Tousled-like kinases phosphorylate Asf1 to promote histone supply during DNA replication

Abstract

During DNA replication, nucleosomes are rapidly assembled on newly synthesized DNA to restore chromatin organization. Asf1, a key histone H3-H4 chaperone required for this process, is phosphorylated by Tousled-Like Kinases (TLKs). Here, we identify TLK phosphorylation sites by mass spectrometry and dissect how phosphorylation impacts on human Asf1 function. The divergent C-terminal tail of Asf1a is phosphorylated at several sites and this is required for timely progression through S phase. Consistent with this, biochemical analysis of wild-type and phosphomimetic Asf1a shows that phosphorylation enhances binding to histones and the downstream chaperones CAF-1 and HIRA. Moreover, we find that TLK phosphorylation of Asf1a is induced in cells experiencing deficiency of new histones and that TLK interaction with Asf1a involves its histone-binding pocket. We thus propose that TLK signaling promotes histone supply in S phase by targeting histone-free Asf1 and stimulating its ability to shuttle histones to sites of chromatin assembly.

Tousled‐like kinase 2 regulates recovery from a DNA damage‐induced G2 arrest

“…Antibodies directed against the following proteins were used: Tlk2, SMC1 (all Bethyl), MPM‐2 (Millipore), α‐tubulin (Sigma), γH2AX, histone H3‐pS10 (Upstate), actin, Chk1, Plk1, cyclin B, p53, HSP90α/β (all Santa Cruz), Tlk1, Asf1A, Asf1B, Chk1‐pS317, Cdk1‐pY15 (all Cell Signaling), FLAG (Sigma), and histone H3 (Abcam). The antibody directed at Asf1A‐pS66 was a gift from A. Groth . Secondary antibodies Alexa‐488, Alexa‐568, and Alexa‐633 were from Molecular Probes and horseradish peroxidase‐coupled secondary antibodies from Dako.…”

Histone chaperone networks shaping chromatin function

“…ASF1 phosphorylation by TLK1 and TLK2 promotes histone binding and interaction with downstream chaperones 102,103 to ensure efficient histone supply during DNA replication. Phosphorylation of CAF1 p150 by the CDC7–DBF4 kinase disrupts dimerization, which in turn facilitates binding to PCNA 104 and chromatin assembly.…”

Discovery of Stromal Regulatory Networks that Suppress Ras-Sensitized Epithelial Cell Proliferation

“…TLK1 and TLK2 constitute a family of serine/threonine kinases that phosphorylate the histone chaperone ASF1 and histone H3 to coordinately regulate chromatin assembly and remodeling (Carrera et al, 2003; Klimovskaia et al, 2014; Li et al, 2001; Sillje and Nigg, 2001; Sillje et al, 1999). To rigorously evaluate their roles in vivo , we used standard homologous recombination approaches to introduce conditional LoxP -flanked Tlk1 and Tlk2 alleles into the mouse genome ( Tlk1 Neo and Tlk2 Neo , respectively; Figure 4A, B).…”

“…TLKs phosphorylate histone H3 and ASF1A/B, two DNA-replication dependent and independent histone chaperones that facilitate chromatin assembly (Carrera et al, 2003; Klimovskaia et al, 2014). The scaffolding protein SYMPLEKIN (SYMPK) is a core component of a cytoplasmic polyadenylation complex that includes CPEB1 (also identified in this screen) and CPSF (Barnard et al, 2004).…”