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

Abstract

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.

Introduction

HIC (Human I-mfa domain-Containing protein) is a 246 amino acid protein with a prominent cytoplasmic distribution [1]. The C-terminal 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), which is associated with chromosomal instability [2], [3], [4]. Deletions in this region have been associated with a range of malignancies, including leukemia [2], [3], [4], [5], [6], [7], [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 Tat-mediated 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 to modulate their function and/or associated signaling pathways [10], [12], [13].

HIC was first cloned at the cDNA level and identified in the T-cell 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.