 5A). Supershift analysis demonstrated
that DNA-bound NF-␬B complexes mainly comprised RelA, p50,
and RelB (Figure 5B). Interestingly, P20 exhibited weak Aiolos
expression levels, whereas all others CLL patients tested presented
a high level of Aiolos protein expression (Figure 1C), thus
suggesting a correlation between NF-␬B activity and Aiolos
expression. To further investigate the influence of NF-␬B on Aiolos
expression, we analyzed the effect of NF-␬B inhibition.
All pharmacologic NF-␬B inhibitors potentially suffer from a
lack of selectivity and off-target effects, and we therefore used
2 distinct NF-␬B pathway inhibitors, BAY 11-7082 (referred as
BAY) and NBD peptide. The pharmacologic inhibitor BAY inhibits
the tumor necrosis factor-␣–inductible phosphorylation of I␬B␣,
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1922
BLOOD, 10 FEBRUARY 2011 䡠 VOLUME 117, NUMBER 6
BILLOT et al
DAUDI
DAPI
AIO
Merge
DAPI
Merge
DAPI
AIO
AIO
Merge
CLL B-cells
HD B-cells
DAPI
Figure 2. Localization of Aiolos protein in B cells from HDs and CLL
patients. Samples were stained with anti-Aiolos antibody (green) and
DAPI (blue) and analyzed by confocal microscopy. Similar results were
obtained in 8 CLL patients. Daudi and U937 cells were used as positive
and negative control for Aiolos expression, respectively.
U937
AIO
Merge
P21
P1
P17
of NF-␬B activation in Daudi cells upon treatment with BAY or
NBD peptide. As expected, treatment with DMSO and the mNBD
peptide had no significant effect on NF-␬B activation. We then
analyzed the expression of Aiolos and several antiapoptotic molecules upon treatment of the Daudi cell line (Figure 5D) and
B-CLL cells (Figure 5E) with BAY (Figure 5D-E, left) or NBD
whereas the NEMO binding domain peptide disrupts the association of NEMO (or IKK␥) with IKK␤ and therefore blocks tumor
necrosis factor-␣–induced NF-␬B activation. The inhibitors efficiency was tested on the human Burkitt lymphoma Daudi cells,
which present a strong NF-␬B DNA binding activity and a high
level of Aiolos expression. Figure 5C shows the specific inhibition
A
CpG
Island
-1000 pb
-500 pb
a
b
0
500 pb
c
e
d
H3K4 Dimethylation
*
*
**
20
CLL
HD **
*
0
a
c
b
d
*
e
f
0
40
*
a
b
c
d
e
f
20
0
40
a
b
c
d
e
f
CLL
HD
H3K9 Acetylation
a
b
c
d
e
f
CLL
HD
H3K9 Trimethylation
20
0
G
20
0
CLL
HD
H3K27 Trimethylation
a
SMYD3 / β-Actin (x 10 - 4)
In vivo enrichment
CLL *
HD
H3K4 Trimethylation
*
20
40
F
D
In vivo enrichment
Figure 3. ChIP analysis of histone modifications within the Aiolos
promoter in CLL and normal B cells. (A) Schematic representation of
the Aiolos locus. CpG island is represented by a gray box, and the arrows
indicate the positions of the primer pairs used to analyze chromatin
remodeling at Aiolos promoter. (B-F) ChIP analysis of H3K4 dimethylation
(B), H3K4 trimethylation (C), H3K9 acetylation (D), H3K27 trimethylation
(E), and H3K9 trimethylation (F) in B cells from HD (black bars) and CLL
patients (white bars). qPCR was performed with primers a-f. The gray
portions of the graphs correspond to the CpG island. The graph shows the
average percent immunoprecipitation with SEM for 8 HD and 14 CLL
patients (P1, P2, P17-P28) calculated for each position. (G) Quantitative
analysis of SMYD3 mRNA levels in 14 HD and 16 CLL patients (P1, P2,
P8, P17, P20-P23, P25, P26, and P28-P33). Data are expressed as
normalized expression by use of the 2⫺⌬CT calculation method
(Ct indicates cycle threshold) and ␤-actin as reference gene. Differences
between groups were tested by use of Mann-Whitney U test (Prism5.0c
software; *P ⬍ .05; **P ⬍ .01; ***P ⬍ .001).
In vivo enrichment
C
40
f
E
In vivo enrichment
In vivo enrichment
B
40
1500 pb
1000 pb
b
c
d
e
P = .0031
7.5
**
6.0
4.5
3.5
2.5
1.5
HD
CLL
f
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BLOOD, 10 FEBRUARY 2011 䡠 VOLUME 117, NUMBER 6
DEREGULATION OF AIOLOS IN CLL
A
% of Input
60
DAUDI
U937
CpG Methylation
30
0
TSH2B GAPDH a
b
c
d
e
f
B
% of Input
30
CLL
HD
CpG Methylation
15
0
TSH2B GAPDH c
d
1923
expression. The effect on Bfl-1 and TRAF1 expression was not
significant (Figure 6A right). We further analyzed the effect of
Aiolos overexpression on apoptosis by PI and AV/PI staining.
Transfection per se with pcDNA-Flag vector (control) or pcDNAFlag-Aio-1 vector (Aiolos expression) in PBMC cells from CLL
patients induced approximately 16% and 14% of apoptosis,
respectively. When transfected cells were stimulated with PMA/
ionomycin, the average level of apoptosis reached approximately
26% in cells with pcDNA3.1-Flag vector and 14% in cells with
pcDNA3.1-Flag-Aio-1 vector. At same time, the proportion of
viable cells was more elevated in cells with pcDNA3.1-Flag-Aio-1
vector (41% vs 54%; Figure 6B). These results should be verified
on a larger group of CLL patients. They demonstrate that Aiolos
overexpression confers CLL cells resistance to PMA/ionomycininduced apoptosis. These results were confirmed by measurement
of caspase activity (data not shown). Taken together, our data
suggest that Aiolos may be involved in apoptosis regulation of
CLL cells.
f
Figure 4. MeDIP analysis of DNA methylation within the Aiolos promoter
containing a CpG rich region. (A) Aiolos promoter methylation status was analyzed
in Daudi and U937 human cell lines, positive and negative for Aiolos expression,
respectively. CpG island is marked in gray. (B) Aiolos promoter methylation status
was analyzed in CLL patients and HD. The graph shows the average percent
immunoprecipitation with SEM for HD (n ⫽ 3) and CLL patients (n ⫽ 3) calculated for
each position. GAPDH and TSH2B loci were used as negative and positive control for
MeDIP, respectively.
peptide (Figure 5D-E, right). All the analyzed genes, Bcl-XL, Bcl-2,
Bfl-1, TRAF1, TRAF2, XIAP, and Bcl-w, have been reported to be
NF-␬B target genes. Upon treatment with BAY, a down-regulation
of Aiolos, Bcl-w, Bcl-XL, Bcl-2, Bfl-1, TRAF1, and TRAF2
expression was observed in the Daudi cells (Figure 5C, left).
Similarly, Aiolos, Bcl-2, Bfl-1, TRAF1, and Bcl-XL expression was
down-regulated upon treatment with NBD (Figure 5D, right).
Treatment of B cells from 2 CLL patients by the inhibitor BAY
strongly inhibits expression of Aiolos, Bcl-2, Bfl-1, Bcl-w, TRAF1,
and Bcl-xL (Figure 5E, left). Similarly, expression of Aiolos, Bcl-2,
TRAF1, and Bcl-XL was down-regulated upon NBD treatment of
cells from 3 CLL patients (Figure 5E, right). The inhibition of
Aiolos expression upon BAY treatment was also confirmed at the
protein level in nuclear extracts (N) of Daudi cells (Figure 5F).
Taken together, these results strongly suggest the implication of
NF-␬B in the control of Aiolos expression in CLL cells.
Aiolos is involved in the survival of cells from CLL patients
NF-␬B is responsible for up-regulating gene products that control
cell survival. Furthermore, we have previously shown that Aiolos is
involved in the control of Bcl-2 expression in a lymphokinedependent murine T-cell line.25 To analyze the implication of
Aiolos in the control of apoptosis in CLL cells, we inhibited Aiolos
expression in PBMCs from CLL patients by specific Aiolos siRNA
followed by analysis of the expression of antiapoptotic genes.
Aiolos siRNA did not completely abolish Aiolos expression, but
attenuated Aiolos message levels by approximately 50%. Interestingly, Aiolos knockdown also resulted in decreased levels of
Bcl-XL, Bcl-2, Bfl-1, TRAF1, and Bcl-w transcripts in PBMCs
from CLL patients (Figure 6A left). We then analyzed the effect of
Aiolos overexpression on these antiapoptotic genes. Ectopic overexpression of Aio-1 wild-type isoform in PBMCs from CLL
patients induces a strong increase of Bcl-2 expression and a more
modest but significant induction of Bcl-w and Bcl-XL mRNA
Discussion
Aiolos is a transcription factor mainly expressed in mature B cells.
Given its expression pattern, we decided to study the contribution
of this transcription factor to CLL pathogenesis. This disease is
characterized by the monoclonal expansion of B lymphocytes in
the peripheral blood, bone marrow, and lymphoid organs with an
indolent course that can become aggressive or even fatal.12 Current
knowledge of the pathogenesis is limited because no specific
genetic alteration has yet been associated with this disease.26 In this
study, we showed an overexpression of total Aiolos transcripts and
proteins in cells from CLL patients compared with HDs. The
increase in Aiolos expression in CLL does not seem to be related to
patients’ age. Aiolos expression levels do not correlate with the
well-known CLL clinical markers such as IgVH gene status, high
ZAP-70 expression, or high CD38 expression. It is known that
Aiolos proteins exhibit an isoform-specific cell distribution, the
dominant-negative isoform being mainly localized in the cytoplasm.4 We observed by confocal microscopy that the Aiolos
protein show a nuclear localization both in normal and B cells from
CLL patients. Taken together, these data show that Aiolos deregulation in CLL, in contrast to Ikaros in ALL,27 CML,28 and AML,29
might be caused by an overexpression of the all Aiolos isoforms
and not to an overexpression of a dominant-negative isoform,
which would preferentially localize in the cytoplasm.
We have recently proposed that epigenetic mechanisms, such as
DNA methylation and histone modifications, could control the
activity of its promoter.22 Our previous study showed that different
mechanisms trigger Aiolos repression in tumor cell lines and
hematopoietic primary cells. DNA methylation seems to be an
important epigenetic modification controlling Aiolos expression
and chromatin modifications in tumor cell lines whereas histone
modifications are the main epigenetic modification in hematopoietic primary cells. In a preliminary work, on the basis of 4 CLL
patients and 1 HD,9 we observed no significant modifications of the
H3K4me3 and H3K9ac throughout the Aiolos promoter between
CLL patients and HDs. In this study, realized on a new larger group
of CLL patients, we were able to correlate Aiolos expression in
CLL cells with an increase of enriched euchromatin associated
markers, such as H3K4me2, H3K4me3, and H3K9ac. The statistically significant enrichment in H3K4me2 and H3K4me3 confers an
open chromatin status at the Aiolos promoter, resulting in an
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1924
BLOOD, 10 FEBRUARY 2011 䡠 VOLUME 117, NUMBER 6
BILLOT et al
A
HEALTHY
DONORS
CLL
P34 P36
P13
P35 P20
P32
P33
A
B
C
NF-κB
B
P35
Antibodies
-
P32
RelA RelB p50 c-Rel p52
RelA RelB p50 c-Rel
-
p52
RelA / p50
RelB / p50
C
DAUDI
DMSO
BAY
mNBD NBD
NF-κB
Figure 5. Effect of NF-␬B inhibition on Aiolos expression.
(A) Analysis of NF-␬B DNA-binding activity in B cells from CLL.
Electrophoretic mobility shift assays were performed with total
extracts from PBMC of 7 CLL patients (P13, P20, and P32-P36)
and 3 HD using a 32P-labeled human immunodeficiency virus-long
terminal repeat tandem ␬B oligonucleotide as a probe. (B) For
supershift analysis, total extracts from 2 CLL patients (P35 and
P32) were incubated with the indicated antibodies before incubation with the labeled probe. (C) Analysis of NF-␬B DNA-binding
activity in Daudi cells. Electrophoretic mobility shift assays were
performed with nuclear extracts from Daudi cells in 3 independent
experiments. Daudi cells were treated for 8 hours with the NF-␬B
inhibitor BAY 11-7082 at 5␮M (referred as BAY, left) or for 2 hours
with the wild-type NEMO binding domain peptide at 20␮M (referred as
NBD, right). DMSO or mNBD was used as a control. (D-E) Expression
of Aiolos and antiapoptotic molecules in NF-␬B inhibitor-treated
Daudi and CLL cells. The expression of different genes was
analyzed by qRT-PCR and normalized by use of the 2⫺⌬Ct
calculation method (Ct indicates cycle threshold) and ␤-actin as
reference gene. Daudi and CLL cells were treated with BAY
11-7082 (5␮M, 8 hours; panels D-E left) or NBD peptide (20␮M,
RelA / p50
RelB / p50 2 hours; panels D-E right). CLL cells from 2 (P1 and P37) and
4 (P1 and P38-40) patients were treated with BAY and NBD,
respectively. DMSO or mNBD was used as controls. Results are
expressed as fold down- or up-regulation of inhibitor-treated cells
compared with control cells. Data are mean ⫾ SEM of at least
3 independent experiments. (F) Aiolos expression at the protein
level was analyzed by Western blotting in nuclear (N) or cytosolic
(C) extracts isolated from Daudi cells treated or not with BAY
(5␮M, 8 hours). Fibrillarin and ␤-actin expression were used as
internal control of the nuclear and cytosolic fraction purity, respectively. Densitometric analysis of nuclear proteins and molecular
weight of the proteins are shown. Similar results were obtained in
2 independent experiments.
D
Fold regulation
Fold regulation
DAUDI
1.4
1.2 BAY 11-7082
1.0
0.8
0.6
0.4
0.2
0.0
1.4
1.2 NBD PEPTIDE
1.0
0.8
0.6
0.4
0.2
0.0
Bcl-X L Bcl-2 Bfl-1 TRAF1 TRAF2 XIAP Bcl-w Aiolos
Bcl-2
Bfl-1
TRAF1
Bcl-w
Aiolos
CLL
1.4
1.2 BAY 11-7082
1.0
0.8
0.6
0.4
0.2
0.0
Bcl-XL Bcl-2
Fold regulation
Fold regulation
E
Bcl-XL
1.4
1.2 NBD PEPTIDE
1.0
0.8
0.6
0.4
0.2
0.0
Bfl-1 TRAF1 Bcl-w Aiolos
Bcl-XL Bcl-2
DMSO
BAY
DMSO
AIOLOS
58 kDa
FIBRILLARIN
34 kDa
ACTIN
45 kDa
3.0
(Densitometric analysis)
C
N
BAY
AIOLOS / FIBRILLARIN
F
Bfl-1 TRAF1 Bcl-w Aiolos
2.5
2.0
1.5
1.0
0.5
0.0
BAY
increase of its transcriptional activity in CLL cells compared with
HDs. Furthermore, we observed for the first time an up-regulation
of the SMYD3 transcripts in B cells from CLL patients compared
with HDs. SMYD3 is a H3K4 specific dimethyltransferase and
trimethyltransferase and plays an important role in oncogenesis.30-32
DMSO
The epigenetic mechanisms are very important in the Aiolos
activity, not only at the level of its transcriptional regulation,22 but
also as means to exercise its own function.33 Recently, it was shown
that Aio-1 wild-type isoform interacts with the deacetylation and
chromatin-remodeling complex Mi2/NuRD, colocalizing either
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BLOOD, 10 FEBRUARY 2011 䡠 VOLUME 117, NUMBER 6
DEREGULATION OF AIOLOS IN CLL
A
Bcl-XL
Bcl-2
Fold regulation
Fold regulation
1.4
1.2 Aiolos siRNA
1.0
0.8
0.6
0.4
0.2
0.0
TRAF1
Bfl-1
Bcl-w
Aiolos
20.0
10.0
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
pcDNA3.1-Flag-Aio-1
Bcl-XL
Bcl-2
Bfl-1
TRAF1
Bcl-w
Aiolos
B
0.3 39.2
43.2 17.3
0.7 40.6
46.9 11.8
1.2 42.2
23.8 32.9
100
PBMC CLL (%)
+ PMA / Ionomycin
0.2 39.6
50.2 10
pcDNA-Flag
Propidium Iodide
P44
- PMA / Ionomycin
pcDNA-Flag-Aio-1
Figure 6. Effect of Aiolos on apoptosis in B cells from CLL
patients. (A) Influence of Aiolos knockdown and Aiolos overexpression on antiapoptotic gene expression analyzed by qRT-PCR and
normalized by use of the 2⫺⌬Ct calculation method (Ct indicates
cycle threshold) and ␤-actin as reference gene. Results are
expressed as fold down- or up-regulation of transfected cells by
pcDNA3.1-Flag-Aio-1 vector or Aiolos siRNA compared with
transfected cells by pcDNA3.1-Flag vector or nontargeting negative control siRNA, respectively. The histograms represent the
mean ⫾ SD of genes down-regulation (n ⫽ 3, left) or upregulation (n ⫽ 2, right). (B) PMA/ionomycin-induced cell death
assayed by AV/PI double staining and flow cytometry. B cells from
CLL patients transfected with pcDNA-Flag-Aio-1 or pcDNA-Flag
vector. Eighteen hours after transfection, cells were stimulated by
PMA and ionomycin (10 ng/mL and 1 ␮g/mL) for 8 hours before
apoptosis analysis. Representative dot plots show AV/PI staining
on B cells from patient P44. Percentages of viable (AV⫺/PI⫺),
apoptotic (AV⫹/PI⫺), secondary apoptotic/necrotic (AV⫹/PI⫹), and
necrotic (AV⫺/PI⫹) populations are indicated (left). The histograms
represent the mean proportions of AV⫺/PI⫺, AV⫹/PI⫺, AV⫹/PI⫹,
and AV⫺/PI⫹ cells obtained with cells from 5 patients (P37-39,
P43, P44; right).
1925
75
AV+/ PI+ &
AV - / PI +
50
AV+/ PI -
25
AV - / PI -
0
+
+
-
+
+
+
-
+
PMA / Ionomycin
pcDNA3.1-Flag
pcDNA3.1-Flag-Aio-1
Annexin V
with the HDAC1 or with the MTA2 subfraction. However, Aio-1
associates specifically with the promoter of SIRT1, a class III
nicotinamide adenine dinucleotide–dependent histone deacetylase
that is thought to participate in the formation of facultative
heterochromatin.4 Furthermore, induction of Aio-1 expression in
Aiolos negative cell line results in a global acetylation decrease of
histones H3 and H4 and of specific lysine residues, such as K8 and
K16 of histone H4 and K9 of histone H3.4 The Aiolos overexpression could therefore have a global impact on acetylation levels and
contribute to epigenetic alterations in CLL cells. Global and
gene-specific aberrant DNA methylation34 has been described for
genes that are specifically deregulated in CLL such as those
encoded for Bcl-2,35 TCL1 (T cell leukemia/lymphoma1),36
ZAP70,37 and DAPK1 (ie, Death-associated protein kinase 1).38
However, it is difficult to determine whether the epigenetic
deregulations in CLL are the cause or the consequence of the
tumoral transformation.
The open chromatin status at the Aiolos promoter in CLL might
allow its upstream effectors to gain access to promoter (Figure 7).
The increased activity or amount of the factors controlling, directly
or indirectly, Aiolos expression in CLL patients may be the origin
of Aiolos up-regulation. In a previous study, we showed the role of
B-CLL cell
Normal B cell
NF- B
SMYD3
Ikaros
Ikaros
Ikaros
?
NF- B
SMYD3
NF- B
NF- B
Aiolos ++
homeostasis
Aiolos promoter
Ikaros and NF-␬B transcription factors, which have binding sites
on the Aiolos promoter, in the control of Aiolos expression.24
Contrary to the data observed in ALL,27 CML28 and AML,29 Ikaros
does not seem deregulated at expression level regarding its total
transcripts. Modifications in its cellular localization or in its
association with chromatin-remodeling complexes could however
be involved in controlling Aiolos expression.
Aiolos promoter possesses also binding sites for NF-␬B transcription factor that is constitutively activated in B-CLL cells,23,39-41
and the binding activity of p65 was found to be a prognostic marker
in CLL.42 We confirmed the constitutive activation of p65/p50 in
6 of 7 CLL patients studied. The strong NF-␬B activity was
associated with a high expression of the Aiolos protein. The
inhibition of NF-␬B activity resulted in a down-regulation of the
Aiolos expression. These results suggest that Aiolos may be a direct
or indirect target of NF-␬B transcription factor and acts in
survival/proliferation mechanisms.
Several evidence strongly support the view that Aiolos is
involved in regulation of B-cell apoptosis. Aiolos overexpression
rescues the number of viable cells, decreases the number of
apoptotic cells and induces overexpression of some Bcl-2 family
members. Aiolos knockdown down-regulates expression of some
Unmethylated cytosines
SMYD3
Ikaros
Aiolos +++
cell survival
Euchromatin
Heterochromatin
Figure 7. Hypothetical model of the mechanisms involved in regulation of Aiolos transcription and consequence of its overexpression
on the cell survival in leukemogenesis of CLL. The chromatin status at
the Aiolos promoter in CLL is defined by the demethylation of DNA and an
enrichment of euchromatin associated histone markers, compared with
normal B cells. These epigenetic modifications should allow its upstream
effectors, such as NF-␬B, constitutively activated in CLL, to gain access to
promoter, resulting overexpression of Aiolos. Ikaros does not seem
deregulated at its expression level in CLL. This Aiolos deregulation could
participate in the survival of cells from CLL patients.
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1926
BLOOD, 10 FEBRUARY 2011 䡠 VOLUME 117, NUMBER 6
BILLOT et al
Bcl-2 family members and Aiolos has been implicated in the direct
control of the Bcl-2 gene promoter activity in murine T cells.25
Furthermore, the absence of Aiolos accelerates avian premature
B-cell apoptosis mediated by B-cell antigen receptor (BCR)
signaling.43,44 CLL is considered to be a disease in which apoptosis
was deregulated, suggesting that Aiolos deregulation could contribute to leukemogenesis by suppressing B-cells apoptosis, through
up-regulation of the Bcl-2 antiapoptotic proteins. The antiapoptotic
effect of Aiolos protein in CLL cells is in contradiction to the data
observed in Aiolos-deficient mice that develop B lymphoma7 and
to the confirmed function of the wild-type isoform Ik-1 as a tumor
suppressor gene.45 Activity of the wild-type isoform Aio-1 in CLL
is more similar to that of the dominant-negative Ik-6.
Induction of apoptosis in tumoral cells is an efficient antitumoral strategy. Several preclinical studies indicate that NF-␬B is a
relevant target in CLL, because inhibition of its activation, leads to
apoptosis of CLL cells.46,47 Although the exact mechanism of the
aberrant NF-␬B activity in CLL remains unresolved, it is well
known that NF-␬B is activated downstream of BCR activation. The
BCR components are weakly expressed on the surface of CLL
cells, one of the main characteristics of this malignant hemopathy.48,49 Aiolos is required for the pre-BCR control by regulating
␭5 expression at the transition from the pre-BI to pre-BII stage50
and for the inhibition of the threshold of BCR activation in part by
modulating tyrosine kinase Btk in murine model.7 Unlocking the
mechanism by which Aiolos affects gene expression could be
critical for improving antitumoral therapy. Moreover, a better
understanding of the molecular consequences of Aiolos overexpression can provide insight into the network of deregulated gene
expression and signaling pathways that contribute to CLL
pathogenesis.
Acknowledgments
This work was supported by Inserm and Spanish Ministry of
Science. K.B. and F.C. are supported by a doctoral fellowship from
the French Minister of Education. J.S. and M.-E.H. are supported
by CNRS and Institut Curie. V.B. is supported by Agence Nationale
pour la Recherche, Association pour la recherche sur le Cancer,
Belgian InterUniversity Attraction Pole, Cancéropole Ile-deFrance, Institut National du Cancer, and Université Paris Descartes.
Authorship
Contribution: K.B. designed the study, performed experiments,
analyzed data, and wrote the original draft of the manuscript; J.S.
performed the functional study on apoptosis by flow cytometry;
F.C. performed EMSA; I.A. contributed to the discussion; H.M.-B.
provided the clinical samples of CLL patients; H.M.B., M.-E.H.,
D.M., and V.B. contributed to scientific discussion and improving
the manuscript; and A.R. initiated the study, analyzed the results,
participated in the discussion, and improved the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Angelita Rebollo, Hôpital Pitié Salpêtrière,
Bâtiment CERVI, Inserm UMR-S 945, 83, bd de l’hôpital 75013
Paris, France; e-mail: [email protected].
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From www.bloodjournal.org by guest on September 19, 2016. For personal use only.
2011 117: 1917-1927
doi:10.1182/blood-2010-09-307140 originally published
online December 7, 2010
Deregulation of Aiolos expression in chronic lymphocytic leukemia is
associated with epigenetic modifications
Katy Billot, Jérémie Soeur, Fanny Chereau, Issam Arrouss, Hélène Merle-Béral, Meng-Er Huang,
Dominique Mazier, Véronique Baud and Angelita Rebollo
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