Tlk Function and Regulation in Chromatin Replication

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. Our recent work identified Tousled-like kinases 2 (TLK2) as a factor required for maintaining replication fork stability (Lee et al, Nuclear Acid Research under-consideration). TLKs are a family of 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. Ongoing project aims to dissect the TLK signaling network and unveil its role in chromosomal stability and tumor suppression (MOST 105-2311-B-038-002). We have identified a novel TLK partner Repo-Man and shown that TLKs may regulate replication initiation. Thus, we apply a connected funding to support the further investigation on TLK functions and regulations during chromatin replication. Through cutting-edge technologies like single molecule assay, multi-protein complex proteomics and tumor analysis, the proposed research should provide the foundation to evaluate TLKs as new therapeutic targets. Building on our unpublished findings that TLK activity is required for DNA replication and genome maintenance along with a large toolbox of reagents, we have set the following objectives: Objective #1: Dissection of TLK function in chromatin replication and genome maintenance To unravel the role of TLK in maintenance of chromatin stability during replication, we will analyze the effects of TLK dysregulation on dynamics of chromatin structure and kinetics of DNA replication by using quantitative micrococcal nuclease digestion analysis, SNAP-tag histone deposition analysis, DNA combing and nascent chromatin capture. In addition, the role of p53 in protecting TLK-depleted cells from chromosomal damage will also be explored. These cell-based analyses will provide important insight into the functions of TLKs. Objective #2: Identification and characterization of TLK targets We will determine whether Repo-Man and RAD9 are the TLK downstream target to control chromatin replication. We will also continue purifying TLK complexes with the adjusted condition and perform orthogonal affinity purification for subtract identification. Molecular characterization of new targets will provide mechanistic insight into how TLKs regulate chromatin replication. Objective #3: The TLK upstream signaling network & deregulation in cancer We will test whether TLK auto-phosphorylation regulates its own activity. To seek out the mechanisms modulating TLK activity, we will use pathway-specific characterization approaches. Moreover, we will investigate how TLK activity is regulated during cancer development. Characterization of TLK upstream pathways will open new avenues to understand how chromatin replication is controlled by nuclear signaling.