Arabidopsis thaliana RNase H2 deficiency counteracts the needs for the WEE1 checkpoint kinase but triggers genome instability
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina HiSeq 2000
ORGANISM(S): Arabidopsis thaliana
SUBMITTER: Lieven De Veylder
PROVIDER: E-MTAB-2590 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Col-0 and WEE1KO (wee1-1 and wee1-2) were germinated on control medium on a nylon mesh and transferred 5 days after germination to control medium. Samples were harvested at three different time points after transfer: 0 h, 5 h, and 24 h. All sampling points were performed in four independent experiments for Col-0, two independent experiments for wee1-1 and two independent experiments for wee1-2. For each time point in each experiment ﾱ50 root tips were collected and frozen in liquid nitrogen. RNA was extracted from root tissue with TriZol reagent (Invitrogen) and purified with RNneasy kit (Qiagen). The RNA of two independent experiments for Col-0 and WEE1KO were subsequently pooled and used for microarray analysis.
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Project description:The G2 DNA damage checkpoint inhibits Cdc2 and mitotic entry through the dual regulation of Wee1 and Cdc25 by the Chk1 effector kinase. Up-regulation of Chk1 by mutation or overexpression bypasses the requirement for up-stream regulators or DNA damage to promote a G2 cell cycle arrest. We screened in fission yeast for mutations that rendered cells resistant to overexpressed Chk1. We identified a mutation in tra1, which encodes one of two homologs of TRRAP, an ATM/R-related pseudokinase that scaffolds several histone acetyltransferase (HAT) complexes. Inhibition of histone deacetylases reverts the resistance to overexpressed Chk1, suggesting this phenotype is due to a HAT activity, though expression of checkpoint and cell cycle genes is not greatly affected. Cells with mutant or deleted tra1 activate Chk1 normally and are checkpoint proficient. However, these cells are semi-wee even when overexpressing Chk1, and accumulate inactive Wee1 protein. The Cdr (changed division response) kinases Cdr1 and Cdr2 are negative regulators of Wee1, and while best characterized in the cellular response to limited nutrition, we show that they are required for the Tra1-dependent alterations to Wee1 function. This identifies Tra1 as another component controlling the timing of entry into mitosis via Cdc2 activation.
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Project description:Background: Atherosclerosis leads high mortality, highlighting an urgent need for new therapeutic strategies. Protein kinases orchestrate multiple cellular events in atherosclerosis and may provide new therapeutic targets for atherosclerosis. Here, we identified a protein kinase, WEE1 G2 checkpoint kinase (WEE1), promoting inflammatory response in atherosclerosis. Methods: The ApoE-/- mice without macrophage-specific WEE1 deletion (ApoE-/-WEE1f/f) and ApoE-/- mice with macrophage-specific WEE1 deletion (ApoE-/-WEE1MCKO) were generated using bone marrow transplantation. These mice and WEE1 inhibitor MK1775 were used in a high-fat diet (HFD)-induced atherosclerotic model. Human atherosclerotic tissues, mouse primary peritoneal macrophages (MPMs), 293T cells, and recombinant proteins were utilized to investigate the pathogenic role and underlying mechanisms of WEE1. Results: We identified up-regulated p-WEE1 in macrophages in atherosclerotic lesions of HFD-fed ApoE-/- mice. Transcriptome sequencing analysis indicated that WEE1 promotes oxLDL-induced inflammation in macrophages. We then demonstrated that macrophage-specific deletion or pharmacological inhibition of WEE1 attenuates atherosclerosis by reducing inflammation both in vivo and in vitro. The overexpression of wild-type WEE1 or activating-mutant WEE1, but not inactivating-mutant WEE1, exacerbates inflammation in macrophages. Mechanistically, transcriptome sequencing analysis and co-immunoprecipitation followed by quantitative proteomics analysis identified p65 as a binding protein of WEE1. We confirmed that WEE1 directly interacts with the RHD domain of p65 via kinase domain and phosphorylates p65 at S536, thereby facilitating subsequent NF-κB activation and inflammatory response in macrophages. Conclusions: Our findings demonstrated that macrophage WEE1 promotes inflammatory atherosclerosis by directly binding to p65 and phosphorylate it at S536. This study provides WEE1 as a new p65 regulator in inflammation and a potential therapeutic target for atherosclerosis.
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