The p values and FC are computed by fitting a linear model with the R package limma. Supplemental Information mmc8.pdf (6.6M) GUID:?D4E40D92-0BC8-481B-B5FF-9A7F3916487D Data Availability StatementThe accession number for the datasets reported in this paper (ATAC-seq (U-2-OS and HeLa LT), H3.3 ChIP-seq (U-2-OS) and RNA-seq (U-2-OS)) are GEO: “type”:”entrez-geo”,”attrs”:”text”:”GSE131023″,”term_id”:”131023″GSE131023. https://www.ncbi.nlm.nih.gov/geo/ The computer code generated during the current study is available on reasonable request. Summary The Tousled-like kinases 1 and 2 (TLK1/2) control histone deposition through the ASF1 histone chaperone and influence cell cycle progression and genome maintenance, yet the mechanisms underlying TLK-mediated genome stability remain uncertain. Here, we show that TLK loss results in severe chromatin decompaction and altered genome accessibility, particularly affecting heterochromatic regions. Failure to maintain heterochromatin increases spurious transcription of repetitive elements and induces features of alternative lengthening of telomeres (ALT). TLK depletion culminates in a cGAS-STING-TBK1-mediated innate immune response that is independent of replication-stress signaling and attenuated by the depletion of factors required to produce extra-telomeric DNA. Analysis of human cancers reveals that chromosomal instability correlates with high TLK2 and low STING levels in many cohorts. Based on these findings, we propose that high TLK levels contribute to immune evasion in chromosomally unstable and ALT+ cancers. and are maintained in most cancers and often amplified. In some cases, their increased expression correlates with poor prognosis, suggesting they may be potential cancer targets (Kim et?al., 2016; Lee et?al., 2018b). DNA damage inducing chemotherapy elicits an inflammatory response involving the induction and secretion of cytokines (Li and Chen, 2018; Rodier et?al., 2009). This relies on the detection of cytosolic DNA by Cyclic GMP-AMP Synthase (cGAS) and Stimulator of Interferon Reponse cGAMP Interactor 1 (STING) signaling. Rabbit polyclonal to FOXQ1 Several nuclease activities, including TREX1, SAMHD1, and MRE11, were implicated in suppression of the interferon (IFN) response (Pasero and Vindigni, 2017). SAMHD1 localizes to replication forks where it recruits the MRE11 nuclease to KRP-203 regulate replication fork progression and prevent IFN activation in response to RS (Coquel et?al., 2018). In addition to the so-far poorly defined substrates generated at replication forks, both micronucleiwhich can result from RSand extrachromosomal telomeric repeats (ECTRs) that are produced in ALT+ cancer cellssuch as partially single-stranded C-rich telomeric circles (C-circles)are inducers of cGAS-STING signaling (Chen et?al., 2017; Harding et?al., 2017; Mackenzie et?al., 2017). We previously demonstrated that TLK depletion impaired histone deposition and led to RS (Lee et?al., 2018b). To understand the genome-wide impact of TLK depletion, we conducted the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) and RNA sequencing (RNA-seq) to examine chromatin accessibility and transcription. We found that TLK loss increased chromatin accessibility at heterochromatic regions and caused spurious transcription of silenced repetitive elements and telomeres. In ALT+ U-2-OS cells and in ALT? HeLa LT cells, TLK depletion hyperactivated or induced hallmarks of ALT, respectively, and robustly activated cGAS-STING-TBK1-mediated innate immune responses. Innate immune responses were attenuated by limiting the production of KRP-203 ECTRs but not KRP-203 by modulating RS signaling. Finally, we show that expression correlates with CIN and anti-correlates with innate and adaptive immune responses in many human tumors, suggesting that high TLK levels may prevent innate immune responses induced by CIN and ALT, thus limiting immune KRP-203 recognition in cancer. Results Loss of TLK Activity Compromises Heterochromatin Maintenance Given that TLK depletion reduced histone deposition and provoked RS (Lee et?al., 2018b), we sought to see whether particular genomic regions may be hypersensitive to TLK1/2 depletion. To map genome-wide ramifications of TLK depletion on chromatin ease of access, we utilized the ATAC (Buenrostro et?al., 2013) accompanied by deep sequencing (ATAC-seq) or quantitative real-time PCR (ATAC-qPCR). We examined chromatin ease of access in U-2-Operating-system, pursuing depletion of TLK1, TLK2, or both TLK2 and TLK1. To recognize relative chromatin ease of access changes, we regarded consensus peaks as those within at least among four biological circumstances (Amount?1A). From consensus peaks within the annotated genome, 10% from the genome exhibited differential ease of access upon TLK1/2 depletion (Amount?1B). These significant adjustments involved locations depleted of available peaks (detrimental fold transformation [FC]); generally promoter-transcriptional begin site (TSS) locations; and locations enriched with available peaks (positive FC), that have been generally intronic and intergenic (Statistics 1B, 1C, and S1ACS1C). One depletion of TLK1 or TLK2 triggered similar tendencies, but differences had been more evident pursuing depletion of both (Amount?S1B). Open up in another window Amount?1 Lack of TLK Activity Compromises Heterochromatin Maintenance (A) Schematic depicting experimental style of ATAC experiments in U-2-OS. (B) Chromatin ease of access adjustments in U-2-Operating-system upon TLK depletion are statistically significant in 10% from the genome (n?= 2) (still left -panel). Genome annotation.