《自然》专题：表观遗传学

IC

在基因组中除了DNA和RNA序列以外，还有许多调控基因的信息，它们虽然本身不改变基因的序列，但是可以通过基因修饰，蛋白质与蛋白质、DNA和其它分子的相互作用，而影响和调节遗传的基因的功能和特性，并且通过细胞分裂和增殖周期影响遗传。

这就是表观遗传学（epigenetics），表观遗传学又称为实验遗传学、化学遗传学、特异性遗传学、后遗传学、表遗传学和基因外调节系统，它是生命科学中一个普遍而又十分重要的新的研究领域。它不仅对基因表达、调控、遗传有重要作用，而且在肿瘤、免疫等许多疾病的发生和防治中亦具有十分重要的意义。它是生命科学中近年来的一个突出进展，具有十分广泛深刻研究和应用前景。

5月24日出版的《Nature》的副刊就以Epigenetics为核心展开了论述，在Alex Eccleston等人撰写的社论中介绍到，表观遗传学（epigenetics）是一种关于区别于DNA序列改变的基因表达可遗传变化的研究，近年来有关这一方面的研究越来越受到科学家们的注目。

表观遗传改变对于生物体各种细胞类型的发育与分化具有关键性的作用，在正常细胞过程，比如雌性哺乳动物X染色体失活，以及酵母中配对型位点的沉默方面也有重要作用。但是表观遗传状态会受到环境的影响或衰老的干扰，并且癌症和其它疾病发生过程中表观遗传的变化也需要更深入的研究。

这一专题围绕目前表观遗传学研究的热点展开了讨论，为表观遗传学领域研究拓宽了视野。目前表观遗传学的研究热点是在发育和疾病发生过程中，基因表达相关的染色质和染色体结构对表观遗传学机制的影响，并且对表观遗传学进行更深入的定义。希望通过这一专题能让这一研究领域，或对表观遗传学感兴趣的研究人员颇有收获。

http://www.nature.com/nature/supplements/insights/epigenetics/index.html

IC

Research directions

www.clo.cuhk.edu.hk/research_2.html

1. Cancer epigenetics of major tumours in Hong Kong

Epigenetic regulation of transcription through promoter CpG methylation is a fundamental regulatory process of gene expression. CpG methylation strongly represses promoter activity, and normally is a cellular defence mechanism against invasive DNA materials. Methylation is important for embryonic development, tissue-specific gene expression and genetic imprinting. In tumours, aberrant hypermethylation of tumour suppressor gene (TSG) promoters silences their expression, resulting in the loss of TSG functions and contributing to tumorigenesis, similar to genetic mutations.

Hong Kong is a developed and densely-populated city. Cancer has been the leading cause of death in Hong Kong for years, which causes serious social and economic problems. It is well established that at molecular levels, carcinogenesis is a multi-step process involving the disruption of multiple genes by either genetic or epigenetic mechanisms. The presence of epigenetic abnormalities provides us with not only potential epigenetic tumour markers for molecular diagnosis and therapeutics, but also a novel way of identifying candidate TSGs. Theoretically, frequent epigenetic inactivation of a candidate gene specifically in tumours but not in the corresponding normal tissues indicates that it is probably a genuine TSG. For some TSGs, only the epigenetic inactivation can be found in tumours, while their genetic alterations are rare, such as RASSF1A. This type of genes is now called as Class two (new-age) tumour suppressor genes.

Hypermethylation of TSG promoters has been detected not only in primary tumours, but also in serum, sputum, stool, bodily fluids, ductal lavage fluid and saliva of cancer patients. Thus, it can be used as a non-invasive molecular diagnostic marker. Moreover, promoter methylation can be reversed by using DNA methyltransferase (DNMT) inhibitors (5-aza-2'-deoxycytidine - decitabine or 5-aza-cytidine). Clinical trials using these agents have been carried out in several research centres including ours, on myelodysplastic syndrome patients and cancer patients with colon, head, neck, renal, lung tumours and nasopharyngeal carcinoma (NPC). These trials were designed to demethylate and reactivate hypermethylated and silenced genes and thus restore normal cell growth control or induce apoptosis in tumour cells.

My major research interest is the identification of new TSG candidates, which are inactivated by hypermethylation in major tumours in Hong Kong (including NPC, nasal lymphoma and other common carcinomas), by using various subtractive, cancer genomic and epigenomic, and microarray analyses. I further aim to study the regulation and functions of these identified genes, and use them to develop molecular diagnostic and therapeutic strategies towards these tumours (Fig. 1).

Fig.1 Identification of a candidate TSG – G8 at 16q in NPC. The promoter of G8 is a CpG island, which is frequently hypermethylated in tumours.

G8 can inhibit tumour cell colony formation, same as p53, thus is a functional TSG.

2. Molecular pathogenesis of Epstein-Barr virus (EBV) in NPC and nasal lymphoma

EBV is a ubiquitous herpes virus with worldwide infection. EBV is a transforming virus which can immortalize B-cells and cause lymphomas in animal models. It is strongly associated with a variety of tumours, including Burkitt's lymphoma, NPC, nasal lymphoma, posttransplant lymphoma, Hodgkin's disease, and AIDS-lymphoma. Both NPC and nasal lymphoma are prevalent tumours in Hong Kong.
NPC is a major tumour killer for Chinese in a few places in Asia, including Southern China (Hong Kong) and Southeast Asia (Singapore), reaching an incidence of 30-50/100,000/year. It is the fourth most common tumour in Hong Kong. Each year, around 1,100 new cases of NPC were diagnosed in Hong Kong, and about 400 patients died of this disease. Histologically, this tumour usually belongs to the WHO types II and III: non-keratinising and undifferentiated carcinoma. Various studies on its aetiology suggest that both environmental (such as diet, EBV) and genetic/epigenetic factors contribute to its pathogenesis. As for EBV, it is associated with virtually all NPC, regardless of geographic distribution, ethnic origin and local prevalence of the disease. The presence of EBV in NPC provides us with novel diagnostic and therapeutic targets.

Nasal NK/T cell lymphoma (NL) occurs in the nose and midfacial region, and clinically used to be called as "lethal midline granuloma" to describe its necrotic and ulcerative features. In Western countries, nasal lymphoma is uncommon, comprising only 0.44% of all extranodal lymphoma and 0.17% of all malignant lymphoma. However, it is relatively more common in Oriental populations. In Hong Kong, it is the second most frequent group of extranodal lymphoma after gastrointestinal lymphoma, constituting around 7%-10% of all non-Hodgkin's lymphoma. NL often shows histologic features of polymorphic cellular infiltration, necrosis and some extent of angiocentricity and angioinvasion, so terms "polymorphic reticulosis" and "angiocentric immunoproliferative lesion" have been used to describe this disease. Clinically, NL often has a poor prognosis.

Both NPC and nasal lymphoma are strongly associated with EBV, with almost 100% viral positivity in tumours. Moreover, EBV exists as a clonal population in these tumours. However, the molecular mechanisms of EBV pathogenesis in these tumours are poorly understood. I am interested in the EBV gene expression and regulation in these tumour models, especially the epigenetic regulation of EBV promoters by CpG methylation. Dense CpG methylation silences the transcription of EBV promoters and plays an important role in the regulation of EBV latency. There are four EBV promoters driving the expression of the indispensable viral protein EBNA1. The promoters (Cp and Wp) which drive both EBNA1 and other immunodominant viral proteins are often suppressed by hypermethylation in tumours and meanwhile, EBNA1 expression from the hypomethylated Qp is constitutively maintained. Through this way, EBV lives as latency I and II and escapes from host immune surveillance in tumour patients (Fig. 2).

Fig. 2 The four EBV promoters which drive the transcription of EBNA1.

The potential translational and clinical implications of this line of research are the development of cancer treatment strategies to pharmacologically manipulate the methylation status of viral promoters in tumour patients, thus enhance tumour cell antigenicity and target them for killing by immune system. Our clinical trials in NPC patients have managed to demethylate EBV promoters and reactivate the expression of EBV proteins in NPC tumour cells (Fig. 3).

Fig.3 Pharmacological demethylation of EBV promoters and induction of EBV antigen expression

Representative publications:

1. Tao Q, Srivastava G, Loke SL, Liang R, Liu YT, Ho FCS. Epstein-Barr virus (EBV)-related lymphoproliferative disorder with subsequent EBV-negative T-cell lymphoma. Int J Cancer 1994; 58:33-39.
2. Tao Q, Srivastava G, Loke SL, Ho FCS. Lack of correlation between the expression of Epstein-Barr virus (EBV) latent membrane protein and bcl-2 in vivo. J Clin Pathol 1994; 47:589-591.
3. Tao Q, Srivastava G, Chan ACL, Ho FCS. Epstein-Barr virus-infected nasopharyngeal intraepithelial lymphocytes. Lancet 1995; 345:1309-1310.
4. Tao Q, Srivastava G, Chan ACL, Chung LP, Loke SL, Ho FCS. Evidence for lytic infection by Epstein-Barr virus in mucosal lymphocytes instead of nasopharyngeal epithelial cells in normal individuals. J Med Virol 1995; 45:71-77.
5. Tao Q, Ho FCS, Loke SL, Srivastava G. Epstein-Barr virus is localized in the tumor cells of nasal lymphomas of NK, T or B cell type. Int J Cancer 1995; 60:315-320.
6. Tao Q, Srivastava G, Dickens P, Ho FCS. Detection of Epstein-Barr virus-infected mucosal lymphocytes in nasal polyps. Am J Pathol 1996; 149:1111-1118.
7. Chiang AKS*, Tao Q*, Srivastava G, Ho FCS. Nasal NK- and T-cell lymphomas share the same type of Epstein-Barr virus latency as nasopharyngeal carcinoma and Hodgkin's disease. Int J Cancer 1996; 68:285-290. (*: co-first author)
8. Tao Q, Robertson KD, Manns A, Hildesheim A, Ambinder RF. Epstein-Barr virus (EBV) in endemic Burkitt's lymphoma: molecular analysis of primary tumor tissue. Blood 1998; 91:1373-1381.
9. Tao Q, Robertson KD, Manns A, Hildesheim A, Ambinder RF. The Epstein-Barr virus major latent promoter Qp is constitutively active, hypomethylated, and methylation-sensitive. J Virol 1998; 72:7075-7083.
10. Tao Q, Swinnen L, Yang J, Srivastava G, Robertson KD, Ambinder RF. Methylation status of the Epstein-Barr Virus major latent promoter C in iatrogenic B-cell lymphoproliferative disease: application of PCR-based analysis. Am J Pathol 1999; 155:619-625.
11. Ambinder RF, Robertson KD, Tao Q. DNA methylation and the Epstein-Barr virus (Review). Semin Cancer Biol 1999; 9:369-375.
12. Srivastava G, Wong KY, Chiang AKS, Lam KY, Tao Q. Coinfection of multiple strains of Epstein-Barr virus in immunocompetent normal individuals: reassessment of the viral carrier state. Blood 2000; 95:2443-2445.
13. Yang J, Tao Q, Flinn IW, Murray PG, Post L, Ma H, Piantadosi S, Caligiuri MA, Ambinder RF. Characterization of EBV-infected B cells in patients with PTLD; disappearance after Rituximab therapy does not predict clinical response. Blood 2000; 96:4055-4063.
14. Tao Q, Yang J, Huang H, Swinnen L, Ambinder RF. Conservation of Epstein-Barr virus cytotoxic T cell epitopes in posttransplant lymphomas: implications for immune therapy. Am J Pathol 2002; 160:1839-1845.
15. Tao Q, Huang H, Geiman TM, Lim CY, Fu L, Qiu GH, Robertson KD. Defective de novo methylation of viral and cellular DNA sequences in ICF syndrome cells. Human Mol Genetics 2002; 11:2091-2102.
16. Tao Q and Robertson KD. Stealth Technology: How Epstein-Barr virus (EBV) utilizes DNA methylation to cloak itself from immune detection (Review). Clinical Immunol 2003; 109:53-63.
17. Ying J, Srivastava G, Gao Z, Zhang XH, Murray P, Ambinder R, Tao Q. Promoter hypermethylation of the Cyclin-dependent kinase inhibitor (CDKI) gene p21WAF1/CIP1/SDI1 is rare in various lymphomas and carcinomas. Blood 2004; 103:743-746.
18. Murray PG, Qiu GH, Fu L, Waites ER, Srivastava G, Agathanggelou A, Latif F, Grundy RG, Mann JR, Starczynski J, Crocker J, Parkes SE, Ambinder RF, Young LS, Tao Q. Frequent epigenetic inactivation of the RASSF1A tumor suppressor gene in Hodgkin’s lymphoma. Oncogene 2004; 23:1326-1331.
19. Chan AT*, Tao Q*, Robertson KD, Flinn IW, Mann RB, Klencke B, Leung T, Johnson PJ, Ambinder RF (*: Co-first author). Azacitidine Induces Demethylation of the Epstein-Barr Virus Genome in Tumors in Patients. J Clin Oncol 2004; 22:1373-1381. (Editorial by Dowell JE & Minna JD. Cancer Chemotherapy Targeted at Reactivating the Expression of Epigenetically Inactivated Genes. JCO 2004; 22: 1353–1355).
20. Qiu GH, Tan LKS, Loh KS, Lim CY, Srivastava G, Tsai ST, Tsao SW, Tao Q. The candidate tumor suppressor gene BLU, located at the commonly deleted region 3p21.3, is an E2F-regulated, stress-responsive gene, and inactivated by both epigenetic and genetic mechanisms in nasopharyngeal carcinoma. Oncogene 2004; 23:4793-4806.
21. Ying J, Srivastava G, Hsieh WS, Gao Z, Murray P, Liao SK, Ambinder R, Tao Q. The Stress-responsive Gene GADD45G Is a Functional Tumor Suppressor, with Its Response to Environmental Stresses Frequently Disrupted Epigenetically in Multiple Tumors. Clin Cancer Res 2005; 11:6442-6449. (Editorial by Zerbini LF & Libermann TA. GADD45 Deregulation in Cancer. Clin Cancer Res. 2005; 11:6409-13)
22. Zhou L, Jiang W, Ren C, Yin Z, Feng X, Liu W, Tao Q, Yao K. Frequent hypermethylation of RASSF1A and TSLC1 and high viral load of Epstein-Barr virus DNA in nasopharyngeal carcinoma and matched tumor-adjacent tissues. Neoplasia 2005; 7:809-815.
23. Ying J, Li H, Seng TJ, Langford C, Srivastava G, Tsao SW, Putti T, Murray P, Chan ATC, Tao Q. Functional epigenetics identifies a protocadherin - PCDH10 as a tumor suppressor for nasopharyngeal, esophageal and multiple other carcinomas with frequent methylation. Oncogene 2006; 25:1070-80.
24. Wong MLY, Tao Q*, Fu L, Wong KY, Qiu GH, FBF Law, Tin PC, Cheung WL, Lee PY, Tang JCO, Tsao GSW, Lam KY, Law S, Wong J, Srivastava G*. (*Corresponding authors). Aberrant promoter hypermethylation and silencing of the critical 3p21 tumor suppressor gene RASSF1A in Chinese esophageal squamous cell carcinoma. Int J Oncol 2006; 28:767-773.
25. Tao Q, Young LS, Woodman CBJ, Murray PG. Epstein-Barr virus (EBV) and its associated human cancers - Genetics, epigenetics, pathobiology and novel therapeutics (Invited review). Front Biosci 2006; 11:2672-2713.
26. Ai L, Tao Q, Zhong S, Fields CR, Kim WJ, Lee MW, Cui Y, Brown KD, Robertson KD. Inactivation of Wnt inhibitory factor-1 (WIF1) expression by epigenetic silencing is a common event in breast cancer. Carcinogenesis 2006; 27:1341-1348.
27. Ying J, Li H, Cui Y, Wong AHY, Langford C, Tao Q. Epigenetic disruption of two proapoptotic genes MAPK10/JNK3 and PTPN13/FAP-1 in multiple lymphomas and carcinomas through hypermethylation of a common bidirectional promoter. Leukemia 2006; 20:1173-5.
28. Chen YW, Hu X, Liang ACT, Au WY, Wong MLY, Shen L, Tao Q, Chu KM, Kwong YL, Liang R, Srivastava G. High BCL6 expression predicts better prognosis independent of BCL6 translocation status or presence of Ig/BCL6 or non-Ig/BCL6 translocations or BCL6 deregulating mutations in non-coding exon 1 in gastric lymphoma. Blood 2006; 108:2373-83.
29. Soo RA, Wu J, Aggarwal A, Tao Q, Hsieh W, Putti T, Tan KB, Low JSW, Lai YF, Mow B, Hsu S, Loh KS, Tan L, Tan P, Goh BC. Celecoxib reduces microvessel density in patients treated with nasopharyngeal carcinoma and induces changes in gene expression. Ann Oncol 2006; 17:1625-30.
30. Seng TJ, Low JSW, Li H, Cui Y, Goh HK, Wong MLY, Srivastava G, Sydransky D, Califano J, Steenbergen RDM, Rha SY, Tang J, Hsieh WS, Ambinder R, Lin X, Chan ATC, Tao Q. The major 8p22 tumor suppressor DLC1 is frequently silenced by methylation in both endemic and sporadic nasopharyngeal, esophageal, and cervical carcinomas, and inhibits tumor cell colony formation. Oncogene 2007; 26:934-44.
31. Zhang Q, Ying J, Zhang K, Li H, Ng KM, Zhao Y, He Q, Yang X, Xin D, Liao SK, Tao Q*, Jin J*. (*Corresponding authors). Aberrant methylation of the 8p22 tumor suppressor gene DLC1 in renal cell carcinoma. Cancer Letter 2007; 249:220-226.
32. Tao Q, Chan ATC. Nasopharyngeal carcinoma - Molecular pathogenesis and therapeutic developments. (Invited review) Expert Rev Mol Med. 2007; In press.
33. Ying J, Gao Z, Li H, Srivastava G, Murray PG, Goh HK, Lim CY, Wang Y, Marafioti T, Mason DY, Ambinder RF, Chan ATC, Tao Q. Frequent epigenetic silencing of protocadherin 10 by methylation in multiple hematologic malignancies. Br J Hematol 2007; 136:829-832.
34. Sung FL, Hui EP, Tao Q, Li H, Tsui NBY, Lo YMD, Ma BBY, To KF, Harris AL, Chan ATC. Genome-wide expression analysis using microarray identified complex signaling pathways modulated by hypoxia in nasopharyngeal carcinoma. Cancer Letter 2007; Feb 21, on line.
    Table S1 Table S2
35. Ying J, Li H, Murray P, Gao Z, Chen YW, Wang Y, Lee KY, Chan ATC, Ambinder RF, Srivastava G*, Tao Q*. (*Corresponding authors). Tumor-specific methylation of the 8p22 tumor suppressor gene DLC1 is an epigenetic biomarker for Hodgkin, nasal NK/T-cell and other types of lymphomas. Epigenetics 2007; on line.
36. Chan SL, Cui Y, van Hasselt A, Li H, Srivastava G, Jin H, Ng KM, Wang Y, Lee KY, Tsao GSW, Zhong S, Robertson KD, Rha SY, Chan ATC, Tao Q. Identification of the Wnt inhibitory factor-1 (WIF1) as a tumor suppressor for nasopharyngeal and esophageal carcinomas with frequent epigenetic inactivation. Lab Invest 2007; Mar 26, on line.
37. Law FBF, Chen YW, Wong KY, Ying J, Tao Q, Langford C, Lee PY, Law S, Cheung RWL, Tsao GSW, Lam KY, Wong J, Srivastava G, Tang JCO. Identification of a novel tumor transforming gene GAEC1 at 7q22 which encodes a nuclear protein and is frequently amplified and overexpressed in esophageal squamous cell carcinoma. Oncogene 2007; Mar 26, on line.
38. Shao L, Cui Y, Li H, Liu Y, Zhao H, Wang Y, Zhang Y, Ng KM, Han W, Ma D*, Tao Q*. CMTM5 has tumor suppressor functions and is frequently silenced by methylation in multiple tumor cell lines. (*Corresponding authors). Clin Cancer Res 2007; In press.
39. Jin H, Wang X, Ying J, Wong AHY, Li H, Lee KY, Srivastava G, Chan ATC, Yeo W, Ma BBY, Putti TC, Lung ML, Shen ZY, Xu LY, Langford C, Tao Q. Epigenetic identification of ADAMTS18 as a novel 16q23.1 tumor suppressor frequently silenced in esophageal, nasopharyngeal and multiple other carcinomas. Oncogene 2007; In press.
40. Yu J, Tao Q*, Jin H, Poon FF, Wang X, Li H, Cheng YY, Yeo W, Ebert MPA, Srivastava G, Rha SY, Chan ATC, Sung JJY. (*Corresponding author). Frequent silencing of the ubiquitin carboxyl-terminal hydrolase L1 gene (UCHL1) by promoter methylation in multiple digestive tumors. Gut 2007; In revision.
41. Jin H, Wang X, Ying J, Wong AHY, Srivastava G, Shen ZY, Li EM, Zhang Q, Jin J, Kupzig S, Chan ATC, Cullen PJ, Tao Q. Epigenetic silencing of a Ras GTPase-activating protein RASAL acts as a new mechanism of Ras activation in human cancers. (PNAS, in revision)
42. Tedoldi S, Mottok A, Ying J, Paterson, J, Cui Y, Facchetti F, van Krieken JH, Ponzoni M, Özkal S, Masir N, Natkunam Y, Pileri SA, Hansmann ML, *Mason DY, *Tao Q, *Marafioti T. Selective loss of B cell phenotype in lymphocyte predominant Hodgkin’s disease. (*equal contribution, submitted)
43. Qiu GH, Salto-Tellez M, Ross JA, Yeo W, Cui Y, Wheelhouse N, Chen GG, Harrison D, Lai P, Tao Q*, Hooi SC*. (*Corresponding authors). The candidate tumor suppressor gene DLEC1 is frequently silenced by DNA methylation in hepatocellular carcinoma and induces G1 cell cycle arrest through down-regulation of c-Myc and cyclin D2. (Submitted)