**Transcription Regulation and Epigenetic Control of Expression of Natural Killer Cell Receptors and Their Ligands**

Zhixia Zhou, Cai Zhang, Jian Zhang and Zhigang Tian *Institute of Immunopharmacology & Immunotherapy, School of Pharmaceutical Sciences, Shandong University China* 

### **1. Introduction**

426 Advances in Cancer Therapy

Rietz, C. & Chen, L. (2004). New B7 family members with positive and negative

Rincon-Orozco, B., Kunzmann, V., Wrobel, P., Kabelitz, D., Steinle, A. & Herrmann, T.

Saenz, S. A., Noti, M. & Artis, D. (2010). Innate immune cell populations function as initiators and effectors in Th2 cytokine responses. *Trends Immunol* 31(11): 407-413. Simone, R., Barbarat, B., Rabellino, A., Icardi, G., Bagnasco, M., Pesce, G., Olive, D. &

Tanaka, Y., Morita, C. T., Nieves, E., Brenner, M. B. & Bloom, B. R. (1995). Natural and

Tokuyama, H., Hagi, T., Mattarollo, S. R., Morley, J., Wang, Q., Fai-So, H., Moriyasu, F.,

Wesch, D., Beetz, S., Oberg, H. H., Marget, M., Krengel, K. & Kabelitz, D. (2006). Direct

Yamashiro, H., Yoshizaki, S., Tadaki, T., Egawa, K. & Seo, N. (2010). Stimulation of human

Zhu, Y. & Chen, L. (2009). Turning the tide of lymphocyte costimulation. *J Immunol* 182(5):

derived dendritic cells. *Molecular Immunology* 48(1-3): 109-118.

Ward, S. G. (1996). CD28: a signalling perspective. *Biochem J* 318 ( Pt 2): 361-377.

(2005). Activation of V gamma 9V delta 2 T cells by NKG2D. *J Immunol* 175(4): 2144-

Saverino, D. (2010). Ligation of the BT3 molecules, members of the B7 family, enhance the proinflammatory responses of human monocytes and monocyte-

synthetic non-peptide antigens recognized by human gamma delta T cells. *Nature*

Nieda, M. & Nicol, A. J. (2008). V gamma 9 V delta 2 T cell cytotoxicity against tumor cells is enhanced by monoclonal antibody drugs--rituximab and

costimulatory effect of TLR3 ligand poly(I:C) on human gamma delta T

butyrophilin 3 molecules results in negative regulation of cellular immunity.

costimulatory function. *Am J Transplant* 4(1): 8-14.

trastuzumab. *Int J Cancer* 122(11): 2526-2534.

lymphocytes. *J Immunol* 176(3): 1348-1354.

*Journal of Leukocyte Biology* 88(4): 757-767.

2151.

375(6527): 155-158.

2557-2558.

Throughout the life of an individual organism, a successful host defense relies on the coordination of innate and adaptive immunity (McQueen and Parham, 2002). Natural killer (NK) cells are characteristic cytolytic cells that are integral components of innate immunity and play a major role both in the direct destruction of infected or transformed cells and in the production of cytokines and chemokines that mediate inflammatory responses and exert a regulatory effect on the adaptive immune responses (McQueen and Parham, 2002; Moretta et al., 2006). Cells become susceptible to NK cell-mediated killing following downregulation of cell surface MHC class I expression after virus infection, which ultimately leads to escape from the MHC-restricted adaptive immune system; a phenomenon also seen in metastasized tumor cells. MHC class I-specific NK receptors (NKR) have acquired the ability to detect immune escape variants and in rodents and primates can be grouped into two distinct classes of MHC class I-specific receptor families based on protein structure: the C-type lectin superfamily (Cl-SF) and the immunoglobulin superfamily (Ig-SF). Humans possess a large family of killer cell immunoglobulin-like receptors (KIR) that belong to the Ig superfamily, whereas mice express lectin-like Ly49 receptors that are now not present in humans, except for a single nonfunctional gene fragment (Lanier, 1998, 2001; Vilches and Parham, 2002; Takei et al., 2001). A third family of NKR, the lectin-like CD94/NKG2 heterodimers, are structurally and functionally conserved between rodents and primates and interact with their ligands: nonclassical MHC class I molecules and some specific ligands that are differentially expressed in different tissues in response to different stresses (McQueen and Parham, 2002; Moretta et al., 2006; Raulet et al., 2001; Uhrberg et al., 1997; Valiante et al., 1997). The actions of NK cells, therefore, are thought to be mediated by the complex interactions between inhibitory and activating signals sent by cell-surface receptors following ligation (Lanier, 2005; Ravetch and Lanier, 2000).

Malignant cells in tumor growth frequently demonstrate alterations in MHC class I expression that play a major role in their ability to escape immune recognition and killing (Dunn et al., 2002; Campoli et al., 2005). NK cells enhance their cytotoxic function and immune regulation by using their stimulatory and inhibitory receptors to maintain the constant balance in the immune system (Chang and Ferrone, 2006). There are major

Transcription Regulation and Epigenetic

**2.2 Epigenetic control(** 

Jacobsen, 2010).

families and their ligands.

**3.1 NKG2 receptor family** 

**NK-cell receptors and their ligands**

Control of Expression of Natural Killer Cell Receptors and Their Ligands 429

Transcription is also controlled by epigenetic regulation that is defined as gene-regulation activity that does not involve changes in the underlying DNA code and includes DNA methylation and a variety of histone protein posttranslational modifications (Feng et al., 2010; Furumatsu and Ozaki, 2010). DNA methylation usually occurs in CpG islands, CGrich regions that are "upstream" of the promoter region. The letter "p" here signifies that C and G are connected by a phosphodiester bond. In humans, DNA methylation is carried out by a group of enzymes called DNA methyltransferases. These enzymes not only determine DNA methylation patterns during early development, but are also responsible for copying these patterns to the strands generated from DNA replication. DNA methylation can alter condensed chromatin structure by influencing histone–DNA and histone–histone contact. Methylation of the promoter and 5′ region of the gene and methylation of histone are associated with transcriptional silencing (Bird, 2002; Bird and Wolffe, 1999; Law and

Acetylation and methylation of histones are the most general and important histone modifications associated with transcriptional activation. Histone acetylation is catalysed by histone acetyl transferases (HATs), whereas the reverse reaction is performed by histone deacetylases (HDACs) (Furumatsu and Ozaki, 2010). HATs and HDACs are classified into many families that are often conserved throughout evolution from yeast to humans. Histone H3 is primarily acetylated at several lysine residues, Lys9, 14, 18, 23, 27 and 56, whereas histone H4 is acetylated at Lys5, 8, 12 and 16 (Su et al., 2004). Interestingly, methylation is often modified at lysine and arginine residues in histones H3 and H4. It has been suggested that these modifications constitute a "histone code"; the "code" hypothesis speculates that this modification pattern is read by proteins that recognize distinct modifications specifically and then regulate chromatin dynamics and genome function (Wang et al., 2004; Kisliouk et al., 2010). Whether these modifications form a true code is still under discussion. The processes of histone modification and DNA methylation can both influence each other. Moreover, DNA methylation and suppressive histone modifications may prevent transcription, even when transcription factors are abundant. In turn, transcription factors may cause alterations in DNA methylation and histone modifications (Furumatsu and Ozaki, 2010; Fernandez-Morera et al., 2010; Strahl and Allis, 2000). Molecular details of the transcriptional regulators involved in expression of many NK-cell receptors and their ligands are becoming increasingly available, especially for the NKG2 and KIR receptor

**3. Transcription factor regulation and epigenetic control of the expression of** 

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

questions, however, regarding the molecular mechanisms that govern the shape of the NKcell receptors and MHC class I molecules or other ligands that exhibit an exceptionally high degree of genetic polymorphism in a clonally distributed fashion. Studies, therefore, that focus on the molecular mechanisms that govern the expression of NK-cell receptors and their ligands may provide improved strategies of active-specific immunotherapy for the treatment of cancer, infection and other diseases (Campoli and Ferrone, 2008; Krukowski et al., 2011; Gao et al., 2009). Some stimulators, including viruses, tumor cells and heat shock, could promote the expression of NK-cell receptors and their ligands via activation of certain transcription factors that are capable of regulating activity of NKG2 promoters. Epigenetic mechanisms, including DNA methylation and histone posttranslational modification, are also critical for expression of NK-cell receptors and their ligands, and may control the clonal distribution of some NK-cell receptors. These effects may influence the performance of NKcell functions. In this review, we will discuss the recent advances in transcriptional regulation and epigenetic control of the expression of NK-cell receptors and their ligands (and of KIR and NKG2 receptor families in particular).

## **2. Regulation of gene expression – An introduction in the transcriptional level**

Mechanisms that underlie the control of gene expression, which drives the processes of gene morphogenesis and distribution, are complex. Transcription regulation at every step of the process is subject to dynamic regulation in the cell (Beckett, 2009; Pan et al., 2010). This regulation includes structural changes in the chromatin to make a particular gene accessible for transcription, transcription of DNA into RNA, splicing of RNA into mRNA, editing and other covalent modifications of the mRNA, translation of mRNA into protein, and, finally, posttranslational modification of the protein into its mature functional form. Information on molecular details of each of these regulatory steps is becoming increasingly available (Fry and Peterson, 2002; Mitchell and Tjian, 1989).

#### **2.1 Transcriptional factor regulation**

Regulation at the transcriptional level is a critical mechanism of controlling gene expression. Transcription is controlled by trans-acting factors that regulate spatiotemporal gene expression by associating with cis-acting elements such as promoters, enhancers, and other regulatory regions. Transcription factors perform this function either alone or with other proteins in a complex and by promoting or blocking the transcription of genetic information from DNA to RNA (Mitchell and Tjian, 1989; Alexander and Beggs, 2010; Brivanlou and Darnell, 2002; Karin, 1990). A defining feature of transcription factors is that they contain one or more DNA-binding domains (DBDs), which attach to specific DNA sequences adjacent to the genes that they regulate. In eukaryotes, an important class of transcription factors, called general transcription factors (GTFs), is necessary for transcription to occur. Many of these GTFs do not actually bind to DNA but are parts of the large transcription preinitiation complex that interact directly with RNA polymerase. The most common GTFs are transcription factor II members (TFIIA/B/D/E/F and TFIIH). There are also some nonclassical transcription factors that play crucial roles in gene regulation, including coactivators, chromatin remodelers, histone acetylases, deacetylases, kinases, and methylases, which lack DNA-binding domains (Brivanlou and Darnell, 2002; Karin, 1990; Dowell, 2010; van Nimwegen, 2003).

### **2.2 Epigenetic control(**

428 Advances in Cancer Therapy

questions, however, regarding the molecular mechanisms that govern the shape of the NKcell receptors and MHC class I molecules or other ligands that exhibit an exceptionally high degree of genetic polymorphism in a clonally distributed fashion. Studies, therefore, that focus on the molecular mechanisms that govern the expression of NK-cell receptors and their ligands may provide improved strategies of active-specific immunotherapy for the treatment of cancer, infection and other diseases (Campoli and Ferrone, 2008; Krukowski et al., 2011; Gao et al., 2009). Some stimulators, including viruses, tumor cells and heat shock, could promote the expression of NK-cell receptors and their ligands via activation of certain transcription factors that are capable of regulating activity of NKG2 promoters. Epigenetic mechanisms, including DNA methylation and histone posttranslational modification, are also critical for expression of NK-cell receptors and their ligands, and may control the clonal distribution of some NK-cell receptors. These effects may influence the performance of NKcell functions. In this review, we will discuss the recent advances in transcriptional regulation and epigenetic control of the expression of NK-cell receptors and their ligands

**2. Regulation of gene expression – An introduction in the transcriptional** 

Mechanisms that underlie the control of gene expression, which drives the processes of gene morphogenesis and distribution, are complex. Transcription regulation at every step of the process is subject to dynamic regulation in the cell (Beckett, 2009; Pan et al., 2010). This regulation includes structural changes in the chromatin to make a particular gene accessible for transcription, transcription of DNA into RNA, splicing of RNA into mRNA, editing and other covalent modifications of the mRNA, translation of mRNA into protein, and, finally, posttranslational modification of the protein into its mature functional form. Information on molecular details of each of these regulatory steps is becoming increasingly available (Fry

Regulation at the transcriptional level is a critical mechanism of controlling gene expression. Transcription is controlled by trans-acting factors that regulate spatiotemporal gene expression by associating with cis-acting elements such as promoters, enhancers, and other regulatory regions. Transcription factors perform this function either alone or with other proteins in a complex and by promoting or blocking the transcription of genetic information from DNA to RNA (Mitchell and Tjian, 1989; Alexander and Beggs, 2010; Brivanlou and Darnell, 2002; Karin, 1990). A defining feature of transcription factors is that they contain one or more DNA-binding domains (DBDs), which attach to specific DNA sequences adjacent to the genes that they regulate. In eukaryotes, an important class of transcription factors, called general transcription factors (GTFs), is necessary for transcription to occur. Many of these GTFs do not actually bind to DNA but are parts of the large transcription preinitiation complex that interact directly with RNA polymerase. The most common GTFs are transcription factor II members (TFIIA/B/D/E/F and TFIIH). There are also some nonclassical transcription factors that play crucial roles in gene regulation, including coactivators, chromatin remodelers, histone acetylases, deacetylases, kinases, and methylases, which lack DNA-binding domains (Brivanlou and Darnell, 2002; Karin, 1990;

(and of KIR and NKG2 receptor families in particular).

and Peterson, 2002; Mitchell and Tjian, 1989).

**2.1 Transcriptional factor regulation** 

Dowell, 2010; van Nimwegen, 2003).

**level** 

Transcription is also controlled by epigenetic regulation that is defined as gene-regulation activity that does not involve changes in the underlying DNA code and includes DNA methylation and a variety of histone protein posttranslational modifications (Feng et al., 2010; Furumatsu and Ozaki, 2010). DNA methylation usually occurs in CpG islands, CGrich regions that are "upstream" of the promoter region. The letter "p" here signifies that C and G are connected by a phosphodiester bond. In humans, DNA methylation is carried out by a group of enzymes called DNA methyltransferases. These enzymes not only determine DNA methylation patterns during early development, but are also responsible for copying these patterns to the strands generated from DNA replication. DNA methylation can alter condensed chromatin structure by influencing histone–DNA and histone–histone contact. Methylation of the promoter and 5′ region of the gene and methylation of histone are associated with transcriptional silencing (Bird, 2002; Bird and Wolffe, 1999; Law and Jacobsen, 2010).

Acetylation and methylation of histones are the most general and important histone modifications associated with transcriptional activation. Histone acetylation is catalysed by histone acetyl transferases (HATs), whereas the reverse reaction is performed by histone deacetylases (HDACs) (Furumatsu and Ozaki, 2010). HATs and HDACs are classified into many families that are often conserved throughout evolution from yeast to humans. Histone H3 is primarily acetylated at several lysine residues, Lys9, 14, 18, 23, 27 and 56, whereas histone H4 is acetylated at Lys5, 8, 12 and 16 (Su et al., 2004). Interestingly, methylation is often modified at lysine and arginine residues in histones H3 and H4. It has been suggested that these modifications constitute a "histone code"; the "code" hypothesis speculates that this modification pattern is read by proteins that recognize distinct modifications specifically and then regulate chromatin dynamics and genome function (Wang et al., 2004; Kisliouk et al., 2010). Whether these modifications form a true code is still under discussion. The processes of histone modification and DNA methylation can both influence each other. Moreover, DNA methylation and suppressive histone modifications may prevent transcription, even when transcription factors are abundant. In turn, transcription factors may cause alterations in DNA methylation and histone modifications (Furumatsu and Ozaki, 2010; Fernandez-Morera et al., 2010; Strahl and Allis, 2000). Molecular details of the transcriptional regulators involved in expression of many NK-cell receptors and their ligands are becoming increasingly available, especially for the NKG2 and KIR receptor families and their ligands.
