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DNA Topoisomerases a
a
Laura Baranello , Fedor Kouzine & David Levens
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Laboratory of Pathology; National Cancer Institute; Bethesda, MD USA Published online: 30 Sep 2013.
To cite this article: Laura Baranello, Fedor Kouzine & David Levens (2013) DNA Topoisomerases, Transcription, 4:5, 232-237, DOI: 10.4161/trns.26598 To link to this article: http://dx.doi.org/10.4161/trns.26598
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Review
Transcription 4:5, 232–237; September–December 2013; © 2013 Landes Bioscience
DNA Topoisomerases Beyond the standard role Laura Baranello, Fedor Kouzine* and David Levens
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Keywords: DNA topology, DNA Topoisomerases, DNA structure, transcriptional initiation, transcriptional elongation, chromatin
Chromatin is dynamically changing its structure to accommodate and control DNA-dependent processes inside of eukaryotic cells. These changes are necessarily linked to changes of DNA topology, which might itself serve as a regulatory signal to be detected by proteins. Thus, DNA Topoisomerases may contribute to the regulation of many events occurring during the transcription cycle. In this review we will focus on DNA Topoisomerase functions in transcription, with particular emphasis on the multiplicity of tasks beyond their widely appreciated role in solving topological problems associated with transcription elongation.
Introduction Higher organisms have evolved sophisticated mechanisms to orchestrate gene expression and modulate transcription output. The concerted and coordinated action of transcription factors, chromatin remodeling proteins, histones, and RNA polymerase (RNAP) sets the basis for different regulatory programs. However, traditional models of eukaryotic gene regulation rarely consider that DNA-protein interactions may promote dynamic changes in the structure of the double helix and that DNA stiffness constrains the assembly of multi-protein complexes. The transcription cycle consists of a defined sequence of events: chromatin opening and nucleosome mobilization, recruitment of RNAP and Pre-Initiation Complex (PIC) assembly, promoter escape and promoter-proximal pausing, pause release, elongation and termination.1 In addition to the loading of transcription factors at promoters, transcriptional regulation involves chromatin loop formation between the promoter and enhancers or other remote elements that play an important role in the gene activation.2 All these transactions involve the unwinding, bending or writhing of DNA and so are associated with changes in DNA topology that may feedback and contribute to the control of gene output.3 DNA Topoisomerases (Topos), a family of enzymes that regulate DNA topology, are fundamental to the modulation of this system.4 As these proteins are the targets of many anti-cancer
drugs,5 there is considerable interest in elucidating their complex functions. Despite long standing effort, the picture of Topos involvement in gene regulation remains incomplete. In this review, our goal is to highlight some crucial, but less studied issues concerning the roles of Topos in the process of transcription.
Topoisomerases and the TwinSupercoiled-Domain Model In general Topos are divided in two classes, type I and type II, depending on whether they cleave one or two strands of DNA, respectively.4,6,7 DNA Topoisomerase I (Topo I) relaxes supercoiled template by nicking a single strand of duplex DNA and allowing one end to rotate with respect to the other around the intact strand (Topo IB) or by passing one strand through the break (Topo IA). DNA Topoisomerase II (Topo II) cleaves both strands of a DNA duplex and passes a second intact duplex through the transient break. The type of topological problem to be solved, the architecture of the surrounding chromatin environment and the presence of accessory factors likely dictate which Topos are used for a particular purpose. Due to its stiffness, DNA undergoes several sorts of deformation in response to forces applied by genetic processes.8 Thus, overwinding or underwinding of the helix changes the twist, a parameter describing the number of times the individual strands coil around the helical axis. When the twist reaches a critical density, the molecule bends to form plectonemic structures in which the double helix coils about itself, a property known as writhe. Writhe can also be accommodated via solenoid-like wrapping of the double helix on a spool such as occurs around nucleosomes. The plectonemic or solenoidal coiling of the double helix is more commonly known as supercoiling. The “twin-supercoiled-domain model” describes the interplay between DNA topology and Topos activity and predicts that genomic transactions forcing DNA to circle around its axis generates local domains of DNA supercoiling. The best example of this prediction is presented by transcriptional elongation in which the RNAP generates positive supercoils ahead and leaves negative supercoils behind, as it moves along the DNA.9
*Correspondence to: Fedor Kouzine; Email: [email protected] Submitted: 08/19/13; Revised: 09/23/13; Accepted: 09/24/13 Citation: Baranello L, Kouzine F, Levens D. DNA Topoisomerases: Beyond the standard role. Transcription 2013; 4:230–235; PMID: 24135702; http://dx.doi.org/10.4161/trns.26598 232 Transcription Volume 4 Issue 5
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Laboratory of Pathology; National Cancer Institute; Bethesda, MD USA
Review
Figure 1. Topos’ participation in the transcription cycle. Numbers indicate Topos regulated steps. Step 1, chromatin opening: Topos cooperate with chromatin remodeling complexes to clear promoters during initial steps of transcription. Step 2, PIC assembly: Topos’ activity and Topos’ interaction with the general transcription factors (TF) favor RNAP recruitment at promoters. Step 3, promoter pausing/escape: Together with negative elongation factors (NEF) and positive elongation factors (PEF) Topos directs the temporary arrest and release of RNAP downstream the TSS. Step 4, elongation: Topos relax helical stress generated by the moving RNAP. Step 5, termination: Topos contribute to the formation of architectural domains. Gene looping between initiator and terminator or between promoter and enhancer (Step 6, chromatin looping) favors transcription regulation. Light-gray area represents Topos roles not related to the twin-domain model. Dark-gray area denotes Topos functions associated to the twin-domain model. RNAP, RNA polymerase; A, activator; ACF, ATP-utilizing chromatin assembly and remodeling factor.
The Classical View of Topoisomerases in Transcription One of the main cellular functions of Topo I is believed to be removal of helical stress generated during elongation in order to ensure processivity of transcription.4 This torque-sensitive enzyme10 is not proficient on nucleosomal template11 and works in front of the elongating RNAP where nucleosomes are displaced with the assistance of chromatin remodeling activities.12,13 In regions where the transcription rate is high, helical tension would deform DNA mostly by the twist regime,11 requiring the “twistase” Topo I to favor elongation.14 Accordingly Topo I recruitment to the Hsp70 genes in Drosophila follows chromatin remodeling; this is consistent with the idea of Topo I operating on accessible DNA.15,16 Topo II works mainly on the nucleosome-free regions near active TSSs.17 As Topo II activity depends on the juxtapositionprobability of DNA segments18 it will relax helical tension as long as DNA deformation occurs in the writhe regime.11 At promoters of highly transcribed genes the RNAP II imposes a 90° bend on the template, therefore as DNA is screwed through the active site, the upstream DNA is translationally rotated, directly generating
writhe.19 Indeed Topo II is the main “writhase” at promoters of highly expressed genes.20 In recent papers the genome mapping of transcription-generated supercoils in human cell lines has provided insight into the DNA dynamics of a transcribed locus and into Topos role at 5′ end of genes.20,21 Negative supercoiling generated during transcription is transmitted locally upstream of promoters; highly expressed genes rely upon Topo II to dissipate torsional stress around the TSS, whereas moderately expressed genes depend on Topo I.20 This indicates that instead of working randomly at promoters, Topos are able to distinguish between different types of DNA juxtapositions and collaborate with different partners that direct their action. Moreover the presence of a sturdy level of negative supercoiling within promoter areas of active genes suggests that rather than simply untwisting or unknotting DNA, these enzymes have the ability to maintain a steady-state level of torsional stress that may support the structural transitions in the double helix that insure the proper transcription.22 Thus according to the classical view Topos operate in transcription by relieving torsional stress that arises during elongation (Fig. 1, step 4). However, specific transcription events such as nucleosome mobilization, pre-initiation complex
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Review
Topo I Regulates Key Steps of Transcription Initiation Experimental evidence indicates that Topo I is a differential activator/repressor of transcription depending upon the specific utilization of the core elements at specific promoters.27 Together with the absence of sequence specificity for the enzymatic activity of Topo I, these features seem to be associated with the capacity of Topo I to interact with different proteins. Once recruited to the targeted promoter, the interplay of Topo I with its partners may modify chromatin structure and/or cooperate with the general transcription machinery. Topo I was found to regulate transcription through the maintenance of an open chromatin state at the promoters, in vivo, by the concerted action with the chromatin remodeler Hrp128 (Fig. 1, step 1). This is consistent with in vitro data showing that in a topologically constrained environment Topos activity contributes to the nucleosomes remodeling at active promoters.29 Accordingly, cells with reduced Topo I levels had increased histone H3 density at promoters that in turn is associated with downregulation of gene expression.28 Additionally, Topo I was identified biochemically as a co-activator able to enhance transcription at a pre-elongation step(s)30-32 (Fig. 1, step 2). The dramatic Topo I-dependent increase in the rate of TFIID-TFIIADNA complex formation in presence of an activator is probably explained by the ability of Topo I to bend or distort DNA upon binding. The enzyme most likely fulfills an architectural role rationalizing why a catalytically inactive mutant of Topo I is also able to promote pre-initiation complex formation and to potentiate activation.
Topo I and the Rate of Transcription Of course, Topo I’s contribution might differ from one context to the other. For example in vivo evidence highlights how promoters associated with paused RNAP II are extremely sensitive to camptothecin (CPT)20 which is a Topo I-selective inhibitor.33 In accordance with this, paused RNAP II is redistributed after
CPT treatment.34,35 Considering that promoter-pausing is one of the most highly regulated steps in transcription initiation,36 these findings indicate that Topo I specifically affects the pool of factors involved in this process (Fig. 1, step 3). The capacity of the Topo I N-terminal segment to bind the serine-phosphorylated carboxy terminal domain (CTD) of RNAP II37,38 further solidifies the idea that this enzyme acts as an interchangeable factor among different constituents of the transcription apparatus. At a single locus, the pattern of Topo I binding resembles RNAP II localization39 and experiments with Topo I inhibitors revealed the importance of Topo I in RNAP II recruitment and movement40 at specific regulatory sites in the genome. The CTD of RNAP II is modified at various stages of transcription: in the pre-initiation complex it is hypo-phosphorylated, it is phosphorylated on serine 5 (Ser5P) during initiation and then on serine 2 (Ser2P) during elongation.13 The peak of Ser5P is located in the promoter-proximal region while the peak of Ser2P is located at a second pause site in the 3′-flanking region of genes, where termination occurs.41 The mechanisms that establish the promoter pause are not fully understood and despite the identification of key regulators, emerging evidence suggests a role for DNA topology in this process.40 Indeed accumulated supercoils can arrest RNAP movement and subsequent DNA relaxation promotes the resumption of transcription.42 The interaction of Topo I with hyper-phosphorylated RNAP II forms and the sensitivity of paused genes to CPT suggest that the enzyme might be part of the mechanism for RNAP II pause-release both at promoters and at transcription termination sites (Fig. 1, step 5). The localization of Topo I is not restricted to promoters as the enzyme was also enriched at enhancers.39 DNA looping that brings distant enhancers in contact with promoters has long been appreciated as source of transcription regulation.2 Topo I was detected by ChIP assay at β-globin locus control region (LCR), one of the best-studied examples of enhancerpromoter regulation,33 and Topo I-associated cleavage sites were shown at rDNA enhancer region in Saccharomyces cerevisiae.43 Although the Topo I function at these remote regions has not been elucidated, one can hypothesize that Topo I activity might resolve topological problems that might arise during the process of looping (Fig. 1, step 6).
Topo II is a Chromatin Regulator Via its enzymatic activity Topo II has been recognized to regulate promoter output and to serve as an activator or repressor depending on the specific chromatin context. Topo II is the primary relaxase of nucleosomal DNA11 and a growing body of experimental evidence supports the idea of a close link between Topo II and nucleosome-interacting proteins. Human and Drosophila Topo II co-purify with the chromatin-remodeling factors ACF and CHRAC44,45 that promote chromatin perturbations near the binding sites of site-specific factors, facilitating transcription initiation.46 Chromosome-wide analysis showed that Topo II binds preferentially to promoter regions containing H3K4 methylation, a feature of active chromatin. This is compatible with the idea of a chromatin-based recruitment mechanism
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formation, recruitment of RNAP, activation or repression of promoters have also been linked to Topos recruitment. The behavior of DNA and Topos at promoters, where architectural restraints and dynamic torsional stress combine in a complex balance, has been less explored. At these regions, in addition to negative supercoiling accumulating as a result of elongation, the stiff double helix resists bending and twisting during PIC assembly,23-25 and constrains the deformation of DNA and chromatin that likely accompanies promoter-enhancers interactions.26 The elastic properties of DNA are likely to influence the efficient recruitment and assembly of transcription factors and RNAP and to complicate attempts to model and predict Topos function beyond their ability to control transcriptionally generated supercoiling. In the following paragraphs we touch on an expanded view of Topos participation in gene expression, broadening our appreciation about how cells could use these enzymes to regulate important steps of transcription.
for Topo II47 that might exert a double role at 5′ termini of genes favoring the targeting of chromatin modifiers to specific regulatory sites and controlling topology during the nucleosomes remodeling (Fig. 1, step 1).
Topo II in Transcription Activation Topo II preferentially associates with the highly transcribed promoters.20 This might be in part the result of the promoter architecture where bending of promoter regions synergize with the propensity of Topo II to bind bent DNA.48 Recent work accurately describes how the cleavage reaction of Topo II goes through three distinct and well-ordered reaction steps: nonspecific enzyme-DNA binding, sequence-specific DNA bending, and finally, cleavage.49 Importantly, the stabilization of a highly curved DNA geometry is critical to the Topo II catalytic cycle.48 DNA curvature at the TSS favors transcription by lowering the energy cost for DNA to interact with transcription factors and RNAP.50,51 Highly expressed promoters may have a higher bendability hence recruit Topo II more easily. Conversely, Topo II might enhance transcription activation because of its bending capability. One of the best and most interesting examples of Topo II regulated gene activation was discovered at the pS2 promoter in the human cell line MCF752 where estrogen-dependent activation requires a promoter intermediate containing a Topo II dependent double-strand break (DSB). Topo II and poly(ADPribose) polymerase (PARP-1) collaborate with factors normally associated with DNA damage response, to alter the molecular composition and nucleosome structure of the promoter. Recruitment of Topo II/PARP-1 complex as well as promoter
cleavage was shown at other promoters upon gene activation16 and evokes the startling concept that DNA cleavage supports transcription activation. Consistently, in a recent paper it was shown that upon gene activation, the transcription machinery assembles sequentially with the nucleotide excision repair (NER) factors in a XPC protein dependent fashion at promoters in absence of exogenous genotoxic attack.53 Although the authors linked this phenomenon with the ability of NER factors to enhance chromatin modifications, an alternative possibility is that this recruitment represents a first-aid kit, just in case the double-strand break resealing by Topo II at promoters fails. The normally transient Topos-DNA cleavage complex can be converted into a potential DNA lesion if prolonged misalignment of the ends occurs at their interface with the enzyme.54,55 Thus, Topos cleavage activity represents a potential source of DNA damage that can affect genomic transactions and the cell fate. Besides controlling RNAP II-dependent promoters, Topo II is also a component of the RNAP I machinery binding the RRN3 component that mediates the interaction between RNAP I and the transcription factor SL1 at the rRNA gene promoter.56 In this context, de novo PIC formation is enhanced when Topo II cleavage/re-ligation activity alleviates elastic DNA constraints thus promoting PIC assembly and stability (Fig. 1, step 2). Interestingly, the enzyme is able to exert tissue specific regulation in the nervous systems. Topo II binding to promoters of developmentally regulated genes in post-mitotic neurons governs the transcriptional program associated with neuronal differentiation and longevity, supporting activation or repression of tissue specific genes but not of the housekeeper ones.57 How the tissue specific regulation is achieved is still poorly understood but it might involve interactions with characteristic DNA structures
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Figure 2. The rigidity of DNA represents a barrier for protein-DNA-protein interactions. (A) To overcome this resistance, Topos introduce DNA breaks favoring conformational changes that bring in contact distal transcriptional regulators. (B) In the absence of cleavage activity Topo I bridges proteinprotein interactions that help to regulate transcription.
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Gene Architecture and Topo II In budding yeast, Topo II sites are detected not only at promoters but also at transcription termination regions58 (Fig. 1, step 5). Considering Topo I as the main relaxase in the body of transcribed genes, the 3` localization of Topo II might reflect a different role. As Topo II has been implicated in DNA looping59 one possibility is that the enzyme contributes to the formation of architectural domains containing gene units. Gene looping between initiation and terminator regions has been proposed to facilitate RNAP II recycling and to increase transcription rates.60,61 Topo II has a significant affinity for the base of a DNA loop structure where two duplexes juxtapose and this feature makes the enzyme an attractive candidate for looping regulation. Likewise Topo II was discovered near the tissue-specific enhancer62 of the kappa immunoglobulin gene, associated with nuclear matrix associated regions (MARs), and additional binding sites were related to other enhancers in the heavy-chain (IgH) locus (Fig. 1, step 6). References 1.
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Conclusions Although DNA Topoisomerases were first discovered more than 40 years ago, the study of these enzymes remains highly vigorous. In higher Eukaryotes the emerging evidence presented in this review suggests that Topoisomerases have evolved to participate at multiple steps in transcription. The ability to perform so many functions through simple binding, cleavage and ligation reactions dictates that rather than acting as mere relaxases of torsionally stressed DNA, Topos may modulate DNA stiffness to facilitate multi-protein complexes assembly (Fig. 2A). The double helix is a rigid polymer and the cell needs strategies and tools that promote contacts between factors at distant sites. The ability to introduce breaks in the DNA is a tool for Topos to weaken its stiffness favoring protein-DNA-protein interactions. Even in the absence of catalysis Topo I may help to assemble such complexes via protein-protein interactions (Fig. 2B). Because the most DNA Topoisomerase sensitive genes are the ones characterized by highly regulated transcription initiation and transcriptional plasticity63 it is reasonable to conclude that Topo I and Topo II can act as transcriptional factors involved in key steps. Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
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or specialized proteins. For example, a similar temporal-spatial expression pattern of Topo II and histone deacetylase 2 in developing neurons was observed and may contribute to Topo II targeting at tissue specific promoters.
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53. Le May N, Mota-Fernandes D, Vélez-Cruz R, Iltis I, Biard D, Egly JM. NER factors are recruited to active promoters and facilitate chromatin modification for transcription in the absence of exogenous genotoxic attack. Mol Cell 2010; 38:54-66; PMID:20385089; http://dx.doi.org/10.1016/j.molcel.2010.03.004 54. Pourquier P, Pommier Y. Topoisomerase I-mediated DNA damage. Adv Cancer Res 2001; 80:189216; PMID:11034544; http://dx.doi.org/10.1016/ S0065-230X(01)80016-6 55. Pommier Y, Barcelo JM, Rao VA, Sordet O, Jobson AG, Thibaut L, Miao ZH, Seiler JA, Zhang H, Marchand C, et al. Repair of topoisomerase I-mediated DNA damage. Prog Nucleic Acid Res Mol Biol 2006; 81:179-229; PMID:16891172; http:// dx.doi.org/10.1016/S0079-6603(06)81005-6 56. Ray S, Panova T, Miller G, Volkov A, Porter AC, Russell J, Panov KI, Zomerdijk JC. Topoisomerase IIα promotes activation of RNA polymerase I transcription by facilitating pre-initiation complex formation. Nat Commun 2013; 4:1598; PMID:23511463; http://dx.doi.org/10.1038/ ncomms2599 57. Lyu YL, Lin CP, Azarova AM, Cai L, Wang JC, Liu LF. Role of topoisomerase IIbeta in the expression of developmentally regulated genes. Mol Cell Biol 2006; 26:7929-41; PMID:16923961; http://dx.doi. org/10.1128/MCB.00617-06 58. Bermejo R, Capra T, Gonzalez-Huici V, Fachinetti D, Cocito A, Natoli G, Katou Y, Mori H, Kurokawa K, Shirahige K, et al. Genome-organizing factors Top2 and Hmo1 prevent chromosome fragility at sites of S phase transcription. Cell 2009; 138:87084; PMID:19737516; http://dx.doi.org/10.1016/j. cell.2009.06.022 59. Li TK, Chen AY, Yu C, Mao Y, Wang H, Liu LF. Activation of topoisomerase II-mediated excision of chromosomal DNA loops during oxidative stress. Genes Dev 1999; 13:1553-60; PMID:10385624; http://dx.doi.org/10.1101/gad.13.12.1553 60. Ansari A, Hampsey M. A role for the CPF 3′-end processing machinery in RNAP II-dependent gene looping. Genes Dev 2005; 19:2969-78; PMID:16319194; http://dx.doi.org/10.1101/gad.1362305 61. O’Sullivan JM, Tan-Wong SM, Morillon A, Lee B, Coles J, Mellor J, Proudfoot NJ. Gene loops juxtapose promoters and terminators in yeast. Nat Genet 2004; 36:1014-8; PMID:15314641; http://dx.doi. org/10.1038/ng1411 62. Cockerill PN, Garrard WT. Chromosomal loop anchorage of the kappa immunoglobulin gene occurs next to the enhancer in a region containing topoisomerase II sites. Cell 1986; 44:273-82; PMID:3002631; http://dx.doi. org/10.1016/0092-8674(86)90761-0 63. Pedersen JM, Fredsoe J, Roedgaard M, Andreasen L, Mundbjerg K, Kruhøffer M, Brinch M, Schierup MH, Bjergbaek L, Andersen AH. DNA Topoisomerases maintain promoters in a state competent for transcriptional activation in Saccharomyces cerevisiae. PLoS Genet 2012; 8:e1003128; PMID:23284296; http://dx.doi.org/10.1371/journal.pgen.1003128
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27. Xu M, Sharma P, Pan S, Malik S, Roeder RG, Martinez E. Core promoter-selective function of HMGA1 and Mediator in Initiator-dependent transcription. Genes Dev 2011; 25:2513-24; PMID:22156211; http:// dx.doi.org/10.1101/gad.177360.111 28. Durand-Dubief M, Persson J, Norman U, Hartsuiker E, Ekwall K. Topoisomerase I regulates open chromatin and controls gene expression in vivo. EMBO J 2010; 29:2126-34; PMID:20526281; http://dx.doi.org/10.1038/emboj.2010.109 29. Gavin I, Horn PJ, Peterson CL. SWI/SNF chromatin remodeling requires changes in DNA topology. Mol Cell 2001; 7:97-104; PMID:11172715; http://dx.doi. org/10.1016/S1097-2765(01)00158-7 30. Merino A, Madden KR, Lane WS, Champoux JJ, Reinberg D. DNA topoisomerase I is involved in both repression and activation of transcription. Nature 1993; 365:227-32; PMID:8396729; http://dx.doi. org/10.1038/365227a0 31. Shykind BM, Kim J, Stewart L, Champoux JJ, Sharp PA. Topoisomerase I enhances TFIID-TFIIA complex assembly during activation of transcription. Genes Dev 1997; 11:397-407; PMID:9030691; http://dx.doi.org/10.1101/gad.11.3.397 32. Kretzschmar M, Meisterernst M, Roeder RG. Identification of human DNA topoisomerase I as a cofactor for activator-dependent transcription by RNA polymerase II. Proc Natl Acad Sci U S A 1993; 90:11508-12; PMID:8265582; http://dx.doi. org/10.1073/pnas.90.24.11508 33. Pommier Y. Topoisomerase I inhibitors: camptothecins and beyond. Nat Rev Cancer 2006; 6:789-802; PMID:16990856; http://dx.doi. org/10.1038/nrc1977 34. Khobta A, Ferri F, Lotito L, Montecucco A, Rossi R, Capranico G. Early effects of topoisomerase I inhibition on RNA polymerase II along transcribed genes in human cells. J Mol Biol 2006; 357:12738; PMID:16427078; http://dx.doi.org/10.1016/j. jmb.2005.12.069 35. Baranello L, Bertozzi D, Fogli MV, Pommier Y, Capranico G. DNA topoisomerase I inhibition by camptothecin induces escape of RNA polymerase II from promoter-proximal pause site, antisense transcription and histone acetylation at the human HIF-1alpha gene locus. Nucleic Acids Res 2010; 38:159-71; PMID:19854946; http://dx.doi. org/10.1093/nar/gkp817 36. Adelman K, Lis JT. Promoter-proximal pausing of RNA polymerase II: emerging roles in metazoans. Nat Rev Genet 2012; 13:720-31; PMID:22986266; http://dx.doi.org/10.1038/nrg3293 37. Wu J, Phatnani HP, Hsieh TS, Greenleaf AL. The phosphoCTD-interacting domain of Topoisomerase I. Biochem Biophys Res Commun 2010; 397:1179; PMID:20493173; http://dx.doi.org/10.1016/j. bbrc.2010.05.081 38. Carty SM, Greenleaf AL. Hyperphosphorylated C-terminal repeat domain-associating proteins in the nuclear proteome link transcription to DNA/ chromatin modification and RNA processing. Mol Cell Proteomics 2002; 1:598-610; PMID:12376575; http://dx.doi.org/10.1074/mcp.M200029-MCP200 39. Rosenberg M, Fan AX, Lin IJ, Liang SY, Bungert J. Cell-cycle specific association of transcription factors and RNA polymerase ii with the human β-globin gene locus. J Cell Biochem 2013; 114:19972006; PMID:23519692; http://dx.doi.org/10.1002/ jcb.24542
Transcription Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ktrn20
DNA Topoisomerases a
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Laura Baranello , Fedor Kouzine & David Levens
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Laboratory of Pathology; National Cancer Institute; Bethesda, MD USA Published online: 30 Sep 2013.
To cite this article: Laura Baranello, Fedor Kouzine & David Levens (2013) DNA Topoisomerases, Transcription, 4:5, 232-237, DOI: 10.4161/trns.26598 To link to this article: http://dx.doi.org/10.4161/trns.26598
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Review
Transcription 4:5, 232–237; September–December 2013; © 2013 Landes Bioscience
DNA Topoisomerases Beyond the standard role Laura Baranello, Fedor Kouzine* and David Levens
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Keywords: DNA topology, DNA Topoisomerases, DNA structure, transcriptional initiation, transcriptional elongation, chromatin
Chromatin is dynamically changing its structure to accommodate and control DNA-dependent processes inside of eukaryotic cells. These changes are necessarily linked to changes of DNA topology, which might itself serve as a regulatory signal to be detected by proteins. Thus, DNA Topoisomerases may contribute to the regulation of many events occurring during the transcription cycle. In this review we will focus on DNA Topoisomerase functions in transcription, with particular emphasis on the multiplicity of tasks beyond their widely appreciated role in solving topological problems associated with transcription elongation.
Introduction Higher organisms have evolved sophisticated mechanisms to orchestrate gene expression and modulate transcription output. The concerted and coordinated action of transcription factors, chromatin remodeling proteins, histones, and RNA polymerase (RNAP) sets the basis for different regulatory programs. However, traditional models of eukaryotic gene regulation rarely consider that DNA-protein interactions may promote dynamic changes in the structure of the double helix and that DNA stiffness constrains the assembly of multi-protein complexes. The transcription cycle consists of a defined sequence of events: chromatin opening and nucleosome mobilization, recruitment of RNAP and Pre-Initiation Complex (PIC) assembly, promoter escape and promoter-proximal pausing, pause release, elongation and termination.1 In addition to the loading of transcription factors at promoters, transcriptional regulation involves chromatin loop formation between the promoter and enhancers or other remote elements that play an important role in the gene activation.2 All these transactions involve the unwinding, bending or writhing of DNA and so are associated with changes in DNA topology that may feedback and contribute to the control of gene output.3 DNA Topoisomerases (Topos), a family of enzymes that regulate DNA topology, are fundamental to the modulation of this system.4 As these proteins are the targets of many anti-cancer
drugs,5 there is considerable interest in elucidating their complex functions. Despite long standing effort, the picture of Topos involvement in gene regulation remains incomplete. In this review, our goal is to highlight some crucial, but less studied issues concerning the roles of Topos in the process of transcription.
Topoisomerases and the TwinSupercoiled-Domain Model In general Topos are divided in two classes, type I and type II, depending on whether they cleave one or two strands of DNA, respectively.4,6,7 DNA Topoisomerase I (Topo I) relaxes supercoiled template by nicking a single strand of duplex DNA and allowing one end to rotate with respect to the other around the intact strand (Topo IB) or by passing one strand through the break (Topo IA). DNA Topoisomerase II (Topo II) cleaves both strands of a DNA duplex and passes a second intact duplex through the transient break. The type of topological problem to be solved, the architecture of the surrounding chromatin environment and the presence of accessory factors likely dictate which Topos are used for a particular purpose. Due to its stiffness, DNA undergoes several sorts of deformation in response to forces applied by genetic processes.8 Thus, overwinding or underwinding of the helix changes the twist, a parameter describing the number of times the individual strands coil around the helical axis. When the twist reaches a critical density, the molecule bends to form plectonemic structures in which the double helix coils about itself, a property known as writhe. Writhe can also be accommodated via solenoid-like wrapping of the double helix on a spool such as occurs around nucleosomes. The plectonemic or solenoidal coiling of the double helix is more commonly known as supercoiling. The “twin-supercoiled-domain model” describes the interplay between DNA topology and Topos activity and predicts that genomic transactions forcing DNA to circle around its axis generates local domains of DNA supercoiling. The best example of this prediction is presented by transcriptional elongation in which the RNAP generates positive supercoils ahead and leaves negative supercoils behind, as it moves along the DNA.9
*Correspondence to: Fedor Kouzine; Email: [email protected] Submitted: 08/19/13; Revised: 09/23/13; Accepted: 09/24/13 Citation: Baranello L, Kouzine F, Levens D. DNA Topoisomerases: Beyond the standard role. Transcription 2013; 4:230–235; PMID: 24135702; http://dx.doi.org/10.4161/trns.26598 232 Transcription Volume 4 Issue 5
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Laboratory of Pathology; National Cancer Institute; Bethesda, MD USA
Review
Figure 1. Topos’ participation in the transcription cycle. Numbers indicate Topos regulated steps. Step 1, chromatin opening: Topos cooperate with chromatin remodeling complexes to clear promoters during initial steps of transcription. Step 2, PIC assembly: Topos’ activity and Topos’ interaction with the general transcription factors (TF) favor RNAP recruitment at promoters. Step 3, promoter pausing/escape: Together with negative elongation factors (NEF) and positive elongation factors (PEF) Topos directs the temporary arrest and release of RNAP downstream the TSS. Step 4, elongation: Topos relax helical stress generated by the moving RNAP. Step 5, termination: Topos contribute to the formation of architectural domains. Gene looping between initiator and terminator or between promoter and enhancer (Step 6, chromatin looping) favors transcription regulation. Light-gray area represents Topos roles not related to the twin-domain model. Dark-gray area denotes Topos functions associated to the twin-domain model. RNAP, RNA polymerase; A, activator; ACF, ATP-utilizing chromatin assembly and remodeling factor.
The Classical View of Topoisomerases in Transcription One of the main cellular functions of Topo I is believed to be removal of helical stress generated during elongation in order to ensure processivity of transcription.4 This torque-sensitive enzyme10 is not proficient on nucleosomal template11 and works in front of the elongating RNAP where nucleosomes are displaced with the assistance of chromatin remodeling activities.12,13 In regions where the transcription rate is high, helical tension would deform DNA mostly by the twist regime,11 requiring the “twistase” Topo I to favor elongation.14 Accordingly Topo I recruitment to the Hsp70 genes in Drosophila follows chromatin remodeling; this is consistent with the idea of Topo I operating on accessible DNA.15,16 Topo II works mainly on the nucleosome-free regions near active TSSs.17 As Topo II activity depends on the juxtapositionprobability of DNA segments18 it will relax helical tension as long as DNA deformation occurs in the writhe regime.11 At promoters of highly transcribed genes the RNAP II imposes a 90° bend on the template, therefore as DNA is screwed through the active site, the upstream DNA is translationally rotated, directly generating
writhe.19 Indeed Topo II is the main “writhase” at promoters of highly expressed genes.20 In recent papers the genome mapping of transcription-generated supercoils in human cell lines has provided insight into the DNA dynamics of a transcribed locus and into Topos role at 5′ end of genes.20,21 Negative supercoiling generated during transcription is transmitted locally upstream of promoters; highly expressed genes rely upon Topo II to dissipate torsional stress around the TSS, whereas moderately expressed genes depend on Topo I.20 This indicates that instead of working randomly at promoters, Topos are able to distinguish between different types of DNA juxtapositions and collaborate with different partners that direct their action. Moreover the presence of a sturdy level of negative supercoiling within promoter areas of active genes suggests that rather than simply untwisting or unknotting DNA, these enzymes have the ability to maintain a steady-state level of torsional stress that may support the structural transitions in the double helix that insure the proper transcription.22 Thus according to the classical view Topos operate in transcription by relieving torsional stress that arises during elongation (Fig. 1, step 4). However, specific transcription events such as nucleosome mobilization, pre-initiation complex
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Review
Topo I Regulates Key Steps of Transcription Initiation Experimental evidence indicates that Topo I is a differential activator/repressor of transcription depending upon the specific utilization of the core elements at specific promoters.27 Together with the absence of sequence specificity for the enzymatic activity of Topo I, these features seem to be associated with the capacity of Topo I to interact with different proteins. Once recruited to the targeted promoter, the interplay of Topo I with its partners may modify chromatin structure and/or cooperate with the general transcription machinery. Topo I was found to regulate transcription through the maintenance of an open chromatin state at the promoters, in vivo, by the concerted action with the chromatin remodeler Hrp128 (Fig. 1, step 1). This is consistent with in vitro data showing that in a topologically constrained environment Topos activity contributes to the nucleosomes remodeling at active promoters.29 Accordingly, cells with reduced Topo I levels had increased histone H3 density at promoters that in turn is associated with downregulation of gene expression.28 Additionally, Topo I was identified biochemically as a co-activator able to enhance transcription at a pre-elongation step(s)30-32 (Fig. 1, step 2). The dramatic Topo I-dependent increase in the rate of TFIID-TFIIADNA complex formation in presence of an activator is probably explained by the ability of Topo I to bend or distort DNA upon binding. The enzyme most likely fulfills an architectural role rationalizing why a catalytically inactive mutant of Topo I is also able to promote pre-initiation complex formation and to potentiate activation.
Topo I and the Rate of Transcription Of course, Topo I’s contribution might differ from one context to the other. For example in vivo evidence highlights how promoters associated with paused RNAP II are extremely sensitive to camptothecin (CPT)20 which is a Topo I-selective inhibitor.33 In accordance with this, paused RNAP II is redistributed after
CPT treatment.34,35 Considering that promoter-pausing is one of the most highly regulated steps in transcription initiation,36 these findings indicate that Topo I specifically affects the pool of factors involved in this process (Fig. 1, step 3). The capacity of the Topo I N-terminal segment to bind the serine-phosphorylated carboxy terminal domain (CTD) of RNAP II37,38 further solidifies the idea that this enzyme acts as an interchangeable factor among different constituents of the transcription apparatus. At a single locus, the pattern of Topo I binding resembles RNAP II localization39 and experiments with Topo I inhibitors revealed the importance of Topo I in RNAP II recruitment and movement40 at specific regulatory sites in the genome. The CTD of RNAP II is modified at various stages of transcription: in the pre-initiation complex it is hypo-phosphorylated, it is phosphorylated on serine 5 (Ser5P) during initiation and then on serine 2 (Ser2P) during elongation.13 The peak of Ser5P is located in the promoter-proximal region while the peak of Ser2P is located at a second pause site in the 3′-flanking region of genes, where termination occurs.41 The mechanisms that establish the promoter pause are not fully understood and despite the identification of key regulators, emerging evidence suggests a role for DNA topology in this process.40 Indeed accumulated supercoils can arrest RNAP movement and subsequent DNA relaxation promotes the resumption of transcription.42 The interaction of Topo I with hyper-phosphorylated RNAP II forms and the sensitivity of paused genes to CPT suggest that the enzyme might be part of the mechanism for RNAP II pause-release both at promoters and at transcription termination sites (Fig. 1, step 5). The localization of Topo I is not restricted to promoters as the enzyme was also enriched at enhancers.39 DNA looping that brings distant enhancers in contact with promoters has long been appreciated as source of transcription regulation.2 Topo I was detected by ChIP assay at β-globin locus control region (LCR), one of the best-studied examples of enhancerpromoter regulation,33 and Topo I-associated cleavage sites were shown at rDNA enhancer region in Saccharomyces cerevisiae.43 Although the Topo I function at these remote regions has not been elucidated, one can hypothesize that Topo I activity might resolve topological problems that might arise during the process of looping (Fig. 1, step 6).
Topo II is a Chromatin Regulator Via its enzymatic activity Topo II has been recognized to regulate promoter output and to serve as an activator or repressor depending on the specific chromatin context. Topo II is the primary relaxase of nucleosomal DNA11 and a growing body of experimental evidence supports the idea of a close link between Topo II and nucleosome-interacting proteins. Human and Drosophila Topo II co-purify with the chromatin-remodeling factors ACF and CHRAC44,45 that promote chromatin perturbations near the binding sites of site-specific factors, facilitating transcription initiation.46 Chromosome-wide analysis showed that Topo II binds preferentially to promoter regions containing H3K4 methylation, a feature of active chromatin. This is compatible with the idea of a chromatin-based recruitment mechanism
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formation, recruitment of RNAP, activation or repression of promoters have also been linked to Topos recruitment. The behavior of DNA and Topos at promoters, where architectural restraints and dynamic torsional stress combine in a complex balance, has been less explored. At these regions, in addition to negative supercoiling accumulating as a result of elongation, the stiff double helix resists bending and twisting during PIC assembly,23-25 and constrains the deformation of DNA and chromatin that likely accompanies promoter-enhancers interactions.26 The elastic properties of DNA are likely to influence the efficient recruitment and assembly of transcription factors and RNAP and to complicate attempts to model and predict Topos function beyond their ability to control transcriptionally generated supercoiling. In the following paragraphs we touch on an expanded view of Topos participation in gene expression, broadening our appreciation about how cells could use these enzymes to regulate important steps of transcription.
for Topo II47 that might exert a double role at 5′ termini of genes favoring the targeting of chromatin modifiers to specific regulatory sites and controlling topology during the nucleosomes remodeling (Fig. 1, step 1).
Topo II in Transcription Activation Topo II preferentially associates with the highly transcribed promoters.20 This might be in part the result of the promoter architecture where bending of promoter regions synergize with the propensity of Topo II to bind bent DNA.48 Recent work accurately describes how the cleavage reaction of Topo II goes through three distinct and well-ordered reaction steps: nonspecific enzyme-DNA binding, sequence-specific DNA bending, and finally, cleavage.49 Importantly, the stabilization of a highly curved DNA geometry is critical to the Topo II catalytic cycle.48 DNA curvature at the TSS favors transcription by lowering the energy cost for DNA to interact with transcription factors and RNAP.50,51 Highly expressed promoters may have a higher bendability hence recruit Topo II more easily. Conversely, Topo II might enhance transcription activation because of its bending capability. One of the best and most interesting examples of Topo II regulated gene activation was discovered at the pS2 promoter in the human cell line MCF752 where estrogen-dependent activation requires a promoter intermediate containing a Topo II dependent double-strand break (DSB). Topo II and poly(ADPribose) polymerase (PARP-1) collaborate with factors normally associated with DNA damage response, to alter the molecular composition and nucleosome structure of the promoter. Recruitment of Topo II/PARP-1 complex as well as promoter
cleavage was shown at other promoters upon gene activation16 and evokes the startling concept that DNA cleavage supports transcription activation. Consistently, in a recent paper it was shown that upon gene activation, the transcription machinery assembles sequentially with the nucleotide excision repair (NER) factors in a XPC protein dependent fashion at promoters in absence of exogenous genotoxic attack.53 Although the authors linked this phenomenon with the ability of NER factors to enhance chromatin modifications, an alternative possibility is that this recruitment represents a first-aid kit, just in case the double-strand break resealing by Topo II at promoters fails. The normally transient Topos-DNA cleavage complex can be converted into a potential DNA lesion if prolonged misalignment of the ends occurs at their interface with the enzyme.54,55 Thus, Topos cleavage activity represents a potential source of DNA damage that can affect genomic transactions and the cell fate. Besides controlling RNAP II-dependent promoters, Topo II is also a component of the RNAP I machinery binding the RRN3 component that mediates the interaction between RNAP I and the transcription factor SL1 at the rRNA gene promoter.56 In this context, de novo PIC formation is enhanced when Topo II cleavage/re-ligation activity alleviates elastic DNA constraints thus promoting PIC assembly and stability (Fig. 1, step 2). Interestingly, the enzyme is able to exert tissue specific regulation in the nervous systems. Topo II binding to promoters of developmentally regulated genes in post-mitotic neurons governs the transcriptional program associated with neuronal differentiation and longevity, supporting activation or repression of tissue specific genes but not of the housekeeper ones.57 How the tissue specific regulation is achieved is still poorly understood but it might involve interactions with characteristic DNA structures
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Figure 2. The rigidity of DNA represents a barrier for protein-DNA-protein interactions. (A) To overcome this resistance, Topos introduce DNA breaks favoring conformational changes that bring in contact distal transcriptional regulators. (B) In the absence of cleavage activity Topo I bridges proteinprotein interactions that help to regulate transcription.
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Gene Architecture and Topo II In budding yeast, Topo II sites are detected not only at promoters but also at transcription termination regions58 (Fig. 1, step 5). Considering Topo I as the main relaxase in the body of transcribed genes, the 3` localization of Topo II might reflect a different role. As Topo II has been implicated in DNA looping59 one possibility is that the enzyme contributes to the formation of architectural domains containing gene units. Gene looping between initiation and terminator regions has been proposed to facilitate RNAP II recycling and to increase transcription rates.60,61 Topo II has a significant affinity for the base of a DNA loop structure where two duplexes juxtapose and this feature makes the enzyme an attractive candidate for looping regulation. Likewise Topo II was discovered near the tissue-specific enhancer62 of the kappa immunoglobulin gene, associated with nuclear matrix associated regions (MARs), and additional binding sites were related to other enhancers in the heavy-chain (IgH) locus (Fig. 1, step 6). References 1.
2.
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Conclusions Although DNA Topoisomerases were first discovered more than 40 years ago, the study of these enzymes remains highly vigorous. In higher Eukaryotes the emerging evidence presented in this review suggests that Topoisomerases have evolved to participate at multiple steps in transcription. The ability to perform so many functions through simple binding, cleavage and ligation reactions dictates that rather than acting as mere relaxases of torsionally stressed DNA, Topos may modulate DNA stiffness to facilitate multi-protein complexes assembly (Fig. 2A). The double helix is a rigid polymer and the cell needs strategies and tools that promote contacts between factors at distant sites. The ability to introduce breaks in the DNA is a tool for Topos to weaken its stiffness favoring protein-DNA-protein interactions. Even in the absence of catalysis Topo I may help to assemble such complexes via protein-protein interactions (Fig. 2B). Because the most DNA Topoisomerase sensitive genes are the ones characterized by highly regulated transcription initiation and transcriptional plasticity63 it is reasonable to conclude that Topo I and Topo II can act as transcriptional factors involved in key steps. Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
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