ENGLISH |中文

 

Poster session

20-22, April

Poster session


1, EZH2 promotes hepatocellular carcinoma cell metastasis via epigenetic silencing of gap junction proteins

Alfred S.L. Cheng, Suki S. Lau, Daisy P. Tsang, Ka F. To, Joseph J.Y. Sung
Institute of Digestive Disease, The Chinese University of Hong Kong 

The Polycomb protein Enhancer of zeste homolog 2 (EZH2) causes epigenetic silencing of tumor-suppressor genes by trimethylating histone H3 lysine-27 (H3K27me3) and is over-expressed in many cancer types including hepatocellular carcinoma (HCC). By chromatin immunoprecipitation (ChIP) and gene expression profiling of HCC cells, we have recently uncovered an EZH2-mediated epigenetic mechanism that constitutively activates β-catenin signaling via concordant silencing of the Wnt antagonists, providing a functional link between EZH2 and Wnt/β-catenin pathways in the initiation and development of HCC (Cancer Res 2011;71:4028-39). However, the biological role and mechanistic contribution of EZH2 in HCC progression remains unknown. Here, through an immunohistochemical analysis in a HCC tissue microarray that contains 172 pairs of tumor and adjacent non-tumoral liver, we found that EZH2 protein was expressed in 42.4% (73/172) of HCC cases. Over-expression of EZH2 significantly correlated with macrovascular invasion characterized by formation of grossly visible tumoral thrombi (Pearson’s Chi-Square test, P = 0.001). To investigate whether EZH2 contributes to the motile phenotype of HCC cells, we performed siRNA knockdown of EZH2 in invasive MHCC97 and PLC5 HCC cells and examined cell invasiveness using Boyden chamber assay. Consistent with the notion that EZH2 correlates with the metastatic potential of HCC cells in vivo, knockdown of EZH2 significantly reduced the associated repressive chromatin mark, H3K27me3, and decreased HCC cell invasion (P < 0.05). In order to characterize direct targets of EZH2 that confer this oncogenic property in HCC, we performed ChIP coupled with microarray in HCC cells using promoter arrays of ~17,000 best-defined human transcripts and found that 4-5% promoters were significantly bound by EZH2 in HCC cells. Around 30 percent of the identified promoters were known targets of polycomb complex and/or H3K27me3, which in turn confirms the validity of our array finding. To develop a comprehensive model of altered pathways that mediate EZH2 functions, we integrated the novel direct EZH2 targets to pathway analysis based on Kyoto Encyclopedia of Genes and Genomes classification and revealed that EZH2 may involve in the regulation of metastasis-related pathways, including tight and gap junctions (P = 0.0029 and 0.0096, respectively). Notably, connexin 43 (GJA1), a gap junction protein known to inhibit cancer development, growth and metastasis, was found to be regulated by EZH2. Down-regulation of EZH2 in both MHCC97 and PLC5 cells significantly increased GJA1 mRNA and protein expression. Since epigenetic gene repression in cancer cells can be associated with both H3K27 and DNA methylation, we treated a panel of HCC cell lines with DNA demethylating agent 5-aza-2’-deoxycytidine (5-aza) which resulted in GJA1 gene reactivation. Moreover, concomitant treatment with EZH2 siRNA and 5-aza synergistically reactivated GJA1 (up to 15-fold) in Hep3B, Huh7 and PLC5 cells, suggesting that reversible polycomb-mediated repression can result in stable silencing of the metastasis-suppressor gene. In conclusion, our study delineates a novel pathway by which EZH2-mediated epigenetic silencing contributes to increased invasiveness of HCC cells, where the loss of cell–cell contacts mediated by the gap junction protein might facilitate escape of tumor cells from the primary tumor mass in early steps of metastasis. Understanding this molecular pathway in HCC will present new opportunities for deriving therapeutic strategies to halt HCC metastasis, which accounts for most of the mortality of this dreadful malignancy. This study was supported by the General Research Fund (Ref No. CUHK4623/09M) from the Research Grants Council.  

2.Aberrant TGF-beta/SMAD4 signaling contributes to epigenetic silencing of a putative tumor suppressor, RunX1T1 in ovarian cancer

Kun-Tu Yeh1,12,13*, Tze-Ho Chen2,*, Hui-Wen Yang3,4, Jian-Liang Chou3,4, Lin-Yu Chen3,4, Chia-Ming Yeh3,4, Yu-Hsin Chen3, Ru-Inn Lin6, Her-Young Su7,8, Gary C.-W. Chen3,4, Daniel E. Deatherage9, Yi-Wen Huang9, Pearlly S. Yan9, Huey-Jen Lin9,10, Kenneth P. Nephew11, Tim H.-M. Huang9, Hung-Cheng Lai7,8 and Michael W.Y. Chan3,5 ‡
1Department of Pathology and 2Department of Obstetrics and Gynecology, Changhua Christian Hospital, Changhua, Taiwan, ROC
3Department of Life Science and 4Institute of Molecular Biology and 5Human Epigenomics Center, National Chung Cheng University, Min-Hsiung, Chia-Yi, Taiwan, ROC
6Department of Radiation Oncology, Buddist Tzu Chi General Hospital, Dalin Branch, Taiwan
7Graduate Institute of Medical Sciences, and 8Department of Obstetrics and Gynecology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, ROC
9Human Cancer Genetics Program, Department of Molecular Virology, Immunology, and Medical Genetics, Comprehensive Cancer Center; 10Division of Medical Technology, School of Allied Medical Professions, the Ohio State University, Columbus, Ohio, USA
11Medical Sciences, Indiana University School of Medicine, Bloomington, IN, USA
12Institute of Clinical Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan, ROC
13School of Medicine, Chung Shan Medical University, Taichung, Taiwan, ROC
*these authors contribute equally to this work
‡Correspondence author  

Aberrant TGF-beta signaling pathway may alter the expression of down-stream targets and promotes ovarian carcinogenesis. However, the mechanism of this impairment is not fully understood. Our previous study has identified RunX1T1 as a putative SMAD4 target in an immortalized ovarian surface epithelial cell line, IOSE. In this study, we report that transcription of RunX1T1 was confirmed to be positively regulated by SMAD4 in IOSE cells and epigenetically silenced in a panel of ovarian cancer cell lines by promoter hypermethylation and histone methylation at H3 lysine 9. SMAD4 depletion increased repressive histone modifications of RunX1T1 promoter without affecting promoter methylation in IOSE cells. Epigenetic treatment can restore RunX1T1 expression by reversing its epigenetic status in MCP3 ovarian cancer cells. When transiently treated with a demethylating agent, the expression of RunX1T1 was partially restored in MCP3 cells, but gradual re-silencing through promoter re-methylation was observed after the treatment.  Interestingly, SMAD4 knockdown accelerated this re-silencing process, suggesting that normal TGF-beta signaling is essential for the maintenance of RunX1T1 expression. In vivo analysis confirmed that hypermethylation of RunX1T1 was detected in 35.7% (34/95) of ovarian tumors with high clinical stages (P=0.035) and in 83% (5/6) of primary ovarian cancer-initiating cells. Additionally, concurrent methylation of RunX1T1 and another SMAD4 target, FBXO32 which was previously found to be hypermethylated in ovarian cancer was observed in this same sample cohort (P<0.05). Restoration of RunX1T1 inhibited cancer cell growth. Taken together, dysregulated TGF-beta/SMAD4 signaling may lead to epigenetic silencing of a putative tumor suppressor, RunX1T1, during ovarian carcinogenesis.

3, DSC3 expression is regulated by p53, and methylation of DSC3 DNA is a prognostic marker in human colorectal cancer

Yuan Chen1, Tiantian Cui1, Linlin Yang1, Thomas Knosel1, Kristin Zoller1, Otmar Huber2, and Iver Petersen1
1Institute of Pathology, University Hospital Jena, Ziegelmühlenweg 1, 07743 Jena, Germany
2Institute of Biochemistry II, University Hospital Jena, Nonnenplan 4, 07743 Jena, Germany
Aims: Desmocollin 3 (DSC3), a member of the cadherin superfamily and integral component of desmosomes, is involved in carcinogenesis. However, the role of DSC3 in human colorectal cancer (CRC) has not yet been established.
Methods: DSC3 expression in CRC cell lines was analyzed by RT-PCR and western blotting. Methylation status of DSC3 was examined by demethylation tests, methylation-specific PCR, and bisulfite sequencing (BS). The regulatory role of p53 was investigated by transfection.
RESULTS: DSC3 was downregulated in CRC cells at both mRNA and protein levels. DSC3 expression was restored in five out of seven cell lines after 5-aza-2’-deoxycytidine (DAC) treatment. A heterogeneous methylation pattern was detected by BS in promoter region and exon 1 of DSC3. Methylation of DSC3 genomic sequences was found in 41% (41 out of 99) of primary CRC, being associated with poor prognosis (P = 0.002). Transfection of p53 alone or in combination of DAC increased the DSC3 expression. Similarly, treatment with p53 inducer adriamycin alone or in combination with DAC enhanced DSC3 expression.
CONCLUSIONS: DNA methylation contributes to downregulation of DSC3 in CRC cell lines. Methylation status of DSC3 DNA is a prognostic marker for CRC. P53 appears to play an important role in regulating DSC3 expression.

4, Elongator regulates cell cycle by promoting chromatin replication in Arabidopsis

Deyang Xu, Weihua Huang, Yang Li, Hua Wang, Hai Huang, Xiaofeng Cui*
National Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China
*Corresponding author
E-mail: xiaofeng@sippe.ac.cn
Phone: 86-21-54924086
Fax: 86-21-54924015

Elongator is a multiunit histone acetyltransferase complex widely present in eukaryotes from yeast to human, implying that Elongator may be fundamentally required for the life cycle. Here, we report that Elongator plays an important role in cell cycle progression in Arabidopsis. Mutations in genes encoding Elongator subunits resulted in aberrant cell cycle progression, and the altered cell division affects leaf polarity formation. The defective cell cycle progression is caused by the aberrant DNA replication and increased DNA damage, which activate DNA replication checkpoint to arrest cell cycle. Elongator interacts with proliferating cell nuclear antigen (PCNA) and is required for efficient histone3 (H3) and H4 acetylation coupled with DNA replication. Levels of chromatin-bound H3K56Ac and H4K5Ac known to associate with replicons during DNA replication were reduced in the mutants of both Elongator and CAF-1 (chromatin assembly factor 1), another protein complex that physically interacts with PCNA for DNA replication-coupled chromatin assembly. Disruptions of CAF-1 also led to severe leaf polarity defects, indicating that Elongator and CAF-1 at least partially act in the same pathway to promote cell cycle progression.Taken together, our results reveal a previously unrecognized Elongator function in chromatin replication in the context of plant development. 

5, Hepatitis B virus X protein represses PDCD4 via increasing miR-21 in hepatocellular carcinoma  

Xuemei Qiu1, Suisui Dong2, Fengchang Qiao1, Sen Lu3, Yunwei Song1, Yingbin Lao1, Hong Fan1*
The Key Laboratory of Developmental Genes and Human Diseases, Ministry of Education, Department of Medical Genetics and Developmental Biology, Medical School of Southeast University, Nanjing, China
Department of Clinical Oncology, The University of Hong Kong, Pokfulam, Hong Kong, China;
The First Affiliated Hospital with Nanjing Medical University, Nanjing, 210029, China
* Correspondence: H Fan, The Key Laboratory of Developmental Genes and Human Diseases, Ministry of Education, Department of Genetics and Developmental Biology, Medical School of Southeast University, Nanjing 210009, China. E-mail: fanh@seu.edu.cn

PDCD4, a novel tumor suppressor gene, was recently reported to be down-regulated in Hepatocellular carcinoma (HCC), but little is known about the underlying molecular mechanism of its down-regulation. HCC is a common and aggressive cancer that is strongly associated with chronic infection by the hepatitis B virus (HBV). The HBV X protein (HBx), a protein encoded by HBV, is thought to play a key role in the molecular pathogenesis of HBV-related HCC. The aim of our study is to investigate whether the down-regulation of PDCD4 is related to HBx and how HBx deregulates PDCD4 expression.
Our results showed that PDCD4 is down-regulated in HCC and is negative correlates with HBx. In order to verify whether the down-regulation of PDCD4 was regulated by HBx, QSG-7701, SMMC-7721 and L02 cells were transfected with HBx expressing vector. The results showed that PDCD4 was down-regulated in HBx transfected cells. Knockdown of HBx through RNAi increased PDCD4 expression. HBx expression also induced DNMT1 and DNMT3B expression. Methylation-specific PCR (MSP) implied that HBx failed to induces hypermethylation of the PDCD4 promoter. However, several studies have previously demonstrated that PDCD4 is a direct target gene of miR-21 in several cancers. We next confirmed the regulation of PDCD4 by miR-21 in HCC cell line 97H. After transfected miR-21 inhibitor or the negative control into 97H cells, we found inhibition of miR-21 upregulated PDCD4 expression. In clinical cases,when the HCC patients were sub-grouped based on the relative miR-21 status, the frequency of PDCD4 down-expression were higher in samples which miR-21 expression was high. While in miR-21 expression was low samples, the frequency of PDCD4 down-expression were 46%. These data suggest that PDCD4 is negatively associated with the expression of miR-21. It supports that PDCD4 is regulated by miR-21 in HCC.
Given that HBx could deregulate the expression of cellular miRNAs, whether HBx deregulate a specific candidate miRNA, miR-21, and furthermore regulate PDCD4 expression was explored further. HBx expression construct and HBx siRNA were employed in HCC cell lines. In the present study, we find enforcing HBx induces miR-21 expression, while inhibiting HBx reduces the miR-21 expression. In clinical HCC tissues, the relative expression levels of miR-21 were examined by qRT-PCR and the results showed miR-21 was overexpressioned in HCC tissues compared to corresponding non-tumor liver tissues. Moreover, its expression was positively correlated with the status of the HBx in the paired tumor versus non-tumorous tissues.  qPCR analysis of miR-21 expression level in 13 liver cell lines also showed that the expression of miR-21 was relatively high in HBx positive cells, including 97H, 97L and HepG2.2.15 cells that constitutively replicate HBV. Hence, this significant positive correlation between the expression of miR-21 and the expression of the HBx protein in HCC patients and liver cell lines confirms the clinical relevance of our observations. Further, we knocked down miR-21 with a miR-21 inhibitor in L02-HBx cells which were stably transfected with HBx-expressing constructs and L02-Vector cells, and found that knock-down of miR-21 was able to abolish the downregulation of PDCD4 of L02-HBx cells mediated by HBx. Taken together, our data demonstrate that HBx down-regulates PDCD4 expression at least partially through miR-21.
In summary, we first report that the downregulation of PDCD4 in HCC is correlated with HBx, and HBx down-regulates PDCD4 expression, at least partly, through miR-21. Notably, a significant positive relationship between the expression of the HBx and the expression of miR-21 was observed in HCC patients highlighting the clinical relevance of our observations. Our findings thus offer a new regulation mechanism of PDCD4 expression in HCC and enhance the understanding of both the pleiotropic nature of HBx viral protein and its contribution to HCC.

6, Epigenetic editing: towards gene-specific overwriting of epigenetic marks 

Hui Chen1,3,Marloes L. de Groote1,Christian Huisman1,Fahimeh Falahi1,G.Bea A. Wisman2,Guo-Liang Xu3,Marianne G. Rots1
1 Epigenetic Editing, Department of Medical Biology and Pathology, 2 Department of Gynecological Oncology, University Medical Center Groningen, University of Groningen, The Netherlands
3 Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China
Many diseases, including certain genetic diseases, metabolic, autoimmune, and neurological disorders and tumors are accompanied by aberrant DNA methylation and/or histone modification profiles. In contrast to genetic mutations, such epi-mutations are reversible. Therefore, the overwriting of epigenetic marks to modulate gene expression in a gene-specific way (Epigenetic Editing) would offer a novel approach in biomedical research to develop epigenetic interventions. In Epigenetic Editing, DNA binding domains are designed to bind a specific region of a target gene and fused to indirect transcriptional modulators or to epigenetic enzymes. As DNA sequence specific domains, we engineer Zinc Finger Proteins (ZFPs) and Triplex Forming Oligos (TFOs) and fuse these with effector domains which include the catalytic domains of epigenetic enzymes like DNA methyltransferases1. Here, we will show the feasibility of the targeted rewriting of epigenetic marks (TREMs) as a novel gene-specific approach towards permanent normalisation of aberrantly expressed genes.
First, the critical transcription regulatory sites of the target gene are determined, followed by the construction of ZFPs to specifically bind these sites. The engineered ZFPs are first validated for affinity and specificity by fusing to a transcriptional repressor (KRAB) or activator (VP64), then coupled to the appropriate epigenetic effector domains. Finally, the engineered constructs will be tested for their efficiency to overwrite epigenetic signatures, to modulate gene expression as well as for their biological potency through a series of experiments, such as ChIP, bisulfite sequencing, qRT-PCR, FACS, and MTT assay. Our results show that ZFPs can be easily engineered to bind target genes, including epigenetically silenced genes, and that subsequent fusing of transcription modules results in a flexible bidirectional gene expression modulation approach. Moreover, TREM-induced overwriting of epigenetic marks was demonstrated in cancer cell lines, and this was associated with gene expression modulation. Subsequent cell proliferation assays validated intended effects on cancer cells growth.
Various advantages exist for controlling the expression of endogenous genes by epigenetic editing. First, ZFPs show great potential as tools to up- as well as down-regulate the transcription of virtually any (undruggable) gene. ZFPs have great potential of being gene-specific, not affecting expression of other genes like current epigenetic (FDA-approved) drugs, and not interfering with endogenous cellular pathways as some siRNA approaches. Second, as the appropriate combination of epigenetic marks is mitotically stable, prolonged effects can be achieved while the effect of siRNA is generally transient. Third, introducing an “open” epigenetic signature allows natural expression regulation mechanisms to induce all the isoforms of the targeted gene. In addition, these editors are very small, which allows the incorporation of multiple gene-targeting factors in one gene transfer vector. In conclusion, epigenetic editing is a very promising approach in the field of epigenetic and biomedical research.  

7, MicroRNA-940 inhibits cell growth and promotes apoptosis by down-regulating intermediate filament protein Nestin 

Ji Ma, Central Lab,Shanghai Tenth People’s Hospital,No. 301, Middle Yanchang Road,Shanghai, 200072, P. R. China,E-mail: miaomiaotangtang@163.com,

MiRNAs, a class of small non-coding RNAs that primarily function as gene regulators, are becoming an emerging theme in cancer research. Here we found that microRNA-940 (miR-940) significantly suppresses the proliferation of nasopharyngeal carcinoma cells, and its overexpression induces cell apoptosis, arrests cells into mitosis, and inhibits xenograft tumor formation. We further show that miR-940 directly binds to 3’-UTR of Nestin mRNA and promotes its degradation. Depletion of Nestin leads to activation of caspase3/7 and caspase8, inhibition of tumor cell proliferation and suppression of xenograft tumor formation in vivo. Moreover, the functions of miR-940 can be reversed by overexpressing Nestin, suggesting that miR-940 regulates cell growth and survival through Nestin. We analyzed tumor tissue samples and found the down-regulation of miR-940 as well as the up-regulation of Nestin in NPC tissues. Protein microarray assays also revealed that knockdown of Nestin can alter protein phosphorylation involved in DNA damage and repair signaling, which subsequently increase the radiation sensitivity in NPC cells. Collectively, our data demonstrate that miR-940 suppresses tumorigenesis by regulating the novel oncogene Nestin, and the expression level of miR-940 may be indicative for cancer classification and prognositication.

8, Control of leaf patterning by polycomb-mediated H3K27me3

Xiaofan Chen, Hua Wang, Jiqin Li, Hai Huang, Lin Xu*
National Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant
Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese
Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China.

The Polycomb group (PcG) epigenetic pathway is conserved in plants and animals, and represses genes by modification of H3K27me3. Axial patterning at primordial stages is central to leaf morphogenesis, especialiy to the establishment of adaxial-abaxial polarity. ASYMMETRIC LEAVES2 (AS2) is one of the key polarity genes and specifies leaf adaxial identity. The expression pattern of AS2 is only in the adaxial leaf domain, becausee AS2 is directly repressed by an abaxially-located transcription factor KANADI1 (KAN1). Here we show that different from the spatial repression by KAN1, the PcG pathway quantitatively represses AS2. as2-5D and isoginchaku-2D (iso-2D) , two gain-of-function AS2 mutants, has the similar AS2-overexpressional phenotypes, which reflect two kinds of repression of AS2. While as2-5D causes disruption of a KAN1-binding site at the AS2 promoter leading to derepression of AS2 in the abaxial side, iso-2D results in defective PcG repression of AS2 in the adaxial leaf domain. Leaves of the PcG double mutant curly leaf swinger also show polarity defective leaves, where the adaxially featured epidermal cells appears on the abaxial side, resembling those observed in the as2-5D and iso-2D leaves. The conclusion is that the quantitative repression of AS2 by PcG is critical for leaf patterning beyond the spatial repression by KAN1. 

*Corresponding author
E-mail: xulin@sippe.ac.cn
Phone: 86-21-54924086

9, Epigenetic Effect on Immunity in Preeclampsia

Fang Xie 1, Joe Nadeau1
Institute for Systems Biology, 401 Terry Avenue North, Seattle, WA 98019
Contact: fang.cindy.xie@gmail.com

Preeclampsia (PE) is a life-threatening, pregnancy-induced disease and a leading cause of maternal and fetal morbidity and mortality. It has recently been recognized that innate immune system is activated with PE and can contribute to evolving systemic inflammation. The emerging awareness of the contribution of epigenetic processes to immune genome function in placenta is central to successful pregnancy. Such influences can alter the appropriate genetic programming needed to allow for sustained healthy pregnancy and appropriate fetal development. Here, we summarize the epigenetic regulations of immune system— through changes in promoter DNA methylation of inflammatory genes, modulation of innate immune signaling and subsequent of various immune cell activation in PE. We have reviewed studies in both animals and humans that made it increasingly clear that proper epigenetic regulation of immunity in pregnancy trophoblast implantation. Its disturbance, which can be caused by environmental infectious factors, can lead to abnormal placental development and immune dysfunction with possible consequences for PE, fetal development and chronic disease susceptibility in later life

10, Development and application of a novel technique for microscopic visualization of locus-specific DNA methylation in individual cell 

Yufeng Li1, Yusuke Miyanari2, Hirohisa Nitta1, Takeo Kubota3, Hirofumi Ohashi4,
Akimitsu Okamoto5 and Hiroyuki Sasaki1
(1Division of Epigenomics, Medical Institute of Bioregulation, Kyushu University, 2Department of Cell Biology and Development, IGBMC, France, 3Department of Epigenetics Medicine, Interdisciplinary Graduate School of Medicine and Engineering, Yamanashi University, 4Division of Medical Genetics, Saitama Children's Medical Center, Saitama, 5RIKEN Advanced Science Institute and Japan Science and Technology Agency)

DNA methylation at CpG sites is a major epigenetic modification of genome DNA that plays important roles in normal development, aging, and a variety of disease conditions. Immuno-fluorescence microscopy with anti-5-methylcytosine antibodies has been used to visualize global DNA methylation patterns. However, methods that allow visualization of DNA methylation at specific loci have been lacking to date. Recently, it was reported that reactivities of methylated and unmethylated cytosines for interstrand complex formation with osmium and bipyridine-containing nucleic acid (ICON) are markedly different in vitro. Based on this chemistry, we have developed an experimental protocol, named MeFISH, to observe the DNA methylation status of specific loci in individual cells in situ. Cell nuclei or chromosomes were hybridized with fluorescence-labeled ICON probes for major and minor satellite repeats, treated with osmium and then deprobed. Specific retention of fluorescent signals was observed in wild type ES cells but not in Dnmt1/3a/3b triple knockout ES cells, which had virtually no DNA methylation. Therefore, the signals observed in wild type ES cells most likely resulted from specific interstrand crosslinks formed between the ICON probes and methylated cytosines of the target sequences. MeFISH was also successfully used to detect DNA hypomethylation of satellite repeats in lymphoblast cells of ICF syndrome patients. Moreover, MeFISH was used to detect DNA methylation of satellite repeats in mouse germ cells during embryonic and postnatal testis development, with a combination of immunostaining. MeFISH will have a wide application in epigenetics research.

11, Genome-wide Modifications of Bovine H3K27me3 and Their Effects on Genes Expression in Peripheral Blood Lymphocytes

Yanghua He, Ying Yu*, Yuan Zhang*
(Key Laboratory of Agricultural Animal Genetics and Breeding & National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, 100193, Beijing, P.R. China)
*Corresponding authors: yuying@cau.edu.cn, changy@cau.edu.cn

INTRODUCTION
Several large-scale studies have revealed interesting insights into the complex relationship between gene expression and histone methylations. In bovine, histone methylations were only studied in bovine oocyte and embryos by using immunostaining approach but they are still not completed at an extensive genome coverage and resolution in bovine lymphocytes. Therefore, the high resolution pictures of histone methylation for the entire bovine genome are required to construct for understanding their functional correlation with gene expressions.
MATERIALS AND METHODS
Lymphocytes were isolated from peripheral blood of Chinese Holstein cows with different parities. The distribution and regulation pattern of histone H3 trimethylation on lysine 27 (H3K27me3) were investigated in bovine lymphocytes by high-throughput ChIP-Seq and DGE techniques. The enrichment profile of the ChIP-seq mapping and gene expression levels were confirmed with ChIP-qPCR and qRT-PCR approach. GO annotations of significant genes were functionally categorized by BGI WEGO (Web Gene Ontology Annotation Plotting).
RESULTS AND ANALYSIS
As consistent with previous reports, bovine H3K27me3 marks tended to repress expressions of target genes via acting on 5’ and 3’ regulatory regions. The regions surrounding the TSSs, especially the upstream 2Kb of TSS, were crucial H3K27me3 enrichment locations in bovine genome. Moreover, lower H3K27me3 levels were observed in the promoter regions of cytokine and transcription factor genes. In addition, H3K27me3 has parity-dependent features which may be associated with udder health of dairy cattle.
CONCLUSIONS
Our data suggest that bovine H3K27me3 is important epigenetic marks repressing gene expression and deep functional studies of bovine lymphocytes is warranted through histone modification regulation.

12, DNA methylation phenotypes of genes reveal a regulatory mechanism of human gene expression

Jianzhong Su1, Haidan Yan1, Zixiu Li1, Hongbo Liu1 and Yan Zhang1*
College of Bioinformatics Science and Technology, Harbin Medical University, Harbin, China
*Corresponding author Email: yanyou1225@yahoo.com.cn

Bisulfite sequencing is a powerful technology to measure cytosines methylation at single-base resolution, which permits genome-wide analysis of the regulatory mechanism of DNA methylation. However, the DNA methylation patterns and the regulatory function of DNA methylation in different genomic regions remain unclear. In this study, DNA methylation patterns of six categories of genomic regions relative to genes were investigated in human embryo stem cells (H1) and fibroblast cells (IMR90).We found that their distributions of DNA methylation level and DNA methylation patterns in each gene were significant difference. DNA methylation phenotypes of genes were determined through computing the DNA methylation level in the six genomic regions of genes. Nine kinds of methylation phenotypes of genes with significantly different methylation patterns in the corresponding genomic regions were extracted by K-means clustering from H1 and IMR90. The genes with the same methylation phenotypes show the similar gene expression level even in different cell types, while genes with different methylation phenotypes of genes show significant difference. Numerous genes differential expression possess the changes of DNA methylation phenotypes from H1 transient to IMR90. Our studies provide a new prospective for systemic revealing the regulatory mechanism of gene expression through introducing DNA methylation phenotypes of genes.

13, Prioritizing cancer-related genes with aberrant methylation based on a weighted protein-protein interaction network
Hui Liu1, Jianzhong Su1, and Yan Zhang1*

1College of Bioinformatics Science and Technology, Harbin Medical University, Harbin, China
*Corresponding author Email: yanyou1225@yahoo.com.cn

As an important epigenetic modification, DNA methylation plays a crucial role in the development of mammals and in the occurrence of complex diseases. Genes that interact directly or indirectly may have the same or similar functions in the biological processes in which they are involved and together contribute to the related disease phenotypes. The complicated relations between genes can be clearly represented using network theory. A protein-protein interaction (PPI) network offers a platform from which to systematically identify disease-related genes from the relations between genes with similar functions. We constructed a weighted human PPI network (WHPN) using DNA methylation correlations based on human protein-protein interactions. WHPN represents the relationships of DNA methylation levels in gene pairs for four cancer types. A cancer-associated subnetwork (CASN) was obtained from WHPN by selecting genes associated with seed genes which were known to be methylated in the four cancers. We found that CASN had a more densely connected network community than WHPN, indicating that the genes in CASN were much closer to seed genes. We prioritized 154 potential cancer-related genes with aberrant methylation in CASN by neighborhood-weighting decision rule. A function enrichment analysis for GO and KEGG indicated that the optimized genes were mainly involved in the biological processes of regulating cell apoptosis and programmed cell death. An analysis of expression profiling data revealed that many of the optimized genes were expressed differentially in the four cancers. By examining the PubMed co-citations, we found 43 optimized genes were related with cancers and aberrant methylation, and 10 genes were validated to be methylated aberrantly in cancers. Of 154 optimized genes, 27 were as diagnostic markers and 20 as prognostic markers previously identified in literature for cancers and other complex diseases by searching PubMed manually. We found that 31 of the optimized genes were targeted as drug response markers in DrugBank. Here we have shown that network theory combined with epigenetic characteristics provides a favorable platform from which to identify cancer-related genes. We prioritized 154 potential cancer-related genes with aberrant methylation that might contribute to the further understanding of cancers.

14, EpiDiff: entropy-based quantitative identification of differential epigenetic modification regions from epigenomes

Hongbo Liu1, Yanjun Wei1, and Yan Zhang1,*
1College of Bioinformatics Science and Technology, Harbin Medical University, Harbin 150081, China
*Corresponding author Email: yanyou1225@yahoo.com.cn

Genome-wide epigenetic modification dynamics, including DNA methylation and chromatin modification, are involved in biological processes such as development, aging, and disease. Quantitative identification of differential epigenetic modification regions (DEMRs) from various temporal and spatial epigenomes is a crucial step towards investigating the relationship between epigenotype and phenotype. Here, we describe EpiDiff (http://bioinfo.hrbmu.edu.cn/epidiff/), an integrated software platform that supports quantification of epigenetic difference and identification of DEMRs by Shannon entropy. Two main modules, quantitative differential chromatin modification region (QDCMR) and quantitative differentially methylated region (QDMR) are provided for bioinformatic analysis of chromatin modifications and DNA methylation data, respectively. The third module, quantitative differential expressed gene (QDEG), can be used to identify differentially expressed genes. The platform-free and species-free nature of EpiDiff makes it potentially applicable to a wide variety of epigenomes at an unprecedented scale and resolution. The graphical user interface provides biologists with a practicable and reliable way to analyze and visualize epigenetic difference.