Mechanisms And Functions Of Cdyl-mediated Histone Crotonylation In Autosomal Dominant Polycystic Kidney Disease

Autosomal dominant polycystic kidney disease(ADPKD)is the most common inherited kidney disease.However,the molecular mechanisms underlying ADPKD progression remain poorly understood,and safe and effective treatments are still lacking.Accumulating evidence suggest that epigenetic regulation plays a key role in ADPKD.Histone modification is an important part of epigenetic regulation.In recent years,with the application of high-resolution mass spectrometry in the field of histone modification,novel histone modifications are continuously identified,especially shortchain lysine acylation.However,the current researches focus on biochemical identification,the regulatory mechanisms and functions of these modifications in diseases are still unclear.We assessed a variety of histone acylations,such as crotonylation,acetylation,propionylation,butylation,β-hydroxybutylation,lactylation and succinylation in the ADPKD mouse kidneys.We found that histone crotonylation increased markedly in mouse ADPKD kidneys,and histone β-hydroxybutyrylation was decreased.Histone lysine crotonylation is a newly discovered and evolutionarily conserved histone acylation modification.Crotonyl-Co A acts as a donor for crotonylation and regulates the level of lysine crotonylation in cells.The chromodomain Y-like protein(Cdyl)acts as a crotonyl-Co A hydratase and inhibits histone crotonylation by reducing crotonylCo A.We found that Cdyl expression was substantially decreased in mouse ADPKD kidneys,indicating that the increase of histone crotonylation in ADPKD may due to the loss of Cdyl.To further explore the role of Cdyl in the progression of ADPKD,we overexpressed Cdyl in ADPKD mouse models and found that it inhibited histone crotonylation and slowed ADPKD progression.In order to explore the molecular mechanism of Cdyl in ADPKD,we first identified the principle sites of Cdyl-regulated histone crotonylation through proteomic analysis and biochemical experiments.We found substantial increase of H3K9 cr and H3K18 cr in Cdyl KO cells compared to Cdyl intact cells.To unravel the molecular basis of Cdyl function,we first analyzed the amino acid sequence of Cdyl.We found a predicated intrinsically disordered regions(IDRs)in amino acid 52-233.Recent studies demonstrated that many proteins with IDRs have the ability to undergo phase separation,among which are many transcription regulators.Phase separation of these transcription regulators have been implicated in the regulation of gene expression.We observed that Cdyl formed phase-separated droplets in vitro and in vivo.We also found that lysines(K)and arginines(R)residues in IDRs are necessary for the phase separation of Cdyl.Further,by overexpressing wild-type Cdyl and Cdyl KR-A mutant in WT9-12 cells,a well-known ADPKD cell line,we demonstrated that the phaseseparation capacity of Cdyl is essential for Cdyl-regulated histone crotonylation.Integrative analysis of Ch IP-seq and RNA-seq data revealed that Cdyl inhibited the expression of ADPKD cystogenic genes through a phase separation dependent mechanism.Finally,in order to explore the role of Cdyl phase separation in vivo,we microinjected wild-type Cdyl and Cdyl KR-A mutant m RNAs into a zebrafish ADPKD model,and found that phase separation of Cdyl slowed ADPKD progression in the zebrafish model.In this study,we established the important role of Cdyl-mediated histone crotonylation in ADPKD.This work may not only help us better understand the pathophysiology function of Cdyl-mediated histone crotonylation,the role of epigenetic regulation in the pathogenesis of ADPKD,but also may have important implications in developing new therapeutic strategies for ADPKD treatment.