We report the identification of a new type of histone mark, lysine 
2-hydroxyisobutyrylation (Khib), and identify the mark at 63 human and mouse 
histone Khib sites, including 27 unique lysine sites that are not known to be 
modified by lysine acetylation (Kac) and lysine crotonylation (Kcr). This 
histone mark was initially identified by MS and then validated by chemical and 
biochemical methods. Histone Khib shows distinct genomic distributions from 
histone Kac or histone Kcr during male germ cell differentiation. Using 
chromatin immunoprecipitation sequencing, gene expression analysis and 
immunodetection, we show that in male germ cells, H4K8hib is associated with 
active gene transcription in meiotic and post-meiotic cells. In addition, 
H4K8ac-associated genes are included in and constitute only a subfraction of 
H4K8hib-labeled genes. The histone Khib mark is conserved and widely 
distributed, has high stoichiometry and induces a large structural change. These 
findings suggest its critical role on the regulation of chromatin functions.