Intermittent High Glucose Elevates Nuclear Localization of EZH2 to Cause H3K27me3-Dependent Repression of KLF2 Leading to Endothelial Inflammation
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture and High Glucose Treatment Condition
2.2. Animal Dissection and Treatment Conditions
2.3. Inhibitor Treatment Condition and RNA Silencing in Cultured Endothelial Cells
2.4. Cell Viability Assay
2.5. RNA Isolation
2.6. cDNA Synthesis and Quantitative Analysis through Reverse Transcriptase-Quantitative Polymerase Chain Reaction
2.7. Subcellular Fractionation
2.8. Co-Immunoprecipitation
2.9. Immunoblotting
2.10. Immunofluorescence Imaging and Analysis
2.11. Chromatin Immunoprecipitation (ChIP) and Subsequent Quantitative PCR
2.12. Primer Sequences for Transcript and Promoter Primers
2.13. Statistics
3. Results
3.1. Intermittent High Glucose Treatment Maximally Induced Endothelial Inflammation In Vitro and Ex Vivo
3.2. Intermittent High Glucose Causes Nuclear Localization of EZH2 through Its Threonine 367 Phosphorylation, Thereby Elevating H3K27me3 Level
3.3. In Endothelial Cells Treated with Intermittent High Glucose, Nuclear EZH2 Assembles PRC2 That Endorses H3K27me3 Enrichment on KLF2 and KLF4 Promoters and Further Suppresses Their Expression
3.4. Inhibition of EZH2′s Methyltransferase Activity or siRNA-Mediated Knockdown of EZH2 Reverses Intermittent High Glucose-Dependent Endothelial Inflammation
3.5. Inhibition of EZH2 Blocks Intermittent High Glucose-Driven Endothelial Inflammation in Ex Vivo Rat Aortic Ring Model
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Gene Name | Sequence of Forward Primer | Sequence of Reverse Primer |
---|---|---|
ICAM1 transcript | 5′TTCGTGTCCTGTATGGCCC3′ | 5′CACATTGGAGTCTGCTGGGA3′ |
P-Selectin transcript | 5′CCAACCTGCAAAGGCATAGC3′ | 5′GCGTTGCAGCCAAAGTAACA3′ |
VCAM1 transcript | 5′ACGAATGAGGGGACCACATC3′ | 5′TCCAGAGGGCCACTCAAATG3′ |
UTX transcript | 5′GCAACAGTTAGGTTGGATGC3′ | 5′AGGCATCCTGAACTTTCCCA3′ |
JMJD3 transcript | 5′GGTCTGTTGTACCCCACTGC3′ | 5′CCGCCTCAGTAACAGCCAGA3′ |
KLF2 transcript | 5′CGGCAAGACCTACACCAAGA3′ | 5′TGGTAGGGCTTCTCACCTGT3′ |
KLF4 transcript | 5′CCACCTTCTTCACCCCTAGA3′ | 5′AAGGTTTCTCACCTGTGTGG3′ |
GAPDH transcript | 5′TCGGAGTCAACGGATTTGGT3′ | 5′TTCCCGTTCTCAGCCTTGAC3′ |
KLF2 Promoter Primer | 5′TCCCATCCATCCAGGGTTCT3′ | 5′TCAGAGACTCTCAGGGGAGC3′ |
KLF4 Promoter Primer | 5′TAGAGGGATTCCTGGCGTTG3′ | 5′GATTTTTCCACTCCTCGCCG3′ |
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Thakar, S.; Katakia, Y.T.; Ramakrishnan, S.K.; Pandya Thakkar, N.; Majumder, S. Intermittent High Glucose Elevates Nuclear Localization of EZH2 to Cause H3K27me3-Dependent Repression of KLF2 Leading to Endothelial Inflammation. Cells 2021, 10, 2548. https://doi.org/10.3390/cells10102548
Thakar S, Katakia YT, Ramakrishnan SK, Pandya Thakkar N, Majumder S. Intermittent High Glucose Elevates Nuclear Localization of EZH2 to Cause H3K27me3-Dependent Repression of KLF2 Leading to Endothelial Inflammation. Cells. 2021; 10(10):2548. https://doi.org/10.3390/cells10102548
Chicago/Turabian StyleThakar, Sumukh, Yash T Katakia, Shyam Kumar Ramakrishnan, Niyati Pandya Thakkar, and Syamantak Majumder. 2021. "Intermittent High Glucose Elevates Nuclear Localization of EZH2 to Cause H3K27me3-Dependent Repression of KLF2 Leading to Endothelial Inflammation" Cells 10, no. 10: 2548. https://doi.org/10.3390/cells10102548