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Topic Introduction
Measuring Chromatin Structure in Budding Yeast Jon-Matthew Belton and Job Dekker1 Program in Systems Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605
Chromosome conformation capture (3C) has revolutionized the ways in which the conformation of chromatin and its relationship to other molecular functions can be studied. 3C-based techniques are used to determine the spatial arrangement of chromosomes in organisms ranging from bacteria to humans. In particular, they can be applied to the study of chromosome folding and organization in model organisms with small genomes and for which powerful genetic tools exist, such as budding yeast. Studies in yeast allow the mechanisms that establish or maintain chromatin structure to be analyzed at very high resolution with relatively low cost, and further our understanding of these fundamental processes in higher eukaryotes as well. Here we provide an overview of chromatin structure and introduce methods for performing 3C, with a focus on studies in budding yeast. Variations of the basic 3C approach (e.g., 3C-PCR, 5C, and Hi-C) can be used according to the scope and goals of a given experiment.
INTRODUCTION
Since chromosomes were first described, understanding the structure of these enormous molecules has been a fundamental topic in biology. For decades, light and electron microscopy have been instrumental in studying the relevance of chromosome structure to genome functions such as gene expression, DNA replication, and chromosome transmission. In 2002, a molecular approach called chromosome conformation capture (3C) (Dekker et al. 2002) was developed to complement these imaging-based methods. 3C relies on formaldehyde cross-linking to detect the physical association of genomic loci in three-dimensional (3D) space (Fig. 1A). Briefly, chromatin is first cross-linked with formaldehyde and then digested with a restriction enzyme. Cross-linked pairs of restriction fragments are ligated together and purified, resulting in a library of chimeric DNA molecules which represent 3D spatial interactions between the fragments. The library can be analyzed in a variety of ways, including via polymerase chain reaction (PCR) and deep sequencing. The frequency with which a pair of restriction fragments is found cross-linked and ligated indicates the likelihood that the two fragments of chromatin interact in the cell. Detailed methods for performing 3C and its related techniques are provided in the protocols that accompany this introduction (see below). 3C-based studies of chromatin conformation have revealed many aspects of chromatin behavior. It is clear that chromatin is not randomly dispersed in the eukaryotic nucleus. In higher eukaryotes (e.g., mouse and human), the genome is organized hierarchically. The highest level of organization is that of chromosome territories, which are on the order of tens to hundreds of megabases in size and are formed because the chromosomes only rarely intermingle with other chromosomes. The next level of chromatin hierarchy is compartments, which are formed by the association of large regions (one to tens of megabases) that have similar properties (active and inactive) and are spatially separated from 1
Correspondence: [email protected]
© 2015 Cold Spring Harbor Laboratory Press Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top077552
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Downloaded from http://cshprotocols.cshlp.org/ at HARVARD UNIVERSITY on July 7, 2015 - Published by Cold Spring Harbor Laboratory Press
Measuring Chromatin Structure in Budding Yeast
A
B
Cross-linking
3C PCR
Digestion
Ligation
5C
Purification
Hi-C Biotinylation
Fragmentation
Strepavidin
Enrichment
FIGURE 1. Schematic overview of the 3C, 5C, and Hi-C techniques. (A) Common steps in 3C-based techniques. First, cells are cross-linked with formaldehyde. This process covalently links protein–DNA complexes in the cell nucleus. The DNA is then digested with the restriction enzyme of choice, breaking up the cross-linked chromatin and freeing covalently linked protein–DNA complexes into solution. These complexes are diluted in a ligation reaction. The dilution favors ligation between two chromatin fragments that are attached to the same complex. After ligation the DNA is purified. (B) There are different ways to detect 3C ligation products. Locus-specific PCR primers can be designed to detect specific interactions of interest. To detect many interactions at once, 5C uses pools of hundreds to millions of short oligonucleotides which hybridize to ligation products of interest and are then ligated together. The “carbon-copies” are analyzed by high-throughput sequencing to quantify interaction frequencies. For unbiased and genome-wide analysis using Hi-C, ends of chromatin fragments are biotinylated after digestion but before ligation to mark the ligation junctions. After ligation, DNA is fragmented by sonication and the molecules that contain a biotin at the ligation junction are enriched by pulling down with streptavidin beads. The resulting short molecules are analyzed directly by high-throughput DNA sequencing.
each other in the genome (Lieberman-Aiden et al. 2009; Zhang et al. 2012). Compartments are further subdivided into topologically associating domains (TADs), which are regions (100 kb to >1 Mb) in which chromatin is found preferentially interacting with itself but not with neighboring regions (Dixon et al. 2012; Nora et al. 2012). These hierarchical levels of chromatin structure play a role in many biological functions, such as gene expression (Bau et al. 2011; Sanyal et al. 2012; Jin et al. 2013; Symmons et al. 2014) and DNA repair (Zhang et al. 2012). Defects in this organization have also been implicated in human pathology (e.g., in the premature aging disease Hutchinson–Gilford progeria syndrome [McCord et al. 2013]). CHROMATIN ORGANIZATION IN YEAST
Interestingly, budding yeast (Saccharomyces cerevisiae) chromosomes, which range from hundreds of kilobases to >1 Mb in size, have a very different chromatin topological landscape than mammalian chromosomes. Studies employing 3C-based techniques, live cell imaging, and polymer simulations have revealed global and some local features of yeast nuclear organization. For instance, very strong interactions between pairs of centromeres have been observed (Berger et al. 2008; Duan et al. 2010), initially by microscopy (Jin et al. 1998). These interactions are caused by clustering of centromeres at one pole of the nucleus adjacent to the spindle pole body (SPB). Yeast nuclei display prominent centromere clustering that is reduced in nondividing cells and in meiotic prophase (Jin et al. 1998, 2000). Centromere clustering is also a major determinant of yeast interphase nuclear organization (Jin et al. 2000). Strong interactions between pairs of telomeres located on separate chromosomes have Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top077552
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J.-M. Belton and J. Dekker
also been observed (Berger et al. 2008; Duan et al. 2010). This phenomenon is consistent with observations that telomeres are tethered to the nuclear periphery (Gotta et al. 1996). The clustering of telomeres coincides with colocalization with Rap1, Sir3, and Sir4 proteins in wild-type S. cerevisiae (Gotta et al. 1996; Trelles-Sticken et al. 2000). Meiotic telomere protein Ndj1p is required for meiosisspecific telomere distribution, bouquet formation and efficient homolog pairing (Trelles-Sticken et al. 2000). A limiting of mobility from a 3D volume to a 2D surface, or direct interactions between telomeres, may explain their colocalization with one another. The conformational biology of yeast Chromosome XII is also unique. The rDNA array is located on the right arm of Chromosome XII. Usually one or two copies of the rDNA locus is included in most assemblies, but in reality there are approximately 150 copies per cell (Kobayashi et al. 1998). This massive array of rDNA repeats forms a crescent-shaped nucleolus which is located at the pole opposite of the SPB (Berger et al. 2008). Therefore, unique constraints are placed on the two portions of the right arm of Chromosome XII. The portion of the right arm upstream of the rDNA array is tethered to both the SPB and the nucleolus, making it the only portion of a chromosome which is tethered to both poles of the nucleus. The portion of the right arm downstream from the rDNA array is the only chromosome section which is anchored at the opposite pole from the SPB and thus projects into the nucleus from a different pole than all of the other chromosome arms. It has also been observed that there is somewhat of a difference in the behavior of short arms compared to long arms of yeast chromosomes (Tjong et al. 2012). Short arms seem to occupy a volume very close to the SPB, whereas long arms stretch out in the rest of the nucleus. Polymer simulations suggest this is an effect of volume exclusion near the SPB (Tjong et al. 2012). Some aspects of yeast nuclear organization are not constitutive but rather are acquired, depending on specific cell conditions. For example, SAGA-dependent genes are confined to the nuclear periphery when activated (Cabal et al. 2006). Likewise, it has been shown that a double-stranded break at the MAT locus on Chromosome III, which initiates mating type switching, also becomes confined to the nuclear periphery (Oza et al. 2009). In addition, the hidden MAT left (HML) and hidden MAT right (HMR) loci on the ends of Chromosome III have been shown to not only be confined to the nuclear periphery but to colocalize with one another (Miele et al. 2009). The properties of chromatin at finer scales (e.g., the sub-TAD scale in mammals) are less well characterized, in part because it is costly to perform comprehensive chromatin interaction studies at the resolution of single restriction fragments in organisms with large genomes. Because the number of possible pairwise chromatin interactions scales with the square of the number of restriction fragments in a genome, organisms with small genomes present a unique opportunity to study the conformation of chromatin at high resolution in a cost-effective manner. S. cerevisiae has a genome size of 12 Mb distributed over 16 chromosomes, it is genetically tractable, and its genome can easily be manipulated. Coupled with the vast depth of knowledge about the physiology of budding yeast, these features allow for the analysis of chromatin interactions in contexts that are not readily available in higher eukaryotes, including an array of unique environmental conditions. Further, the roles of specific proteins suspected to be involved in chromatin organization can be straightforwardly studied. In addition, yeast cultures are easily synchronized to study changes in chromosome conformation during the cell cycle. 3C-BASED TECHNIQUES
3C-based techniques have revolutionized the way we study and think about the structure of chromosomes. The type of technique that should be used for a given study depends on the question and scope of the experiment. All rely on formaldehyde cross-linking of chromatin and subsequent proximity ligation of cross-linked restriction fragments to detect chromatin interactions; the only difference is the manner in which the products are detected (Fig. 1B). In 3C-PCR, locus-specific PCR primers designed to target chromatin fragments of interest are used to detect and quantify specific interactions one at a time. 3C-PCR is applicable when there are only a few (key) regions/loci of interest 616
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Measuring Chromatin Structure in Budding Yeast
for which one would like to study physical associations. Typically, experiments with less than 50 restriction fragments to be analyzed can be readily performed using 3C-PCR. Classical 3C-PCR is described in Protocol: Chromosome Conformation Capture (3C) in Budding Yeast (Belton and Dekker 2015a). High-throughput versions of 3C have also been described. 3C-on-ChIP/Circular 3C (4C) (Simonis et al. 2006; Zhao et al. 2006) employs inverse PCR to detect interactions for single loci genome-wide. 4C is suitable for studies of the genome-wide interactions of a single locus of interest, e.g., a centromere. For more comprehensive studies of target regions (e.g., whole chromosomes) that are not genome-wide, chromosome conformation capture carbon copy (5C) is appropriate; see Protocol: Chromosome Conformation Capture Carbon Copy (5C) in Budding Yeast (Belton and Dekker 2015b). 5C allows for the high-throughput detection of many chromatin interactions in a single reaction (Dostie et al. 2006). In brief, short oligonucleotide probes are first hybridized to ligation junctions of interest in a 3C library. The probes are then ligated together by Taq ligase, in effect making “carbon-copies” of the 3C ligation products. The 5C products are detected by highthroughput sequencing. The types of questions that can be addressed with 5C depend on how the probes are designed. The probes can be arranged for all restriction fragments along a chromosome; this yields comprehensive information on the spatial distribution of the entire chromosome. Such information can be used to generate 3D models of the chromosome (Bau and Marti-Renom 2011; Bau et al. 2011). The probes can also be designed so that one set hybridizes to restriction fragments containing regulatory elements and the other set hybridizes to fragments which overlap genes. This design allows for a comprehensive analysis of the spatial interactions between genes and their regulatory elements (Sanyal et al. 2012). Many other variations of these two generic probe designs are possible. 5C experiments typically use 50–5000 probes to detect hundreds to millions of chromatin interactions in parallel. Note that for both 3C and 5C, production of a random control library is essential to account for intrinsic biases in restriction fragments, probes, and primers; see Protocol: Randomized Ligation Control for Chromosome Conformation Capture (Belton and Dekker 2015c). When the goal of an experiment is to obtain information regarding the spatial organization of a complete genome, Hi-C is the technique of choice; see Protocol: Hi-C in Budding Yeast (Belton and Dekker 2015d). Hi-C (Lieberman-Aiden et al. 2009) is an unbiased whole-genome conformation capture assay which uses biotin incorporation to enrich for ligation junctions. In brief, the ends left after restriction digestion are filled in with biotinylated nucleotides before ligation. As a result, ligation junctions are labeled with biotin and can be purified using streptavidin-coated agarose beads. Purified ligation junctions obtained using Hi-C can be analyzed and quantified using high-throughput sequencing to generate a genome-wide interaction map.
REFERENCES Bau D, Marti-Renom MA. 2011. Structure determination of genomic domains by satisfaction of spatial restraints. Chromosome Res 19: 25–35. Bau D, Sanyal A, Lajoie BR, Capriotti E, Byron M, Lawrence JB, Dekker J, Marti-Renom MA. 2011. The three-dimensional folding of the α-globin gene domain reveals formation of chromatin globules. Nat Struct Mol Biol 18: 107–114. Belton J-M, Dekker J. 2015a. Chromosome conformation capture (3C) in budding yeast. Cold Spring Harb Protoc doi: 10.1101/pdb.prot085175. Belton J-M, Dekker J. 2015b. Chromosome conformation capture carbon copy (5C) in budding yeast. Cold Spring Harb Protoc doi: 10.1101/pdb .prot085191. Belton J-M, Dekker J. 2015c. Randomized ligation control for chromosome conformation capture. Cold Spring Harb Protoc doi: 10.1101/pdb .prot085183. Belton J-M, Dekker J. 2015d. Hi-C in budding yeast. Cold Spring Harb Protoc doi: 10.1101/pdb.prot085209. Berger AB, Cabal GG, Fabre E, Duong T, Buc H, Nehrbass U, Olivo-Marin J-C, Gadal O, Zimmer C. 2008. High-resolution statistical mapping reveals gene territories in live yeast. Nat Meth 5: 1031–1037.
Cabal GG, Genovesio A, Rodriguez-Navarro S, Zimmer C, Gadal O, Lesne A, Buc H, Feuerbach-Fournier F, Olivo-Marin J-C, Hurt EC, et al. 2006. SAGA interacting factors confine sub-diffusion of transcribed genes to the nuclear envelope. Nature 441: 770–773. Dekker J, Rippe K, Dekker M, Kleckner N. 2002. Capturing chromosome conformation. Science 295: 1306–1311. Dixon JR, Selvaraj S, Yue F, Kim A, Li Y, Shen Y, Hu M, Liu JS, Ren B. 2012. Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature 485: 376–380. Dostie J, Richmond TA, Arnaout RA, Selzer RR, Lee WL, Honan TA, Rubio ED, Krumm A, Lamb J, Nusbaum C, et al. 2006. Chromosome Conformation Capture Carbon Copy (5C): A massively parallel solution for mapping interactions between genomic elements. Genome Res 16: 1299–1309. Duan Z, Andronescu M, Schutz K, McIlwain S, Kim YJ, Lee C, Shendure J, Fields S, Blau CA, Noble WS. 2010. A three-dimensional model of the yeast genome. Nature 465: 363–367. Gotta M, Laroche T, Formenton A, Maillet L, Scherthan H, Gasser SM. 1996. The clustering of telomeres and colocalization with Rap1, Sir3, and Sir4
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proteins in wild-type Saccharomyces cerevisiae. J Cell Biol 134: 1349–1363. Jin Q-W, Trelles-Sticken E, Scherthan H, Loidl J. 1998. Yeast nuclei display prominent centromere clustering that is reduced in nondividing cells and in meiotic prophase. J Cell Biol 141: 21–29. Jin QW, Fuchs J, Loidl J. 2000. Centromere clustering is a major determinant of yeast interphase nuclear organization. J Cell Sci 113: 1903–1912. Jin F, Li Y, Dixon JR, Selvaraj S, Ye Z, Lee AY, Yen C-A, Schmitt AD, Espinoza CA, Ren B. 2013. A high-resolution map of the threedimensional chromatin interactome in human cells. Nature 503: 290–294. Kobayashi T, Heck DJ, Nomura M, Horiuchi T. 1998. Expansion and contraction of ribosomal DNA repeats in Saccharomyces cerevisiae: Requirement of replication fork blocking (Fob1) protein and the role of RNA polymerase I. Genes Dev 12: 3821–3830. Lieberman-Aiden E, van Berkum NL, Williams L, Imakaev M, Ragoczy T, Telling A, Amit I, Lajoie BR, Sabo PJ, Dorschner MO, et al. 2009. Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science 326: 289–293. McCord RP, Nazario-Toole A, Zhang H, Chines PS, Zhan Y, Erdos MR, Collins FS, Dekker J, Cao K. 2013. Correlated alterations in genome organization, histone methylation, and DNA–lamin A/C interactions in Hutchinson-Gilford progeria syndrome. Genome Res 23: 260–269. Miele A, Bystricky K, Dekker J. 2009. Yeast silent mating type loci form heterochromatic clusters through silencer protein-dependent longrange interactions. Plos Genet 5: e1000478. doi: 10.1371/journal .pgen.1000478. Epub 2009 May 8. Nora EP, Lajoie BR, Schulz EG, Giorgetti L, Okamoto I, Servant N, Piolot T, van Berkum NL, Meisig J, Sedat J, et al. 2012. Spatial partitioning of
618
the regulatory landscape of the X-inactivation centre. Nature 485: 381–385. Oza P, Jaspersen SL, Miele A, Dekker J, Peterson CL. 2009. Mechanisms that regulate localization of a DNA double-strand break to the nuclear periphery. Genes Dev 23: 912–927. Sanyal A, Lajoie BR, Jain G, Dekker J. 2012. The long-range interaction landscape of gene promoters. Nature 489: 109–113. Simonis M, Klous P, Splinter E, Moshkin Y, Willemsen R, de Wit E, van Steensel B, de Laat W. 2006. Nuclear organization of active and inactive chromatin domains uncovered by chromosome conformation captureon-chip (4C). Nat Genet 38: 1348–1354. Symmons O, Uslu VV, Tsujimura T, Ruf S, Nassari S, Schwarzer W, Ettwiller L, Spitz F. 2014. Functional and topological characteristics of mammalian regulatory domains. Genome Res 24: 390–400. Tjong H, Gong K, Chen L, Alber F. 2012. Physical tethering and volume exclusion determine higher-order genome organization in budding yeast. Genome Res 22: 1295–1305. Trelles-Sticken E, Dresser ME, Scherthan H. 2000. Meiotic telomere protein Ndj1p is required for meiosis-specific telomere distribution, bouquet formation and efficient homologue pairing. J Cell Biol 151: 95–106. Zhang Y, McCord Rachel P, Ho Y-J, Lajoie Bryan R, Hildebrand Dominic G, Simon Aline C, Becker Michael S, Alt Frederick W, Dekker J. 2012. Spatial organization of the mouse genome and its role in recurrent chromosomal translocations. Cell 148: 908–921. Zhao Z, Tavoosidana G, Sjolinder M, Gondor A, Mariano P, Wang S, Kanduri C, Lezcano M, Singh Sandhu K, Singh U, et al. 2006. Circular chromosome conformation capture (4C) uncovers extensive networks of epigenetically regulated intra- and interchromosomal interactions. Nat Genet 38: 1341–1347.
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Topic Introduction
Measuring Chromatin Structure in Budding Yeast Jon-Matthew Belton and Job Dekker1 Program in Systems Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605
Chromosome conformation capture (3C) has revolutionized the ways in which the conformation of chromatin and its relationship to other molecular functions can be studied. 3C-based techniques are used to determine the spatial arrangement of chromosomes in organisms ranging from bacteria to humans. In particular, they can be applied to the study of chromosome folding and organization in model organisms with small genomes and for which powerful genetic tools exist, such as budding yeast. Studies in yeast allow the mechanisms that establish or maintain chromatin structure to be analyzed at very high resolution with relatively low cost, and further our understanding of these fundamental processes in higher eukaryotes as well. Here we provide an overview of chromatin structure and introduce methods for performing 3C, with a focus on studies in budding yeast. Variations of the basic 3C approach (e.g., 3C-PCR, 5C, and Hi-C) can be used according to the scope and goals of a given experiment.
INTRODUCTION
Since chromosomes were first described, understanding the structure of these enormous molecules has been a fundamental topic in biology. For decades, light and electron microscopy have been instrumental in studying the relevance of chromosome structure to genome functions such as gene expression, DNA replication, and chromosome transmission. In 2002, a molecular approach called chromosome conformation capture (3C) (Dekker et al. 2002) was developed to complement these imaging-based methods. 3C relies on formaldehyde cross-linking to detect the physical association of genomic loci in three-dimensional (3D) space (Fig. 1A). Briefly, chromatin is first cross-linked with formaldehyde and then digested with a restriction enzyme. Cross-linked pairs of restriction fragments are ligated together and purified, resulting in a library of chimeric DNA molecules which represent 3D spatial interactions between the fragments. The library can be analyzed in a variety of ways, including via polymerase chain reaction (PCR) and deep sequencing. The frequency with which a pair of restriction fragments is found cross-linked and ligated indicates the likelihood that the two fragments of chromatin interact in the cell. Detailed methods for performing 3C and its related techniques are provided in the protocols that accompany this introduction (see below). 3C-based studies of chromatin conformation have revealed many aspects of chromatin behavior. It is clear that chromatin is not randomly dispersed in the eukaryotic nucleus. In higher eukaryotes (e.g., mouse and human), the genome is organized hierarchically. The highest level of organization is that of chromosome territories, which are on the order of tens to hundreds of megabases in size and are formed because the chromosomes only rarely intermingle with other chromosomes. The next level of chromatin hierarchy is compartments, which are formed by the association of large regions (one to tens of megabases) that have similar properties (active and inactive) and are spatially separated from 1
Correspondence: [email protected]
© 2015 Cold Spring Harbor Laboratory Press Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top077552
614
Downloaded from http://cshprotocols.cshlp.org/ at HARVARD UNIVERSITY on July 7, 2015 - Published by Cold Spring Harbor Laboratory Press
Measuring Chromatin Structure in Budding Yeast
A
B
Cross-linking
3C PCR
Digestion
Ligation
5C
Purification
Hi-C Biotinylation
Fragmentation
Strepavidin
Enrichment
FIGURE 1. Schematic overview of the 3C, 5C, and Hi-C techniques. (A) Common steps in 3C-based techniques. First, cells are cross-linked with formaldehyde. This process covalently links protein–DNA complexes in the cell nucleus. The DNA is then digested with the restriction enzyme of choice, breaking up the cross-linked chromatin and freeing covalently linked protein–DNA complexes into solution. These complexes are diluted in a ligation reaction. The dilution favors ligation between two chromatin fragments that are attached to the same complex. After ligation the DNA is purified. (B) There are different ways to detect 3C ligation products. Locus-specific PCR primers can be designed to detect specific interactions of interest. To detect many interactions at once, 5C uses pools of hundreds to millions of short oligonucleotides which hybridize to ligation products of interest and are then ligated together. The “carbon-copies” are analyzed by high-throughput sequencing to quantify interaction frequencies. For unbiased and genome-wide analysis using Hi-C, ends of chromatin fragments are biotinylated after digestion but before ligation to mark the ligation junctions. After ligation, DNA is fragmented by sonication and the molecules that contain a biotin at the ligation junction are enriched by pulling down with streptavidin beads. The resulting short molecules are analyzed directly by high-throughput DNA sequencing.
each other in the genome (Lieberman-Aiden et al. 2009; Zhang et al. 2012). Compartments are further subdivided into topologically associating domains (TADs), which are regions (100 kb to >1 Mb) in which chromatin is found preferentially interacting with itself but not with neighboring regions (Dixon et al. 2012; Nora et al. 2012). These hierarchical levels of chromatin structure play a role in many biological functions, such as gene expression (Bau et al. 2011; Sanyal et al. 2012; Jin et al. 2013; Symmons et al. 2014) and DNA repair (Zhang et al. 2012). Defects in this organization have also been implicated in human pathology (e.g., in the premature aging disease Hutchinson–Gilford progeria syndrome [McCord et al. 2013]). CHROMATIN ORGANIZATION IN YEAST
Interestingly, budding yeast (Saccharomyces cerevisiae) chromosomes, which range from hundreds of kilobases to >1 Mb in size, have a very different chromatin topological landscape than mammalian chromosomes. Studies employing 3C-based techniques, live cell imaging, and polymer simulations have revealed global and some local features of yeast nuclear organization. For instance, very strong interactions between pairs of centromeres have been observed (Berger et al. 2008; Duan et al. 2010), initially by microscopy (Jin et al. 1998). These interactions are caused by clustering of centromeres at one pole of the nucleus adjacent to the spindle pole body (SPB). Yeast nuclei display prominent centromere clustering that is reduced in nondividing cells and in meiotic prophase (Jin et al. 1998, 2000). Centromere clustering is also a major determinant of yeast interphase nuclear organization (Jin et al. 2000). Strong interactions between pairs of telomeres located on separate chromosomes have Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top077552
615
Downloaded from http://cshprotocols.cshlp.org/ at HARVARD UNIVERSITY on July 7, 2015 - Published by Cold Spring Harbor Laboratory Press
J.-M. Belton and J. Dekker
also been observed (Berger et al. 2008; Duan et al. 2010). This phenomenon is consistent with observations that telomeres are tethered to the nuclear periphery (Gotta et al. 1996). The clustering of telomeres coincides with colocalization with Rap1, Sir3, and Sir4 proteins in wild-type S. cerevisiae (Gotta et al. 1996; Trelles-Sticken et al. 2000). Meiotic telomere protein Ndj1p is required for meiosisspecific telomere distribution, bouquet formation and efficient homolog pairing (Trelles-Sticken et al. 2000). A limiting of mobility from a 3D volume to a 2D surface, or direct interactions between telomeres, may explain their colocalization with one another. The conformational biology of yeast Chromosome XII is also unique. The rDNA array is located on the right arm of Chromosome XII. Usually one or two copies of the rDNA locus is included in most assemblies, but in reality there are approximately 150 copies per cell (Kobayashi et al. 1998). This massive array of rDNA repeats forms a crescent-shaped nucleolus which is located at the pole opposite of the SPB (Berger et al. 2008). Therefore, unique constraints are placed on the two portions of the right arm of Chromosome XII. The portion of the right arm upstream of the rDNA array is tethered to both the SPB and the nucleolus, making it the only portion of a chromosome which is tethered to both poles of the nucleus. The portion of the right arm downstream from the rDNA array is the only chromosome section which is anchored at the opposite pole from the SPB and thus projects into the nucleus from a different pole than all of the other chromosome arms. It has also been observed that there is somewhat of a difference in the behavior of short arms compared to long arms of yeast chromosomes (Tjong et al. 2012). Short arms seem to occupy a volume very close to the SPB, whereas long arms stretch out in the rest of the nucleus. Polymer simulations suggest this is an effect of volume exclusion near the SPB (Tjong et al. 2012). Some aspects of yeast nuclear organization are not constitutive but rather are acquired, depending on specific cell conditions. For example, SAGA-dependent genes are confined to the nuclear periphery when activated (Cabal et al. 2006). Likewise, it has been shown that a double-stranded break at the MAT locus on Chromosome III, which initiates mating type switching, also becomes confined to the nuclear periphery (Oza et al. 2009). In addition, the hidden MAT left (HML) and hidden MAT right (HMR) loci on the ends of Chromosome III have been shown to not only be confined to the nuclear periphery but to colocalize with one another (Miele et al. 2009). The properties of chromatin at finer scales (e.g., the sub-TAD scale in mammals) are less well characterized, in part because it is costly to perform comprehensive chromatin interaction studies at the resolution of single restriction fragments in organisms with large genomes. Because the number of possible pairwise chromatin interactions scales with the square of the number of restriction fragments in a genome, organisms with small genomes present a unique opportunity to study the conformation of chromatin at high resolution in a cost-effective manner. S. cerevisiae has a genome size of 12 Mb distributed over 16 chromosomes, it is genetically tractable, and its genome can easily be manipulated. Coupled with the vast depth of knowledge about the physiology of budding yeast, these features allow for the analysis of chromatin interactions in contexts that are not readily available in higher eukaryotes, including an array of unique environmental conditions. Further, the roles of specific proteins suspected to be involved in chromatin organization can be straightforwardly studied. In addition, yeast cultures are easily synchronized to study changes in chromosome conformation during the cell cycle. 3C-BASED TECHNIQUES
3C-based techniques have revolutionized the way we study and think about the structure of chromosomes. The type of technique that should be used for a given study depends on the question and scope of the experiment. All rely on formaldehyde cross-linking of chromatin and subsequent proximity ligation of cross-linked restriction fragments to detect chromatin interactions; the only difference is the manner in which the products are detected (Fig. 1B). In 3C-PCR, locus-specific PCR primers designed to target chromatin fragments of interest are used to detect and quantify specific interactions one at a time. 3C-PCR is applicable when there are only a few (key) regions/loci of interest 616
Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top077552
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Measuring Chromatin Structure in Budding Yeast
for which one would like to study physical associations. Typically, experiments with less than 50 restriction fragments to be analyzed can be readily performed using 3C-PCR. Classical 3C-PCR is described in Protocol: Chromosome Conformation Capture (3C) in Budding Yeast (Belton and Dekker 2015a). High-throughput versions of 3C have also been described. 3C-on-ChIP/Circular 3C (4C) (Simonis et al. 2006; Zhao et al. 2006) employs inverse PCR to detect interactions for single loci genome-wide. 4C is suitable for studies of the genome-wide interactions of a single locus of interest, e.g., a centromere. For more comprehensive studies of target regions (e.g., whole chromosomes) that are not genome-wide, chromosome conformation capture carbon copy (5C) is appropriate; see Protocol: Chromosome Conformation Capture Carbon Copy (5C) in Budding Yeast (Belton and Dekker 2015b). 5C allows for the high-throughput detection of many chromatin interactions in a single reaction (Dostie et al. 2006). In brief, short oligonucleotide probes are first hybridized to ligation junctions of interest in a 3C library. The probes are then ligated together by Taq ligase, in effect making “carbon-copies” of the 3C ligation products. The 5C products are detected by highthroughput sequencing. The types of questions that can be addressed with 5C depend on how the probes are designed. The probes can be arranged for all restriction fragments along a chromosome; this yields comprehensive information on the spatial distribution of the entire chromosome. Such information can be used to generate 3D models of the chromosome (Bau and Marti-Renom 2011; Bau et al. 2011). The probes can also be designed so that one set hybridizes to restriction fragments containing regulatory elements and the other set hybridizes to fragments which overlap genes. This design allows for a comprehensive analysis of the spatial interactions between genes and their regulatory elements (Sanyal et al. 2012). Many other variations of these two generic probe designs are possible. 5C experiments typically use 50–5000 probes to detect hundreds to millions of chromatin interactions in parallel. Note that for both 3C and 5C, production of a random control library is essential to account for intrinsic biases in restriction fragments, probes, and primers; see Protocol: Randomized Ligation Control for Chromosome Conformation Capture (Belton and Dekker 2015c). When the goal of an experiment is to obtain information regarding the spatial organization of a complete genome, Hi-C is the technique of choice; see Protocol: Hi-C in Budding Yeast (Belton and Dekker 2015d). Hi-C (Lieberman-Aiden et al. 2009) is an unbiased whole-genome conformation capture assay which uses biotin incorporation to enrich for ligation junctions. In brief, the ends left after restriction digestion are filled in with biotinylated nucleotides before ligation. As a result, ligation junctions are labeled with biotin and can be purified using streptavidin-coated agarose beads. Purified ligation junctions obtained using Hi-C can be analyzed and quantified using high-throughput sequencing to generate a genome-wide interaction map.
REFERENCES Bau D, Marti-Renom MA. 2011. Structure determination of genomic domains by satisfaction of spatial restraints. Chromosome Res 19: 25–35. Bau D, Sanyal A, Lajoie BR, Capriotti E, Byron M, Lawrence JB, Dekker J, Marti-Renom MA. 2011. The three-dimensional folding of the α-globin gene domain reveals formation of chromatin globules. Nat Struct Mol Biol 18: 107–114. Belton J-M, Dekker J. 2015a. Chromosome conformation capture (3C) in budding yeast. Cold Spring Harb Protoc doi: 10.1101/pdb.prot085175. Belton J-M, Dekker J. 2015b. Chromosome conformation capture carbon copy (5C) in budding yeast. Cold Spring Harb Protoc doi: 10.1101/pdb .prot085191. Belton J-M, Dekker J. 2015c. Randomized ligation control for chromosome conformation capture. Cold Spring Harb Protoc doi: 10.1101/pdb .prot085183. Belton J-M, Dekker J. 2015d. Hi-C in budding yeast. Cold Spring Harb Protoc doi: 10.1101/pdb.prot085209. Berger AB, Cabal GG, Fabre E, Duong T, Buc H, Nehrbass U, Olivo-Marin J-C, Gadal O, Zimmer C. 2008. High-resolution statistical mapping reveals gene territories in live yeast. Nat Meth 5: 1031–1037.
Cabal GG, Genovesio A, Rodriguez-Navarro S, Zimmer C, Gadal O, Lesne A, Buc H, Feuerbach-Fournier F, Olivo-Marin J-C, Hurt EC, et al. 2006. SAGA interacting factors confine sub-diffusion of transcribed genes to the nuclear envelope. Nature 441: 770–773. Dekker J, Rippe K, Dekker M, Kleckner N. 2002. Capturing chromosome conformation. Science 295: 1306–1311. Dixon JR, Selvaraj S, Yue F, Kim A, Li Y, Shen Y, Hu M, Liu JS, Ren B. 2012. Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature 485: 376–380. Dostie J, Richmond TA, Arnaout RA, Selzer RR, Lee WL, Honan TA, Rubio ED, Krumm A, Lamb J, Nusbaum C, et al. 2006. Chromosome Conformation Capture Carbon Copy (5C): A massively parallel solution for mapping interactions between genomic elements. Genome Res 16: 1299–1309. Duan Z, Andronescu M, Schutz K, McIlwain S, Kim YJ, Lee C, Shendure J, Fields S, Blau CA, Noble WS. 2010. A three-dimensional model of the yeast genome. Nature 465: 363–367. Gotta M, Laroche T, Formenton A, Maillet L, Scherthan H, Gasser SM. 1996. The clustering of telomeres and colocalization with Rap1, Sir3, and Sir4
Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top077552
617
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J.-M. Belton and J. Dekker
proteins in wild-type Saccharomyces cerevisiae. J Cell Biol 134: 1349–1363. Jin Q-W, Trelles-Sticken E, Scherthan H, Loidl J. 1998. Yeast nuclei display prominent centromere clustering that is reduced in nondividing cells and in meiotic prophase. J Cell Biol 141: 21–29. Jin QW, Fuchs J, Loidl J. 2000. Centromere clustering is a major determinant of yeast interphase nuclear organization. J Cell Sci 113: 1903–1912. Jin F, Li Y, Dixon JR, Selvaraj S, Ye Z, Lee AY, Yen C-A, Schmitt AD, Espinoza CA, Ren B. 2013. A high-resolution map of the threedimensional chromatin interactome in human cells. Nature 503: 290–294. Kobayashi T, Heck DJ, Nomura M, Horiuchi T. 1998. Expansion and contraction of ribosomal DNA repeats in Saccharomyces cerevisiae: Requirement of replication fork blocking (Fob1) protein and the role of RNA polymerase I. Genes Dev 12: 3821–3830. Lieberman-Aiden E, van Berkum NL, Williams L, Imakaev M, Ragoczy T, Telling A, Amit I, Lajoie BR, Sabo PJ, Dorschner MO, et al. 2009. Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science 326: 289–293. McCord RP, Nazario-Toole A, Zhang H, Chines PS, Zhan Y, Erdos MR, Collins FS, Dekker J, Cao K. 2013. Correlated alterations in genome organization, histone methylation, and DNA–lamin A/C interactions in Hutchinson-Gilford progeria syndrome. Genome Res 23: 260–269. Miele A, Bystricky K, Dekker J. 2009. Yeast silent mating type loci form heterochromatic clusters through silencer protein-dependent longrange interactions. Plos Genet 5: e1000478. doi: 10.1371/journal .pgen.1000478. Epub 2009 May 8. Nora EP, Lajoie BR, Schulz EG, Giorgetti L, Okamoto I, Servant N, Piolot T, van Berkum NL, Meisig J, Sedat J, et al. 2012. Spatial partitioning of
618
the regulatory landscape of the X-inactivation centre. Nature 485: 381–385. Oza P, Jaspersen SL, Miele A, Dekker J, Peterson CL. 2009. Mechanisms that regulate localization of a DNA double-strand break to the nuclear periphery. Genes Dev 23: 912–927. Sanyal A, Lajoie BR, Jain G, Dekker J. 2012. The long-range interaction landscape of gene promoters. Nature 489: 109–113. Simonis M, Klous P, Splinter E, Moshkin Y, Willemsen R, de Wit E, van Steensel B, de Laat W. 2006. Nuclear organization of active and inactive chromatin domains uncovered by chromosome conformation captureon-chip (4C). Nat Genet 38: 1348–1354. Symmons O, Uslu VV, Tsujimura T, Ruf S, Nassari S, Schwarzer W, Ettwiller L, Spitz F. 2014. Functional and topological characteristics of mammalian regulatory domains. Genome Res 24: 390–400. Tjong H, Gong K, Chen L, Alber F. 2012. Physical tethering and volume exclusion determine higher-order genome organization in budding yeast. Genome Res 22: 1295–1305. Trelles-Sticken E, Dresser ME, Scherthan H. 2000. Meiotic telomere protein Ndj1p is required for meiosis-specific telomere distribution, bouquet formation and efficient homologue pairing. J Cell Biol 151: 95–106. Zhang Y, McCord Rachel P, Ho Y-J, Lajoie Bryan R, Hildebrand Dominic G, Simon Aline C, Becker Michael S, Alt Frederick W, Dekker J. 2012. Spatial organization of the mouse genome and its role in recurrent chromosomal translocations. Cell 148: 908–921. Zhao Z, Tavoosidana G, Sjolinder M, Gondor A, Mariano P, Wang S, Kanduri C, Lezcano M, Singh Sandhu K, Singh U, et al. 2006. Circular chromosome conformation capture (4C) uncovers extensive networks of epigenetically regulated intra- and interchromosomal interactions. Nat Genet 38: 1341–1347.
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