Expression profile and differential regulation of the Human I-mfa domain-Containing protein (HIC) gene in immune cells

Immunology Letters 123 (2009) 179–184

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Expression profile and differential regulation of the Human I-mfa domain-Containing protein (HIC) gene in immune cells Lili Gu, Jonathan Dean, André L.A. Oliveira, Noreen Sheehy, William W. Hall, Virginie W. Gautier ∗ School of Medicine and Medical Science, Centre for Research in Infectious Diseases, University College Dublin, Belfield, Dublin 4, Ireland

a r t i c l e

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Article history: Received 11 December 2008 Received in revised form 5 March 2009 Accepted 18 March 2009 Available online 28 March 2009 Keywords: HIC Gene expression profile Immune expression IL-2 responsive gene Relative quantification real-time RT-PCR

a b s t r a c t The Human I-mfa domain-Containing protein, HIC, is a 246 amino acid protein that functions as a transcriptional regulator. Although the precise function of HIC remains to be clarified, the association of the HIC gene locus with myeloid neoplasms, its interactions with lymphotropic viruses such as EBV, HIV-1 and HTLV-1 and its expression in immune tissues suggest that HIC might have a modulatory role in immune cells. To further characterise the HIC functional relationship with the immune system, we sought to analyse the HIC gene expression profile in immune cells and to determine if immunomodulatory cytokines, such as interleukin (IL)-2, could regulate the expression of HIC mRNA. Relative quantitative real-time RT-PCR revealed that HIC mRNA is highly expressed in PBMCs and in various hematopoietic cell lines. The immunomodulatory cytokine IL-2 up-regulated HIC gene expression in PBMCs, CEM, MT-2 and U937 but markedly reduced HIC gene expression in Raji. Addition of cycloheximide indicated that the IL-2 effects were independent of de novo protein synthesis and that the HIC gene is a direct target of IL-2. Two cell lines (Jurkat and BJAB) displayed a distinct loss in HIC gene expression. However, when these cell lines were subjected to a combination of DNA methyltransferase and histone-deacetylase inhibitors, (5-aza-2-deoxycytidine and trichostatin A, respectively), HIC expression was de-repressed, indicating possible epigenetic control of HIC expression. Overall, our study describes that the immune expression of HIC is cell-specific, dynamic, and identifies the HIC gene as an IL-2 responsive gene. Furthermore, our de-repression studies support the hypothesis that HIC might represent a candidate tumor suppressor gene. Overall, this report provides new insights for a putative role of HIC in the modulation of immune and inflammatory responses and/or hematological malignancies. © 2009 Elsevier B.V. All rights reserved.

1. Introduction HIC (Human I-mfa domain-Containing protein) is a 246 amino acid protein with a prominent cytoplasmic distribution [1]. The Cterminal domain of HIC encompasses a cysteine-rich region termed the I-mfa domain because of its 74% homology with the C-terminal domain of the inhibitor of MyoD family a (I-mfa) [1]. The I-mfa domains of both HIC and I-mfa are essential for their activities. HIC is encoded by a gene located on the long arm of chromosome 7 and maps to the locus 7q31.1-q31.2, spanning a region of 100,000 kb. The HIC gene encompasses four exons and three introns; the ORF is encoded by all exons and starts within exon one, following an UTR of 263 nucleotides (Fig. 1A and B). Interestingly, this locus has been identified as a fragile region (FRA7G),

∗ Corresponding author. Tel.: +353 1 7161229; fax: +353 1 7161236. E-mail addresses: [email protected] (L. Gu), [email protected] (J. Dean), [email protected] (A.L.A. Oliveira), [email protected] (N. Sheehy), [email protected] (W.W. Hall), [email protected] (V.W. Gautier). 0165-2478/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2009.03.010

which is associated with chromosomal instability [2–4]. Deletions in this region have been associated with a range of malignancies, including leukemia [2–8]. Furthermore, because of its location in a region frequently deleted in myeloid neoplasms, HIC has been considered as a strong candidate tumor suppressor gene (TSG) [5]. However, no mutations within the HIC coding sequence itself have been identified. HIC displays a complex yet incompletely understood functional profile, but is generally described as a transcriptional regulator. Indeed, HIC was first identified as a protein that enhances Tax-mediated expression of HTLV-1 promoters and represses Tatmediated expression of HIV-1 promoters [1]. Subsequently, it was demonstrated that HIC physically interacts with the viral transactivator, HIV-1 Tat, which results in Tat cytoplasmic sequestration and down-regulation of Tat-mediated transcription of the virus promoter [9,10]. HIC has also been shown to interact with the Epstein-Barr viral protein RK-BARF0, although the functional relevance of this interaction has not been investigated [11]. In parallel, HIC has also been shown to interact with several cellular transcription factors, including Axin, cyclin T1 and TCF1, and

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to modulate their function and/or associated signaling pathways [10,12,13]. HIC was first cloned at the cDNA level and identified in the Tcell line MT-2 [1]. However, the protein exhibits a wide but specific tissue distribution. Northen Blot analyses have shown that in addition to prostate, uterus, and small intestine, HIC mRNA is expressed in human lymphoid organs including thymus, spleen and peripheral blood leukocytes but is almost absent in testis and colon [1]. End-point RT-PCR have shown that HIC is differentially expressed in various cell lines including cell lines of immune origin [13]. The precise function of HIC remains to be clarified. Nevertheless, the association of the HIC gene locus with myeloid neoplasms, its interactions with lymphotropic viruses such as EBV, HIV-1 and HTLV-1 and its expression in immune tissues suggest that HIC might have a modulatory role in immune cells. To further characterise HIC activity and its functional relationship with the immune system, we conducted this study which describes (i) HIC mRNA expression profiles in selected human primary immune cells (PBMCs and distinct subsets of PBMCs), (ii) analysis of the control of HIC immune cell expression at the epigenetic level in certain malignant states, as represented by B and T cell lines, and (iii) examination of how stimulation with IL-2, a potent immunomodulatory cytokine, can regulate the expression of HIC in immune cells. 2. Methods 2.1. In silico analysis of the HIC gene promoter A genomic sequence spanning 1.2 kbp upstream and 483 bp downstream of the transcription start site (+1) of HIC gene (NC 000007.12) was retrieved from the NCBI human genome database (Fig. 1A and B). Transcription factor binding sites were identified by the TESS software (http://www.cbil.upenn.edu/cgibin/tess/tess?RQ = SEA-FR-Query) and combined search option TRANSFAC and Jaspar databases were selected [14]. We manually selected the transcription factors by using the following

critical parameters: (i) Matrix similarity (Sm) was limited at 0.8 (maximum 1.0) for TRANSFAC data. (ii) Log-likelihood score divided by length of the site (La/) was limited at 1.7 (maximum 2.0) for Jaspar databases. (iii) Only sense not anti-sense sequences of the corresponding transcription factor binding sites were chosen. CpG islands were retrieved using the FirstEF software (http://rulai.cshl.edu/tools/FirstEF). 2.2. Cell culture and treatment THP-1, Jurkat, MT-2, BJAB, Raji and CEM cell lines were cultured in RPMI 1640 medium containing 10% fetal calf serum and supplemented with 0.3 mg/l of l-Glutamine (GIBCO) and antibiotics. HeLa and 293T cell lines were cultured in DMEM medium containing 10% fetal calf serum and supplemented with 0.3 mg/l of l-Glutamine (GIBCO) and antibiotics. Human PBMCs from healthy donors were isolated using Ficoll-PaqueTM Plus (Amersham Biosciences) density gradient separation. Helper T-lymphocytes were isolated by positive selection using CD4 MicroBeads (Miltenyi Biotec), following an initial step of monocyte depletion using CD14 MicroBeads (Miltenyi Biotec). Cytotoxic T-lymphocytes, monocytes, B cells and NK cells were isolated by positive selection using CD8, CD14, CD19 and CD56 MicroBeads, respectively. All separations were performed using an AutoMAC Pro cell separator (Miltenyi Biotec), following the manufacturer’s guidelines. The purity of isolated subsets was examined by flow cytometry (FACSCalibur, Becton Dickinson) using mouse anti-human CD4, CD8, CD14, CD19 and CD56 monoclonal antibodies labeled with APC, PerCP or PE (BD Pharmingen). For IL-2 stimulation studies, cells (1 × 106 /ml) were seeded in 12-well tissue culture plates and stimulated with 10 U/ml of IL-2 (Sigma) with or without 1 ␮g/ml of Cycloheximide (Sigma) for 3 h. For derepression studies, Jurkat, BJAB, Raji and CEM cells (0.1 × 106 /ml) were seeded in 12-well tissue culture plates. Cells were treated either with 500 nM Trichostatin A (TSA) (Sigma) for 48 h or with 2 ␮M 2 -Deoxy-5-azacytidine (AZA) (Sigma) for 72 h or a combination of both, TSA being added for the last 48 h, following

Fig. 1. Maps of the HIC gene. (A) Chromosomal map of the HIC gene locus on human chromosome 7. (B) Structure of the HIC gene: intron-exon organisation with each of the 4 exons depicted as a box. The corresponding coding region (cds) is colored in black. (C) Schematic of the HIC promoter region, showing the relative location of transcription factor binding sites as determined by the TESS software. The transcription start site is indicated by an arrow. The location of the CpG window, as determined by the FirstEF software, is indicated by a double arrow.

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24 h culture with AZA alone. AZA concentration was maintained by addition of fresh AZA each day. Cells were re-suspended in fresh medium with appropriate drugs every 48 h. 2.3. RNA extraction and real-time RT-PCR Total RNA was extracted using the RNeasy mini Kit (Qiagen). RNA concentration was measured by NanoDrop® ND-1000 Spectrophotometer. RNA quality was analyzed using Agilent 2100 Bioanalyser (Agilent Technologies). 600 ng of total RNA extracted from the cell lines or 200 ng of total RNA extracted from isolated PBMCs subsets was reverse transcribed with 200 units of M-MLV Reverse Transcriptase (Promega) and Random Primers (Promega) according to the manufacturer’s instructions. HIC Primers were designed using the HIC cDNA sequence (GenBank accession no. AY196485). Primer sequences were chosen to prevent homologies to other genes, in particular to I-mfa. Furthermore, to avoid possible genomic DNA contamination, the primers were designed to overlap the first intron-spanning region. The sequences for HIC forward and reverse primers are the following: HIC F 5 -CCAATAGCCACTTCACACATG-3 ; HIC R 5 GAAACAGGTGCTGAAAGTTG-3 . The resulting PCR product was isolated, sequenced and was found to be identical to the expected HIC sequence (data not shown). Candidate housekeeping genes, including 18s rRNA, ␤2 M and PBGD, were tested for their Ct, PCR efficiency, and mRNA expression level (data not shown). Their corresponding primer pairs were described previously [15]. 18s rRNA, whose mRNA expression remained relatively consistent throughout the cell types and experimental conditions and which has a similar Ct and PCR efficiency to the target gene HIC, was selected as the endogenous reference gene. Real-time PCR was performed on the Roche LightCycler platform using QuantiTect SYBR Green PCR Kit (QIAGEN). 2 ␮l template cDNA was added to the final volume of 20 ␮l reaction mix. Realtime PCR cycle parameters included 15 min at 95 ◦ C followed by 45 cycles involving denaturation at 95 ◦ C for 15 s, annealing at 60 ◦ C for 20 s and elongation at 72 ◦ C for 20 s. All experiments were performed in duplicate and the data expressed as the mean of at least two independent experiments. 2.4. Relative quantification The relative quantification of HIC mRNA expression ratio was calculated based on the Pfaffl mathematical model but with PCR efficiency (E) correction of each individual sample [16]. The relative expression ratio (R) of HIC mRNA, compared to the reference gene 18s rRNA, was calculated for each sample (S) versus control (C) cells (either 293T or untreated PBMC subsets) using the formula:

Ratio =

CTHIC(C) CTHIC(S) / EHIC(C) EHIC(S) CT18s rRNA(S) CT18s rRNA(C) / E18s rRNA(C) E18s rRNA(S)

The PCR efficiency (E) of each individual sample was calculated by LinRegPCR Software which performed linear regression analysis of the raw fluorescent data [17]. CT (Crossing Threshold) which recorded the cycle when sample fluorescence exceeds a chosen threshold above background fluorescence was achieved from LightCycler software version 3.5 (Roche). 3. Results 3.1. In silico analysis of the HIC gene promoter To examine if the promoter of HIC could determine the tissue and cell-specific regulated pattern of HIC gene expression, we anal-

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ysed its promoter sequence for putative cis-regulatory elements. A sequence spanning 1.2 kbp upstream and 483 bp downstream of the transcription start site (+1) of the HIC gene was retrieved from the NCBI human genome database (NC 000007.12). Searches of transcription factor binding sites were performed using the combined search option of the TESS software, which includes both the Transfac and Jaspar databases. The search resulted in the identification of multiple cis-acting regulatory elements and their cognate protein factors including a consensus TATA sequence (−527) and a CAAT box (−761), which were associated with several putative cis-acting elements for both general and cell-specific transcription factors (Fig. 1C). In particular, a large number of these transcription factors are specifically involved in the regulation of immune/inflammatory processes and hematopoiesis, and include NF-␬B, NF-AT, GATA -1/2/-3, c-myb, LEF-1, Ikaros (Lyf-1), c-Ets, STAT6, MZF1, Fra-1, AREB6 and AML-1 factors (Fig. 1C and Supplementary Material 1). Collectively, they constitute a potential cluster of cis-acting elements which could control HIC gene expression in hematopoietic cells. In addition, in silico analysis of the genomic structure revealed one putative CpG island, located within exon one (+159 to +360) of the HIC gene (Fig. 1C). 3.2. HIC is highly expressed in primary human immune cells. To characterise the expression of HIC in primary immune cells, we performed real-time RT-PCR in PBMCs and specific PBMC subsets. The purities of isolated subsets ranged from 90% to 99.5% and were determined by FACS analysis (Fig. 2A). The relative expression of HIC was calculated employing the expression of HIC in a nonimmune cell line, 293T, as control. HIC mRNA expression profiles in PBMCs were performed in duplicate, from six different healthy donors and demonstrated that HIC is highly expressed (Fig. 2B). CD4+ T-cells and CD8+ T-cells displayed the similar levels of HIC expression compared to PBMCs. NK cells (CD56+) and monocytes (CD14+) both exhibited a greater expression of HIC mRNA relative to PBMCs. Conversely, a significantly lower level of HIC expression was observed in B-cells (CD19+), with only one third of HIC mRNA expression relative to the PBMCs. 3.3. IL-2 directly up-regulates HIC mRNA expression in primary human immune cells As described above, the HIC expression level in primary immune cells varied depending on the PBMC subsets investigated. This variability suggests that HIC expression may be regulated by specific conditions, such as differentiation stage or activation status. We therefore investigated whether HIC mRNA expression is regulated in PBMCs following stimulation with IL-2, a potent immunomodulatory cytokine. To examine the direct effects of IL-2 stimulation, cells were incubated with IL-2 for 3 h prior to RNA extraction, in the presence or absence of cycloheximide (CHX), a translation inhibitor. IL-2 treatment did not modulate the expression of the reference gene 18s rRNA but remarkably, it did induce an immediate and significant up-regulation of HIC gene expression (up to 3-fold increase P < 0.001) (Fig. 2C). In addition, we examined whether HIC mRNA expression is differentially regulated in distinct PBMCs subsets following stimulation with IL-2 (Fig. 2D). HIC gene expression was highly induced by IL-2 treatment in CD4+ T-cells and NK cells (CD56+) (up to 3.5 and 2.5 fold increase, respectively, P < 0.001). CD8+ T-cells and monocytes (CD14+) both displayed similar induction of HIC gene expression (up to 2-fold increase, P < 0.01). Conversely, HIC gene expression did not respond to IL-2 treatment in B-cells (CD19+), which do not express functional IL-2 receptor [18,19]. HIC gene expression was similarly increased by IL-2 in the presence of CHX, strengthening the evidence for a direct effect of the IL-2. Meanwhile, CHX did

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Fig. 2. HIC expression profile in human immune primary cells. (A) Isolated cell subsets purity was evaluated by FACS. Circles indicate purity of cell isolates from each of 6 healthy individuals. Triangles indicate the purity of cell isolates from each of 3 individuals which were used in IL-2 treatment experiments. Bars represent mean purity observed for each all subsets. Dot plots illustrate subset purity observed in one representative sample. (B) Relative expression of HIC mRNA measured by real-time RT-PCR in PBMCs and PBMC subsets (CD4, CD8, CD14, CD19, CD56) isolated from 6 individual donors. HIC mRNA expression was compared to 293T cells. HIC mRNA expression in (C) total PBMCs and (D) PBMC subsets from three individual donors treated with IL-2 for 3 h with or without cycloheximide (CHX). Expression is presented relative to untreated cells (BL). Corresponding 18s rRNA expression levels are shown. Values are mean ± standard deviation for a minimum of two independent experiments performed in duplicate. * P < 0.05; ** P < 0.01; *** P < 0.001.

not affect HIC gene expression on its own. These results identify the HIC gene as an IL-2 inducible gene in PBMCs and more specifically in CD4+ T-cells, NK cells, CD8+ T-cells and monocytes. 3.4. Distribution of HIC mRNA expression in hematopoeitic cell lines

IL-2R␣, ␤ and ␥ chains. With the exception of THP-1 and BJAB, the hematopoietic cell lines tested in this study, (Raji,CEM,U937) express the low affinity receptor containing the ␣ and ␥ chains, the intermediate affinity receptor containing the ␤ and ␥ chains (Jurkat), and the high affinity receptor containing the ␣, ␤ and ␥ chains (MT2) [20–25]. As expected, IL-2 did not modulate HIC mRNA

Next, we studied the expression of HIC in several cell lines derived from hematological malignancies, including lymphoid and monocytic cell lines (Fig. 3). Expression levels in the two nonhematopoietic cell lines 293T and HeLa are comparable and at an intermediate level (data not shown). The levels in 293T cells were given the arbitrary value of 1 and used as a reference. The cell lines MT-2 (T cell leukemia), CEM (T cell leukemia), THP-1 (acute monocytic leukemia) and U937 (monocytic leukemia) all showed greater expression of HIC mRNA, relative to 293T (up to 4 times greater). On the other hand, the B lymphoblastoid cell lines Raji and BJAB showed lower or no HIC expression, relative to 293T cells, and no signal was detected in Jurkats, a T cell leukemia-derived cell line, in agreement with a previous study by Wang et al. (2007) [13]. 3.5. Differential regulation of HIC expression by IL-2 We subsequently measured the immediate effect of IL-2 stimulation for 3 h with or without CHX on HIC gene expression in the different cell lines (Fig. 3). IL-2 is a highly potent stimulus for cells that express the IL-2 multimeric receptor (IL-2R), consisting of the

Fig. 3. HIC expression profile in human immune cell lines. Relative expression of HIC mRNA measured by real-time RT-PCR in various cell lines treated with or without IL2 and/or CHX treatment for 3 h. HIC mRNA expression was compared to 293T cells. Corresponding 18s rRNA expression levels are shown. Values are mean ± standard deviation for a minimum of two independent experiments performed in duplicate. * P < 0.05; ** P < 0.01; *** P < 0.001.

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4. Discussion

Fig. 4. Combination of TSA and AZA but not TSA alone reactivate HIC gene expression. Relative expression of HIC mRNA measured by real-time RT-PCR in Jurkat, BJAB, Raji and CEM cells treated with TSA for 48 h and/or AZA for 72 h. HIC mRNA expression was compared to 293T cells. Corresponding 18s rRNA expression levels are shown. Values are mean ± standard deviation for a minimum of two independent experiments performed in duplicate. * P < 0.05; ** P < 0.01; *** P < 0.001.

expression in the non-hematopoietic cell lines 293T and HeLa, which do not express functional IL-2 receptors (data not shown). Similarily, HIC expression remained constant following IL-2 stimulation of THP-1 cells (data not shown). However, HIC expression was significantly up-regulated by IL-2 in MT-2, CEM and U937, with a maximum of 2.77 fold for CEM. Interestingly, we could observe IL-2-mediated HIC transcriptional repression in Raji cells (2-fold decrease). Both observed effects were the result of an immediate and direct action of IL-2, since HIC gene expression was similarily modulated by IL-2 in the presence of CHX (Fig. 3). Finally, IL-2 did not induce HIC expression in Jurkat and BJAB, which remained negative for HIC expression (data not shown). Under these conditions 18sRNA expression remained stable. 3.6. Derepression of HIC gene expression upon AZA and TSA treatment The repression of HIC gene expression in Jurkat and BJAB cell lines could be the result of abnormal epigenetic mechanisms, which can be associated with hematological malignancies. Epigenetic alterations include DNA methylation at CpG islands and post-translational modifications of histones followed by chromatin remodelling [26]. These modifications are mediated by DNA methyltransferases (DNMTs) and histone deacetylases (HDACs), and are reversible upon treatment with DNMT inhibitors, (AZA) and/or HDAC inhibitors including Trichostatin (TSA). To investigate which epigenetic mechanisms may alter HIC expression, we treated Jurkat and BJAB cells, both negative for HIC expression, and CEM and Raji cells expressing high and low levels of HIC, respectively, with TSA, AZA or a combination of both (Fig. 4). Remarkably, in both Jurkat and BJAB cells, silenced HIC gene expression could be reversed by the combination of TSA and AZA, where a synergistic effect was observed, and to a lesser degree by AZA alone in Jurkat but not BJAB cells. In contrast, TSA treatment alone had no effect on HIC expression in either cell line. Similarly, in CEM and Raji, HIC expression was up-regulated (up to 3.5 fold for CEM) by treating the cells with AZA alone and with the combination of AZA and TSA. However we did not observe a synergistic action when both drugs were employed. Remarkably, HIC expression was dramatically reduced (up to 17 fold) in both cell lines treated with TSA alone. Under these conditions 18sRNA expression remained stable.

To examine the relationship of HIC expression with immune function, we characterised HIC mRNA expression profiles in primary cells (PBMCs and distinct subsets of PBMCs) and cell lines. It could be shown that HIC expression is highly dynamic, and celltype and environment specific. HIC gene expression levels changed significantly following stimulation by IL-2. Furthermore, we have provided evidence supporting a putative epigenetic control of HIC gene expression in specific immune cell lines. These features of HIC gene expression are associated with the promoter sequence, which harbors putative binding sites for transcription factors known to be critical for the development and regulation of the immune system. Finally, the identification of cell lines expressing or not expressing HIC constitutes a valuable tool for testing the activities of HIC in functional studies. Overall, our study provides new insights into the possible role(s) for HIC in immuno-regulatory functions. Prior to this report, little was known about the regulation of HIC expression during immunomodulatory conditions. Here, we show that the HIC gene could be directly regulated by IL-2 treatment. Interestingly, IL-2 regulates both cellular proliferation, survival and activation induced cell death (AICD) [27,28]. These two opposing processes are mediated by downstream signaling events triggered by the interaction of IL-2 with its multimeric receptor (IL-2R␣, ␤ and ␥ chains), and include the Ras-MAP kinase, JAK-STAT, phosphoinositol 3-kinase/akt/p70 S6 kinase and the Fas-FasL pathways [27–30]. They ultimately result in the up-regulation of specific genes involved in anti-apoptotic pathways (bcl-2), cell cycle progression (cyclin D2), production of cytokines and their receptors (TNF␥, TNF␣, IL-4R, IL-2R), and production of transcription factors (c-myb) [27–29]. Our study places HIC on the specific list of genes regulated by IL-2. Interestingly, the analysis of the promoter revealed that it contains binding sites for Ikaros/Aiolos, STAT6 and NF-␬B, which are mediators of the signaling pathways triggered by IL-2 and could form the basis of the molecular mechanisms involved [27] [31]. However, the specific involvement of these factors and their interactions remains to be established. To investigate whether other cytokines could interfere with HIC expression, we also treated PBMCs with IFN-␥, however this did not result in any variation in HIC gene expression level, suggesting that the IL-2 effect is specific (data not shown). To examine the molecular mechanisms involved in HIC gene silencing observed in Jurkat cells, which remained negative for HIC expression following IL-2 stimulation, we treated the cells with TSA, AZA or a combination of both. The silencing of HIC gene expression could be overcome in both cell lines, with a synergistic effect when both drugs were employed. While we did not assess the methylation status of the promoter, these results support the possibility that methylation of the putative CpG island identified within exon 1 could play a role in the silencing of HIC expression in both Jurkat and BJAB cells. However, since many genes can be reactivated by AZA and TSA, it is not impossible that HIC reactivation could result from the indirect effect during the 72 h of combined treatment. The de-repression of HIC gene expression by the synergistic action of AZA and TSA is a characteristic of candidate tumor suppressor genes (TSG) and other cancer-related genes displaying aberrant cytosine methylation [32]. Interestingly, the HIC gene locus is associated within a fragile region (FRA7G), which can result in chromosomal rearrangement and/or loss of heterozygocity and could ultimately affect HIC expression [2–4]. Accordingly, because of its position in a region encompassed in frequent chromosomal deletions associated with myeloid neoplasms, HIC has been previously identified as a candidate tumor suppressor gene (TSG) [5]. Our study strengthens this hypothesis and it would be of interest to characterise the HIC expression profile in these cells and to

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determine if HIC gene promoter region is the target of abherant methylation leading to gene silencing. In this study, we have also described the TSA-mediated downregulation of HIC gene expression in CEM and Raji cells. This effect was observed following 48 h incubation with TSA, making it possible that both direct and indirect effects might be involved. TSA targets and inhibits HDAC activity and is usually employed to characterise the effect of histone deacetylation on gene expression. One possible mechanism of TSA inhibition of HIC gene expression could be that TSA de-repressed the expression of transcriptional repressors, which in turn targeted the HIC promoter. Conversely, TSA has been shown to down-regulate transcription factors such as the cmyc proto-oncongene [33]; these could be essential in mediating HIC gene transcription. A number of proteins other than histones are targets for HDAC activity, including transcription factors such as p53 and GATA-1, GATA-2 [34–36], and de-acetylation of these factors interferes with their DNA binding activity [36,37]. We have identified putative binding sites for these factors within the HIC gene promoter. Finally, TSA could modulate signaling pathways relevant for HIC promoter activation. Indeed, TSA has been shown to block IL-2 signaling and IL-2 induced gene expression [33]. In conclusion, this report describes three features of HIC immune expression: cell-specificity, modulation and silencing. These properties are corroborated by the specific cis-acting elements identified on HIC gene promoter by our in silico studies and support putative role(s) for HIC in the development, maturation, homeostasis and/or response of the immune system.

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