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0 | PMC-1310901-00-TIAB | [
{
"id": "PMC-1310901-00-TIAB__text",
"type": "abstract",
"text": [
"Down-regulation of interferon regulatory factor 4 gene expression in leukemic cells due to hypermethylation of CpG motifs in the promoter region \nAlthough 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. Promoter methylation of CpG target sites or direct deletions/insertions of genes are mechanisms of a reversible or permanent silencing of gene expression, respectively. Therefore, we investigated whether IRF-4 promoter methylation or mutation may be involved in the regulation of IRF-4 expression in leukemia cells. 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. 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. 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. 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. Together, these data suggest CpG site-specific IRF-4 promoter methylation as a putative mechanism of down-regulated IRF-4 expression in leukemia. \n"
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"interferon regulatory factor 4"
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"type": "Protein",
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"text": [
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{
"id": "PMC-1310901-00-TIAB_E1",
"type": "Negative_regulation",
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"Down-regulation"
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"role": "Theme",
"ref_id": "PMC-1310901-00-TIAB_E2"
}
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"type": "Gene_expression",
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"expression"
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"role": "Theme",
"ref_id": "PMC-1310901-00-TIAB_T1"
}
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{
"id": "PMC-1310901-00-TIAB_E3",
"type": "Regulation",
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"text": [
"deregulation"
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"role": "Theme",
"ref_id": "PMC-1310901-00-TIAB_T4"
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"id": "PMC-1310901-00-TIAB_E4",
"type": "Regulation",
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"regulation"
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"role": "Theme",
"ref_id": "PMC-1310901-00-TIAB_E5"
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"type": "Gene_expression",
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"expression"
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"role": "Theme",
"ref_id": "PMC-1310901-00-TIAB_T7"
}
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"altered"
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"expression"
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"role": "Theme",
"ref_id": "PMC-1310901-00-TIAB_T8"
}
]
},
{
"id": "PMC-1310901-00-TIAB_E8",
"type": "Regulation",
"trigger": {
"text": [
"influence"
],
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},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-00-TIAB_E9"
}
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"role": "Theme",
"ref_id": "PMC-1310901-00-TIAB_T10"
}
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"id": "PMC-1310901-00-TIAB_E10",
"type": "Gene_expression",
"trigger": {
"text": [
"negative"
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]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-00-TIAB_T11"
}
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},
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"id": "PMC-1310901-00-TIAB_E11",
"type": "Positive_regulation",
"trigger": {
"text": [
"resulted"
],
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"ref_id": "PMC-1310901-00-TIAB_E12"
}
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},
{
"id": "PMC-1310901-00-TIAB_E12",
"type": "Positive_regulation",
"trigger": {
"text": [
"increase"
],
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"ref_id": "PMC-1310901-00-TIAB_T12"
}
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"text": [
"negative"
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}
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"text": [
"positive"
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}
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},
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"type": "Negative_regulation",
"trigger": {
"text": [
"down-regulation"
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}
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"down-regulated"
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}
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"type": "Gene_expression",
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"expression"
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"role": "Theme",
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}
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}
] | [
{
"id": "PMC-1310901-00-TIAB_1",
"entity_ids": [
"PMC-1310901-00-TIAB_T4",
"PMC-1310901-00-TIAB_T5"
]
}
] | [] |
1 | PMC-1310901-01-INTRODUCTION | [
{
"id": "PMC-1310901-01-INTRODUCTION__text",
"type": "abstract",
"text": [
"INTRODUCTION\nChronic 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). 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). Treatment with interferon alpha (IFN-alpha) prolongs survival of CML patients and is associated with a complete cytogenetic response in 5-33% of CML patients (1,2). Recently, we described an impaired expression of the interferon regulatory factor 4 (IRF-4) in CML, correlating with poor response to IFN-alpha treatment (3). The cause of the silencing of IRF-4 level remained unclear. \nInterferon regulatory factors (IRFs) are a family of transcriptional regulators defined by a characteristic homology in their DNA-binding domain. 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). IRF-4 (ICSAT/Pip/MUM1/LSIRF) is one member with very restricted expression pattern: Predominately B- and activated T-lymphocytes are IRF-4 positive (7-11). 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). 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). These data suggest a crucial role for IRF-4 in the function of immune cells. \nMethylation 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). As opposed to normal cells, hypermethylation of CpG islands is a frequently observed phenomenon in every cancer type. 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). Examples for such aberrated genes are MGMT, DAPK, p14ARF, p15INK4b, p16INK4a, BRCA1, CDH13 and APAF-1 (17-19). 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). Hypermethylation due to overexpression of DNA methyltransferases (DNMTs) remains one possible explanation for de novo methylation in tumorigenesis. Recently, DNMTs have been shown to be up-regulated in hematopoietic malignancies (25). 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). \nIn this work, we studied mechanisms of IRF-4 gene expression silencing in leukemic cells. We analyzed the IRF-4 promoter region for genetic aberrations and methylational status in IRF-4-positive and -negative hematopoietic cells. \n"
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"type": "Protein",
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"text": [
"interferon alpha"
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"type": "Protein",
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"IFN-alpha"
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"type": "Protein",
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"MGMT"
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"DAPK"
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"type": "Protein",
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"type": "Protein",
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"type": "Protein",
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"type": "Protein",
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"text": [
"APAF-1"
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{
"id": "PMC-1310901-01-INTRODUCTION_T26",
"type": "Protein",
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"c-abl"
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"type": "Protein",
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"bcr"
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"text": [
"IRF-4"
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"normalized": []
}
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"trigger": {
"text": [
"impaired"
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"role": "Theme",
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}
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"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-01-INTRODUCTION_T4"
}
]
},
{
"id": "PMC-1310901-01-INTRODUCTION_E5",
"type": "Negative_regulation",
"trigger": {
"text": [
"silencing"
],
"offsets": [
[
806,
815
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-01-INTRODUCTION_T7"
}
]
},
{
"id": "PMC-1310901-01-INTRODUCTION_E6",
"type": "Gene_expression",
"trigger": {
"text": [
"expression"
],
"offsets": [
[
1217,
1227
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-01-INTRODUCTION_T8"
}
]
},
{
"id": "PMC-1310901-01-INTRODUCTION_E7",
"type": "Gene_expression",
"trigger": {
"text": [
"positive"
],
"offsets": [
[
1292,
1300
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-01-INTRODUCTION_T13"
}
]
},
{
"id": "PMC-1310901-01-INTRODUCTION_E8",
"type": "Gene_expression",
"trigger": {
"text": [
"expression"
],
"offsets": [
[
1336,
1346
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-01-INTRODUCTION_T14"
}
]
},
{
"id": "PMC-1310901-01-INTRODUCTION_E9",
"type": "Positive_regulation",
"trigger": {
"text": [
"induced"
],
"offsets": [
[
1366,
1373
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-01-INTRODUCTION_E8"
}
]
},
{
"id": "PMC-1310901-01-INTRODUCTION_E10",
"type": "Negative_regulation",
"trigger": {
"text": [
"deletion"
],
"offsets": [
[
1570,
1578
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-01-INTRODUCTION_T15"
}
]
},
{
"id": "PMC-1310901-01-INTRODUCTION_E11",
"type": "Gene_expression",
"trigger": {
"text": [
"expression"
],
"offsets": [
[
1681,
1691
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-01-INTRODUCTION_T16"
}
]
},
{
"id": "PMC-1310901-01-INTRODUCTION_E12",
"type": "Regulation",
"trigger": {
"text": [
"regulate"
],
"offsets": [
[
2488,
2496
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-01-INTRODUCTION_E13"
}
]
},
{
"id": "PMC-1310901-01-INTRODUCTION_E14",
"type": "Regulation",
"trigger": {
"text": [
"regulate"
],
"offsets": [
[
2488,
2496
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-01-INTRODUCTION_E15"
}
]
},
{
"id": "PMC-1310901-01-INTRODUCTION_E13",
"type": "Gene_expression",
"trigger": {
"text": [
"expression"
],
"offsets": [
[
2497,
2507
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-01-INTRODUCTION_T26"
}
]
},
{
"id": "PMC-1310901-01-INTRODUCTION_E15",
"type": "Gene_expression",
"trigger": {
"text": [
"expression"
],
"offsets": [
[
2497,
2507
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-01-INTRODUCTION_T27"
}
]
},
{
"id": "PMC-1310901-01-INTRODUCTION_E16",
"type": "Gene_expression",
"trigger": {
"text": [
"expression"
],
"offsets": [
[
3165,
3175
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-01-INTRODUCTION_T29"
}
]
},
{
"id": "PMC-1310901-01-INTRODUCTION_E17",
"type": "Negative_regulation",
"trigger": {
"text": [
"silencing"
],
"offsets": [
[
3176,
3185
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-01-INTRODUCTION_E16"
}
]
},
{
"id": "PMC-1310901-01-INTRODUCTION_E18",
"type": "Gene_expression",
"trigger": {
"text": [
"positive"
],
"offsets": [
[
3301,
3309
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-01-INTRODUCTION_T31"
}
]
},
{
"id": "PMC-1310901-01-INTRODUCTION_E19",
"type": "Gene_expression",
"trigger": {
"text": [
"negative"
],
"offsets": [
[
3315,
3323
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-01-INTRODUCTION_T31"
}
]
}
] | [
{
"id": "PMC-1310901-01-INTRODUCTION_1",
"entity_ids": [
"PMC-1310901-01-INTRODUCTION_T2",
"PMC-1310901-01-INTRODUCTION_T3"
]
},
{
"id": "PMC-1310901-01-INTRODUCTION_2",
"entity_ids": [
"PMC-1310901-01-INTRODUCTION_T4",
"PMC-1310901-01-INTRODUCTION_T5"
]
},
{
"id": "PMC-1310901-01-INTRODUCTION_3",
"entity_ids": [
"PMC-1310901-01-INTRODUCTION_T8",
"PMC-1310901-01-INTRODUCTION_T10",
"PMC-1310901-01-INTRODUCTION_T11",
"PMC-1310901-01-INTRODUCTION_T9",
"PMC-1310901-01-INTRODUCTION_T12"
]
}
] | [] |
2 | PMC-1310901-02-MATERIALS_AND_METHODS-01 | [
{
"id": "PMC-1310901-02-MATERIALS_AND_METHODS-01__text",
"type": "abstract",
"text": [
"Cell lines\nK-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). All cell lines, except BV-173, SD-1 and RPMI-8226, were IRF-4-negative. \n"
],
"offsets": [
[
0,
341
]
]
}
] | [
{
"id": "PMC-1310901-02-MATERIALS_AND_METHODS-01_T1",
"type": "Protein",
"offsets": [
[
324,
329
]
],
"text": [
"IRF-4"
],
"normalized": []
}
] | [
{
"id": "PMC-1310901-02-MATERIALS_AND_METHODS-01_E1",
"type": "Gene_expression",
"trigger": {
"text": [
"negative"
],
"offsets": [
[
330,
338
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-02-MATERIALS_AND_METHODS-01_T1"
}
]
}
] | [] | [] |
3 | PMC-1310901-03-MATERIALS_AND_METHODS-02 | [
{
"id": "PMC-1310901-03-MATERIALS_AND_METHODS-02__text",
"type": "abstract",
"text": [
"Cell culture and stimulation\nAll 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). When indicated, cells were treated with 5-aza-2-deoxycytidine (AzadC) or 5-azacytidine (AzaC) (Sigma, Taufkirchen, Germany) for different time periods. Owing to their chemical instability fresh substances were re-added every 24 h. \n"
],
"offsets": [
[
0,
480
]
]
}
] | [] | [] | [] | [] |
4 | PMC-1310901-04-MATERIALS_AND_METHODS-03 | [
{
"id": "PMC-1310901-04-MATERIALS_AND_METHODS-03__text",
"type": "abstract",
"text": [
"Sequencing of the IRF-4 promoter\nFor 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. The primers were 1-forward: 5'-TTGAGATGGAGTCTTGCTCTGT-3', 1-reverse: 5'-CCAGGACCTCAGGAGGCCAGTCA-3'; 2-forward: 5'-AGCGGTGAAACTGAGAGTGCGAGGT-3', 2-reverse: 5'-GCCACATCGCTGCAGTTTAG-3'. The products were cloned with the 'TOPO TA cloning kit' (Invitrogen, Groningen, The Netherlands). 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. \n"
],
"offsets": [
[
0,
837
]
]
}
] | [
{
"id": "PMC-1310901-04-MATERIALS_AND_METHODS-03_T1",
"type": "Protein",
"offsets": [
[
18,
23
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-04-MATERIALS_AND_METHODS-03_T2",
"type": "Protein",
"offsets": [
[
53,
58
]
],
"text": [
"IRF-4"
],
"normalized": []
}
] | [] | [] | [] |
5 | PMC-1310901-05-MATERIALS_AND_METHODS-04 | [
{
"id": "PMC-1310901-05-MATERIALS_AND_METHODS-04__text",
"type": "abstract",
"text": [
"Expression analysis\nTo analyze the IRF-4 transcriptional level, RNA was extracted from cells using the commercial RNAzol-kit (Paesel, Frankfurt, Germany). An aliquot of 1 mug total RNA was used for cDNA synthesis as described previously (27). RNA expression analysis for IRF-4 and the reference gene beta-actin was carried out by semi-quantitative PCR as described previously (3,27). PCR products were verified by automated sequencing. PCR primers and conditions for expression analysis of DNMT or MBP (DNMT1 DNMT3A, DNMT3B, MeCP, MBD1, MBD2 and MBD4) were published elsewhere (28). \nFor analysis of IRF-4 protein expression, a standard immunoblotting assay was performed as described previously (29). Briefly, protein lysates were generated by incubating 1 x 106 cells in 100 microl RIPA buffer (1% NP-40, 0.5% sodiumdesoxycholate, 0.1% SDS, 100 microg/ml phenylmethylsulfonyl fluoride, 10 microl/ml protease-inhibitory-mix, 1 micromol/ml sodiumorthovanadate in phosphate-buffered saline) for 30 min on ice. After centrifugation, protein concentration of the supernatant was determined by BCA-method (Pierce, Rockford, IL) as recommended. Protein lysates (70-100 microg) were electrophoresed on polyacrylamide gels and transferred to a PVDF-membrane (Immobilon P, 0.45 microm; Millipore, Eschborn, Germany). 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 microl/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. The membranes were then developed with an ECL detection kit (Amersham Pharmacia Biotech, Freiburg, Germany). The primary antibodies were goat anti-IRF-4/ICSAT (M-17) (Santa Cruz Biotechnology, Santa Cruz, CA) and mouse anti-beta-actin (AC-74) (Sigma). \n"
],
"offsets": [
[
0,
1944
]
]
}
] | [
{
"id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T1",
"type": "Protein",
"offsets": [
[
35,
40
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T2",
"type": "Protein",
"offsets": [
[
271,
276
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T3",
"type": "Protein",
"offsets": [
[
300,
310
]
],
"text": [
"beta-actin"
],
"normalized": []
},
{
"id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T4",
"type": "Protein",
"offsets": [
[
502,
508
]
],
"text": [
"(DNMT1"
],
"normalized": []
},
{
"id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T5",
"type": "Protein",
"offsets": [
[
509,
515
]
],
"text": [
"DNMT3A"
],
"normalized": []
},
{
"id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T6",
"type": "Protein",
"offsets": [
[
517,
523
]
],
"text": [
"DNMT3B"
],
"normalized": []
},
{
"id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T7",
"type": "Protein",
"offsets": [
[
525,
529
]
],
"text": [
"MeCP"
],
"normalized": []
},
{
"id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T8",
"type": "Protein",
"offsets": [
[
531,
535
]
],
"text": [
"MBD1"
],
"normalized": []
},
{
"id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T9",
"type": "Protein",
"offsets": [
[
537,
541
]
],
"text": [
"MBD2"
],
"normalized": []
},
{
"id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T10",
"type": "Protein",
"offsets": [
[
546,
550
]
],
"text": [
"MBD4"
],
"normalized": []
},
{
"id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T11",
"type": "Protein",
"offsets": [
[
600,
605
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T12",
"type": "Protein",
"offsets": [
[
1838,
1843
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T13",
"type": "Protein",
"offsets": [
[
1844,
1849
]
],
"text": [
"ICSAT"
],
"normalized": []
},
{
"id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T14",
"type": "Protein",
"offsets": [
[
1851,
1855
]
],
"text": [
"M-17"
],
"normalized": []
},
{
"id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T15",
"type": "Protein",
"offsets": [
[
1915,
1925
]
],
"text": [
"beta-actin"
],
"normalized": []
}
] | [
{
"id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_E1",
"type": "Transcription",
"trigger": {
"text": [
"transcriptional"
],
"offsets": [
[
41,
56
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T1"
}
]
},
{
"id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_E2",
"type": "Transcription",
"trigger": {
"text": [
"RNA expression"
],
"offsets": [
[
243,
257
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T2"
}
]
},
{
"id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_E3",
"type": "Transcription",
"trigger": {
"text": [
"RNA expression"
],
"offsets": [
[
243,
257
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T3"
}
]
},
{
"id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_E4",
"type": "Gene_expression",
"trigger": {
"text": [
"expression"
],
"offsets": [
[
467,
477
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T4"
}
]
},
{
"id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_E5",
"type": "Gene_expression",
"trigger": {
"text": [
"expression"
],
"offsets": [
[
467,
477
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T5"
}
]
},
{
"id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_E6",
"type": "Gene_expression",
"trigger": {
"text": [
"expression"
],
"offsets": [
[
467,
477
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T6"
}
]
},
{
"id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_E7",
"type": "Gene_expression",
"trigger": {
"text": [
"expression"
],
"offsets": [
[
467,
477
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T7"
}
]
},
{
"id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_E8",
"type": "Gene_expression",
"trigger": {
"text": [
"expression"
],
"offsets": [
[
467,
477
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T8"
}
]
},
{
"id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_E9",
"type": "Gene_expression",
"trigger": {
"text": [
"expression"
],
"offsets": [
[
467,
477
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T9"
}
]
},
{
"id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_E10",
"type": "Gene_expression",
"trigger": {
"text": [
"expression"
],
"offsets": [
[
467,
477
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T10"
}
]
},
{
"id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_E11",
"type": "Gene_expression",
"trigger": {
"text": [
"expression"
],
"offsets": [
[
614,
624
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T11"
}
]
}
] | [
{
"id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_1",
"entity_ids": [
"PMC-1310901-05-MATERIALS_AND_METHODS-04_T12",
"PMC-1310901-05-MATERIALS_AND_METHODS-04_T14",
"PMC-1310901-05-MATERIALS_AND_METHODS-04_T13"
]
}
] | [] |
6 | PMC-1310901-06-MATERIALS_AND_METHODS-05 | [
{
"id": "PMC-1310901-06-MATERIALS_AND_METHODS-05__text",
"type": "abstract",
"text": [
"Methylation-specific restriction-PCR-assay\nDNA was extracted with a commercial kit (Qiagen) as recommended. 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). As control the methylation-resistant enzyme MspI and an enzyme with no recognition site in the target promoter, EcoRI, were used. DNA (0.8 microg) was digested by 40 U the respective enzyme for 6 h and, to ensure complete cleavage, additional 20 U for 16 h. 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). The sequences of the primers were F1-forward: 5'-TTGAGATGGAGTCTTGCTCTGT-3', F1-reverse: ATCACTTCCAGACTTCAGTTCACCT-3' (341 bp); F2-forward: 5'-AAGGTGAACTGAAGTCTGGAAGTGA-3', F2-reverse: 5'-CCAGGACCTCAGGAGGCCAGTCA-3' (474 bp). The PCR conditions were described elsewhere (3). PCR was performed with an annealing temperature of 62degreesC and 35 cycles. 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. The PCR products were electrophoresed on a 3% agarose gel, were stained with ethidium bromide and photographed. PCR products were verified by automated sequencing. \n"
],
"offsets": [
[
0,
1495
]
]
}
] | [
{
"id": "PMC-1310901-06-MATERIALS_AND_METHODS-05_T1",
"type": "Protein",
"offsets": [
[
726,
731
]
],
"text": [
"IRF-4"
],
"normalized": []
}
] | [] | [] | [] |
7 | PMC-1310901-07-MATERIALS_AND_METHODS-06 | [
{
"id": "PMC-1310901-07-MATERIALS_AND_METHODS-06__text",
"type": "abstract",
"text": [
"Bisulfite treatment\nDNA was extracted as described above. 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. Briefly, 1 microg of DNA and 2 microg of poly(dA-dT)(poly(dA-dT) copolymers (Amersham Pharmacia Biotech) were denaturated for 20 min at 42degreesC in 0.3 M NaOH in a volume of 50 microl. Fresh solutions of 30 microl of 10 mM hydrochinon (Sigma) and 530 microl 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 50degreesC. The aqueous phase was recovered using the 'Wizard DNA clean-up system' (Promega, Mannheim, Germany). The purified DNA was subsequently mixed with 1 M NaOH to a final concentration of 0.3 M and incubated for 20 min at 37degreesC to ensure complete desulfonisation. DNA was ethanol precipitated in the presence of 1/10 vol of 3 M sodium acetate, washed with 70% ethanol and resuspended in 50 microl H2O. Subsequent PCR amplification of 4 microl 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). 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). 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). \n"
],
"offsets": [
[
0,
1900
]
]
}
] | [
{
"id": "PMC-1310901-07-MATERIALS_AND_METHODS-06_T1",
"type": "Protein",
"offsets": [
[
1171,
1176
]
],
"text": [
"IRF-4"
],
"normalized": []
}
] | [] | [] | [] |
8 | PMC-1310901-08-MATERIALS_AND_METHODS-07 | [
{
"id": "PMC-1310901-08-MATERIALS_AND_METHODS-07__text",
"type": "abstract",
"text": [
"In vitro methylation and reporter gene assays\nThe IRF-4 promoter-reporter gene construct was generously provided by J.Hiscott (31). 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). Reporter gene assays using the dual luciferase assay (Promega) were performed similar to previous reports (29). Briefly, 5 nM of the reporter construct and the transfection control construct expressing the renilla luciferase gene were transiently co-expressed via electroporation. The control construct served as an internal reference for transfection efficiency. Forty-eight hours after transfection, luciferase activity was measured with a LB 96 P microlumat (EG&G Berthold, Bad Wildbad, Germany). IRF-4 promoter activation was quantified as a ratio of measured firefly light units (flu) relative to renilla (rlu). Each experiment was carried out at least three times. \n"
],
"offsets": [
[
0,
992
]
]
}
] | [
{
"id": "PMC-1310901-08-MATERIALS_AND_METHODS-07_T1",
"type": "Protein",
"offsets": [
[
50,
55
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-08-MATERIALS_AND_METHODS-07_T2",
"type": "Protein",
"offsets": [
[
173,
186
]
],
"text": [
"CpG Methylase"
],
"normalized": []
},
{
"id": "PMC-1310901-08-MATERIALS_AND_METHODS-07_T3",
"type": "Protein",
"offsets": [
[
188,
195
]
],
"text": [
"M.Sss I"
],
"normalized": []
},
{
"id": "PMC-1310901-08-MATERIALS_AND_METHODS-07_T4",
"type": "Protein",
"offsets": [
[
356,
366
]
],
"text": [
"luciferase"
],
"normalized": []
},
{
"id": "PMC-1310901-08-MATERIALS_AND_METHODS-07_T5",
"type": "Protein",
"offsets": [
[
534,
544
]
],
"text": [
"luciferase"
],
"normalized": []
},
{
"id": "PMC-1310901-08-MATERIALS_AND_METHODS-07_T6",
"type": "Protein",
"offsets": [
[
722,
732
]
],
"text": [
"luciferase"
],
"normalized": []
},
{
"id": "PMC-1310901-08-MATERIALS_AND_METHODS-07_T7",
"type": "Protein",
"offsets": [
[
820,
825
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-08-MATERIALS_AND_METHODS-07_T10",
"type": "Entity",
"offsets": [
[
826,
834
]
],
"text": [
"promoter"
],
"normalized": []
}
] | [
{
"id": "PMC-1310901-08-MATERIALS_AND_METHODS-07_E1",
"type": "Gene_expression",
"trigger": {
"text": [
"expressing"
],
"offsets": [
[
511,
521
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-08-MATERIALS_AND_METHODS-07_T5"
}
]
},
{
"id": "PMC-1310901-08-MATERIALS_AND_METHODS-07_E2",
"type": "Gene_expression",
"trigger": {
"text": [
"expressed"
],
"offsets": [
[
570,
579
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-08-MATERIALS_AND_METHODS-07_T5"
}
]
},
{
"id": "PMC-1310901-08-MATERIALS_AND_METHODS-07_E3",
"type": "Positive_regulation",
"trigger": {
"text": [
"activation"
],
"offsets": [
[
835,
845
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-08-MATERIALS_AND_METHODS-07_T7"
},
{
"role": "Site",
"ref_id": "PMC-1310901-08-MATERIALS_AND_METHODS-07_T10"
}
]
}
] | [
{
"id": "PMC-1310901-08-MATERIALS_AND_METHODS-07_1",
"entity_ids": [
"PMC-1310901-08-MATERIALS_AND_METHODS-07_T2",
"PMC-1310901-08-MATERIALS_AND_METHODS-07_T3"
]
}
] | [] |
9 | PMC-1310901-09-RESULTS-01 | [
{
"id": "PMC-1310901-09-RESULTS-01__text",
"type": "abstract",
"text": [
"Absence of IRF-4 expression in leukemia cells is not due to promoter alterations\nWe have previously demonstrated a lack of IRF-4 expression in leukemia patients and specifically in CML T-cells (3). 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). 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. 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). 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). Consequently, these alterations are unlikely to affect IRF-4 expression. \n"
],
"offsets": [
[
0,
1274
]
]
}
] | [
{
"id": "PMC-1310901-09-RESULTS-01_T1",
"type": "Protein",
"offsets": [
[
11,
16
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-09-RESULTS-01_T2",
"type": "Protein",
"offsets": [
[
123,
128
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-09-RESULTS-01_T3",
"type": "Protein",
"offsets": [
[
234,
239
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-09-RESULTS-01_T4",
"type": "Protein",
"offsets": [
[
333,
340
]
],
"text": [
"bcr-abl"
],
"normalized": []
},
{
"id": "PMC-1310901-09-RESULTS-01_T5",
"type": "Protein",
"offsets": [
[
372,
379
]
],
"text": [
"bcr-abl"
],
"normalized": []
},
{
"id": "PMC-1310901-09-RESULTS-01_T6",
"type": "Protein",
"offsets": [
[
452,
459
]
],
"text": [
"bcr-abl"
],
"normalized": []
},
{
"id": "PMC-1310901-09-RESULTS-01_T7",
"type": "Protein",
"offsets": [
[
505,
512
]
],
"text": [
"bcr-abl"
],
"normalized": []
},
{
"id": "PMC-1310901-09-RESULTS-01_T8",
"type": "Protein",
"offsets": [
[
608,
615
]
],
"text": [
"bcr-abl"
],
"normalized": []
},
{
"id": "PMC-1310901-09-RESULTS-01_T9",
"type": "Protein",
"offsets": [
[
682,
687
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-09-RESULTS-01_T10",
"type": "Protein",
"offsets": [
[
735,
740
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-09-RESULTS-01_T11",
"type": "Protein",
"offsets": [
[
910,
915
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-09-RESULTS-01_T12",
"type": "Protein",
"offsets": [
[
940,
945
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-09-RESULTS-01_T13",
"type": "Protein",
"offsets": [
[
1255,
1260
]
],
"text": [
"IRF-4"
],
"normalized": []
}
] | [
{
"id": "PMC-1310901-09-RESULTS-01_E1",
"type": "Negative_regulation",
"trigger": {
"text": [
"Absence"
],
"offsets": [
[
0,
7
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-09-RESULTS-01_E2"
}
]
},
{
"id": "PMC-1310901-09-RESULTS-01_E2",
"type": "Gene_expression",
"trigger": {
"text": [
"expression"
],
"offsets": [
[
17,
27
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-09-RESULTS-01_T1"
}
]
},
{
"id": "PMC-1310901-09-RESULTS-01_E3",
"type": "Positive_regulation",
"trigger": {
"text": [
"due"
],
"offsets": [
[
53,
56
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-09-RESULTS-01_E1"
}
]
},
{
"id": "PMC-1310901-09-RESULTS-01_E4",
"type": "Negative_regulation",
"trigger": {
"text": [
"lack"
],
"offsets": [
[
115,
119
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-09-RESULTS-01_E5"
}
]
},
{
"id": "PMC-1310901-09-RESULTS-01_E5",
"type": "Gene_expression",
"trigger": {
"text": [
"expression"
],
"offsets": [
[
129,
139
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-09-RESULTS-01_T2"
}
]
},
{
"id": "PMC-1310901-09-RESULTS-01_E6",
"type": "Gene_expression",
"trigger": {
"text": [
"expression"
],
"offsets": [
[
240,
250
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-09-RESULTS-01_T3"
}
]
},
{
"id": "PMC-1310901-09-RESULTS-01_E7",
"type": "Gene_expression",
"trigger": {
"text": [
"positive"
],
"offsets": [
[
341,
349
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-09-RESULTS-01_T4"
}
]
},
{
"id": "PMC-1310901-09-RESULTS-01_E8",
"type": "Gene_expression",
"trigger": {
"text": [
"positve"
],
"offsets": [
[
380,
387
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-09-RESULTS-01_T5"
}
]
},
{
"id": "PMC-1310901-09-RESULTS-01_E9",
"type": "Gene_expression",
"trigger": {
"text": [
"positve"
],
"offsets": [
[
460,
467
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-09-RESULTS-01_T6"
}
]
},
{
"id": "PMC-1310901-09-RESULTS-01_E10",
"type": "Gene_expression",
"trigger": {
"text": [
"positive"
],
"offsets": [
[
513,
521
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-09-RESULTS-01_T7"
}
]
},
{
"id": "PMC-1310901-09-RESULTS-01_E11",
"type": "Gene_expression",
"trigger": {
"text": [
"positive"
],
"offsets": [
[
616,
624
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-09-RESULTS-01_T8"
}
]
},
{
"id": "PMC-1310901-09-RESULTS-01_E12",
"type": "Negative_regulation",
"trigger": {
"text": [
"absence"
],
"offsets": [
[
724,
731
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-09-RESULTS-01_E13"
}
]
},
{
"id": "PMC-1310901-09-RESULTS-01_E13",
"type": "Gene_expression",
"trigger": {
"text": [
"expression"
],
"offsets": [
[
741,
751
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-09-RESULTS-01_T10"
}
]
},
{
"id": "PMC-1310901-09-RESULTS-01_E14",
"type": "Gene_expression",
"trigger": {
"text": [
"positive"
],
"offsets": [
[
916,
924
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-09-RESULTS-01_T11"
}
]
},
{
"id": "PMC-1310901-09-RESULTS-01_E15",
"type": "Gene_expression",
"trigger": {
"text": [
"negative"
],
"offsets": [
[
946,
954
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-09-RESULTS-01_T12"
}
]
},
{
"id": "PMC-1310901-09-RESULTS-01_E16",
"type": "Regulation",
"trigger": {
"text": [
"affect"
],
"offsets": [
[
1248,
1254
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-09-RESULTS-01_E17"
}
]
},
{
"id": "PMC-1310901-09-RESULTS-01_E17",
"type": "Gene_expression",
"trigger": {
"text": [
"expression"
],
"offsets": [
[
1261,
1271
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-09-RESULTS-01_T13"
}
]
}
] | [] | [] |
10 | PMC-1310901-10-RESULTS-02 | [
{
"id": "PMC-1310901-10-RESULTS-02__text",
"type": "abstract",
"text": [
"Increase of IRF-4 expression in hematopoietic cells after demethylating treatment\nWe next analyzed whether promoter methylation could be responsible for down-regulation of IRF-4 expression. A region including exon1 in the IRF-4 promoter exhibited a large number of CpG-rich sequences (Figure 3A). 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). We used AzadC to generate unmethylated DNA. 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). 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). 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). 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). 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). 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). 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). \n"
],
"offsets": [
[
0,
1646
]
]
}
] | [
{
"id": "PMC-1310901-10-RESULTS-02_T1",
"type": "Protein",
"offsets": [
[
12,
17
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-10-RESULTS-02_T2",
"type": "Protein",
"offsets": [
[
172,
177
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-10-RESULTS-02_T3",
"type": "Protein",
"offsets": [
[
222,
227
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-10-RESULTS-02_T4",
"type": "Protein",
"offsets": [
[
613,
618
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-10-RESULTS-02_T5",
"type": "Protein",
"offsets": [
[
722,
727
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-10-RESULTS-02_T6",
"type": "Protein",
"offsets": [
[
929,
934
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-10-RESULTS-02_T7",
"type": "Protein",
"offsets": [
[
1139,
1144
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-10-RESULTS-02_T8",
"type": "Protein",
"offsets": [
[
1207,
1212
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-10-RESULTS-02_T9",
"type": "Protein",
"offsets": [
[
1287,
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]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-10-RESULTS-02_T10",
"type": "Protein",
"offsets": [
[
1400,
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]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-10-RESULTS-02_T11",
"type": "Protein",
"offsets": [
[
1510,
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]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-10-RESULTS-02_T12",
"type": "Protein",
"offsets": [
[
1619,
1624
]
],
"text": [
"IRF-4"
],
"normalized": []
}
] | [
{
"id": "PMC-1310901-10-RESULTS-02_E1",
"type": "Positive_regulation",
"trigger": {
"text": [
"Increase"
],
"offsets": [
[
0,
8
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-10-RESULTS-02_E2"
}
]
},
{
"id": "PMC-1310901-10-RESULTS-02_E2",
"type": "Gene_expression",
"trigger": {
"text": [
"expression"
],
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[
18,
28
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-10-RESULTS-02_T1"
}
]
},
{
"id": "PMC-1310901-10-RESULTS-02_E3",
"type": "Gene_expression",
"trigger": {
"text": [
"expression"
],
"offsets": [
[
178,
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]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-10-RESULTS-02_T2"
}
]
},
{
"id": "PMC-1310901-10-RESULTS-02_E4",
"type": "Positive_regulation",
"trigger": {
"text": [
"activation"
],
"offsets": [
[
599,
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]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-10-RESULTS-02_E5"
}
]
},
{
"id": "PMC-1310901-10-RESULTS-02_E5",
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"transcription"
],
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619,
632
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]
},
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{
"role": "Theme",
"ref_id": "PMC-1310901-10-RESULTS-02_T4"
}
]
},
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"text": [
"transcription"
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{
"role": "Theme",
"ref_id": "PMC-1310901-10-RESULTS-02_T5"
}
]
},
{
"id": "PMC-1310901-10-RESULTS-02_E7",
"type": "Positive_regulation",
"trigger": {
"text": [
"induced"
],
"offsets": [
[
746,
753
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-10-RESULTS-02_E6"
}
]
},
{
"id": "PMC-1310901-10-RESULTS-02_E8",
"type": "Positive_regulation",
"trigger": {
"text": [
"increased"
],
"offsets": [
[
846,
855
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-10-RESULTS-02_E6"
}
]
},
{
"id": "PMC-1310901-10-RESULTS-02_E9",
"type": "Transcription",
"trigger": {
"text": [
"transcripts"
],
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[
935,
946
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-10-RESULTS-02_T6"
}
]
},
{
"id": "PMC-1310901-10-RESULTS-02_E10",
"type": "Gene_expression",
"trigger": {
"text": [
"positive"
],
"offsets": [
[
1213,
1221
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-10-RESULTS-02_T8"
}
]
},
{
"id": "PMC-1310901-10-RESULTS-02_E11",
"type": "Regulation",
"trigger": {
"text": [
"effect"
],
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[
1277,
1283
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-10-RESULTS-02_E12"
}
]
},
{
"id": "PMC-1310901-10-RESULTS-02_E12",
"type": "Gene_expression",
"trigger": {
"text": [
"expression"
],
"offsets": [
[
1293,
1303
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-10-RESULTS-02_T9"
}
]
},
{
"id": "PMC-1310901-10-RESULTS-02_E13",
"type": "Positive_regulation",
"trigger": {
"text": [
"increased"
],
"offsets": [
[
1386,
1395
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-10-RESULTS-02_E14"
}
]
},
{
"id": "PMC-1310901-10-RESULTS-02_E14",
"type": "Transcription",
"trigger": {
"text": [
"mRNA level"
],
"offsets": [
[
1406,
1416
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-10-RESULTS-02_T10"
}
]
},
{
"id": "PMC-1310901-10-RESULTS-02_E15",
"type": "Regulation",
"trigger": {
"text": [
"control"
],
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[
1502,
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]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-10-RESULTS-02_E16"
}
]
},
{
"id": "PMC-1310901-10-RESULTS-02_E16",
"type": "Gene_expression",
"trigger": {
"text": [
"expression"
],
"offsets": [
[
1516,
1526
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-10-RESULTS-02_T11"
}
]
}
] | [] | [] |
11 | PMC-1310901-11-RESULTS-03 | [
{
"id": "PMC-1310901-11-RESULTS-03__text",
"type": "abstract",
"text": [
"Methylation-sensitive enzymes do not cut specific sites in the IRF-4 promoter in hematopoietic cells\nTo 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. 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. EcoRI, which has no recognition site within the IRF-4 promoter, and the methylation-resistant enzyme MspI served as controls. Two separate amplification reactions were performed, generating two fragments, F1 and F2 (Figure 3A). 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). Notably, in IRF-4-positive SD-1 cells digestion with the methylation-sensitive enzymes completely inhibited amplification of F1 and F2. 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). 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. \n"
],
"offsets": [
[
0,
1564
]
]
}
] | [
{
"id": "PMC-1310901-11-RESULTS-03_T1",
"type": "Protein",
"offsets": [
[
63,
68
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-11-RESULTS-03_T2",
"type": "Protein",
"offsets": [
[
174,
179
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-11-RESULTS-03_T3",
"type": "Protein",
"offsets": [
[
567,
572
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-11-RESULTS-03_T4",
"type": "Protein",
"offsets": [
[
856,
861
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-11-RESULTS-03_T5",
"type": "Protein",
"offsets": [
[
1046,
1051
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-11-RESULTS-03_T6",
"type": "Protein",
"offsets": [
[
1173,
1178
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-11-RESULTS-03_T7",
"type": "Protein",
"offsets": [
[
1385,
1390
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-11-RESULTS-03_T8",
"type": "Protein",
"offsets": [
[
1541,
1546
]
],
"text": [
"IRF-4"
],
"normalized": []
}
] | [
{
"id": "PMC-1310901-11-RESULTS-03_E1",
"type": "Gene_expression",
"trigger": {
"text": [
"expression"
],
"offsets": [
[
185,
195
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-11-RESULTS-03_T2"
}
]
},
{
"id": "PMC-1310901-11-RESULTS-03_E2",
"type": "Gene_expression",
"trigger": {
"text": [
"negative"
],
"offsets": [
[
862,
870
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-11-RESULTS-03_T4"
}
]
},
{
"id": "PMC-1310901-11-RESULTS-03_E3",
"type": "Gene_expression",
"trigger": {
"text": [
"positive"
],
"offsets": [
[
1052,
1060
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-11-RESULTS-03_T5"
}
]
},
{
"id": "PMC-1310901-11-RESULTS-03_E4",
"type": "Gene_expression",
"trigger": {
"text": [
"positive"
],
"offsets": [
[
1179,
1187
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-11-RESULTS-03_T6"
}
]
},
{
"id": "PMC-1310901-11-RESULTS-03_E5",
"type": "Transcription",
"trigger": {
"text": [
"transcription"
],
"offsets": [
[
1391,
1404
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-11-RESULTS-03_T7"
}
]
},
{
"id": "PMC-1310901-11-RESULTS-03_E6",
"type": "Gene_expression",
"trigger": {
"text": [
"negative"
],
"offsets": [
[
1547,
1555
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-11-RESULTS-03_T8"
}
]
}
] | [] | [] |
12 | PMC-1310901-12-RESULTS-04 | [
{
"id": "PMC-1310901-12-RESULTS-04__text",
"type": "abstract",
"text": [
"Specific CpG sites in the IRF-4 promoter are methylated in hematopoietic cells\nIn 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). This technique is especially useful for detection of unknown methylation patterns. 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). 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). 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). 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). \nIntriguingly, 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). 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. In addition, two CpG sites located in a NFkappaB (#48) and a SP1element (#45) are less methylated in IRF-4-positive than in IRF-4-negative cells (mean number of methylated clones: 1/8 versus 8/8). These results indicate the involvement of CpG methylation in the regulation of IRF-4 expression in leukemic cells. \n"
],
"offsets": [
[
0,
2222
]
]
}
] | [
{
"id": "PMC-1310901-12-RESULTS-04_T1",
"type": "Protein",
"offsets": [
[
26,
31
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-12-RESULTS-04_T2",
"type": "Protein",
"offsets": [
[
136,
141
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-12-RESULTS-04_T3",
"type": "Protein",
"offsets": [
[
766,
771
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-12-RESULTS-04_T4",
"type": "Protein",
"offsets": [
[
788,
793
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-12-RESULTS-04_T5",
"type": "Protein",
"offsets": [
[
831,
836
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-12-RESULTS-04_T6",
"type": "Protein",
"offsets": [
[
857,
862
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-12-RESULTS-04_T7",
"type": "Protein",
"offsets": [
[
930,
935
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-12-RESULTS-04_T8",
"type": "Protein",
"offsets": [
[
950,
955
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-12-RESULTS-04_T9",
"type": "Protein",
"offsets": [
[
1012,
1017
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-12-RESULTS-04_T10",
"type": "Protein",
"offsets": [
[
1561,
1566
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-12-RESULTS-04_T11",
"type": "Protein",
"offsets": [
[
1675,
1680
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-12-RESULTS-04_T12",
"type": "Protein",
"offsets": [
[
1891,
1896
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-12-RESULTS-04_T13",
"type": "Protein",
"offsets": [
[
2010,
2015
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-12-RESULTS-04_T14",
"type": "Protein",
"offsets": [
[
2033,
2038
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-12-RESULTS-04_T15",
"type": "Protein",
"offsets": [
[
2185,
2190
]
],
"text": [
"IRF-4"
],
"normalized": []
}
] | [
{
"id": "PMC-1310901-12-RESULTS-04_E1",
"type": "Gene_expression",
"trigger": {
"text": [
"positive"
],
"offsets": [
[
794,
802
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-12-RESULTS-04_T4"
}
]
},
{
"id": "PMC-1310901-12-RESULTS-04_E2",
"type": "Gene_expression",
"trigger": {
"text": [
"negative"
],
"offsets": [
[
863,
871
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-12-RESULTS-04_T6"
}
]
},
{
"id": "PMC-1310901-12-RESULTS-04_E3",
"type": "Gene_expression",
"trigger": {
"text": [
"positive"
],
"offsets": [
[
956,
964
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-12-RESULTS-04_T8"
}
]
},
{
"id": "PMC-1310901-12-RESULTS-04_E4",
"type": "Gene_expression",
"trigger": {
"text": [
"negative"
],
"offsets": [
[
1018,
1026
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-12-RESULTS-04_T9"
}
]
},
{
"id": "PMC-1310901-12-RESULTS-04_E5",
"type": "Gene_expression",
"trigger": {
"text": [
"negative"
],
"offsets": [
[
1567,
1575
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-12-RESULTS-04_T10"
}
]
},
{
"id": "PMC-1310901-12-RESULTS-04_E6",
"type": "Gene_expression",
"trigger": {
"text": [
"positive"
],
"offsets": [
[
1681,
1689
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-12-RESULTS-04_T11"
}
]
},
{
"id": "PMC-1310901-12-RESULTS-04_E7",
"type": "Negative_regulation",
"trigger": {
"text": [
"absence"
],
"offsets": [
[
1880,
1887
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-12-RESULTS-04_E8"
}
]
},
{
"id": "PMC-1310901-12-RESULTS-04_E8",
"type": "Gene_expression",
"trigger": {
"text": [
"expression"
],
"offsets": [
[
1897,
1907
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-12-RESULTS-04_T12"
}
]
},
{
"id": "PMC-1310901-12-RESULTS-04_E9",
"type": "Gene_expression",
"trigger": {
"text": [
"positive"
],
"offsets": [
[
2016,
2024
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-12-RESULTS-04_T13"
}
]
},
{
"id": "PMC-1310901-12-RESULTS-04_E10",
"type": "Gene_expression",
"trigger": {
"text": [
"negative"
],
"offsets": [
[
2039,
2047
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-12-RESULTS-04_T14"
}
]
},
{
"id": "PMC-1310901-12-RESULTS-04_E11",
"type": "Regulation",
"trigger": {
"text": [
"regulation"
],
"offsets": [
[
2171,
2181
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-12-RESULTS-04_E12"
}
]
},
{
"id": "PMC-1310901-12-RESULTS-04_E12",
"type": "Gene_expression",
"trigger": {
"text": [
"expression"
],
"offsets": [
[
2191,
2201
]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1310901-12-RESULTS-04_T15"
}
]
}
] | [] | [] |
13 | PMC-1310901-13-RESULTS-05 | [
{
"id": "PMC-1310901-13-RESULTS-05__text",
"type": "abstract",
"text": [
"In vitro methylation of an IRF-4 promoter-reporter construct decreases its activity\nTo 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. A complete methylation of these constructs was checked via restriction assays with methylation-sensitive endonucleases (Figure 5A). Intriguingly, methylation of the IRF-4 promoter significantly decreased promoter activity in IRF-4-positive SD-1 cells by 85.0% (Figure 5B). 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). 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). These data proved a direct association between methylation and activity of the IRF-4 promoter. \n"
],
"offsets": [
[
0,
1030
]
]
}
] | [
{
"id": "PMC-1310901-13-RESULTS-05_T1",
"type": "Protein",
"offsets": [
[
27,
32
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-13-RESULTS-05_T2",
"type": "Protein",
"offsets": [
[
151,
156
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-13-RESULTS-05_T3",
"type": "Protein",
"offsets": [
[
214,
219
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-13-RESULTS-05_T4",
"type": "Protein",
"offsets": [
[
450,
455
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-13-RESULTS-05_T5",
"type": "Protein",
"offsets": [
[
510,
515
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-13-RESULTS-05_T6",
"type": "Protein",
"offsets": [
[
620,
625
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-13-RESULTS-05_T7",
"type": "Protein",
"offsets": [
[
719,
724
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
"id": "PMC-1310901-13-RESULTS-05_T8",
"type": "Protein",
"offsets": [
[
844,
848
]
],
"text": [
"FasL"
],
"normalized": []
},
{
"id": "PMC-1310901-13-RESULTS-05_T9",
"type": "Protein",
"offsets": [
[
1013,
1018
]
],
"text": [
"IRF-4"
],
"normalized": []
},
{
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{
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"decreased"
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}
] | [] | [] |
14 | PMC-1310901-14-RESULTS-06 | [
{
"id": "PMC-1310901-14-RESULTS-06__text",
"type": "abstract",
"text": [
"mRNA expression of DNA methyltransferases and methyl-CpG-binding proteins may not be associated with IRF-4 promoter methylation\nSince 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. 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). 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. 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. \n"
],
"offsets": [
[
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879
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] | [
{
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{
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174,
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],
"text": [
"promoter"
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}
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"regulation"
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193
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"mRNA levels"
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"text": [
"mRNA levels"
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}
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"mRNA levels"
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"mRNA levels"
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"mRNA levels"
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"text": [
"expression"
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"expression"
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"expression"
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"expression"
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"negative"
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] | [] | [] |
15 | PMC-1310901-15-DISCUSSION | [
{
"id": "PMC-1310901-15-DISCUSSION__text",
"type": "abstract",
"text": [
"DISCUSSION\nMany genetic lesions are known to influence gene expression of tumor suppressor genes. Whereas mutations and deletions or insertions have permanent effects, reversible mechanisms are gene methylation, or expression and activation of transcription factors, respectively. We studied a putative cause for absent IRF-4 expression in leukemia cells and first focused on genetic aberrations of the promoter. We observed no genetic alterations in the IRF-4 promoter, which can account for the lack of IRF-4 expression: 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. 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. 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. Since IRF-4 expression in cell lines and CML can be induced by demethylation and successful IFN-alpha therapy (3), respectively, the existence of such genetic aberrations seems unlikely. \nWe 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. 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. 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. We found that IRF-4 expression could indeed be connected to the methylation status of distinct CpG motifs in the IRF-4 promoter. 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. One of them (#54) is adjacent to an identified regulatory element (NFkappaB-site), indicating a possible involvement of this site. At two further CpG sites (#48, 45) the methylation status in IRF-4-positive was lower than that of IRF-4-negative cells. These CpG sites are located in an NFkappaB and an SP1 element (31) and thus may also play a role in regulation of IRF-4 expression. It has been shown that NFkappaB 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). Furthermore, methylation of the central CpG in the NFkappaB element inhibits binding of the NFkappaB protein complexes (38), promoting the significance of the observed methylation differences in IRF-4-positive and -negative cells. \nVia 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. One possible cause for the aberrant methylation in tumorigenesis is an increased level of DNMTs during the pathogenetic process. In colon, lung and hematologic malignancies, overexpression of DNMT1, a maintenance DNMT, has been detected (39-41). 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). Interestingly, a positive correlation between DNMT1 expression levels and hypermethylation of p15INK4b has been detected in AML (25). 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. \nThe 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. 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). \nThe expression of another IRF, IFN consensus sequence binding protein (ICSBP/IRF-8), is impaired in myeloid leukemias especially CML (27,46,47). 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). These data suggest a distinct regulatory mechanism for these two IRFs. \nIRF-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. Still, the obvious functional diversity of IRF-4 remains remarkable and cannot be fully explained by the IRF-4 promoter methylation status. For example, IRF-4 is primarily known for its oncogenic features. 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). 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). This draws a more complex picture of the role of IRF-4. 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). \nTaken 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. \n"
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] | [] |
16 | PMC-1447668-00-TIAB | [
{
"id": "PMC-1447668-00-TIAB__text",
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"Foxp3 Represses Retroviral Transcription by Targeting Both NF-kappaB and CREB Pathways \nForkhead box (Fox)/winged-helix transcription factors regulate multiple aspects of immune responsiveness and Foxp3 is recognized as an essential functional marker of regulatory T cells. Herein we describe downstream signaling pathways targeted by Foxp3 that may negatively impact retroviral pathogenesis. Overexpression of Foxp3 in HEK 293T and purified CD4+ T cells resulted in a dose-dependent and time-dependent decrease in basal levels of nuclear factor-kappaB (NF-kappaB) activation. Deletion of the carboxyl-terminal forkhead (FKH) domain, critical for nuclear localization and DNA-binding activity, abrogated the ability of Foxp3 to suppress NF-kappaB activity in HEK 293T cells, but not in Jurkat or primary human CD4+ T cells. We further demonstrate that Foxp3 suppressed the transcription of two human retroviral promoters (HIV-1 and human T cell lymphotropic virus type I [HTLV-I]) utilizing NF-kappaB-dependent and NF-kappaB-independent mechanisms. Examination of the latter identified the cAMP-responsive element binding protein (CREB) pathway as a target of Foxp3. Finally, comparison of the percent Foxp3+CD4+CD25+ T cells to the HTLV-I proviral load in HTLV-I-infected asymptomatic carriers and patients with HTLV-I-associated myelopathy/tropical spastic paraparesis suggested that high Foxp3 expression is associated with low proviral load and absence of disease. These results suggest an expanded role for Foxp3 in regulating NF-kappaB- and CREB-dependent cellular and viral gene expression. \n"
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17 | PMC-1447668-01-Synopsis | [
{
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"Synopsis\nOver the past several years, mounting evidence has shown that immune tolerance in healthy individuals can be maintained by a population of T lymphocytes known as regulatory T cells (Tregs). As a component of this system, a protein known as Foxp3 has been shown to be absolutely required for the development and function of Tregs. While Foxp3 plays an important role in maintaining immune tolerance by blocking T cell proliferation and production of inflammatory proteins known as cytokines, little is known about the molecular mechanisms that are used by Foxp3 to accomplish these events. The present study expands our understanding of how Foxp3 maintains a check on inappropriate immune responses by demonstrating that Foxp3 can block activation of key inducible proteins such as nuclear factor kappaB (NF-kappaB) and cAMP-responsive element binding protein (CREB). Since NF-kappaB and CREB are integrally involved in controlling cell cycle progression, inflammatory cytokine production, and the replication of numerous viruses at the level of transcription, understanding the mechanisms by which Foxp3 functions to regulate cellular and viral gene expression may aid in the discovery of therapeutic approaches designed to rescue the expression and/or function of Foxp3, which have been found to be deficient in several autoimmune diseases and virus-induced disorders. \n"
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18 | PMC-1447668-02-Introduction | [
{
"id": "PMC-1447668-02-Introduction__text",
"type": "abstract",
"text": [
"Introduction\nImmunological tolerance to self-antigens is the result of the deletion of self-reactive T lymphocytes in the thymus (central tolerance) and suppression of the activation of potentially self-reactive T lymphocytes in the periphery (peripheral tolerance) [1]. Suppression of pathogenic T cell responses is mediated by naturally arising CD4+CD25+ T regulatory cells (Tregs) [2,3]. Deficiencies in Treg development and function have been linked to the severe autoimmune disorder known as immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) [4]. In addition, recent studies have provided strong evidence that dysregulation of Treg development and/or function may be a significant factor in the pathogenesis of several autoimmune disorders (e.g., multiple sclerosis [5], myasthenia gravis [6], and type 1 diabetes [7]) and virus-induced immunologic disorders (e.g., human T lymphotropic virus type I [HTLV-I]-associated myelopathy/tropical spastic paraparesis [HAM/TSP], and HIV-induced AIDS [8-10]). \nThe transcription factor Foxp3 is a 431-amino acid (48-kDa) protein expressed at very high levels in CD4+CD25hi T cells and has previously been shown to be absolutely critical for Treg development and function [11-14]. Foxp3 contains a proline-rich amino-terminal domain reported to function as a nuclear factor of activated T cells (NF-AT) and nuclear factor-kappaB (NF-kappaB) binding domain, a central region containing a zinc finger and leucine zipper potentially important for protein-protein interactions, and a carboxyl-terminal forkhead (FKH) domain required for nuclear localization and DNA-binding activity [14-16]. Functional inactivation of Foxp3 by genetic mutations affecting the Foxp3 coding region, as demonstrated in IPEX, or repression of Foxp3 expression by the HTLV-I-encoded transactivator protein Tax, as recently reported in patients with HAM/TSP, results in loss of regulatory activity in CD4+CD25hi T cells [4,8,17]. Although it is clear that Foxp3 regulates T cell proliferation and cytokine production, very little is known concerning the molecular mechanisms of Foxp3 function. \nThe first evidence to indicate how Foxp3 promotes the development and function of regulatory T cells came from a report by Ziegler and colleagues [16], which suggested that Foxp3 could inhibit transcriptional activation by physically interacting with forkhead binding sites located immediately adjacent to critical cis-acting NF-AT binding sites found in various cytokine promoters (e.g., IL-2 promoter). That study also demonstrated that Foxp3 could repress activation of a synthetic reporter vector containing an SV40 promoter and three tandem copies of a forkhead binding site. These results provided additional evidence suggesting that Foxp3 transcriptional repression was mediated by binding in a sequence-specific manner to promoters containing forkhead binding sites. A recent study by Bettelli and colleagues [15] further demonstrated that Foxp3 could inhibit NF-AT as well as NF-kappaB activation, although the mechanism of suppression was shown to involve direct protein-protein interactions between NF-AT or NF-kappaB and Foxp3 rather than binding of Foxp3 to promoter elements adjacent to cis-acting NF-AT or NF-kappaB sites. Collectively, these data suggested that Foxp3 may function as a transcriptional repressor, potentially through the formation of both DNA-protein and protein-protein interactions. \nIn the present study, we expanded upon these observations by defining additional requirements of Foxp3-mediated repression of NF-kappaB activation, and investigated whether Foxp3 could target additional signaling pathways by examining transcriptional activation of NF-kappaB-dependent and NF-kappaB-independent retroviral pathogens. The characterization of the molecular targets of Foxp3 and the mechanism(s) utilized by Foxp3 to support Treg development and function will aid in our understanding of the role Tregs play in the pathogenesis of human autoimmune disease. \n"
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}
] | [] | [] |
19 | PMC-1447668-03-Results-01 | [
{
"id": "PMC-1447668-03-Results-01__text",
"type": "abstract",
"text": [
"Foxp3 Suppresses NF-kappaB Dependent Transcriptional Activation\nTo ascertain the molecular mechanisms by which Foxp3 functions to promote the regulatory function of CD4+CD25hi T cells, we first confirmed the function of Foxp3 as a repressor of activation of NF-kappaB, previously implicated as a target of other forkhead/winged-helix family transcription factors (e.g., Foxj1 and Foxo3a) [19,20]. We analyzed the effect of Foxp3 overexpression on NF-kappaB activation in HEK 293T cells in dose-response and time course analyses. Transfection of HEK 293T cells with an NF-kappaB luciferase reporter vector in the presence or absence of increasing concentrations of a Foxp3 expression vector or a control vector (enhanced green fluorescent protein [EGFP]) was performed, and cells were harvested after 24 h to assay for luciferase activity and Foxp3 mRNA expression. Results indicated that as the concentration of Foxp3 transfected into cells increases (from 50 to 2,400 ng), the level of NF-kappaB activation decreases proportionally (Figure 1A). Foxp3 mRNA was also assayed to monitor activity of the Foxp3 expression vector (Figure 1B). Since NF-kappaB activation was partially affected by transfection of high concentrations of the control vector, we determined the fold inhibition of NF-kappaB activation by Foxp3 compared to the control vector at each concentration (Figure 1A). Fold inhibition of NF-kappaB activation was directly proportional to the level of Foxp3 mRNA expression detected by real-time RT-PCR. To determine the level of Foxp3-mediated suppression of NF-kappaB activation over time, HEK 293T cells were transfected with an NF-kappaB luciferase reporter vector and an expression vector encoding Foxp3 or EGFP (control vector) and harvested over 4 d. As shown in Figure 1C, NF-kappaB activation was suppressed by overexpression of Foxp3 at all time points. Extending these results from established, in vitro HEK cell lines to primary human lymphocytes, overexpression of Foxp3 in purified CD4+ T cells from three healthy donors also down-regulated the steady-state level of NF-kappaB activation (Figure 1D). These results recapitulate those from Bettelli and colleagues [15] demonstrating that Foxp3 functions, in part, to block NF-kappaB-dependent transcription in human cell lines as well as in primary human CD4+ T cells. \n"
],
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] | [
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}
] | [] | [] |
20 | PMC-1447668-04-Results-02 | [
{
"id": "PMC-1447668-04-Results-02__text",
"type": "abstract",
"text": [
"The Carboxyl-Terminal FKH Domain Is Not Required for Suppression of NF-kappaB Activation in T Cells\nTo define the requirements of Foxp3 with respect to inhibition of NF-kappaB-dependent transcription, we utilized a mutant of Foxp3 lacking the FKH domain (Figure 2A) [16], similar to the scurfy mutant Foxp3 of mice, and a mutant Foxp3 protein from a patient with IPEX [4,11,14,17]. Unlike full-length Foxp3, which localizes almost exclusively to the nucleus and can bind in a sequence-specific manner to forkhead binding sites, the DeltaFKH mutant fails to localize to the nucleus and thus cannot interact with promoter elements or nuclear proteins [16]. Therefore, we utilized the DeltaFKH mutant to determine whether nuclear localization (or other function associated with the FKH domain) of Foxp3 was a prerequisite for inhibition of NF-kappaB activation. Although Foxp3 interaction with NF-kappaB presumably takes place in the nucleus, it may also be possible for a cytoplasmic Foxp3 protein to bind to NF-kappaB in the cytoplasm and prevent localization to the nucleus following an activation stimulus. Overexpression of full-length Foxp3, but not of DeltaFKH, was able to suppress activation of a cotransfected NF-kappaB reporter vector in HEK 293T cells (Figure 2B). Both Foxp3 and DeltaFKH were expressed at very high levels following transfection as detected by real-time RT-PCR (unpublished data). These data appear to suggest that the carboxyl-terminal FKH domain is critically important for Foxp3 to down-regulate NF-kappaB-dependent transcription. However, NF-kappaB activation was blocked to a similar extent by both full-length Foxp3 and DeltaFKH in Jurkat T cells (Figure 2C) and primary human CD4+ T cells (Figure 2D). Western blot analysis of NF-kappaB p65 expression demonstrated that Foxp3 and DeltaFKH does not block NF-kappaB activation at the level of p65 protein expression (Figure 2E). These results are very interesting with respect to Foxp3 function, because they suggest that the carboxyl-terminal FKH domain, and possibly nuclear localization, are dispensable for Foxp3 function in T cell populations. Alternative interpretations may include the possibility that the localization of DeltaFKH differ between epithelial cells and T cells. In either case, these results suggest a cell type-specific mechanism of action for this Foxp3 mutant. \n"
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] | [] | [] |
21 | PMC-1447668-05-Results-03 | [
{
"id": "PMC-1447668-05-Results-03__text",
"type": "abstract",
"text": [
"Foxp3 Suppresses HIV-1 Gene Expression in Part through Blocking Activation of NF-kappaB\nIf Foxp3 functions as a repressor of NF-kappaB-dependent gene expression, then we hypothesized that Foxp3 overexpression could selectively down-regulate transcription from promoters previously shown to be responsive to NF-kappaB. To address this question, we examined the transcriptional activation of the HIV-1 LTR, which contains two tandem cis-acting NF-kappaB binding sites located between positions -102 and -81 with respect to the transcription initiation site [21]. NF-kappaB plays a crucial role in regulating gene expression directed from the HIV-1 LTR in CD4+ T cells [21]. Overexpression of full-length Foxp3, but not DeltaFKH, in HEK 293T cells was able to inhibit basal activation of the HIV-1 LTR (Figure 3A), similar to what was previously demonstrated with the synthetic NF-kappaB reporter vector (Figure 2B). Furthermore, HIV-1 LTR activation was suppressed by full-length Foxp3 and DeltaFKH in Jurkat T cells (Figure 3B). To demonstrate that Foxp3-mediated HIV-1 LTR repression was associated with interactions with NF-kappaB bound to the HIV-1 LTR, we compared basal activation of the HIV-1 LTR or an identical HIV-1 LTR lacking the NF-kappaB sites located between -102 and -81 (HIV-1 Delta-kappaB LTR) (Figure 3C). This mutant HIV-1 LTR construct exhibited reduced levels of transcription compared to the parental HIV-1 LTR in purified healthy donor CD4+ T cells (unpublished data). However, directly comparing the effect of Foxp3 overexpression on the activation of these two viral promoters demonstrated that Foxp3 was more capable of suppressing transcriptional activation of the HIV-1 LTR (Figure 3D) compared to the mutated HIV-1 LTR (Figure 3E). These results suggest that Foxp3 down-regulation of HIV-1 LTR activation was mediated at least in part by cis-acting NF-kappaB binding sites. Residual levels of inhibition of the HIV-1 Delta-kappaB LTR by Foxp3 may be due to NF-AT binding sites located upstream of the NF-kappaB sites within the HIV-1 LTR [22,23]. \n"
],
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}
] | [] | [] |
22 | PMC-1447668-06-Results-04 | [
{
"id": "PMC-1447668-06-Results-04__text",
"type": "abstract",
"text": [
"The Transactivation Functions of HTLV-I Tax Are Suppressed by Foxp3\nPrevious studies by Bettelli and colleagues have demonstrated that Foxp3 can repress both the basal levels of NF-kappaB activation as well as tumor necrosis factor-alpha-stimulated NF-kappaB activation [15]. Our next step was to determine whether Foxp3 could also suppress the activation of NF-kappaB caused by a strong viral transactivator protein. HTLV-I encodes a multifunctional transactivator protein, Tax, capable of activating both the NF-kappaB and CREB pathways [24-29]. Since the HTLV-I Tax protein can function at multiple levels in both the cytoplasm and the nucleus to stimulate activation of NF-kappaB [28,29], we hypothesized that overexpression of Foxp3 may interfere with this process. However, since Tax-dependent HTLV-I gene expression is independent of NF-kappaB [18], we also hypothesized that Foxp3 would not affect Tax-dependent activation of the HTLV-I LTR. To test these hypotheses, we overexpressed HTLV-I Tax, full-length Foxp3, and/or DeltaFKH in HEK 293T cells cotransfected with an HTLV-I LTR or NF-kappaB reporter vector. As shown in Figure 4A, HTLV-I Tax strongly up-regulated NF-kappaB-dependent transcriptional activation (~60-fold). Interestingly, overexpression of Foxp3, but not DeltaFKH, suppressed Tax-mediated activation of NF-kappaB-dependent transcription. These observations further suggest that the carboxyl-terminal FKH domain is required for inhibiting activation of NF-kappaB in the presence of Tax in HEK 293T cells, strikingly similar to the requirements of Foxp3 inhibition of basal NF-kappaB activation shown in Figure 2B. Transactivation of the HTLV-I LTR was stimulated about 55-fold by overexpression of Tax (Figure 4B), while transfection of Foxp3 suppressed Tax-dependent HTLV-I LTR activation, although HTLV-I LTR activation in the presence or absence of Tax is independent of NF-kappaB or NF-AT (another transcriptional activator known to interact with Foxp3). Furthermore, overexpression of DeltaFKH also led to suppression of HTLV-I transactivation by Tax to a similar extent as full-length Foxp3 (Figure 4B). The suppressive effects shown in Figure 4A and 4B were not the result of Foxp3 down-regulating the expression of the transfected Tax plasmid as determined by real-time RT-PCR (Figure 4C). These results strongly suggest that Foxp3 interacts with transcriptional regulators in addition to NF-kappaB and NF-AT, and that the carboxyl-terminal FKH domain, and therefore localization to the nucleus, are not required for inhibition of Tax-mediated HTLV-I LTR activation (even in HEK 293T cells). \nTo determine whether Foxp3 inhibited the transactivation functions of Tax by directly associating with this viral protein, we generated an expression vector in which HTLV-I Tax was fused in-frame to the carboxyl terminus of the Gal4 DNA-binding domain (Gal4-BD). This Gal4-BD-Tax fusion protein activated transcription of a synthetic promoter containing five Gal4 binding sites, while Gal4-BD was insufficient to stimulate transcription by itself (Figure 4D). Transactivation of the Gal4-resposive promoter by Gal4-BD-Tax remained relatively unaffected by overexpression of either EGFP (control), Foxp3, or DeltaFKH, suggesting that Foxp3 does not repress Tax transactivation by directly interfacing with the HTLV-I Tax protein. To confirm that Foxp3 had a direct effect on HTLV-I replication, we transfected HEK 293T cells with a well-characterized HTLV-I infectious molecular clone (termed ACH) [30] in the presence of full-length Foxp3, DeltaFKH, or control vector. ACH has been previously shown to direct the expression of viral antigens, produce infectious virus, and transform CD4+ T cells both in vitro and in vivo [31,32]. After 24 h, the amount of viral antigen expression, in this case Tax mRNA, was detected by a sensitive real-time RT-PCR assay. As illustrated in Figure 5, the level of Tax mRNA synthesized from ACH was down-regulated in the presence of Foxp3 compared to the level produced in the presence of the control vector. DeltaFKH did not have a discernable affect on Tax expression. These data indicate that Foxp3 is capable of repressing the expression of Tax from an infectious HTLV-I molecular clone. \n"
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"interfacing"
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{
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},
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}
]
},
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"expression"
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}
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}
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"id": "PMC-1447668-06-Results-04_E21",
"type": "Negative_regulation",
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"text": [
"down-regulated"
],
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]
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{
"role": "Theme",
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}
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}
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"affect"
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}
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}
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"repressing"
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}
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]
}
] | [] | [] |
23 | PMC-1447668-07-Results-05 | [
{
"id": "PMC-1447668-07-Results-05__text",
"type": "abstract",
"text": [
"Increased Foxp3 Protein Expression Is Associated with Low HTLV-I Proviral Load\nSince Foxp3 is expressed almost exclusively within CD4+CD25+ T cells, a major viral reservoir for HTLV-I [33], it was important to determine whether there was an association between Foxp3 and HTLV-I replication in infected patients. We therefore quantitated the Foxp3 protein expression in CD4+CD25+ T cells by flow cytometry and the HTLV-I proviral load (a surrogate marker of viral replication) by real-time PCR from eight patients with HAM/TSP and eight asymptomatic carriers (ACs). The data from this analysis is summarized in Table 1. As expected, patients with HAM/TSP exhibited significantly higher proviral loads (indicated as HTLV-I proviral DNA copies/100 cells) (34.68 +/- 23.19) compared to ACs (4.75 +/- 5.47) (p = 0.0008). The percentage of Foxp3+ cells within the CD4+CD25+ T cell population was significantly greater in ACs (43.23 +/- 12.95) than in HAM/TSP patients (18.59 +/- 5.77) (p = 0.0033). These data suggest that high levels of Foxp3 protein expression are associated with reduced HTLV-I replication in vivo. They also support our recent reports that high proviral loads, which have been shown to correlate with high Tax mRNA in HTLV-I-infected patients, are associated with reduced Foxp3 expression [8,34]. \n"
],
"offsets": [
[
0,
1313
]
]
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] | [
{
"id": "PMC-1447668-07-Results-05_T1",
"type": "Protein",
"offsets": [
[
10,
15
]
],
"text": [
"Foxp3"
],
"normalized": []
},
{
"id": "PMC-1447668-07-Results-05_T2",
"type": "Protein",
"offsets": [
[
85,
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"text": [
"Foxp3"
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{
"id": "PMC-1447668-07-Results-05_T3",
"type": "Protein",
"offsets": [
[
130,
133
]
],
"text": [
"CD4"
],
"normalized": []
},
{
"id": "PMC-1447668-07-Results-05_T4",
"type": "Protein",
"offsets": [
[
134,
138
]
],
"text": [
"CD25"
],
"normalized": []
},
{
"id": "PMC-1447668-07-Results-05_T5",
"type": "Protein",
"offsets": [
[
261,
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]
],
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"Foxp3"
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"type": "Protein",
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{
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"type": "Protein",
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"CD4"
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},
{
"id": "PMC-1447668-07-Results-05_T8",
"type": "Protein",
"offsets": [
[
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],
"text": [
"CD25"
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},
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"id": "PMC-1447668-07-Results-05_T9",
"type": "Protein",
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"text": [
"Foxp3"
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"type": "Protein",
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"text": [
"CD4"
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},
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"id": "PMC-1447668-07-Results-05_T11",
"type": "Protein",
"offsets": [
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],
"text": [
"CD25"
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},
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"id": "PMC-1447668-07-Results-05_T12",
"type": "Protein",
"offsets": [
[
1032,
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]
],
"text": [
"Foxp3"
],
"normalized": []
},
{
"id": "PMC-1447668-07-Results-05_T13",
"type": "Protein",
"offsets": [
[
1221,
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]
],
"text": [
"Tax"
],
"normalized": []
},
{
"id": "PMC-1447668-07-Results-05_T14",
"type": "Protein",
"offsets": [
[
1287,
1292
]
],
"text": [
"Foxp3"
],
"normalized": []
}
] | [
{
"id": "PMC-1447668-07-Results-05_E1",
"type": "Positive_regulation",
"trigger": {
"text": [
"Increased"
],
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0,
9
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]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1447668-07-Results-05_E2"
}
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"id": "PMC-1447668-07-Results-05_E2",
"type": "Gene_expression",
"trigger": {
"text": [
"Expression"
],
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24,
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]
]
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"ref_id": "PMC-1447668-07-Results-05_T1"
}
]
},
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"id": "PMC-1447668-07-Results-05_E3",
"type": "Gene_expression",
"trigger": {
"text": [
"expressed"
],
"offsets": [
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]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1447668-07-Results-05_T2"
}
]
},
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"id": "PMC-1447668-07-Results-05_E4",
"type": "Gene_expression",
"trigger": {
"text": [
"expression"
],
"offsets": [
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355,
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]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1447668-07-Results-05_T6"
}
]
},
{
"id": "PMC-1447668-07-Results-05_E5",
"type": "Positive_regulation",
"trigger": {
"text": [
"high"
],
"offsets": [
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]
]
},
"arguments": [
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"role": "Theme",
"ref_id": "PMC-1447668-07-Results-05_E6"
}
]
},
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"id": "PMC-1447668-07-Results-05_E6",
"type": "Gene_expression",
"trigger": {
"text": [
"expression"
],
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]
},
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}
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},
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"id": "PMC-1447668-07-Results-05_E7",
"type": "Positive_regulation",
"trigger": {
"text": [
"high"
],
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]
},
"arguments": [
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"role": "Theme",
"ref_id": "PMC-1447668-07-Results-05_T13"
}
]
},
{
"id": "PMC-1447668-07-Results-05_E8",
"type": "Negative_regulation",
"trigger": {
"text": [
"reduced"
],
"offsets": [
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1279,
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]
]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1447668-07-Results-05_E9"
}
]
},
{
"id": "PMC-1447668-07-Results-05_E9",
"type": "Gene_expression",
"trigger": {
"text": [
"expression"
],
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]
},
"arguments": [
{
"role": "Theme",
"ref_id": "PMC-1447668-07-Results-05_T14"
}
]
}
] | [] | [] |
24 | PMC-1447668-08-Results-06 | [
{
"id": "PMC-1447668-08-Results-06__text",
"type": "abstract",
"text": [
"CREB Is a Target for Transcriptional Repression by Foxp3\nAlthough Foxp3 could down-regulate Tax-dependent transactivation of the HTLV-I LTR (Figure 4B) and inhibit Tax expression from an infectious molecular clone (Figure 5), Foxp3 failed to modulate Tax function in the absence of the viral promoter (Figure 4D). These results led us to hypothesize that Foxp3 acts on HTLV-I gene expression by interacting with proteins important for driving HTLV-I LTR activity in vivo. Previous studies have demonstrated that the Tax-responsive elements within the HTLV-I LTR play a crucial role in driving Tax-mediated transactivation of the HTLV-I LTR [18]. The Tax-responsive elements have been shown to resemble CREB binding sites, bind CREB in vitro and in vivo, and facilitate HTLV-I LTR activation both in the presence and in the absence of Tax [35,36]. Ching and colleagues [18] demonstrated that addition of a dominant-negative CREB expression vector resulted in nearly complete inhibition of Tax-mediated activation of the HTLV-I LTR, while blocking NF-kappaB activation by addition of a dominant-negative IKKbeta expression vector had no effect on Tax transactivation of the HTLV-I LTR. Therefore, we hypothesized that Foxp3 may inhibit Tax transactivation of the HTLV-I LTR via disruption of the CREB signaling pathway. To test this possibility, HEK 293T cells were transfected with an HTLV-I LTR or synthetic CREB reporter vector along with a control expression vector (EGFP) or expression vectors encoding Foxp3 or DeltaFKH. As shown in Figure 6A, Foxp3 down-regulated basal activation of the HTLV-I LTR and transcription of a synthetic CREB reporter vector, suggesting that Foxp3 down-regulates HTLV-I LTR activation by targeting the CREB pathway. Deletion of the FKH domain of Foxp3 dampened the suppressive effect of Foxp3, but did not completely abrogate suppression, as is seen with NF-kappaB-responsive promoters in HEK 293T cells. Like NF-kappaB activation, CREB transcriptional activation was also suppressed by expression of Foxp3, and to a similar extent DeltaFKH, in healthy donor CD4+ T cells (Figure 6B). Similarly, Foxp3 and DeltaFKH also repressed basal HTLV-I LTR activation in primary human CD4+ T cells (Figure 6C). To our knowledge, this is the first evidence implicating CREB as a molecular target of Foxp3. As observed with NF-kappaB activation, DeltaFKH was a more potent inhibitor of CREB activation in CD4+ T cells than in HEK 293T cells, further indicating that a cell type-specific mechanism of action may govern the function of this Foxp3 mutant. \nTo determine whether Foxp3 functioned by directly signaling through CREB, we utilized expression vectors encoding CREB-1 or c-Jun (a member of the activator protein 1 family of transcription factors) fused in-frame to the Gal4-BD (Gal4-BD-CREB-1 and Gal4-BD-c-Jun). As shown in Figure 6D, activation of a Gal4-responsive reporter vector by Gal4-BD-CREB-1 was down-regulated by Foxp3 compared to control vector (EGFP), indicating that Foxp3 functions by directly or indirectly interacting with CREB-1. However, Foxp3 failed to markedly affect transcriptional activation of Gal4-BD-c-Jun (c-Jun has also been demonstrated to bind to the HTLV-I LTR) and Gal4-BD-Tax (see Figure 5). Importantly, the mechanism of Foxp3-mediated inhibition of CREB-dependent transcription was not due to a block in CREB-1 protein expression, as determined by Western blot analysis (Figure 6E). Although these results demonstrate that Foxp3 functions as a co-repressor of CREB activation (in addition to NF-kappaB and NF-AT), we were unable to detect a direct physical interaction between CREB-1 and Foxp3 by coimmunoprecipitation or mammalian two-hybrid analysis (unpublished data). Therefore, our data suggest that Foxp3 may interfere with CREB signaling at an upstream event, such as phosphorylation of CREB or recruitment/function of coactivator proteins CREB-binding protein (CBP)/p300. \n"
],
"offsets": [
[
0,
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}
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{
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"type": "Protein",
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51,
56
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{
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66,
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92,
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"type": "Protein",
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"type": "Protein",
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226,
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"Foxp3"
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"type": "Protein",
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251,
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"Tax"
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"type": "Protein",
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355,
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"text": [
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"type": "Protein",
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"text": [
"CREB-1"
],
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},
{
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"type": "Protein",
"offsets": [
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"text": [
"c-Jun"
],
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{
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"type": "Protein",
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"text": [
"activator protein 1"
],
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],
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] | [] |
25 | PMC-1447668-09-Results-07 | [
{
"id": "PMC-1447668-09-Results-07__text",
"type": "abstract",
"text": [
"Foxp3 Antagonizes CREB Transcriptional Activation by Disrupting Coactivator Recruitment\nStimulation of CREB-dependent transcription by reagents that activate adenylate cyclase and increase cAMP levels (e.g., forskolin) increase the transactivation potential of CREB through phosphorylation of serine 133 by protein kinase A, which permits binding and recruitment of coactivators CBP/p300 to the promoter [37,38]. Phosphorylation of serine 133 does not, however, affect the DNA-binding activity of CREB in most cases [39-41]. Addition of forskolin to HEK 293T cells stimulated activation of a CREB reporter vector about 65 fold (Figure 7A). Overexpression of Foxp3 was capable of down-regulating forskolin-induced CREB transcriptional activation. The functional interaction between Foxp3 and CREB did not affect the DNA-binding activity of CREB-1, but did show a modest decrease in activating transcription factor 2 (ATF-2) DNA-binding activity in the presence of forskolin as determined by transcription factor ELISA (Figure 7B). \nWhile Foxp3 has been shown to bind to and repress activation of both NF-kappaB and NF-AT, exactly how Foxp3 functions to bring about this affect has not been elucidated. To determine how Foxp3 blocks CREB-dependent transcription, we examined whether Foxp3 was capable of (1) disrupting the recruitment of coactivator proteins and/or (2) preventing phosphorylation of CREB at serine 133 (which is a prerequisite for coactivator recruitment). Since both of these events are required for CREB-dependent gene expression, we hypothesized that Foxp3 may affect CREB activation at both steps. To determine whether Foxp3 can disrupt the function/recruitment of the coactivator protein p300, we introduced a Gal4 reporter vector and a Gal4-BD-CREB-1 expression vector into HEK 293T cells in the absence or presence of Foxp3, p300, and/or control expression vectors (Figure 7C). As expected, p300 overexpression stimulated transcription of the Gal4-BD-CREB-1 fusion protein. Foxp3, again, repressed basal levels of Gal4-BD-CREB-1 activation by more than 2-fold, while effectively neutralizing Gal4-BD-CREB-1 activation in the presence of p300. We next analyzed the effect of Foxp3 on phosphorylation of CREB at serine 133 in forskolin-treated HEK 293T cells by Western blot analysis. Overexpression of Foxp3 failed to reduce the detectable levels of CREB phosphorylation using a phosphospecific antibody for CREB-1 (unpublished data). However, when we attempted to determine whether Foxp3 could physically interact with the coactivator protein p300, we found that p300 immunoprecipitated Foxp3 when both proteins were overexpressed in HEK 293T cells (Figure 7D). Collectively, these results suggest that Foxp3 antagonizes CREB-dependent gene expression by directly interacting with coactivator p300 and interfering with its function and/or recruitment to CREB-responsive promoter sequences. \n"
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] | [] |
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Dataset Card for BioNLP 2011 GE
The BioNLP-ST GE task has been promoting development of fine-grained information extraction (IE) from biomedical documents, since 2009. Particularly, it has focused on the domain of NFkB as a model domain of Biomedical IE. The GENIA task aims at extracting events occurring upon genes or gene products, which are typed as "Protein" without differentiating genes from gene products. Other types of physical entities, e.g. cells, cell components, are not differentiated from each other, and their type is given as "Entity".
Citation Information
@inproceedings{10.5555/2107691.2107693,
author = {Kim, Jin-Dong and Wang, Yue and Takagi, Toshihisa and Yonezawa, Akinori},
title = {Overview of Genia Event Task in BioNLP Shared Task 2011},
year = {2011},
isbn = {9781937284091},
publisher = {Association for Computational Linguistics},
address = {USA},
abstract = {The Genia event task, a bio-molecular event extraction task,
is arranged as one of the main tasks of BioNLP Shared Task 2011.
As its second time to be arranged for community-wide focused
efforts, it aimed to measure the advance of the community since 2009,
and to evaluate generalization of the technology to full text papers.
After a 3-month system development period, 15 teams submitted their
performance results on test cases. The results show the community has
made a significant advancement in terms of both performance improvement
and generalization.},
booktitle = {Proceedings of the BioNLP Shared Task 2011 Workshop},
pages = {7–15},
numpages = {9},
location = {Portland, Oregon},
series = {BioNLP Shared Task '11}
}
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