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UV-B Radiation Induces Mu Element Somatic Transposition in Maize

2013; Elsevier BV; Volume: 6; Issue: 6 Linguagem: Inglês

10.1093/mp/sst112

1674-2052

Julia I. Qüesta, Virginia Walbot, Paula Casati,

CRISPR and Genetic Engineering

Dear Editor, The maize Mutator (MuDR/Mu) transposon family is the most active DNA transposon in plants (Lisch, 2002Lisch D Mutator transposons.Trends Plant Sci. 2002; 7: 498-504Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). The Mu family contains diverse elements, all sharing similar ~215-bp terminal inverted repeats (TIRs). MuDR is the autonomous element, and a transcriptionally active MuDR is required for transposition of the non-autonomous Mu elements (Chomet et al., 1991Chomet P. Lisch D. Hardeman K.J. Chandler V.L. Freeling M Identification of a regulatory transposon that controls the Mutator transposable element system in maize.Genetics. 1991; 129: 261-270PubMed Google Scholar). MuDR contains two genes, mudrA and mudrB (Figure 1A); mudrA encodes the MURA transposase, and mudrB encodes a protein with unknown function. To avoid the deleterious effects of transposons, plants have acquired mechanisms to epigenetically silence them. Silenced Mu elements become heavily methylated, and their reactivation is very rare. To date, only UV-B radiation treatments have reactivated silenced Mu (Walbot, 1999Walbot V UV-B damage amplified by transposons in maize.Nature. 1999; 397: 398-399Crossref PubMed Scopus (57) Google Scholar). We previously demonstrated that UV-B radiation modifies the chromatin at MuDR/Mu loci, triggering the expression of the transposase (Questa et al., 2010Questa J.I. Walbot V. Casati P Mutator transposon activation after UV-B involves chromatin remodeling.Epigenetics. 2010; 5: 352-363Crossref PubMed Scopus (30) Google Scholar). MURA transposase interacts in vitro with a 32-bp domain, highly conserved in all mobile Mu TIRs (Benito and Walbot, 1997Benito M.I. Walbot V Characterization of the maize Mutator transposable element MURA transposase as a DNA-binding protein.Mol. Cell Biol. 1997; 17: 5165-5175PubMed Google Scholar). Furthermore, MURA can bind unmethylated, methylated, and hemimethylated forms of its target sequence; therefore, the production of MURA is the rate-limiting step in reactivation of silent Mu elements. In the present work, we aim to elucidate the in vivo binding of MURA to its target sequence and whether this interaction is affected by UV-B radiation. We used a Mutator active line with bz2-mu4 reporter allele (Figure 1D) and an epigenetically silencing sister line. Silencing plants are in the process of losing activity and exhibit a reduced frequency of somatic excision from reporter alleles. Plants were exposed to UV-B lamps over 8h, and leaf samples were collected after treatment. Control plants were treated in parallel using lamps covered with a UV-B filter. We performed Chromatin Immunoprecipitation (ChIP) using an antibody specific for MURA (Rudenko and Walbot, 2001Rudenko G.N. Walbot V Expression and post-transcriptional regulation of maize transposable element MuDR and its derivatives.Plant Cell. 2001; 13: 553-570Crossref PubMed Scopus (47) Google Scholar) followed by quantitative PCR (qPCR) to assess the binding of MURA to MuDR TIRs in somatic leaf tissues (Figure 1A–1C). Under normal growing conditions, MURA is bound to some MuDR TIRs in Mutator active plants (Figure 1B). However, in silencing plants, we could not detect enrichment of MURA bound to MuDR TIRs (Figure 1C). Interestingly, UV-B radiation induces a significant increase in the number of TIRs occupied by MURA, in both active and silencing plants. Transposase binding is highly sensitive to its concentration (Lavoie et al., 1991Lavoie B.D. Chan B.S. Allison R.G. Chaconas G Structural aspects of a higher order nucleoprotein complex: induction of an altered DNA structure at the Mu-host junction of the Mu type 1 transpososome.EMBO J. 1991; 10: 3051-3059Crossref PubMed Scopus (138) Google Scholar); thus, we wondered whether the increase of TIRs occupied by MURA reflected a higher amount after UV-B exposure. We failed to detect higher levels of MURA after 8-h UV-B treatment (Supplemental Figure 1), thereby excluding an effect of UV-B in elevating protein concentration. Alternatively, we propose that UV-B radiation modulates chromatin in the vicinity of Mu elements as a first step and that this is a pre-requisite for MURA binding. Additionally, MURA transposase may be undergoing posttranslational modifications that influence in vivo binding avidity to its target sites. After we demonstrated that UV-B induces the enrichment of MURA in chromatin where Mu elements reside in somatic tissues, we next asked whether this led to transposition. It was previously proposed that MuDR/Mu elements transpose by a cut-and-paste mechanism in somatic cells, in which each insertion is preceded by a corresponding excision (Raizada et al., 2001Raizada M.N. Nan G.L. Walbot V Somatic and germinal mobility of the RescueMu transposon in transgenic maize.Plant Cell. 2001; 13: 1587-1608Crossref PubMed Scopus (82) Google Scholar). We first tested whether UV-B induced Mu somatic excisions in tissues that are directly exposed to the radiation treatment. For this aim, we used two different maize reporter alleles that have either Mu1 or MuDR elements inserted into the bz2 gene, bz2-mu2, and bz2-mu4, respectively (Figure 1D). We confirmed the locations of Mu1 and MuDR in these reporter alleles by PCR, using specific oligonucleotides represented with arrows in Figure 1D (Supplemental Figure 2). bz2-mu2//bz2 and bz2-mu4//bz2 heterozygous plants were exposed to UV-B lamps for 8 h and leaf samples were collected 24 h after the end of the treatment. The excision frequency of Mu from its location in each reporter allele was analyzed by qPCR and a genomic sequence of a thiorredoxin H-type gene (GRMZM2G082886) was used as an internal control to normalize the amount of genomic DNA. The occupancy of Mu1 and MuDR elements inserted into bz2 decreases in a statistically significant manner after UV-B treatment (Figure 1E), demonstrating that UV-B increases Mu element somatic excision frequency. In addition, we investigated whether UV-B also induced somatic insertions. For this purpose, we used plants that carry a copy of a modified Mu1 element, RescueMu (Raizada et al., 2001Raizada M.N. Nan G.L. Walbot V Somatic and germinal mobility of the RescueMu transposon in transgenic maize.Plant Cell. 2001; 13: 1587-1608Crossref PubMed Scopus (82) Google Scholar). The pBluescript-containing RescueMu transposon (Supplemental Figure 3A) can be readily recovered by a procedure called plasmid rescue (Nan and Walbot, 2009Nan G.L. Walbot V Plasmid rescue: recovery of flanking genomic sequences from transgenic transposon insertion sites.Methods Mol. Biol. 2009; 526: 101-109Crossref PubMed Scopus (7) Google Scholar). In brief, this is a technique for recovering bacterial plasmids from transgenic eukaryotic genomic DNA utilizing antibiotic selection in a prokaryotic host. Total maize DNA was first digested with restriction enzyme(s), ligated, and then transformed into Escherichia coli cells plated on selective media (100 mg L–1 carbenicillin). For this study, we selected plants which had RescueMu inserted in a known genomic region called GP1 (Supplemental Figure 3A). We corroborated that RescueMu was inserted in the expected location by PCR using specific oligonucleotides that bind to RescueMu and to the flanking GP1 region (Supplemental Figure 3B). We then tested whether individual plants were Mutator active by measuring mudrA transcript levels by qRT–PCR; active plants have higher levels of mudrA transcripts than silencing plants (Supplemental Figure 3C). Plants confirmed to be active Mutator and carriers of the RescueMu allele were irradiated with UV-B for 8 h. Leaf samples were taken before (control) and immediately after the end of the treatment (UV-B) and then processed for plasmid rescue. A crucial step in a successful rescue is minimizing contamination. Therefore, we tested that the colonies obtained contained RescueMu plasmids. We randomly selected colonies and performed plasmid extraction; plasmids were then digested with BglII, BamHI, and MluI. As shown in Figure 1F, MluI releases a 4.323-kbp fragment containing most of the RescueMu construct. Once we determined that the plasmid rescue was successful, we performed Southern blots with the digestion products shown in Figure 1F using a probe that binds specifically to the GP1 region (Figure 1G and Supplemental Figure 4A). While all lanes show the 4.323-kbp fragment containing the RescueMu construct, the amount of plasmids containing the GP1 insert was reduced after UV-B treatment, suggesting that RescueMu somatic insertion to different genome sites is induced by UV-B. Moreover, we performed colony lift hybridization using the same GP1 probe (Supplemental Figure 4B–4D). With this technique, we could differentiate the colonies containing the original GP1 insertion from those that corresponded to RescueMu inserted in other regions. Briefly, a nylon membrane disc was laid on top of the agar plates containing RescueMu colonies to permit transfer of a colony sample to the membrane (Supplemental Figure 4B). The nylon disc was then hybridized with the GP1 probe and exposed to an X-ray film to visualize those colonies that carry the GP1 sequence (Supplemental Figure 4C). We then counted and compared the total number of colonies obtained and the number of colonies that were recognized by the probe (GP1 colonies; Supplemental Figure 4D). In this comparison, we counted the number of colonies that contained new insertions (cold colonies; Supplemental Figure 4D) different from GP1. Similarly to what we observed in the Southern blots, we found a higher number of new RescueMu insertion events after 8-h UV-B. The sequence analysis of some RescueMu plasmids, isolated from both GP1 and cold colonies (Supplemental Figure 5), confirmed that the cold colonies carried diverse maize nuclear genomic sequences into which RescueMu had inserted. In summary, our data demonstrate that UV-B radiation increases the binding of MURA to its target site within MuDR TIRs, and this is true in both active and silencing Mutator lines. UV-B radiation is a major source of DNA damage and therefore chromatin dynamics must be accurately synchronized to assure DNA repair. We hypothesize that this remodeling during DNA repair increases the likelihood that MURA can bind to its target sequence. On the other hand, transposable element mobilization is typically deleterious, causing deletions, genomic rearrangements, and gene misregulation. Rarely, excisions and insertions generate selectively advantageous novelty in the somatic cells or gametes. Considering plants are sessile organisms, we can also understand transposition as an important tool in evolutionary adaptation. The amount of variation conferred by transposon mutagenic effects may allow plants to adapt to adverse environments. In this context, we can speculate that UV-B-induced Mutator transposition is being maintained through selection as a source of genomic variation in maize. Supplementary Data are available at Molecular Plant Online. J.I.Q. was awarded a Fulbright Commission–Bounge & Born Foundation fellowship for a research stay at Stanford University, USA. This research was supported by FONCyT grants PICT 2007-00711 and 2010-00105 to P.C.