RBBP5 is an epigenetic regulator of mammalian hepatic 12h oscillator [ChIP-seq]

Proteostasis is vital for cellular health, with disruptions leading to pathologies including aging, neurodegeneration and metabolic disorders. Traditionally, proteotoxic stress responses were studied as acute reactions to various noxious factors; however, recent evidence reveals that many stress-response genes exhibit ~12-hour ultradian rhythms under physiological conditions in mammals. These rhythms, driven by an XBP1s-dependent 12h oscillator, are crucial for managing proteostasis. By exploring the chromatin landscape of the murine 12h hepatic oscillator, we identified RBBP5, a key subunit of the COMPASS complex, as an essential epigenetic regulator of proteostasis. RBBP5 is indispensable for regulating both the hepatic 12h oscillator and transcriptional response to acute proteotoxic stress, acting as a co-activator for master proteostasis transcription factor XBP1s. RBBP5 ablation leads to increased sensitivity to proteotoxic stress, chronic inflammation, and hepatic steatosis in mice, along with impaired autophagy and reduced cell survival in vitro. In humans, lower RBBP5 expression is associated with dampened adaptive stress-response gene expression and hepatic steatosis. Our findings establish RBBP5 as a critical regulator of proteostasis, essential for maintaining mammalian organismal health. All mice used for biological rhythm study are in C57BL/6J background, male and between 3 and 4 months of age.Wild-type C57BL/6J mice (n=12) were first entrained under LD12:12 conditions for 2 weeks before transferred to constant darkness for 24hrs. Mice were then sacrificed via cervical dislocation at a 4h interval for a total of 48 hours under constant darkness. Mice were fed ad libitum during the entire experiment. Liver were taken. ChIP for RBBP5 was performed using anti-RBBP5 antibody (Bethyl, A300-109A) as previously described (Pan et al., 2020). Briefly, mouse liver samples were submerged in PBS + 1% formaldehyde, cut into small (~1 mm3) pieces with a razor blade and incubated at room temperature for 15 minutes. Fixation was stopped by the addition of 0.125 M glycine (final concentration). The tissue pieces were then treated with a TissueTearer and finally spun down and washed twice in PBS. Chromatin was isolated by the addition of lysis buffer, followed by disruption with a Dounce homogenizer. The chromatin was enzymatically digested with MNase. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, Proteinase K and heated for reverse-crosslinking, followed by ethanol precipitation. Pellets were resuspended and the resulting DNA was quantified on a NanoDrop spectrophotometer. An aliquot of chromatin (30 μg) was precleared with protein A agarose beads (Invitrogen). Genomic DNA regions of interest were isolated using 4 μg of antibody. Complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinking was reversed by incubation overnight at 65 °C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. The DNA libraries were prepared at the University of Pittsburgh and sequenced at Gene by Gene, Ltd per standard protocols. DNA libraries were prepared with Ovation® Ultralow V2 DNA-Seq library preparation kit (NuGen) using 1ng input DNA. The size selection for libraries was performed using SPRIselect beads (Beckman Coulter) and purity of the libraries were analyzed using the High Sensitivity DNA chip on Bioanalyzer 2100 (Agilent). The prepared libraries pooled and sequenced using Nova-Seq 6000 (Illumina), generating ~30 million 75 bp single-end reads per sample.