Intersectin­1s: a novel nucleo­cytoplasmic endocytic protein

Poster Sessions

Table of Contents Poster Session 1

Sunday 5 July & Monday 6 July 08:30–19:30, Foyer Convention Center

56 Gen EX S1, Chromatin Structure and Epigenetic Modifications and Maintenance of the Genome 70 Gen Ex S2, Turning Signals into Messages – the

Complexity of Gene Regulation

89 Gen Ex S3, Translational Control and Protein Turnover 98 Mem Biol S1, Organelle Dynamics and Communication 107 Mem Biol S2, Autophagy and Degradation

110 Mem Biol S3, Redox-Regulation of Biological Activities 129 Chem Biol S1, Probing Cellular Function with Small

Molecules

158 Chem Biol S2, Targeted Cancer Therapy

160 Chem Biol S4, RNA-Based Disease Mechanism and Therapy 166 Mol Neu S1, Neuronal Ion Channels and their Role in

Disease

168 Mol Neu S2, Mechanisms of Nervous System Development and Regeneration

172 Mol Neu S3, Degeneration and Ageing of the Nervous System 184 Sys Biol S2, Molecular Clocks

187 Sys Biol S3, Comprehensive Models of Metabolism and Signaling

198 Struct Biol S1, Mechanisms of Membrane Transport 205 Struct Biol S2, Channels and Transporters

206 Struct Biol S3, Protein-Mediated Membrane Deformation and Penetration

Poster Session 2

Tuesday 7 July & Wednesday 8 July 08:30–19:30, Foyer Convention Center

209 Gen Ex S4, RNA Processing and Modifications 215 Gen Ex S5, Non-Coding RNAs in Gene Regulation 220 Mem Biol S4, Extrinsic and intrinsic regulation of cellular

growth control

228 Mem Biol S5, Lipid Signaling & Dynamics 240 Chem Biol S2, Targeted Cancer Therapy

274 Chem Biol S3, Functional Glycobiology – from Mechanism to Disease

281 Chem Biol S5, Signal Transduction in Tumor Development, Differentiation and Immune Escape

293 Mol Neu S4, Molecular Architecture and Assembly of the Synapse

297 Mol Neu S5, Control of Neuronal Function by Regulating Protein Homeostasis

302 Sys Biol S1, Interspecies Communications

304 Sys Biol S4, Functional Networks Regulating Cellular Stress Response and Ageing

315 Sys Biol S5, Systems Biology in Stem Cells 316 Struct Biol S2, Channels and Transporters 324 Struct Biol S4, Monitoring Protein Conformational

Dynamics and Movement

329 Struct Biol S5, Advances in Structural Biology – from Subcellular to Molecular Resolution

353 FEBS Education Session

380 Late-breaking abstracts

Volume 282 Supplement 1 July 2015

Each poster has been given a unique number and the first part of the poster number relates to the session in which the poster was presented. Gen EX S1 = P02; Gen EX S2 = P03; Gen EX S3 = P04; Gen EX S4 = P05; Gen EX S5 = P06; Mem Biol S1 = P08; Mem Biol S2 = P09; Mem Biol S3 = P10; Mem Biol S4 = P11; Mem Biol S5 = P12; Chem Biol S1 = P14; Chem Biol S2 = P15; Chem Biol S3 = P16; Chem Biol S4 = P17; Chem Biol S5 = P18; Mol Neu S1 = P20; Mol Neu S2 = P21; Mol Neu S3 = P22; Mol Neu S4 = P23; Mol Neu S5 = P24; Sys Biol S1 = P26; Sys Biol S2 = P27; Sys Biol S3 = P28; Sys Biol S4 = P29; Sys Biol S5 = P30; Struct Biol S1 = P32; Struct Biol S2 = P33; Struct Biol S3 = P34; Struct Biol S4 = P35; Struct Biol S5 = P36; FEBS Education Session = P38; Late-breaking abstracts = LB.

FEBS Journal 282 (Suppl. 1) (2015) 55 55

© 2015 The Authors. FEBS Journal © 2015 FEBS

POSTER SESSIONS

Poster Session 1

Sunday 5 July & Monday 6 July

08:30–19:30, Foyer Convention Center

Gen EX S1, Chromatin Structure and Epigenetic

Modifications and Maintenance of the Genome

P02-005-SP

Investigation of the G4 interactome using

human protein microarrays

S. Severov1, A. Varizhuk1,2_{, I. Smirnov}1_{, G. Pozmogova}1 1_{SRI of Physical-Chemical Medicine, Molecular Genetics}

Department, Moscow, Russian Federation,2_{Department of}

Structure-Functional Analysis of Biopolymers, Engelhardt Institute of Molecular Biology, Moscow, Russian Federation

We report the detailed analysis of G-quadruplex (G4) interactions with human proteins. Recently, great attention has been paid toward the role of noncanonical DNA structures in genomic regu-lation. A large body of data supports the significance of such struc-tures for transcription and translation control, recombination, alternative splicing and other processes. G4s have gained particu-lar interest since their cell-cycle progression-dependant formation was quantitatively visualized in vivo. The present knowledge about G4-binding proteins is insufficient for elucidating all the diverse mechanisms of G4-mediated regulation of gene expression. To complement the current data on the G4 interactome, we profiled several model oligonucleotides, which represent different types of G4 architectures (parallel and antiparallel; 2- and 4-tetrad, etc.) with microarrays that contain over 9000 immobilized human pro-teins. The protoarray approach has been previously employed for analyzing sequence-specific DNA-protein binding. This is the first example of a protoarray-based study of conformation-specific binding. We identified several dozens of proteins affine to a partic-ular G4 topology or all G4 topologies, including those reported in the literature and some new G4-recognising proteins. Predictably, the majority of them are related to nucleic acid processing. The new G4-recognising proteins include transcription factors, splicing factors, chromatin remodeling regulators and others. Although evaluation of their specificity for G4s in comparison with other nucleic acid structures is still underway, our preliminary data pro-vide a more integrative view on the G4 interactome. The implica-tions of the newly identified protein interacimplica-tions for some G4-associated gene expression modulation mechanisms are discussed.

Supported by RSF [14-25-00013].

P02-006-SP

Analysis of XCI mosaicism in the liver from a

patient with OTC deficiency

D. Musalkova1, L. Dvorakova1, O. Luksan2, M. Jirsa2, J. Sikora1_{, M. Hrebicek}1

1_{First Faculty of Medicine, Institute of Metabolic Disorders,}

Charles University in Prague, Prague, Czech Republic,

2_{Laboratory of Experimental Hepatology, Institute for Clinical and}

Experimental Medicine (IKEM), Prague, Czech Republic Ornithine transcarbamylase (OTC) deficiency is an X-linked inborn error of metabolism of the urea cycle associated with

severe hyperammonemia and significant morbidity and mortality. OTCgene is located on Xp21.1 (subject to X-chromosome inacti-vation, XCI) and it is expressed mainly in the liver. Clinical symptoms of the carrier females are highly variable both in onset and severity.

Here we report a case of a female patient heterozygous for the mutation c.583G>C exon 6 (p.G195R) in the OTC gene. The patient underwent successful liver transplantation at the age of 14 years that was performed because of worsening of metabolic control on dietary therapy with increased frequency of hyperam-monemic episodes during puberty. She has not manifested any symptoms of the disease after the transplantation.

We have examined 25 DNA and RNA samples isolated from the liver and 1 DNA sample from the blood to determine the XCI ratios and the expression ratios of the individual alleles. The standard XCI method (HUMARA) was not informative but three of our novel assays published previously were usable and we found skewing of X-inactivation in the liver ranging from 45:55 to 82:18 (mean 70:30). The XCI ratio in blood was 57:43. The results show the intra-organ variation of XCI ratios and a significant difference from the ratio in blood, however the aver-age values were not dissimilar. X-inactivation in the liver samples obtained by biopsy in carriers may vary from the average, further research is needed to decide whether XCI ratio in blood is a use-ful surrogate.

P02-007-SP

DNA structural transitions upon dehydration

of DNA solutions revealed by FTIR

spectroscopy

S. V. Paston, O. V. Shulenina, S. A. Tankovskaya, A. M. Polyanichko

Molecular Biophysics, Saint Petersburg State University, St. Petersburg, Russian Federation

The DNA secondary structure is very important for genome functioning. B- to A-DNA transition occurs at manifold natural processes such as replication, transcription, DNA-protein interac-tions, etc. Aqueous environment and metal ions play the key role in DNA secondary structure maintenance. We investigated fast structural transition in DNA using attenuated total reflectance FTIR spectroscopy of drying drop of DNA solution. The main aspects we studied were the velocity of water release, the amount of water in the hydrations shells of DNA, and the manifestations of B- and A-forms in IR spectra.

We observed some indicative alterations in IR spectra of DNA confirming B to A transition during the sample dehydra-tion. The disturbance of native DNA structure in the solution, such as partial denaturation or UVC irradiation, lead to the soft-ening of A-form IR markers and to the increase in the amount of residual water in DNA films. In the native DNA the time of full dehydration process and the amount of the water in the hydration shells depended on the salt concentration in the sam-ple. The effects observed in the experiment can be attributed to the ions penetration into the DNA hydration shell and dismissal of water molecules.

Part of research was performed at the Center for Optical and Laser Materials Research of Research park of St.Petersburg State University.

This work was supported by the Russian Foundation for Basic Research (RFBR 13-03-01192 and 15-08-06876) and SPbSU (11.38.644.2013).

P02-008-SP

PRE-PIK3C2B: a Human PRE with a difference?

Thakur2_{, V. Brahmachari}1

1_{Dr. B.R. Ambedkar Center for Biomedical Research, University}

of Delhi, Delhi, India,2_{Centre for Cellular & Molecular Biology,}

Hyderabad, India

The transcriptional layout determined by gene specific regulators during early development is maintained by cellular memory mod-ules consisting of Polycomb and Trithorax complexes and the polycomb/trithorax response elements (PRE/TRE). We have identified a hPRE/TRE which maps in PIK3C2B (PRE-PIK3C2B; Bengani et al. 2013, PLoS One) and HOXB3 (CE-HOXB3: Unpublished). PRE-PIK3C2B functions as a PRE in transgenic Drosophila and interacts with both polycomb and tri-thoraxgenes. In this background, we have delineated the interac-tion of PRC2 complex with PRE-PIK3C2B in human cells. Following the screening of different cell lines for the expression level of PIK3C2B and its neighbours, we analysed the probable TRE-like function of PRE-PIK3C2B. Concurrently, we carried out de novo search for PRE-PIK3C2B interacting proteins in human cells, using DNA based affinity columns followed by mass spectrometry, which led to the identification of MLL protein among others. Validation of the interaction of both MLL and BRM proteins with PRE-PIK3C2B strongly suggests TRE activ-ity of PRE-PIK3C2B. We have identified the potential targets of PRE-PIK3C2B as MDM4 and PPP1R15B based on their coordi-nated expression and the effect of knock-down of PRC2 mem-bers on them. Furthermore, we have performed the Circular Chromosomal Conformation Capture assay to identify long range interaction of PRE-PIK3C2B.

P02-009

The toxic effect of calcium carbide on DNA

damage in banana

K. S. Ali, M. M. Odah

Biology, University of Aden, Aden, Yemen

This study was conducted to evaluate the toxic effects of ripening accelerator (CaC2) on DNA damage in banana. Samples were

taken after 0, 4, 8, and 16 days of CaC2treatment and DNA was

extracted from banana’s leaves, peels, and fruits. We were used comet assay to detected DNA damage by measuring the head DNA (H-DNA), tail DNA (T-DNA), and olive tail moment (OTM) of the comets. The levels of DNA damage were assessed and quantified by computer image analysis. The values of H-DNA, T-DNA, and OTM exhibited significant differences from the control at 4, 8 and 16 days of CaC2 treatment. The DNA

damage was induced by CaC2 treatment in a time-dependent

manner.

P02-010

Oxidative stress induces LINE-1

hypomethylation through depletion of

S-adenosylmethionine

C. Boonla1, C. Kloypan1_{, M. Srisa-art}2_{, A. Mutirangura}3 1_{Department of Biochemistry, Faculty of Medicine, Chulalongkorn}

University, Bangkok, Thailand,2_{Department of Chemistry, Faculty}

of Science, Chulalongkorn University, Bangkok, Thailand,

3_{Department of Anatomy, Faculty of Medicine, Chulalongkorn}

University, Bangkok, Thailand

Increased oxidative stress and hypomethylation of long-inter-spersed nuclear element-1 (LINE-1) are demonstrated in patients with bladder cancer. Induction of LINE-1 hypomethylation by reactive oxygen species (ROS) is demonstrated in a bladder can-cer cell line with unknown mechanism. We hypothesized that ROS-induced LINE-1 hypomethylation was mediated through the depletion of the methyl donor, S-adenosylmethionine (SAM). Bladder cancer (UM-UC-3 and TCCSUP) and normal human kidney (HK-2) cell lines were used in the experiments. No signifi-cant change of cell viability was observed in cells exposed to 20 lM H2O2. Intracellular ROS production and protein carbonyl

content were significantly increased, but LINE-1 methylation was significantly decreased, in the H2O2-exposed cells. LINE-1

hy-pomethylation was significantly restored by a-tocopheryl acetate (TA), N-acetylcysteine (NAC), methionine, SAM, and folic acid. SAM level in H2O2-treated cells was significantly decreased while

total glutathione was significantly increased. The depleted SAM in H2O2-treated cells was restored by NAC, methionine, SAM,

and folic acid, whereas, an increased total glutathione was nor-malized by TA and NAC. Homocysteine (Hcy) was significantly decreased in the H2O2-treated cells, which was reinstated by

NAC. Conclusion, SAM and Hcy were depleted, but total gluta-thione was raised, in bladder cancer and normal kidney cells exposed to H2O2. These changes were restored by antioxidants

(TA and NAC), and one-carbon metabolites (SAM, methionine, and folic acid). The present findings suggest that exposure of cells to ROS activates glutathione synthesis via the transsulfuration pathway leading to deficiency of Hcy to be used for SAM synthe-sis. This consequently causes SAM depletion, and hence hypome-thylation of LINE-1.

P02-011

Cell cycle arrest mediates Rb gene methylation

patterns in APL patients

A. Khaleghian

Department of Biochemistry, School of Medicine Semnan, University of Medical Sciences, Semnan, Iran

The retinoblastoma tumor suppressor (Rb) protein binds to the E2F transcription factors to control gene expression.

Overexpres-sion of E2F will drive quiescent cells to reenter the cell cycle. Rb

is a phosphoprotein that regulates cell cycle progression from the G1 to S phase by reversibly inhibiting E2F-mediated

transcrip-tion of genes required for S-phase entry. Epigenetic regulatranscrip-tion is critical for mammalian development and cellular differentiation and dysregulation of this process causes human developmental diseases and cancer. DNA methylation is the most frequent epi-genetic alteration seen in mammalian genomes. In cancer the gen-ome in general is hypermethylated.

In this study, aberrant DNA methylation of target promoters in APL patients that were treated with ATO (As2O3) and the

NB4 cell line was confirmed using Methylation Specific PCR (MSP) and was linked to changes in the expression of the Rb gene, as assessed by real time RT-PCR. We further addressed the

hypothesis that Rb gene methylation might be of value in the detection of APL.Therefore, it could be suggested that hyperme-thylation of the Rb promoter is one of the epigenetic factors affecting the progress of sporadic APL carcinogenesis in patients.

P02-012

Epigenetic regulation is a determinant of the

cell line specific expression of the UDP

glycosyltransferase 3A1 and 3A2 genes

Adelaide, Australia,2Flinders Centre for Innovation in Cancer, Flinders University, Adelaide, Australia

The UDP glycosyltransferases (UGTs) are a super-family of enzymes involved in the metabolism of xenobiotics and endoge-nous molecules. Variations in UGT expression have been associ-ated with disease states and inter-individual variation in response to drug therapy. The UGT3A family was discovered and charac-terised by our laboratory. Upon investigating UGT3A expression in a range of cell lines, we found a large number in which UGT3A1 was not expressed. To determine if epigenetic mecha-nisms are responsible for this lack of expression, cell lines were treated with the demethylating agent 5-aza-20-deoxycytidine (5-aza-dC), and the histone deacetylase inhibitor trichostatin A (TSA), and levels of UGT3A mRNA measured. In three cell lines (T47D, MCF-7 and ZR75.1), 5-aza-dC induced UGT3A1 expres-sion from 8.9-fold (ZR75.1, p= 0.024) up to 12.2-fold (T47D, p= 0.007). TSA alone did not induce UGT3A1 expression, but in some cases, was synergistic with 5-aza-dC. Neither chemical had an effect on UGT3A2 expression in these three cell lines. In MDA-MB-453 cells, only 5-aza-dC with TSA induced UGT3A1 expression (14.5-fold, p_{= 0.036), while UGT3A2 was induced by} 5-aza-dC (27.8-fold, p= 0.002), and 5-aza-dC with TSA (23.9-fold, p= 0.012). Thus our preliminary data indicates that epige-netic mechanisms, particularly DNA methylation, are a determi-nant of cell line specific expression of UGT3A1 and UGT3A2. Bisulfite sequence analysis will be performed to identify the meth-ylation status of predicted promoter CpG islands. This should provide further novel insights into the epigenetic regulation of the UGT3A family.

P02-013

Epigenetic changes over long-term evolution

of breast tumor

D. li

Tongji University, Shanghai, China

The key feature separating cancer from genetically inherited dis-orders is that cancer is continuously evolving during the course of the disease. Histone modifications are well-established media-tors of transcriptional programs that control the cell evolution process. Thus, an epigenetically perspective is critical for under-standing the initiation and progression of cancer.

The present understanding of the relationship of dynamic epi-genetic signature and cancer evolution is unclear and fragmented because comprehensive epigenetic data related to long-term evo-lution of cancer and clinical samples covering the whole life his-tory of a human tumor from initiation to metastasis have been unavailable.

The mouse-based xenograft model has long been used to study the genetics underlying tumourigenesis, tumor progression and metastasis, and has been proven highly successful in human can-cer biology.

In our study, we used serial passages of a human cell-derived xenograft tumor in mice to study the profiling of epigenetic dynamics in breast cancer evolution, including tumourigenesis, tumor progression and metastasis.

Our current study initially identified that the study of the dynamics of epigenetic signature is a potentially productive approach that could help us to understand the mechanisms of cancer evolution, especially tumor progression and metastasis. In our further study, we will study predicted transcription factor motifs that are enriched in a subset of dynamic H3K27ac clus-ters, which might link to functional pathways, and could further assist to understand the linkage between transcriptional regula-tory elements and biological process of cancer evolution.

P02-014

Acetylation on the nucleoprotein of influenza

A virus

D. Hatakeyama1, M. Shoji1, R. Yoh1, N. Ohmi1, S. Takenaka1, S. Yamayoshi2_{, Y. Arakaki}1_{, T. Komatsu}1_{, A. Masuda}1_{, M.}

Nakano2_{, T. Noda}2_{, Y. Kawaoka}2_{, T. Kuzuhara}1

1_{Faculty of Pharmaceutical Sciences, Tokushima Bunri University,}

Tokushima City, Japan,2_{Institute of Medical Science, The}

University of Tokyo, Tokyo, Japan

Posttranslational acetylation of lysine residues is most extensively studied in histones, and is known to play crucial roles in gene expression. This modification is also found in many other pro-teins and is implicated in a wide range of biological processes. Recently, acetylation was reported to occur on the non-structural protein 1 protein in H3N2 strain of influenza A virus, and this modification selectively suppressed inducible gene expression for antiviral response. Here, we report that nucleoprotein (NP), his-tone-like viral protein, is acetylated. First, to screen (a) novel viral acetylated protein(s), the A549 cells infected with H1N1 and H3N2 strains of influenza A viruses were homogenized and sepa-rated by SDS-PAGE. Western blotting analysis using anti-acetyl-lysine antibody indicated the positive signals around 50 kDa. The combination analysis of immunoprecipitation using anti-NP antibody and western blotting using anti-acetyl-lysine antibody showed that this acetylated protein was NP, and LC-MS/MS analysis confirmed this result. This acetylation was also detected on the NP-expressed cells, suggesting that an endogenous transferase acetylated NP. Next, we investigated which acetyl-transferase acetylates NP, using the recombinant NP and various acetyltransferases. We found that recombinant GCN5, pCAF and HBO1 proteins acetylated NP upon incubation with [14 C]-acetyl-CoA. NP in ribonucleoprotein purified from virus particles was also acetylated by GCN5 and pCAF. This enzymatic reac-tion was inhibited by embelin, which was a known inhibitor of pCAF. These results indicated that pCAF and GCN5 acetylated NP of influenza A virus in infected cells. We will discuss from various aspects in the Congress.

P02-015

The impact of mm-waves on the level of DNA

methylation on the plant model

L. Minasbekyan, P. O. Vardevanyan Yerevan State University, Yerevan, Armenia

The rapid increase in the use of mobile phones (MPs), as well as the wide use of mm-waves-therapy and diagnostic equipments in medicine in recent years, has raised the problem of health risks connected with high-frequency electromagnetic fields in our envi-ronment. DNA methylation is one of the most studied epigenetic modifications in biological systems.

We explore a plant model for investigating the impact of mm-waves on the cell nuclei and DNA methylation. We have chosen wheat seeds for the model, a plant very well investigated by us. We separate DNA from control samples and samples treated by EHF (Extremely High Frequencies) EMI (Electromagnetic Irradi-ation) seedlings on the 4th day. After that some of the treated seedlings are grown in soil till to get a harvest (as a second gener-ation). Investigation is carried out in the range of 45–51.8 GHz frequencies of EMI. EHF EMI can bring some changes in the DNA methylation level and aqua environment, which lead to changes in chromatin architecture. The data indicate that 5mC changes depend on EHF EMI frequencies and exposition. After that we have investigated the level of DNA methylation from 4th-day seedlings by growing seeds from the new harvest (second generation). Data obtained in our study shows that the changes in the level of DNA methylation in the first generation of seeds during the plant ontogeny are partially conserved and pass to the seedlings of second generation seeds. So we have shown in the plant model that the changes in biological systems under the influence of EHF EMI have rather epigenetic character and par-tially pass to the next generation.

P02-016

Investigation of DNA methylation and H4

hyperacetylation dynamics in the 5S rRNA

genes family by chromatin

immunoprecipitation assay

C. C. Burcea1_{, I. Suciu}2_{, P. Armean}3_{, N. Cucu}2_{, C. Domnariu}4_,

L. Burlibasa2 1

UMF Carol Davila, Bucharest, Romania,2Faculty of Biology, University of Bucharest, Bucharest, Romania,3University of Medicine and Pharmacy “Carol Davila”, Bucharest, Romania,

4

University of Sibiu Lucian Blaga, Sibiu, Romania

Oogenesis is an important event in the formation of female gam-ete, whose role in development is to transfer genomic information to the next generation. During this process, the gene expression pattern changes concomitant with genome remodeling, while genomic information is stably maintained. Active and silent genes are distinct from one another with respect to their chromatin configurations. Two 5S RNA gene families are transcribed in oo-cytes, firstly the major oocyte type and secondly the somatic type. In somatic cells, only the somatic 5S RNA genes are transcribed, while the oocyte genes are repressed.

The aim of the study was to investigate the presence of H4 acetylation and DNA methylation of the oocyte and somatic 5S rRNA genes in Triturus cristatus, using chromatin immunopre-cipitation assay (ChIP and RE-ChIP). Our findings suggest that histone acetylation and DNA methylation are critical mecha-nisms involved in transcriptional regulation of 5S rRNA genes family.

P02-017

Retinoic acid induced Hoxa5 is negatively

regulated by CTCF in F9 teratocarcinoma cells

Department of Anatomy, Embryology Laboratory, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea

Hoxgenes are essential for anterior–posterior body patterning at early stage embryonic development. In mammals, 39 Hox genes are divided into four clusters called HoxA, B, C and D on four different chromosomes. The combinatorial expression of Hox plays an important role in the process of mammalian

develop-ment. However, the precise mechanisms by which signal path-ways are stimulated to regulate the expression of Hox are not clear. In the previous study, retinoic acid (RA) has been identi-fied as a modulator of cell survival, proliferation and differentia-tion in the developing embryo. Interestingly, RA induces Hox expression in F9 cells. Furthermore, CCCTC-binding factor (CTCF) was reported as a controller of Hox expression. Here, we provide relationship of RA, CTCF and Hox expression in F9 teratocarcinoma cells. In order to investigate the expression pat-tern of Hox in response to the RA, we performed the RT-PCR in the RA treated F9 cells. The result showed that the anterior Hoxa mRNA levels were up-regulated in RA treated F9 cells. However, RA induced Hoxa5 expression level was decreased in CTCF over-expressed F9 cells. This experiment demonstrated that CTCF negatively associated with up-regulation of Hoxa5 in response to retinoic acid. In addition, to investigate whether the RA regulates the CTCF binding at Hoxa5 promoter region, we carried out the chromatin immunoprecipitation assay. When RA was present, CTCF was dissociated from the binding site. These results altogether indicate that RA might regulate the expression of Hoxa5 by modulating the binding of CTCF at the Hoxa5 pro-moter region.

P02-019

DNA methylation contributes to consitutive

telomerase gene expression by inhibition of

KLF2 binding to a promoter element in

huuman T cells

M. Nakamura

Human Gene Sciences Center, Tokyo Medical and Dental University, Tokyo, Japan

Telomerase is critical for the life span of normal and tumor cells. The telomerase limiting subunit hTERT (human telomerase reverse transcriptase) is strictly regulated in its transcription and highly expressed even in normal human T lymphocytes when they grow. We previously identified a novel element in the hTERT promoter, which was unmethylated and functioned as a repressor in normal resting T cells, and have recently published that the transcription factor Kr€uppel-like factor 2 (KLF2) bound to the element, resulting in repression of hTERT transcription. During our studies, we noticed that KLF2 bound to the exo-geously introduced unmethylated element but not to the endoge-nous element in a resting human T leukemic cell line (Kit 225), in which KLF2 was expressed and the endogenous element was DNA-methylated. Demethylation treatment with Zebularine restored KLF2 binding to the endogeous element in Kit 225 cells. We thus assumed that DNA methylation inhibited KLF2 bind-ing, leading to consitutive expression hTERT that is one of the common signatures of tumor cells. Transcriptional repressive mark of histone H3 lysin nine trimethylation (H3K9me3) was associated with KLF2 binding to the promoter, indicating KLF2-mediated epigenetic silencing of the hTERT promoter. Another mark of H3K27me3 was related to cell growth but inde-pendent of KLF2 binding. Our findings demonstrate that KLF2 regulates strict transcription of the hTERT gene by direct bind-ing to the promoter in normal T cells, while in tumor cells these important mechnisms are modulated by DNA methylation.

P02-020

CTCF regulates HOXA10 gene expression in

breast cancer cell lines

M. Mustafa, J. Y. Lee, M. H. Kim

Department of Anatomy, Embryology Laboratory and Brain Korea 21 PLUS project for Medical Sciences Yonsei University College of Medicine, Seoul, Korea

CTCF (or CCCTC-binding factor), a ubiquitous 11-zinc finger multifunctional protein has distinct molecular functions such as transcriptional activation, transcriptional repression, or enhancer blocking activity, in a locus-specific manner. Identification of somatic mutations in CTCF in different cancers and its involve-ment in cellular growth, differentiation and apoptosis point towards its role in cancer progression. The HOX genes, a family of transcription factors, not only play a key role in body pattern-ing durpattern-ing embryonic development but also regulate cell cycle, cell adhesion, migration and differentiation in adults. Many HOXare found to be dysregulated in different cancers. Among HOX, HOXA10 is an emerging tumor suppressor for its role in activation of p53 and in countering tumorigenesis in breast can-cer. HOXA10 silencing is associated with different cancers but the underlying mechanism is still elusive. We investigated the effect of CTCF on the expression pattern of HOX genes and identified HOXA10 as one of the genes directly regulated by CTCF. Our data defines the putative promoter region of HOXA10and CTCF binding site, using dual luciferase reporter assays and ChIP analysis, respectively. Analysis of histone modi-fication reveals that the presence of CTCF is associated with decreased active histone marks H3K4me3 and increased repres-sive histone marks H3K27me3 on HOXA10 locus. Based on the evidence in our study, we propose that enrichment of CTCF on the promoter region of HOXA10 opposes the recruitment of transcription factors and alters the histone modification. Epige-netic silencing of HOXA10 may contribute towards tumorigenesis by decreasing apoptosis and promoting metastasis.

P02-021

HOXB gene upregulation is associated with

tamoxifen-resistance in MCF7 breast cancer

cells

S. Y. Yang1, J. Y. Lee1, H. Hur2, M. H. Kim1

1

Department of Anatomy, Embryology Laboratory and Brain Korea 21 PLUS project for Medical Sciences Yonsei University College of Medicine, Seoul, Korea,2_{Department of Surgery,}

National Health Insurance Corporation Ilsan Hosital, Goyang, Korea

Endocrine therapy, such as tamoxifen and aromatase inhibitors, has been used to treat both early and advanced estrogen receptor a (ER)-positive breast cancer. Despite improvements in treat-ment, resistance to the current therapeutics can occur in up to one quarter of all cases and thus presents a serious therapeutic challenge. Multiple mechanisms responsible for endocrine resis-tance have been proposed, however, the molecular events under-lying resistance to therapeutic agents are not clearly understood. Therefore, a better understanding of gene expression alterations associated with the resistance would suggest alternative regimens that overcome endocrine resistance. HOX transcription factors have recently been implicated as strong candidates to control cancer progression and metastasis. Previously we have demon-strated HOX gene dysregulation in human breast cancer samples as well as breast cancer cell lines. To identify HOX genes involved in tamoxifen resistance, here we have generated in vitro model of acquired tamoxifen resistance using MCF breast cancer

cells (MCF7-TamR) and analyzed expression pattern of HOX genes. MCF7-TamR cells were more resistant to tamoxifen in MTT assay and exhibited up-regulation of HOXB including HOXB4, HOXB5, and HOXB6. ChIP analysis of histone modifi-cation revealed that the activation of midcluster HOXB in MCF7-TamR cells is associated with the loss of H3K27me3. Meanwhile, Kaplan-Meier analysis of the overall survival for all patients treated with only endocrine therapy showed the correla-tion of high HOXB5, HOXB6 expression with a poor response to endocrine therapy. These results suggest a functional role of epi-genetically regulated HOXB in the development of aquired tamoxifen resistance in breast cancer.

P02-022

O-GlcNAc transferase impact on

EZH2-dependent FOXC1 and FOXA1 gene expression

in breast cancer cells

E. Forma, P. Jozwiak, P. Ciesielski, M. Brys, A. Krzeslak Department of Cytobiochemistry, University of Lodz, Lodz, Poland

EZH2 is involved in transcriptional repression by methylation of histone H3 at lysine 27. EZH2 is associated with progression of breast cancer and its expression correlates with breast cancer aggressiveness. O-GlcNAc transferase (OGT) is an enzyme which catalyzes the addition of N-acetylglucosamine moiety to serine/ threonine residues of cellular proteins. Recent studies have sug-gested that OGT may be involved in transcriptional repression caused by Polycomb proteins. In this study we analyzed the effect of OGT and EZH2 on FOXC1 and FOXA1 expression as well as migration and invasion of breast cancer cells. FOXC1 and FOXA1 are expressed in a vast majority of cancers, including breast cancer, in which high expression is associated with a good prognosis. To investigate regulatory effects of EZH2 and OGT on gene expression the breast cancer cells were transfected with OGT and EZH2 siRNA. We found that OGT down-regulation caused decrease in EZH2 protein and H3K27 methylation levels as well as increased level of FOXC1 and FOXA1 expression. Similar increase of the two transcription factors expression was observed in cells with down-regulated expression of EZH2. We examined the binding of EZH2 and OGT at different regions of FOXC1 and FOXA1 promotors. Chromatin immunoprecipita-tion analysis was performed using EZH2, OGT and H3K27 spe-cific antibodies. Enrichment of the FOXC1 and FOXA1 was quantified using qRT-PCR. We have found that the OGT occu-pancy coincides with EZH2 and the H3K27me3 at promoters. Our studies suggest the role of O-GlcNAc transferase in EZH2-dependent repression of genes activity in breast cancer cells.

P02-023

Global methylation profiles in lung tissues of

silicosis patients

M. Ye, X. N. Zhang, X. W. Jia, W. J. Hu, L. Y. Mei, M. Zheng, C. Yu

Chinese Center for Disease Control and Prevention, Key Laboratory of Chemical Safety and Health, National Institute for Occupational Health and Poison Control, Beijing, China

A previous study has demonstrated that silica mediates the acti-vation of the PI3K/PTEN/AKT/MAPK/AP-1 pathway in human embryo lung fibroblasts (HELFs).The purpose of this study is to identify genome-wide aberrant DNA methylation profiling in lung tissues from silicosis patients. We performed Illumina Human Methylation 450K Beadchip arrays to investigate the methylation alteration in formalin-fixed, paraffin-embedded

(FFPE) lung specimens from six early-stage silicosis patients and four advanced-stage patients. Immunohistochemistry was used to confirm the level of PI3K/PTEN/AKT/MAPK/AP-1 protein in FFPE samples. MS-PCR was used to investigate the methylation of PTEN and c-Jun. We found 86,770 CpG sites and 79,660 CpG sites significantly differed in methylation status in early-stage and advanced early-stage compared with normal lung methyla-tion data from GEO, respectively. Analysis of the Kyoto Ency-clopedia of Genes and Genomes (KEGG) revealed the MAPK signaling pathway was considered significant, indicating that the MAPK pathway was regulated by DNA methylation. The CpG promoter sites of PTEN and c-Jun were shown to be increased in advanced-stage cases. Early-stage cases showed the positive expression of c-Jun and PTEN protein and negative or mild expression in advanced-stage cases using immunohistochemistry. The PTEN promoter was no differentially methylated and the c-Jun promoter differed at 12 and 24 h in HELFs detected by MSP-PCR. These results suggested that abnormal DNA methyla-tion on a genome-scale was implicated in silicosis, and PTEN promoter hypermethylation might be associated with the decrease of PTEN protein.

P02-024

The association between preeclampsia and

K55R polymorphism and methylation levels of

the soluble epoxide hydrolase gene (EPHX2)

1_{Department of Biochemistry, School of Medicine, Cumhuriyet}

University, Sivas, Turkey,2Department of Biochemistry, School of Medicine, Halic University, _Istanbul, Turkey,3School of Medicine, Cumhuriyet University, Sivas, Turkey

Preeclampsia is a multifactorial disease characterized by new onset of hypertension and either proteinuria or end-organ dys-function after 20 weeks of gestation. It is a leading cause of maternal and fetal mortality and morbidity and its etiology is not yet understood. Soluble epoxide hydrolase (sEH) is involved in metabolism of epoxyeicosatrienoic acids (EETs) to their less bioactive corresponding diols. In this study the association between K55R polymorphism and methylation levels of the EPHX2 promoter region and preeclampsia was investigated in 520 individuals including 260 preeclamptic patients and 260 healthy pregnant women.

K55R polymorphism and methylation levels of the EPHX2 gene promoter were determined by real time PCR using double-dye hydrolysis probes and methylation-sensitive high-resolution melting analysis, respectively.

The presence of the K55R polymorphism was significantly higher in cases than controls, and was associated with increased risk of preeclampsia (OR 1.86; 95% CI 1.09–2.63). Methylation levels of the EPHX2 promoter region in cases were significantly lower than controls. 2.83 times increased preeclampsia risk was observed in pregnant women with EPHX2 promoter methylation levels of<25%.

In conclusion, hypomethylation of the promoter region of the EPHX2gene and K55R polymorphism were associated with sig-nificant increased risk of preeclampsia. sEH enzyme may play a role in the pathogenesis of preeclampsia by contributing to reduc-tion of the vasodilatator, anti-hypertensive and anti-inflammatory effects of EETs by rapid degradation of these molecules.

P02-025

Short telomere length and increase expression

of its related proteins were associated with

the level of benzo(a)pyrene exposure in

human bronchial epithelial cell

P. Bin, X. Li, Y. Wang, H. Yang

National Institute for Occupational Health and Poison Control, Chinese Center for Disease Control and Prevention, Beijing, China Telomeres play a key role in the maintenance of chromosome integrity and stability. Shortened telomere length (TL) is thought to been associated with genomic instability. External stressors from environmental exposures might accelerate shortening of TL and induce expression of its related proteins, e.g. telomeric repeat binding factor 2 (TRF2) and telomerase. Benzo(a)pyrene is the main form of carcinogenic polycyclic aromatic hydrocarbons which could cause lung cancer. In the present study, we investi-gated the association of TL and its related proteins with benzo(a) pyrene exposure in vitro. Human bronchial epithelial cells were treated with three concentrations of benzo(a)pyrene (1, 4 and 16 lmol/l). The TL of genomic DNA, the TRF2 and the telo-merase in cells were evaluated, and were detected by real-time polymerase chain reaction, western-blotting and TRAP-ELISA, respectively. The positive control was 4 lg/ml bleomycin. Com-pared to the 0 lmol/l group, the TL in the 1, 4 lmol/l and posi-tive control groups were significantly shorter (p< 0.05), but no significant difference was found between the 16 and 0 lmol/l groups. There was high expression of TRF2 in the 1 lmol/l and positive control groups,but there was low expression in the 16 lmol/l group, as well as the activity of telomerase. The results indicate that short TL is associated with the benzo(a)pyrene exposure and induce the expression of TRF2 and telomerase, and suggest that benzo(a)pyrene might have influence on the genomic instability by acting on telomeres in human bronchial epithelial cell.

P02-026

Functional requirement of zinc finger motif(s)

in Helicobacter pylori Topoisomerase I

function

S. M. Kondekar, R. N. Desirazu

Department of Biochemistry, Indian Institute of Science, Bangalore, India

Helicobacter pylori, a human pathogen dominating the gastric microbial population, displays differential gene expression during various stages of stomach colonization. Topoisomerases play a crucial role in maintaining DNA superhelicity and thus gene expression. H. pylori has only two topoisomerases: DNA gyrase and Topoisomerase I, as opposed to four in most other prokary-otes. Biochemical characterization of Topoisomerase I from H. pylori (HpTopoI) revealed that HpTopoI has properties dis-tinct from Escherichia coli Topoisomerase I (EcTopoI). While Ec-TopoI prefers ssDNA over dsDNA, HpEc-TopoI binds both ssDNA and dsDNA with similar affinities. Although both enzymes have comparable DNA-relaxation activity, sequence comparison of HpTopoI with EcTopoI shows that there are four zinc finger motifs (ZFs) at the carboxy terminal domain (CTD) unlike three in EcTopoI. Moreover, Thermotoga maritima Topo-isomerase I (TmTopoI) has one non-essential ZF and Mycobacte-rium tuberculosisTopoI lacks ZFs. To gain an insight on the role of ZFs in HpTopoI function, we sequentially deleted the ZFs from carboxy terminus. We observed that third and fourth ZFs are dispensable for HpTopoI function whereas deletion of distal three ZFs hampered DNA-relaxation activity with no effect on

DNA-binding. Deletion of all ZFs, however, drastically reduced DNA-binding and abolished DNA-relaxation. Thus, we hypothe-size the role of first two ZFs in strand passage activity of HpTo-poI. Intriguingly, atomic-spectroscopy data suggested that HpTopoI ZFs failed to co-ordinate with Zn2+ as documented for TmTopoI but the observation is in contrast with that of Ec-TopoI. Our observations would further help to understand the variability in number of ZFs in Topoisomerase I function

P02-027

Harmonious pattern of HOXA10 gene

expression with epigenetic aberration of its

regulatory region in eutopic endometrium and

ectopic endometriotic lesion of endometriosis

patient during the menstrual cycle

Y. Samadieh1, M. Shahhosein1, S. Mahdian2, F. Ramezanali2, P. Afsharian1, R. Aflatoonian2

1

Genetics, Royan institute, Tehran, Iran,2Endocrinology and Female Infertility, Royan institute, Tehran, Iran

Epigenetic aberrations such as DNA methylation and histone modifications appear to be involved in various diseases such as Endometriosis. Here, we investigate the epigenetic regulation of the HOXA10 promoter, as a crucial gene responsible for uterine organogenesis, functional endometrial differentiation and endo-metrial receptivity, and its correlation with mRNA expression of this gene in eutopic, ectopic and normal endometrium, during the menstrual cycle.

For this respect, chromatin immunoprecipitation using anti-MeCP2, H3K9ac, H3K9me2, H3K4me3, H3K27me3 and the real-time PCR technique were performed. Ectopic endometriotic lesions and eutopic endometrium samples were collected through laparoscopy from 36 women with endometriosis. Also, endome-trial biopsies were obtained from 20 fertile women as control group. Ethical consent was gained from patients.

Epigenetic data showed a coordinate pattern with expression data. In the secretory phase, in eutopic tissues, reduction of HOXA10gene expression was shown along with lower H3K9ac and higher H3K9me2 incorporation on HOXA10 gene promoter. In return in ectopic endometriotic lesions in the secretory phase, induction of HOXA10 gene was correlated with higher H3K9ac, H3K27me3 and H3K4me3 incorporation on HOXA10 gene pro-moter, all in comparison with the control group. In the prolifera-tive phase, in the ectopic endometrium up-regulation of HOXA10 coincides with higher incorporation of H3K4me3 on its promoter region compared with the control group.

Since HOXA10 aberrant expression in the endometrium is involved in endometriosis pathogenesis according to previous data, epigenetics can play an important role in this gene aberrant expression.

P02-028

Epigenetic disruption of CRE transcriptional

activity pathway in human spermatogenic

disorders

R. Favaedi1, M. Shahhoseini1, M. A. Sadighi Gilani2

1

Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran,2Department of Andrology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran

One of the most interesting epigenetic regulations occurs through compaction of sperm chromatin. Expression of sperm chromatin

condensing genes including transition proteins (TNPs) and prota-mins (PRMs) are activated by a testis specific transcription factor named CREM, a DNA binding regulator necessary for sper-matogenesis in mammals. CREM or cAMP response element modulator in combination with its co-factor ACT (activator of cAMP-responsive element modulator) binds to cAMP response elements (CRE) in regulatory regions of chromatin condensing genes and regulates their expression.

Regarding the critical role of compaction of sperm chromatin in male fertility, the potential role of CREM and ACT on regula-tion of these genes was the aim of the study.

For this reason, consent was obtained from azoospermic men referred to the Royan Institute, according to local ethical approval and then testes tissue samples were collected from three groups including complete maturation arrest, Sertoli cell only syndrome, and hypospermatogenesis (as positive control), based on pathological features. Expression levels of TNPs, PRMs, CREMand ACT were evaluated by qRT-PCR. Also, chromatin immunoprecipitation coupled with real-time PCR was performed to evaluate the incorporation of CREM and ACT into the CRE regions of TNPs and PRMs.

The results showed a significant decrease in expression of CREM and ACT as well as TNPs and PRMs genes, in two groups with spermatogenesis failure versus positive control. Also our findings revealed decreased incorporation of CREM and ACT into the CRE regions of the mentioned genes in the two mentioned groups versus control.

This study implies association of epigenetic disruption of the CRE transcriptional activity pathway and male (in)fertility.

P02-029

Evaluation of telomere length and TERRA

transcription level in PCOS patients

N. Ghobadi1,2, M. Shahhoseini2, R. Favaedi2, F. Hassani3, B. Movaghar3, L. Karimian3, P. Eftekhari Yazdi3

1

Faculty of Basic Sciences and Advanced Technologies in Biology at University of Science and Culture, ACECR, Tehran, Iran,

2_{Department of Genetics at Reproductive Biomedicine Research}

Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran,3_{Department of Embryology at Reproductive}

Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran

Telomeres are essential structures at the ends of all eukaryotic chromosomes. In human, telomeres and their transcripts, TERRA, are composed of hexa nucleotide tandem repeats which play an important role in chromosomes protection. By each cell division telomere length is shortened but this shortening is coun-teracted by telomerase, a ribonucleoprotein enzyme that can extend the 30 ends of chromosomes. Some studies showed that increasing both the expression and activity of telomerase is effected by some hormones such as androgens. Based on the Rot-terdam consensus, clinical or biochemical hyperandrogenism is one of the three diagnostic criteria for Polycystic Ovarian Syn-drome (PCOS). Therefore, we aimed our attention on study of TERRA expression in ovary of PCOS patients parallel with eval-uation of telomere length of leukocyte cells (LTL) of the same patients. For this respect, cumulus cells and blood samples were collected from PCOS patients and healthy women with male fac-tor infertility as positive control. TERRA expression and telo-mere length were measured by quantitative real time PCR. Results showed significant increase of TERRA expression in cumulus cells of PCOS versus control group, but there is no dif-ference in telomere length of blood cells between two groups.

This finding implies a considerable association between alter-ation of TERRA transcription and PCOS. Also, no difference of

LTL in PCOS versus control group may be interpretable by importance of TL measuring in cumulus cells.

P02-030

Histone deacetylase inhibitor, CG200745,

attenuates transcriptional activity of

mineralocorticoid receptor through its

acetylation

E. J. Lee1,2,3_{, M.-J. Song}1_{, I. K. Kim}1,2,3

1_{Pharmacology, Kyungpook National University School of}

Medicine, Daegu, Korea,2Cell and Matrix Research Institute, Kyungpook National University School of Medicine, Daegu, Korea,3BK21 Plus KNU Biomedical Convergence Program, Kyungpook National University School of Medicine, Daegu, Korea HDAC inhibitors attenuate fibrosis, hypertrophy, inflammation, and hypertension in several animal models. CG200745 {(E)-2-(Naphthalen-1-yloxymethyl)-oct-2-enedioic acid 1-[(3-dimethyla-mino-propyl)-amide] 8-hydroxyamide]} (CG) is a novel HDAC inhibitor that is being evaluated in phase II clinical trials for its anticancer effect. However, the antihypertension effect of CG remains unknown. We hypothesized that CG attenuates tran-scriptional activity of the mineralocorticoid receptor (MR) through its acetylation. Expression of MR target genes was mea-sured by quantitative real-time polymerase chain reaction. Recruitment of MR and RNA polymerase II on target genes was analyzed by chromatin immunoprecipitation. MR acetylation was determined by western blot with an acetyl-lysine anti-body after immunoprecipitation with an anti-MR antianti-body. In f-hMR-HEK293 cells, treatment with CG increased MR acetyla-tion and decreased expression of MR target genes. The down-regulation of target genes coincided with a decrease in the recruitment of MR and RNA polymerase II to specific hormone response element (HRE). These results demonstrate that CG can attenuate transcriptional activity of MR through its acetylation. Our data strongly suggest that CG could be used for a novel an-tihypertension drug.

P02-031

Aberrant methylation of CYP19A1 gene in

human endometrium throughout the

menstrual cycle in endometriosis patients

Afsharian2

1_{Department of Molecular and Cellular Science, Faculty of}

Advanced Sciences & Technology, Pharmaceutical Sciences Branch, Islamic Azad University, Tehran, Iran,2_{Department of}

Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran,

3_{Department of Endocrinology and Female Infertility at}

Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran

Endometriosis is defined as the presence of endometrium-like tis-sue outside of the uterus. It has been proved that endometriosis is an estrogen-dependent disease and one of the key enzymes in estrogen biosynthesis is CYP19A1. Regarding the important role of epigenetic mechanisms such as DNA methylation in many dis-eases including endometriosis, epigenetic evaluation of the CYP19A1gene was the aim of this study.

For this reason, ectopic endometriotic lesions and eutopic endometrium samples were collected through laparoscopy from 10 women with endometriosis in the proliferative phase. Also endometrial biopsies were obtained from 10 fertile women as a

control group. Ethical approval and informed patient consent was gained for the use of tissue samples. Epigenetic analysis of the PII promoter of CYP19A1 in collected tissues was assayed by Chromatin Immunoprecipitation (ChIP), using antibody against MeCP2, a protein which specifically binds to methylated DNA. Also, quantitative expression analysis of this gene was performed using the real-time PCR technique.

Data showed a harmonious pattern between mRNA expres-sion of CYP19A1 and methylation level of its promoter region (PII). Expression of CYP19A1 was significantly higher in ectopic and eutopic endometrium of patients with endometriosis versus control group. The incorporation epigenetic mark of MeCP2 induced in both ectopic and eutpoic groups in comparison with control group.

Our findings imply that higher expression of CYP19A1 as well as epigenetic alteration of this gene can contribute to the etiology and progression of endometriosis.

Keywords: CYP19A1 gene, Epigenetics, Endometriosis

P02-032

Protein arginine methyltransferase five

regulated encystation of Acanthamoeba

E.-K. Moon1, H.-H. Kong2_{, D.-I. Chung}1_{, Y.-K. Goo}1_{, Y. Hong}1 1_{Kyungpook National University School of Medicine, Daegu,}

Korea,2_{Dong-A University College of Medicine, Busan, Korea}

Acanthamoebais an opportunistic protozoan pathogen that can cause granulomatous encephalitis and keratitis. Under harsh con-ditions or chemotherapeutic drugs, Acanthamoeba transforms to a resistant cyst form, and it remains a significant problem in the treatment of Acanthamoeba infections. Recently, several encysta-tion mediating factors were studied to inhibit the encystaencysta-tion of Acanthamoeba. However, the regulation mechanisms of encysta-tion mediating factors are still unknown. We were interested in the inhibition of encystation in Acanthamoeba by the hypomethy-lating agent, azacitidine. We first found a protein arginine meth-yltransferase five from Acanthamoeba castellanii (AcPRMT5). AcPRMT5 showed increased expression levels during encystation of Acanthamoeba. EGFP fused recombinant protein of Ac-PRMT5 localized in the nucleus. Acanthamoeba transfected with siRNA against AcPRMT5 failed to form mature cysts. The infor-mation about this methyltransferase in Acanthamoeba may open the way to further study on understanding the encystation mech-anism and improving the therapeutic efficacy of amoebicidal drugs by interrupting encystation of Acanthamoeba.

P02-033

G4 structures and reparation efficiency: the

focus on DNA end processing

S. Severov1_{, A. Varizhuk}1,2_{, A. Sekridova}1_{, I. Smirnov}1_{, G.}

Pozmogova1 1

Molecular Genetics Department, SRI of Physical-Chemical Medicine, Moscow, Russian Federation,2Department of Structure-Functional Analysis of Biopolymers, Engelhardt Institute of Molecular Biology, Moscow, Russian Federation

G-quadruplexes (G4s) are noncanonical nucleic acid structures consisting of planar guanine tetrad arrangements. They have diverse functions in the human genome, which include gene expression control and the maintenance of genome stability. G4s are regarded as the hallmarks of DNA fragile sites and recombi-nation hotspots. The role of such structures in DNA reparation is a rather complex and somewhat controversial matter. We dis-cuss here the possible implications of G4 formation in the prox-imity of DNA single-strand or double-strand breaks (SSB, DSB)

for the efficiency of excision reparation and non-homologous end-joining. Regulation of both processes suggests the interplay between a number of proteins. One key DNA damage response protein is poly-(ADP-ribose) polymerase (PARP-1). In the case of low damage levels (e.g., local SSB/DSB), it promotes DNA repair by recruiting reparation proteins to the sites of damage and activating other response proteins, including the ATM kinase and p53. PARP-1 has been shown to interact with noncanonical DNA structures, in particular G4s, and induce their unwinding. We studied the impact of noncanonical structures, which are rec-ognized and unfolded by PARP-1, on the rate of DNA end phos-phorilation using model 50-mer oligonucleotides with 50-terminal G4 or hairpin motifs and control ss-oligonucleotides. We show that G4/hairpin formation inhibits polynucleotide kinase function and may hamper ligation. We also analyzed binding of several model G4s with PARP and the extent of PARP activation. Col-lectively, our findings suggest that G4-unwinding by PARP-1 near SSB/DSB sites may be essential for efficient DNA end-pro-cessing during the reparation.

Supported by RSF:14-25-00013.

P02-034

High glucose induce overall DNA

hypomethylation in Human Endothelial

Progenitor Cells

P. C. Fernandez1,2_{, M. A. Hinojosa}1_{, N. A. Schnake}1_{, S. E.}

Gutierrez1

1_{Department of Biochemistry and Molecular Biology, Universidad}

de Concepcion, Concepcion, Chile,2Medical Technology, Medical Science, Universidad San Sebastian, Concepcion, Chile

Endothelial Progenitor Cells play important functions in postna-tal vasculogenesis, specifically in the repair and angiogenesis of damaged tissue, however, both in vitro studies and studies in dia-betic patients have shown that high glucose condition alters their potential for vascular repair in damaged tissue.

In this research work, mononuclear cells from healthy donors were cultured and differentiated to Endothelial Progenitor Cells, evidenced through changes in expression of cell surface markers Oct-4, CD34+ characteristics of immature cell and CD31+ and KDR which are endothelial cell markers. Cell viability assays showed that the Endothelial Progenitor Cells are able to survive in high concentrations of D-glucose (20 mM) without affecting their immunophenotype, however, overall DNA methylation was significantly reduced in stem cells cultured in high glucose media compared to control cells. This phenomenon may be involved in the loss of angiogenic functions of Endothelial Progenitors Cells in diabetic patients. Studies using changes in gene expression could help to explain the alteration in vascular repair mecha-nisms observed in patients with permanent hyperglycemia.

P02-035

Epigenetic regulation of endothelin-1

expression by histone acetylation/

deacetylation in diabetes

S. A. Manea, I. M. Fenyo, A. Manea

Institute of Cellular Biology and Pathology “Nicolae Simionescu” of the Romanian Academy, Bucharest, Romania

Hyperglycemia-induced endothelin-1 (ET-1) synthesis plays a major role in the development of diabetic vasculopathies. The mechanisms of ET-1 regulation are not completely elucidated. Histone acetylation is an important epigenetic mechanism that regulates gene transcription. We have aimed at elucidating the role of histone acetylation in mediating ET-1 up-regulation in

diabetes. Human umbilical vein endothelial cells EAhy926 (ECs) were exposed to normal (5.5 mM) or high levels of glucose (11– 25 mM) in the absence/presence of pharmacological inhibitors of histone acetyltransferase (HAT-CPTH2) or histone deacetylase (HDAC-SAHA). Male C57BL/6J mice were rendered diabetic with streptozotocin. The animals were randomized into three groups: (i) non-diabetic, (ii) diabetic, and (iii) diabetic+SAHA, and were follow-up for 4 weeks. Real-time PCR, ELISA, and Western blot analysis were used to investigate the regulation of histone acetylation, HAT, HDAC, and ET-1. We found that high glucose induced a dose-dependent increase in H3K27ac and pro-moted a steady up-regulation of HAT1 and HDAC1 protein lev-els in ECs. Inhibition of HAT or HDAC greatly reduced the up-regulated ET-1 mRNA and protein expression levels in high glu-cose-exposed ECs. Treatment of diabetic animals with SAHA diminished significantly the aortic mRNA expression and the plasma levels of ET-1, and the mRNA of MCP-1, ICAM-1, and VCAM-1. Histone acetylation plays a role in the regulation of ET-1 in diabetes. HAT/HDAC may represent attractive pharma-cological targets to counteract the deleterious effects of ET-1 in diabetes.

Work supported by grants of the Romanian National Author-ity for Scientific Research (ID-PCE-2011-3-0548, PN-II-RU-TE-2011-3-0142). Simona A. Manea acknowledges the sup-port of POSDRU/159/1.5/S/133391.

P02-036

The investigation of genetic analysis of genes

encoding sperm nuclear proteins with the

effects on fertility in infertile men

O. S. Aydos1, B. Altinok2, T. Ozkan1, _I. Yukselen1, F. Kaplan1, Y. Hekmatshoar1, O. Sakiragaoglu1, M. Taspinar1, A. Sunguroglu1, K. Aydos3

1

Medical Biology, Ankara University School of Medicine, Ankara, Turkey,2Medical Laboratory Techniques, Ankara University Vocational School of Health Services, Ankara, Turkey,

3

Reproductive Health Research Center, Ankara University School of Medicine, Ankara, Turkey

Infertility is a progressively increasing disease under control of environmental and genetic conditions. Investigation of the factors that cause infertility may play important role on patients with this disease and stimulate the developments in in vitro fertiliza-tion techniques.Protamines are specialized proteins which only exist in sperm DNA. These proteins act as packaging of sperm DNA and help to protect it until fertilization. Over 20 single nucleotide polymorphism (SNPs) have been reported for the protamine 1 and 2. The aim of the study is to investigate the association between the changes in three polymorphic regions in PRM1, PRM2 genes and infertility. Also we analyzed the sperm DNA damage in these regions and correlated them with poly-morphisms. We made genotype analyses of the PRM1 and PRM2genes in 90 infertile and 70 fertile Turkish men; and three genetic markers (PRM1190C?A, PRM1G197T and PRM2C248T) were analyzed by PCR-RFLP analysis. In 90 patients, DNA damage was studied with the Comet assay. Obser-vations were made at magnification 4009 using an epifluorescent microscope. Each image was classified according to nucleus scale and tail length given a value of 0, 1, 2, 3 or 4 (from undamaged class 0 to maximally damaged class 4). Although no correlation was found between PRM1190C?A (p = 0.40; p > 0.05), PRM1G197T(p= 0.17; p > 0.05) and PRM2C248T (p = 0.23; p> 0.05) polymorphisms in infertile patients compared to fertile control groups, we found statistical significant association between the 190C?A and C248T SNP regions on PRM1–PRM2 genes respectively and sperm DNA fragmentation in patients

(p< 0.05). The results of the study suggested that, the protamine polymorphisms which were associated with sperm DNA fragmen-tation may be important in infertility treatment, recurrent IVF failures, recurrent pregnancy loss, healthy pregnancy and healthy development of the new born.

P02-037

High glucose-induced NADPH oxidase

expression and activity is mediated by histone

acetylation/deacetylation mechanisms in

vascular smooth muscle cells

A. Manea, I. M. Fenyo, S. A. Manea

Institute of Cellular Biology and Pathology “Nicolae Simionescu” of the Romanian Academy, Bucharest, Romania

High glucose-induced vascular smooth muscle cell (SMC) pheno-typic alterations in diabetes are considered to be partially medi-ated by oxidative stress genermedi-ated by activmedi-ated NADPH oxidase (Nox). Still, the molecular mechanisms are not completely defined. In this study we have aimed to investigate the role of histone acetyltransferase (HAT) and histone deacetylase (HDAC) in mediating Nox regulation in diabetes.

Human aortic SMCs were exposed to glucose (5.5–25 mM) in the absence/presence of selective pharmacological inhibitors of HAT (CPTH2) or HDAC (SAHA). Streptozotocin-induced dia-betic C57BL/6J mice were used: (i) non-diadia-betic, (ii) diadia-betic, (iii) diabetic+ CPTH2, and (iv) diabetic + SAHA. The animals were followed up for 4 weeks. Lucigenin-enhanced chemiluminescence, dichlorofluorescein assay, real-time PCR, and Western blot analysis were employed to investigate epigenetic changes and Nox regulation. Exposure of cultured SMCs to increasing con-centrations of glucose led to a dose-dependent up-regulation of H3K27ac, HAT1, HDAC1, and HDAC2 protein levels. Pharma-cological inhibition of either HAT or HDAC reduced signifi-cantly the up-regulated Nox activity, as well as the mRNA and protein expression levels of Nox1, Nox4, and Nox5 subtypes in high glucose-exposed SMCs. Treatment with either CPTH2 or SAHA decreased the Nox1, Nox2, and Nox4 mRNA and protein levels in the aorta of diabetic mice. These data indicate the exis-tence of a new epigenetic mechanism whereby a complex inter-play among HAT and HDAC converges to Nox up-regulation in SMCs in diabetes.

Work supported by grants of the Romanian National Author-ity for Scientific Research (ID-PCE-2011-3-0548, PN-II-RU-TE-2011-3-0142). Simona A. Manea acknowledges the sup-port of POSDRU/159/1.5/S/133391.

P02-038

Association of Contrin (YBX2) 187T

>C and

1095

+ 16A>G single nucleotide

polymorphisms with male factor infertility

1

Medical Biology, Ankara University School of Medicine, Ankara, Turkey,2Medical Laboratory Techniques, Ankara University Vocational School of Health Services, Ankara, Turkey,

3

Reproductive Health Research Center, Ankara University School of Medicine, Ankara, Turkey

Environmental factors or infections contribute to infertility to some extent, but genetic factors also play a pivotal role in etiol-ogy of male infertility. A number of such SNPs have been reported recently, some of these SNPs are associated with repro-ductive functions, such as sperm production. Y-box proteins, a

highly conserved family expressed in organisms from bacteria to humans, with nucleic acid binding activity by way of the cold-shock domain, function in regulating both transcription and translation, especially in germ cells. As a member of the Y-box proteins, the YBX2 gene is located on chromosome 17 and encodes a protein, called Contrin, with specific expression in the testis. With the essential role of YBX2 in male infertility, it was reasonable to postulate that the YBX2 gene might be associated with human idiopathic infertility. The aim of the study is to investigate the association between the changes in two polymor-phic regions in the YBX2 gene and infertility. We extracted gen-ome DNA, genotyped the polymorphisms of the YBX2 gene in 90 infertile and 70 fertile Turkish men, and two YBX2 genetic markers (187T>C and 1095 + 16A>G) were analyzed by PCR-RFLP analysis, compared the genotype frequencies between the case and control groups. We did not find statistical correlation between the YBX2 gene 187T>C (p = 0.442; p > 0.05) and 1095+ 16A>G (p = 0.51; p > 0.05) polymorphisms in infertile patients compared to fertile control groups. Further research in a large group of men is required to clarify the role of Contrin in male fertility and these data might be correlated with sperm motility, sperm count and sperm morphology.

P02-039

Βacterial mutagenicity, oxidative stress and

DNA damage caused by airborne particulate

matter PM collected from Thessaloniki

Choli-Papadopoulou1, C. Samara2

1

Laboratory of Biochemistry, Chemistry, Aristotle University of Thessaloniki, Thessaloniki, Greece,2Environmental Pollution Control Laboratory, Chemistry, Aristotle University of Thessaloniki, Thessaloniki, Greece

Τhe size segregated airborne particulate matter PM (d < 0.49, 0.49–0.95, 0.95–3, >3–7.2 lm) collected in Thessaloniki during winter and summer of 2013 was investigated to evaluate their possible genotoxic potencies. Their mutagenicity was evaluated by Ames test on Salmonella typhimurium T100 tester strain, in presence and in absence of the metabolic activation system S9. Most samples increased the number of revertant colonies, proba-bly due to its organic components. PM can cross the membranes through ion channels and/or with the aid of transporter proteins, as well as via endocytosis. After entering into cell, they can directly interact with DNA and oxidative organelles such as mitochondria, redox active proteins, which stimulate ROS pro-duction in bacterial cells. PM charged with organic compounds induces reactive oxygen species (ROS) by various chemical reac-tions, resulting in DNA strand breaks. The ROS created intracel-lularly in the bacterial strain E. coli as result of organic PM was measured based on Nitroblue tetrazolium (NBT) reduction pro-tocol and showed a dose-dependent response. DNA damage inside bacterial cells was monitored using plasmid based reporter gene assay. Blue colonies (due to the hydrolysis of X-gal by b-galactosidase enzyme) reduced significally for the bacterial cells treated with organic PM. Finally, malondialdehyde (MDA) equivalents (nM), which is an endogenous genotoxic product of enzymatic and oxygen radical-induced lipid peroxidation and a potentially important contributor to DNA damage and mutation, were measured.