Physiological hypoxia can trigger transcriptional events that influence many developmental processes during mammalian embryogenesis. One way that hypoxia affects transcription is by engaging chromatin-remodeling complexes. We now report that chromodomain helicase DNA binding protein 4 (CHD4), an enzyme belonging to the nucleosome remodeling and deacetylase (NuRD) chromatin-remodeling complex, is required for transcriptional repression of the receptor-interacting protein kinase 3 (Ripk3)—a critical executor of the necroptosis cell death program—in hypoxic murine embryonic endothelial cells. Genetic deletion of Chd4 in murine embryonic endothelial cells in vivo results in upregulation of Ripk3 transcripts and protein prior to vascular rupture and lethality at midgestation, and concomitant deletion of Ripk3 partially rescues these phenotypes. In addition, CHD4 binds to and prevents acetylation of the Ripk3 promoter in cultured endothelial cells grown under hypoxic conditions to prevent excessive Ripk3 transcription. These data demonstrate that excessive RIPK3 is detrimental to embryonic vascular integrity and indicate that CHD4 suppresses Ripk3 transcription when the embryonic environment is particularly hypoxic prior to the establishment of fetal-placental circulation at midgestation. Altogether, this research provides new insights into regulators of Ripk3 transcription and encourages future studies into the mechanism by which excessive RIPK3 damages embryonic blood vessels.
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We thank K. Georgopoulos for Chd4-floxed mice, V. Dixit for Ripk3-/- mice, W. Alexander for Mlkl-/- mice, R. Adams for Cdh5(PAC)-CreERT2 mice, Genentech for anti-RIPK3 antibody, B. Frank and the OMRF Microarray Core for technical assistance with microarrays, T. Griffin for help with and use of his hypoxic culture chamber system, W. Freeman and the OUHSC Targeted DNA Methylation & Mitochondrial Heteroplasmy Core for help with bisulfite sequencing, and Griffin lab members for helpful discussions.
This study was supported in part by NIH grants HL111178, HL134778, HL144605, and GM103441 awarded to CTG and GM114731 awarded to CTG and FL, by grants from the Oklahoma Center for the Advancement of Science and Technology (HR11-013 and HR15-078) awarded to CTG, by grants from the American Heart Association (15GRNT25090015) awarded to CTG, (16POST31300013) awarded to MM, and (19PRE343800708) awarded to SG, and by a grant from the Presbyterian Health Foundation awarded to CTG.
Conflict of interest
The authors declare that they have no conflict of interest.
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Edited by J. Silke