The radioresistance kinase TLK1B protects the cells by promoting repair of double strand breaks
© Sunavala-Dossabhoy et al; licensee BioMed Central Ltd. 2005
Received: 15 March 2005
Accepted: 12 September 2005
Published: 12 September 2005
The mammalian protein kinase TLK1 is a homologue of Tousled, a gene involved in flower development in Arabidopsis thaliana. The function of TLK1 is not well known, although knockout of the gene in Drosophila or expression of a dominant negative mutant in mouse cells causes loss of nuclear divisions and missegregation of chromosomes probably, due to alterations in chromatin remodeling capacity. Overexpression of TLK1B, a spliced variant of the TLK1 mRNA, in a model mouse cell line increases it's resistance to ionizing radiation (IR) or the radiomimetic drug doxorubicin, also likely due to changes in chromatin remodeling. TLK1B is translationally regulated by the availability of the translation factor eIF4E, and its synthesis is activated by IR. The reason for this mechanism of regulation is likely to provide a rapid means of promoting repair of DSBs. TLK1B specifically phosphorylates histone H3 and Asf1, likely resulting in changes in chromatin structure, particularly at double strand breaks (DSB) sites.
In this work, we provide several lines of evidence that TLK1B protects the cells from IR by facilitating the repair of DSBs. First, the pattern of phosphorylation and dephosphorylation of H2AX and H3 indicated that cells overexpressing TLK1B return to pre-IR steady state much more rapidly than controls. Second, the repair of episomes damaged with DSBs was much more rapid in cells overexpressing TLK1B. This was also true for repair of genomic damage. Lastly, we demonstrate with an in vitro repair system that the addition of recombinant TLK1B promotes repair of a linearized plasmid incubated with nuclear extract. In addition, TLK1B in this in vitro system promotes the assembly of chromatin as shown by the formation of more highly supercoiled topomers of the plasmid.
In this work, we provide evidence that TLK1B promotes the repair of DSBs, likely as a consequence of a change in chromatin remodeling capacity that must precede the assembly of repair complexes at the sites of damage.
The Tousled gene of Arabidopsis thaliana encodes a protein kinase which, when mutated, results in abnormal flower development characterized by a stochastic loss of floral meristem and organs . Two mammalian Tousled-like kinases (TLK1 and TLK2) were cloned by Sillje et al., 1999  during a PCR-based search for human kinases, who also reported that the activity of these kinases is maximal in S phase, and more recently, these kinases were reported to be targets of checkpoint kinases, ATM and Chk1 . Specifically, it was reported that TLK1 is inhibited by Chk1 by direct phosphorylation at S695. These findings identify a functional cooperation between ATM and Chk1 in propagation of a checkpoint response to DNA damage and suggest that through transient inhibition of TLK1 the ATM-CHK1-TLK pathway may regulate processes involved in chromatin assembly . Indeed, in AT cells (cells deficient in ATM protein) TLK1 was not inhibited after genotoxic stress . Since ATM and Chk1 are involved in the DNA damage checkpoint upon radiation, this suggests that TLKs may be involved in some aspect of genome surveillance, particularly chromatin remodeling concurrent with DNA repair (see below). The function of TLK1 is not well known, although knockout of the gene in Drosophila or expression of a dominant negative mutant in mouse cells causes loss of nuclear divisions and missegregation of chromosomes [5, 6] likely through changes in chromatin remodeling capacity. The importance of TLK1 in chromosome segregation was recently confirmed in C. elegans embryos .
We recently cloned a cDNA encoding a mammalian Tousled-like kinase (TLK1B) through a very different scheme than the one used by Sillje et al. , based upon polysomal redistribution of weakly translated transcripts that become preferentially recruited upon overexpression of eIF4E . Indeed, the human TLK1B mRNA (a splice variant of the TLK1 mRNA cloned by Sillje et al.) contains a 5'UTR 1088-nt-long with two upstream AUG codons, which was found to be very inhibitory for translation [8, 9]. The inhibition of translation could be relieved by either overexpressing eIF4E, or by deleting a large section of the 5'UTR . We subsequently discovered that TLK1B overexpression protects the cells from the genotoxic effects of ionizing radiation (IR) or the radiomimetic drug, doxorubicin. TLK1B probably exerts these effects by phosphorylating histone H3 [8, 10] and the histone H3 chaperone Asf1 [10, 11], and thereby promoting chromatin remodeling concurrent with repair of DNA damage. Interestingly, synthesis of TLK1B is induced at the translation level by the presence of double strand breaks [DSBs; ].
The discovery that TLK1B is a kinase that phosphorylates histone H3 came first in a series of experiments aimed at identifying the potential substrates of TLK1B. We carried out kinase assays with recombinant GST-TLK1B and various typical substrates, only a few of which were phosphorylated efficiently. However, we found that TLK1B phosphorylated very well histone H3 at S10 but not the other core histones or H1 . We subsequently confirmed that the MM3MG cells overexpressing TLK1B had a higher constitutive level of H3 phosphorylation in asynchronous cells . We could also show genetically that TLK1B is a histone H3 kinase. We showed that inducible expression of TLK1B in a yeast strain carrying a temperature-sensitive allele of the major H3 kinase [Ipl-1; ] could rescue growth at the non-permissive temperature . In addition, it restored normal levels of histone H3 phosphorylation . Significantly, expression of TLK1B in yeast increased radioresistance, indicating a conservation of function and substrates. Interestingly, we also found that IR results in a loss of H3 phosphorylation, but the significance of this result was unclear. The dephosphorylation of H3 after IR was reported also by another group . Recently, a possible explanation for this effect was described [3, 4]. These authors showed that TLK1 (the larger isoform) is inhibited by γ-radiation. The inhibition is presumably mediated by ATM and Chk1 by direct phosphorylation at S695 . It seemed possible that physiologically the increased TLK1B synthesis following IR can help offset the loss of TLK1 activity resulting from IR and restore appropriate levels of histone H3 phosphorylation.
In this paper we present evidence that overexpression of TLK1B protects the cells from IR by facilitating the repair of DSBs, and that the steady state phosphorylation of H3 and H2AX is restored to pre-IR much more rapidly in TLK1B overexpressing cells.
TLK1B protects cells from IR
Inverse phosphorylation of H2AX and H3 after IR
Blocking ATM activity with wortmannin prevents the loss of H3 phosphorylation after IR
Episomal vectors as reporters for DSBs
To overexpress TLK1B, we used an episomal vector called BK-Shuttle . An important advantage is that these plasmids can be easily rescued from mammalian cells by the Hirt's supernatant protocol . Most stable cell lines carrying these vectors typically have hundreds of copies of the episomes, which have the typical structure of mini-chromosomes and thus behave like genomic DNA [20, 21]. We previously showed that the extraction of episomes is a very simple and efficient protocol that yields very consistent amounts of plasmids that are linear with respect to cell numbers 
Episomal vectors as reporters for DSB repair
Enhanced repair of genomic damage in TLK1B cells
RECOVERY TIME (hr)
MM3MG (number of breaks)
TLK1B (number of breaks)
13 +/- 0.35
11 +/- 2.83
11 +/- 0.64
5 +/- 1.06
7 +/- 1.91
4 +/- 1.27
Rapid repair and chromatin assembly in vitro
In Fig. 7B, we show an assay of supercoiling activity, which is a measure of chromatin assembly. In this assay, the plasmid Bluescript (nearly 100% supercoiled, input) is used as a template for the deposition of core histones in the presence of nuclear extract and an energy mix. In the absence of histones, the extract causes the bacterially supercoiled form to convert to mostly relaxed forms due to endogenous topoisomerases (data not shown). However, after incubation in the presence of histones, the plasmid migrates as a series of discrete supercoiled forms due to the formation of nucleosomes, which decrease the linking number by one integer per nucleosome. The addition of recombinant TLK1B stimulated the formation of the more highly supercoiled forms, particularly the form that runs like bacterially supercoiled plasmid.
In this work, we have provided four lines of evidence that the Tousled kinase, TLK1B, protects the cells from IR by facilitating the repair of DSBs. First, the pattern of phosphorylation/dephosphorylation of H2AX and H3 indicated that cells overexpressing TLK1B return to pre-IR phosphorylation state much more rapidly than controls. Second, the repair of episomes damaged with DSBs was much more rapid and complete by 8 hr of recovery in cells overexpressing TLK1B. Third, we have found that the repair of genomic breaks occurs more rapidly in cells overexpressing TLK1B, and with kinetics that are similar to those of repair of episomes. Lastly, we demonstrated with an in vitro repair system that the addition of recombinant TLK1B promotes repair of a linearized plasmid incubated with nuclear extract. Consistent with the results published by Groth  and Kodym  we found that TLK1 activity is inhibited by IR, as shown by loss of phosphorylation of histone H3, which is one of the best substrates of TLK1. Nonetheless, when TLK1B is overexpressed (about 6-fold in our stably transfected MM3MG cells), the recovery of H3 phosphorylation was quite rapid (about 4 hr) probably because of mass action due to higher levels of the kinase.
The role of TLK1B in radioresistance is particularly intriguing based on the recent findings that Asf1 is also a specific TLK1 substrate  known to participate with Rad53 in chromatin remodeling at sites of DSBs . Furthermore, the importance of histone H3 kinases cannot be overstated. Phosphorylation of H3 at S10 is becoming one of the most intensely studied aspects of chromatin remodeling, both during segregation of chromosomes at mitosis, and in aspects of transcription [13, 26–28]. Therefore, studies of TLK1B and the family of Tousled kinases are bound to become the center of much attention. Elevated phosphorylation of H3 has also been reported in several lines of oncogenically transformed fibroblasts , although the underlying mechanism is unknown. We have found that elevated expression of TLK1B did not oncogenically transform MM3MG cells, but the cells became highly resistant to IR or doxorubicin . We have preliminary results that demonstrate that TLK1B is elevated in some breast carcinomas and it is possible that this may result in a disease refractory to treatment . We currently favor a mechanism by which TLK1B protects the cells from DSBs, by promoting repair-coupled chromatin remodeling which depends on the phosphorylation of histone H3 and Asf1.
Radiation-induced damage has been shown to result in rapid phosphorylation of histone H2AX. Phosphorylated foci of H2AX co-localize with DNA repair and signaling proteins, and H2AX has been demonstrated to be involved in their recruitment to sites of DSBs . Knockout of H2AX in mice resulted in defects in concentration of repair proteins (53BP1, BRCA1, NBS1) at the sites of DNA damage, and H2AX-deficient cells are IR sensitive demonstrating the requirement of H2AX in DNA repair . The importance of radiation-induced phosphorylation of H2AX in repair of DSBs has been shown by the fact that H2AX deficiency results in genomic instability . In yeast, H2AX is not involved in activation of the S-phase checkpoint by DSBs, but rather in the efficiency of DNA repair . The importance of histone H3 phosphorylation in DNA damage has not been investigated as well as that of H2AX, but the overall effect of these modifications is likely to be chromatin remodeling and recruitment of repair proteins. Recently, H2AX was found to recruit the chromatin remodeling protein INO80 .
Our current model is that in normal cells TLK1 (the constitutively expressed larger isoform) performs the normal functions of this kinase, which may have a role in chromatin remodeling and genome surveillance in unstressed conditions. Following DNA damage by IR or doxorubicin, synthesis of TLK1B is induced through a translational control mechanism . TLK1B can then facilitate repair of DNA damage. This would greatly accelerate the response of those cells to DNA damage and the efficiency with which repair is implemented, significantly increasing their resistance to IR.
An alternative that we considered for a role of TLK1B in radioprotection is that TLK1B functions in a signaling pathway that protects cells from undergoing apoptosis. There are two compelling reasons why this is not likely (at least not directly). First, overexpression of TLK1B did not change the transcriptome in MM3MG cells (microarray analysis, data not shown), indicating that its protective effect is post-transcriptional. There were no changes in the expression of pro- and anti-apoptotic genes, or cell cycle regulators. Second, expression of TLK1B conferred protection against IR even in yeast. Whereas proteins involved in sensing and repairing DNA damage are conserved between mammals and yeast, prototypical proteins involved in the apoptotic and antiapoptotic pathways are not found in yeast.
Studies of the Tousled kinases are only now beginning to shed light upon their function, despite the early discovery of a role in flower and leaf morphology inferred by mutations in plants. In this work, we have provided four lines of evidence that the Tousled kinase, TLK1B, protects the cells from IR by facilitating the repair of DSBs. First, the pattern of phosphorylation/dephosphorylation of H2AX and H3 indicated that cells overexpressing TLK1B return to pre-IR phosphorylation state much more rapidly than controls. Second, the repair of episomes damaged with DSBs was much more rapid and complete by 8 hr of recovery in cells overexpressing TLK1B. Third, we have found that the repair of genomic breaks occurs more rapidly in cells overexpressing TLK1B, and with kinetics that are similar to those of repair of episomes. Lastly, we demonstrated with an in vitro repair system that the addition of recombinant TLK1B promotes repair of a linearized plasmid incubated with nuclear extract. Therefore, it appears that TLK1 and TLK1B have a role in genome surveillance, particularly upon genotoxic stress, which induces the expression of TLK1B.
Cell lines and tissue culture
Normal breast epithelial cells, MM3MG, transfected or not with TLK1B were cultured as described in Li et al. .
Control MM3MG cells and the cells overexpressing TLK1B [MM3MG-TLK1B; ] were harvested with PBS/EDTA and adjusted to 10,000 cells/tube in DMEM/10% FCS. Cells were irradiated in the Radiation department at LSUHSC with Elekta Precise linear accelerator at 6 MV. For each radiation dose levels (0 to 8 Gy), aliquots of serially diluted cells (100–5000) were plated on 6-well plates in triplicate. After a period of 10 days of incubation, the wells were rinsed with PBS and stained with crystal violet, and the colonies counted. The experiment was repeated thrice, and the results were expressed as the fraction of surviving cells compared to the number of colonies formed in the non-irradiated samples (plating efficiency).
The anti-histone H3 phosphorylated at Ser-10 and anti-histone H2AX phosphorylated at Ser-139 were from Upstate Cell Signaling (Lake Placid, NY). For Western blots, 30 μg of protein of each sample was separated on a 15% SDS/PAGE gel. The proteins were transferred to Immobilon-P membranes (Millipore, Bedford, MA) and incubated overnight with primary antiserum and for 1 hr with secondary antisera (1:1000 dil.). Finally, the membranes were washed three times and developed with Opti-4CN reagent (Bio-Rad, Hercules, CA).
Extraction of episomes
Episomes were isolated from 2 × 107 cells (90% confluent flasks) stably transfected with empty vector (BK-Shuttle) or vector carrying TLK1B. Two methods were used to extract the episomes: either the standard Hirt's supernatant protocol , or alkaline lysis. Briefly, the cells were resuspended in 0.1 ml TE, lysed at room temperature with solution 1 (0.2 ml of 0.2 M NaOH, 1% SDS), which was then neutralized with solution 2 (0.15 ml of 3 M K-Acetate/glacial acetic acid). After a brief centrifugation at 10,000 × g to remove insoluble material (including genomic DNA), remaining nucleic acids were extracted with Phenol/Chloroform (1:1), and precipitated with cold EtOH. The episomes were analyzed by gel electrophoresis on 1% agarose/TAE, and stained with EtBr.
Assay of genomic repair
To assess the repair of DNA damage in vivo, the modified TUNEL assay (terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling) was applied. MM3MG or TLK1B cells were grown to 50% confluence on tissue culture slides prior to exposing them to ionizing irradiation (20 Gy). After radiation, the cells were allowed to recover for varying times (0, 2, 8 hr). Subsequently, cells were fixed in 4% formalin/PBS and permeabilized in 0.2% Triton-X100/PBS. For labeling DNA breaks in situ, the DeadEnd Colorimetric TUNEL System (Promega, Madison, WI) was used according to the manufacturer's protocol. The biotinylated nucleotides incorporated at 3'OH ends were reacted with horseradish peroxidase labeled-streptavidin, which was then detected by diaminobenzidine (DAB). The nuclear DNA-labeled sites (brown spots) within each cell were counted under a light microscope (40 × magnification), and the average (± SD) number of DNA breaks per cell was calculated. At least 10 cells per dose were counted.
Assay of DSB repair in vitro
Nuclear extract from MM3MG cells was prepared as described in . Repair assays were carried out as described by Mello . Briefly, repair/nucloesome assembly was carried out on 0.1 μg of Bluescript plasmid (per reaction) that was linearized with EcoRI. We monitored simultaneously ligation of the ends and superhelicity of the plasmid by the formation of nucleosomes on the template. Reactions contain 20 μg of nuclear extract, 5 mM MgCl2, 40 mM Hepes, pH7.8, 0.5 mM DTT, 4 mM ATP, 20 μM dNTPs, 4 mM phosphocreatine, 2 u of creatine phosphokinase, and additional recombinant TLK1B. After incubation at 37°C for the indicated amount of time, the reactions were deproteinized with phenol and the plasmid was re-precipitated with cold EtOH.
Assay of chromatin assembly
Nucloesomes assembly was carried out on 2 μg of Bluescript plasmid. Reactions contained 15 μg of MM3MG cell extract (which already contains sufficient amounts of topoisomerases), 5 mM MgCl2, 40 mM Hepes, pH 7.8, 0.5 mM DTT, 4 mM ATP, 20 μM dNTPs, 4 mM phosphocreatine, 2 u of creatine phosphokinase, and additional purified proteins (200 ng TLK1B and 2 μg supplemental HeLa histones). The reactions were incubated at 37°C for 0.5 hr. The plasmid was re- extracted with GeneClean III kit (Bio 101, Vista, CA), separated on an agarose gel and subsequently stained with EtBr.
This work was supported by an LSUHC-S Office of Research Institutional award. GSD is supported by funds from the Louisiana Gene Therapy Consortium, and SKB and SS are supported by the Feist-Weiller Cancer Center.
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