Tlk Function and Regulation in Chromatin Replication (2nd Years)

During each cell division, DNA sequence and epigenetic information embedded in chromatin must be accurately transmitted to ensure genome integrity. Failure to keep this information leads to genomic instability and can jeopardize the programs controlling the cell fates. Whereas signaling networks that control genome integrity has been extensively characterized, we know little about mechanisms that ensure chromatin and epigenome integrity in dividing cells. We have identified Tousled-like kinase 2 (TLK2) as a factor required for maintaining replication fork stability (Lee et al, Cancer Research, revision complete). TLK1 and 2 are kinases highly active in S phase and directly regulated by DNA integrity checkpoints. These enzymes are specific to higher eukaryotes, and loss of TLK function leads to chromosomal aberrations and developmental defects in worms and flies. Yet, except for one well-established downstream target, the histone chaperone ASF1, the functions of TLKs in mammals remain relatively unexplored. We have obtained the first year funding from MOST to dissect TLK signaling network and unveil its role in regulating chromosomal stability (MOST 106-2311-B-038-001). Our recent works reveal that TLK2 facilitates chromatin assembly on newly synthesized DNA and hereby regulates fork progression and integrity. In addition, we show that TLK2 may also regulate replication initiation. Importantly, our proteomics screen identified a novel TLK2 partner Repo-Man and we further demonstrated that it is also required for chromatin replication. Based on our achievements, we thus apply the extended funding for the 2nd and 3rd years to further investigate TLK2 functions and regulations during chromatin replication. Through cutting-edge technologies like single molecule assay, multi-protein complex proteomics and high-throughput analysis, the proposed research should provide the foundation to evaluate TLK2 as new therapeutic target for cancers. Next 2 years, we expect to accomplish the preset tasks of following objectives:Objective #1: Dissection of TLK2 function in chromatin replication and genome maintenanceTo unravel the role of TLK2 in maintenance of chromatin stability during replication, we will analyze the effects of TLK2 dysregulation on kinetics of DNA replication by using the single-molecule analysis DNA combing and the replisome isolation technology Nascent Chromatin Capture (NCC). These cell-based analyses will provide important insight into the functions of TLK2.Objective #2: Identification and characterization of TLK2 targetsWe will determine whether the known TLK substrates Repo-Man and RAD9 are the TLK2 downstream target to control chromatin replication. In addition, we will purifying TLK2 complexes with the adjusted condition to identify new substrates. Molecular characterization of new targets will provide mechanistic insight into how TLK2 regulates chromatin replication.Objective #3: The TLK2 upstream signaling networkWe will test whether TLK2 auto-phosphorylation regulates its own activity. To seek out the mechanisms modulating TLK2 activity, we will use pathway-specific characterization approaches. Characterization of TLK2 upstream pathways will open new avenues to understand how chromatin replication is controlled by nuclear signaling.