The Study Of Histone Crotonylation On The Pluripotency Of Mouse Embryonic Stem Cell

Crotonylation is a new type of posttranslational modification that is catalyzed by crotonyltransferases that transfer crotonyl group from crotonyl-CoA to lysine residue in protein.Previous studies from others and our laboratory demonstrate that CBP/p300 can catalyze histone crotonylation and that class ? deacetylases HDAC1/2/3/8 and SIRT1 can catalyze decrotonylation.In this study,I first performed embryoid body(EB)differentiation with CGR8 embryonic stem cells(ESCs)and then compared the levels of histone acetylation and crotonylation between CGR8 and differentiated EB.I observed markedly reduced levels of histone acetylation and crotonylation in EB.To investigate the potential role of histone crotonylation in ES cells,we made use of a HDAC1 mutant(HDAC1-VRPP)previously identified in our laboratory.This mutant is defective in histone deacetylase activity but maintains a robust histone decrotonylase activity.This mutant provides an important tool for studying the effect of selective histone decrotonylation on the pluripotency of ESCs.To this aim,I first established wild-type HDAC1 and HDAC1-VRPP inducible stable cell lines in ESCs.After treating HDAC1 and HDAC1-VRPP stable cell lines with Dox at different time,I performed WB to examine the protein levels of Oct4,Sox2 and Nanog and qRT-PCR the transcriptional levels of the marker genes of differentiation.The results demonstrate that induction of both HDAC1 and HDAC1-VRPP mutant leads to reduced levels of ESC key transcription factors Oct4,Sox2 and Nanog and elevated levels of differentiation marker genes.The effect of induced expression of both HDAC1 and HDAC1-VRPP on ESC differentiation is further confirmed by morphological examination of ESC colonies.Together our study reveals that ESCs are enriched of histone crotonylation and that enriched histone crotonylation is critically important for maintenance of ESC pluripotency.