Allele-Specific Engineering of Methyllysine Writers and Readers for Controlling Chromatin-Dependent Processes

	One of the key players in regulating the gene pattern is the post-translational modifications (PTMs) of histone proteins. Histone modifications regulate the transcriptional potential of genes by interacting with reader/effector protein domains. Post-translational modifications on methyllysine are ubiquitous in biological systems and critical for mammalian development. Specific perturbation of such interactions has remained a challenging endeavor. We hypothesized that incorporation of an unnatural modification with the aid of an engineered writer domain and its recognition by reader domain would regulate the downstream genes (epigenetic editing) leading to modification of the epigenetic landscape. The engineered orthogonal pairs together with catalytically inactive Cas9 would specifically modulate the expression of a gene of interest, thereby providing control on transcription machinery. We employed the allele-specific strategy towards engineering the epigenetic landscape at protein-protein interface orthogonal to the human proteome. We generated a hole-modified methyltransferase (writer) that would install an aryllysine moiety on histones in-cellulo. We established the orthogonality of the engineered system, overcame the permeability issue of SAM analogues, developed an antibody and established the applicability of the system in cells. Our data confirms successful benzylation of histone proteins in mammalian cells at sites known to be regulated by SUV39h2 (writer protein) in cellulo. Further we engineered a chromodomain (reader) with a pocket to accommodate the bulky modifications. We established the biochemical integrity of the engineered interface, provided structural evidence for domain integrity, demonstrated the generality of the approach, and validated its applicability to identify transcriptional regulators. We have shown that the orthogonal reader domain on binding to the unnatural modification remains functionally intact and to regulate the epigenetic landscape similar engineering can be translated to other reader-histone proteins as well.