MGMT expression: insights into its regulation. 1. Epigenetic factors

MGMT expression: insights into its regulation. 1. Epigenetic factors

O⁶-methylguanine-DNA methyltransferase (MGMT) is the DNA repair enzyme responsible for removing of alkylation adducts from the O6-guanine in DNA. Despite MGMT prevents mutations and cell death, this enzyme can provide resistance of cancer cells to alkylating agents of chemotherapy. The high intra- a...

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Дата:2013
Автор: Iatsyshyna, A.P.
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Опубліковано: Інститут молекулярної біології і генетики НАН України 2013
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Цитувати:MGMT expression: insights into its regulation. 1. Epigenetic factors / A.P. Iatsyshyna // Вiopolymers and Cell. — 2013. — Т. 29, №. 2. — С. 99-106. — Бібліогр.: 52 назв. — англ.

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spelling nasplib_isofts_kiev_ua-123456789-1524972025-02-09T13:30:46Z MGMT expression: insights into its regulation. 1. Epigenetic factors Експресія гена MGMT: розуміння її регуляції. 1. Епігенетичні фактори Экспрессия гена MGMT: понимание ее регуляции. 1. Эпигенетические факторы Iatsyshyna, A.P. Reviews O⁶-methylguanine-DNA methyltransferase (MGMT) is the DNA repair enzyme responsible for removing of alkylation adducts from the O6-guanine in DNA. Despite MGMT prevents mutations and cell death, this enzyme can provide resistance of cancer cells to alkylating agents of chemotherapy. The high intra- and inter-individual variations in the human MGMT expression level have been observed indicating to a complicated regulation of this gene. This review is focused on the study of epigenetic factors which could be potentially involved in regulation of the human MGMT gene expression. These include chromatin remodeling via histone modifications and DNA methylation of promoter region and gene body, as well as RNA-based mechanisms, alternative splicing, protein post- translational modifications, and other. О⁶-метилгуанін-ДНК метилтрансфераза (MGMT) – це репаративний фермент, який видаляє алкільні адукти з О6-гуаніну в ДНК. Незважаючи на те, що MGMT запобігає появі мутацій і клітинній смерті, він також забезпечує стійкість ракових клітин до алкілувальних сполук за хіміотерапії. Спостерігають значні внутрішньо- та міжіндивідуальну коливання у рівнях експресії MGMT, що вказує на складну систему регуляції даного гена. Представлений огляд присвячений вивченню епігенетичних факторів, які можуть бути потенційно залучені до регуляції експресії гена MGMT людини. До них належать ремоделювання хроматину за рахунок модифікацій гістонів і метилювання ДНК промоторної ділянки та тіла гена, а також РНК-регуляторні механізми, альтернативний сплайсинг, посттрансляційні модифікації білка тощо. О⁶-метилгуанин-ДНК метилтрансфераза (MGMT) – это фермент репарации ДНК, ответственный за удаление алкильных аддуктов из О6-гуанина в ДНК. Несмотря на то, что MGMT предохраняет от появления мутаций и клеточной гибели, этот фермент может также обеспечивать устойчивость раковых клеток к алкилирующим соединениям при химиотерапии. Обнаружены высокие внутри- и межиндивидуальные вариации в уровнях экспрессии MGMT, что указывает на сложную систему регуляции этого гена. Данный обзор посвящен изучению эпигенетических факторов, которые потенциально могут участвовать в регуляции экспрессии гена MGMT человека. Среди них ремоделирование хроматина с помощью модификации гистонов и метилирования ДНК промоторного участка и тела гена, а также РНК- регуляторные механизмы, альтернативный сплайсинг, посттрансляционные модификаии белка и др. 2013 Article MGMT expression: insights into its regulation. 1. Epigenetic factors / A.P. Iatsyshyna // Вiopolymers and Cell. — 2013. — Т. 29, №. 2. — С. 99-106. — Бібліогр.: 52 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.00080C https://nasplib.isofts.kiev.ua/handle/123456789/152497 577.21:577.218 en Вiopolymers and Cell application/pdf Інститут молекулярної біології і генетики НАН України
institution Digital Library of Periodicals of National Academy of Sciences of Ukraine
collection DSpace DC
language English
topic Reviews
Reviews
spellingShingle Reviews
Reviews
Iatsyshyna, A.P.
MGMT expression: insights into its regulation. 1. Epigenetic factors
Вiopolymers and Cell
description O⁶-methylguanine-DNA methyltransferase (MGMT) is the DNA repair enzyme responsible for removing of alkylation adducts from the O6-guanine in DNA. Despite MGMT prevents mutations and cell death, this enzyme can provide resistance of cancer cells to alkylating agents of chemotherapy. The high intra- and inter-individual variations in the human MGMT expression level have been observed indicating to a complicated regulation of this gene. This review is focused on the study of epigenetic factors which could be potentially involved in regulation of the human MGMT gene expression. These include chromatin remodeling via histone modifications and DNA methylation of promoter region and gene body, as well as RNA-based mechanisms, alternative splicing, protein post- translational modifications, and other.
format Article
author Iatsyshyna, A.P.
author_facet Iatsyshyna, A.P.
author_sort Iatsyshyna, A.P.
title MGMT expression: insights into its regulation. 1. Epigenetic factors
title_short MGMT expression: insights into its regulation. 1. Epigenetic factors
title_full MGMT expression: insights into its regulation. 1. Epigenetic factors
title_fullStr MGMT expression: insights into its regulation. 1. Epigenetic factors
title_full_unstemmed MGMT expression: insights into its regulation. 1. Epigenetic factors
title_sort mgmt expression: insights into its regulation. 1. epigenetic factors
publisher Інститут молекулярної біології і генетики НАН України
publishDate 2013
topic_facet Reviews
url https://nasplib.isofts.kiev.ua/handle/123456789/152497
citation_txt MGMT expression: insights into its regulation. 1. Epigenetic factors / A.P. Iatsyshyna // Вiopolymers and Cell. — 2013. — Т. 29, №. 2. — С. 99-106. — Бібліогр.: 52 назв. — англ.
series Вiopolymers and Cell
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fulltext UDC 577.21:577.218 MGMT expression: insights into its regulation. 1. Epigenetic factors A. P. Iatsyshyna Institute of Molecular Biology and Genetics, NAS of Ukraine 150, Akademika Zabolotnogo Str., Kyiv, Ukraine, 03680 a.p.iatsyshyna@imbg.org.ua O6-methylguanine-DNA methyltransferase (MGMT) is the DNA repair enzyme responsible for removing of alky- lation adducts from the O6-guanine in DNA. Despite MGMT prevents mutations and cell death, this enzyme can provide resistance of cancer cells to alkylating agents of chemotherapy. The high intra- and inter-individual va- riations in the human MGMT expression level have been observed indicating to a complicated regulation of this gene. This review is focused on the study of epigenetic factors which could be potentially involved in regulation of the human MGMT gene expression. These include chromatin remodeling via histone modifications and DNA me- thylation of promoter region and gene body, as well as RNA-based mechanisms, alternative splicing, protein post- translational modifications, and other. Keywords: O6-methylguanine-DNA methyltransferase (MGMT), epigenetic regulation of gene expression, me- thylation, chromatin remodeling. Introduction. O6-methylguanine-DNA methyltransfe- rase (MGMT) is the DNA repair enzyme responsible for removing alkylation adducts from the O6-position of guanine in DNA by mechanism of so-called «suicidal» reaction [1–3]. MGMT is irreversibly inactivated after binding of alkyl group to cysteine, so the synthesis of molecules of MGMT de novo is required for further DNA repair. Despite this enzyme prevents mutations and cell death, it can provide resistance of cancer cells to alkylating agents, which are frequently used in the che- motherapy of many types of cancer [3, 4]. Thus, the ex- pression of MGMT and its activity in human tumors can determine cell response to therapies with alkylating agents. However, levels of the MGMT expression are highly variable among normal tissues within an indivi- dual, among different tumors, between cells within a tis- sue or tumor, as well as between individuals within one tissue [2, 5]. The MGMT expression variation with re- versible loss has been also revealed during long-term in vitro cultivation of cells [5–9]. Observed variations of the MGMT expression level indicate a complicated regulation of this gene, but mo- lecular basis of intra- and inter-individual variations is still not fully defined. Therefore, the aim of this paper is to review epigenetic factors which could be poten- tially involved in regulation of the human MGMT ex- pression. This article is the first part of a thematic series on re- gulation of MGMT expression and is devoted to the epigenetic regulation. The next two parts are about genetic factors regulating MGMT expression. Epigenetic regulation of gene expression includes several mechanisms, in particular DNA methylation, histone packaging and modifications, RNA-based me- chanisms, post-translational modifications of protein, and other factors. To date, the Encyclopedia of DNA Elements (ENCODE) Consortium aims to build a com- prehensive data list of functional regulatory elements in the human genome which is freely available for down- load and analysis [10]. Most of discussed in this review experimental data, related to regulatory elements wi- thin human MGMT, were taken from ENCODE project 99 ISSN 0233–7657. Biopolymers and Cell. 2013. Vol. 29. N 2. P. 99–106 doi: 10.7124/bc.00080C  Institute of Molecular Biology and Genetics, NAS of Ukraine, 2013 100 at the UCSC Genome Browser (the University of Cali- fornia Santa Cruz, http://genome.ucsc.edu/), Genome Reference Consortium Human Build 37 (GRCh37, or hg19 assembly, submitted in February 2009) [10, 11]. O6-alkylguanine-DNA alkyltransferases. During lifespan cells are under the influence of different endo- genous and exogenous stress factors, which can damage the genomic DNA including the alkylation. The various sites of alkylation have been revealed, but alkylation of guanine at the O6-position, in despite of its minority, is cytotoxic, has the strongest mutagenic potential, as well as can cause tumor development [2, 12, 13]. Diffe- rent pathways of DNA repair were evolved in mamma- lian cells for self-defence from toxic and mutagenic ef- fects [14]. O6-Alkylguanine-DNA alkyltransferase (of- ficial name O6-methylguanine-DNA methyltransfera- se, MGMT) is the DNA repair enzyme responsible for removing alkylation adducts from DNA [1–3]. The al- kyltransferases are widely expanded among over 100 different species from Archaea and Bacteria to many organisms of Eukarya including humans, but not in Plan- tae, fission yeast Schizosaccharomyces pombe and bac- terium Deinococcus radiodurans [15]. At the same time MGMT is highly conservative that evidences this alkyl- transferase has a great importance for genomic integri- ty (see alignments of sequences of the alkyltransferase and alkyltransferase like proteins in reviews [5, 15]). This enzyme belongs to the direct repair pathway, it removes alkyl groups (such as methyl-, ethyl-, chlo- roethyl-group a. o.) without DNA lesion transferring them to an own cysteine residue by mechanism of the so- called «suicidal» reaction, namely MGMT is irreversib- ly inactivated after binding alkyl group to cysteine, so the synthesis of MGMT molecules de novo is required for further DNA repair. The capacity of cells to repair the O6-alkylguanine depends on the level of MGMT in cell or the rate at which a cell can synthesize this enzyme. Though MGMT prevents mutations and cell death, this enzyme can provide resistance of cancer cells to alkyla- ting agents, which are frequently used in the chemothe- rapy of many types of cancer [3, 4]. Thus, the expression of MGMT and its activity in human tumors can determine cell response to therapies with alkylating agents. Intra- and inter-individual variations of MGMT expression. The MGMT gene is ubiquitously expres- sed in mammals, but the level of its expression widely varies depending on the type of cell or tissue, cell cycle phase, developmental stage of organism, as well as on species [2, 5, 16]. For example, a high level of the inter- and intra-individual variability of the MGMT expression has been revealed in peripheral blood mononuclear cells from healthy individuals [17]. A substantial variation of the amount of MGMT activity has been also revea- led in all examined normal and tumor tissues from rats and humans, as well as individual MGMT variations in the human samples [16]. Variation of the MGMT acti- vity level in human normal and tumor tissues, as well as in human lymphocytes is reviewed in [5]. The tissue-specific variation of the human MGMT activity has been shown. For example, the highest level of expression was usually detected in the liver, follow- ed by the colon and lung, and low – in the brain and myeloid tissues [5, 16, 18], while the lowest expression was detected in hematopoietic (CD34+) progenitor cells [2]. In tumors the highest level of MGMT expression was observed in the breast, colon, lung and ovarian tu- mors, and the lowest – in the gliomas, malignant mela- nomas, pancreatic carcinomas and testis tumors [2]. The MGMT expression is widely varied in immorta- lized mammalian cells. It has been supposed that this va- riability in cultered cells reflects the MGMT expression level in the tissue from which they were obtained [5, 8]. For example, such correlation was revealed for the cell lines of lymphoblastoid cells derived from the peri- pheral blood lymphocytes [6]. Moreover, it has been shown that levels of the MGMT vary during long-term in vitro cultivation of cells [6, 9], and decrease in the MGMT expression can be reversible [7]. In particular, the lymphoblastoid cell lines are loosing the MGMT activity compared with the cells at early passages of in vitro cultivation [8]. Cell cycle dependent variation of MGMT in cultured mammalian cells is discussed in [5]. In our investigations fluctuations of MGMT pro- tein level during long-term in vitro cultivation of the cell lines derived from mouse embryonic germ cells has been also revealed [9]. Epigenetic regulation of the MGMT expression. Observed variations of the MGMT expression indicate a complicated regulation of this gene. Genetic, epige- netic and environmental factors may affect the gene ex- pression, and the human MGMT gene is no exception. IATSYSHYNA A. P. Epigenetic changes can concern chromatin structure via histone modifications and methylation of promoter region and gene body, as well as RNA interference, al- ternative splicing, and post-translational modifications of the MGMT protein. DNA methylation and chromatin remodeling. T h e MGMT p r o m o t e r c o n t a i n s t h e CpG i s l a n d (CGI). A predicted CGI is located at position chr10: 131264949-131265710 in hg19/Human [10]. The size of CGI is 762 bps. It contains 75 CpG dinucleotides and overlaps with the transcription start site (TSS) and 5'- flanking sequence of the gene (Fig. 1). The location of TSSs helps to define the promoter regions. TSS of the human MGMT gene (Fig. 1) was determined by Switch Gear Genomics by integrating experimental data using an empirically derived scoring function [10]. This TSS (CHR10_P0807_R1) has position chr10:131265479 in the plus-strand DNA of 10q26 chromosome band, whe- reas the RefSeq MGMT gene is located at chr10: 131265454-131565783 (NM_002412) [10]. CGIs are typically common near or overlapping TSSs and may be associated with promoter regions [19]. In case of the MGMT promoter TSS is located within detec- ted CGI. Approximately 70 % of promoters of vertebra- tes contain CGI, mainly they are promoters of virtually all housekeeping genes, as well as a part of tissue-speci- fic genes and developmental regulator genes [20]. Ac- cording to promoter types indicated in [19], so-called housekeeping genes with broad expression throughout organismal cycle have promoters of ubiquitous type, which are characterized by dispersed TSSs and ordered nucleosome configuration; they are TATA-depleted and have CGIs. The promoter region of human MGMT gene is known to be GC-rich and TATA-free [21]. Since pro- moters function as sites of transcription initiation by binding and correct positioning of the transcription ini- tiation complex [19], the binding of transcription factors (TFs) with cis-regulatory elements within promoter al- ters the local chromatin structure, creating open chroma- tin regions [22]. These regions can be identified by de- oxyribonuclease I (DNaseI) hypersensitive site (DHS) mapping [22]. DHS tracks in Fig. 1 display the location of active cis-regulatory elements identified as open chromatin within promoter region of the MGMT gene in different cell lines [10]. Three DNase clusters are shown from this track in Fig. 1 [10]. Thus, as shown in Fig. 1, the promoter region of hu- man MGMT gene contains TSS, which is located within CGI, DHS and exon 1 of the gene. Overlapping of CGI and DHS marks the open chromatin region and the loca- tion of active cis-regulatory elements. The ENCODE studies of different cell lines demonstrated that MGMT promoter can be targeted by several TFs in this region (Fig. 1). The track of TF ChIP-seq shows regions where TFs bind to DNA as assayed by ChIP-seq (chromatin immunoprecipitation (ChIP) assay in combination with sequencing). The track in Fig. 2, B (see inset), shows nucleo- some position by MNase-seq in the MGMT promoter region in K562 cell line [10]. DHS, which overlaps with CGI and TFs binding regions, lies in a linker DNA area and is framed by nucleosomes (Fig. 1, Fig. 2, see in- set), thus, providing accessibility of DNA to regulatory proteins in the cells expressing gene. 101 MGMT EXPRESSION: INSIGHTS INTO ITS REGULATION. 1. EPIGENETIC FACTORS A B Fig. 1. Cis-regulatory elements within the human MGMT promoter region: A – chromosome 10 ideogram; B – integrated regulation tracks from ENCODE (1 – RefSeq Gene; 2 – the location of TSS of the human MGMT gene on track from SwitchGear Genomics (CHR10_P0807_R1); 3 – DNaseI hypersensitive regions, marked as gray and dark boxes, darkness of which is proportional to the maximum signal strength observed in any cell line; the number to the left of the box shows how many cell lines are hypersensitive in the region; 4 – the track of transcription factor ChIP-seq, which shows regions where TFs bind to DNA as assayed in different cell lines; the darkness of the box is proportional to the maximum signal strength observed in any cell line; 5 – CGI, shown as dark green box) ISSN 0233-7657. Biopolymers and Cell. 2013. Vol. 29. N 2 F ig . 3. H is to ne M o di fi ca ti o ns w it hi n th e hu m an M G M T p ro m ot er r eg io n in H ep G 2 ce ll s by C hI P -s eq f ro m E N C O D E /B ro ad I ns ti tu te [ 10 ]. R ef S eq hu m an M G M T g en e is m ar ke d b y da rk b lu e co lo r. T h e lo ca ti on o f T S S o n tr ac k fr om S w it ch G ea r G en om ic s is m ar ke d by r ed . D N as eI h yp er se ns it iv e si te s (D H S s) a re m ar ke d as g ra y an d da rk b ox es , da rk n es s of w h ic h is pr op or ti on al t o th e m ax im um s ig na l st re n gt h o b se rv ed i n an y ce ll l in e. T he nu m be r to th e le ft o f th e bo x sh o w s h ow m an y ce ll li n es a re h y pe rs en si ti ve in th e re gi on . C G I is s h ow n as d ar k g re en b ox . H is to ne m od if ic at io n tr ac ks a re co lo re d in p in k. H 3K 4m e3 , H 3K 9a c, a nd H 3 K 27 ac a re a ss oc ia te d w it h tr an sc ri pt io na l in it ia ti o n an d op en c h ro m at in s tr uc tu re , an d m ar k ac ti v e or po is ed t o be a ct iv at ed p ro m ot er s. H 3 K 9 m e3 a nd H 3K 27 m e3 a re a ss oc ia te d w it h re pr es si ve h et er oc hr om at ic s ta te (m ar k si le nc ed c hr o m at in re gi on s) . F ig u re s to a rt ic le b y Ia ts ys hy n a A . P . A B K 56 2 H eL a- S 3 H ep G 2 M C F -7 H E K 29 3 H ep at o cy te H e p G 2 1 S c a le c h r1 0 : D N a s e C lu s te rs T x n F a c to r C h IP C p G I s la n d s K 5 6 2 1 M C F – 7 1 H e p a to c y te s 1 H e L a – S 3 1 K 5 6 2 S ig 8 0 S c a le c h r1 0 : C p G I s la n d s 1 3 1 ,2 6 4 ,0 0 0 1 3 1 ,3 0 0 ,0 0 0 1 3 1 ,2 6 4 ,5 0 0 1 3 1 ,4 0 0 ,0 0 0 1 3 1 ,3 5 0 ,0 0 0 1 3 1 ,4 5 0 ,0 0 0 1 3 1 ,5 0 0 ,5 0 0 1 3 1 ,5 5 0 ,5 0 0 C p G I s la n d s ( Is la n d s < 3 0 0 B a s e s a re l ig h t G re e n ) H e p G 2 M e h y l- R R B S R e p 1 f ro m E N C O D E /H u d s o n A 1 p h a H e p G 2 M e th y la ti o n 4 5 0 K B e a d A rr a y f ro m E N C O D E /H A IB R e fS e g G e n e s R e fS e g G e n e s T ra n s c ri p ti o n L e v e ls A s s a y e d b y R N A -s e q o n 9 C e ll L in e s f ro m E N C O D E T ra n s c ri p ti o n F a c to r C h IP -s e q f ro m E N C O D E D N A M e th y la ti o n b y R e d u c e d R e p re s e n ta ti o n B is u lf it e S e q f ro m E N C O D E /H u d s o n A lp h a D ig it a l D N a s e I H y p e rs e n s it iv it y C lu s te rs i n 1 2 5 c e ll t y p e s f ro m E N C O D E C p G M e th y la ti o n b y M e th y l 4 5 0 K B e a d A rr a y s f ro m E N C O D E /H A IB K 5 6 2 N u c le o s o m e S ig n a l fr o m E N C O D E /S ta n fo rd /B Y U C p G I s la n d s ( Is la n d s < 3 0 0 B a s e s a re l ig h t G re e n ) 1 3 1 ,2 6 5 ,0 0 0 1 3 1 ,2 6 5 ,5 0 0 1 3 1 ,2 6 6 ,0 0 0 1 k b h g 1 9 1 0 0 k b h g 1 9 1 3 1 ,2 5 0 ,0 0 0 1 kb hg 1 9 M G M T T S S D H S s 4 8 12 1 4 0 C G I H 3 K 4m e3 H 3K 9a c H 3K 27 ac H 3 K 9m e3 H 3K 27 m e3 5 0 5 0 5 0 5 0 5 01 1 1 1 1 F ig . 2. M et hy la ti on d at a tr ac ks o f th e hu m an M G M T g en e fr om E N C O D E p ro je ct [ 10 ]: A – m et hy la ti on d at a on t he g en e in H ep G 2 ce ll s; B – m et hy la ti on s ta tu s o f pr om o te r re gi on . M et hy la ti on t ra ck s di sp la y th e m et hy la ti on s ta tu s of s pe ci fi c C pG d in uc le ot id es i de nt if ie d by R ed uc ed R ep re se nt at io n B is ul fi te S eq ue nc in g (R R B S , m et hy la ti o n is r ep re se n te d w it h an 11 – co lo r gr ad ie nt u si ng th e fo ll ow in g co nv en ti on : r ed – 1 00 % o f m ol ec ul es s eq ue nc ed a re m et hy la te d, y el lo w – 5 0 % , g re en – 0 % ) an d by th e Il lu m in a In fi ni um H um an M et hy la ti on 4 5 0 B ea d A rr ay p la tf or m (m et hy la ti on s ta tu s is c ol or -c od ed a s: o ra ng e – m et hy la te d, p ur pl e – p ar ti al ly m et hy la te d, b ri gh t b lu e – u nm et hy la te d ) T he tr ac k o f t ra ns cr ip ti o n le v el s is s ho w n fo r H eL a, H ep G 2 an d K 56 2 ce ll li ne s as a ss ay ed b y hi gh -t hr ou gh pu t s eq ue nc in g of p ol ya de ny la te d R N A ( R N A -s eq ). E ac h of th e ce ll li ne s is a ss oc ia te d w it h a p ar ti cu la r co lo r, in p ar ti cu la r ye ll o w – H eL a- S 3, g re en – H ep G 2, b lu e – K 56 2. D N as eI h yp er se ns it iv it y cl us te rs a re m ar ke d as g re y an d da rk b ox es , d ar kn es s of w hi ch is p ro po rt io na l t o th e m ax im um s ig n al s tr en gt h ob se rv ed in a ny ce ll li ne . T he tr ac k of T F C hI P -s eq s ho w s re gi on s w he re T F s bi nd to D N A . C G I i s m ar ke d as d ar k bo x. T he tr ac k of N uc le os om e S ig na l b y M N as e- se q is s ho w n fo r K 56 2 ce ll s It is well known that DNA methylation at the CGI of promoters plays a key role in the epigenetic silencing of tumor suppressor genes. It was shown that MGMT is epigenetically silenced in various human cancer types, too. It is demonstrated on an example of more than 500 primary human tumors, cancer cell lines, and normal tis- sues that hypermethylation of the MGMT promoter, asso- ciated with the loss of expression, is frequent [23, 24]. Among examined human neoplasias, the MGMT hyper- methylation was found in about 40 % of brain tumors and colorectal carcinomas. Approximately 25 % of non- small cell lung carcinomas, lymphomas, and head and neck carcinomas also showed the MGMT hypermethy- lation, while some types of tumors had infrequent the MGMT promoter methylation (e. c. pancreatic carcino- mas, melanomas, renal carcinomas, acute leukemias, bladder carcinomas) or had not at all the MGMT methy- lation in other cancer types (e. c. carcinomas of breast, endometrium, ovary, liver, small cells of lung) [24, 25]. It has been shown that immortal cell lines with the methylated MGMT promoter are more sensitive to al- kylating agents [26], and the MGMT-deficient tumors with the methylated promoter also show an increased sensitivity to such drugs [27]. The MGMT promoter me- thylation was revealed to be associated with the tumor regression and prolonged overall and disease-free sur- vival of 40 % of patients with gliomas, which had the methylated promoter and were treated with the alkyla- ting agent carmustine (or BCNU) [27]. The MGMT pro- moter hypermethylation was shown to correlate with a statistically significant increase in survival of patients with diffuse large B-cell lymphoma after treatment with cyclophosphamide as part of multidrug regimen, too [28]. Thereby, it seems that the MGMT promoter methylation can be a useful clinical predictive marker of the responsiveness of tumors to alkylating agents and patient survival [28–30]. However, the MGMT gene si- lencing in tumors causes an accumulation of point muta- tions resulting in genomic instability and determining disease outcome [31]. For example, it has been revealed that the MGMT promoter methylation was significant- ly associated with point mutations of the K-ras gene in patients with gastric carcinoma, as well as with patient survival [32]. Methylation of cytosines in the body of the MGMT gene and in its promoter results in opposite effects. For example, methylation of promoter region is associated with the loss of MGMT expression [23, 33, 34], whilst methylation of downstream sequences in the gene body correlates with an increased expression [33, 35, 36]. It has been shown that most gene bodies are CpG-poor and extensively methylated, that is a feature of transcri- bed genes and is not associated with repression of trans- cription elongation [37]. The MGMT body does not con- tain any predicted CGI (accordingly to the general CGI criteria) and is methylated, for example, in HepG2 cell line expressing this gene (Fig. 2, A, see inset), and this observation is consistent with a feature of actively trans- cribed genes [37]. Possible functions of the gene body methylation are discussed in [37]. Also, it has been demonstrated that the methylation of CGI of MGMT is associated with the gene suppres- sion, and it is generally incomplete, localizing in a core promoter region around TSS, creating hot spots of me- thylation [23]. C h r o m a t i n s t r u c t u r e r e l a t e d t o t h e m e t h y l a t i o n o f t h e MGMT p r o m o t e r. Silencing the CGI-containing genes is correlated with increased cytosine methylation, closed chromatin struc- ture, and exclusion of TF binding in the CGI of promo- ters [37]. Many studies indicate that the methylation of CpGs represses the transcription via recruitment of his- tone deacetylase complexes by methyl-CpG binding pro- teins, and as consequence the chromatin condensation [37]. It has been reported that histone deacetylation plays a role in the MGMT silencing, too [38, 39]. The importance of histone modifications in epige- netic regulation of the human MGMT gene, such as ace- tylation and methylation of residues, due to chromatin structure changes was shown in many studies. In parti- cular, inactivation of the MGMT gene transcription was demonstrated to be associated with the loss of open chro- matin structure and exclusion of TFs from Sp1-like bin- ding sites within the CGI, but not with methylation of the TF binding region [40]. The chromatin condensa- tion after CGI methylation in promoter region and con- sequent complete blockage of the MGMT gene expres- sion were shown in other studies by using a luciferase reporter system [38], an analysis of accessibility of rest- riction enzymes to the MGMT promoter [41, 42] a. o. All of them are consistent with a model of methylation- related silencing of gene expression, which involves 102 IATSYSHYNA A. P. binding of methyl-CpG binding protein to methylated DNA, recruitment by this protein of histone deacetyla- se complexes, and as consequence histone deacetylation and chromatin condensation [38]. It has been shown by using ChIP assay that a higher level of acetylation of histones H3 and H4 bound to the promoter region was associated with the MGMT gene expression, while transcriptional inactivation of the gene was caused by formation of condensed chromatin after binding of a greater amount of methyl-CpG binding domain containing proteins (such as MeCP2, MBD1, and CAF-1) to the methylated promoter and histone de- acetylation [39]. It has been revealed that associated with open chro- matin and active transcription acetylated H3K and H4K, as well as methylated H3K4, were localized outside of the unmethylated CGI whithin minimal promoter in the MGMT-expressing cells, whilst closed chromatin was associated with methylated CGI and hypermethylated lysine 9 in histone H3 (H3K9) throughout this island [43, 44]. This observation is consistent with a model for the aberrant silencing of the human MGMT gene, in ac- cordance with which the open chromatin structure of CGI in MGMT-expressing cells consists of an approxi- mately 250-bp nucleosome-free, TF binding and nuc- lease-accessible region of DNA, and is formed by at least four flanking precisely positioned nucleosome-like struc- tures [42]. Such positioning of nucleosomes is lost and random in MGMT non-expressing cells causing the clo- sed chromatin structure [42]. Thus, in MGMT non-ex- pressing cells the closed chromatin structure is associa- ted with methylated CGI and hypermethylated H3K9 throughout this island [43]. Dimethylation of H3K9 and binding of methyl-CpG binding protein have been shown to be common and essential for MGMT silencing in cases with hypomethylated promoter region [44]. An example of open chromatin state is shown in Fig. 3 (see inset). Histone modification tracks from ENCODE project [10] display signals for markers of ac- tive promoter, such as H3K4me3, H3K9ac, and H3K27ac in MGMT-expressing HepG2 cells, whereas there is no signal for markers of silenced chromatin regions (H3K9me3 and H3K27me3). A causality between the MGMT promoter methyla- tion and its transcriptional silencing was demonstrated also in cultured cells [26, 45, 46]. It was shown that logarithmically growing normal human fibroblasts dis- played approximately 15 % of CpG dinucleotide methy- lation in CGI of the MGMT promoter, compared with approximately 50 % of CGI methylation in confluent growth-arrested cells [45]. The MGMT promoter me- thylation was shown to be not permanent and reversed at logarithmic growth of cells [45]. Progressive increa- sing of the CGI methylation of the MGMT promoter region was demonstrate with increasing of cell culture passage number, so established immortalized cell lines often completely lack of the gene expression [26, 46]. These findings probably could explain our and other researchers observations of fluctuations of MGMT le- vels at different stages of in vitro cultivation and estab- lishment of cell lines [9]. RNA i n t e r f e r e n c e is evolutionarily conserved and widespread mechanism regulation of gene expres- sion by small noncoding RNAs [47, 48]. MicroRNAs (miRNAs) have been identified as one of the most wide- spread class of endogenous small RNAs in mammalian cells [48]. Sites for many miRNA from different fami- lies are predicted in the 3'UTR of MGMT transcript NM_002412 by using TargetScanHuman 5.1 on its web- page [47], as well as in the UCSC Genome Browser (Table). To date there is no evidence about regulation of the human MGMT gene expression in vivo via RNA interference mechanism, but silencing of the MGMT protein biosynthesis via RNA interference belongs to the strategies to increase the sensitivity of cancer cells to alkylating drugs. Small double-stranded RNA mole- cules can be introduced exogenously as short interfe- ring RNAs [14]. The clinical use of such strategy is sug- gested, but it is prevented by significant problems, 103 MGMT EXPRESSION: INSIGHTS INTO ITS REGULATION. 1. EPIGENETIC FACTORS miRNA Position in 3'UTR Seed match hsa-miR-1197 96–102 7mer-m8 hsa-miR-4436a 97–103 7mer-m8 hsa-miR-3607-3p 138–144 7mer-m8 hsa-miR-4718 140–146 7mer-1A hsa-miR-4539 152–159 8mer hsa-miR-3121-3p 209–215 7mer-m8 hsa-miR-3911 362–368 7mer-m8 Conserved sites for miRNA predicted in the 3'UTR of the human MGMT transcript among which are incomplete silencing of a target gene and so-called off-target effects, non-specific immune responses, and a major challenge – in vivo delivery. A l t e r n a t i v e s p l i c i n g of genes is an im- portant mechanism of the post-transcriptional regulation of gene expression in metazoan, generating different transcripts from a single pre-mRNA [49]. The alterna- tive MGMT transcripts were predicted by Swiss Insti- tute of Bioinformatics (NC_000010_1953). The UCSC alternative splicing track is constructed by analyzing ex- perimental RNA transcripts (Fig. 4), but no one alternatively spliced isoform was transcribed from the MGMT gene [50] according to the Alternative Splicing Annotation Project [51]. Thus, the expression of alter- native MGMT transcripts must be shown in experiments of transcriptome profiling by using not only hybridiza- tion-based microarrays, but next generation sequencing based approach, RNA-seq, and so on [52]. Post-translational modifications of the MGMT pro- tein and their influence upon the enzyme fate are exten- sively reviewed and discussed in [50]. Conclusions. The DNA repair enzyme MGMT be- longs to the direct reversal repair system, i. e. removes alkyl groups from the O6-position guanine without DNA lesion by mechanism of so-called «suicidal» reaction, and the capacity of cells to repair O6-alkylguanine de- pends on the level of MGMT in cell or the rate at which a cell can synthesize it. However, MGMT not only pre- vents mutations and cell death, but the enzyme can pro- vide resistance of cancer cells to alkylating chemothera- py. The high intra- and inter-individual variations in the human MGMT expression level have been observed indicating a complicated regulation of this gene. In this study the epigenetic factors, which could be potentially involved in regulation of the MGMT expression, have been reviewed. Among them are DNA methylation of promoter region and gene body, chromatin remodeling via histone modifications, RNA interference, alternative splicing, and protein post-translational modifications. It has been shown that methylation of the MGMT pro- moter is often associated with the loss of its expression in tumor cells, whilst methylation of the gene body cor- relates with an increased expression. The data on methy- lation of the MGMT promoter and gene body in many different cell lines from ENCODE are consistent with the obtained data, providing additional indirect evidence of a control function of these epigenetic factors in the MGMT transcription. Histone modification markers of open chromatin structure have been also revealed within promoter region of different MGMT-expressing cells. This promoter region spans the CGI and DHS, contains TSS and binding sites for several TFs. Also, histone markers of closed chromatin have been revealed within methylated promoter in cells not expressing this alkyl- transferase. Whereas predicted within 3'UTR of MGMT sites for many miRNA, as well alternative transcripts have no experimental evidence and are needed to be analyzed. Acknowledgement. The study was supported by grant for Young Scientists from the National Academy of Sciences of Ukraine (0111U008220). А. П. Яци ши на Експресія гена MGMT: ро зуміння її ре гу ляції. 1. Епіге не тичні фак то ри Ре зю ме О6-ме тил гу анін-ДНК ме тил тран сфе ра за (MGMT) – це ре па ра тив- ний фер мент, який ви да ляє алкільні адук ти з О6-гуаніну в ДНК. Нез ва жа ю чи на те, що MGMT за побігає появі му тацій і клітин- 104 IATSYSHYNA A. P. A B Fig. 4. Graphs of predicted alternative splicing transcripts from Swiss Institute of Bioinformatics [10]. (NC_000010_1953, chr10:131265453- 131566301): A – plot of the alt-splicing drawn to scale; B – plot of alt-splicing drawn with exons enlarged. The graphs of predicted alternative splicing transcripts are constructed by analyzing experimental RNA transcripts. The black blocks represent exons; lines indicate introns ній смерті, він та кож за без пе чує стійкість ра ко вих клітин до алкілу валь них спо лук за хіміот е рапії. Спос теріга ють значні внут- рішньо- та міжіндивіду аль ну ко ли ван ня у рівнях експресії MGMT, що вка зує на склад ну сис те му ре гу ляції да но го гена. Пред став ле - ний огляд при свя че ний вив ченню епіге не тич них фак торів, які мо - жуть бути по тенційно за лу чені до ре гу ляції експресії гена MGMT лю ди ни. До них на ле жать ре мо де лю ван ня хро ма ти ну за раху нок мо дифікацій гістонів і ме ти лю ван ня ДНК про мо тор ної ділянки та тіла гена, а та кож РНК-ре гу ля торні ме ханізми, аль тер на тив - ний сплай синг, по сттран сляційні мо дифікації білка тощо. Клю чові сло ва: О6-ме тил гу анін-ДНК-ме тил тран сфе ра за (MGMT), епіге не тич на ре гу ляція експресії гена, ме ти лю ван ня, ре мо де лю ван ня хро ма ти ну. А. П. Яцы ши на Экспрес сия гена MGMT: по ни ма ние ее ре гу ля ции. 1. Эпи ге не ти чес кие фак то ры Ре зю ме О6-ме тил гу а нин-ДНК ме тил тран сфе ра за (MGMT) – это фер- мент ре па ра ции ДНК, от ве тствен ный за уда ле ние ал киль ных ад - дук тов из О6-гу а ни на в ДНК. Нес мот ря на то, что MGMT предо- хра ня ет от по яв ле ния му та ций и кле точ ной ги бе ли, этот фер- мент мо жет так же об ес пе чи вать устой чи вость ра ко вых кле - ток к ал ки ли ру ю щим со е ди не ни ям при хи ми о те ра пии. Обна ру же - ны вы со кие внут ри- и меж инд и ви ду аль ные ва ри а ции в уров нях экс прес сии MGMT, что ука зы ва ет на слож ную сис те му ре гу ля - ции это го гена. Дан ный об зор по свя щен из уче нию эпи ге не ти че- ских фак то ров, ко то рые по тен ци аль но мо гут учас тво вать в ре - гу ля ции экс прес сии гена MGMT че ло ве ка. Сре ди них ре мо де ли ро - ва ние хро ма ти на с по мощью мо ди фи ка ции гис то нов и ме ти лиро- ва ния ДНК про мо тор но го учас тка и тела гена, а так же РНК-ре- гуля тор ные ме ханизмы, аль тер на тив ный сплай синг, по сттранс- ляци он ные мо ди фи ка ии бел ка и др. 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