C-methods to study 3D organization of the eukaryotic genome

C-methods to study 3D organization of the eukaryotic genome

В последнее время становится все более очевидным, что пространственная организация эукариотического генома играет важную роль в регуляции экспрессии генов. Трехмерную (3D) организацию генома можно исследовать с помощью различных видов микроскопии, в частности, совмещенных с техникой флуоресцентной i...

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Date:2012
Main Authors: Gavrilov, A.A., Razin, S.V., Iarovaia, O.V.
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Published: Інститут молекулярної біології і генетики НАН України 2012
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Cite this:C-methods to study 3D organization of the eukaryotic genome / A.A. Gavrilov, S.V. Razin, O.V. Iarovaia // Вiopolymers and Cell. — 2012. — Т. 28, № 4. — С. 245-251. — Бібліогр.: 41 назв. — англ.

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spelling irk-123456789-1569372019-06-20T01:27:41Z C-methods to study 3D organization of the eukaryotic genome Gavrilov, A.A. Razin, S.V. Iarovaia, O.V. Reviews В последнее время становится все более очевидным, что пространственная организация эукариотического генома играет важную роль в регуляции экспрессии генов. Трехмерную (3D) организацию генома можно исследовать с помощью различных видов микроскопии, в частности, совмещенных с техникой флуоресцентной in situ гибридизации (FISH). Однако когда речь заходит об анализе пространственных взаимодействий между специфическими участками генома, намного более эффективными оказываются методы фиксации конформации хромосомы (3С). Они основаны на предпочтительном лигировании фрагментов ДНК, сшитых через белковые мостики в живых клетках посредством формальдегидной фиксации. Предполагается, что такие мостики связывают фрагменты ДНК, расположенные в непосредственной близости в ядре. В обзоре описаны существующие на сегодня методы фиксации конформации хромосомы – от 3С и ChIP-loop до Hi-C и ChiA- PET, объединенные под общим названием «С»-методы. Клюевые слова: фиксация конформации хромосомы (3С), пространственная организация генома. Останнім часом стає все очевиднішим, що просторова організація еукаріотичного геному відіграє важливу роль у регуляції експресії генів. Тривимірну (3D) організацію геному можна досліджувати за допомогою різних видів мікроскопії, зокрема, сумісних з технікою флуоресцентної in situ гібридизації (FISH). Однак коли йдеться про аналіз просторових взіємодій між специфічними ділянками геному, набагато ефективнішими виявляються методи фіксації конформації хромосоми (3С). Вони засновані на переважаючому лігуванні фрагментів ДНК, зшитих через білкові містки у живих клітинах за посередництвом формальдегідної фіксації. Передбачається, що такі містки зв’язують фрагменти ДНК, розміщені у безпосередній близькості у ядрі. В огляді описано існуючі на сьогодні методи фіксації конформації хромосоми – від 3С і ChIP-loop до Hi-C і ChiA-PET, об’єднані під загальною назвою «С»-методи. Ключові слова: фіксація конформації хромосоми (3С), просторова організація геному. It is becoming increasingly evident that spatial organization of the eukaryotic genome plays an important role in regulation of gene expression. The three-dimensional (3D) genome organization can be studied using different types of microscopy, in particular those coupled with fluorescence in situ hybridization. However, when it comes to the analysis of spatial interaction between specific genome regions, much higher performance demonstrate chromosome conformation capture (3C) methods. They are based on the proximity ligation approach which consists in preferential ligation of the ends of DNA fragments joined via protein bridges in living cells by formaldehyde fixation. It is assumed that such bridges link DNA fragments that are located in close spatial proximity in the cell nucleus. In this review we describe current 3C-based approaches, from 3C and ChiP-loop to Hi-C and ChiA-PET, going under the collective name of C-methods. Keywords: chromosome conformation capture, genome spatial organization. 2012 Article C-methods to study 3D organization of the eukaryotic genome / A.A. Gavrilov, S.V. Razin, O.V. Iarovaia // Вiopolymers and Cell. — 2012. — Т. 28, № 4. — С. 245-251. — Бібліогр.: 41 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.000056 http://dspace.nbuv.gov.ua/handle/123456789/156937 577.21 en Вiopolymers and Cell 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
Gavrilov, A.A.
Razin, S.V.
Iarovaia, O.V.
C-methods to study 3D organization of the eukaryotic genome
Вiopolymers and Cell
description В последнее время становится все более очевидным, что пространственная организация эукариотического генома играет важную роль в регуляции экспрессии генов. Трехмерную (3D) организацию генома можно исследовать с помощью различных видов микроскопии, в частности, совмещенных с техникой флуоресцентной in situ гибридизации (FISH). Однако когда речь заходит об анализе пространственных взаимодействий между специфическими участками генома, намного более эффективными оказываются методы фиксации конформации хромосомы (3С). Они основаны на предпочтительном лигировании фрагментов ДНК, сшитых через белковые мостики в живых клетках посредством формальдегидной фиксации. Предполагается, что такие мостики связывают фрагменты ДНК, расположенные в непосредственной близости в ядре. В обзоре описаны существующие на сегодня методы фиксации конформации хромосомы – от 3С и ChIP-loop до Hi-C и ChiA- PET, объединенные под общим названием «С»-методы. Клюевые слова: фиксация конформации хромосомы (3С), пространственная организация генома.
format Article
author Gavrilov, A.A.
Razin, S.V.
Iarovaia, O.V.
author_facet Gavrilov, A.A.
Razin, S.V.
Iarovaia, O.V.
author_sort Gavrilov, A.A.
title C-methods to study 3D organization of the eukaryotic genome
title_short C-methods to study 3D organization of the eukaryotic genome
title_full C-methods to study 3D organization of the eukaryotic genome
title_fullStr C-methods to study 3D organization of the eukaryotic genome
title_full_unstemmed C-methods to study 3D organization of the eukaryotic genome
title_sort c-methods to study 3d organization of the eukaryotic genome
publisher Інститут молекулярної біології і генетики НАН України
publishDate 2012
topic_facet Reviews
url http://dspace.nbuv.gov.ua/handle/123456789/156937
citation_txt C-methods to study 3D organization of the eukaryotic genome / A.A. Gavrilov, S.V. Razin, O.V. Iarovaia // Вiopolymers and Cell. — 2012. — Т. 28, № 4. — С. 245-251. — Бібліогр.: 41 назв. — англ.
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fulltext 245 REVIEWS UDC 577.21 C-methods to study 3D organization of the eukaryotic genome A. A. Gavrilov1, 3, S. V. Razin1, 2, O. V. Iarovaia1 1Institute of Gene Biology, Russian Academy of Sciences 34/5, Vavilova Str., Moscow, Russian Federation, 119334 2Department of Molecular Biology, Faculty of Biology, M. V. Lomonosov Moscow State University Leninskie Gory, Moscow, Russian Federation, 119991 3University of Oslo, Centre for Medical Studies in Russia 34/5, Vavilova Str., Moscow, Russian Federation, 119334 aleksey.gavrilov@mail.ru It is becoming increasingly evident that spatial organization of the eukaryotic genome plays an important role in regulation of gene expression. The three-dimensional (3D) genome organization can be studied using different types of microscopy, in particular those coupled with fluorescence in situ hybridization. However, when it comes to the analysis of spatial interaction between specific genome regions, much higher performance demonstrate chromosome conformation capture (3C) methods. They are based on the proximity ligation approach which con- sists in preferential ligation of the ends of DNA fragments joined via protein bridges in living cells by formalde- hyde fixation. It is assumed that such bridges link DNA fragments that are located in close spatial proximity in the cell nucleus. In this review we describe current 3C-based approaches, from 3C and ChiP-loop to Hi-C and ChiA-PET, going under the collective name of C-methods. Keywords: chromosome conformation capture, genome spatial organization. Introduction. A characteristic feature of the eukaryotic genome, in comparison with the prokaryotic, is its pa- ckaging into chromatin – a complex of DNA and various proteins placed in a special cellular compartment – cell nucleus. Genomic DNA whose total length can achieve several meters should be folded hundreds of thousands of times to fit within comparatively small volume of the nucleus. There are several levels of such DNA compac- tion which include wrapping naked DNA around his- tone octamers to form nucleosomes, organizing them into a 30-nm chromatin fiber, and its further folding in loop domains of 50–200 kb attached to the nuclear mat- rix [1–3]. Besides reduction of linear size, packaging the genome into chromatin provides for a fundamental- ly new way of regulation of DNA transcription, replica- tion, repair and recombination. It consists in the ability to change the relative positions of genomic elements in the nucleus and the degree of DNA compaction which influences accessibility of different genomic regions for regulatory factors. The problem of genome spatial organization is tight- ly linked to the problem of functional compartmentali- zation of the nucleus, in particular to the existence of ac- tive and repressive nuclear compartments. The latter in- cludes perilamellar compartment and heterochromatin regions (for example, centromeric heterochromatin). So- me nuclear compartments can be observed in a light mic- roscope (for example, nucleolus and heterochromatin) or electronic microscope (replication factories). How- ever most of the compartments can be visualized only by immunofluorescence microscopy (speckles, Cajal bo- dies, perinucleolar compartment, transcription factories [4]. Immunofluorescence methods also allow for the ana- lysis of spatial organization of replication. Many DNA-dependent processes are controlled by cis-acting DNA-regulatory elements. For example, acti- ISSN 0233–7657. Biopolymers and Cell. 2012. Vol. 28. N 4. P. 245–251 Ó Institute of Molecular Biology and Genetics, NAS of Ukraine, 2012 246 GAVRILOV A. A., RAZIN S. V., IAROVAIA O. V. ve transcriptional status of a gene frequently depends on direct interaction of its promoter with upstream enhan- cers resulting in the assembly of an active chromatin hub [5–7]. On the other hand, interaction between in- sulators would place a gene in a chromatin loop contri- buting to functional isolation of this gene from external regulatory signals [8]. In addition to enhancer and insu- lator loops, recent studies have demonstrated the exis- tence of interactions between the start and end of a gene [9, 10]. There are more distant spatial interactions of different genomic regions, for example interchromoso- mal associations between a gene and regulatory region situated on another chromosome [11] or juxtaposition of certain genes located far apart in the genome to share a common transcription factory [12] or become partners for malignant translocations [13]. Not long ago only fluorescence in situ hybridiza- tion (FISH) was a tool to study interactions of distant genomic elements in the nuclear space. Using this expe- rimental approach a researcher was able to address a ra- ther limited number of questions. Indeed, although FISH is helpful in identifying contacts between very remote genome regions, it cannot be used to probe medium and short-range interactions, which means that many regula- tory interactions (like promoter-enhancer communica- tion) could not be studied. This limitation is due to the fact that in FISH experiments any DNA sites located less than 150 kb apart in DNA sequence will produce a merged signal even if these sites do not interact with each other [14]. The problem was solved with the in- vention of chromosome conformation capture (3C) tech- nique [15]. It relies on the idea that digestion and reli- gation of cross-linked chromatin, followed by the quan- tification of ligation junctions, allows for the determi- nation of interaction frequencies of different pairs of ge- nomic elements. Since development in 2002, the tech- nique has given rise to a bunch of derivative methods now making a powerful apparatus to study the genome spatial organization and its functional output. Below we describe and compare different 3C-based methods and provide examples of biological questions addres- sed by these methods. Searching interactions within certain loci. 3C. The 3C protocol includes the following steps: Cells are treated with formaldehyde to cross-link proteins to other proteins nearby and DNA. After lysis of nuclei by SDS and solubilization of proteins that were not cross- linked, the resulting DNA-protein network is subjected to cleavage with a restriction enzyme(s), which is fol- lowed by ligation at a low DNA concentration. Under such conditions, ligations between DNA fragments cross-linked via protein bridges are strongly favored over ligations between random fragments. After liga- tion, the cross-links are reversed, and ligation products, one by one, are detected and quantified by polymerase chain reaction. Primers for PCR are designed to anneal at the ends of restriction fragments of interest facing outwards (Figure, A). The current 3C protocols suggest using real-time PCR with TaqMan probes to improve performance of the assay [16]. The cross-linking fre- quency of two specific restriction fragments, as measu- red by the amount of corresponding ligation product, is, to a first approximation, proportional to the frequency with which these two genomic sites interact. Thus, 3C analysis provides information about the spatial organi- zation of chromosomal regions in vivo [15, 17]. Developed to analyze conformation of chromoso- mes in yeasts [15], the 3C technology in short time was adopted to study spatial organization of genomic loci in higher eukaryotes. Now it is a routine method to perform studies on chromatin, transcription and gene regulation. RNA-TRAP. At about the same time that 3C was de- veloped, a very different biochemical approach was described for analyzing the spatial proximity of geno- me elements. RNA-TRAP (tagging and recovery of as- sociated proteins), as it was called, involves targeting horseradish peroxidase activity to the primary trans- cripts associated with an actively transcribed gene. This is achieved by in situ hybridization of a gene-specific intron probe labeled with digoxigenin to primary trans- cripts followed by immunodetection of the probe with digoxigenin-specific antibodies conjugated to horsera- dish peroxidase. After addition of biotin-tyramide, the localized horseradish peroxidase activates tyramid which mediates the covalent deposition of the linked biotin tag on chromatin proteins in the immediate vici- nity of the gene. The DNA fragments linked to bioti- nylated proteins are isolated by affinity chromato- graphy and analyzed using real-time PCR [18]. The technique was originally applied to an actively trans- cribed mouse b-globin gene, and, in agreement with 3C analysis of the same locus [17], a peak of biotin depo- 247 C-METHODS TO STUDY 3D ORGANIZATION OF THE EUKARYOTIC GENOME sition was observed 50 kb away at hypersensitive site 2 (HS2) of the LCR. This implied that HS2 was in close spatial proximity to the actively transcribed b-globin gene [18]. An apparent disadvantage of RNA-TRAP compa- red to 3C is that it can be applied only to transcribed sequences. Moreover, RNA-TRAP technique seems to be limited to genes such as b-globin that are transcribed at a very high level (so that there are sufficient primary transcripts at the locus for efficient biotin deposition). This partly explains why this technique has not been widely used. Also it should be noted here that RNA- TRAP is not based on the proximity ligation and so does not fall into the category of C-methods. ChIP-loop assay. ChIP-loop assay represents a com- bination of 3C and Chromatin Immunoprecipitation (ChIP). It allows one at the same time to determine which genomic sites interact in the nucleus and to sug- gest candidate proteins mediating the interaction. In this method, after formaldehyde fixation and lysis of cells, the cross-linked chromatin is purified of free pro- teins by urea gradient ultracentrifugation [19]. Purified chromatin is digested with a restriction enzyme and sub- jected to precipitation with specific antibodies follow- ing a standard ChIP protocol. The beads with precipi- tated chromatin are then resuspended in ligation buffer, and the chromatin is ligated directly on beads. Ligation products are then purified and analyzed as in usual 3C experiments (Figure, F). ChIP-loop assay allows one to identify from a panel of tested proteins the ones that may take part in DNA loop organization. For example, it was shown that Mecp2 transcriptional repressor is important for organization of silent chromatin loops [19]. On the contrary, SATB1 protein participates in formation of the high-order struc- ture of active chromatin [20]. However, it should be un- derstood that protein being crosslinked to interacting DNA fragments is not sufficient for assuming protein participation in DNA loop formation: the protein may bind DNA nearby interacting sites but do not mediate the interaction. To that end, additional experiments may be helpful, for example analyzing if a temporal knock- down of this protein synthesis affects the characteristic spatial configuration of the DNA region under study [21]. And nevertheless, in some aspects ChIP-loop assay provides a better insight than 3C and ChIP do when used apart. It concerns the situation when a positive ChIP sig- nal originates from a cell subpopulation where the geno- A schematic representations of the principles of different C-methods: A – 3C; B – 5C; C – 4C (circular chromosome conformation capture); D – 4C (chromosome conformation capture on chip); E – Hi-C; F – ChIP-loop; G – ChIA-PET; H – M3C (see the text for details). Encircled are the basic steps of the 3C procedure appearing in various forms in all C-methods («C-core»). The question marks in sections C and D indicate DNA fragments to be analyzed by deep sequencing mic locus under study has a linear configuration, whe- reas a positive 3C signal originates from another cell sub- population in which the protein does not bind correspon- ding DNA sites. M3C. In our laboratory we developed a variant of 3C allowing analysis of spatial proximity of DNA frag- ments bound to the nuclear matrix [22]. The nuclear matrix is an operationally-defined skeletal structure that underlies the nucleus [23]. Many reports indicate that multi-enzyme complexes and different DNA regu- latory elements are associated with the nuclear matrix [24, 25]. Our goal was to check, using the new approach, the possibility that the nuclear matrix constitutes a plat- form for genomic elements interaction and chromatin hub assembly. The protocol, referred to as Matrix 3C (M3C), inclu- des a high salt extraction of nuclei (which removes his- tones and unfolds DNA loops bound to the nuclear matrix), removal of distal parts of DNA loops using res- triction enzyme treatment, ligation of the nuclear mat- rix-bound DNA fragments and a subsequent analysis of ligation frequencies (Figure, H). Importantly, in cont- rast to 3C, M3C protocol does not include formaldehy- de fixation. Using the M3C procedure, we demonstrated that promoters of at least three housekeeping genes that surround the chicken a-globin gene domain were as- sembled into a complex (presumably, a transcription factory) integrated in the nuclear matrix. In erythroid cells, the regulatory elements of the a-globin genes we- re attracted to this complex. Based on these observa- tions, we proposed a model according to which mixed transcription factories that mediate the transcription of both housekeeping and tissue-specific genes are compo- sed of a permanent compartment containing integrated into the nuclear matrix promoters of housekeeping ge- nes and a «guest» compartment where promoters and regulatory elements of tissue-specific genes can be tem- porarily recruited [22]. Searching interactions throughout the genome. 4C. 4C was the first of the C-methods allowing a full ge- nome analysis of DNA-DNA interactions. It was inde- pendently developed in two variants differing in names but not in abbreviations. The first one is designated as Circular Chromosome Conformation Capture. The stra- tegy is aimed at amplification of circular DNA mole- cules originated from cross-ligation of both ends of cross-linked restriction fragments (Figure, D). Two PCR primers are designed to anneal at the opposite ends of a restriction fragment of interest, facing outwards. In such a way, all DNA fragments ligated with the frag- ment of interest at both ends are amplified. The resul- ting 4C DNA library representing the whole set of part- ners of the DNA fragment of interest is analyzed by clo- ning and sequencing [26]. The second variant of 4C that has gained more po- pularity among the researchers is designated «Chromo- some Conformation Capture on Chip». In this very simi- lar technique ligation products obtained as in standard 3C procedure are digested with a frequently cutting se- condary restriction enzyme and then ligated to form small circular DNA molecules that are amplified with primers specific to the restriction fragment of interest called «bait» or «viewpoint» (Figure, C). Originally, the resulting 4C DNA library was analyzed by the DNA micro-array (chip) technology [27]. Currently deep se- quencing is employed for this purpose. For this reason the method is more frequently referred to as 4C-seq. 4C technology has proved its potential in solving many biological questions. With the aid of this method the DNA interaction profiles of tissue-specific and house- keeping genes were analyzed [27]. 4C was also used to find out how transcription or a presence of an enhancer affect the position of a locus in the nucleus [28–30]. Using an allele-specific 4C strategy, the phenomena of dosage compensation of the mammalian X chromoso- me was studied. It was shown that the inactive X chro- mosome adopts a unique three-dimensional configura- tion that is dependent on Xist RNA [31]. 5C. 5C designates «Chromosome Conformation Capture Carbon Copy». In this protocol the 3C ligation products are mixed with a set of special primers that are designed to anneal at the very ends of all restriction frag- ments from the genomic region under study, ones facing outwards and the others – inwards, so that an end (either 5' or 3') of each primer covers exactly a half of a rest- riction site. In such a way outward and inward primers anneal tail-to-head across ligated junction of definite li- gation products and are then ligated (Figure, B). Addi- tionally these primers contain universal tails for ampli- fication. Such amplification having been done, resulting 5C DNA library is analyzed using either micro-arrays 248 GAVRILOV A. A., RAZIN S. V., IAROVAIA O. V. or deep sequencing. The original 3C library determines the spectrum and frequencies of occurrence of the final 5C products. As a result, the 5C library is a quantitative «carbon copy» of a part of the 3C library, as determined by the collection of 5C primers [32]. 5C technology was successfully applied to study the configuration of the human a-globin locus [32], the human b-globin locus [33], and the human HOX gene cluster [34]. It is worth noting that although 5C pro- vides a matrix of interaction frequencies for many pairs of sites, it cannot embrace the whole genome, only its selected regions, and therein resembles 3C. Truly who- le genome C-approaches will be described below. Hi-C. In this modified 3C procedure an extra step is introduced between restriction enzyme digestion and li- gation – filling DNA ends with nucleotides one of which is biotinylated. After blunt end ligation, DNA is purifi- ed and sheared, and ligation junctions marked by biotin are isolated by affinity chromatography on streptavidin beads followed by deep sequencing analysis (Figure, E). Thus, Hi-C data allow a matrix of ligation frequencies between all fragments in the genome to be constructed [35]. Hi-C was successfully used to analyze the general principles of the genome folding in different taxonomic groups of organisms, from yeasts [36] to mice [37] and humans [35]. ChIA-PET. ChIA-PET stands for Chromatin Inter- action Analysis by Paired-End Tag sequencing. It is a genome-wide version of ChIP-loop assay. In ChIA- PET soluble cross-linked chromatin complexes are ob- tained by sonication instead of restriction enzyme diges- tion. The complexes containing a protein of interest are separated by immunoprecipitation (as in ChIP-loop as- say). Specially designed linkers containing the MmeI recognition site at one end are then ligated to the ends of DNA fragments (Figure, G). At this first ligaion step only one end of the linker (close to the MmeI recogni- tion site) is phosphorylated. Thus, after ligation, the MmeI sites are always situated close to the junction of the linker and a DNA fragment. The linkers contain bio- tinylated nucleotide residues to facilitate subsequent purification. The ligation of linkers having been perfor- med, their free ends are phosphorylated and the proxi- mity ligation is carried out in a highly diluted solution. The so-called PET fragments (Paired-End Tags) are then released by MmeI digestion. This enzyme cuts DNA at a distance of 20 bp downstream of the recognition site. For this reason the PET fragments contain a common in- ternal part (joined linkers) and two 20 bp DNA sequen- ce tags originated from DNA fragments that have been joined by proximity ligation. After affinity purification on a streptavidin column, the PET fragments are analy- zed by deep sequencing. The resulting ChIA-PET se- quences are mapped to reference genomes to reveal rela- tionships between remote chromosomal regions brought together in close spatial proximity by protein factors [38]. ChIA-PET was employed to map the chromatin in- teraction network mediated by oestrogen receptor alpha in the human genome [38]. It was also used to reveal CTCF-mediated chromatin interactome in mouse emb- ryonic stem cells [39]. Concluding remarks and outlook. Beyond ques- tion, development of the 3C and derivative methods has brought the field of genome spatial organization to a new level. It has become evident that the 3D organi- zation of the genome can bring together distant regula- tory elements and thus plays and important role in cont- rol of the genome functions. Disclosing the general principles of the genome folding can provide insight in- to the complex relationships between chromatin structu- re, gene activity, and the functional state of the cell. Still, 3C has a number of weak points. It is recog- nized to be a rather complicated method that is based on many assumptions, and the correct interpretation of the results requires that numerous control experiments be performed [16, 40]. In addition, this method (and other C-methods based on the proximity ligation) has a num- ber of important limitations. First, it does not enable the estimation of the proportion of the cells in which two particular DNA sequences are in close proximity. Se- cond, 3C cannot directly demonstrate simultaneous in- teraction of several genomic elements. Results obtai- ned using 4C analysis suggested that active chromatin hubs that include more than two chromatin elements might exist in the cell [26]. However, it is still unclear whether hub formation occurs in a considerable propor- tion of the cells in which a particular locus is activated. Thus, the active chromatin hub model remains hypothe- tical. Finally, 3C can only determine an average inter- action pattern for a given cell population. To gain fur- 249 C-METHODS TO STUDY 3D ORGANIZATION OF THE EUKARYOTIC GENOME ther insight into the nature of chromatin long-range in- teractions, the studies should be redirected from a my- thic «average cell» [41] to individual cells and even to individual chromosomes. New approaches would be of help in studying these questions. Acknowledgements. This work was supported by the Ministry of Science and Education of the Russian Federation (contracts 16.740.11.0353, 14.740.12.1344 and 16.740.11.0483), by Russian Foundation for Support of Basic Researches (grants 11-04-00361-à, 12-04- 00036-à, 12-04-00313-à and 11-04-91334-NNIO_à), by the Presidium of the Russian Academy of Sciences (grants from the Program on Molecular and Cellular Bio- logy) and by a grant of President of the Russian Fede- ration for young scientists (MK-3813.2012.4). Î. À. Ãàâ ðè ëîâ, Ñ. Â. Ðàç³í, Î. Â. ßðî âà «Ñ»-ìå òî äè äîñë³äæåí íÿ òðè âèì³ðíî¿ îðãàí³çàö³¿ åó êàð³îò è÷ íî ãî ãå íî ìó Ðå çþ ìå Îñòàíí³ì ÷à ñîì ñòຠâñå î÷å âèäí³øèì, ùî ïðî ñòî ðî âà îðãàí³çà- ö³ÿ åó êàð³îò è÷ íî ãî ãå íî ìó â³ä³ãðຠâàæ ëè âó ðîëü ó ðå ãó ëÿö³¿ åêñ- ïðåñ³¿ ãåí³â. Òðè âèì³ðíó (3D) îðãàí³çàö³þ ãå íî ìó ìîæ íà äîñë³ä- æó âà òè çà äî ïî ìî ãîþ ð³çíèõ âèä³â ì³êðîñ êîﳿ, çîê ðå ìà, ñóì³ñíèõ ç òåõí³êîþ ôëó î ðåñ öåí òíî¿ in situ ã³áðè äè çàö³¿ (FISH). Îäíàê êî- ëè éäåòü ñÿ ïðî àíàë³ç ïðî ñòî ðî âèõ â糺ìîä³é ì³æ ñïå öèô³÷íè ìè ä³ëÿí êà ìè ãå íî ìó, íà áà ãà òî åôåê òèâí³øèìè âè ÿâ ëÿ þòü ñÿ ìå òî - äè ô³êñàö³¿ êîí ôîð ìàö³¿ õðî ìî ñî ìè (3Ñ). Âîíè çà ñíî âàí³ íà ïå ðå - âà æà þ ÷î ìó ë³ãó âàíí³ ôðàã ìåíò³â ÄÍÊ, çøè òèõ ÷å ðåç á³ëêîâ³ ì³- ñòêè ó æè âèõ êë³òèíàõ çà ïî ñå ðåä íèö òâîì ôîð ìàëü äåã³äíî¿ ô³ê- ñàö³¿. Ïåðåä áà ÷àºòüñÿ, ùî òàê³ ì³ñòêè çâ’ÿ çó þòü ôðàã ìåí òè ÄÍÊ, ðîçì³ùåí³ ó áåç ïî ñå ðåäí³é áëèçü êîñò³ ó ÿäð³.  îãëÿä³ îïè ñà íî ³ñíó þ÷³ íà ñüî ãîäí³ ìå òî äè ô³êñàö³¿ êîí ôîð ìàö³¿ õðî ìî ñî ìè – â³ä 3Ñ ³ ChIP-loop äî Hi-C ³ ChiA-PET, îá’ºäíàí³ ï³ä çà ãàëü íîþ íà çâîþ «Ñ»-ìå òî äè. Êëþ ÷îâ³ ñëî âà: ô³êñàö³ÿ êîí ôîð ìàö³¿ õðî ìî ñîìè (3Ñ), ïðî ñòî - ðî âà îðãàí³çàö³ÿ ãå íîìó. À. À. Ãàâ ðè ëîâ, Ñ. Â. Ðà çèí, Î. Â. ßðî âàÿ «Ñ»-ìå òî äû èç ó÷å íèÿ òðåõ ìåð íîé îðãà íè çà öèè ýó êà ðè î òè ÷åñ êî ãî ãå íî ìà Ðå çþ ìå  ïî ñëåä íåå âðå ìÿ ñòà íî âèò ñÿ âñå áî ëåå î÷å âèä íûì, ÷òî ïðî ñò- ðà íñòâåí íàÿ îðãà íè çà öèÿ ýó êà ðè î òè ÷åñ êî ãî ãå íî ìà èã ðà åò âàæ - íóþ ðîëü â ðå ãó ëÿ öèè ýêñ ïðåñ ñèè ãå íîâ. Òðåõ ìåð íóþ (3D) îðãà íè - çà öèþ ãå íî ìà ìîæ íî èñ ñëå äî âàòü ñ ïî ìîùüþ ðàç ëè÷ íûõ âè äîâ ìèê ðîñ êîïèè, â ÷àñ òíîñ òè, ñî âìå ùåí íûõ ñ òåõ íè êîé ôëó î ðåñ öåíò- íîé in situ ãèá ðè äè çà öèè (FISH). 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Received 10.03.12 251 C-METHODS TO STUDY 3D ORGANIZATION OF THE EUKARYOTIC GENOME

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