Lysine crotonylation (Kcr) is a newly identified histone modification that is associated with active transcription in mammalian cells. Here we report that the chromodomain Y-like transcription corepressor CDYL negatively regulates histone Kcr by acting as a crotonyl-CoA hydratase to convert crotonyl-CoA to β-hydroxybutyryl-CoA. We showed that the negative regulation of histone Kcr by CDYL is intrinsically linked to its transcription repression activity and functionally implemented in the reactivation of sex chromosome-linked genes in round spermatids and genome-wide histone replacement in elongating spermatids. Significantly, Cdyl transgenic mice manifest dysregulation of histone Kcr and reduction of male fertility with a decreased epididymal sperm count and sperm cell motility. Our study uncovers a biochemical pathway in the regulation of histone Kcr and implicates CDYL-regulated histone Kcr in spermatogenesis, adding to the understanding of the physiology of male reproduction and the mechanism of the spermatogenic failure in AZFc (Azoospermia Factor c)-deleted infertile men.
Previously, we reported that chromodomain Y-like (CDYL) acts as a crotonyl-coenzyme A hydratase and negatively regulates histone crotonylation (Kcr). However, the global CDYL-regulated crotonylome remains unclear. Here, we report a large-scale proteomics analysis for protein Kcr. We identify 14,311 Kcr sites across 3734 proteins in HeLa cells, providing by far the largest crotonylome dataset. We show that depletion of CDYL alters crotonylome landscape affecting diverse cellular pathways. Specifically, CDYL negatively regulated Kcr of RPA1, and mutation of the Kcr sites of RPA1 impaired its interaction with single-stranded DNA and/or with components of resection machinery, supporting a key role of RPA1 Kcr in homologous recombination DNA repair. Together, our study indicates that protein crotonylation has important implication in various pathophysiological processes. reveals CDYL-regulated RPA1 crotonylation in homologous recombination-mediated DNA repair.
Summary
During seed germination, cells embark on extensive post‐transcriptional and post‐translational modifications (PTM), providing a perfect platform to study these events in embryo rebooting from relative quiescenct to highly active state. PR‐619, a deubiquitylase inhibitor, delayed the rice seed germination and resulted in the accumulation of ubiquitylated proteins, which indicated the protein ubiquitylation is involved in this process. Using the K‐Ɛ‐GG antibody enrichment method integrated with high‐resolution mass spectrometry, a list of 2576 lysine ubiquitylated (Kub) sites in 1171 proteins was compiled for rice embryos at 0, 12 and 24 h after imbibition (HAI). Of these, the abundance of 1419 Kub sites in 777 proteins changed significantly. Most of them substantially increased within the first 12 HAI, which is similar to the dynamic state previously observed for protein phosphorylation, implying that the first 12 HAI are essential for subsequent switch during rice seed germination. We also quantitatively analyzed the embryo proteome in these samples. Generally, a specific protein’s abundance in the ubiquitylome was uncorrelated to that in the proteome. The differentially ubiquitinated proteins were greatly enriched in the categories of protein processing, DNA and RNA processing/regulation related, signaling, and transport. The DiGly footprint of the Kub sites was significantly reduced on K48, a linkage typically associated with proteasome‐mediated degradation. These observations suggest ubiquitylation may modulate the protein function more than providing 26S degradation signals in the early stage of rice seed germination. Revealing this comprehensive ubiquitylome greatly increases our understanding of this critical PTM during seed germination.
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