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Id Subject Object Predicate Lexical cue
T105 0-144 Sentence denotes Down-regulation of interferon regulatory factor 4 gene expression in leukemic cells due to hypermethylation of CpG motifs in the promoter region
T1 0-144 Sentence denotes Down-regulation of interferon regulatory factor 4 gene expression in leukemic cells due to hypermethylation of CpG motifs in the promoter region
T2 147-155 Sentence denotes Abstract
T106 156-464 Sentence denotes Although the bcr-abl translocation has been shown to be the causative genetic aberration in chronic myeloid leukemia (CML), there is mounting evidence that the deregulation of other genes, such as the transcription factor interferon regulatory factor 4 (IRF-4), is also implicated in the pathogenesis of CML.
T3 156-464 Sentence denotes Although the bcr-abl translocation has been shown to be the causative genetic aberration in chronic myeloid leukemia (CML), there is mounting evidence that the deregulation of other genes, such as the transcription factor interferon regulatory factor 4 (IRF-4), is also implicated in the pathogenesis of CML.
T107 465-633 Sentence denotes Promoter methylation of CpG target sites or direct deletions/insertions of genes are mechanisms of a reversible or permanent silencing of gene expression, respectively.
T4 465-633 Sentence denotes Promoter methylation of CpG target sites or direct deletions/insertions of genes are mechanisms of a reversible or permanent silencing of gene expression, respectively.
T108 634-780 Sentence denotes Therefore, we investigated whether IRF-4 promoter methylation or mutation may be involved in the regulation of IRF-4 expression in leukemia cells.
T5 634-780 Sentence denotes Therefore, we investigated whether IRF-4 promoter methylation or mutation may be involved in the regulation of IRF-4 expression in leukemia cells.
T109 781-1013 Sentence denotes Whereas promoter mutations or structural rearrangements could be excluded as a cause of altered IRF-4 expression in hematopoietic cells, the IRF-4 promoter methylation status was found to significantly influence IRF-4 transcription.
T6 781-1013 Sentence denotes Whereas promoter mutations or structural rearrangements could be excluded as a cause of altered IRF-4 expression in hematopoietic cells, the IRF-4 promoter methylation status was found to significantly influence IRF-4 transcription.
T110 1014-1235 Sentence denotes First, treatment of IRF-4-negative lymphoid, myeloid and monocytic cell lines with the methylation-inhibitor 5-aza-2-deoxycytidine resulted in a time- and concentration-dependent increase of IRF-4 mRNA and protein levels.
T7 1014-1235 Sentence denotes First, treatment of IRF-4-negative lymphoid, myeloid and monocytic cell lines with the methylation-inhibitor 5-aza-2-deoxycytidine resulted in a time- and concentration-dependent increase of IRF-4 mRNA and protein levels.
T111 1236-1397 Sentence denotes Second, using a restriction-PCR-assay and bisulfite-sequencing we identified specifically methylated CpG sites in IRF-4-negative but not in IRF-4-positive cells.
T8 1236-1397 Sentence denotes Second, using a restriction-PCR-assay and bisulfite-sequencing we identified specifically methylated CpG sites in IRF-4-negative but not in IRF-4-positive cells.
T112 1398-1650 Sentence denotes Third, we clearly determined promoter methylation as a mechanism for IRF-4 down-regulation via reporter gene assays, but did not detect an association of methylational status and mRNA expression of DNA methyltransferases or methyl-CpG-binding proteins.
T9 1398-1650 Sentence denotes Third, we clearly determined promoter methylation as a mechanism for IRF-4 down-regulation via reporter gene assays, but did not detect an association of methylational status and mRNA expression of DNA methyltransferases or methyl-CpG-binding proteins.
T113 1651-1796 Sentence denotes Together, these data suggest CpG site-specific IRF-4 promoter methylation as a putative mechanism of down-regulated IRF-4 expression in leukemia.
T10 1651-1796 Sentence denotes Together, these data suggest CpG site-specific IRF-4 promoter methylation as a putative mechanism of down-regulated IRF-4 expression in leukemia.
T11 1798-1810 Sentence denotes INTRODUCTION
T1054 1811-2082 Sentence denotes Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder with a typical three phased course (chronic, accelerated and blastic phase) reflecting the loss of differentiation and malignant progress which inevitably leads to death after the blastic phase (1,2).
T12 1811-2082 Sentence denotes Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder with a typical three phased course (chronic, accelerated and blastic phase) reflecting the loss of differentiation and malignant progress which inevitably leads to death after the blastic phase (1,2).
T1055 2083-2262 Sentence denotes The hallmark genetic aberration of CML is a reciprocal chromosomal translocation t(9;22) leading to expression of a bcr-abl fusion gene, an aberrant activated tyrosine kinase (2).
T13 2083-2262 Sentence denotes The hallmark genetic aberration of CML is a reciprocal chromosomal translocation t(9;22) leading to expression of a bcr-abl fusion gene, an aberrant activated tyrosine kinase (2).
T1056 2263-2419 Sentence denotes Treatment with interferon α (IFN-α) prolongs survival of CML patients and is associated with a complete cytogenetic response in 5–33% of CML patients (1,2).
T14 2263-2419 Sentence denotes Treatment with interferon α (IFN-α) prolongs survival of CML patients and is associated with a complete cytogenetic response in 5–33% of CML patients (1,2).
T1057 2420-2574 Sentence denotes Recently, we described an impaired expression of the interferon regulatory factor 4 (IRF-4) in CML, correlating with poor response to IFN-α treatment (3).
T15 2420-2574 Sentence denotes Recently, we described an impaired expression of the interferon regulatory factor 4 (IRF-4) in CML, correlating with poor response to IFN-α treatment (3).
T1058 2575-2634 Sentence denotes The cause of the silencing of IRF-4 level remained unclear.
T16 2575-2634 Sentence denotes The cause of the silencing of IRF-4 level remained unclear.
T1059 2635-2780 Sentence denotes Interferon regulatory factors (IRFs) are a family of transcriptional regulators defined by a characteristic homology in their DNA-binding domain.
T17 2635-2780 Sentence denotes Interferon regulatory factors (IRFs) are a family of transcriptional regulators defined by a characteristic homology in their DNA-binding domain.
T1060 2781-2937 Sentence denotes They play an important role in the regulation of various genes (such as IFNs, interleukins, MHC class I/II), apoptosis and differentiation/maturation (4–6).
T18 2781-2937 Sentence denotes They play an important role in the regulation of various genes (such as IFNs, interleukins, MHC class I/II), apoptosis and differentiation/maturation (4–6).
T1061 2938-3021 Sentence denotes IRF-4 (ICSAT/Pip/MUM1/LSIRF) is one member with very restricted expression pattern:
T19 2938-3021 Sentence denotes IRF-4 (ICSAT/Pip/MUM1/LSIRF) is one member with very restricted expression pattern:
T1062 3022-3093 Sentence denotes Predominately B- and activated T-lymphocytes are IRF-4 positive (7–11).
T20 3022-3093 Sentence denotes Predominately B- and activated T-lymphocytes are IRF-4 positive (7–11).
T1063 3094-3272 Sentence denotes In contrast to other IRFs, expression of IRF-4 cannot be induced by IFNs, but by antigen stimulation, crosslinking of T- or B-cell receptors or phorbol-myristate-acetate (10,11).
T21 3094-3272 Sentence denotes In contrast to other IRFs, expression of IRF-4 cannot be induced by IFNs, but by antigen stimulation, crosslinking of T- or B-cell receptors or phorbol-myristate-acetate (10,11).
T1064 3273-3532 Sentence denotes Consistent with the restriction of expression to immunocompetent cells, mice with deletion of IRF-4 failed to develop mature and functionally active B- and T-lymphocytes (12), and the impaired expression of IRF-4 in CML was predominately found in T-cells (3).
T22 3273-3532 Sentence denotes Consistent with the restriction of expression to immunocompetent cells, mice with deletion of IRF-4 failed to develop mature and functionally active B- and T-lymphocytes (12), and the impaired expression of IRF-4 in CML was predominately found in T-cells (3).
T1065 3533-3609 Sentence denotes These data suggest a crucial role for IRF-4 in the function of immune cells.
T23 3533-3609 Sentence denotes These data suggest a crucial role for IRF-4 in the function of immune cells.
T1066 3610-3846 Sentence denotes Methylation of dinucleotide cytosine-guanosine motifs (CpG), especially in CpG islands located in promoter regions, is one of the mechanisms of gene regulation in mammals and a common event of gene silencing in human neoplasias (13,14).
T24 3610-3846 Sentence denotes Methylation of dinucleotide cytosine-guanosine motifs (CpG), especially in CpG islands located in promoter regions, is one of the mechanisms of gene regulation in mammals and a common event of gene silencing in human neoplasias (13,14).
T1067 3847-3964 Sentence denotes As opposed to normal cells, hypermethylation of CpG islands is a frequently observed phenomenon in every cancer type.
T25 3847-3964 Sentence denotes As opposed to normal cells, hypermethylation of CpG islands is a frequently observed phenomenon in every cancer type.
T1068 3965-4128 Sentence denotes De novo DNA methylation of genes such as cell cycle, DNA repair, apoptosis and tumor suppressor genes is therefore thought to be involved in tumorigenesis (15–17).
T26 3965-4128 Sentence denotes De novo DNA methylation of genes such as cell cycle, DNA repair, apoptosis and tumor suppressor genes is therefore thought to be involved in tumorigenesis (15–17).
T1069 4129-4239 Sentence denotes Examples for such aberrated genes are MGMT, DAPK, p14ARF, p15INK4b, p16INK4a, BRCA1, CDH13 and APAF-1 (17–19).
T27 4129-4239 Sentence denotes Examples for such aberrated genes are MGMT, DAPK, p14ARF, p15INK4b, p16INK4a, BRCA1, CDH13 and APAF-1 (17–19).
T1070 4240-4450 Sentence denotes In CML, methylation is known to regulate expression of the c-abl, the bcr gene and others (20–23), and the extent of methylation in the c-abl promoter has been shown to be associated with advanced disease (24).
T28 4240-4450 Sentence denotes In CML, methylation is known to regulate expression of the c-abl, the bcr gene and others (20–23), and the extent of methylation in the c-abl promoter has been shown to be associated with advanced disease (24).
T1071 4451-4598 Sentence denotes Hypermethylation due to overexpression of DNA methyltransferases (DNMTs) remains one possible explanation for de novo methylation in tumorigenesis.
T29 4451-4598 Sentence denotes Hypermethylation due to overexpression of DNA methyltransferases (DNMTs) remains one possible explanation for de novo methylation in tumorigenesis.
T1072 4599-4685 Sentence denotes Recently, DNMTs have been shown to be up-regulated in hematopoietic malignancies (25).
T30 4599-4685 Sentence denotes Recently, DNMTs have been shown to be up-regulated in hematopoietic malignancies (25).
T1073 4686-4897 Sentence denotes Methyl-CpG-binding proteins (MBPs) are thought to inhibit the binding of transcriptional factors to the promoter and are therefore discussed as one mechanism of transcription inhibition by hypermethylation (26).
T31 4686-4897 Sentence denotes Methyl-CpG-binding proteins (MBPs) are thought to inhibit the binding of transcriptional factors to the promoter and are therefore discussed as one mechanism of transcription inhibition by hypermethylation (26).
T1074 4898-4987 Sentence denotes In this work, we studied mechanisms of IRF-4 gene expression silencing in leukemic cells.
T32 4898-4987 Sentence denotes In this work, we studied mechanisms of IRF-4 gene expression silencing in leukemic cells.
T1075 4988-5127 Sentence denotes We analyzed the IRF-4 promoter region for genetic aberrations and methylational status in IRF-4-positive and -negative hematopoietic cells.
T33 4988-5127 Sentence denotes We analyzed the IRF-4 promoter region for genetic aberrations and methylational status in IRF-4-positive and -negative hematopoietic cells.
T34 5129-5150 Sentence denotes MATERIALS AND METHODS
T2707 5152-5162 Sentence denotes Cell lines
T35 5152-5162 Sentence denotes Cell lines
T2708 5163-5419 Sentence denotes K-562, Jurkat and U-937 were obtained from the ATCC (American Type Culture Collection, Rockville, USA) and EM-2, LAMA-84, CML-T1, BV-173, SD-1 and RPMI-8226 from the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany).
T36 5163-5419 Sentence denotes K-562, Jurkat and U-937 were obtained from the ATCC (American Type Culture Collection, Rockville, USA) and EM-2, LAMA-84, CML-T1, BV-173, SD-1 and RPMI-8226 from the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany).
T2709 5420-5491 Sentence denotes All cell lines, except BV-173, SD-1 and RPMI-8226, were IRF-4-negative.
T37 5420-5491 Sentence denotes All cell lines, except BV-173, SD-1 and RPMI-8226, were IRF-4-negative.
T2858 5493-5521 Sentence denotes Cell culture and stimulation
T38 5493-5521 Sentence denotes Cell culture and stimulation
T2859 5522-5740 Sentence denotes All cell lines were maintained at 5% CO2 in RPMI 1640 medium with 1% glutamine (Gibco/BRL Eggenstein, Germany) supplemented with 10% fetal calf serum (Gibco/BRL), 1% penicillin/streptomycin (Biochrom, Berlin, Germany).
T39 5522-5740 Sentence denotes All cell lines were maintained at 5% CO2 in RPMI 1640 medium with 1% glutamine (Gibco/BRL Eggenstein, Germany) supplemented with 10% fetal calf serum (Gibco/BRL), 1% penicillin/streptomycin (Biochrom, Berlin, Germany).
T2860 5741-5892 Sentence denotes When indicated, cells were treated with 5-aza-2-deoxycytidine (AzadC) or 5-azacytidine (AzaC) (Sigma, Taufkirchen, Germany) for different time periods.
T40 5741-5892 Sentence denotes When indicated, cells were treated with 5-aza-2-deoxycytidine (AzadC) or 5-azacytidine (AzaC) (Sigma, Taufkirchen, Germany) for different time periods.
T2861 5893-5971 Sentence denotes Owing to their chemical instability fresh substances were re-added every 24 h.
T41 5893-5971 Sentence denotes Owing to their chemical instability fresh substances were re-added every 24 h.
T3053 5973-6005 Sentence denotes Sequencing of the IRF-4 promoter
T42 5973-6005 Sentence denotes Sequencing of the IRF-4 promoter
T3054 6006-6272 Sentence denotes For analysis of the IRF-4 promoter region for permanent aberrations such as insertions/deletions or mutation, we PCR-amplified two fragments from genomic DNA, which was extracted from depicted cell lines with a commercial kit (Qiagen, Hilde, Germany) as recommended.
T43 6006-6272 Sentence denotes For analysis of the IRF-4 promoter region for permanent aberrations such as insertions/deletions or mutation, we PCR-amplified two fragments from genomic DNA, which was extracted from depicted cell lines with a commercial kit (Qiagen, Hilde, Germany) as recommended.
T3055 6273-6300 Sentence denotes The primers were 1-forward:
T44 6273-6300 Sentence denotes The primers were 1-forward:
T3056 6301-6341 Sentence denotes 5′-TTGAGATGGAGTCTTGCTCTGT-3′, 1-reverse:
T45 6301-6341 Sentence denotes 5′-TTGAGATGGAGTCTTGCTCTGT-3′, 1-reverse:
T3057 6342-6383 Sentence denotes 5′-CCAGGACCTCAGGAGGCCAGTCA-3′; 2-forward:
T46 6342-6383 Sentence denotes 5′-CCAGGACCTCAGGAGGCCAGTCA-3′; 2-forward:
T3058 6384-6427 Sentence denotes 5′-AGCGGTGAAACTGAGAGTGCGAGGT-3′, 2-reverse:
T47 6384-6427 Sentence denotes 5′-AGCGGTGAAACTGAGAGTGCGAGGT-3′, 2-reverse:
T3059 6428-6455 Sentence denotes 5′-GCCACATCGCTGCAGTTTAG-3′.
T48 6428-6455 Sentence denotes 5′-GCCACATCGCTGCAGTTTAG-3′.
T3060 6456-6553 Sentence denotes The products were cloned with the ‘TOPO TA cloning kit’ (Invitrogen, Groningen, The Netherlands).
T49 6456-6553 Sentence denotes The products were cloned with the ‘TOPO TA cloning kit’ (Invitrogen, Groningen, The Netherlands).
T3061 6554-6808 Sentence denotes After bacterial amplification of the cloned PCR fragments by standard procedures, at least three clones from each sample were sequenced with an automated sequencer (ABI Prism 377, Applied Bio-systems, Foster City, USA) as recommended by the manufacturer.
T50 6554-6808 Sentence denotes After bacterial amplification of the cloned PCR fragments by standard procedures, at least three clones from each sample were sequenced with an automated sequencer (ABI Prism 377, Applied Bio-systems, Foster City, USA) as recommended by the manufacturer.
T3408 6810-6829 Sentence denotes Expression analysis
T51 6810-6829 Sentence denotes Expression analysis
T3409 6830-6964 Sentence denotes To analyze the IRF-4 transcriptional level, RNA was extracted from cells using the commercial RNAzol-kit (Paesel, Frankfurt, Germany).
T52 6830-6964 Sentence denotes To analyze the IRF-4 transcriptional level, RNA was extracted from cells using the commercial RNAzol-kit (Paesel, Frankfurt, Germany).
T3410 6965-7051 Sentence denotes An aliquot of 1 μg total RNA was used for cDNA synthesis as described previously (27).
T53 6965-7051 Sentence denotes An aliquot of 1 μg total RNA was used for cDNA synthesis as described previously (27).
T3411 7052-7189 Sentence denotes RNA expression analysis for IRF-4 and the reference gene β-actin was carried out by semi-quantitative PCR as described previously (3,27).
T54 7052-7189 Sentence denotes RNA expression analysis for IRF-4 and the reference gene β-actin was carried out by semi-quantitative PCR as described previously (3,27).
T3412 7190-7241 Sentence denotes PCR products were verified by automated sequencing.
T55 7190-7241 Sentence denotes PCR products were verified by automated sequencing.
T3413 7242-7388 Sentence denotes PCR primers and conditions for expression analysis of DNMT or MBP (DNMT1 DNMT3A, DNMT3B, MeCP, MBD1, MBD2 and MBD4) were published elsewhere (28).
T56 7242-7388 Sentence denotes PCR primers and conditions for expression analysis of DNMT or MBP (DNMT1 DNMT3A, DNMT3B, MeCP, MBD1, MBD2 and MBD4) were published elsewhere (28).
T3414 7389-7506 Sentence denotes For analysis of IRF-4 protein expression, a standard immunoblotting assay was performed as described previously (29).
T57 7389-7506 Sentence denotes For analysis of IRF-4 protein expression, a standard immunoblotting assay was performed as described previously (29).
T3415 7507-7797 Sentence denotes Briefly, protein lysates were generated by incubating 1 × 106 cells in 100 µl RIPA buffer (1% NP-40, 0.5% sodiumdesoxycholate, 0.1% SDS, 100 µg/ml phenylmethylsulfonyl fluoride, 10 µl/ml protease-inhibitory-mix, 1 µmol/ml sodiumorthovanadate in phosphate-buffered saline) for 30 min on ice.
T58 7507-7797 Sentence denotes Briefly, protein lysates were generated by incubating 1 × 106 cells in 100 µl RIPA buffer (1% NP-40, 0.5% sodiumdesoxycholate, 0.1% SDS, 100 µg/ml phenylmethylsulfonyl fluoride, 10 µl/ml protease-inhibitory-mix, 1 µmol/ml sodiumorthovanadate in phosphate-buffered saline) for 30 min on ice.
T3416 7798-7928 Sentence denotes After centrifugation, protein concentration of the supernatant was determined by BCA-method (Pierce, Rockford, IL) as recommended.
T59 7798-7928 Sentence denotes After centrifugation, protein concentration of the supernatant was determined by BCA-method (Pierce, Rockford, IL) as recommended.
T3417 7929-8089 Sentence denotes Protein lysates (70–100 µg) were electrophoresed on polyacrylamide gels and transferred to a PVDF-membrane (Immobilon P, 0.45 µm; Millipore, Eschborn, Germany).
T60 7929-8089 Sentence denotes Protein lysates (70–100 µg) were electrophoresed on polyacrylamide gels and transferred to a PVDF-membrane (Immobilon P, 0.45 µm; Millipore, Eschborn, Germany).
T3418 8090-8467 Sentence denotes Membranes were blocked with 2.5% blocking reagent (Boehringer Mannheim, Germany) in TBST buffer (4.44 g/l Tris–HCL, 2.65 g/l TrisOH, 8.07 g/l NaCl, 0.2 g/l KCl and 500 µl/l Tween-20 in H2O) and subsequently incubated with primary antibody as indicated and horseradish peroxidase-conjugated secondary antibody, anti-mouse or anti-goat IgG (DAKO, Hamburg, Germany), respectively.
T61 8090-8467 Sentence denotes Membranes were blocked with 2.5% blocking reagent (Boehringer Mannheim, Germany) in TBST buffer (4.44 g/l Tris–HCL, 2.65 g/l TrisOH, 8.07 g/l NaCl, 0.2 g/l KCl and 500 µl/l Tween-20 in H2O) and subsequently incubated with primary antibody as indicated and horseradish peroxidase-conjugated secondary antibody, anti-mouse or anti-goat IgG (DAKO, Hamburg, Germany), respectively.
T3419 8468-8576 Sentence denotes The membranes were then developed with an ECL detection kit (Amersham Pharmacia Biotech, Freiburg, Germany).
T62 8468-8576 Sentence denotes The membranes were then developed with an ECL detection kit (Amersham Pharmacia Biotech, Freiburg, Germany).
T3420 8577-8716 Sentence denotes The primary antibodies were goat anti-IRF-4/ICSAT (M-17) (Santa Cruz Biotechnology, Santa Cruz, CA) and mouse anti-β-actin (AC-74) (Sigma).
T63 8577-8716 Sentence denotes The primary antibodies were goat anti-IRF-4/ICSAT (M-17) (Santa Cruz Biotechnology, Santa Cruz, CA) and mouse anti-β-actin (AC-74) (Sigma).
T4350 8718-8760 Sentence denotes Methylation-specific restriction-PCR-assay
T64 8718-8760 Sentence denotes Methylation-specific restriction-PCR-assay
T4351 8761-8825 Sentence denotes DNA was extracted with a commercial kit (Qiagen) as recommended.
T65 8761-8825 Sentence denotes DNA was extracted with a commercial kit (Qiagen) as recommended.
T4352 8826-9069 Sentence denotes Since the restriction ability of several endonucleases is inhibited by methylation of their target sequence, we used methylation-sensitive enzymes HpaII and HaeII-isochizomer Bsp143II and Bsh1236I (MBI Fermentas, St Leon-Rot, Germany) (20,24).
T66 8826-9069 Sentence denotes Since the restriction ability of several endonucleases is inhibited by methylation of their target sequence, we used methylation-sensitive enzymes HpaII and HaeII-isochizomer Bsp143II and Bsh1236I (MBI Fermentas, St Leon-Rot, Germany) (20,24).
T4353 9070-9199 Sentence denotes As control the methylation-resistant enzyme MspI and an enzyme with no recognition site in the target promoter, EcoRI, were used.
T67 9070-9199 Sentence denotes As control the methylation-resistant enzyme MspI and an enzyme with no recognition site in the target promoter, EcoRI, were used.
T4354 9200-9323 Sentence denotes DNA (0.8 µg) was digested by 40 U the respective enzyme for 6 h and, to ensure complete cleavage, additional 20 U for 16 h.
T68 9200-9323 Sentence denotes DNA (0.8 µg) was digested by 40 U the respective enzyme for 6 h and, to ensure complete cleavage, additional 20 U for 16 h.
T4355 9324-9492 Sentence denotes Thereafter 100 ng of digested DNA was used to a PCR amplification of two fragments (F1 and F2) spanning part of the IRF-4 promoter (30) (GenBank U52683; see Figure 3A).
T69 9324-9492 Sentence denotes Thereafter 100 ng of digested DNA was used to a PCR amplification of two fragments (F1 and F2) spanning part of the IRF-4 promoter (30) (GenBank U52683; see Figure 3A).
T4356 9493-9538 Sentence denotes The sequences of the primers were F1-forward:
T70 9493-9538 Sentence denotes The sequences of the primers were F1-forward:
T4357 9539-9631 Sentence denotes 5′-TTGAGATGGAGTCTTGCTCTGT-3′, F1-reverse: ATCACTTCCAGACTTCAGTTCACCT-3′ (341 bp); F2-forward:
T71 9539-9631 Sentence denotes 5′-TTGAGATGGAGTCTTGCTCTGT-3′, F1-reverse: ATCACTTCCAGACTTCAGTTCACCT-3′ (341 bp); F2-forward:
T4358 9632-9676 Sentence denotes 5′-AAGGTGAACTGAAGTCTGGAAGTGA-3′, F2-reverse:
T72 9632-9676 Sentence denotes 5′-AAGGTGAACTGAAGTCTGGAAGTGA-3′, F2-reverse:
T4359 9677-9716 Sentence denotes 5′-CCAGGACCTCAGGAGGCCAGTCA-3′ (474 bp).
T73 9677-9716 Sentence denotes 5′-CCAGGACCTCAGGAGGCCAGTCA-3′ (474 bp).
T4360 9717-9765 Sentence denotes The PCR conditions were described elsewhere (3).
T74 9717-9765 Sentence denotes The PCR conditions were described elsewhere (3).
T4361 9766-9836 Sentence denotes PCR was performed with an annealing temperature of 62°C and 35 cycles.
T75 9766-9836 Sentence denotes PCR was performed with an annealing temperature of 62°C and 35 cycles.
T4362 9837-10037 Sentence denotes When DNA was methylated at specific sites, the sensitive enzymes were not able to digest the DNA and amplification took place; in case of no methylation, DNA was digested and no product was generated.
T76 9837-10037 Sentence denotes When DNA was methylated at specific sites, the sensitive enzymes were not able to digest the DNA and amplification took place; in case of no methylation, DNA was digested and no product was generated.
T4363 10038-10149 Sentence denotes The PCR products were electrophoresed on a 3% agarose gel, were stained with ethidium bromide and photographed.
T77 10038-10149 Sentence denotes The PCR products were electrophoresed on a 3% agarose gel, were stained with ethidium bromide and photographed.
T4364 10150-10201 Sentence denotes PCR products were verified by automated sequencing.
T78 10150-10201 Sentence denotes PCR products were verified by automated sequencing.
T4932 10203-10222 Sentence denotes Bisulfite treatment
T79 10203-10222 Sentence denotes Bisulfite treatment
T4933 10223-10260 Sentence denotes DNA was extracted as described above.
T80 10223-10260 Sentence denotes DNA was extracted as described above.
T4934 10261-10440 Sentence denotes Bisulfite treatment of DNA, leading to conversion of unmethylated cytosine to uracil residues and no change of methylated cytosine residues, was performed as described as follows.
T81 10261-10440 Sentence denotes Bisulfite treatment of DNA, leading to conversion of unmethylated cytosine to uracil residues and no change of methylated cytosine residues, was performed as described as follows.
T4935 10441-10609 Sentence denotes Briefly, 1 µg of DNA and 2 µg of poly(dA–dT)(poly(dA–dT) copolymers (Amersham Pharmacia Biotech) were denaturated for 20 min at 42°C in 0.3 M NaOH in a volume of 50 µl.
T82 10441-10609 Sentence denotes Briefly, 1 µg of DNA and 2 µg of poly(dA–dT)(poly(dA–dT) copolymers (Amersham Pharmacia Biotech) were denaturated for 20 min at 42°C in 0.3 M NaOH in a volume of 50 µl.
T83 10610-10831 Sentence denotes Fresh solutions of 30 µl of 10 mM hydrochinon (Sigma) and 530 µl of 3 M sodium bisulfite (pH 5.0; Sigma) were added, the solution was gently mixed, overlayed with mineral oil and incubated in the dark for 12–13 h at 50°C.
T4936 10610-10932 Sentence denotes Fresh solutions of 30 µl of 10 mM hydrochinon (Sigma) and 530 µl of 3 M sodium bisulfite (pH 5.0; Sigma) were added, the solution was gently mixed, overlayed with mineral oil and incubated in the dark for 12–13 h at 50°C. The aqueous phase was recovered using the ‘Wizard DNA clean-up system’ (Promega, Mannheim, Germany).
T84 10832-10932 Sentence denotes The aqueous phase was recovered using the ‘Wizard DNA clean-up system’ (Promega, Mannheim, Germany).
T4937 10933-11089 Sentence denotes The purified DNA was subsequently mixed with 1 M NaOH to a final concentration of 0.3 M and incubated for 20 min at 37°C to ensure complete desulfonisation.
T85 10933-11089 Sentence denotes The purified DNA was subsequently mixed with 1 M NaOH to a final concentration of 0.3 M and incubated for 20 min at 37°C to ensure complete desulfonisation.
T4938 11090-11223 Sentence denotes DNA was ethanol precipitated in the presence of 1/10 vol of 3 M sodium acetate, washed with 70% ethanol and resuspended in 50 µl H2O.
T86 11090-11223 Sentence denotes DNA was ethanol precipitated in the presence of 1/10 vol of 3 M sodium acetate, washed with 70% ethanol and resuspended in 50 µl H2O.
T4939 11224-11439 Sentence denotes Subsequent PCR amplification of 4 µl bisulfite-treated DNA was used for cloning of two fragments of the IRF-4 promoter (BS-I and BS-II) into pCR2.1 vector with the ‘TOPO TA cloning kit’ (Invitrogen) (see Figure 3A).
T87 11224-11439 Sentence denotes Subsequent PCR amplification of 4 µl bisulfite-treated DNA was used for cloning of two fragments of the IRF-4 promoter (BS-I and BS-II) into pCR2.1 vector with the ‘TOPO TA cloning kit’ (Invitrogen) (see Figure 3A).
T4940 11440-11863 Sentence denotes The primers used for PCR amplification of the BS-I and BS-II fragments contain the putative altered sequence of the sense strand due to bisulfite treatment (converted cytosine residues are written in bold letters): BS-I-forward 5′-TATTTGGATTTTTAGGGAGTTTTTTTT-3′, BS-I-reverse 5′-ACCCAACTCCCTTAAACTATTAAACT-3′ (187 bp); BS-II-forward 5′-AGTTTAATAGTTTAAGGGAGTTGGGT-3′, BS-II-reverse 5′-CTCACCCTAAACTCAAAACTAAAAAC-3′ (674 bp).
T88 11440-11863 Sentence denotes The primers used for PCR amplification of the BS-I and BS-II fragments contain the putative altered sequence of the sense strand due to bisulfite treatment (converted cytosine residues are written in bold letters): BS-I-forward 5′-TATTTGGATTTTTAGGGAGTTTTTTTT-3′, BS-I-reverse 5′-ACCCAACTCCCTTAAACTATTAAACT-3′ (187 bp); BS-II-forward 5′-AGTTTAATAGTTTAAGGGAGTTGGGT-3′, BS-II-reverse 5′-CTCACCCTAAACTCAAAACTAAAAAC-3′ (674 bp).
T4941 11864-12055 Sentence denotes After bacterial amplification of the cloned PCR fragments by standard procedures, eight clones from each sample were sequenced with an automated sequencer (ABI Prism 377, Applied Biosystems).
T89 11864-12055 Sentence denotes After bacterial amplification of the cloned PCR fragments by standard procedures, eight clones from each sample were sequenced with an automated sequencer (ABI Prism 377, Applied Biosystems).
T5684 12057-12102 Sentence denotes In vitro methylation and reporter gene assays
T90 12057-12102 Sentence denotes In vitro methylation and reporter gene assays
T91 12103-12175 Sentence denotes The IRF-4 promoter-reporter gene construct was generously provided by J.
T5685 12103-12189 Sentence denotes The IRF-4 promoter-reporter gene construct was generously provided by J. Hiscott (31).
T92 12176-12189 Sentence denotes Hiscott (31).
T5686 12190-12377 Sentence denotes Constructs were methylated in vitro with CpG Methylase (M.Sss I) as recommended by the manufacturer (NE Biolabs) and complete methylation was checked via restriction analysis (Figure 5A).
T93 12190-12377 Sentence denotes Constructs were methylated in vitro with CpG Methylase (M.Sss I) as recommended by the manufacturer (NE Biolabs) and complete methylation was checked via restriction analysis (Figure 5A).
T5687 12378-12489 Sentence denotes Reporter gene assays using the dual luciferase assay (Promega) were performed similar to previous reports (29).
T94 12378-12489 Sentence denotes Reporter gene assays using the dual luciferase assay (Promega) were performed similar to previous reports (29).
T5688 12490-12657 Sentence denotes Briefly, 5 nM of the reporter construct and the transfection control construct expressing the renilla luciferase gene were transientlyco-expressed via electroporation.
T95 12490-12657 Sentence denotes Briefly, 5 nM of the reporter construct and the transfection control construct expressing the renilla luciferase gene were transientlyco-expressed via electroporation.
T5689 12658-12740 Sentence denotes The control construct served as an internal reference for transfection efficiency.
T96 12658-12740 Sentence denotes The control construct served as an internal reference for transfection efficiency.
T5690 12741-12876 Sentence denotes Forty-eight hours after transfection, luciferase activity was measured with a LB 96 P microlumat (EG&G Berthold, Bad Wildbad, Germany).
T97 12741-12876 Sentence denotes Forty-eight hours after transfection, luciferase activity was measured with a LB 96 P microlumat (EG&G Berthold, Bad Wildbad, Germany).
T5691 12877-12993 Sentence denotes IRF-4 promoter activation was quantified as a ratio of measured firefly light units (flu) relative to renilla (rlu).
T98 12877-12993 Sentence denotes IRF-4 promoter activation was quantified as a ratio of measured firefly light units (flu) relative to renilla (rlu).
T5692 12994-13047 Sentence denotes Each experiment was carried out at least three times.
T99 12994-13047 Sentence denotes Each experiment was carried out at least three times.
T100 13049-13056 Sentence denotes RESULTS
T6214 13058-13138 Sentence denotes Absence of IRF-4 expression in leukemia cells is not due to promoter alterations
T101 13058-13138 Sentence denotes Absence of IRF-4 expression in leukemia cells is not due to promoter alterations
T6215 13139-13255 Sentence denotes We have previously demonstrated a lack of IRF-4 expression in leukemia patients and specifically in CML T-cells (3).
T102 13139-13255 Sentence denotes We have previously demonstrated a lack of IRF-4 expression in leukemia patients and specifically in CML T-cells (3).
T6216 13256-13715 Sentence denotes Here, we demonstrate the absence of IRF-4 expression in various hematopoietic cell lines, such as Jurkat, a T-cell leukemia, CML-T1, a bcr-abl-positive T-cell line, K-562, a bcr-abl-positve erythroleukemia, U-937, a monocytic leukemia, EM-2 and LAMA-84, bcr-abl-positve myeloid leukemia, but not in SD-1, a bcr-abl-positive acute lymphoblastic leukemia (pre B-ALL), RPMI-8226, a multiple myeloma and BV-173, a bcr-abl-positive B-cell line (Figures 1A and 5D).
T103 13256-13715 Sentence denotes Here, we demonstrate the absence of IRF-4 expression in various hematopoietic cell lines, such as Jurkat, a T-cell leukemia, CML-T1, a bcr-abl-positive T-cell line, K-562, a bcr-abl-positve erythroleukemia, U-937, a monocytic leukemia, EM-2 and LAMA-84, bcr-abl-positve myeloid leukemia, but not in SD-1, a bcr-abl-positive acute lymphoblastic leukemia (pre B-ALL), RPMI-8226, a multiple myeloma and BV-173, a bcr-abl-positive B-cell line (Figures 1A and 5D).
T6217 13716-13872 Sentence denotes After sequencing of the IRF-4 promoter, it could be excluded that absence of IRF-4 expression in any of the above cell lines was due to genetic aberrations.
T104 13716-13872 Sentence denotes After sequencing of the IRF-4 promoter, it could be excluded that absence of IRF-4 expression in any of the above cell lines was due to genetic aberrations.
T6218 13873-14045 Sentence denotes However, 2 bp changes (nucleotide −1081, T→C and −1068, A→C) could be detected in both the IRF-4-positive BV-173 and the IRF-4-negative LAMA-84, EM-2 and K-562 (Figure 1B).
T105 13873-14045 Sentence denotes However, 2 bp changes (nucleotide −1081, T→C and −1068, A→C) could be detected in both the IRF-4-positive BV-173 and the IRF-4-negative LAMA-84, EM-2 and K-562 (Figure 1B).
T6219 14046-14251 Sentence denotes At position −116 an A→C substitution was found in EM-2, K-562 and CML-T1, whereas Jurkat, BV-173 and SD-1 exhibited a mixed A/C sequence and U-937, LAMA-84 and RPMI-8226 no substitution at all (Figure 1B).
T106 14046-14251 Sentence denotes At position −116 an A→C substitution was found in EM-2, K-562 and CML-T1, whereas Jurkat, BV-173 and SD-1 exhibited a mixed A/C sequence and U-937, LAMA-84 and RPMI-8226 no substitution at all (Figure 1B).
T6220 14252-14324 Sentence denotes Consequently, these alterations are unlikely to affect IRF-4 expression.
T107 14252-14324 Sentence denotes Consequently, these alterations are unlikely to affect IRF-4 expression.
T7004 14326-14407 Sentence denotes Increase of IRF-4 expression in hematopoietic cells after demethylating treatment
T108 14326-14407 Sentence denotes Increase of IRF-4 expression in hematopoietic cells after demethylating treatment
T7005 14408-14515 Sentence denotes We next analyzed whether promoter methylation could be responsible for down-regulation of IRF-4 expression.
T109 14408-14515 Sentence denotes We next analyzed whether promoter methylation could be responsible for down-regulation of IRF-4 expression.
T7006 14516-14622 Sentence denotes A region including exon1 in the IRF-4 promoter exhibited a large number of CpG-rich sequences (Figure 3A).
T110 14516-14622 Sentence denotes A region including exon1 in the IRF-4 promoter exhibited a large number of CpG-rich sequences (Figure 3A).
T7007 14623-14819 Sentence denotes Several chemical substances such as 5-aza-2-deoxycytidine (AzadC) or 5-azacytidine (AzaC) inhibit de novo and maintenance methylation, and thus can be used to discern promoter methylation (32,33).
T111 14623-14819 Sentence denotes Several chemical substances such as 5-aza-2-deoxycytidine (AzadC) or 5-azacytidine (AzaC) inhibit de novo and maintenance methylation, and thus can be used to discern promoter methylation (32,33).
T7008 14820-14863 Sentence denotes We used AzadC to generate unmethylated DNA.
T112 14820-14863 Sentence denotes We used AzadC to generate unmethylated DNA.
T7009 14864-15047 Sentence denotes A 72 h AzadC-treatment resulted in a concentration-dependent activation of IRF-4 transcription in Jurkat and CML-T1 T-cells as well as in U-937, K-562 and EM-2 cell lines (Figure 2A).
T113 14864-15047 Sentence denotes A 72 h AzadC-treatment resulted in a concentration-dependent activation of IRF-4 transcription in Jurkat and CML-T1 T-cells as well as in U-937, K-562 and EM-2 cell lines (Figure 2A).
T7010 15048-15215 Sentence denotes IRF-4 transcription was induced in a time-dependent manner and was observed as early as 24 h after treatment with AzadC and increased over time until 72 h (Figure 2B).
T114 15048-15215 Sentence denotes IRF-4 transcription was induced in a time-dependent manner and was observed as early as 24 h after treatment with AzadC and increased over time until 72 h (Figure 2B).
T7011 15216-15366 Sentence denotes Time and strength of the appearance of IRF-4 transcripts varied among cell lines, i.e. CML-T1 responded strongest to AzadC-treatment (data not shown).
T115 15216-15366 Sentence denotes Time and strength of the appearance of IRF-4 transcripts varied among cell lines, i.e. CML-T1 responded strongest to AzadC-treatment (data not shown).
T7012 15367-15502 Sentence denotes In line with this, AzadC-treatment of CML-T1 and LAMA-84 cells also translated in an induction of IRF-4 protein expression (Figure 2C).
T116 15367-15502 Sentence denotes In line with this, AzadC-treatment of CML-T1 and LAMA-84 cells also translated in an induction of IRF-4 protein expression (Figure 2C).
T7013 15503-15642 Sentence denotes Accordingly, treatment of the IRF-4-positive cell line BV-173, SD-1 and RPMI-8226 with AzadC had no effect on IRF-4 expression (Figure 2D).
T117 15503-15642 Sentence denotes Accordingly, treatment of the IRF-4-positive cell line BV-173, SD-1 and RPMI-8226 with AzadC had no effect on IRF-4 expression (Figure 2D).
T7014 15643-15784 Sentence denotes There was no difference in the effects of AzaC versus AzadC, as both increased the IRF-4 mRNA level in CML-T1 cells as well (data not shown).
T118 15643-15784 Sentence denotes There was no difference in the effects of AzaC versus AzadC, as both increased the IRF-4 mRNA level in CML-T1 cells as well (data not shown).
T7015 15785-15970 Sentence denotes This implied that promoter methylation may control IRF-4 expression, but an alternative explanation may be activation of positive transcriptional regulators of IRF-4 by AzadC (or AzaC).
T119 15785-15970 Sentence denotes This implied that promoter methylation may control IRF-4 expression, but an alternative explanation may be activation of positive transcriptional regulators of IRF-4 by AzadC (or AzaC).
T7831 15972-16072 Sentence denotes Methylation-sensitive enzymes do not cut specific sites in the IRF-4 promoter in hematopoietic cells
T120 15972-16072 Sentence denotes Methylation-sensitive enzymes do not cut specific sites in the IRF-4 promoter in hematopoietic cells
T7832 16073-16312 Sentence denotes To further investigate promoter methylation as a regulatory mechanism of IRF-4 gene expression, restriction-PCR-assays were performed (20,24), where only methylated DNA would not be cut enabling subsequent PCR amplification and vice versa.
T121 16073-16312 Sentence denotes To further investigate promoter methylation as a regulatory mechanism of IRF-4 gene expression, restriction-PCR-assays were performed (20,24), where only methylated DNA would not be cut enabling subsequent PCR amplification and vice versa.
T7833 16313-16490 Sentence denotes Genomic DNA from leukemic cells Jurkat, CML-T1, U-937, K-562, EM-2 and BV-173 was digested with the methylation-sensitive enzymes HpaII, Bsh1236I and HaeII-isochizomer Bsp143II.
T122 16313-16490 Sentence denotes Genomic DNA from leukemic cells Jurkat, CML-T1, U-937, K-562, EM-2 and BV-173 was digested with the methylation-sensitive enzymes HpaII, Bsh1236I and HaeII-isochizomer Bsp143II.
T7834 16491-16616 Sentence denotes EcoRI, which has no recognition site within the IRF-4 promoter, and the methylation-resistant enzyme MspI served as controls.
T123 16491-16616 Sentence denotes EcoRI, which has no recognition site within the IRF-4 promoter, and the methylation-resistant enzyme MspI served as controls.
T7835 16617-16718 Sentence denotes Two separate amplification reactions were performed, generating two fragments, F1 and F2 (Figure 3A).
T124 16617-16718 Sentence denotes Two separate amplification reactions were performed, generating two fragments, F1 and F2 (Figure 3A).
T7836 16719-17005 Sentence denotes After digestion with HpaII and Bsp143II a sufficient PCR amplification of F1 and F2 was detected in DNA from IRF-4-negative Jurkat, CML-T1, U-937, K-562 and EM-2 cells, suggesting a promoter methylation (and restriction protection) at the respective recognition sites (Figure 3B and C).
T125 16719-17005 Sentence denotes After digestion with HpaII and Bsp143II a sufficient PCR amplification of F1 and F2 was detected in DNA from IRF-4-negative Jurkat, CML-T1, U-937, K-562 and EM-2 cells, suggesting a promoter methylation (and restriction protection) at the respective recognition sites (Figure 3B and C).
T7837 17006-17141 Sentence denotes Notably, in IRF-4-positive SD-1 cells digestion with the methylation-sensitive enzymes completely inhibited amplification of F1 and F2.
T126 17006-17141 Sentence denotes Notably, in IRF-4-positive SD-1 cells digestion with the methylation-sensitive enzymes completely inhibited amplification of F1 and F2.
T7838 17142-17338 Sentence denotes In IRF-4-positive BV-173 cells a HpaII, but not a Bsh1236I digestion, significantly reduced the amplifiable DNA message of F2 (Figure 3C), whereas amplification of F1 was not affected (Figure 3B).
T127 17142-17338 Sentence denotes In IRF-4-positive BV-173 cells a HpaII, but not a Bsh1236I digestion, significantly reduced the amplifiable DNA message of F2 (Figure 3C), whereas amplification of F1 was not affected (Figure 3B).
T7839 17339-17534 Sentence denotes This implied that IRF-4 transcription in SD-1 and BV-173 cells is associated with less promoter methylation (in BV-173 especially at HpaII sites) as compared with the tested IRF-4-negative cells.
T128 17339-17534 Sentence denotes This implied that IRF-4 transcription in SD-1 and BV-173 cells is associated with less promoter methylation (in BV-173 especially at HpaII sites) as compared with the tested IRF-4-negative cells.
T8559 17536-17614 Sentence denotes Specific CpG sites in the IRF-4 promoter are methylated in hematopoietic cells
T129 17536-17614 Sentence denotes Specific CpG sites in the IRF-4 promoter are methylated in hematopoietic cells
T8560 17615-17946 Sentence denotes In order to exactly map the methylation sites within the IRF-4 promoter, we treated DNA of Jurkat, CML-T1, U-937, K-562 and EM-2 cells as well as of SD-1, RPMI-8226 and BV-173 control cells with bisulfite, which chemically converts unmethylated cytosine to uracil, whereas it has no effect on methylated cytosine, i.e. in CpG (34).
T130 17615-17946 Sentence denotes In order to exactly map the methylation sites within the IRF-4 promoter, we treated DNA of Jurkat, CML-T1, U-937, K-562 and EM-2 cells as well as of SD-1, RPMI-8226 and BV-173 control cells with bisulfite, which chemically converts unmethylated cytosine to uracil, whereas it has no effect on methylated cytosine, i.e. in CpG (34).
T8561 17947-18029 Sentence denotes This technique is especially useful for detection of unknown methylation patterns.
T131 17947-18029 Sentence denotes This technique is especially useful for detection of unknown methylation patterns.
T8562 18030-18209 Sentence denotes PCR amplification, cloning and sequencing of the bisulfite-treated DNA showed a specific methylation pattern of the analyzed 62 CpG sites in all cell lines (Figure 4 and Table 1).
T132 18030-18209 Sentence denotes PCR amplification, cloning and sequencing of the bisulfite-treated DNA showed a specific methylation pattern of the analyzed 62 CpG sites in all cell lines (Figure 4 and Table 1).
T8563 18210-18409 Sentence denotes In general, the methylational status ranged from one cell line with a nearly non-methylated IRF-4 promoter (SD-1, IRF-4-positive) to a completely methylated IRF-4 promoter in CML-T1 (IRF-4-negative).
T133 18210-18409 Sentence denotes In general, the methylational status ranged from one cell line with a nearly non-methylated IRF-4 promoter (SD-1, IRF-4-positive) to a completely methylated IRF-4 promoter in CML-T1 (IRF-4-negative).
T8564 18410-18604 Sentence denotes Interestingly, the percentage of CpG methylation in the IRF-4 promoter from IRF-4-positive cells was very low (mean 24%) as compared with IRF-4-negative cells (mean 94%) (Figure 4A and Table 1).
T134 18410-18604 Sentence denotes Interestingly, the percentage of CpG methylation in the IRF-4 promoter from IRF-4-positive cells was very low (mean 24%) as compared with IRF-4-negative cells (mean 94%) (Figure 4A and Table 1).
T8565 18605-18971 Sentence denotes A 5′-region (R1) with 13 hypermethylated CpG sites (mean number of methylated clones 5.5 of 8 with 77% methylated CpGs) was found in most cells (except SD-1 and RPMI-8226) and a 3′-region (R3) of 6 hypomethylated CpG sites (mean number of methylated clones 1.7 of 8 with 33% methylated CpGs) was found in most cells (except CML-T1 and U-937) (Figure 4A and Table 1).
T135 18605-18971 Sentence denotes A 5′-region (R1) with 13 hypermethylated CpG sites (mean number of methylated clones 5.5 of 8 with 77% methylated CpGs) was found in most cells (except SD-1 and RPMI-8226) and a 3′-region (R3) of 6 hypomethylated CpG sites (mean number of methylated clones 1.7 of 8 with 33% methylated CpGs) was found in most cells (except CML-T1 and U-937) (Figure 4A and Table 1).
T8566 18972-19249 Sentence denotes Intriguingly, a stretch of 13 CpG sites (#10–22; R2) was detected in between these regions, which were highly methylated in IRF-4-negative (mean number of methylated clones 7.1 of 8 with 89% methylated CpGs) but totally non-methylated in IRF-4-positive cells (Figure 4A and B).
T136 18972-19249 Sentence denotes Intriguingly, a stretch of 13 CpG sites (#10–22; R2) was detected in between these regions, which were highly methylated in IRF-4-negative (mean number of methylated clones 7.1 of 8 with 89% methylated CpGs) but totally non-methylated in IRF-4-positive cells (Figure 4A and B).
T8567 19250-19443 Sentence denotes Furthermore, three CpG sites at the 5′ end (#54, 56, 58) and two CpG motifs at the 3′ end (#1, 2) showed this direct correlation between high methylation status and absence of IRF-4 expression.
T137 19250-19443 Sentence denotes Furthermore, three CpG sites at the 5′ end (#54, 56, 58) and two CpG motifs at the 3′ end (#1, 2) showed this direct correlation between high methylation status and absence of IRF-4 expression.
T8568 19444-19620 Sentence denotes In addition, two CpG sites located in a NFκB (#48) and a SP1 element (#45) are less methylated in IRF-4-positive than in IRF-4-negative cells (mean number of methylated clones:
T138 19444-19620 Sentence denotes In addition, two CpG sites located in a NFκB (#48) and a SP1 element (#45) are less methylated in IRF-4-positive than in IRF-4-negative cells (mean number of methylated clones:
T8569 19621-19637 Sentence denotes 1/8 versus 8/8).
T139 19621-19637 Sentence denotes 1/8 versus 8/8).
T8570 19638-19752 Sentence denotes These results indicate the involvement of CpG methylation in the regulation of IRF-4 expression in leukemic cells.
T140 19638-19752 Sentence denotes These results indicate the involvement of CpG methylation in the regulation of IRF-4 expression in leukemic cells.
T9690 19754-19837 Sentence denotes In vitro methylation of an IRF-4 promoter-reporter construct decreases its activity
T141 19754-19837 Sentence denotes In vitro methylation of an IRF-4 promoter-reporter construct decreases its activity
T9691 19838-20038 Sentence denotes To provide evidence for a direct effect of methylational status on IRF-4 promoter activity we performed reporter gene assays with IRF-4 promoter constructs before and after their in vitro methylation.
T142 19838-20038 Sentence denotes To provide evidence for a direct effect of methylational status on IRF-4 promoter activity we performed reporter gene assays with IRF-4 promoter constructs before and after their in vitro methylation.
T9692 20039-20170 Sentence denotes A complete methylation of these constructs was checked via restriction assays with methylation-sensitive endonucleases (Figure 5A).
T143 20039-20170 Sentence denotes A complete methylation of these constructs was checked via restriction assays with methylation-sensitive endonucleases (Figure 5A).
T9693 20171-20311 Sentence denotes Intriguingly, methylation of the IRF-4 promoter significantly decreased promoter activity in IRF-4-positive SD-1 cells by 85.0% (Figure 5B).
T144 20171-20311 Sentence denotes Intriguingly, methylation of the IRF-4 promoter significantly decreased promoter activity in IRF-4-positive SD-1 cells by 85.0% (Figure 5B).
T9694 20312-20513 Sentence denotes The silencing effect of CpG methylation was not restricted to IRF-4-positive cells, since in vitro methylation led to a 92.9% abrogation of promoter activity in IRF-4-negative Jurkat cells (Figure 5C).
T145 20312-20513 Sentence denotes The silencing effect of CpG methylation was not restricted to IRF-4-positive cells, since in vitro methylation led to a 92.9% abrogation of promoter activity in IRF-4-negative Jurkat cells (Figure 5C).
T9695 20514-20687 Sentence denotes In contrast, control methylation of a reporter construct with a different promoter (FasL) as well as an empty vector had no effect on the reporter activity (data not shown).
T146 20514-20687 Sentence denotes In contrast, control methylation of a reporter construct with a different promoter (FasL) as well as an empty vector had no effect on the reporter activity (data not shown).
T9696 20688-20782 Sentence denotes These data proved a direct association between methylation and activity of the IRF-4 promoter.
T147 20688-20782 Sentence denotes These data proved a direct association between methylation and activity of the IRF-4 promoter.
T10244 20784-20911 Sentence denotes mRNA expression of DNA methyltransferases and methyl-CpG-binding proteins may not be associated with IRF-4 promoter methylation
T148 20784-20911 Sentence denotes mRNA expression of DNA methyltransferases and methyl-CpG-binding proteins may not be associated with IRF-4 promoter methylation
T10245 20912-21138 Sentence denotes Since abundance of DNMT and MBP contribute to promoter regulation via methylation (25,26,28), we studied their mRNA expression to investigate a possible mechanism for the observed methylation differences in the IRF-4 promoter.
T149 20912-21138 Sentence denotes Since abundance of DNMT and MBP contribute to promoter regulation via methylation (25,26,28), we studied their mRNA expression to investigate a possible mechanism for the observed methylation differences in the IRF-4 promoter.
T10246 21139-21336 Sentence denotes To this end, we did not detect a significant difference in DNMT (DNMT1, DNMT3A and DNMT3B) or MBP (MBD1, MBD2, MBD4 and MeCP) mRNA expression between IRF-4-positive and -negative cells (Figure 5D).
T150 21139-21336 Sentence denotes To this end, we did not detect a significant difference in DNMT (DNMT1, DNMT3A and DNMT3B) or MBP (MBD1, MBD2, MBD4 and MeCP) mRNA expression between IRF-4-positive and -negative cells (Figure 5D).
T10247 21337-21492 Sentence denotes In fact, all analyzed cells had moderate to high mRNA levels of these tested DNMT/MBPs and differences in expression were not correlated with IRF-4 status.
T151 21337-21492 Sentence denotes In fact, all analyzed cells had moderate to high mRNA levels of these tested DNMT/MBPs and differences in expression were not correlated with IRF-4 status.
T10248 21493-21661 Sentence denotes These results indicate a distinct cause of the methylation differences in IRF-4-positive and -negative cells rather than changes in the DNMT and MBP mRNA transcription.
T152 21493-21661 Sentence denotes These results indicate a distinct cause of the methylation differences in IRF-4-positive and -negative cells rather than changes in the DNMT and MBP mRNA transcription.
T153 21663-21673 Sentence denotes DISCUSSION
T11278 21674-21760 Sentence denotes Many genetic lesions are known to influence gene expression of tumor suppressor genes.
T154 21674-21760 Sentence denotes Many genetic lesions are known to influence gene expression of tumor suppressor genes.
T11279 21761-21943 Sentence denotes Whereas mutations and deletions or insertions have permanent effects, reversible mechanisms are gene methylation, or expression and activation of transcription factors, respectively.
T155 21761-21943 Sentence denotes Whereas mutations and deletions or insertions have permanent effects, reversible mechanisms are gene methylation, or expression and activation of transcription factors, respectively.
T11280 21944-22075 Sentence denotes We studied a putative cause for absent IRF-4 expression in leukemia cells and first focused on genetic aberrations of the promoter.
T156 21944-22075 Sentence denotes We studied a putative cause for absent IRF-4 expression in leukemia cells and first focused on genetic aberrations of the promoter.
T11281 22076-22185 Sentence denotes We observed no genetic alterations in the IRF-4 promoter, which can account for the lack of IRF-4 expression:
T157 22076-22185 Sentence denotes We observed no genetic alterations in the IRF-4 promoter, which can account for the lack of IRF-4 expression:
T11282 22186-22597 Sentence denotes The detected base pair changes at position −1081 (T→C substitution), at position −1068 (A→C substitution) and at position −116 (A→C substitution) are unlikely responsible for absent IRF-4-expression since the first two mutations were found both in IRF-4-positive and -negative cells whereas the latter change was not detected consistently in all IRF-4-negative or -positive cells and may thus be a polymorphism.
T158 22186-22597 Sentence denotes The detected base pair changes at position −1081 (T→C substitution), at position −1068 (A→C substitution) and at position −116 (A→C substitution) are unlikely responsible for absent IRF-4-expression since the first two mutations were found both in IRF-4-positive and -negative cells whereas the latter change was not detected consistently in all IRF-4-negative or -positive cells and may thus be a polymorphism.
T11283 22598-22787 Sentence denotes All three substitutions did not change any known putative transcription factor binding sites (30,31) and also do not affect any restriction sites or primer binding sites of the used assays.
T159 22598-22787 Sentence denotes All three substitutions did not change any known putative transcription factor binding sites (30,31) and also do not affect any restriction sites or primer binding sites of the used assays.
T11284 22788-22956 Sentence denotes However, permanent genetic variations in the IRF-4 coding sequence, such as deletions or mutations resulting in stop-codons have not been excluded by sequence analysis.
T160 22788-22956 Sentence denotes However, permanent genetic variations in the IRF-4 coding sequence, such as deletions or mutations resulting in stop-codons have not been excluded by sequence analysis.
T11285 22957-23139 Sentence denotes Since IRF-4 expression in cell lines and CML can be induced by demethylation and successful IFN-α therapy (3), respectively, the existence of such genetic aberrations seems unlikely.
T161 22957-23139 Sentence denotes Since IRF-4 expression in cell lines and CML can be induced by demethylation and successful IFN-α therapy (3), respectively, the existence of such genetic aberrations seems unlikely.
T11286 23140-23459 Sentence denotes We then investigated whether the previously described down-regulation of IRF-4 expression in human myeloid leukemias was due to a differential hypermethylation of the promoter, since the presented re-expression due to AzadC-treatment might also be a result of activation of positive transcriptional regulators of IRF-4.
T162 23140-23459 Sentence denotes We then investigated whether the previously described down-regulation of IRF-4 expression in human myeloid leukemias was due to a differential hypermethylation of the promoter, since the presented re-expression due to AzadC-treatment might also be a result of activation of positive transcriptional regulators of IRF-4.
T11287 23460-23641 Sentence denotes Methylation of CpG sites is a common mechanism of silencing genes in leukemia and has also been shown for another IRF, IRF-7 (35) and for PU.1 (36), an interacting partner of IRF-4.
T163 23460-23641 Sentence denotes Methylation of CpG sites is a common mechanism of silencing genes in leukemia and has also been shown for another IRF, IRF-7 (35) and for PU.1 (36), an interacting partner of IRF-4.
T11288 23642-23844 Sentence denotes To elucidate the relevance of this mechanism for the regulation of IRF-4 expression, various leukemic cells were treated with demethylating agents and promoters were sequenced after bisulfite treatment.
T164 23642-23844 Sentence denotes To elucidate the relevance of this mechanism for the regulation of IRF-4 expression, various leukemic cells were treated with demethylating agents and promoters were sequenced after bisulfite treatment.
T11289 23845-23973 Sentence denotes We found that IRF-4 expression could indeed be connected to the methylation status of distinct CpG motifs in the IRF-4 promoter.
T165 23845-23973 Sentence denotes We found that IRF-4 expression could indeed be connected to the methylation status of distinct CpG motifs in the IRF-4 promoter.
T11290 23974-24124 Sentence denotes In Figure 4A, those CpG sites are shown (bottom line), whose hypermethylation may account for the absence of IRF-4 expression in the respective cells.
T166 23974-24124 Sentence denotes In Figure 4A, those CpG sites are shown (bottom line), whose hypermethylation may account for the absence of IRF-4 expression in the respective cells.
T11291 24125-24251 Sentence denotes One of them (#54) is adjacent to an identified regulatory element (NFκB-site), indicating a possible involvement of this site.
T167 24125-24251 Sentence denotes One of them (#54) is adjacent to an identified regulatory element (NFκB-site), indicating a possible involvement of this site.
T11292 24252-24372 Sentence denotes At two further CpG sites (#48, 45) the methylation status in IRF-4-positive was lower than that of IRF-4-negative cells.
T168 24252-24372 Sentence denotes At two further CpG sites (#48, 45) the methylation status in IRF-4-positive was lower than that of IRF-4-negative cells.
T11293 24373-24500 Sentence denotes These CpG sites are located in an NFκB and an SP1 element (31) and thus may also play a role in regulation of IRF-4 expression.
T169 24373-24500 Sentence denotes These CpG sites are located in an NFκB and an SP1 element (31) and thus may also play a role in regulation of IRF-4 expression.
T11294 24501-24695 Sentence denotes It has been shown that NFκB elements play an important role in IRF-4 induction as IRF-4 expression depends on binding of the transactivator c-Rel to these elements in the IRF-4 promoter (31,37).
T170 24501-24695 Sentence denotes It has been shown that NFκB elements play an important role in IRF-4 induction as IRF-4 expression depends on binding of the transactivator c-Rel to these elements in the IRF-4 promoter (31,37).
T11295 24696-24918 Sentence denotes Furthermore, methylation of the central CpG in the NFκB element inhibits binding of the NFκB protein complexes (38), promoting the significance of the observed methylation differences in IRF-4-positive and -negative cells.
T171 24696-24918 Sentence denotes Furthermore, methylation of the central CpG in the NFκB element inhibits binding of the NFκB protein complexes (38), promoting the significance of the observed methylation differences in IRF-4-positive and -negative cells.
T11296 24919-25117 Sentence denotes Via in vitro methylation and reporter gene assays we could clearly appoint the silencing of the IRF-4 promoter to a methylation effect, which may thus be the mechanism of IRF-4 deregulation in vivo.
T172 24919-25117 Sentence denotes Via in vitro methylation and reporter gene assays we could clearly appoint the silencing of the IRF-4 promoter to a methylation effect, which may thus be the mechanism of IRF-4 deregulation in vivo.
T11297 25118-25246 Sentence denotes One possible cause for the aberrant methylation in tumorigenesis is an increased level of DNMTs during the pathogenetic process.
T173 25118-25246 Sentence denotes One possible cause for the aberrant methylation in tumorigenesis is an increased level of DNMTs during the pathogenetic process.
T11298 25247-25363 Sentence denotes In colon, lung and hematologic malignancies, overexpression of DNMT1, a maintenance DNMT, has been detected (39–41).
T174 25247-25363 Sentence denotes In colon, lung and hematologic malignancies, overexpression of DNMT1, a maintenance DNMT, has been detected (39–41).
T11299 25364-25594 Sentence denotes Furthermore, it has been shown that CML cells in the acute phase exhibited elevated levels of the three known DNMTs, while CML cells in chronic phase expressed normal levels of DNMTs if compared with normal bone marrow cells (25).
T175 25364-25594 Sentence denotes Furthermore, it has been shown that CML cells in the acute phase exhibited elevated levels of the three known DNMTs, while CML cells in chronic phase expressed normal levels of DNMTs if compared with normal bone marrow cells (25).
T11300 25595-25728 Sentence denotes Interestingly, a positive correlation between DNMT1 expression levels and hypermethylation of p15INK4b has been detected in AML (25).
T176 25595-25728 Sentence denotes Interestingly, a positive correlation between DNMT1 expression levels and hypermethylation of p15INK4b has been detected in AML (25).
T11301 25729-25939 Sentence denotes In this work, we did not detect significant mRNA expression differences of selected DNMT or MBP, making it an unlikely cause for the observed methylation and thus IRF-4 expression differences in leukemia cells.
T177 25729-25939 Sentence denotes In this work, we did not detect significant mRNA expression differences of selected DNMT or MBP, making it an unlikely cause for the observed methylation and thus IRF-4 expression differences in leukemia cells.
T11302 25940-26117 Sentence denotes The finding that IRF-4 expression is silenced by promoter hypermethylation might represent a mechanism that accounts for the previously observed loss of IRF-4 expression in CML.
T178 25940-26117 Sentence denotes The finding that IRF-4 expression is silenced by promoter hypermethylation might represent a mechanism that accounts for the previously observed loss of IRF-4 expression in CML.
T11303 26118-26310 Sentence denotes Indeed, several clinical trials with leukemia patients and patients with myelodysplastic syndromes demonstrated the potential clinical benefit of a treatment with demethylating agents (42–45).
T179 26118-26310 Sentence denotes Indeed, several clinical trials with leukemia patients and patients with myelodysplastic syndromes demonstrated the potential clinical benefit of a treatment with demethylating agents (42–45).
T11304 26311-26455 Sentence denotes The expression of another IRF, IFN consensus sequence binding protein (ICSBP/IRF-8), is impaired in myeloid leukemias especially CML (27,46,47).
T180 26311-26455 Sentence denotes The expression of another IRF, IFN consensus sequence binding protein (ICSBP/IRF-8), is impaired in myeloid leukemias especially CML (27,46,47).
T11305 26456-26704 Sentence denotes But in contrast to IRF-4, the loss of this IRF could not be reverted in ICSBP-negative cell lines (EM-2, CML-T1, K-562 and LAMA-84) by treatment with AzadC (Figure 6) and AzadC has no effect on ICSBP levels in ICSBP-positive U-937 cells (Figure 6).
T181 26456-26704 Sentence denotes But in contrast to IRF-4, the loss of this IRF could not be reverted in ICSBP-negative cell lines (EM-2, CML-T1, K-562 and LAMA-84) by treatment with AzadC (Figure 6) and AzadC has no effect on ICSBP levels in ICSBP-positive U-937 cells (Figure 6).
T11306 26705-26775 Sentence denotes These data suggest a distinct regulatory mechanism for these two IRFs.
T182 26705-26775 Sentence denotes These data suggest a distinct regulatory mechanism for these two IRFs.
T11307 26776-27038 Sentence denotes IRF-4, similar to many other classical tumor suppressor genes p15INK4b, p16INK4a or p53, may thus be a subject of alterations in the promoter methylation status leading to expression changes, which might contribute to the initiation and/or progression of cancer.
T183 26776-27038 Sentence denotes IRF-4, similar to many other classical tumor suppressor genes p15INK4b, p16INK4a or p53, may thus be a subject of alterations in the promoter methylation status leading to expression changes, which might contribute to the initiation and/or progression of cancer.
T11308 27039-27178 Sentence denotes Still, the obvious functional diversity of IRF-4 remains remarkable and cannot be fully explained by the IRF-4 promoter methylation status.
T184 27039-27178 Sentence denotes Still, the obvious functional diversity of IRF-4 remains remarkable and cannot be fully explained by the IRF-4 promoter methylation status.
T11309 27179-27244 Sentence denotes For example, IRF-4 is primarily known for its oncogenic features.
T185 27179-27244 Sentence denotes For example, IRF-4 is primarily known for its oncogenic features.
T11310 27245-27446 Sentence denotes In multiple myeloma (MM) a translocation on chromosome 14q was reported to lead to a fusion gene of immunoglobulin heavy-chain (IgH) and IRF-4 resulting in a subsequent overexpression of IRF-4 (48,49).
T186 27245-27446 Sentence denotes In multiple myeloma (MM) a translocation on chromosome 14q was reported to lead to a fusion gene of immunoglobulin heavy-chain (IgH) and IRF-4 resulting in a subsequent overexpression of IRF-4 (48,49).
T11311 27447-27680 Sentence denotes In addition, abundant IRF-4 expression was found to be a marker for various subsets of lymphomas, such as diffuse large B-cell lymphomas, primary effusion lymphoma, and marginal zone lymphoma, and adult T-cell leukemia (11,31,50–52).
T187 27447-27680 Sentence denotes In addition, abundant IRF-4 expression was found to be a marker for various subsets of lymphomas, such as diffuse large B-cell lymphomas, primary effusion lymphoma, and marginal zone lymphoma, and adult T-cell leukemia (11,31,50–52).
T11312 27681-27736 Sentence denotes This draws a more complex picture of the role of IRF-4.
T188 27681-27736 Sentence denotes This draws a more complex picture of the role of IRF-4.
T11313 27737-27976 Sentence denotes Down-regulation of IRF-4 may promote leukemogenesis in myeloid cell context (3), which was recently confirmed in IRF-4−/− ICSBP−/− double knock-out mice (53), while IRF-4 up-regulation may induce a growth advantage in lymphomas or MM (48).
T189 27737-27976 Sentence denotes Down-regulation of IRF-4 may promote leukemogenesis in myeloid cell context (3), which was recently confirmed in IRF-4−/− ICSBP−/− double knock-out mice (53), while IRF-4 up-regulation may induce a growth advantage in lymphomas or MM (48).
T11314 27977-28193 Sentence denotes Taken together, our data suggest that IRF-4 promoter methylation regulates IRF-4 expression, and that aberrant expression of IRF-4 in certain types of leukemia may be a consequence of IRF-4 promoter hypermethylation.
T190 27977-28193 Sentence denotes Taken together, our data suggest that IRF-4 promoter methylation regulates IRF-4 expression, and that aberrant expression of IRF-4 in certain types of leukemia may be a consequence of IRF-4 promoter hypermethylation.