How do cells create enhancers? A biochemical model of enhancer activation using protein trans-splicing

Abstract

Histone post-translational modifications (hPTMs) play a critical rule in the functional and structural organization of chromatin. Increasing evidence suggests that aberrant gene expression patterns associated with cancer may result from chromatin dysregulation at the hPTM level. Moreover, disruptions of the molecular machinery involved in enhancer activation have also been implicated in several human cancers, as well as numerous developmental diseases. Recent studies have identified two hPTMs –H3K4me1 and H3K27ac– as the epigenetic signature of active enhancers. However, the specific sequence of events that creates this signature remains unknown, while available technologies to investigate hPTMs in live cells are limited. Here, we hypothesize that H3K4me1 induces H3K27ac deposition to activate genomic enhancers. First, we use an ultra-fast split intein to develop a protein trans-splicing (PTS) technology for N-terminal protein modification in live cells. We then apply this method to semi-synthesize the N-terminal tail of histone H3 with different PTMs at K4 and K27. We also report the first semi-synthesis of a dually modified protein in live cells. We have thus established live cell PTS as a powerful chemical tool for precise protein modification in biologically complex settings. Next, we employ this PTS approach to test the hypothesized H3K4me1-H3K27ac crosstalk in its native context. Namely, we explore whether selective installation of H3K4me1 with PTS increases H3K27ac in live cells. We present preliminary data suggesting the epigenetic crosstalk of interest may occur in trans in isolated nuclei. Future studies will require a more sensitive readout of local H3K27ac levels to complement the global measurements performed in this work. Further characterization of the relationship between H3K4me1 and H3K27ac will elucidate the exact mechanism of enhancer activation and inform us of the molecular basis of many human diseases.