A distinct epigenetic signature at targets of a leukemia protein

  • Stefano Rossetti1,

    Affiliated with

    • André T Hoogeveen2,

      Affiliated with

      • Ping Liang1,

        Affiliated with

        • Cornel Stanciu1,

          Affiliated with

          • Peter van der Spek3 and

            Affiliated with

            • Nicoletta Sacchi1Email author

              Affiliated with

              BMC Genomics20078:38

              DOI: 10.1186/1471-2164-8-38

              Received: 04 October 2006

              Accepted: 01 February 2007

              Published: 01 February 2007

              Abstract

              Background

              Human myelogenous leukemia characterized by either the non random t(8; 21)(q22; q22) or t(16; 21)(q24; q22) chromosome translocations differ for both their biological and clinical features. Some of these features could be consequent to differential epigenetic transcriptional deregulation at AML1 targets imposed by AML1-MTG8 and AML1-MTG16, the fusion proteins deriving from the two translocations. Preliminary findings showing that these fusion proteins lead to transcriptional downregulation of AML1 targets, marked by repressive chromatin changes, would support this hypothesis. Here we show that combining conventional global gene expression arrays with the power of bioinformatic genomic survey of AML1-consensus sequences is an effective strategy to identify AML1 targets whose transcription is epigenetically downregulated by the leukemia-associated AML1-MTG16 protein.

              Results

              We interrogated mouse gene expression microarrays with probes generated either from 32D cells infected with a retroviral vector carrying AML1-MTG16 and unable of granulocyte differentiation and proliferation in response to the granulocyte colony stimulating factor (G-CSF), or from 32D cells infected with the cognate empty vector. From the analysis of differential gene expression alone (using as criteria a p value < 0.01 and an absolute fold change > 3), we were unable to conclude which of the 37 genes downregulated by AML1-MTG16 were, or not, direct AML1 targets. However, when we applied a bioinformatic approach to search for AML1-consensus sequences in the 10 Kb around the gene transcription start sites, we closed on 17 potential direct AML1 targets. By focusing on the most significantly downregulated genes, we found that both the AML1-consensus and the transcription start site chromatin regions were significantly marked by aberrant repressive histone tail changes. Further, the promoter of one of these genes, containing a CpG island, was aberrantly methylated.

              Conclusion

              This study shows that a leukemia-associated fusion protein can impose a distinct epigenetic repressive signature at specific sites in the genome. These findings strengthen the conclusion that leukemia-specific oncoproteins can induce non-random epigenetic changes.

              Background

              Nuclear hormone receptors and transcription factors can regulate the transcription of their target genes by inducing chromatin changes. Paradigmatic are the retinoic acid receptor alpha (RARα) and the transcription factor core binding factor (CBF), which regulate in this way the transcription of target genes involved in hematopoietic processes [1, 2]. Differently from RARα, which epigenetically activates its targets by recruiting coactivator protein complexes with histone acetyl transferase (HAT) activity only when bound to retinoic acid, CBF can directly recruit HAT-containing complexes to activate its targets [36]. One of the two CBF subunits, CBFα or AML1, can bind target genes endowed with the AML1-consensus sequence TG(T/C)GGT via its N-terminal DNA-binding domain [7]. AML1, encoding a master hematopoietic transcription factor, is frequently affected by different chromosome translocations in leukemic cells [8]. Moreover, AML1 haploinsufficiency was found to be associated with familial platelet disorder, a condition predisposing to acute myeloid leukemia [9].

              Two leukemia-associated chromosome translocations, the t(8;21)(q22;q22) and the t(16;21)(q24;q22), result in the fusion between the N-terminal region of AML1 and the C-terminal regions of two almost identical chromatin corepressors, MTG8 and MTG16, leading to the formation of AML1-MTG8 and AML1-MTG16, respectively [1013]. Upon fusion with either MTG8 or MTG16, AML1 is converted from a transcriptional activator into a transcriptional repressor of AML1-targets. Specific MTG domains in the wild type, as well as in the MTG fusion proteins, can interact, directly or via other corepressors such as NCoR and Sin3A, with histone deacetylases (HDACs), thus creating a repressive chromatin state at AML1 target sites (reviewed in [14, 15]). Repression at these sites is further enhanced by the formation of oligomers between the fusion proteins and wild-type MTG proteins [1618].

              Myeloid cell differentiation systems, such as the 32D mouse myeloid cell line, ectopically expressing either AML1-MTG8 or AML1-MTG16, were used as models to simulate some of the effects of these fusion proteins in myelogenesis and leukemogenesis. Both fusion proteins, when exogenously expressed in the 32D background, were shown to affect granulocytic differentiation and produce distinct effects on cell proliferation [1921]. In a preliminary study, we found that AML1-MTG16, when exogenously expressed in 32D cells, can induce aberrant myeloid phenotypes in association with repressive modifications at the chromatin of the Colony stimulating factor 1 receptor (Csf1r), an AML1-target gene encoding the macrophage colony stimulating factor receptor [19]. Based on this finding, we hypothesize that the comparative epigenetic analysis of the changes induced by different AML1-MTG fusion proteins in an identical cell context (e.g. the 32D context) might provide a lead to elucidating the differences observed in leukemic cells carrying either one of the two proteins [8]. The objective of this study was to demonstrate whether AML1-MTG16 induces epigenetic changes at AML1-target genes in the 32D myeloid cell genome. Only by coupling global gene expression array analysis with a bioinformatic genomic survey for the AML1-consensus sequence, we were able to close onto AML1-targets downregulated by AML1-MTG16. AML1-MTG16-induced transcriptional downregulation was marked by the acquisition of a distinct repressive chromatin signature.

              Results

              Global gene expression array analysis of AML1-MTG16-expressing cells

              To study the molecular and biological consequences of AML1-MTG16 expression in a myeloid differentiation cell model, we previously developed, by infecting 32D mouse myeloblasts with retroviral particles carrying either the pLNCX2 vector containing the AML1-MTG16 cDNA or the cognate empty vector, stable independent clones expressing AML1-MTG16 (hereafter called A16 clones) and stable independent control clones (hereafter called "mock" clones), respectively (Figure 1A). Upon treatment with granulocyte colony stimulating factor (G-CSF), A16 clones do not undergo granulocytic differentiation and proliferate significantly less than mock clones (Figure 1B). Global gene expression analysis (setting the p-value at < 0.05 and the absolute fold change at > 1.5) of a prototypic A16 clone and a prototypic mock clone grown either with interleukin 3 (IL-3) or G-CSF for 16 h, was combined with bioinformatic analysis of the proteins encoded by all the differentially expressed genes with the Ingenuity software (see Methods). This analysis clearly revealed a network comprising proteins critical for platelet function in A16 cells (see Additional file 1). The identification of this protein network strongly supports the biological data, indicating the occurrence of functional AML1 haploinsufficiency in A16 cells [9].
              http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-8-38/MediaObjects/12864_2006_Article_751_Fig1_HTML.jpg
              Figure 1

              Global gene expression analysis of AML1-MTG16-expressing cells. A. The 32D cell model, comprising clones expressing the AML1-MTG16 protein (A16 clones) and control clones ("mock" clones), which do not express the fusion protein. B. A16 clones, differently from mock clones, do not undergo granulocytic differentiation and display an impaired proliferation in the presence of G-CSF. C. Most of the genes whose expression is significantly affected in A16 cells were found previously implicated in biological processes.

              Further analysis of the gene expression data (setting the p-value at < 0.01 and the absolute fold change at > 3) enabled us to identify 138 differentially expressed genes, of which 66 differentially expressed genes in cells grown with IL-3, 67 differentially expressed genes in cells grown with G-CSF, and 5 differentially expressed genes in both cells grown with IL-3 and G-CSF (Figure 1C, left, and Table 1 and Table 2). According to the Ingenuity software, the differentially expressed genes in A16 cells were mostly implicated in tumorigenesis, cell proliferation, and hematopoiesis (Figure 1C, right). Since from this analysis alone we were unable to conclude whether, or not, these genes were AML1-MTG16 direct targets, we devised a bioinformatic approach aimed at identifying the AML1-consensus sequence in the 10 Kb region around the transcription start site of these genes.
              Table 1

              Selection of genes differentially expressed in AML1-MTG16-positive cells versus AML1-MTG16-negative cells grown in the presence of IL-3.

              Affymetrix ID

              NCBI acc. number

              Gene Symbol

              Gene Title

              GO/Ingenuity annotations

              Fold change

              1450042_at

              BB322201

              Arx

              aristaless related homeobox gene (Drosophila)

              regulation of transcription

              16.4

              1460300_a_at

              NM_008523

              Ltk

              leukocyte tyrosine kinase

              kinase signaling pathway

              15.9

              1423869_s_at

              AF349659

              Txnrd3

              thioredoxin reductase 3

              electron transport

              12.9

              1418796_at

              NM_009131

              Scgf

              stem cell growth factor

              cell adhesion/cell proliferation

              9.6

              1427329_a_at

              AI326478

              Igh-6

              immunoglobulin heavy chain 6 (heavy chain of IgM)

              immune response

              8.6

              1418588_at

              NM_009513

              Vmp

              vesicular membrain protein p24

              ---

              7.4

              1450652_at

              NM_007802

              Ctsk

              cathepsin K

              proteolysis

              7.2

              1428439_at

              BG066220

              Nyren18-pending

              NY-REN-18 antigen

              ---

              6.4

              1419416_a_at

              NM_011244

              Rarg

              retinoic acid receptor, gamma

              regulation of transcription

              6.3

              1426800_at

              BM214169

              D330025I23Rik (Cbfb)

              RIKEN cDNA D330025I23 gene (core-binding factor beta subunit)

              regulation of transcription

              6.2

              1419136_at

              NM_134066

              Akr1c18

              aldo-keto reductase family 1, member C18

              electron transport

              6.1

              1425432_at

              AF260307

              Oprm

              opioid receptor, mu

              G-protein signaling pathway

              6.0

              1418346_at

              NM_013754

              Insl6

              insulin-like 6

              physiological processes

              6.0

              1449426_a_at

              NM_011922

              Anxa10

              annexin A10

              ---

              5.9

              1423029_at

              NM_008236

              Hes2

              hairy and enhancer of split 2 (Drosophila)

              regulation of transcription

              5.8

              1454007_a_at

              AK020384

              Zfp142

              zinc finger protein 142

              electron transport

              5.8

              1423313_at

              BG070255

              Pde7a

              phosphodiesterase 7A

              signal transduction

              5.8

              1451915_at

              L20509

              Cct3

              chaperonin subunit 3 (gamma)

              protein folding

              5.7

              1452487_x_at

              BB133664

              Pirb

              paired-Ig-like receptor B

              ---

              5.7

              1422030_at

              AF326316

              Atp6v0a4

              ATPase, H+ transporting, lysosomal V0 subunit A isoform 4

              ATP hydrolysis/proton transport

              5.6

              1427753_at

              Z95479

              Igh-4

              immunoglobulin heavy chain 4 (serum IgG1)

              immune response

              5.5

              1437235_x_at

              BB218844

              Lpp

              LIM domain containing preferred translocation partner in lipoma

              cytoskeleton organization/transcriptional regulation

              5.4

              1426938_at

              BB627486

              Nova1

              neuro-oncological ventral antigen 1

              mRNA splicing

              5.0

              1460416_s_at

              M55219

              Csprs

              component of Sp100-rs

              G-protein signaling pathway

              4.9

              1427884_at

              AW550625

              Col3a1

              procollagen, type III, alpha 1

              cell adhesion

              4.9

              1450453_a_at

              NM_012065

              Pde6g

              phosphodiesterase 6G, cGMP-specific, rod, gamma

              vision

              4.8

              1455957_x_at

              AV034167

              Ceacam11

              CEA-related cell adhesion molecule 11

              ---

              4.7

              1450215_at

              NM_009038

              Rcvrn

              recoverin

              vision

              4.7

              1452489_at

              BC016258

              Vps11

              vacuolar protein sorting 11 (yeast)

              protein transport

              4.4

              1421705_at

              NM_018732

              Scn3a

              sodium channel, voltage-gated, type III, alpha polypeptide

              ion transport

              4.4

              1421375_a_at

              NM_011313

              S100a6

              S100 calcium binding protein A6 (calcyclin)

              cell proliferation

              4.4

              1433658_x_at

              AV300794

              Pcbp4

              poly(rC) binding protein 4

              apoptosis

              4.2

              1418136_at

              NM_009365

              Tgfb1i1

              transforming growth factor beta 1 induced transcript 1

              regulation of transcription

              4.2

              1450629_at

              AV114522

              Eplin-pending

              epithelial protein lost in neoplasm

              ---

              3.9

              1455421_x_at

              AW490145

              Clcn1

              chloride channel 1

              ion transport

              3.7

              1418451_at

              BB522409

              Gng2

              guanine nucleotide binding protein (G protein), gamma 2 subunit

              G-protein signaling pathway

              3.7

              1450709_at

              NM_007851

              Defcr5

              defensin related cryptdin 5

              defense response

              3.5

              1423561_at

              AI838010

              Nell2

              nel-like 2 homolog (chicken)

              cell adhesion

              3.4

              1452279_at

              BB800282

              Pfc

              properdin factor, complement

              complement activation

              3.4

              1424531_a_at

              BC010807

              Tcea3

              transcription elongation factor A (SII), 3

              regulation of transcription

              3.4

              1419325_at

              NM_019515

              Nmu

              neuromedin

              neuropeptide signaling pathway

              3.4

              1422945_a_at

              AI844677

              Kif5c

              kinesin family member 5C

              protein transport

              3.3

              1460280_at

              NM_010815

              Mona

              monocytic adaptor

              intracellular signaling cascade

              3.3

              1448529_at

              NM_009378

              Thbd

              thrombomodulin

              blood coagulation

              3.2

              1449830_at

              NM_013766

              Prlpi

              prolactin-like protein I

              ---

              3.2

              1423596_at

              BB528391

              Nek6

              NIMA (never in mitosis gene a)-related expressed kinase 6

              kinase signaling pathway/cell proliferation

              3.2

              1450435_at

              NM_008478

              Slc7a2

              solute carrier family 7 (cationic amino acid transporter, y+ system), member 2

              amino acid transport

              3.2

              1420373_at

              BI249549

              Foxj2

              forkhead box J2

              regulation of transcription

              3.1

              1436769_at

              AV101011

              Psma1

              proteasome (prosome, macropain) subunit, alpha type 1

              ubiquitin-dependent protein catabolism

              3.1

              1421778_at

              NM_011911

              V1rb2

              vomeronasal 1, receptor B2

              chemosensory perception/G-protein signaling pathway

              3.0

              1448416_at

              NM_008597

              Mglap

              matrix gamma-carboxyglutamate (gla) protein

              ---

              -3.0

              1419012_at

              NM_011766

              Zfpm2

              zinc finger protein, multitype 2

              regulation of transcription

              -3.0

              1449833_at

              NM_011472

              Sprr2f

              small proline-rich protein 2F

              ---

              -3.1

              1424814_a_at

              BC025541

              9030625M01Rik (Bclg)

              RIKEN cDNA 9030625M01 gene (apoptosis regulator Bclg)

              apoptosis

              -3.1

              1417338_at

              U03487

              Epb4.2

              erythrocyte protein band 4.2

              structural function

              -3.3

              1448152_at

              NM_010514

              Igf2

              insulin-like growth factor 2

              cell proliferation

              -3.6

              1429947_a_at

              AK008179

              Zbp1

              Z-DNA binding protein 1

              ---

              -3.7

              1420394_s_at

              U05264

              Gp49b

              glycoprotein 49 B

              immune response?

              -3.7

              1424898_at

              BC021154

              Slc10a1

              solute carrier family 10 (sodium/bile acid cotransporter family), member 1

              ion transport

              -3.8

              1416822_at

              BC013711

              Es2el

              expressed sequence 2 embryonic lethal

              ---

              -4.0

              1420779_at

              NM_010213

              Fhl3

              four and a half LIM domains 3

              cytoskeleton organization

              -4.3

              1419124_at

              NM_133829

              AW212394

              expressed sequence AW212394

              ---

              -4.4

              1425597_a_at

              AW060288

              Qk

              quaking

              apoptosis

              -4.6

              1422416_s_at

              NM_016983

              Vpreb2

              Pre-B lymphocyte gene 2

              hematopoiesis

              -4.7

              1425863_a_at

              AF295638

              Ptpro

              protein tyrosine phosphatase, receptor type, O

              phosphatase signaling pathway

              -4.8

              1418177_at

              AF233778

              Gabrg2

              gamma-aminobutyric acid (GABA-A) receptor, subunit gamma 2

              synaptic transmission

              -4.8

              1421309_at

              NM_008598

              Mgmt

              O-6-methylguanine-DNA methyltransferase

              DNA repair

              -8.2

              1421288_at

              NM_007975

              F2rl3

              coagulation factor II (thrombin) receptor-like 3

              blood coagulation/G-protein signaling pathway

              -14.2

              1449347_a_at

              NM_021365

              Xlr4

              X-linked lymphocyte-regulated 4

              chromatin remodeling?

              -16.9

              1448511_at

              NM_016933

              Ptprcap

              protein tyrosine phosphatase, receptor type, C polypeptide-associated protein

              phosphatase signaling pathway

              -17.7

              1421775_at

              NM_010184

              Fcer1a

              Fc receptor, IgE, high affinity I, alpha polypeptide

              signal transduction

              -27.2

              Limits: p-value < 0.01; absolute fold change > 3.

              In bold are the AML1-MTG16-downregulated genes searched for AML1-consensus motifs.

              Table 2

              Selection of genes differentially expressed in AML1-MTG16-positive cells versus AML1-MTG16-negative cells grown in the presence of G-CSF for 16 h.

              Affymetrix ID

              NCBI acc. number

              Gene Symbol

              Gene Title

              GO/Iingenuity annotations

              Fold change

              1437100_x_at

              BB206220

              Pim3

              proviral integration site 3

              kinase signaling pathway

              24.5

              1460300_a_at

              NM_008523

              Ltk

              leukocyte tyrosine kinase

              kinase signaling pathway

              19.9

              1416257_at

              NM_009794

              Capn2

              calpain 2

              proteolysis/cell migration

              17.7

              1417314_at

              NM_008198

              H2-Bf

              histocompatibility 2, complement component factor B

              cell proliferation/complement activation

              14.7

              1425380_at

              AF331457

              Rasgrp4

              RAS guanyl releasing protein 4

              intracellular signaling cascade

              10.4

              1450322_s_at

              NM_011409

              Slfn3

              schlafen 3

              cell proliferation

              10.2

              1421793_at

              NM_010198

              Fgf11

              fibroblast growth factor 11

              signal transduction/cell proliferation

              9.5

              1420348_at

              NM_008499

              Lhx5

              LIM homeobox protein 5

              regulation of transcription

              8.8

              1419605_at

              NM_010796

              Mgl1

              macrophage galactose N-acetyl-galactosamine specific lectin 1

              cell adhesion

              8.6

              1420360_at

              NM_010051

              Dkk1

              dickkopf homolog 1 (Xenopus laevis)

              signal transduction/apoptosis

              6.7

              1425647_at

              BG069740

              Rnf33

              ring finger protein 33

              ---

              6.4

              1434851_s_at

              AU015319

              Crb3

              crumbs homolog 3 (Drosophila)

              intercellular junction assembly

              6.1

              1427102_at

              AF099975

              Slfn4

              schlafen 4

              cell proliferation

              5.9

              1437218_at

              BM234360

              Fn1

              fibronectin 1

              cell adhesion

              5.5

              1417777_at

              BC014865

              Ltb4dh

              leukotriene B4 12-hydroxydehydrogenase

              metabolism

              5.5

              1419406_a_at

              NM_016707

              Bcl11a

              B-cell CLL/lymphoma 11A (zinc finger protein)

              T/B-cell differentiation/corepressor

              5.5

              1418358_at

              NM_008574

              Mcsp

              mitochondrial capsule selenoprotein

              sperm motility

              5.4

              1450499_at

              NM_009124

              Sca1

              spinocerebellar ataxia 1 homolog (human)

              ---

              5.2

              1418257_at

              BB732135

              Slc12a7

              solute carrier family 12, member 7

              ion transport

              5.1

              1424744_at

              BC021950

              Sds

              serine dehydratase

              amino acid metabolism

              5.1

              1456305_x_at

              BB702568

              Obox1

              oocyte specific homeobox 1

              regulation of transcription

              5.0

              1449707_at

              C80272

              Nr5a2

              nuclear receptor subfamily 5, group A, member 2

              regulation of transcription

              4.9

              1421504_at

              NM_009239

              Sp4

              trans-acting transcription factor 4

              regulation of transcription

              4.8

              1427079_at

              U51204

              Mapre3

              microtubule-associated protein, RP/EB family, member 3

              cytoskeleton organization

              4.8

              1429626_at

              AV024301

              Sftpa

              surfactant associated protein A

              cell adhesion

              4.8

              1452793_at

              AI509011

              Cldn10

              claudin 10

              cell adhesion

              4.7

              1419507_at

              NM_013713

              Krtap15

              keratin associated protein 15

              ---

              4.7

              1421375_a_at

              NM_011313

              S100a6

              S100 calcium binding protein A6 (calcyclin)

              cell proliferation

              4.5

              1419517_at

              NM_028408

              2900075G08Rik

              RIKEN cDNA 2900075G08 gene

              intracellular signaling cascade

              4.4

              1454736_at

              BM119297

              4921515A04Rik

              RIKEN cDNA 4921515A04 gene

              regulation of transcription

              4.3

              1436244_a_at

              AU067681

              Tle2

              transducin-like enhancer of split 2, homolog of Drosophila E(spl)

              regulation of transcription/signal transduction

              4.2

              1420594_at

              NM_007525

              Bard1

              BRCA1 associated RING domain 1

              DNA repair/regulation of transcription/apoptosis

              4.2

              1426093_at

              AF220141

              Trim34

              tripartite motif protein 34

              ---

              4.2

              1424748_at

              BC021504

              Galnt11

              UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 11

              metabolism

              4.1

              1416855_at

              BB550400

              Gas1

              growth arrest specific 1

              cell cycle arrest///programmed cell death

              4.0

              1422310_at

              NM_009223

              Snn

              stannin

              ---

              4.0

              1452463_x_at

              BG966217

              Igk-V8

              immunoglobulin kappa chain variable 8 (V8)

              immune response

              4.0

              1450415_at

              NM_008805

              Pde6a

              phosphodiesterase 6A, cGMP-specific, rod, alpha

              signal transduction

              3.9

              1418792_at

              AF326561

              Sh3gl2

              SH3-domain GRB2-like 2

              ---

              3.9

              1451759_at

              BC013893

              Masp2

              mannan-binding lectin serine protease 2

              cell adhesion/complement activation

              3.9

              1418921_at

              AY059393

              Necl1-pending

              nectin-lke 1

              cell adhesion

              3.9

              1416188_at

              BC004651

              Gm2a

              GM2 ganglioside activator protein

              sphingolipid metabolism

              3.8

              1448392_at

              NM_009242

              Sparc

              secreted acidic cysteine rich glycoprotein

              cell proliferation

              3.8

              1419485_at

              BB759833

              Foxc1

              forkhead box C1

              regulation of transcription

              3.7

              1419602_at

              NM_010451

              Hoxa2

              homeo box A2

              regulation of transcription

              3.7

              1427358_a_at

              BC026671

              Dapk1

              death associated protein kinase 1

              apoptosis

              3.6

              1450827_at

              NM_024245

              Kif23

              kinesin family member 23

              mitosis

              3.6

              1421280_at

              Z36357

              Gabra1

              gamma-aminobutyric acid (GABA-A) receptor, subunit alpha 1

              synaptic transmission

              3.5

              1452279_at

              BB800282

              Pfc

              properdin factor, complement

              complement activation

              3.5

              1415854_at

              BB815530

              Kitl

              kit ligand

              cell proliferation/cell adhesion

              3.4

              1417513_at

              AI255184

              Evi5

              ecotropic viral integration site 5

              ---

              3.3

              1431379_a_at

              AK005153

              Slc13a1

              solute carrier family 13 (sodium/sulphate symporters), member 1

              ion transport

              3.2

              1418476_at

              NM_018827

              Crlf1

              cytokine receptor-like factor 1

              ---

              3.2

              1416009_at

              NM_019793

              Tm4sf8-pending

              transmembrane 4 superfamily member 8

              signal transduction/cell proliferation

              3.1

              1451633_a_at

              BC025929

              Gng1

              guanine nucleotide binding protein (G protein), gamma 1 subunit

              G-protein signaling pathway

              -3.0

              1425978_at

              AF384055

              Srfcp-pending

              SRF co-factor protein (cardiac and smooth muscle)

              regulation of transcription/positive regulation of cell proliferation

              -3.0

              1425153_at

              BC008538

              Myh2

              myosin, heavy polypeptide 2, skeletal muscle, adult

              cytoskeleton organization

              -3.1

              1448755_at

              AF011450

              Col15a1

              procollagen, type XV

              cell adhesion

              -3.2

              1433888_at

              AV343478

              Atp2b2

              ATPase, Ca++ transporting, plasma membrane 2

              metabolism

              -3.5

              1426868_x_at

              AK003174

              Lmna

              lamin A

              cell morphology

              -3.5

              1423292_a_at

              BG072867

              Prx

              periaxin

              intracellular signaling cascade

              -3.6

              1449891_a_at

              NM_028523

              Esdn-pending

              endothelial and smooth muscle cell-derived neuropilin-like molecule

              ---

              -3.6

              1425708_at

              AF285585

              Rnf17

              ring finger protein 17

              ---

              -4.2

              1449836_x_at

              NM_007546

              Biklk

              Bcl2-interacting killer-like

              apoptosis

              -4.6

              1448710_at

              D87747

              Cxcr4

              chemokine (C-X-C motif) receptor 4

              defense response/hematopoiesis

              -4.8

              1419227_at

              NM_009839

              Cct6b

              chaperonin subunit 6b (zeta)

              protein folding

              -5.0

              1455853_x_at

              BB768303

              2700085A14Rik (Sas)

              RIKEN cDNA 2700085A14 gene (Sarcoma amplified sequence)

              cell proliferation/signal transduction

              -5.3

              1416822_at

              BC013711

              Es2el

              expressed sequence 2 embryonic lethal

              ---

              -5.4

              1422473_at

              BM246564

              Pde4b

              phosphodiesterase 4B, cAMP specific

              signal transduction

              -7.8

              1418499_a_at

              NM_020574

              Kcne3

              potassium voltage-gated channel, Isk-related subfamily, gene 3

              ion transport

              -8.3

              1419537_at

              NM_031198

              Tcfec

              transcription factor EC

              regulation of transcription

              -20.6

              1449347_a_at

              NM_021365

              Xlr4

              X-linked lymphocyte-regulated 4

              chromatin remodeling?

              -34.4

              Limits: p-value < 0.01; absolute fold change > 3.

              In bold are the AML1-MTG16-downregulated genes searched for AML1-consensus motifs.

              Identification of genes containing the AML1-consensus sequence by bioinformatic analysis

              Since the AML1-MTG proteins have a transcriptionally repressive function (reviewed in [14]), we focused our bioinformatic analysis on the 37 genes downregulated by AML1-MTG16 (see genes in bold in Table 1 and Table 2). Specifically, we searched the 10 Kb around the transcription start site of each gene for either the AML1-binding consensus sequence TG(T/C)GGT or, this sequence in reverse orientation, ACC(G/A)CA. With the MEME software (see Methods) we identified a conserved motif, hereafter called AML1-consensus motif (Figure 2A), encompassing the AML1-consensus sequence in seventeen out of the 37 genes (Figure 2B and Table 3). We focused on five of these genes, Fcer1a, Tcfec, Ptprcap, F2rl3, and Mgmt (Figure 2B, right), because they were among the most significantly downregulated genes. Fcer1a, Tcfec, Ptprcap, F2rl3, and Mgmt encode for known proteins. Specifically, Fcer1a is the Fc fragment of IgE and is involved in the immune response [22]; Tcfec is a transcription factor that induces, among other genes, the G-CSF receptor gene [23, 24]; Ptprcap is a transmembrane protein associated with CD45, a key regulator of lymphocytes activation [25]; F2rl3 is a member of G protein-coupled protease-activated receptors (PARs) of the coagulation factor II (thrombin) and plays an important role in platelet activation [26]; Mgmt is a DNA repair enzyme that is frequently lost in cancer due to epigenetic silencing [27]. Downregulation of these genes was confirmed by real time RT-PCR (Figure 2C).
              http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-8-38/MediaObjects/12864_2006_Article_751_Fig2_HTML.jpg
              Figure 2

              AML1-MTG16-induced downregulation of putative AML1-targets. A. The AML1-consensus motif, containing the AML1-consensus sequence (framed), found by bioinformatic analysis of the genes significantly downregulated in A16 cells. The height of the columns associated with each nucleotide is proportional to the conservation level. The "logo" representation of the motif instead indicates in which proportion the single bases are present at each position. B. Seventeen out of the 37 downregulated genes are putative AML1-targets. The fold-changes of five of the most significantly downregulated genes are reported at right. C. Real time RT-PCR confirmed the significant (p < 0.01) downregulation of the five genes.

              Table 3

              Selection of putative AML1-target genes downregulated in AML1-MTG16-positive cells versus AML1-MTG16-negative cells.

              Affymetrix ID

              NCBI acc. number

              Gene Symbol

              Gene Title

              GO/Ingenuity annotations

              Fold change IL-3

              Fold change G-CSF

              1417338_at

              U03487

              Epb4.2

              erythrocyte protein band 4.2

              structural function

              -3.3

              ---

              1433888_at

              AV343478

              Atp2b2

              ATPase, Ca++ transporting, plasma membrane 2

              metabolism

              ---

              -3.5

              1426868_x_at

              AK003174

              Lmna

              lamin A

              cell morphology

              ---

              -3.5

              1423292_a_at

              BG072867

              Prx

              periaxin

              intracellular signaling cascade

              ---

              -3.6

              1449891_a_at

              NM_028523

              Esdn-pending

              endothelial and smooth muscle cell-derived neuropilin-like molecule

              ---

              ---

              -3.6

              1425708_at

              AF285585

              Rnf17

              ring finger protein 17

              ---

              ---

              -4.2

              1419124_at

              NM_133829

              AW212394

              expressed sequence AW212394

              ---

              -4.4

              ---

              1425597_a_at

              AW060288

              Qk

              quaking

              apoptosis

              -4.6

              ---

              1419227_at

              NM_009839

              Cct6b

              chaperonin subunit 6b (zeta)

              protein folding

              ---

              -5.0

              1455853_x_at

              BB768303

              2700085A14Rik (Sas)

              RIKEN cDNA 2700085A14 gene (Sarcoma amplified sequence)

              cell proliferation/signal transduction

              ---

              -5.3

              1422473_at

              BM246564

              Pde4b

              phosphodiesterase 4B, cAMP specific

              signal transduction

              ---

              -7.8

              1421309_at

              NM_008598

              Mgmt

              O-6-methylguanine-DNA methyltransferase

              DNA repair

              -8.2

              ---

              1421288_at

              NM_007975

              F2rl3

              coagulation factor II (thrombin) receptor-like 3

              blood coagulation/G-protein signaling pathway

              -14.2

              ---

              1449347_a_at

              NM_021365

              Xlr4

              X-linked lymphocyte-regulated 4

              chromatin remodelling?

              -16.9

              -34.4

              1448511_at

              NM_016933

              Ptprcap (1)

              protein tyrosine phosphatase, receptor type, C polypeptide-associated protein

              phosphatase signaling pathway

              -17.7

              ---

              1419537_at

              NM_031198

              Tcfec

              transcription factor EC

              regulation of transcription

              ---

              -20.6

              1421775_at

              NM_010184

              Fcer1a

              Fc receptor, IgE, high affinity I, alpha polypeptide

              signal transduction

              -27.2

              ---

              Motif conservation significance: p < 10E-5.

              (1) The Ptprcap AML1-consensus motif is located in an intron of a 5' adjacent gene (Coro1b).

              Fcer1a, Tcfec, Ptprcap, F2rl3, and Mgmt are direct AML1-MTG16 targets

              Quantitative chromatin immunoprecipitation (ChIP) with an anti-AML1 specific antibody, but not with an anti-MTG16 antibody (data not shown), showed significant (p < 0.05) enrichment of the region encompassing the AML1-consensus motif (see bars in figure 3A, left) relative to an arbitrary control region without the AML1-consensus motif in the mock clone chromatin for all five genes, indicating endogenous AML1 binding at these regions (Figure 3B). ChIP with an anti-MTG16 antibody showed instead a significant enrichment of exogenous AML1-MTG16 in the same chromatin regions in the A16 clones (Figure 3B). The human homologues of these genes also contain an AML1-consensus sequence(s) in the 10Kb region surrounding the transcription start site, pointing to these five genes as novel, bona fide direct AML1-targets genes.
              http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-8-38/MediaObjects/12864_2006_Article_751_Fig3_HTML.jpg
              Figure 3

              AML1-target gene validation. A. Relative position of the AML1-consensus motifs (left) and their sequence (right) in the five putative AML1-target genes that were analyzed by ChIP. B. Quantitative ChIP analysis with antibodies either against AML1 or MTG16 showing a significant (p < 0.05) enrichment of chromatin containing AML1-consensus motifs vs. chromatin containing a control region in AML1-MTG16-negative and AML1-MTG16-positive cells, respectively.

              Repressive chromatin changes at AML1-MTG16-downregulated targets

              We previously demonstrated that AML1-MTG16 interacts with both HDAC1 and HDAC3 [28]. Further, we found that AML1-MTG16 can induce downregulation marked by repressive histone hypoacetylation at the Csf1r chromatin [19]. Here we show that, in A16 cells, the chromatin associated with both the region containing the AML1-consensus motif and the region encompassing the transcription start site of Fcer1a, Tcfec, Ptprcap, F2rl3, and Mgmt (Figure 3A) displays a significant (p < 0.05) decrease of acetylated histone H4 (Ac-H4), and a significant (p < 0.05) increase of H3K9 tri-methylation (Tri-Met-H3-K9) (Figure 4A), supporting the acquisition of a repressive chromatin state [2931].
              http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-8-38/MediaObjects/12864_2006_Article_751_Fig4_HTML.jpg
              Figure 4

              Repressive epigenetic changes at the AML1-targets. A. ChIP with antibodies against either acetylated histone H4 or tri-methylated histone H3 Lysine 9 (tri-Met-H3-K9) followed by quantitative PCR with primers amplifying a region encompassing either the transcription start site (+1) or the AML1-consensus detected a different level of repressive histone changes in all five genes in A16 cells. B. In silico analysis identified a CpG island only in the Mgmt promoter. This CpG island is hypermethylated in A16 cells (bottom, right).

              Repressive histone modifications are often associated with aberrant hypermethylation at CpG islands present in the 5' regulatory regions of many genes [32, 33] and references within). By using the CpG island searcher [34], a software for the identifying CpG islands, we could identify a CpG island only in the Mgmt promoter region [35] (Figure 4B). Bisulfite sequencing analysis of this region detected hypermethylation in AML1-MTG16-positive cells (Figure 4B).

              The overall epigenetic analysis indicates that downregulation of AML1-targets by AML1-MTG16 can be achieved, even in the absence of DNA methylation, when there is a critical quantitative level of repressive histone changes.

              Discussion

              In this study we show the effectiveness of integrating global gene expression array analysis with a bioinformatic approach aimed at detecting AML1-consensus sequences for identifying novel putative direct AML1-targets downregulated by AML1-MTG16 in 32D cells. Downregulation of these genes is marked by a distinct repressive chromatin profile.

              When we surveyed the 37 most significantly downregulated genes for the presence of the AML1-consensus motif(s) in the 10 Kb region encompassing the transcription start site, we closed on seventeen putative direct AML1-MTG16 targets. For five of these genes, Fcer1a, Tcfec, Ptprcap, F2rl3 and Mgmt, which were among the most significantly downregulated, we were able to demonstrate, using ChIP analysis, the binding of both AML1 and AML1-MTG16 to the gene regions containing the AML1-motifs. Thus, our two-tier approach, combining gene expression array analysis with bioinformatic survey for transcription factor-consensus sequences, seems to be a powerful strategy for identifying transcription factor targets, which would otherwise be missed when using conventional gene expression array analysis alone.

              The chromatin of the five downregulated genes, Fcer1a, Tcfec, Ptprcap, F2rl3, and Mgmt, was marked not only by significant levels of histone H4 hypoacetylation, but also by significant levels of repressive histone H3-K9 trimethylation, suggesting that AML1-MTG16 might induce the recruitment of both histone deacetylases [28] and histone methyltransferases. Apparently, a critical quantity of repressive histone modifications, even in the absence of CpG methylation, might per se be sufficient to "lock in" a transcriptionally downregulated state. In the case of Mgmt, which has a CpG island, it is instead possible that the accumulation of histone repressive changes preceded CpG hypermethylation [[36], and references within].

              It is noteworthy that all the genes for which we demonstrated AML1-MTG16-induced epigenetic downregulation encode for functions relevant to either hematopoiesis and/or leukemogenesis. We would like to underline that downregulation of two of the genes that we identified might be relevant to AML1-MTG16-induced leukemogenesis. One of these genes is Tcfec, whose human counterpart encodes a transcription factor that induces the granulocyte colony stimulating factor receptor G-CSFR [23, 24]. Remarkably, Tcfec downregulation in A16 cells is paralleled by a significant downregulation of G-csfr (data not shown), indicating that AML1-MTG16 might have triggered a coordinated cascade of transcriptional downregulation, as we observed in other differentiation model systems [37, 38]. The second gene is Mgmt, encoding the DNA repair enzyme O6-Methylguanine-DNA-methyltransferase, which is frequently silenced and hypermethylated in leukemia [39]. MGMT epigenetic silencing is thought to lead to random mutations in cancer [40]. A recent study has shown that expression of different acute myeloid leukemia fusion proteins, including AML1-MTG8, leads to downregulation of several DNA repair genes [41]. Thus, the induction of a "mutator phenotype" might be a common consequence of leukemia fusion protein expression.

              A few global gene expression studies on cells expressing exogenous AML1-MTG8 have been recently described [4244]. Given the use of different cell systems, it is difficult to compare the differentially expressed genes in AML1-MTG16-positive 32D cells with the differentially expressed genes reported for AML1-MTG8. Nevertheless, we could identify a few gene families (e.g. S100 Calcium-binding proteins) that are similarly affected by both AML1-MTG8 and AML1-MTG16 even in different cell contexts. Extending our study to the comparison of the epigenetic signatures imposed by either exogenous AML1-MTG16 or exogenous AML1-MTG8 in the very same cell context (e.g. 32D cells) might enable us to narrow down additional critical epigenetic signatures consequent to t(8;21) and t(16;21) translocations.

              Conclusion

              In this study, we show that AML1-MTG16, the leukemia fusion protein associated with the non-random chromosome translocation t(16;21)(q24;q22), can impose transcriptional downregulation marked by a distinct epigenetic signature at specific AML1-target sites in the genome. Thus, our findings further support the hypothesis that non-random genetic abnormalities can lead to non-random epigenetic changes in leukemia cells [19, 45].

              Methods

              Cell cultures

              Stable clones obtained from mouse myeloid 32D cells infected either with pLNCX2-AML1-MTG16 (A16 clones) or the empty vector pLNCX2 (mock clones) were previously described [19]. Two prototypic A16 clones and two prototypic mock clones were used in this study. Cells were maintained in the presence of 10 ng/ml of murine IL-3 (BD Biosciences, San Jose, CA, USA) in RPMI 1640 medium supplemented with 10% fetal calf serum, 1% antibiotics (penicillin/streptomycin), adjusting the cell density to 2 × 105 cells/ml daily. To induce granulocyte differentiation, cells were washed in RPMI medium, and IL-3 was replaced with 10 ng/ml human G-CSF (Amgen, Thousand Oaks, CA, USA). Differentiation was microscopically evaluated on cytospin preparations stained with May-Grünwald-Giemsa.

              RNA extraction and microarray hybridization

              Total RNA was extracted with RNeasy mini kit (Qiagen, Hilden, Germany) and treated with DNase (Qiagen). Double stranded cDNA was generated from 5 μg RNA using Superscript ds cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA) and T7-oligo(dT) primers. The cDNA was purified with GeneChip Sample Cleanup Module (Affymetrix, Santa Clara, CA, USA) and used to synthesize biotin-labeled cRNA with Enzo RNA transcript Labeling Kit (Enzo Life Science, Farmingdale, NY, USA). Purified cRNA was quantified by spectrophotometric methods and the concentration was adjusted in order to exclude the carryover of unlabeled RNA. 11 μg of cRNA were then fragmented in fragmentation buffer (Affymetrix) at 95°C for 35 minutes and hybridized for 16 h at 45°C onto MOE430A microarrays (Affymetrix). After washing and staining, the chips were scanned in a Hewett-Packard/Affymetrix scanner at 570 nm. For all the samples the 5'/3' ratios of Gapdh were 0.7 - 0.9. In comparative experiments the scaling factor, noise and presence calls were similar. Gene expression data represent the average of two independent experiments.

              Microarray data analysis

              The arrays were normalized by geometric mean intensity for each probe set and scaled using log2 transformation for further analysis. Comparison between the A16 and mock clones grown with either IL-3 or G-CSF was done using Spotfire Decision Site. This comparison generated a p-value from a t-test to statistically extract significant changes in mRNA expression levels between the groups. p-values < 0.05 were considered significant. The null hypothesis is that the samples between the groups are derived from the same population i.e. there is no significant differential expression. The t-test looks at the variance within the groups as well as between them. To be considered significantly differentially expressed the variance had to be greater between than within the groups to a level of p < 0.05. Ratios were generated by dividing the average of the unlogged control data by the average of the unlogged AML1-MTG16 data. Ratios were then portrayed as positive or negative fold change between A16 and mock. To confirm statistical significance of these ratios the differentially expressed genes had to satisfy an arbitrary cut-off ratio as well as having a p-value < 0.05 (see Results section). Analysis of the protein networks was performed by using Ingenuity Pathways Analysis (Ingenuity Systems, Redwood City, CA), software able to identify molecular networks based on known functional or physical interactions among the proteins encoded by the differentially expressed genes.

              Search of AML1-consensus sequence in differentially expressed genes

              The well-annotated genes differentially expressed in the A16 clone versus the mock clone either in the presence of IL-3 or G-CSF (p < 0.01 and absolute fold change >3) were searched for the AML1-consensus sequence "5'-TG(T/C)GGT-3"' in the 10 kb region surrounding the transcription initiation sites (from -5000 bp to +5000 bp) using an in-house built PERL script. A 400 bp sequence flanking the potential AML1-binding sites (200 bp on each side) was extracted and analyzed with MEME, which is a software package to discover motifs in groups of related DNA sequences [46], and with multiple sequence alignment to test whether additional conserved motifs in the surrounding regions could be identified and to assess the sequence conservation extending the potential AML1-binding sites.

              Real-time RT-PCR

              Total RNA was obtained using Trizol (Invitrogen), treated with DNase I (Ambion, Austin, TX, USA), retrotranscribed with SuperScript™ First-Strand Synthesis System (Invitrogen) and amplified by Real-time RT-PCR on an iCycler (Bio-Rad, Hercules, CA, USA) by using iQ SYBR Green Supermix (Bio-Rad) and primers specific for γ actin, F2rl3, Fcer1a, Ptprcap, Tcfec, and Mgmt (Table 4). Transcript levels of the genes of interest were quantitated by the Delta-delta Ct method, using the house keeping gene γ-actin for normalization. The amplification efficiency, evaluated from the sample slopes, was similar for all the samples analyzed in the same experiment. Two independent experiments were performed in triplicate using two mock clones and two A16 clones. Significance was determined by using the Student t-test.
              Table 4

              Primers used for real time RT-PCR, quantitative ChIP, and bisulfite sequencing.

              Primer name

              Orientation

              Sequence

              Real time PCR primers

                

              γ-Actin

              sense

              5'-GCCGGCTTACACTGCGCTTCTT-3'

               

              antisense

              5'-TTCTGGCCCATGCCCACCAT-3'

              F2rl3

              sense

              5'-GCTTCTGATCCTGGCAGCATG-3'

               

              antisense

              5'-GTGTCACTGTCGTTGGCACAG-3'

              Fcer1a

              sense

              5'-CCCTTTCCTGCTATGGGAACA-3'

               

              antisense

              5'-GCAGCCAATCTTGCGTTACATT-3'

              Ptprcap

              sense

              5'-GGATGAAGAGGATGCAGAAGAT-3'

               

              antisense

              5'-CTGACTCCTATAGTGCAGTGAC-3'

              Tcfec

              sense

              5'-AGTCTAATGATCCTGATATGCGC-3'

               

              antisense

              5'-TCCTGAATCCGGAGCCTAAGC-3'

              Mgmt

              sense

              5'-GAACTTGGCAGAATGGCTGAG-3'

               

              antisense

              5'-GGTGATGGAGAGCAGGCAA-3'

              ChIP primers

                

              Ptprcap- AML1-consensus

              sense

              5'-GTCCTGCAGCTGGTGTTTACAG-3'

               

              antisense

              5'-CTGGTCTCTGAGTGGCTGCA-3'

              Ptprcap-transcription start

              sense

              5'-GAGGTCTGACAAGTTAGCTGTA-3'

               

              antisense

              5'-ACCCTGTAACTCACTTCTCACT-3'

              Tcfec- AML1-consensus

              sense

              5'AGAGCTTGACTAGAATGGATTT-3'

               

              antisense

              5'-GGTGCAACCCATTCATGGCTT-3'

              Tcfec-transcription start

              sense

              5'-AGTCACACCACTGGAGTAGTTTT-3'

               

              antisense

              5'-CCCTCGTCTCATAACCTAAGCA-3'

              Fcer1a- AML1-consensus

              sense

              5'-GGCCACTGACTTCAGTGTGAA-3'

               

              antisense

              5'-TGCATTCCAGTTCTCTGCAAGA-3'

              Fcer1a-transcription start

              sense

              5'-AGGTGTCAGCTGAAGGTACAATA-3'

               

              antisense

              5'-CCCACCATGACACTCTCTAAAT-3'

              F2rl3-AML1-consensus

              sense

              5'-AGGGTGTCTCTCTGAATCTGGA-3'

               

              antisense

              5'-GGCAAGTCTGTTATCTCAGCAT-3'

              F2rl3-transcription start

              sense

              5'-TTGGAGGAAGGCTGGATTGTTAT-3'

               

              antisense

              5'-CCCATTGGGATCTGCTTGCTCA-3'

              Mgmt-AML1-consensus

              sense

              5'-GAGCTGCACACTGGGAAGATG-3'

               

              antisense

              5'-GTGTACCAGATGCTGTGCAGG-3'

              Mgmt-basic promoter

              sense

              5'-CAGTTTCAGGTCTGGAAGAAGAG-3'

               

              antisense

              5'-AGCTGTGGGCTTGTAGTCCGAG-3'

              Control region

              sense

              5'-ATGCAACACACAACAAAGCAAA-3'

               

              antisense

              5'-GGCCAAATGAGGTTGTGTCCT-3'

              Bisulfite sequencing primers

                

              Mgmt-CpG-1st PCR

              sense

              5'-TAGTGATTGGATTTTTAGTGGGT-3'

               

              antisense

              5'-CTATCTCCCTAAACTTCAACTC-3'

              Mgmt-CpG-2nd PCR

              sense

              5'-GTGAGAAGGTGTAGTTTAGTTT-3'

               

              antisense

              5'-CTCACCAACTTACAAACTACAA-3'

              Quantitative chromatin immunoprecipitation (ChIP)

              ChIP was performed using reagents purchased from Upstate (Charlottesville, VA, USA) following the manufacturer's protocol. AML1 and AML1-MTG16 binding was assessed by ChIP with antibodies against either the AML1 C-terminus (Santa Cruz Biotechnology, Santa Cruz, CA, USA), or the MTG16 C-terminus [28], respectively. Histone hallmarks of repressive chromatin were assessed by ChIP with antibodies against acetyl-histone H4 (Upstate) and trimethyl-K9 at histone H3 (Upstate). Control ChIPs were performed without the respective antibodies. The immunoprecipitated DNA was amplified by real-time PCR with primers specific for regions encompassing the AML1-consensus, the transcription start site, or a control region (Table 4). The DNA relative enrichment was calculated by using the Delta-delta Ct method. The PCR signals obtained for each gene region were normalized to the PCR signal obtained from the input DNA (total chromatin fraction) and compared to a control region approximately 15 kb downstream of F2rl3 transcription start site. Two independent experiments were performed in triplicate, and significance was calculated by using the Student t-test.

              Bisulfite sequencing

              Genomic DNA was extracted with DNAzol (Invitrogen) according to the manufacturer's instructions. DNA was modified by sodium bisulfite treatment as previously described [47]. Mgmt CpG island was amplified by nested PCR by using the primers indicated in Table 4. The PCR fragments were subcloned into pGEM-T (Promega, San Luis Obispo, CA, USA) and 20 clones for each PCR fragment were sequenced.

              Declarations

              Acknowledgements

              We wish to thank Frank Staal, Justine Peeters, Violeta Stoyanova, Leontine van Unen (ErasmusMC, Rotterdam, The Netherlands), and Alan Hutson (Roswell Park Cancer Institute, Buffalo, NY) for technical support and critical discussions. This work was supported through Erasmus MC funds (ATH) and RPCI institutional funds (NS).

              Authors’ Affiliations

              (1)
              Department of Cancer Genetics, Roswell Park Cancer Institute
              (2)
              Department of Clinical Genetics, Erasmus MC
              (3)
              Department of Bioinformatics, Erasmus MC

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              © Rossetti et al. 2007

              This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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