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. 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.