Epigenetic regulation by Polycomb Repressive Complex 1 promotes Cerebral Cavernous Malformations

Cerebral cavernous malformations (CCMs) are anomalies that develop mainly in the cerebral vasculature. They result from mutations in CCM1/KRIT1, CCM2, or CCM3/PDCD10. Loss of CCM proteins triggers a MAPK-Krüppel-like factor 2 (KLF2) signaling cascade, which induces a pathophysiological pattern of gene expression within endothelial cells. The downstream target genes that are activated by KLF2 are mostly unknown. Here we show that Chromobox Protein Homolog 7 (CBX7), component of the Polycomb Repressive Complex 1, contributes to pathophysiological KLF2 signaling during zebrafish cardiovascular development. CBX7/cbx7a mRNA is strongly upregulated in lesions of CCM patients, and in human, mouse, and zebrafish CCM-deficient endothelial cells. The silencing or pharmacological inhibition of CBX7/Cbx7a suppresses pathological CCM phenotypes in ccm2 zebrafish, CCM2-deficient HUVECs, and in a pre-clinical murine CCM3 disease model. Whole-transcriptome datasets from zebrafish cardiovascular tissues and human endothelial cells reveal that CBX7/Cbx7a plays a role in the activation of KLF2 targets including genes encoding TEK, Angiopoietin1, WNT9, and endoMT proteins. Our findings uncover an intricate interplay in the regulation of Klf2-dependent biomechanical signaling by CBX7 in CCM. This work also provides insights for therapeutic strategies in the pathogenesis of CCM. Overall design: In this experiment, we want to compare the RNA transcripts of different genetic background of zebrafish heart (which is considerred as enriched endothelial tissue). We have pooled heart samples from 56-59 hour post fertilized hour zebrafish embryos with 3 conditions (4 groups): wildtype (wt), ccm2 mutant (ccm2), double knockout mutant (DKOwBF). We prepared 4 biological samples for each group. Each sample consisting of 15-60 heart was RNA extracted, DNase treatment using Zymo Quick-RNA Microprep Kit. RNA later was checked integrity and concentration by D1000 Agilent ScreenTape. Samples with RIN values ranged from 7.2-9.3 were further librarized with NebNext Ultra II kit, with 30 ng of RNA as starting point.