Active Versus Accessible: Engineering Open Chromatin in Mammalian Cells

Chromatin contributes to variability in mammalian genome engineering. Controlling chromatin states allows us to overcome chromosomal context dependence to effectively manipulate gene expression and editing in eukaryotic chassis. We designed, built, and are currently testing a panel of synthetic pioneer factors (SPiFs) to open condensed, repressive chromatin. Pioneer factors are are a unique class of activators able to bind DNA even in closed chromatin. Our SPiFs are composed of a chromatin-modifying peptide fused to a Gal4 DNA binding domain and mCherry tag. We used 16 distinct peptides with different functions including posttranslational histone modification, PolII recruitment, and nucleosome remodeling. To test our SPiFs, we used a doxycycline-inducible system that induces accumulation of PRC2 heterochromatin at a luciferase reporter in HEK293 cells. We targeted the induced heterochromatin with each SPiF and measured enhancement of expression in closed chromatin. Consistent with previous work, we found that PolII recruitment factors significantly increase expression in PRC2-enriched heterochromatin. We also discovered that the active site from proto-oncogene MYB comparably increases expression. Transient induction by these SPiFs yields a sustained increase in relative expression over 12 cell divisions, suggesting an open chromatin state with exposed DNA. To further investigate DNA accessibility, we tested the ability of SPiFs to improve Cas9 editing, which is inhibited by PRC2 heterochromatin. Deep sequencing data show that SPiFs that induce gene expression fail to increase chromatin accessibility for CRISPR/Cas9. We found that high levels of transcription after pioneer treatment inhibit Cas9-mediated repair in active chromatin, most likely because of polymerase crowding. How then can we engineer accessible, expression-neutral chromatin? Other SPiFs in our panel do not increase gene expression (neutral effect), but are expected to impact local chromatin by altering histone modification. Tests are underway to determine if the neutral SPiFs alter nucleosome occupancy and enhance Cas9 editing in closed chromatin.