`ISRN Molecular BiologyVolume 2012 (2012), Article ID 627596, 9 pagesdoi:10.5402/2012/627596`
Review Article

## The Tousled-Like Kinases as Guardians of Genome Integrity

Department of Biochemistry and Molecular Biology and Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130, USA

Received 10 April 2012; Accepted 2 May 2012

Academic Editors: Y.-K. Jang, Y. B. Lebedev, and A. J. Molenaar

Copyright © 2012 Arrigo De Benedetti. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### Abstract

The Tousled-like kinases (TLKs) function in processes of chromatin assembly, including replication, transcription, repair, and chromosome segregation. TLKs interact specifically (and phosphorylate) with the chromatin assembly factor Asf1, a histone H3-H4 chaperone, histone H3 itself at Ser10, and also Rad9, a key protein involved in DNA repair and cell cycle signaling following DNA damage. These interactions are believed to be responsible for the action of TLKs in double-stranded break repair and radioprotection and also in the propagation of the DNA damage response. Hence, I propose that TLKs play key roles in maintenance of genome integrity in many organisms of both kingdoms. In this paper, I highlight key issues of the known roles of these proteins, particularly in the context of DNA repair (IR and UV), their possible relevance to genome integrity and cancer development, and as possible targets for intervention in cancer management.

#### 1. General Information on Tousled-Like Kinases

The Tousled locus was originally identified in A. thaliana and Antirrhinum majus during a study of mutations leading to defects in meristem expansion. Mutations of Tousled produce a complex phenotype characterized by specific defects in development of leaf and floral organs [1]. This was proposed to be linked to a replicative defect during organogenesis, but it may also result from failure to protect the genomefrom DNA damage [2, 3], resulting in developmental aberrations [4, 5]. Highly related Tousled-like genes can be found in many organisms in both kingdoms, several of which encode multiple transcripts resulting in different protein isoforms [6]. It was originally proposed that Tousled (TSL) may be a component in a signal transduction pathway controlling cell proliferation and DNA synthesis during organogenesis, and this immediately prompted a search for its substrates. However, unlike most kinases that usually display a propensity to phosphorylate numerous substrates, after many years of study, only a few direct “interacting” substrates of TLKs have been identified, namely, the histone chaperone Asf1 [7], histone H3-S10 [8], Aurora B [5], and more recently Rad9 [9]. This suggested a function for TLKs in chromatin assembly [9, 10], during transcription [2, 11], DNA repair [3, 9, 12], and condensation of chromosomes at mitosis [4, 5]. The latter function, which was found critical for proper chromosome segregation, prompted a search for additional “indirect” substrates and functions and resulted in the identification of an activity on myosin II in mammalian cells [13] and on the chromosome passenger complex in trypanosomes [14]. The search for TLKs functions at mitosis and meiosis is currently a very active pursuit in several labs in more genetically tractable organisms like Drosophila [15] and C. elegans (Jill Schumacher, personal communication). In addition, whereas only nuclear functions were initially proposed for these proteins, some splice variants localize also to the cytoplasm [8], perhaps due to their reported interaction with 14-3-3 proteins [16] with their shuttling function and hence could play additional roles in potential cytoplasmic substrates, one of which was identified as the DEAD-box p68 RNA helicase [17]. More emphasis is presented next for three of the most important substrates of TLKs: Asf1, Rad9, and histone H3.

##### 1.1. The Chromatin Assembly Factor Asf1

Asf1 is a histone H3-H4 chaperone [18] that is essential in mammals [19] and other organisms [20, 21] but not in S. cerevisiae, although such cells deleted for Asf1 are sensitive to genotoxins [22]. A recent review on Asf1 and other histone chaperones can be found in [23] and its critical importance for epigenome maintenance in [24]. Asf1, in conjunction with another chaperone called CAF1, promotes the assembly of nucleosomes onto newly replicated DNA, but it can also promote nucleosome eviction at activated promoters [2527]. Thus, Asf1 is generally involved in chromatin remodeling, which also entails DNA repair [22, 28]. The crystal structure of Asf1 in complex with H3-H4 was solved at high resolution [29], and Asf1 was found to cover the dorsal side of the H3-H4 dimer, thereby sterically preventing formation of the core tetramer. This is thought to be important for disrupting nucleosomes during transcription [30] or remodeling of chromatin in damaged DNA [3, 9], and the role of TLK1B in radioprotection was initially attributed to its effect on Asf1, presumed to be via its phosphorylation [12]. More recently, however, I showed that TLK1B can stimulate chromatin assembly in vitro in conjunction with Asf1 regardless of its phosphorylation [31]. This suggested that TLK1/1B act as chaperones in chromatin assembly, in addition to their kinase function. Hence, an important role of TLKs via Asf1 is to promote nucleosomes eviction at DSBs and access of the repair machinery to unencumbered DNA.

##### 1.3. Histone H3

Phosphorylation of histone H3 at Ser10 was recognized as the first substrate of TLK1B [8], as demonstrated both biochemically and by direct mass-spec measurements; this was further confirmed by genetic complementation of a yeast strain defective in Ipl1 (Aurora kinase of S. cerevisiae) that is the main H3-S10 kinase in this organism [8]. The significance of this phosphorylation remains unclear. Tousled (TSL) could phosphorylate histone H3 in vitro, just like mammalian TLK1B, but the recombinant C. elegans TLK was not effective at phosphorylating directly H3 but was highly stimulatory in conjunction with Air-1 (Aurora Kinase) [5], which was found to be an interacting target of the single TLK protein in C. elegans [5]. Phosphorylation of H3 by Aurora kinase is a hallmark of mitosis, and that specific phosphorylation is proposed to be critical for chromosome condensation at mitosis [54]. In that context, it is not clear if the role of TLK-mediated phosphorylation of H3 is central to mitosis, although the phosphorylation of H3-S10 is reduced in nonsynchronized cells expressing the KD, and the condensation of chromosomes and phosphorylation of H3 is reduced at mitosis [4]. But this of course could be also an indirect effect on Aurora kinase activity [5, 55]. More importantly, however, inhibition of TLK activity by genotoxic stress (see below) by either IR or UV results in reduced levels of H3-S10 in unsynchronized cells [3, 8]. Since mitotic cells represent only a small minority of cycling cells, even the large ~5-fold increase in H3P(S10) seen during mitosis would account for only a very small amount of the H3P from the total population. Hence, it would seem logical to assume that TLK-mediated H3P phosphorylation probably accounts for some other function in chromatin maintenance. If for example TLK1/1B is the main H3 kinase involved in a “chromosomal response” to DNA damage, then ATM-mediated inhibition of TLK1 (see below) is expected to result in a loss of H3 phosphorylation by endogenous phosphatases and in altered kinetics of chromatin assembly during replication and/or repair. It is possible that physiologically the increased TLK1B synthesis following IR [56] can help offset the loss of TLK activity resulting from IR and restore appropriate levels of H3P later on during the recovery. The reduction of H3P following genotoxic stress (IR) was previously reported also by another group [57]. In any case, there are other situations where H3-S10 phosphorylation is induced beside mitosis, and a clear case is that of the “nucleosomal response” at the early-response genes following mitogenic stimulation. Gene disruption in murine embryonic stem cells, and genetic evidence from Coffin-Lowry syndrome, has implicated Rsk-2 as the kinase directly responsible for phosphorylating H3 following mitogenic stimulation [58]. On the other hand, another kinase (MSK1) was reported to phosphorylate H3 more efficiently and be sensitive to the kinase inhibitor H89, which impairs the nucleosomal response, whereas Rsk-2 was insensitive to this inhibitor [59]. However, it now seems clear that several families of H3-S10 kinases exist (e.g., Ipl1/Aurora and NIMA) and may be involved in different or partially overlapping functions [60, 61]. We have clearly shown that recombinant TLK1B phosphorylates H3 at S10 and could complement a temperature-sensitive mutant of Ipl1 in yeast and restore H3P in those cells at the nonpermissive temperature. Furthermore, it could do so with high specificity in a mix of core histones, and in cells overexpressing TLK1B we found increased levels of H3 phosphorylation [8]. These findings, as well as the genetic complementation data, strongly suggest the inclusion of the Tousled family of kinases to the list of H3-S10 kinases, even though their precise role during the cell cycle or perturbations of it (inhibitory or stimulatory) has not been fully elucidated. The use of newly identified specific chemical inhibitors of TLKs could perhaps shed light on the role of TLK-mediated phosphorylation of H3-S10 and its significance in chromatin assembly during normal division or after DNA damage.

#### 2. TLKs in Man, as Guardians of Genome Stability, and Their Possible Involvement in Cancer

The first human TLK cDNA to be cloned, what we later referred to as the TLK1B splice variant (KIAA0137), was first identified during the random cloning of novel cDNAs from the human myeloid cell line KG-1 [62]. The cDNAs for TLK1 (Chr 2) and TLK2 (Chr 17) were later cloned during a PCR-based search for human kinases [63] and independently from an expression library screened on the basis of autophosphorylation activity ([64]; named PKUβ and PKUα by these authors). Instead, we have independently cloned the TLK1B splice variant with a completely different screen, based upon polysomal redistribution of weakly translated transcripts that become preferentially recruited upon overexpression of eIF4E [8]. We subsequently found that TLK1B is synthesized efficiently in several cell lines overexpressing the translation factor/oncogene eIF4E, and we then presented several lines of evidence to confirm its translational regulation, particularly after genotoxic stress [56]. The significance of this translational regulation is discussed later when I emphasize the role of TLKs in DNA repair and protection from genotoxic agents, including IR and UV. Below, we propose that an important role for TLKs is as guardian of the genome, and we implicate a function in cancer development and progression. This derivation seems obvious given their role both in basic aspects of chromatin assembly, transcription, replication, and repair and also for their distinct role in chromosome segregation into daughter cells.

A high percentage of human tumors, including cancer of the prostate (CaP) and breast (BCA), show mutations in DNA repair genes and checkpoint functions that make them overly dependent on alternative pathways for survival. Unfortunately, this can result in carcinomas that are highly resistant to radiation therapy (XRT) or radiomimetic therapy (RMT) from failsafe repair mechanisms also designed to contain excessive genomic instability. Targeting those mechanisms can result in highly specific and effective therapies. We propose that the addition of inhibitors of TLKs to enhance response to radiochemotherapy will greatly benefit CaP and BCA patients’ therapy management. In fact, ameliorating the effects of standard therapy, and possibly reducing its doses while maintaining specific killing, still seems to be the one of most promising course of action for the near future. Certainly, the success of PARP inhibitors for triple negative BCA seem to point in that direction [65].

In contrast to Rad9, there is no report for the direct involvement of either of the two human Asf1 genes in cancer development, perhaps due to the critical importance of these histone H3/H4 chaperones for all mammalian cells (normal and cancer). Nonetheless, a recent report correlated the expression of Asf1b in prediction of BCA relapse, perhaps due to its higher importance for cell proliferation and chromosomes duplication [77].

It is noteworthy that translocations/amplifications involving 17q23 that include the TLK2 gene are not unique to BCA but are also found neuroblastomas [78] and glioblastomas multiforme [79].

#### 3. TLKs in DNA Repair and as Possible Targets for Gene/Molecular Therapy

The fact that overexpression of TLKs (or more specifically the TLK1B splice variant) conferred a high degree of protection against IR in mouse cells was one of the first effects reported for these proteins [8]. It was soon found that the protection involved increased and more efficient repair of DSBs in living cells [9, 12], and then more precisely with in vitro plasmid repair assays with defined components and recombinant proteins [46]. In such reactions, the assembly of nucleosomes on the plasmid was simultaneously monitored as a decrease in the linking number via formation of high-mobility topoisomers in conjunction with repair of a DSB [46] or of excision of UV-induced pyrimidine dimers [3]. Hence, the specific contribution of Asf1 to repair of DSBs or UV damage could be studied in those conditions. For repair of the DSB, depletion of Asf1 had some effect on supercoiling, but it had only modest effect on religation of the ends. Quantitative analysis showed that conversion of the linear form to circular/relaxed and then supercoiled was complete after 20 min in control extract, but not until 40 min in Asf1-depleted. Hence, Asf1 albeit likely involved, was not essential for these repair reactions nor for supercoiling [46], at least for the case of cohesive-ends repair. Similarly, repair of UV-damaged plasmids did not absolutely depend on Asf1, although the kinetics of repair were strongly delayed [3], consistent with a previous report that looked at the contribution of Asf1/CAF1, and even TLKs, in repair of UV-damaged plasmids [80]. The identification of Asf1 [7] and later Rad9 [9] as two main targets of TLKs immediately suggested some plausible mechanisms for their role in DNA repair. We believe that the binding of 9-1-1 and TLK1B to DSBs recruits repair enzymes in conjunction with the chromatin remodeling machinery to create limited repair regions of DNA that is not encumbered by chromatin [9], similar to what has been reported in yeast for the repair of the single DSB at MAT during mating-type switching [81]. We should, however, stress that in such capacity, the role of TLKs as kinases has not been fully elucidated, since for some of these repair functions, expression of the TLK1B-KD was capable of producing effects similar to the catalytically active protein, and in specific reactions of nucleosome assembly, even in the absence of ATP [9, 31, 82]. On the other hand, it seems now clear that the kinase activity of TLKs is very significant in DDR signaling, and most likely during deactivation of the checkpoint. This is the last topic of this paper and is described below.

It would seem obvious that finding inhibitors of TLKs could greatly improve current radio- and chemotherapeutic approaches to cancer treatment. And in fact, silencing TLK1 was highly effective in sensitizing cholangiocarcinoma (a rather incurable disease) cell lines to cisplatin-induced apoptosis [89]. On the other hand, one could envision that exploiting the functions of TLKs in DNA repair could actually produce beneficial effects for normal tissues and organs exposed to the same genotoxic regimens: XRT, radiomimetic chemotherapy, or even daily skin exposure to UV damage. Indeed such cases are being contemplated in our labs, and both gene therapy approaches aimed at sparing salivary glands from the damaging effects of XRT to treat head and neck cancer [90], as well as direct TAT-TLK1 protein delivery to salivary glands [91], have been recently explored with a human clinical trial in sight. Perhaps additional modes of delivery of these proteins, such as a topical skin delivery in a liposomal complex (of either the protein itself or via viral or plasmid gene delivery vehicle), will become feasible in the near future. A model for the participation of TLK in chromatin-remodeling linked to DNA repair is shown in Figure 1.

Figure 1: A DNA repair model involving the 9-1-1 complex, Sunavala-Dossabhoy and De Benedetti [9]. Tousled homolog, TLK1, binds and phosphorylates RAD9 and acts as a molecular chaperone in DNA repair. DNA Repair 8(1):87-102.

#### Acknowledgments

This work was supported by Grant W81XWH-10-1-0120 IDEA Development Award from the Department of Defense Prostate Cancer Research Program. A patent titled “Modulators of Tousled Kinase in Cellular processes” was filed at the US PTO on March 2, 2012. However, no commercialization of the products described therein have been attempted to date. Furthermore, this paper does not contain new research material that was not in the public domain and hence could be viewed as a potential financial COI by the author.

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