Journal: iScience

Article Title: CaMKKβ regulates transcription factor Elf2 gene methylation to maintain endothelial junctional barrier integrity

doi: 10.1016/j.isci.2025.114255

Figure Lengend Snippet: CaMKKβ deficiency leads to the hyper-methylation of the Elf2 transcription factor gene (A) DNA sequence reveals the low levels of CpG sites in the promoter regions of mouse Tie2 and VE-cadherin (Cdh5). (B) Volcano plot shows changes in the methylation level of genes in lungs of Camkkβ +/+ and CamKKβ −/− mice. The ratio of CpG methylated sites in genes (+/− 2 kb of the TSS) of Camkkβ +/+ /Camkkβ −/− mice were presented (fold changes vs. p -value). (C) Quantified data show hyper-methylation of Elf2 gene promoter in CamKKβ −/− mice. N = 3 samples per genotype; ∗ p < 0.05 (unpaired t test). (D) DNA sequence analysis reveals the abundance of CpG islands and CpG sites in the promoter regions of human and mouse Elf2 genes. (E) WT and Camkkβ −/− mice lung mRNA levels of Elf2 and Elf1 were determined by qRT-PCR. N = 3 mice per genotype; ns, not significant, ∗∗∗ p < 0.001 (unpaired t test). (F) DNA methyl transferase inhibition restores Elf2 expression in Camkkβ −/− mice. WT and Camkkβ −/− mice were injected with 5-AZA (1 mg/kg, i.p.) or vehicle for 5 days as described above in C. Lungs were harvested and used for IB. N = 3 mice per group; representative blot is shown. (G) Expression of WT-CaMKKβ in EC of Camkkβ −/− mice rescued Elf2 expression. Camkkβ −/− mice were injected with liposome-CaMKKβ plasmid (1 μg plasmid/g body weight) complexes containing either WT-CaMKKβ or the kinase defective CaMKKβ ( K193A -CaMKKβ) mutant. At 96 h after injection, lungs were harvested and used for IB analysis. N = 3 mice per group; representative blot is shown.

Article Snippet: The purified genomic DNA was sheared and used as input for the Methylated DNA IP kit (Zymo Research), along with a spike-in Control DNA.

Techniques: Methylation, Sequencing, Quantitative RT-PCR, Inhibition, Expressing, Injection, Plasmid Preparation, Mutagenesis

Journal: iScience

Article Title: CaMKKβ regulates transcription factor Elf2 gene methylation to maintain endothelial junctional barrier integrity

doi: 10.1016/j.isci.2025.114255

Figure Lengend Snippet: EC-restricted deletion of Elf2 ( Elf2 ΔEC ) in adult mice reduces VE-cadherin and Tie2 expression and exacerbates sepsis-induced lung vascular injury (A) Depicts the protocol used to create EC-restricted Elf2 knockout ( Elf2 ΔEC ) mice. The mixture of liposome and plasmid expressing sgRNA to target mElf2 or scrambled sg-RNA (Sc-sgRNA) was injected i.v. into the CRISPR/Cas9-cdh5-Cre mice. Four days after injection, mice were used for experiments. (B) LEC isolated from mice injected with Sc-sgRNA (WT) or sgRNA to target mElf2 (Elf2 ΔEC ) were used for IB analysis. (C) IB analysis of lung tissue from WT and Elf2 ΔEC mice. (D) Lung sections from WT and Elf2 ΔEC mice were stained with antibodies specific to VE-cadherin, vWF (EC-marker), and DAPI. Right panels show the magnified images. (Scale bars = 20 μm). (E) H&E staining of lung sections. BR, bronchi; V, vessel. Green arrow heads showed increased perivascular space in Elf2 ΔEC mice indicating basal vascular leak. (Scale bars = 50 μm). (F) WT and Elf2 ΔEC mice were challenged with LPS (5 mg/kg, i.p.) and then used to assess in vivo lung vascular leak by measuring EBA uptake. Augmented lung vascular leak was observed in Elf2 ΔEC mice. N = 4 mice per genotype; ∗∗∗∗ p < 0.0001 (two-way ANOVA). (G) Survival follows LPS (5 mg/kg, i.p.) in WT and Elf2 ΔEC mice. N = 6 in each group. ∗∗ p < 0.01 (log rank test). (H) Model for the epigenetic regulation of Elf2 gene expression in endothelial cells during repair of sepsis-induced lung vascular injury created in https://BioRender.com . In quiescent endothelial cells (ECs), the CpG-rich promoter of the Elf2 gene is methylated by DNA methyltransferases (DNMTs). This methylation recruits the methyl-CpG binding protein MeCP2, which binds to the methylated CpG sites and represses Elf2 transcription. During endotoxemia, activation of TLR4 and/or PAR-1 increases intracellular Ca 2+ levels in EC, leading to loss of endothelial barrier integrity through phosphorylation-driven internalization and ubiquitin-mediated degradation of VE-cadherin. , The rise in intracellular Ca 2+ also activates Ca 2+ /calmodulin-dependent kinase CaMKKβ, which translocates to the nucleus and phosphorylates MeCP2 at Ser 421 . This phosphorylation causes MeCP2 to dissociate from methylated CpG sites, triggering CpG demethylation and reactivation of Elf2 transcription. Once reactivated, Elf2 promotes its own expression and upregulates VE-cadherin and Tie2, facilitating the repair of the disrupted endothelial barrier. SAM, S-adenosyl-methionine; SAHC, S-adenosylhomocysteine; TLR4, toll-like receptor 4; PAR-1, protease-activated receptor 1; Thr, thrombin; orange star, methylated CpGs.

Article Snippet: The purified genomic DNA was sheared and used as input for the Methylated DNA IP kit (Zymo Research), along with a spike-in Control DNA.

Techniques: Expressing, Knock-Out, Plasmid Preparation, Injection, CRISPR, Isolation, Staining, Marker, In Vivo, Gene Expression, Methylation, Binding Assay, Activation Assay, Phospho-proteomics, Ubiquitin Proteomics