Organization and Dynamics of Plant Chromatin Cytogenet Genome Res 2014;143:51–59 DOI: 10.1159/000360774
Published online: March 28, 2014
Drugs for Plant Chromosome and Chromatin Research Ales Pecinka Chun-Hsin Liu
Key Words Chromatin · DNA damage repair · Epigenetics · Inhibitors · Plants
Abstract Eukaryotic genomes are organized into chromosomes. Genetic information regularly becomes damaged and requires repair in order to ensure genome stability. Furthermore, expression of individual genetic elements on the chromosome(s) is controlled by several factors, including chromatin. Understanding the functions of chromatin may provide efficient tools for regulating gene expression. There has been great progress in understanding genome control using genetic mutations, but the use of mutants is sometimes not possible or may require additional interference with DNA or chromatin structure using specific treatments in order to obtain phenotypes. Therefore, chemical genetics has become an integral part of plant genome research. Here, we summarize information on the most commonly used drugs for chromatin and DNA damage repair studies, with the aim of simplifying the choice of drug and the estimation of possible side effects for current and future research. © 2014 S. Karger AG, Basel
Eukaryotic genomes consist of DNA organized into chromosomes. DNA sequence and chromosome integrity are challenged by DNA replication errors and the oc© 2014 S. Karger AG, Basel 1424–8581/14/1433–0051$39.50/0 E-Mail [email protected] www.karger.com/cgr
currence of pre-mutagenic structures such as non-native bonds, bulky adducts, nucleotide modifications, or DNA breaks, which need to be repaired in order to ensure genome functionality. The repair is carried out by partially redundant DNA damage repair pathways that are conserved to some extent among eukaryotes. In animals, loss of function in many individual DNA damage repair genes leads to severe phenotypes. However, in the case of plant orthologs, loss of function yields phenotypes only when agents inducing moderate-to-high levels of DNA damage are applied [Britt, 1996; Bray et al., 2005]. Hence, the external sources of DNA damage have become one of the cornerstones of plant DNA damage repair studies. Although DNA replication is highly conservative and chromosome integrity is strictly controlled, genomes are not static in space and time, and DNA molecules are subject to various processes orchestrated in developmental and environmental context. This is achieved by the combined action of trans-acting factors and local (cis) components comprising DNA sequence and chromatin, i.e. a complex of DNA and associated proteins (mainly histones). Histones occur in specific variants, and their Nterminal tails are post-translationally modified in order to define specific expression-permissive or -repressive chromatin states. In plants, this complex arrangement is set up and controlled by the action of more than 500 chromatin-related regulatory genes [Gendler et al., 2008]. Here, we summarize information on drugs commonly used in chromatin and DNA damage repair studies which Ales Pecinka Department of Plant Breeding and Genetics Max Planck Institute for Plant Breeding Research Carl-von-Linné-Weg 10, DE–50829 Cologne (Germany) E-Mail pecinka @ mpipz.mpg.de
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Department of Plant Breeding and Genetics, Max Planck Institute for Plant Breeding Research, Cologne, Germany
Chemical Inducers of DNA Damage
Chemicals that induce DNA damage have frequently been applied to study DNA repair in both prokaryotes and eukaryotes, and to suppress viruses, neoplastic diseases and the immune system in mammals, including humans [Deans and West, 2011; Fu et al., 2012]. They also provide an alternative tool to study the mechanism of DNA replication, to compensate for the limitations of mutant selection and the complicated process of genetic analysis. The DNA damaging agents most commonly used in plant studies are described below and an overview is provided in table 1. Replication Blocking Agents Deoxyribonucleotide triphosphates (dNTPs) are the building blocks of DNA. Interference with dNTP biosynthesis may limit the progression of DNA replication [Christopherson et al., 2002]. Hydroxyurea (HU) reduces dNTP levels by inhibiting the activity of the small subunits of RIBONUCLEOTIDE REDUCTASE (RNR), an enzyme which catalyzes the reduction of ribonucleotide diphosphates (NDPs) into deoxyribonucleosides (dNDPs), thus affecting the progression of replication fork and inducing DNA replication stress [Roa et al., 2009]. Recently it has been found in Arabidopsis that HU inhibits also CATALASE 2 and thus interferes with the decomposition of hydrogen peroxide and increases oxidative stress [Juul et al., 2010]. In plants, HU transiently arrests the cell cycle at the transition between G1/S and G2/M, providing a useful tool for cell-cycle synchrony [Pan et al., 1993; Cools et al., 2010], and has been utilized to identify the components involved in cell-cycle control [Cools et al., 2011; Spadafora et al., 2011]. The eukaryotic DNA polymerase α is essential for primer synthesis during replication of both DNA strands [Srivastava and Busbee, 2003]. Its inhibition by aphidicolin [Spadari et al., 1982] has been used for cell-cycle synchrony and understanding DNA-replication checkpoints in plants [Francis, 2011; Cook et al., 2013]. Signaling of DNA damage repair in response to both HU and aphidicolin is mainly mediated by ATAXIA TELANGIECTASIA MUTATED AND RAD3-RELATED (ATR) kinase [Culligan et al., 2004; De Schutter et al., 2007]. 52
Cytogenet Genome Res 2014;143:51–59 DOI: 10.1159/000360774
Inhibitor of DNA Synthesis in Organelles DNA gyrase is the bacterial DNA topoisomerase II that negatively supercoils DNA in the presence of adenosine triphosphate (ATP). In plant cells, it occurs in chloroplasts and mitochondria [Wall et al., 2004]. Chemical inhibition of plant gyrase in chloroplasts by novobiocin [Mills et al., 1989] has recently opened the possibility for studying DNA repair mechanisms in plant organelles [Cappadocia et al., 2010]. Alkylating Agents Alkylating agents transfer alkyl groups onto a broad range of biomolecules. They react with the ring nitrogens (N) and extracyclic oxygens (O) in DNA and generate a variety of covalent adducts [Fu et al., 2012]. Traditionally, they are described as either monofunctional or bifunctional based on the number of reactive sites they contain. Monofunctional Alkylating Agents Ethyl methanesulfonate (EMS) ethylates the N7- or O6-position of guanine, which leads to frequent G/ C→A/T transitions [Sega, 1984; Kim et al., 2006]. In plants, EMS is essential for generating mutagenized populations for mutant screens and novel breeding lines [Hartwig et al., 2012]. EMS-induced mutations can be efficiently detected at the genome-wide scale, allowing for rapid and cost-saving mutation mapping [Abe et al., 2012; Nordstrom et al., 2013]. 1-Ethyl-1-nitrosourea (ENU) can transfer an ethyl group to nucleophilic sites of cellular constituents and a carbonyl group to an amino group of a protein. It alkylates the O6-position of guanine and the O4-position of thymine in DNA, leading to AT→TA transversions and AT→GC transitions [Shibuya and Morimoto, 1993]. ENU is frequently used in mammalian mutagenesis, owing to its high-efficiency induction of point mutations in the male germline [Nolan et al., 2002]. In plants, ENU has been used as a positive control substance in genotoxicity studies [Gichner, 2003], and in TILLING (targeting induced local lesions in genomes) approaches [Cooper et al., 2013]. Methyl methanesulfonate (MMS) predominantly methylates DNA on N7-guanine and N3-adenine. The former leads to depurination followed by formation of a toxic apurinic site, while the latter is highly cytotoxic due to its blocking of most DNA polymerases and inhibition of DNA synthesis [Beranek, 1990; Fu et al., 2012]. MMS produces T/A→G/C transversions and A/T→G/C transitions [Lundin et al., 2005]. MMS has been widely used for studying DNA damage repair in plants, particularly in the Pecinka/Liu
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interfere with chromatin structure and/or cause specific types of DNA lesions. We highlight practical aspects of using these compounds in a variety of plant species, molecular mechanisms of their action and known side effects.
Table 1. Major drugs used in plant DNA damage repair studies Drugs Replication blocking agents Hydroxyurea (HU) Aphidicolin
Mechanism of action
Induced damage
Key references
RIBONUCLEOTIDE REDUCTASE and CATALASE 2 inhibition DNA POLYMERASE α inhibition
dNTP pool depletion replication blocking
De Schutter et al., 2007; Cools et al., 2010; Spadafora et al., 2011 Cook et al., 2013
replication blocking
Cappadocia et al., 2010
AT→TA transversions, AT→GC transitions GC→AT transitions
Gichner et al., 2003; Cooper et al., 2013
Inhibitor of DNA synthesis in organelles Novobiocin inhibition of DNA GYRASE in organelles Alkylating agents 1-Ethyl-1-nitrosourea
ethylation of bases (mostly T)
Ethyl methanesulfonate
ethylation of G→O6-ethylG
Methyl methanesulfonate
methylation of N7- and O6-dG, N3-dA and N4-dT G-specific 5′-CpG-3′ double-alkylation
Mitomycin C Cisplatin Radiomimetic drugs Bleomycin (bleocin) Zeocin
1,2-intrastrand d(GpG) and d(ApG) adducts metal-dependent free radical production most likely similar to bleomycin
TA→GC transversions, AT→GC transitions G-G or G-A interstrand crosslinks G-G or G-A intrastrand crosslinks
Kim et al., 2006; Abe et al., 2012; Nordstrom et al., 2013 Bagherieh-Najjar et al., 2005; Mannuss et al., 2010 Bleuyard et al., 2005; Mannuss et al., 2010; Da Ines et al., 2013 Jamieson and Lippard, 1999; Mannuss et al., 2010
DSB, SSB at 5′-GC-3′ or 5′-GT-3′ sequences DSB
Ma et al., 2009; Pecinka et al., 2009; Lang et al., 2012 Adachi et al., 2011
DSB = Double strand break; SSB = single strand break.
Bifunctional Alkylating Agents Bifunctional alkylating agents contain 2 active moieties that can react with separate bases of DNA to form bi-adducts [Fu et al., 2012]. The most commonly used bifunctional alkylating agent in plant research is mitomycin C (MMC). In cells, MMC becomes reduced and transformed into vinylogous quinone methide with high alkylating activity [Tomasz, 1995]. It then reacts with N7- or O6-guanine in DNA to form an MMC-mono-dG adduct that can subsequently react with another DNA base to form guanine-guanine (G-G) and/or guanine-adenine (G-A) interstrand crosslinks [Fu et al., 2012]. Application of MMC (alone or in combination with other drugs) readily induces expression of DNA damage repair genes (AtGenExpress dataset ME00326). MMC has been used as one of the major marker drugs for associating gene functions with DNA damage repair, DNA replication and cell-cycle control. The repair of MMC-induced interstrand crosslinks is thought to be mediated mainly by ATR and RAS ASSOCIATED WITH DIABETES PRODrugs for Chromosome Research
TEIN 51 (RAD51)-dependent HR pathways [Bleuyard et al., 2005; Nezames et al., 2012; Da Ines et al., 2013]. Alkylating-Like Agents These drugs induce crosslinks without DNA alkylation. Cisplatin is composed of a central platinum atom surrounded by 2 chlorine atoms and 2 ammonia groups. Upon hydrolysis of chloride ligands, it forms mainly DNA intrastrand and rarely also interstrand crosslinks between the N7-positions of purine bases [Siddik, 2003]. The lasting activity of cisplatin is hampered by the short 2-hour half-time of hydrolysis and partial cytoplasmic inactivation by interactions with endogenous nucleophiles and proteins [Jamieson et al., 1999]. In plant studies, it is commonly used as an intrastrand DNA crosslinker to investigate the ATR-dependent repair of bulky DNA lesions via HR [Osakabe et al., 2006; Abe et al., 2009; Mannuss et al., 2010; Nezames et al., 2012] and base excision repair [Liang et al., 2006]. Radiomimetic Drugs Short wavelength and high energy radiation (gamma radiation and X-rays) have traditionally been used in mutagenesis and DNA damage repair experiments. These Cytogenet Genome Res 2014;143:51–59 DOI: 10.1159/000360774
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homologous recombination (HR) pathway [BagheriehNajjar et al., 2005; Kim, 2006; Yin et al., 2009; Mannuss et al., 2010].
Table 2. Drugs for chromatin research DNA Histone H3 modificationsa methylation K4me3 K9me2 K27me3
Inhibitors
Chromatin Transposon Gene excondensation expression pression
Pol II Key references activity
Methyl-group biosynthesis inhibitors and methionine antagonists Sulfamethazine n.a. ↓ ↓ S-Dihydroxypropyladenine ↓ ↑ ↓ 3-Deazaneplanocin n.a. n.a. n.a. L-Ethionine n.a. n.a. ↓
n.a. n.a. n.a. n.a.
n.a. n.a. n.a. ↓
↑ ↑ ↑ n.a.
n.a. n.a. n.a. n.a.
n.a. n.a. n.a. n.a.
Zhang et al., 2012 Baubec et al., 2010 Foerster et al., 2011 Fajkus et al., 1992
DNA methylation inhibitors Zebularine 5-Azacytidine
↓ ↓
↑ n.a.
↓ ↓
n.a. n.a.
↓ ↓
↑ ↑
n.a. ↑ 73; ↓ 52b
n.a. n.a.
Baubec et al., 2009 Chang and Pikaard, 2005
−
−
↓
n.a.
n.a.
−
↑ 2; ↓ 34b
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
↑ 138c
n.a.
Chang and Pikaard, 2005; Baubec et al., 2010 Grozinger et al., 2001; Zhao et al., 2003
Inhibitors of RNA polymerase II α-Amanitin Actinomycin D
n.a. n.a.
n.a. n.a.
n.a. n.a.
n.a. n.a.
n.a. n.a.
n.a. n.a.
Unknown mode of action Genistein
↓
n.a.
n.a.
n.a.
n.a.
↑
Histone deacetylase inhibitors Trichostatin A and sodium butyrate Sirtinol (putative)
n.a. n.a. n.a.
↓ ↓
Haag et al., 2012 Narsai et al., 2007
n.a.
Arase et al., 2012
↑ = Increase; ↓ = decrease; − = no change; n.a. = not analyzed. At normally silenced loci. b Out of 7,800 Arabidopsis genes analyzed [Chang and Pikaard, 2005]. c Out of 23,000 Arabidopsis genes analyzed, number of down-regulated genes not specified [Zhao et al., 2003]. a
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Cytogenet Genome Res 2014;143:51–59 DOI: 10.1159/000360774
the same family. The induction of breaks has been described in detail for bleomycin: upon activation by a metal ion, bleomycin abstracts H from C4′ of the 2-deoxyribose moiety to induce single-strand breaks, doublestrand breaks, and abasic sites at 5′-GT-3′ and 5′-GC-3′ sequences [Povirk, 1996; Ma et al., 2009]. Radiomimetic drugs damage DNA independently of cell cycle stage, effectively induce expression of DNA damage repair genes and inhibit cell growth [Houser et al., 2001; De Schutter et al., 2007; Pecinka et al., 2009; Adachi et al., 2011]. They are frequently employed as markers for studying ATAXIA TELANGIECTASIA MUTATED (ATM)-mediated double-strand break repair [Lang et al., 2012].
Inhibitors Interfering with Chromatin
Interference with epigenetic marks and chromatin structure (fig. 1) has been employed in a variety of plant studies (table 2). It has been particularly useful in cases where genetic mutations were not available or caused lethality, conditional interference was needed, or where siPecinka/Liu
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types of radiation induce predominantly DNA strand breaks while chemicals introduce mainly non-native bonds. The effect of radiation is direct, instant and more equally distributed within the plant body [Grigaravičius et al., 2009], compared to the damage caused by chemicals that can be potentially highly dependent on the uptake and distribution of a drug over individual tissues. However, sources of high energy radiation may be costly and inconvenient for some plant researchers. Therefore, many laboratories substitute short wavelength irradiation with radiomimetic (radiation resembling) drugs. By inducing highly specific attack of free radicals, these drugs abstract hydrogen (H) predominantly at the C1-, C4-, or C5-positions, leading to oxidation of 2-deoxyribose in DNA and DNA strand breaks. It should be noted that the lesions induced by radiomimetic drugs are only a small subset of the lesions induced by ionizing radiation, yet their effects on cells are remarkably similar [Povirk, 1996]. The most commonly used radiomimetic drugs in plant research are bleomycin (commercially supplied also as bleocin) and zeocin, glycopeptide-derived antibiotics of
terfere with chromatin marks. The black and the white lollipops show the methyl group (-CH3) and the acetyl group, respectively. S-DHPA = S-dihydroxypropyl adenine; DZnep = 3-deazaneplanocin A; Pol II = RNA polymerase II; HDA = a class I histone deacetylase; SAHH = S-adenosyl homocysteine hydrolase.
multaneous inhibition of multiple enzymes of the same family or several pathways was required. Methyl-Group Biosynthesis Inhibitors and Methionine Antagonists The methyl-group is widely implicated in many biochemical processes in plants. It plays a central role in epigenetic signaling and is used to modify DNA, RNA and proteins. Depending on the context and type of modification, it can have transcription-permissive or -repressive potential (see also DNA Methylation Inhibitors). The methyl-group precursor is synthesized via dihydrofolate and methionine cycles. Sulfamethazine is an inhibitor of DIHYDROPTEROATE SYNTHASE, an enzyme that catalyzes the synthesis of dihydropteroic acid, an early step in methyl-group biosynthesis within the dihydrofolate cycle [Zhang et al., 2012]. The second group of inhibitors includes S-dihydroxypropyl adenine and 3-deazaneplanocin A that block activity of the S-ADENOSYLHOMOCYSTEINE HYDROLASE in the methionine cycle. The third group consists of methionine antagonists, i.e. non-proteinogenic amino acids related to methionine that interfere with the methionine cycle. The most commonly used methionine antagonist is L-ethionine that contains an ethyl group at the position of the Drugs for Chromosome Research
DNA Methylation Inhibitors The methylation of cytosine at the 5th position is a prominent epigenetic mark present in many groups of eukaryotes. In plants, it occurs in all sequence contexts (CG, CHG and CHH, where H is any base but G) and functionally has been implicated mainly in silencing of repetitive DNA [Feng et al., 2010; Stroud et al., 2013]. It is deposited by several DNA methyltransferases that show preference for methylating cytosines in particular DNA sequence contexts and act via different mechanisms [Stroud et al., 2013]. In contrast to mammals where DNA methylation is re-established in early stages of every generation, plant DNA methylation is faithfully transmitted throughout development [reviewed in e.g. Feng et al., 2010]. There are a number of genes known to affect DNA methylation in plants, but many show severe developmental and fitness phenotypes [Miura et al., 2001; Mathieu et al., 2007]. Two drugs are commonly used for interference with DNA methylation in plants: 5-azacytidine and zebularine. Both are non-methylable cytosine analogs with identical mode of action, but their stability differs greatly. While 5-azacytidine has a half-life of only 4 hours and needs to be supplied several times in course of a standard experiment, zebularine’s half-life is over 3 weeks [Champion et al., 2010], making it the preferred inhibitor in plant studies. In planta, cytosine analogs are phosphorylated by uriCytogenet Genome Res 2014;143:51–59 DOI: 10.1159/000360774
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Fig. 1. Schematic representation of drugs (labeled in red) that in-
methionine methyl group and induces DNA demethylation in both plants and animals [Cox and Irving, 1977; Fajkus et al., 1992]. All these inhibitors have broad effects: they reduce DNA methylation in all sequence contexts at a number of tested targets, histone methylation at histone H3 lysine 9 and 27 (and most likely also other methylated residues) and promote expression of otherwise silenced genetic elements [Fajkus et al., 1992; Kovařík et al., 2000; Koukalová et al., 2002; Majerová et al., 2011; Zhang et al., 2012]. The broad range of side-effects limits the use of these drugs, particularly the interference with many nonepigenetic processes that require the methyl-group such as chlorophyll biosynthesis. However, they can be useful in situations when global interference is required. For example, 3-deazaneplanocin A and S-dihydroxypropyl adenine were used to mimic loss of the S-ADENOSYL HOMOCYSTEINE HYDROLASE, since its genetic mutations are normally lethal for plants [Baubec et al., 2010], or to interfere with silencing that required simultaneous knockout of several repressive pathways [Foerster et al., 2011].
Histone Deacetylase Inhibitors Acetylation of histone tails at specific residues is a transcription-promoting epigenetic mark deposited by histone acetyltransferase enzymes and removed by histone deacetylases (HDAs). Hence HDAs contribute to the maintenance of the repressive state. There are 3 types of plant HDAs: type I HDAs of the RPD3-like superfamily, which correspond to classes I and II in mammals; plantspecific type II HDAs, HD-tuins; and type III HDAs, sirtuins [reviewed by Hollender and Liu, 2008]. 56
Cytogenet Genome Res 2014;143:51–59 DOI: 10.1159/000360774
Type I HDAs have been implicated in global transcriptional regulation in developmental fashion, but specific genomic targets of individual enzymes remain a matter of debate. The best-characterized are type I HDAs: HDA6 and HDA19. HDA6 has been associated preferentially with transcriptional gene silencing and control of gene expression at heterochromatic regions, and to a lesser extent within genes [Probst et al., 2004; Tanaka et al., 2008]. In contrast, HDA19 seems to control developmentally regulated genes during specific developmental stages such as germination [Tanaka et al., 2008]. The most commonly used inhibitors of type I HDAs are trichostatin A (TSA) and sodium butyrate. Whole genome transcriptome analysis revealed that there are only few genes deregulated in common between TSA and 5-azacytidine, and that TSA has a weaker potential for activating transcriptionally silenced genetic elements [Chang and Pikaard, 2005]. In contrast, TSA affected developmentally regulated genes, such as those controlling germination and root patterning [Xu et al., 2005; Tanaka et al., 2008], most likely by inhibition of HDA19. This indicates that TSA could potentially interfere with polycomb-based silencing, and its effects on the levels of H3K27me3 should be tested in the future. The target genes of sirtuin HDAs are currently unknown in plants [Hollender and Liu, 2008]. Several sirtuin inhibitors have been found by chemical genetic screening in Saccharomyces cerevisiae, and the initial experiments with the compound sirtinol revealed disturbed body axis formation, vascularization problems and hypocotyl thickening in Arabidopsis [Grozinger et al., 2001]. Furthermore, sirtinol treatment led to upregulation of genes involved in auxin signaling, and several auxin signaling mutants had been found insensitive to this treatment [Zhao et al., 2003; Dai et al., 2005]. However, it has to be noted that the connection of these phenotypes with SIRTUIN (SRT) function is only indirect and so far not substantiated by data produced using srt mutants. An RNAi knockdown of SRT1 in rice led to increased oxidative stress, programmed cell death and release of transposon silencing [Huang et al., 2007] which differs from the sirtinol effects in Arabidopsis. Hence, the use of yeastderived sirtuin inhibitors in plant studies still requires careful testing and comparisons with SRT loss-of-function mutants. Inhibitors of RNA Polymerase II (Pol II) There are 5 DNA-dependent RNA polymerases (Pol I–V) in plants. Pol I and Pol III transcribe rDNA and tRNA genes while Pol II transcribes protein-coding genes. Pecinka/Liu
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dine/cytidine kinase and incorporated into both RNA and DNA, and lead to loss of DNA methylation in all sequence contexts. Drug-mediated interference with DNA methylation results in pleiotropic phenotypes (similar but not identical to genetic mutations) and reduces e.g. levels of H3K9me2 and compromises heterochromatin condensation [Baubec et al., 2009, 2010]. However, genome-wide transcriptome profiling of Arabidopsis plants treated with 5-azacytidine revealed a complex transcriptional response that could not easily be associated with targets controlled by DNA methylation [Chang and Pikaard, 2005]. This is supported by the observations that low doses of zebularine transcriptionally activate transposons without reducing their DNA methylation levels [T. Baubec, A. Finke, O. Mittelsten Scheid, A. Pecinka, unpubl. data]. The DNA de-methylation by zebularine is strongly opposed by de novo DNA methylation activity of the RNA-directed DNA methylation (RdDM) pathway in the apical meristems [Baubec et al., 2014]. This indicates that cytosine analogs may work to some extent by interference with chromatin structure and/or have other previously unnoticed effects. Furthermore, DNA methylation inhibitors reduce plant growth. The most likely cause is the covalent binding of DNA methyltransferases to incorporated DNA methylation inhibitors leading to formation of nucleoprotein adducts [Champion et al., 2010]. This represents a physical barrier to the progression of RNA and DNA polymerases, which is recognized as DNA damage by plants and requires repair [C.-H. Liu, A. Pecinka, unpubl. data]. The negative side effects of DNA methylation inhibitors could potentially be eliminated by use of specific inhibitors of DNA methyltransferases. In mammals, the drug RG108 efficiently inhibits DNA methyltransferase 1 (DNMT1), an ortholog of plant MET1 enzyme [Brueckner et al., 2005]. However, adopting such drugs for the plant field may not be straightforward. For example, RG108 did not activate DNA methylated reporter constructs that are steadily activated by zebularine or 5-azacytidine [A. Pecinka, unpubl. data].
Pol IV and Pol V are plant-specific polymerases that are closely related to Pol II (Pol II-group), and have been implicated in silencing of repetitive DNA by the mechanism of RdDM [reviewed in Haag and Pikaard, 2011]. Since Pol II activity is essential, use of the mutants is not straightforward. Instead, there are several compounds used as Pol II inhibitors. The most common ones are actinomycin D, which blocks polymerase transcriptional elongation, and α-amanitin, which drastically reduces the rate of polymerase processivity. Application of Pol II inhibitors is highly toxic and leads to cell death. Nevertheless, they have been indispensable in studying mRNA decay-related processes in plants [Narsai et al., 2007]. Importantly, α-amanitin does not inhibit transcription of Pol IV. This allowed the nature of transcripts produced by Pol IV and interactions of individual components of the RdDM during double-stranded RNA synthesis to be determined [Haag et al., 2012].
Concluding Remarks
This article reviews the information on the most commonly used chemicals in plant chromatin and chromosome research, with particular focus on those applied in the studies of DNA damage repair and epigenetics. Although chemical inducers of DNA damage or drugs affecting particular chromatin marks have been proven to be useful in plant chromosome and chromatin studies, there are several major points to be considered. First, most of the DNA damaging drugs induce more than one type of DNA damage, which may activate different repair pathways, and estimating the proportions in which different damages occur may be difficult. This may be misleading and therefore requires careful analysis using mul-
tiple methods. Similarly, chromatin inhibitors (in particular those interfering with methyl group biosynthesis) may have a broad effect and affect multiple chromatin marks as well as chromatin structure. However, it has to be noted that such pleiotropic effects are, to some extent, similar to those observed with mutants of the key methylation controlling components such as MET1 and DECREASE IN DNA METHYLATION 1 (DDM1) [Miura et al., 2001; Mathieu et al., 2007]. Therefore, we suggest preferentially using low concentrations of the most specific drugs. In conclusion, it is clear that chemical inhibitors cannot replace the advantage of genetic mutations, but in many cases they can be a complementary or even indispensable tool. Furthermore, chemical inhibitors have been fundamental in discovering novel genes and gene functions in both DNA damage repair and chromatin studies. However, in contrast to human field, where almost all drugs discussed here are used for clinical studies in cancer treatments, only few plant studies went beyond the level of basic research so far [Abe et al., 2012; Zhong et al., 2013]. Another challenge remains in finding compounds that have highly specific effects and/or inhibit only a single enzyme, while having no side effects. Finding novel drugs with specific functions will be useful to expand our understanding of plant genome and chromosome biology.
Acknowledgements We apologize to all colleagues whose work could not be mentioned due to the space limitations and thank M. Fojtova for valuable discussions and comments and T. Harrop for editing the manuscript. The authors were funded by the Max Planck Society.
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Drugs for Chromosome Research
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Drugs for Chromosome Research
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Published online: March 28, 2014
Drugs for Plant Chromosome and Chromatin Research Ales Pecinka Chun-Hsin Liu
Key Words Chromatin · DNA damage repair · Epigenetics · Inhibitors · Plants
Abstract Eukaryotic genomes are organized into chromosomes. Genetic information regularly becomes damaged and requires repair in order to ensure genome stability. Furthermore, expression of individual genetic elements on the chromosome(s) is controlled by several factors, including chromatin. Understanding the functions of chromatin may provide efficient tools for regulating gene expression. There has been great progress in understanding genome control using genetic mutations, but the use of mutants is sometimes not possible or may require additional interference with DNA or chromatin structure using specific treatments in order to obtain phenotypes. Therefore, chemical genetics has become an integral part of plant genome research. Here, we summarize information on the most commonly used drugs for chromatin and DNA damage repair studies, with the aim of simplifying the choice of drug and the estimation of possible side effects for current and future research. © 2014 S. Karger AG, Basel
Eukaryotic genomes consist of DNA organized into chromosomes. DNA sequence and chromosome integrity are challenged by DNA replication errors and the oc© 2014 S. Karger AG, Basel 1424–8581/14/1433–0051$39.50/0 E-Mail [email protected] www.karger.com/cgr
currence of pre-mutagenic structures such as non-native bonds, bulky adducts, nucleotide modifications, or DNA breaks, which need to be repaired in order to ensure genome functionality. The repair is carried out by partially redundant DNA damage repair pathways that are conserved to some extent among eukaryotes. In animals, loss of function in many individual DNA damage repair genes leads to severe phenotypes. However, in the case of plant orthologs, loss of function yields phenotypes only when agents inducing moderate-to-high levels of DNA damage are applied [Britt, 1996; Bray et al., 2005]. Hence, the external sources of DNA damage have become one of the cornerstones of plant DNA damage repair studies. Although DNA replication is highly conservative and chromosome integrity is strictly controlled, genomes are not static in space and time, and DNA molecules are subject to various processes orchestrated in developmental and environmental context. This is achieved by the combined action of trans-acting factors and local (cis) components comprising DNA sequence and chromatin, i.e. a complex of DNA and associated proteins (mainly histones). Histones occur in specific variants, and their Nterminal tails are post-translationally modified in order to define specific expression-permissive or -repressive chromatin states. In plants, this complex arrangement is set up and controlled by the action of more than 500 chromatin-related regulatory genes [Gendler et al., 2008]. Here, we summarize information on drugs commonly used in chromatin and DNA damage repair studies which Ales Pecinka Department of Plant Breeding and Genetics Max Planck Institute for Plant Breeding Research Carl-von-Linné-Weg 10, DE–50829 Cologne (Germany) E-Mail pecinka @ mpipz.mpg.de
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Department of Plant Breeding and Genetics, Max Planck Institute for Plant Breeding Research, Cologne, Germany
Chemical Inducers of DNA Damage
Chemicals that induce DNA damage have frequently been applied to study DNA repair in both prokaryotes and eukaryotes, and to suppress viruses, neoplastic diseases and the immune system in mammals, including humans [Deans and West, 2011; Fu et al., 2012]. They also provide an alternative tool to study the mechanism of DNA replication, to compensate for the limitations of mutant selection and the complicated process of genetic analysis. The DNA damaging agents most commonly used in plant studies are described below and an overview is provided in table 1. Replication Blocking Agents Deoxyribonucleotide triphosphates (dNTPs) are the building blocks of DNA. Interference with dNTP biosynthesis may limit the progression of DNA replication [Christopherson et al., 2002]. Hydroxyurea (HU) reduces dNTP levels by inhibiting the activity of the small subunits of RIBONUCLEOTIDE REDUCTASE (RNR), an enzyme which catalyzes the reduction of ribonucleotide diphosphates (NDPs) into deoxyribonucleosides (dNDPs), thus affecting the progression of replication fork and inducing DNA replication stress [Roa et al., 2009]. Recently it has been found in Arabidopsis that HU inhibits also CATALASE 2 and thus interferes with the decomposition of hydrogen peroxide and increases oxidative stress [Juul et al., 2010]. In plants, HU transiently arrests the cell cycle at the transition between G1/S and G2/M, providing a useful tool for cell-cycle synchrony [Pan et al., 1993; Cools et al., 2010], and has been utilized to identify the components involved in cell-cycle control [Cools et al., 2011; Spadafora et al., 2011]. The eukaryotic DNA polymerase α is essential for primer synthesis during replication of both DNA strands [Srivastava and Busbee, 2003]. Its inhibition by aphidicolin [Spadari et al., 1982] has been used for cell-cycle synchrony and understanding DNA-replication checkpoints in plants [Francis, 2011; Cook et al., 2013]. Signaling of DNA damage repair in response to both HU and aphidicolin is mainly mediated by ATAXIA TELANGIECTASIA MUTATED AND RAD3-RELATED (ATR) kinase [Culligan et al., 2004; De Schutter et al., 2007]. 52
Cytogenet Genome Res 2014;143:51–59 DOI: 10.1159/000360774
Inhibitor of DNA Synthesis in Organelles DNA gyrase is the bacterial DNA topoisomerase II that negatively supercoils DNA in the presence of adenosine triphosphate (ATP). In plant cells, it occurs in chloroplasts and mitochondria [Wall et al., 2004]. Chemical inhibition of plant gyrase in chloroplasts by novobiocin [Mills et al., 1989] has recently opened the possibility for studying DNA repair mechanisms in plant organelles [Cappadocia et al., 2010]. Alkylating Agents Alkylating agents transfer alkyl groups onto a broad range of biomolecules. They react with the ring nitrogens (N) and extracyclic oxygens (O) in DNA and generate a variety of covalent adducts [Fu et al., 2012]. Traditionally, they are described as either monofunctional or bifunctional based on the number of reactive sites they contain. Monofunctional Alkylating Agents Ethyl methanesulfonate (EMS) ethylates the N7- or O6-position of guanine, which leads to frequent G/ C→A/T transitions [Sega, 1984; Kim et al., 2006]. In plants, EMS is essential for generating mutagenized populations for mutant screens and novel breeding lines [Hartwig et al., 2012]. EMS-induced mutations can be efficiently detected at the genome-wide scale, allowing for rapid and cost-saving mutation mapping [Abe et al., 2012; Nordstrom et al., 2013]. 1-Ethyl-1-nitrosourea (ENU) can transfer an ethyl group to nucleophilic sites of cellular constituents and a carbonyl group to an amino group of a protein. It alkylates the O6-position of guanine and the O4-position of thymine in DNA, leading to AT→TA transversions and AT→GC transitions [Shibuya and Morimoto, 1993]. ENU is frequently used in mammalian mutagenesis, owing to its high-efficiency induction of point mutations in the male germline [Nolan et al., 2002]. In plants, ENU has been used as a positive control substance in genotoxicity studies [Gichner, 2003], and in TILLING (targeting induced local lesions in genomes) approaches [Cooper et al., 2013]. Methyl methanesulfonate (MMS) predominantly methylates DNA on N7-guanine and N3-adenine. The former leads to depurination followed by formation of a toxic apurinic site, while the latter is highly cytotoxic due to its blocking of most DNA polymerases and inhibition of DNA synthesis [Beranek, 1990; Fu et al., 2012]. MMS produces T/A→G/C transversions and A/T→G/C transitions [Lundin et al., 2005]. MMS has been widely used for studying DNA damage repair in plants, particularly in the Pecinka/Liu
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interfere with chromatin structure and/or cause specific types of DNA lesions. We highlight practical aspects of using these compounds in a variety of plant species, molecular mechanisms of their action and known side effects.
Table 1. Major drugs used in plant DNA damage repair studies Drugs Replication blocking agents Hydroxyurea (HU) Aphidicolin
Mechanism of action
Induced damage
Key references
RIBONUCLEOTIDE REDUCTASE and CATALASE 2 inhibition DNA POLYMERASE α inhibition
dNTP pool depletion replication blocking
De Schutter et al., 2007; Cools et al., 2010; Spadafora et al., 2011 Cook et al., 2013
replication blocking
Cappadocia et al., 2010
AT→TA transversions, AT→GC transitions GC→AT transitions
Gichner et al., 2003; Cooper et al., 2013
Inhibitor of DNA synthesis in organelles Novobiocin inhibition of DNA GYRASE in organelles Alkylating agents 1-Ethyl-1-nitrosourea
ethylation of bases (mostly T)
Ethyl methanesulfonate
ethylation of G→O6-ethylG
Methyl methanesulfonate
methylation of N7- and O6-dG, N3-dA and N4-dT G-specific 5′-CpG-3′ double-alkylation
Mitomycin C Cisplatin Radiomimetic drugs Bleomycin (bleocin) Zeocin
1,2-intrastrand d(GpG) and d(ApG) adducts metal-dependent free radical production most likely similar to bleomycin
TA→GC transversions, AT→GC transitions G-G or G-A interstrand crosslinks G-G or G-A intrastrand crosslinks
Kim et al., 2006; Abe et al., 2012; Nordstrom et al., 2013 Bagherieh-Najjar et al., 2005; Mannuss et al., 2010 Bleuyard et al., 2005; Mannuss et al., 2010; Da Ines et al., 2013 Jamieson and Lippard, 1999; Mannuss et al., 2010
DSB, SSB at 5′-GC-3′ or 5′-GT-3′ sequences DSB
Ma et al., 2009; Pecinka et al., 2009; Lang et al., 2012 Adachi et al., 2011
DSB = Double strand break; SSB = single strand break.
Bifunctional Alkylating Agents Bifunctional alkylating agents contain 2 active moieties that can react with separate bases of DNA to form bi-adducts [Fu et al., 2012]. The most commonly used bifunctional alkylating agent in plant research is mitomycin C (MMC). In cells, MMC becomes reduced and transformed into vinylogous quinone methide with high alkylating activity [Tomasz, 1995]. It then reacts with N7- or O6-guanine in DNA to form an MMC-mono-dG adduct that can subsequently react with another DNA base to form guanine-guanine (G-G) and/or guanine-adenine (G-A) interstrand crosslinks [Fu et al., 2012]. Application of MMC (alone or in combination with other drugs) readily induces expression of DNA damage repair genes (AtGenExpress dataset ME00326). MMC has been used as one of the major marker drugs for associating gene functions with DNA damage repair, DNA replication and cell-cycle control. The repair of MMC-induced interstrand crosslinks is thought to be mediated mainly by ATR and RAS ASSOCIATED WITH DIABETES PRODrugs for Chromosome Research
TEIN 51 (RAD51)-dependent HR pathways [Bleuyard et al., 2005; Nezames et al., 2012; Da Ines et al., 2013]. Alkylating-Like Agents These drugs induce crosslinks without DNA alkylation. Cisplatin is composed of a central platinum atom surrounded by 2 chlorine atoms and 2 ammonia groups. Upon hydrolysis of chloride ligands, it forms mainly DNA intrastrand and rarely also interstrand crosslinks between the N7-positions of purine bases [Siddik, 2003]. The lasting activity of cisplatin is hampered by the short 2-hour half-time of hydrolysis and partial cytoplasmic inactivation by interactions with endogenous nucleophiles and proteins [Jamieson et al., 1999]. In plant studies, it is commonly used as an intrastrand DNA crosslinker to investigate the ATR-dependent repair of bulky DNA lesions via HR [Osakabe et al., 2006; Abe et al., 2009; Mannuss et al., 2010; Nezames et al., 2012] and base excision repair [Liang et al., 2006]. Radiomimetic Drugs Short wavelength and high energy radiation (gamma radiation and X-rays) have traditionally been used in mutagenesis and DNA damage repair experiments. These Cytogenet Genome Res 2014;143:51–59 DOI: 10.1159/000360774
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homologous recombination (HR) pathway [BagheriehNajjar et al., 2005; Kim, 2006; Yin et al., 2009; Mannuss et al., 2010].
Table 2. Drugs for chromatin research DNA Histone H3 modificationsa methylation K4me3 K9me2 K27me3
Inhibitors
Chromatin Transposon Gene excondensation expression pression
Pol II Key references activity
Methyl-group biosynthesis inhibitors and methionine antagonists Sulfamethazine n.a. ↓ ↓ S-Dihydroxypropyladenine ↓ ↑ ↓ 3-Deazaneplanocin n.a. n.a. n.a. L-Ethionine n.a. n.a. ↓
n.a. n.a. n.a. n.a.
n.a. n.a. n.a. ↓
↑ ↑ ↑ n.a.
n.a. n.a. n.a. n.a.
n.a. n.a. n.a. n.a.
Zhang et al., 2012 Baubec et al., 2010 Foerster et al., 2011 Fajkus et al., 1992
DNA methylation inhibitors Zebularine 5-Azacytidine
↓ ↓
↑ n.a.
↓ ↓
n.a. n.a.
↓ ↓
↑ ↑
n.a. ↑ 73; ↓ 52b
n.a. n.a.
Baubec et al., 2009 Chang and Pikaard, 2005
−
−
↓
n.a.
n.a.
−
↑ 2; ↓ 34b
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
↑ 138c
n.a.
Chang and Pikaard, 2005; Baubec et al., 2010 Grozinger et al., 2001; Zhao et al., 2003
Inhibitors of RNA polymerase II α-Amanitin Actinomycin D
n.a. n.a.
n.a. n.a.
n.a. n.a.
n.a. n.a.
n.a. n.a.
n.a. n.a.
Unknown mode of action Genistein
↓
n.a.
n.a.
n.a.
n.a.
↑
Histone deacetylase inhibitors Trichostatin A and sodium butyrate Sirtinol (putative)
n.a. n.a. n.a.
↓ ↓
Haag et al., 2012 Narsai et al., 2007
n.a.
Arase et al., 2012
↑ = Increase; ↓ = decrease; − = no change; n.a. = not analyzed. At normally silenced loci. b Out of 7,800 Arabidopsis genes analyzed [Chang and Pikaard, 2005]. c Out of 23,000 Arabidopsis genes analyzed, number of down-regulated genes not specified [Zhao et al., 2003]. a
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the same family. The induction of breaks has been described in detail for bleomycin: upon activation by a metal ion, bleomycin abstracts H from C4′ of the 2-deoxyribose moiety to induce single-strand breaks, doublestrand breaks, and abasic sites at 5′-GT-3′ and 5′-GC-3′ sequences [Povirk, 1996; Ma et al., 2009]. Radiomimetic drugs damage DNA independently of cell cycle stage, effectively induce expression of DNA damage repair genes and inhibit cell growth [Houser et al., 2001; De Schutter et al., 2007; Pecinka et al., 2009; Adachi et al., 2011]. They are frequently employed as markers for studying ATAXIA TELANGIECTASIA MUTATED (ATM)-mediated double-strand break repair [Lang et al., 2012].
Inhibitors Interfering with Chromatin
Interference with epigenetic marks and chromatin structure (fig. 1) has been employed in a variety of plant studies (table 2). It has been particularly useful in cases where genetic mutations were not available or caused lethality, conditional interference was needed, or where siPecinka/Liu
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types of radiation induce predominantly DNA strand breaks while chemicals introduce mainly non-native bonds. The effect of radiation is direct, instant and more equally distributed within the plant body [Grigaravičius et al., 2009], compared to the damage caused by chemicals that can be potentially highly dependent on the uptake and distribution of a drug over individual tissues. However, sources of high energy radiation may be costly and inconvenient for some plant researchers. Therefore, many laboratories substitute short wavelength irradiation with radiomimetic (radiation resembling) drugs. By inducing highly specific attack of free radicals, these drugs abstract hydrogen (H) predominantly at the C1-, C4-, or C5-positions, leading to oxidation of 2-deoxyribose in DNA and DNA strand breaks. It should be noted that the lesions induced by radiomimetic drugs are only a small subset of the lesions induced by ionizing radiation, yet their effects on cells are remarkably similar [Povirk, 1996]. The most commonly used radiomimetic drugs in plant research are bleomycin (commercially supplied also as bleocin) and zeocin, glycopeptide-derived antibiotics of
terfere with chromatin marks. The black and the white lollipops show the methyl group (-CH3) and the acetyl group, respectively. S-DHPA = S-dihydroxypropyl adenine; DZnep = 3-deazaneplanocin A; Pol II = RNA polymerase II; HDA = a class I histone deacetylase; SAHH = S-adenosyl homocysteine hydrolase.
multaneous inhibition of multiple enzymes of the same family or several pathways was required. Methyl-Group Biosynthesis Inhibitors and Methionine Antagonists The methyl-group is widely implicated in many biochemical processes in plants. It plays a central role in epigenetic signaling and is used to modify DNA, RNA and proteins. Depending on the context and type of modification, it can have transcription-permissive or -repressive potential (see also DNA Methylation Inhibitors). The methyl-group precursor is synthesized via dihydrofolate and methionine cycles. Sulfamethazine is an inhibitor of DIHYDROPTEROATE SYNTHASE, an enzyme that catalyzes the synthesis of dihydropteroic acid, an early step in methyl-group biosynthesis within the dihydrofolate cycle [Zhang et al., 2012]. The second group of inhibitors includes S-dihydroxypropyl adenine and 3-deazaneplanocin A that block activity of the S-ADENOSYLHOMOCYSTEINE HYDROLASE in the methionine cycle. The third group consists of methionine antagonists, i.e. non-proteinogenic amino acids related to methionine that interfere with the methionine cycle. The most commonly used methionine antagonist is L-ethionine that contains an ethyl group at the position of the Drugs for Chromosome Research
DNA Methylation Inhibitors The methylation of cytosine at the 5th position is a prominent epigenetic mark present in many groups of eukaryotes. In plants, it occurs in all sequence contexts (CG, CHG and CHH, where H is any base but G) and functionally has been implicated mainly in silencing of repetitive DNA [Feng et al., 2010; Stroud et al., 2013]. It is deposited by several DNA methyltransferases that show preference for methylating cytosines in particular DNA sequence contexts and act via different mechanisms [Stroud et al., 2013]. In contrast to mammals where DNA methylation is re-established in early stages of every generation, plant DNA methylation is faithfully transmitted throughout development [reviewed in e.g. Feng et al., 2010]. There are a number of genes known to affect DNA methylation in plants, but many show severe developmental and fitness phenotypes [Miura et al., 2001; Mathieu et al., 2007]. Two drugs are commonly used for interference with DNA methylation in plants: 5-azacytidine and zebularine. Both are non-methylable cytosine analogs with identical mode of action, but their stability differs greatly. While 5-azacytidine has a half-life of only 4 hours and needs to be supplied several times in course of a standard experiment, zebularine’s half-life is over 3 weeks [Champion et al., 2010], making it the preferred inhibitor in plant studies. In planta, cytosine analogs are phosphorylated by uriCytogenet Genome Res 2014;143:51–59 DOI: 10.1159/000360774
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Fig. 1. Schematic representation of drugs (labeled in red) that in-
methionine methyl group and induces DNA demethylation in both plants and animals [Cox and Irving, 1977; Fajkus et al., 1992]. All these inhibitors have broad effects: they reduce DNA methylation in all sequence contexts at a number of tested targets, histone methylation at histone H3 lysine 9 and 27 (and most likely also other methylated residues) and promote expression of otherwise silenced genetic elements [Fajkus et al., 1992; Kovařík et al., 2000; Koukalová et al., 2002; Majerová et al., 2011; Zhang et al., 2012]. The broad range of side-effects limits the use of these drugs, particularly the interference with many nonepigenetic processes that require the methyl-group such as chlorophyll biosynthesis. However, they can be useful in situations when global interference is required. For example, 3-deazaneplanocin A and S-dihydroxypropyl adenine were used to mimic loss of the S-ADENOSYL HOMOCYSTEINE HYDROLASE, since its genetic mutations are normally lethal for plants [Baubec et al., 2010], or to interfere with silencing that required simultaneous knockout of several repressive pathways [Foerster et al., 2011].
Histone Deacetylase Inhibitors Acetylation of histone tails at specific residues is a transcription-promoting epigenetic mark deposited by histone acetyltransferase enzymes and removed by histone deacetylases (HDAs). Hence HDAs contribute to the maintenance of the repressive state. There are 3 types of plant HDAs: type I HDAs of the RPD3-like superfamily, which correspond to classes I and II in mammals; plantspecific type II HDAs, HD-tuins; and type III HDAs, sirtuins [reviewed by Hollender and Liu, 2008]. 56
Cytogenet Genome Res 2014;143:51–59 DOI: 10.1159/000360774
Type I HDAs have been implicated in global transcriptional regulation in developmental fashion, but specific genomic targets of individual enzymes remain a matter of debate. The best-characterized are type I HDAs: HDA6 and HDA19. HDA6 has been associated preferentially with transcriptional gene silencing and control of gene expression at heterochromatic regions, and to a lesser extent within genes [Probst et al., 2004; Tanaka et al., 2008]. In contrast, HDA19 seems to control developmentally regulated genes during specific developmental stages such as germination [Tanaka et al., 2008]. The most commonly used inhibitors of type I HDAs are trichostatin A (TSA) and sodium butyrate. Whole genome transcriptome analysis revealed that there are only few genes deregulated in common between TSA and 5-azacytidine, and that TSA has a weaker potential for activating transcriptionally silenced genetic elements [Chang and Pikaard, 2005]. In contrast, TSA affected developmentally regulated genes, such as those controlling germination and root patterning [Xu et al., 2005; Tanaka et al., 2008], most likely by inhibition of HDA19. This indicates that TSA could potentially interfere with polycomb-based silencing, and its effects on the levels of H3K27me3 should be tested in the future. The target genes of sirtuin HDAs are currently unknown in plants [Hollender and Liu, 2008]. Several sirtuin inhibitors have been found by chemical genetic screening in Saccharomyces cerevisiae, and the initial experiments with the compound sirtinol revealed disturbed body axis formation, vascularization problems and hypocotyl thickening in Arabidopsis [Grozinger et al., 2001]. Furthermore, sirtinol treatment led to upregulation of genes involved in auxin signaling, and several auxin signaling mutants had been found insensitive to this treatment [Zhao et al., 2003; Dai et al., 2005]. However, it has to be noted that the connection of these phenotypes with SIRTUIN (SRT) function is only indirect and so far not substantiated by data produced using srt mutants. An RNAi knockdown of SRT1 in rice led to increased oxidative stress, programmed cell death and release of transposon silencing [Huang et al., 2007] which differs from the sirtinol effects in Arabidopsis. Hence, the use of yeastderived sirtuin inhibitors in plant studies still requires careful testing and comparisons with SRT loss-of-function mutants. Inhibitors of RNA Polymerase II (Pol II) There are 5 DNA-dependent RNA polymerases (Pol I–V) in plants. Pol I and Pol III transcribe rDNA and tRNA genes while Pol II transcribes protein-coding genes. Pecinka/Liu
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dine/cytidine kinase and incorporated into both RNA and DNA, and lead to loss of DNA methylation in all sequence contexts. Drug-mediated interference with DNA methylation results in pleiotropic phenotypes (similar but not identical to genetic mutations) and reduces e.g. levels of H3K9me2 and compromises heterochromatin condensation [Baubec et al., 2009, 2010]. However, genome-wide transcriptome profiling of Arabidopsis plants treated with 5-azacytidine revealed a complex transcriptional response that could not easily be associated with targets controlled by DNA methylation [Chang and Pikaard, 2005]. This is supported by the observations that low doses of zebularine transcriptionally activate transposons without reducing their DNA methylation levels [T. Baubec, A. Finke, O. Mittelsten Scheid, A. Pecinka, unpubl. data]. The DNA de-methylation by zebularine is strongly opposed by de novo DNA methylation activity of the RNA-directed DNA methylation (RdDM) pathway in the apical meristems [Baubec et al., 2014]. This indicates that cytosine analogs may work to some extent by interference with chromatin structure and/or have other previously unnoticed effects. Furthermore, DNA methylation inhibitors reduce plant growth. The most likely cause is the covalent binding of DNA methyltransferases to incorporated DNA methylation inhibitors leading to formation of nucleoprotein adducts [Champion et al., 2010]. This represents a physical barrier to the progression of RNA and DNA polymerases, which is recognized as DNA damage by plants and requires repair [C.-H. Liu, A. Pecinka, unpubl. data]. The negative side effects of DNA methylation inhibitors could potentially be eliminated by use of specific inhibitors of DNA methyltransferases. In mammals, the drug RG108 efficiently inhibits DNA methyltransferase 1 (DNMT1), an ortholog of plant MET1 enzyme [Brueckner et al., 2005]. However, adopting such drugs for the plant field may not be straightforward. For example, RG108 did not activate DNA methylated reporter constructs that are steadily activated by zebularine or 5-azacytidine [A. Pecinka, unpubl. data].
Pol IV and Pol V are plant-specific polymerases that are closely related to Pol II (Pol II-group), and have been implicated in silencing of repetitive DNA by the mechanism of RdDM [reviewed in Haag and Pikaard, 2011]. Since Pol II activity is essential, use of the mutants is not straightforward. Instead, there are several compounds used as Pol II inhibitors. The most common ones are actinomycin D, which blocks polymerase transcriptional elongation, and α-amanitin, which drastically reduces the rate of polymerase processivity. Application of Pol II inhibitors is highly toxic and leads to cell death. Nevertheless, they have been indispensable in studying mRNA decay-related processes in plants [Narsai et al., 2007]. Importantly, α-amanitin does not inhibit transcription of Pol IV. This allowed the nature of transcripts produced by Pol IV and interactions of individual components of the RdDM during double-stranded RNA synthesis to be determined [Haag et al., 2012].
Concluding Remarks
This article reviews the information on the most commonly used chemicals in plant chromatin and chromosome research, with particular focus on those applied in the studies of DNA damage repair and epigenetics. Although chemical inducers of DNA damage or drugs affecting particular chromatin marks have been proven to be useful in plant chromosome and chromatin studies, there are several major points to be considered. First, most of the DNA damaging drugs induce more than one type of DNA damage, which may activate different repair pathways, and estimating the proportions in which different damages occur may be difficult. This may be misleading and therefore requires careful analysis using mul-
tiple methods. Similarly, chromatin inhibitors (in particular those interfering with methyl group biosynthesis) may have a broad effect and affect multiple chromatin marks as well as chromatin structure. However, it has to be noted that such pleiotropic effects are, to some extent, similar to those observed with mutants of the key methylation controlling components such as MET1 and DECREASE IN DNA METHYLATION 1 (DDM1) [Miura et al., 2001; Mathieu et al., 2007]. Therefore, we suggest preferentially using low concentrations of the most specific drugs. In conclusion, it is clear that chemical inhibitors cannot replace the advantage of genetic mutations, but in many cases they can be a complementary or even indispensable tool. Furthermore, chemical inhibitors have been fundamental in discovering novel genes and gene functions in both DNA damage repair and chromatin studies. However, in contrast to human field, where almost all drugs discussed here are used for clinical studies in cancer treatments, only few plant studies went beyond the level of basic research so far [Abe et al., 2012; Zhong et al., 2013]. Another challenge remains in finding compounds that have highly specific effects and/or inhibit only a single enzyme, while having no side effects. Finding novel drugs with specific functions will be useful to expand our understanding of plant genome and chromosome biology.
Acknowledgements We apologize to all colleagues whose work could not be mentioned due to the space limitations and thank M. Fojtova for valuable discussions and comments and T. Harrop for editing the manuscript. The authors were funded by the Max Planck Society.
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