Tousled kinase TLK1B mediates chromatin assembly in conjunction with Asf1 regardless of its kinase activity

<p>Abstract</p> <p>Background</p> <p>The Tousled Like Kinases (TLKs) are involved in chromatin dynamics, including DNA replication and repair, transcription, and chromosome segregation. Indeed, the first two TLK1 substrates were identified as the histone H3 and Asf1 (a histone H3/H4 chaperone), which immediately suggested a function in chromatin remodeling. However, despite the straightforward assumption that TLK1 acts simply by phosphorylating its substrates and hence modifying their activity, TLK1 also acts as a chaperone. In fact, a kinase-dead (KD) mutant of TLK1B is functional in stimulating chromatin assembly in vitro. However, subtle effects of Asf1 phosphorylation are more difficult to probe in chromatin assembly assays. Not until very recently was the Asf1 site phosphorylated by TLK1 identified. This has allowed for probing directly the functionality of a site-directed mutant of Asf1 in chromatin assembly assays.</p> <p>Findings</p> <p>Addition of either wt or non-phosphorylatable mutant Asf1 to nuclear extract stimulates chromatin assembly on a plasmid. Similarly, TLK1B-KD stimulates chromatin assembly and it synergizes in reactions with supplemental Asf1 (wt or non-phosphorylatable mutant).</p> <p>Conclusions</p> <p>Although the actual function of TLKs as mediators of Asf1 activity cannot be easily studied in vivo, particularly since in mammalian cells there are two TLK genes and two Asf1 genes, we were able to study specifically the stimulation of chromatin assembly in vitro. In such assays, clearly the TLK1 kinase activity was not critical, as neither a non-phosphorylatable Asf1 nor use of the TLK1B-KD impaired the stimulation of nucleosome formation.</p

Differential requirements for Tousled-like kinases 1 and 2 in mammalian development

The regulation of chromatin structure is critical for a wide range of essential cellular processes. The Tousled-like kinases, TLK1 and TLK2, regulate ASF1, a histone H3/H4 chaperone, and likely other substrates, and their activity has been implicated in transcription, DNA replication, DNA repair, RNA interference, cell cycle progression, viral latency, chromosome segregation and mitosis. However, little is known about the functions of TLK activity in vivo or the relative functions of the highly similar TLK1 and TLK2 in any cell type. To begin to address this, we have generated Tlk1- and Tlk2-deficient mice. We found that while TLK1 was dispensable for murine viability, TLK2 loss led to late embryonic lethality because of placental failure. TLK2 was required for normal trophoblast differentiation and the phosphorylation of ASF1 was reduced in placentas lacking TLK2. Conditional bypass of the placental phenotype allowed the generation of apparently healthy Tlk2-deficient mice, while only the depletion of both TLK1 and TLK2 led to extensive genomic instability, indicating that both activities contribute to genome maintenance. Our data identifies a specific role for TLK2 in placental function during mammalian development and suggests that TLK1 and TLK2 have largely redundant roles in genome maintenance

Probable neuroimmunological link between Toxoplasma and cytomegalovirus infections and personality changes in the human host

BACKGROUND: Recently, a negative association between Toxoplasma-infection and novelty seeking was reported. The authors suggested that changes of personality trait were caused by manipulation activity of the parasite, aimed at increasing the probability of transmission of the parasite from an intermediate to a definitive host. They also suggested that low novelty seeking indicated an increased level of the neurotransmitter dopamine in the brain of infected subjects, a phenomenon already observed in experimentally infected rodents. However, the changes in personality can also be just a byproduct of any neurotropic infection. Moreover, the association between a personality trait and the toxoplasmosis can even be caused by an independent correlation of both the probability of Toxoplasma-infection and the personality trait with the third factor, namely with the size of living place of a subject. To test these two alternative hypotheses, we studied the influence of another neurotropic pathogen, the cytomegalovirus, on the personality of infected subjects, and reanalyzed the original data after the effect of the potential confounder, the size of living place, was controlled. METHODS: In the case-control study, 533 conscripts were tested for toxoplasmosis and presence of anti-cytomegalovirus antibodies and their novelty seeking was examined with Cloninger's TCI questionnaire. Possible association between the two infections and TCI dimensions was analyzed. RESULTS: The decrease of novelty seeking is associated also with cytomegalovirus infection. After the size of living place was controlled, the effect of toxoplasmosis on novelty seeking increased. Significant difference in novelty seeking was observed only in the largest city, Prague. CONCLUSION: Toxoplasma and cytomegalovirus probably induce a decrease of novelty seeking. As the cytomegalovirus spreads in population by direct contact (not by predation as with Toxoplasma), the observed changes are the byproduct of brain infections rather than the result of manipulation activity of a parasite. Four independent lines of indirect evidence, namely direct measurement of neurotransmitter concentration in mice, the nature of behavioral changes in rodents, the nature of personality changes in humans, and the observed association between schizophrenia and toxoplasmosis, suggest that the changes of dopamine concentration in brain could play a role in behavioral changes of infected hosts

Proteomics and in silico approaches to extend understanding of the glutathione transferase superfamily of the tropical liver fluke Fasciola gigantica

Fasciolosis is an important foodborne, zoonotic disease of livestock and humans, with global annual health and economic losses estimated at several billion US$. Fasciola hepatica is the major species in temperate regions, while F. gigantica dominates in the tropics. In the absence of commercially available vaccines to control fasciolosis, increasing reports of resistance to current chemotherapeutic strategies and the spread of fasciolosis into new areas, new functional genomics approaches are being used to identify potential new drug targets and vaccine candidates. The glutathione transferase (GST) superfamily is both a candidate drug and vaccine target. This study reports the identification of a putatively novel Sigma class GST, present in a water-soluble cytosol extract from the tropical liver fluke F. gigantica. The GST was cloned and expressed as an enzymically active recombinant protein. This GST shares a greater identity with the human schistosomiasis GST vaccine currently at Phase II clinical trials than previously discovered F. gigantica GSTs, stimulating interest in its immuno-protective properties. In addition, in silico analysis of the GST superfamily of both F. gigantica and F. hepatica has revealed an additional Mu class GST, Omega class GSTs, and for the first time, a Zeta class member