Post-translational modification of the histone proteins in chromatin plays a central role in the epigenetic control of DNA-templated processes in eukaryotic cells. Developing methods that enable the structure of histones to be manipulated is, therefore, essential to understand the biochemical mechanisms that underlie genomic regulation. Here we present a synthetic biology method to engineer histones that bear site-specific modifications on cellular chromatin using protein trans-splicing (PTS). We genetically fused the N-terminal fragment of ultrafast split intein to the C terminus of histone H2B, which, on reaction with a complementary synthetic C intein, generated labelled histone. Using this approach, we incorporated various non-native chemical modifications into chromatin in vivo with temporal control. Furthermore, the time and concentration dependence of PTS performed in nucleo enabled us to examine differences in the accessibility of the euchromatin and heterochromatin regions of the epigenome. Finally, we used PTS to semisynthesize a native histone modification, H2BK120 ubiquitination, in isolated nuclei and showed that this can trigger downstream epigenetic crosstalk of H3K79 methylation.