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A2665 - Mechano-Activation of Desmoplakin (DSP) Gene Is Mediated by Ten-Eleven Translocation 1 Oxygenase- and Thymine DNA Glycosylase-Dependent Active DNA Demethylation
Author Block: J. Qu, Z. Zhou, H. Chen, S. Liu, T. Kulkarni, V. J. Thannickal, Y. Zhou; Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, United States.
RATIONALE: We recently reported that desmoplakin (DSP), a GWAS-identified risk allele of IPF, is a matrix stiffness-regulated mechanosensitive gene. Stiff matrix promotes DSP overexpression by induction of DNA demethylation in the DSP promoter, leading to transactivation of the gene expression. CRISPR/dCas9-mediated epigenome editing re-establishes the epigenetic control of the DSP gene and reverses mechanoinductive DSP overexpression. This study provided a potential mechanism underlying aberrant DSP expression in IPF and offered a critical tool for investigation of the functional role of DSP in lung fibrogenesis. The current study aims to determine the mechanisms by which stiff matrix promotes DNA demethylation in the DSP promoter.
METHODS: Matrigel-coated soft (1 kPa) and stiff (20 kPa) polyacrylamide gels were made to simulate the stiffness grades of normal and fibrotic lung matrix. 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) in the regulatory elements of the DSP promoter responsible for mechanoinductive DSP expression were quantified by restriction enzyme digestion- and sequence specific PCR-based approaches. DNA methylation profiles were determined by sodium bisulfite sequencing. Knockdown of gene expression was performed by siRNA.
RESULTS: Stiff matrix promotes expression of α-ketoglutarate (α-KG)-dependent ten-eleven translocation 1 (TET1) and thymine DNA glycosylase (TDG) at both the mRNA and protein levels in lung epithelial cells. 5hmC, an intermediate product of TET1/TDG-dependent active demethylation pathway, is increased on stiff vs. soft matrix stiffness conditions with a concomitant decrease in 5mC. Knockdown of TET1 expression restores DNA methylation in the DSP promoter and blocks stiff matrix-induced DSP overexpression. Furthermore, stiff matrix promotes Rho kinase-dependent phosphorylation of STAT3 and activation of this transcription factor. Inhibition of STAT3 activity abrogates stiff matrix-induced TET1 expression.
CONCLUSIONS: These findings suggest that stiff matrix-dependent demethylation of the DSP promoter occurs by an active DNA demethylation mechanism involved in TET1-mediated oxidation of 5mC and TDG-mediated base excision repair, which together revert 5-methylcytosine to 5-cytosine in the regulatory elements of the DSP promoter and activate DSP gene expression. Our data also suggest that transcription factor STAT3 mediates matrix stiffness-regulated TET1 expression.
RESEARCH FUNDING SOURCE: AHA Postdoctoral Fellowship Award 17POST33660976 to J.Q.; NIH R01HL124076 to Y.Z.; NIH P01HL114470 to V.J.T.