Differential Dynamics of ATR-Mediated Checkpoint Regulators

The ATR-Chk1 checkpoint pathway is activated by UV-induced DNA lesions and replication stress. Little was known about the spatio and temporal behaviour of the proteins involved, and we, therefore, examined the behaviour of the ATRIP-ATR and Rad9-Rad1-Hus1 putative DNA damage sensor complexes and the downstream effector kinase Chk1. We developed assays for the generation and validation of stable cell lines expressing GFP-fusion proteins. Photobleaching experiments in living cells expressing these fusions indicated that after UV-induced DNA damage, ATRIP associates more transiently with damaged chromatin than members of the Rad9-Rad1-Hus1 complex. Interestingly, ATRIP directly associated with locally induced UV damage, whereas Rad9 bound in a cooperative manner, which can be explained by the Rad17-dependent loading of Rad9 onto damaged chromatin. Although Chk1 dissociates from the chromatin upon UV damage, no change in the mobility of GFP-Chk1 was observed, supporting the notion that Chk1 is a highly dynamic protein

Identification of Two Protein-Signaling States Delineating Transcriptionally Heterogeneous Human Medulloblastoma

Summary: The brain cancer medulloblastoma consists of different transcriptional subgroups. To characterize medulloblastoma at the phosphoprotein-signaling level, we performed high-throughput peptide phosphorylation profiling on a large cohort of SHH (Sonic Hedgehog), group 3, and group 4 medulloblastomas. We identified two major protein-signaling profiles. One profile was associated with rapid death post-recurrence and resembled MYC-like signaling for which MYC lesions are sufficient but not necessary. The second profile showed enrichment for DNA damage, as well as apoptotic and neuronal signaling. Integrative analysis demonstrated that heterogeneous transcriptional input converges on these protein-signaling profiles: all SHH and a subset of group 3 patients exhibited the MYC-like protein-signaling profile; the majority of the other group 3 subset and group 4 patients displayed the DNA damage/apoptotic/neuronal signaling profile. Functional analysis of enriched pathways highlighted cell-cycle progression and protein synthesis as therapeutic targets for MYC-like medulloblastoma. : Using peptide phosphorylation profiling, Zomerman et al. identify two medulloblastoma phosphoprotein-signaling profiles that have prognostic value and are potentially targetable. They find that these profiles extend across transcriptome-based subgroup borders. This suggests that diverse genetic information converges on common protein-signaling pathways and highlights protein-signaling as a unique information layer. Keywords: medulloblastoma, protein-signaling, protein synthesis, MYC, TP53, proteome, phosphoproteom

Heterochromatin protein 1 is recruited to various types of DNA damage

Heterochromatin protein 1 (HP1) family members are chromatin-associated proteins involved in transcription, replication, and chromatin organization. We show that HP1 isoforms HP1-α, HP1-β, and HP1-γ are recruited to ultraviolet (UV)-induced DNA damage and double-strand breaks (DSBs) in human cells. This response to DNA damage requires the chromo shadow domain of HP1 and is independent of H3K9 trimethylation and proteins that detect UV damage and DSBs. Loss of HP1 results in high sensitivity to UV light and ionizing radiation in the nematode Caenorhabditis elegans, indicating that HP1 proteins are essential components of DNA damage response (DDR) systems. Analysis of single and double HP1 mutants in nematodes suggests that HP1 homologues have both unique and overlapping functions in the DDR. Our results show that HP1 proteins are important for DNA repair and may function to reorganize chromatin in response to damage

Stochastic and reversible assembly of a multiprotein DNA repair complex ensures accurate target site recognition and efficient repair

Computational modeling and quantitative analysis show that although accumulation of repair complexes can take hours, the individual components rapidly exchange between the nucleoplasm and DNA damage sites

Keeping ribosomal DNA intact: a repeating challenge

More than half of the human genome consists of repetitive sequences, with the ribosomal DNA (rDNA) representing two of the largest repeats. Repetitive rDNA sequences may form a threat to genomic integrity and cellular homeostasis due to the challenging aspects of their transcription, replication, and repair. Predisposition to cancer, premature aging, and neurological impairment in ataxia-telangiectasia and Bloom syndrome, for instance, coincide with increased cellular rDNA repeat instability. However, the mechanisms by which rDNA instability contributes to these hereditary syndromes and tumorigenesis remain unknown. Here, we review how cells govern rDNA stability and how rDNA break repair influences expansion and contraction of repeat length, a process likely associated with human disease. Recent advancements in CRISPR-based genome engineering may help to explain how cells keep their rDNA intact in the near future

The core spliceosome as target and effector of non-canonical ATM signalling

In response to DNA damage, tissue homoeostasis is ensured by protein networks promoting DNA repair, cell cycle arrest or apoptosis. DNA damage response signalling pathways coordinate these processes, partly by propagating gene-expression-modulating signals. DNA damage influences not only the abundance of messenger RNAs, but also their coding information through alternative splicing. Here we show that transcription-blocking DNA lesions promote chromatin displacement of late-stage spliceosomes and initiate a positive feedback loop centred on the signalling kinase ATM. We propose that initial spliceosome displacement and subsequent R-loop formation is triggered by pausing of RNA polymerase at DNA lesions. In turn, R-loops activate ATM, which signals to impede spliceosome organization further and augment ultraviolet-irradiation-triggered alternative splicing at the genome-wide level. Our findings define R-loop-dependent ATM activation by transcription-blocking lesions as an important event in the DNA damage response of non-replicating cells, and highlight a key role for spliceosome displacement in this process

Apc-mutant cells act as supercompetitors in intestinal tumour initiation

A delicate equilibrium of WNT agonists and antagonists in the intestinal stem cell (ISC) niche is critical to maintaining the ISC compartment, as it accommodates the rapid renewal of the gut lining. Disruption of this balance by mutations in the tumour suppressor gene APC, which are found in approximately 80% of all human colon cancers, leads to unrestrained activation of the WNT pathway1,2. It has previously been established that Apc-mutant cells have a competitive advantage over wild-type ISCs3. Consequently, Apc-mutant ISCs frequently outcompete all wild-type stem cells within a crypt, thereby reaching clonal fixation in the tissue and initiating cancer formation. However, whether the increased relative fitness of Apc-mutant ISCs involves only cell-intrinsic features or whether Apc mutants are actively involved in the elimination of their wild-type neighbours remains unresolved. Here we show that Apc-mutant ISCs function as bona fide supercompetitors by secreting WNT antagonists, thereby inducing differentiation of neighbouring wild-type ISCs. Lithium chloride prevented the expansion of Apc-mutant clones and the formation of adenomas by rendering wild-type ISCs insensitive to WNT antagonists through downstream activation of WNT by inhibition of GSK3β. Our work suggests that boosting the fitness of healthy cells to limit the expansion of pre-malignant clones may be a powerful strategy to limit the formation of cancers in high-risk individuals

アズキにおける分子マーカーを利用した遺伝子的変異の解析

博士（農学）神戸大