- Research article
- Open Access
TLK1B promotes repair of UV-damaged DNA through chromatin remodeling by Asf1
© Sen and De Benedetti; licensee BioMed Central Ltd. 2006
- Received: 02 August 2006
- Accepted: 20 October 2006
- Published: 20 October 2006
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 mammary cells causes loss of nuclear divisions and chromosome mis-segregation. TLK1B is a splice variant of TLK1 and it confers radioresistance in a normal mammary mouse cell line possibly due to increased chromatin remodeling capacity, but the mechanism of resistance remains to be fully elucidated.
We now show that TLK1B also affords protection against UV radiation. We find that nuclear extracts isolated from TLK1B-containing mouse cells promote more efficient chromatin assembly than comparable extracts lacking TLK1B. TLK1B-containing extracts are also more efficient in repair of UV-damaged plasmid DNA assembled into nucleosomes. One of the two known substrates of TLK1 (or TLK1B) is the histone chaperone Asf1, and immuno-inactivation experiments suggest that TLK1B increases UV-repair through the action of Asf1 on chromatin assembly/disassembly.
Our studies provide evidence for TLK1B-mediated phosphorylation of Asf1 triggering DNA repair. We suggest that this occurs via Asf1-mediated chromatin assembly at the sites of UV damage.
- Ionize Radiation
- Chromatin Remodel
- Clonogenic Assay
- Chromatin Assembly
- Histone Chaperone
Tousled-like kinases (TLKs) belong to a family of serine-threonine kinases highly conserved in plants as well as animals [1, 2]. Tousled-mutants in Arabidopsis have abnormal flower development, with defects also in leaf morphology and flowering time [3, 4]. Humans have two homologs of Tousled: TLK1 and TLK2 . The exact function of these kinases has not been determined, but they are known to act in a cell-cycle dependent manner. They are maximally active during the S phase, and are also the targets of checkpoint kinases . Specifically, it has been reported that TLK1 is inhibited by ATM-Chk1 by direct phosphorylation at Ser 695 . Knockout of the Tousled gene in Drosophila and C. elegans cause an early arrest in embryonic development [7, 8], while expression of a dominant negative mutant in mouse cells causes loss of nuclear divisions and missegregation of chromosomes . The importance of TLK1 in chromosome segregation was also confirmed by a study on C. elegans embryos . We recently cloned a cDNA encoding a mammalian Tousled-like kinase, through a different scheme, based on the polysomal redistribution of weakly translated transcripts that become preferentially recruited upon overexpression of the translation initiation factor eIF4E . This mRNA is a splice-variant of TLK1 mRNA, and encodes a 60 kDa protein (TLK1B) as compared to the 82 kDa TLK1 protein. TLK1B is translationally regulated by its 5'UTR and both ionizing radiation (IR) and the radiomimetic drug doxorubicin cause an increase in its translation . Unlike TLK1 and TLK2 that are widely expressed in various organs and tissues, TLK1B is expressed at very low levels in normal cells, but is overexpressed in some breast carcinomas .
TLK1 has only two known substrates. One is the histone H3, and it is phosphorylated at serine 10 . The other substrate is the histone chaperone Asf1 , which, in addition to its role in nucleosome assembly [14–16] and disassembly [17, 18], has been shown to have multiple functions. Most importantly, Asf1 has been implicated in DNA repair [14, 19, 20]. Human Asf1 and CAF-1 (another histone chaperone) have been shown to interact and synergize in a repair-coupled nucleosome assembly pathway . The role of Asf1 in DNA repair has been further confirmed by genetic studies in S. cerevisiae . In addition, Asf1 is also associated with checkpoint effectors . In S. cerevisiae, it interacts with the unphosphorylated form of the checkpoint kinase Rad53 (ortholog of mammalian Chk2). Upon DNA damage and replication block, Rad53 is phosphorylated and dissociates from this complex, leaving Asf1 free to interact with acetylated histones H3 and H4 . Despite the association of Asf1 and Rad53 in yeast, an Asf1-Chk2 interaction in mammalian cells has not yet been reported. Thus the mechanism of Asf1 regulation might be different, and could be dependent on its phosphorylation status. However, the consequence of Asf1 phosphorylation in mammalian cells remains unknown.
TLK1B overexpression protects mouse mammary cells (MM3-TLK1B) from the genotoxic effects of ionizing radiation (IR) . (We were unable to make a stable cell line that would express the full-length TLK1 protein). Based on this evidence and the fact that ATM and Chk1 are involved in the DNA damage checkpoint, it can be hypothesized that TLKs are involved in some aspect of genome surveillance, particularly chromatin remodeling concurrent with DNA repair. Accordingly, we showed that TLK1B protected cells from IR by facilitating the repair of double-stranded breaks (DSBs) . Using an in vitro repair system, we showed that addition of recombinant TLK1B promoted the repair of a linearized plasmid incubated with nuclear extracts, possibly by influencing the assembly of chromatin on the DNA template .
In this paper we provide evidence that overexpression of TLK1B also protects cells from the harmful effects of UV-radiation. It is well known that UV-radiation is a potent and ubiquitous carcinogen responsible for the majority of skin cancers . Unlike IR which primarily causes DSBs, UV-induced DNA damage mainly results in the formation of cyclobutane pyrimidine dimers (CPDs) . In humans, CPDs are repaired by a process known as nucleotide-excision repair (NER) . One of the best studied pathways in DNA repair, NER is highly conserved in eukaryotes. There are many differences between prokaryotes and eukaryotes; however, the basic principles are retained. NER is subdivided into two pathways; global genomic repair (GGR) that targets and removes lesions from the whole genome, and transcription-coupled repair (TCR) that preferentially removes lesions from the transcribed strand of expressed genes. It consists of four main steps: 1) Recognition of the damage; 2) Dual excision and removal of the intervening damaged DNA strand; 3) Gap repair synthesis; and 4) Ligation of the nick. In mammalian cells, NER is a complex process involving multiple large protein complexes. Packaging of the eukaryotic DNA into chromatin further complicates the process because in principle, damage accessibility would be dependent on the structural properties of the nucleosomes in and around the site of DNA damage [28–30]. NER is not essential for viability, but defects in the process result in disorders. One example is the genetic disease xeroderma pigmentosum (XP), which is associated with severe light sensitivity and an increased risk of UV-induced skin cancers. These diseases exemplify the importance of UV-induced DNA damage and its repair in humans .
Based on clonogenic assays, we first show that TLK1B-overexpressing cells are more resistant to UV radiation. This is further confirmed by an in vivo assay showing efficient and faster repair of genomic DNA as well as episomes in intact cells exposed to UV-radiation. Finally we use in vitro assays that demonstrate that TLK1B-overexpressing cells promote chromatin remodeling as well as repair of DNA damage, likely by modulating the activity of Asf1.
TLK1B protects the cells from UV
We found that 21% of the MM3MG cells survive at 2 J/m2, 10% of the MM3-KD cells survive, while 63% of the MM3-TLK1B cells survive at the same dose. For the dose of 4 J/m2 also, the survival fraction is considerably higher in MM3-TLK1B cells as compared to MM3MG and MM3-KD cells (19.3% vs. 1.4% vs. 0.7%); (P < 0.01 in both cases). This indicates that overexpression of TLK1B significantly increases the resistance to UV radiation.
In vivo repair of UV damage in MM3MG and MM3-TLK1B cells
The phosphorylation of H3 recovers faster in TLK1B-expressing cells
In vitro assays to study the repair of UV-induced DNA damage
TLK1B stimulates chromatin assembly in vitro
The importance of TLK1 (or TLK1B) in the chromatin assembly is further demonstrated when an antibody that inactivates TLK1 is added to the in vitro chromatin assembly reaction prior to the addition of the plasmid. As is seen in Figure 7B, addition of anti-TLK1 antibody decreased the supercoiling (lane 3), while no appreciable effect was seen when a non-specific antibody was used (lane 4, pre-immune serum). At the same time, addition of recombinant TLK1B protein to the TLK-depleted extract could restore the supercoiling activity considerably, indicating that the recombinant protein could complement the depletion by the antibody (lane 5). Both extracts showed very similar results in this case.
Role of TLK1-Asf1 relationship in DNA repair
When a western blot for Asf1b was done on whole cell extracts of both the cell lines, we clearly detected higher migrating forms of the protein in the MM3-TLK1B extracts as compared to the MM3MG extracts (Figure 9C, compare lanes 1 and 2), indicating that it is more phosphorylated. That the slower migrating forms were due to phosphorylation was confirmed when treatment with calf-intestinal alkaline phosphatase (lane 3) caused the Asf1b in the TLK1B cells to migrate like the Asf1b in the MM3MG cells. The most direct interpretation of these results is that TLK1B extracts have increased activity of Asf1 due to phosphorylation.
The effect of the Asf1-antiserum could also be complemented by adding back recombinant Asf1b protein, indicating that it was specifically interfering with the Asf1 function during the repair process (Figure 10B).
Role of TLK1B in the DNA repair process
TLK1 and TLK2 were originally cloned during a PCR-based search for human kinases . We identified a splice variant of TLK1 (TLK1B) using a completely different approach, based on differential redistribution of transcripts on the polysomes of cells overexpressing the translation factor eIF4E . It stands to reason that the phenotypical changes induced by eIF4E overexpression, which include transformation in several cell lines, would be mediated by a change in the recruitment of mRNAs on the polysomes and the corresponding increase in protein expression. One of the known changes induced by eIF4E is an increase in resistance to genotoxins and resistance to apoptosis . We then set out to uncover that TLK1B was conferring resistance to IR when overexpressed by itself even in the absence of eIF4E. In this report, we set out to study whether the effects of another genotoxin (UV) could also be attenuated by overexpressing TLK1B, and if so, the possible mechanism for UV resistance, which we postulate is mediated through upregulation of Asf1 activity.
In a previous publication we had shown that TLK1B overexpression induced resistance to IR and this was due to more efficient chromatin remodeling coupled to DNA repair . To assess whether TLK1B protected cells from UV-induced DNA damage, we carried out clonogenic assays which showed a highly significant increase in survival, while the converse was observed in a cell line expressing a dominant negative mutant of TLK1B. This indicated that TLK1B might have a more general role in repair of DNA damage than just protection from IR. Indeed, we subsequently found that the repair of genomic DNA following exposure to UV was faster and more efficient in MM3-TLK1B cells as compared to MM3MG cells. In addition, repair of UV damaged episomes in the TLK1B overexpressing cells was also significantly faster and more complete than in control cells. We should point out that the episomes are packed in regular chromatin in mammalian cells and thus mimic repair of genomic DNA . So episomal repair can be taken as an indicator of the repair that occurs on genomic DNA, which is generally more difficult to assess in vivo. To assess the mechanism of TLK1B-induced resistance to UV radiation, we resorted to in vitro assays for a more direct assessment. We assayed UV-damaged plasmids that had been packaged into chromatin for two main reasons. First, chromatin is the authentic substrate of NER in vivo, and second, the effect of TLK1B was expected to be most likely due to a change in chromatin remodeling capacity.
The role of chromatin remodeling in cellular processes like transcription is well established, but only recently is it being studied in the context of DNA repair. Several lines of evidence clearly indicate that the presence of nucleosomes on damaged DNA severely hampers access of the DNA repair machinery [35, 36]. At the same time, chromatin remodeling facilitates the repair process. Recombinant ACF, an ATP-dependent chromatin remodeling factor, was found to facilitate the dual incision on a dinucleosomal template . The Cockayne Syndrome-B (CSB) protein involved in transcription-coupled repair has homology to the SWI/SNF2 family of chromatin remodeling complexes and has chromatin remodeling activity . Although not a classic 'chromatin remodeler', the histone chaperone Asf1 plays an important role in several processes that are very much dependent on the chromatin structure. In addition to its well-studied role in chromatin assembly, yeast Asf1 has also been shown to be important in silencing, transcription, DNA replication and normal cell cycle progression [22, 23, 39, 40]. Most importantly though, Asf1 has been implicated in the maintenance of genomic integrity . Yeast cells deleted for ASF1 are highly sensitive to DNA-damaging agents  and Asf1 mutants have an increased rate of genomic instability . In mammalian cells, Asf1 is phosphorylated by TLK1. In fact, the only two known substrates of TLK1 thus far are histone H3 and Asf1. Also, Asf1 phosphorylation in the S-phase has been shown to correlate with TLK1 activity . This suggests that TLK1 and TLK1B have a function in chromatin remodeling, particularly TLK1B, which may have a function in remodeling during the DNA damage repair pathway by NER.
We have shown in this study that the extract containing TLK1B was far more active, both in repair of UV-damaged chromatin templates, as well as plasmid supercoiling. The role of TLK1/TLK1B was further confirmed by immuno-inactivation/complementation experiments. TLK1B's effect is almost certainly due to modulation of Asf1 activity, as addition of Asf1 alone to control extracts could restore repair activity to the level seen with TLK1B-containing extract, while Asf1 depletion resulted in a considerable decrease in the repair synthesis. It should be noted that depletion of Asf1 resulted in a decrease in the repair synthesis only in the earlier time points, but by one hour, the final extent of repair was equivalent to that seen with extract incubated with a non-specific antibody (Figure 8, lanes 1–5). A recent study showed that Asf1 increased the rate of histone eviction at the PHO5 promoter in S. cerevisiae . In the absence of Asf1 histone eviction was delayed but the final outcome of the chromatin transition was not affected. Our results indicate a similar finding. Asf1 depletion probably results in inefficient disassembly of chromatin at the sites of DNA damage, resulting in a delay in repair. But at later time points (probably because of the action of other chromatin remodeling factors), there is chromatin remodeling followed by repair of the damage.
In conclusion, our results show that TLK1B enhances the repair of UV-damaged DNA in the context of chromatin, and most likely does so through the action of Asf1, though other possible substrates cannot be excluded. The future challenge will be to determine if there are other substrates of this kinase, and if so, whether they have any role in DNA damage-response and repair.
Cell lines and tissue culture
Normal breast epithelial cells, MM3MG, transfected or not with TLK1B were cultured as described in Li et al. . MM3MG cells expressing the kinase dead mutant of TLK1B (MM3-KD) were described in Sunavala-Dossabhoy et al. .
For the clonogenic assay, MM3MG cells, cells overexpressing TLK1B (MM3MG-TLK1B) and cells expressing the kinase dead mutant of TLK1B (MM3-KD) were harvested with PBS/EDTA and adjusted to 10,000 cells/dish in PBS. Cells were then treated with the mentioned doses of UV using a germicidal UV lamp (254 nm) kept at a fixed distance from the dishes. The fluence rate was measured by a UV-meter (UVP Inc., Upland, CA). For each UV-dose level (0 to 6 J/m2), 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, 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).
Analysis of genomic repair by slot blots
80% confluent MM3MG and MM3-TLK1B cultures were irradiated with 5 J/m2 of UV. The cells were then collected at various times, and the genomic DNA was isolated using the Wizard SV kit (Promega, Madison, WI) as described by the manufacturer. 200 ng of DNA from the UV-irradiated as well as mock-irradiated cells was spotted onto Immobilon-N (Millipore, MA) using a slot blot apparatus. The filter was then baked for 2 hours at 80°C. Quantification of the CPDs was carried out using a mouse monoclonal antibody for CPDs (MBL International, MA), by densitometry using the ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Antibody binding was determined using the Opti4CN reagent (Biorad, Hercules, CA).
Analysis of episomal repair by T4 Endonuclease V
MM3MG cells (transfected with the empty BK-shuttle vector) and MM3-TLK1B cells grown to 75% confluence were either sham treated (sample used as non-irradiated control) or UV-irradiated with a dose of 5 J/m2. One dish of each cell line was immediately harvested (0 hr time point). The other flasks were re-incubated to allow DNA repair for 12 hours and the cells were harvested at 2, 4, 8 and 12 hour time points respectively. Episomes were extracted by alkaline lysis. Purified DNA (2 μg) was then either mock treated or digested with T4 endonuclease V (Epicentre, Madison, WI) according to the manufacturer's instructions. T4 endonuclease V specifically incises the unrepaired UV-induced CPDs in DNA. The cleavage products were then separated on a 0.8% agarose gel for one and a half hours at 4°C, and stained with ethidium bromide.
Western blot for H3-Ser10-phosphorylation
The anti-histone H3 phosphorylated at Ser-10 antibody was purchased from Upstate Cell signaling (Lake Placid, NY). For Western blots, 25 μg of protein of each sample was separated on a 15% SDS/PAGE gel. The proteins were transferred to nitrocellulose, incubated with primary antiserum for 1 hour followed by secondary antiserum for 1 hour (1:1000 dilution each). Finally, the membranes were washed and developed using the Opti-4CN reagent (Biorad, Hercules, CA).
Assembly of Bluescript plasmid into a nucleosomal template
Native core histones were purified from HeLa cells by hydroxylapatite based chromatography according to the protocol described by Simon and Felsenfeld . These purified core histones were then used for assembling nucleosomes. The plasmid Bluescript (pBS) was first damaged with 300 J/m2 of UV (a fluence of 100 J/m2 induces roughly 1 pyrimidine dimer photoproduct in 1000 bp) (Wood et al.) , following which it was assembled into a nucleosomal template by salt dialysis according to the protocol described by Jeong et al. . Briefly, supercoiled pBS was incubated with the purified core histones (in a 2 M NaCl containing buffer) and then subjected to three sequential dialysis steps, in which the salt concentration was gradually decreased. Chromatin assembly was then confirmed by doing a micrococcal nuclease (MNase) digestion and processed for electrophoresis on a 1.5% agarose gel.
In vitro DNA repair assays
Nuclear extract from MM3MG and MM3-TLK1B cells was prepared as described by Gaymes et al. . Repair assay was done according to the protocol described by Mello et al. , but with some modifications. Reactions contained the damaged plasmid assembled into nucleosomes, MM3MG or MM3-TLK1B nuclear extract, 5 mM MgCl2, 40 mM Hepes, pH7.8, 0.5 mM DTT, 4 mM ATP, 20 μM dNTPs, 4 mM phosphocreatine and [α]32P-dATP. After incubation at 37°C for varying time periods, the reaction was stopped by transferring on to ice, the plasmid extracted using the Gene Clean kit (Bio 101, Vista, CA) and run on an agarose gel. The gel was then dried and the amount of [α]32P-dATP incorporated determined by phosphor-imager analysis (Molecular Dynamics, Sunnyvale, CA) using the ImageQuant software. Undamaged plasmid incorporated into nucleosomal template was used as a negative control.
Antibodies and recombinant proteins used
The rabbit TLK1-antiserum used in the in vitro repair and supercoiling assays was prepared in our lab (Li et al.) . Antiserum to Asf1a and Asf1b proteins were obtained from the CIM Antibody Core at Arizona State University. TLK1B and Asf1b proteins that were used in our experiments were expressed in E. coli as GST-fusion proteins. The pGEX-CIA plasmid that encodes the full length GST-Asf1b protein was a kind gift from Dr. Masami Horikoshi, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Japan. For figures 9, 10 and 11, the amount of [α]32P-dATP incorporated was determined as described in the in vitro repair assays, by phosphor-imager analysis (Molecular Dynamics, Sunnyvale, CA) using the ImageQuant software.
Assay of chromatin assembly
Nucleosome 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, 100 ng Asf1b and 2 μg supplemental HeLa histones). The reactions were incubated at 37°C for 1 hr. The plasmid was re-extracted with the GeneClean kit (Bio 101, Vista, CA), separated on an agarose gel and subsequently stained with ethidium bromide.
This work was supported by an LSUHSC-S Office of Research Institutional award and the Susan G. Komen BCTR0601319 award. SS is supported by funds from the Feist Weiller Cancer Center and the Louisiana Gene Therapy Consortium.
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