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

Abstract

Background: 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.

Findings: 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).

Conclusions: 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.

Figure 1

TLK1B mediates chromatin assembly with Asf1 regardless of Asf1 phosphorylation. A. In vitro phosphorylation of Asf1 wt and S192Y mutant by TLK1B. B. Chromatin assembly on a plasmid is stimulated equally well by Asf1 wt and mutant, and the addition of wt TLK1B further stimulates plasmid supercoiling. C. Chromatin assembly is stimulated by TLK1B, wt or KD, and the addition of wt or mutant Asf1 synergizes in plasmid supercoiling. D. Western blot for Asf1a in nuclear extract supplemented or not with recombinant Asf1a. The blot was also probed for eIF4E (4E) as a loading control.