**3. Transcription factor regulation and epigenetic control of the expression of NK-cell receptors and their ligands**

#### **3.1 NKG2 receptor family**

In humans, the NKG2 receptor family comprises seven members called NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F and NKG2H; NKG2A/B and NKG2E/H pairs are splice variants of the same gene (Brostjan et al., 2000; Glienke et al., 1998). All members of the NKG2 family, except NKG2D, share substantial sequence homology, are closely linked, and of the same transcriptional orientation; they contain conserved sequences at the transcriptional start site and at other transcription factor-binding sites, such as the TCF-1 (testosterone conversion factor-1) and GATA-1 (GATA-binding factor 1) sites. These

Transcription Regulation and Epigenetic

(Zhang et al., 2009).

**3.3 Ligands of NKG2D-ULBPs** 

Control of Expression of Natural Killer Cell Receptors and Their Ligands 431

MICB contain putative heat shock elements (HSE) that are prototypic transcription inducer sites in heat shock protein-70 (HSP70) genes and that bind activated trimeric heat shock factor protein-1 (HSF1). Cytomegalovirus (CMV) infection results in up to a 10-fold increase in cell surface MIC expression and is associated with induced HSP70 expression. TATA-like elements and Sp1 consensus sites, as with HSE, are located unusually far "upstream" from MIC gene, but can also moderately or profoundly affect stress-induced and proliferationassociated induction. The common integrating conjugative element (ICE), however, appears to be a negative regulator of MICB, but not of MICA. It is an important to note, although the sequences of MICA and MICB promoters are very similar, the baseline transcriptional activity of MICA is higher than that of MICB and they have different regulatory mechanisms for expression. Most MICA transcripts start downstream of the conserved AP-1 TATA-like motif, a region that lacks a recognizable RNA start site. In contrast, most MICB transcripts initiate further upstream, proximal to the HSE–Sp1–ICE elements (Venkataraman et al., 2007). Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) regulates MICA expression on activated T lymphocytes and on HeLa tumor cells by binding to a specific sequence in the long intron 1 region of the MICA gene (Molinero et al., 2004). Histone deacetylase inhibitor (HDAC-I) and sodium valproate (VPA) induced transcription of MICA and MICB in hepatocellular carcinoma cells, leading to increased cell surface, soluble and total MIC protein expression (Armeanu et al., 2005). Similar results have been obtained using histone deacetylase inhibitors FR901228 and SAHA (Skov et al., 2005). In our laboratory, we found that VPA can increase the expression of HSP70 and Sp1 and increase the binding of transcription factors Sp1 and HSF1 to the MICA/B promoter. This activation leads to the upregulation of MICA/B in HeLa and HepG2 tumor cells

All expressed ULBPs (ULBP1–5) have good conservation of the coding sequence, but the 5′ end flanking regions of these genes have little homology. This situation suggests that ULBPs are regulated specifically in response to different stimuli or pathogens (Lopez-Soto et al., 2006). Mechanisms that regulate the expression of ULBP1 have been described previously. In the core region of the ULBP1 promoter, there are many binding sites for transcription factors, including a canonical TATA box, three GC boxes, one overlapping sequence GC(4)/AP-2, two cytokinin response element (CRE)-like sequences (CRE1 and CRE2) (Zeng et al., 1997; Wajapeyee and Somasundaram, 2003), and one NF-κB site. Transcription of ULBP1 depends strictly on binding of Sp1 and Sp3 to a CRE1 site located in the ULBP1 minimal promoter. The mutation or deletion of this Sp1/Sp3 binding site abolished ULBP1 transcription; Sp3 is the main transcription factor that regulates ULBP1 through the CRE1 site. AP-2α, however, repressed the expression of ULBP1 by binding to the GC(4)/AP-2α site and interfering with the binding of Sp3 and Sp1 to the ULBP1 promoter (Lopez-Soto et al., 2006). It has been shown that heat shock and ionizing radiation treatment can induce the expression of ULBP1/2/3 in human cancer cell lines; HSP 70 is induced by heat shock but not by ionizing radiation (Kim et al., 2006). Human cytomegalovirus (HCMV) can also induce the expression of ULBP1/2/3 proteins that are predominantly localized in the endoplasmic reticulum of infected fibroblasts together with UL16 (Rolle et al., 2003). There is interplay, however, between virus and host cells depending on the viral dose. At low viral dose, ULBP1 or ULBP2 surface expression is completely inhibited compared with ULBP3, while at a higher viral doses cell surface expression of ULBP1 and ULBP2 is delayed (Rolle

members of the NKG2 family form disulfide-linked heterodimers with CD94. Each member of the NKG2 family, however, has a unique repeat Alu sequence that functions as a gene promoter. Alu repeats are the putative binding sites for several transcription factors: activator protein-1/3 (AP-1/3), cAMP response element binding/activating transcription factors (CREB/ATF), CCAAT/enhancer binding protein (C/EBP), transcription factor-1 (Sp1), nuclear factor-1/CCAAT transcription factor (NF-1/CTF) and lymphocyte-specific DNA-binding protein-1 (LyF-1), and may contribute to differences in gene regulation among family members (Brostjan et al., 2000; Glienke et al., 1998). The CD94 protein associates with different NKG2 isoforms to form heterodimeric receptors that function either to inhibit or to trigger cytotoxicity of NK cells, depending on the NKG2 isoform. Functionally, therefore, CD94/NKG2 heterodimers are divided into activating (NKG2C, NKG2E/H) or inhibitory (NKG2A/B) isotypes (Glienke et al., 1998). It has also been shown that several cytokines, including interleukin (IL)-12 (Derre et al., 2002), transforming growth factor (TGF)-β (Bertone et al., 1999), IL-15 (Mingari et al., 1998) and IL-10 (Romero et al., 2001), are capable of inducing expression of CD94/NKG2 in human NK or T cells, in which IL-15 and TGF- can upregulate CD94/NKG2A, but not other CD94/NKG2 receptors (Wilhelm et al., 2003). NKG2D is a special activating receptor that is not covalently associated with CD94. NKG2D is downmodulated by TGF-β, but markedly upregulated by IL-15, interferon (IFN)- and IL-12 stimulation in primary NK cells (Dasgupta et al., 2005). IFN- can stimulate the expression of NKG2D, but inhibits the expression of the inhibitory receptor NKG2A, and therefore alters the balance of stimulatory and inhibitory receptors in favor of activation, leading to NK-cell-mediated cytotoxicity. IFN-γ addition exerts the opposite effect (Zhang et al., 2005).

CD94 is a C-type lectin and is required for the dimerization of the CD94/NKG2 family of receptors, which are expressed on NK cells and T-cell subsets. The CD94 and NKG2 genes are located in the center of the NK-gene complex between the NKRP1 and PRB3 loci in the region containing the STS markers D12S77 and D12S1093. All six genes are closely linked and are situated 100 to 200 kb proximal to the D12S77 STS marker (Sobanov et al., 1999). The CD94 gene is placed in the opposite orientation relative to the NKG2 gene family members (which all have the same transcriptional orientation). CD94 gene expression is regulated by distal and proximal promoters that transcribe unique initial exons specific for each promoter. Both promoters contain elements with IFN-γ-activated and E26 transformationspecific (ETS)-binding sites (called GAS and EBS respectively). The two promoters differentially regulate expression of the CD94 gene in response to treatment with IL-2 or IL-15 (Lieto et al., 2003).

#### **3.2 Ligands of NKG2D-MIC**

The two types of NKG2D ligands are MHC class I chain-related proteins (MICA and MICB) and UL16-binding proteins (ULBP) (Sutherland et al., 2001; Bauer et al., 1999). MICA and MICB are located in the MHC complex within the 6q21.3 chromosomal region. The ULBP family contains 10 genes, of which five are expressed (ULBP1–5). ULBPs are located outside the MHC gene complex, in the 6q24.2–25.3 region (Samarakoon et al., 2009). These molecules exhibit highly restricted expression in healthy tissues but are widely expressed on epithelial tumors. In hematological malignancies they are upregulated in nontumor and tumor cells by genotoxic stress (Kim et al., 2006; Venkataraman et al., 2007). Aligned cosmid-derived regions 1.5 kb upstream of the translation start codon (ATG) in MICA and MICB share approximately 90% sequence identity. The 5′-end flanking regions of MICA and MICB contain putative heat shock elements (HSE) that are prototypic transcription inducer sites in heat shock protein-70 (HSP70) genes and that bind activated trimeric heat shock factor protein-1 (HSF1). Cytomegalovirus (CMV) infection results in up to a 10-fold increase in cell surface MIC expression and is associated with induced HSP70 expression. TATA-like elements and Sp1 consensus sites, as with HSE, are located unusually far "upstream" from MIC gene, but can also moderately or profoundly affect stress-induced and proliferationassociated induction. The common integrating conjugative element (ICE), however, appears to be a negative regulator of MICB, but not of MICA. It is an important to note, although the sequences of MICA and MICB promoters are very similar, the baseline transcriptional activity of MICA is higher than that of MICB and they have different regulatory mechanisms for expression. Most MICA transcripts start downstream of the conserved AP-1 TATA-like motif, a region that lacks a recognizable RNA start site. In contrast, most MICB transcripts initiate further upstream, proximal to the HSE–Sp1–ICE elements (Venkataraman et al., 2007). Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) regulates MICA expression on activated T lymphocytes and on HeLa tumor cells by binding to a specific sequence in the long intron 1 region of the MICA gene (Molinero et al., 2004). Histone deacetylase inhibitor (HDAC-I) and sodium valproate (VPA) induced transcription of MICA and MICB in hepatocellular carcinoma cells, leading to increased cell surface, soluble and total MIC protein expression (Armeanu et al., 2005). Similar results have been obtained using histone deacetylase inhibitors FR901228 and SAHA (Skov et al., 2005). In our laboratory, we found that VPA can increase the expression of HSP70 and Sp1 and increase the binding of transcription factors Sp1 and HSF1 to the MICA/B promoter. This activation leads to the upregulation of MICA/B in HeLa and HepG2 tumor cells (Zhang et al., 2009).
