Tousled kinase TLK1B counteracts the effect of Asf1 in inhibition of histone H3–H4 tetramer formation

<p>Abstract</p> <p>Background</p> <p>The <it>T</it>ousled-<it>l</it>ike <it>k</it>inases (TLKs) function in processes of chromatin assembly, including replication, transcription, repair, and chromosome segregation. TLK1 interacts specifically with the chromatin assembly factor Asf1, a histone H3–H4 chaperone, and with Rad9, a protein involved in DNA repair. Asf1 binds to the H3–H4 dimer at the same interface that is used for formation of the core tetramer, and hence Asf1 is implicated in disruption of the tetramer during transcription, although Asf1 also has a function in chromatin assembly during replication and repair.</p> <p>Findings</p> <p>We have used protein crosslinking with purified components to probe the interaction between H3, H4, Asf1, and TLK1B. We found that TLK1B, by virtue of its binding to Asf1, can restore formation of H3–H4 tetramers that is sterically prevented by adding Asf1.</p> <p>Conclusion</p> <p>We suggest that TLK1B binds to Asf1 in a manner that interferes with its binding to the H3–H4 dimer, thereby allowing for H3–H4 tetramerization. A description of the function of TLK1 and Asf1 in chromatin remodeling is presented.</p

TLK1B promotes repair of UV-damaged DNA through chromatin remodeling by Asf1

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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

TLK1B promotes repair of DSBs via its interaction with Rad9 and Asf1 Caroline Canfield, Justin Rains, and Arrigo De Benedetti

<p>Abstract</p> <p>Background</p> <p>The <it>Tousled-like kinases </it>are involved in chromatin assembly, DNA repair, transcription, and chromosome segregation. Previous evidence indicated that TLK1B can promote repair of plasmids with cohesive ends in vitro, but it was inferred that the mechanism was indirect and via chromatin assembly, mediated by its interaction with the chromatin assembly factor Asf1. We recently identified Rad9 as a substrate of TLK1B, and we presented evidence that the TLK1B-Rad9 interaction plays some role in DSB repair. Hence the relative contribution of Asf1 and Rad9 to the protective effect of TLK1B in DSBs repair is not known. Using an adeno-HO-mediated cleavage system in MM3MG cells, we previously showed that overexpression of either TLK1B or a kinase-dead protein (KD) promoted repair and the assembly of Rad9 in proximity of the DSB at early time points post-infection. This established that it is a chaperone activity of TLK1B and not directly the kinase activity that promotes recruitment of 9-1-1 to the DSB. However, the phosphorylation of Rad9(S328) by TLK1B appeared important for mediating a cell cycle checkpoint, and thus, this phosphorylation of Rad9 may have other effects on 9-1-1 functionality.</p> <p>Results</p> <p>Here we present direct evidence that TLK1B can promote repair of linearized plasmids with incompatible ends that require processing prior to ligation. Immunodepletion of Rad9 indicated that Rad9 was important for processing the ends preceding ligation, suggesting that the interaction of TLK1B with Rad9 is a key mediator for this type of repair. Ligation of incompatible ends also required DNA-PK, as addition of wortmannin or immunodepletion of Ku70 abrogated ligation. Depletion of Ku70 prevented the ligation of the plasmid but did not affect stimulation of the fill-in of the ends by added TLK1B, which was attributed to Rad9. From experiments with the HO-cleavage system, we now show that Rad17, a subunit of the "clamp loader", associates normally with the DSB in KD-overexpressing cells. However, the subsequent release of Rad17 and Rad9 upon repair of the DSB was significantly slower in these cells compared to controls or cells expressing wt-TLK1B.</p> <p>Conclusions</p> <p>TLKs play important roles in DNA repair, not only by modulation of chromatin assembly via Asf1, but also by a more direct function in processing the ends of a DSB via interaction with Rad9. Inhibition of Rad9 phosphorylation in KD-overexpressing cells may have consequences in signaling completion of the repair and cell cycle re-entry, and could explain a loss of viability from DSBs in these cells.</p

Translation of the radioresistance kinase TLK1B is induced by γ-irradiation through activation of mTOR and phosphorylation of 4E-BP1

BACKGROUND: 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 chromosome missegregation 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 their resistance to ionizing radiation, also likely through changes in chromatin remodeling. The TLK1B mRNA is translationally repressed by its 5'UTR and is regulated by the availability of eIF4E. We now report that radiation or doxorubicin result in an increase in the translation of TLK1B, and we have uncovered the likely mechanism for this effect. RESULTS: Radiation causes a shift in the polysomal distribution of TLK1B mRNA, from the untranslated region and small polysomes to the large polysomes, concomitant with an increase in the expression of TLK1B protein. This change is preceded by an increase in phosphorylation of the eIF4E inhibitory protein 4E-BP1, which releases eIF4E when it is phosphorylated. The phosphorylation of 4E-BP1 depends on mTOR, since rapamycin blocked the increase in phosphorylation induced by radiation, and prevented the increase in TLK1B protein expression. The activation of mTOR was likely due to the rapid activation of Akt following radiation. The activation of Akt could be inhibited with wortmannin, an inhibitor of PI3 kinase, hence placing PI3 kinase upstream of Akt as a very early event following radiation. Wortmannin also inhibited translation of TLK1B mRNA following activation by IR. This was shown both by western blot and by measuring the initiation capacity of the mRNA, as indicated by its distribution on polysomes. CONCLUSIONS: The translational upregulation of TLK1B elicited by DNA double strand breaks represents an interesting mechanism of translational regulation of a protein involved in radioprotection and highlights a novel mechanism of the stress response following radiation

Nucleosome resection at a double-strand break during Non-Homologous Ends Joining in mammalian cells - implications from repressive chromatin organization and the role of ARTEMIS

<p>Abstract</p> <p>Background</p> <p>The <it>S. cerevisiae </it>mating type switch model of double-strand break (DSB) repair, utilizing the HO endonuclease, is one of the best studied systems for both Homologous Recombination Repair (HRR) and direct ends-joining repair (Non-Homologous Ends Joining - NHEJ). We have recently transposed that system to a mammalian cell culture model taking advantage of an adenovirus expressing HO and an integrated genomic target. This made it possible to compare directly the mechanism of repair between yeast and mammalian cells for the same type of induced DSB. Studies of DSB repair have emphasized commonality of features, proteins and machineries between organisms, and differences when conservation is not found. Two proteins that stand out that differ between yeast and mammalian cells are DNA-PK, a protein kinase that is activated by the presence of DSBs, and Artemis, a nuclease whose activity is modulated by DNA-PK and ATM. In this report we describe how these two proteins may be involved in a specific pattern of ends-processing at the DSB, particularly in the context of heterochromatin.</p> <p>Findings</p> <p>We previously published that the repair of the HO-induced DSB was generally accurate and occurred by simple rejoining of the cohesive 3'-overhangs generated by HO. During continuous passage of those cells in the absence of puromycin selection, the locus appears to have become more heterochromatic and silenced by displaying several features. 1) The site had become less accessible to cleavage by the HO endonuclease; 2) the expression of the puro mRNA, which confers resistance to puromycin, had become reduced; 3) occupancy of nucleosomes at the site (ChIP for histone H3) was increased, an indicator for more condensed chromatin. After reselection of these cells by addition of puromycin, many of these features were reversed. However, even the reselected cells were not identical in the pattern of cleavage and repair as the cells when originally created. Specifically, the pattern of repair revealed discrete deletions at the DSB that indicated unit losses of nucleosomes (or other protein complexes) before religation, represented by a ladder of PCR products reminiscent of an internucleosomal cleavage that is typically observed during apoptosis. This pattern of cleavage suggested to us that perhaps, Artemis, a protein that is believed to generate the internucleosomal fragments during apoptosis and in DSB repair, was involved in that specific pattern of ends-processing. Preliminary evidence indicates that this may be the case, since knock-down of Artemis with siRNA eliminated the laddering pattern and revealed instead an extensive exonucleolytic processing of the ends before religation.</p> <p>Conclusions</p> <p>e have generated a system in mammalian cells where the absence of positive selection resulted in chromatin remodeling at the target locus that recapitulates many of the features of the mating-type switching system in yeast. Specifically, just as for yeast HML and HMR, the locus had become transcriptionally repressed; accessibility to cleavage by the HO endonuclease was reduced; and processing of the ends was drastically changed. The switch was from high-fidelity religation of the cohesive ends, to a pattern of release of internucleosomal fragments, perhaps in search of micro-homology stretches for ligation. This is consistent with reports that the involvement of ATM, DNA-PK and Artemis in DSB repair is largely focused to heterochromatic regions, and not required for the majority of IR-induced DSB repair foci in euchromatin.</p

A dominant negative mutant of TLK1 causes chromosome missegregation and aneuploidy in normal breast epithelial cells

BACKGROUND: In Arabidopsis thaliana, the gene Tousled encodes a protein kinase of unknown function, but mutations in the gene lead to flowering and leaf morphology defects. We have recently cloned a mammalian Tousled-Like Kinase (TLK1B) and found that it phosphorylates specifically histone H3, in vitro and in vivo. We now report the effects that overexpression of a kinase-dead mutant of TLK1B mediates in a normal diploid cell line. RESULTS: Expression of a kinase-dead mutant resulted in reduction of phosphorylated histone H3, which could have consequences in mitotic segregation of chromosomes. When analyzed by FACS and microscopy, these cells displayed high chromosome number instability and aneuploidy. This phenomenon was accompanied by less condensed chromosomes at mitosis; failure of a number of chromosomes to align properly on the metaphase plate; failure of some chromosomes to attach to microtubules; and the occasional presentation of two bipolar spindles. We also used a different method (siRNA) to reduce the level of endogenous TLK1, but in this case, the main result was a strong block of cell cycle progression suggesting that TLK1 may also play a role in progression from G1. This block in S phase progression could also offer a different explanation of some of the later mitotic defects. CONCLUSIONS: TLK1 has a function important for proper chromosome segregation and maintenance of diploid cells at mitosis in mammalian cells that could be mediated by reduced phosphorylation of histone H3 and condensation of chromosomes, although other explanations to the phenotype are possible

The radioresistance kinase TLK1B protects the cells by promoting repair of double strand breaks

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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

NEK1 Phosphorylation of YAP Promotes Its Stabilization and Transcriptional Output

Most prostate cancer (PCa) deaths result from progressive failure in standard androgen deprivation therapy (ADT), leading to metastatic castration-resistant PCa (mCRPC); however, the mechanism and key players leading to this are not fully understood. While studying the role of tousled-like kinase 1 (TLK1) and never in mitosis gene A (NIMA)-related kinase 1 (NEK1) in a DNA damage response (DDR)-mediated cell cycle arrest in LNCaP cells treated with bicalutamide, we uncovered that overexpression of wt-NEK1 resulted in a rapid conversion to androgen-independent (AI) growth, analogous to what has been observed when YAP1 is overexpressed. We now report that overexpression of wt-NEK1 results in accumulation of YAP1, suggesting the existence of a TLK1\u3eNEK1\u3eYAP1 axis that leads to adaptation to AI growth. Further, YAP1 is co-immunoprecipitated with NEK1. Importantly, NEK1 was able to phosphorylate YAP1 on six residues in vitro, which we believe are important for stabilization of the protein, possibly by increasing its interaction with transcriptional partners. In fact, knockout (KO) of NEK1 in NT1 PCa cells resulted in a parallel decrease of YAP1 level and reduced expression of typical YAP-regulated target genes. In terms of cancer potential implications, the expression of NEK1 and YAP1 proteins was found to be increased and correlated in several cancers. These include PCa stages according to Gleason score, head and neck squamous cell carcinoma, and glioblastoma, suggesting that this co-regulation is imparted by increased YAP1 stability when NEK1 is overexpressed or activated by TLK1, and not through transcriptional co-expression. We propose that the TLK1\u3eNEK1\u3eYAP1 axis is a key determinant for cancer progression, particularly during the process of androgen-sensitive to -independent conversion during progression to mCRPC

Tissue microarray analysis of eIF4E and its downstream effector proteins in human breast cancer

Correction to Kleiner HE, Krishnan P, Tubbs J, Smith M, Meschonat C, Shi R, Lowery-Nordberg M, Adegboyega P, Unger M, Cardelli J et al: Tissue microarray analysis of eIF4E and its downstream effector proteins in human breast cancer. J Exp Clin Cancer Res 2009, 28:5