Global Profiling of the Lysine Crotonylome in Different Pluripotent States

Abstract

Pluripotent stem cells (PSCs) can be expanded in vitro in different culture conditions, resulting in a spectrum of cell states with distinct properties. Understanding how PSCs transition from one state to another, ultimately leading to lineage-specific differentiation, is important for developmental biology and regenerative medicine. Although there is significant information regarding gene expression changes controlling these transitions, less is known about post-translational modifications of proteins. Protein crotonylation is a newly discovered post-translational modification where lysine residues are modified with a crotonyl group. Here, we employed affinity purification of crotonylated peptides and liquid chromatography–tandem mass spectrometry (LC–MS/MS) to systematically profile protein crotonylation in mouse PSCs in different states including ground, metastable, and primed states, as well as metastable PSCs undergoing early pluripotency exit. We successfully identified 3628 high-confidence crotonylated sites in 1426 proteins. These crotonylated proteins are enriched for factors involved in functions/processes related to pluripotency such as RNA biogenesis, central carbon metabolism , and proteasome function. Moreover, we found that increasing the cellular levels of crotonyl-coenzyme A (crotonyl-CoA) through crotonic acid treatment promotes proteasome activity in metastable PSCs and delays their differentiation, consistent with previous observations showing that enhanced proteasome activity helps to sustain pluripotency. Our atlas of protein crotonylation will be valuable for further studies of pluripotency regulation and may also provide insights into the role of metabolism in other cell fate transitions.

Global crotonylome reveals hypoxia-mediated lamin A crotonylation regulated by HDAC6 in liver cancer

“…Previous studies on non-histone crotonylation with the application of quantitative proteomics have usually been carried out on cell lines [ 12 , 21 ]. This gives better data stability and uniformity.…”

Qualitative lysine crotonylome analysis in the ovarian tissue of Harmonia axyridis (Pallas)

“…axyridis ovarian tissues, which was consistent with multiple ribosome related terms in cellular component analysis. Previous study in pluripotent stem cells showed a similar observation that RNA binding proteins are widely crotonylation modified [ 35 ]. Moreover, in the biological process level, cytoplasmic translation displayed the highest enrichment score.…”

Crotonylation promotes human embryonic stem cell endodermal lineage differentiation and metabolic switch

“…Taken together, these observations point to GAPDH as a critical player in regulating hESC pluripotency and promoting endoderm differentiation, and in mediating metabolic switching in response to differentiation-inducing signals. Recently, another group showed that other glycolytic enzymes (e.g., hexokinase 2 and phosphoglycerate kinase 1) could also be crotonylated 55 , adding further support to the role of crotonylation in regulating metabolic function in pluripotent stem cells.…”