TonEBP in ER stress-induced autophagy formation and DNA damage response

Abstract

Tonicity-responsive enhancer-binding protein (TonEBP), is defined as transcription regulator in cellular response to hypertonic stress in kidney. Many previous studies have reported about TonEBP function in pleiotropic stress condition. In case of functional study, TonEBP has been reported as regulator for pathogenesis of rheumatoid arthritis, atherosclerosis, diabetic bephropathy, acute kidney injury, hyperlipidaemia, insulin resistance, autoimmune diseases, salt-sensitive hypertension and hepatocellular carcinoma. But interestingly, these pathological actions of TonEBP are opposite from safeguard functions of TonEBP in response to ER stress and DNA damage. Newly defined function of TonEBP is based on interactomes of TonEBP. I used tandem affinity purification (TAP) systems for TonEBP purification, followed by detection of peptides with LC-MS/MS. I identified over 465 proteins that interacts with TonEBP and over 50% of list of proteins are related to acute stress response, such as autophagy formation and DNA damage response. 1) ER stress-induced autophagy formation: Autophagy has protective roles against the endoplasmic reticulum (ER) stress. TonEBP promotes β-cell survival under ER stress. TonEBP is associated with FIP200, essential component for autophagy initiation. TonEBP depletion leads to accumulation of unfolded proteins, followed by reduction of cell survival. Mice with TonEBP deletion in pancreatic endocrine progenitor cells leads to defective glucose homeostasis and loss of islet mass. 2) PCNA polyubiquitination: Polyubiquitination of proliferating cell nuclear antigen (PCNA) regulates the error-free template-switching for the bypass of DNA adducts during DNA replication. PCNA polyubiquitination is essential for the genomic integrity. TonEBP regulates MMS-induced PCNA polyubiquitination. TonEBP was recruited to DNA damage sites and sequentially recruited SHPRH, USP1 to DNA damage sites for regulation of PCNA polyubiquitination, followed by replication fork reversal and template switching. 3) R-loop resolution via m6A RNA methylation: During transcription or genomic threats, the nascent RNA strand can base pair with its template DNA, displacing the non-template strand as ssDNA and forming a structure called an R loop. Accumulation of R loop induces Transcription-replication collision (TRC), followed by replication stress, so regulation for proper level of R loop is critical for the maintenance of genomic integrity. R loop is recognized and resolved by TonEBP via UV-induced N6-methyladenosine (m6A) RNA methylation on R loop. TonEBP was recruited to DNA damage sites and recruited m6A methylase, METTL3 in correlation with m6A RNA methylation. TonEBP had higher binding affinity with highly distorted DNA structure and recognized UV-induced R loop. Depletion of TonEBP and METTL3 reduced R loop resolution. In conclusion, TonEBP has protective roles in response to ER stress via autophagy formation, and in response DNA damage via regulating PCNA polyubiquitination, R-loop recognition, and resolution of R-loop by initiating m6A RNA methylation. These diverse functions of TonEBP are mediated by distinct and specific protein-protein interactions.