A systematic comparison reveals substantial differences in chromosomal versus episomal encoding of enhancer activity

The magnitude and determinants of differences in cis-regulation for regulatory sequences residing in episomes versus chromosomes remain almost completely unknown. To address this question in a systematic manner, we developed and applied a novel lentivirus-based massively parallel reporter assay (lentiMPRA) to directly compare the functional activities of 2,236 candidate liver enhancers in an episomal versus a chromosomally integrated context. We find that the activities of chromosomally integrated sequences are substantially different from the activities of the identical sequences assayed on episomes, and furthermore are correlated with different subsets of ENCODE annotations. The results of chromosomally-based reporter assays are also more reproducible and more strongly predictable by both ENCODE annotations and sequence-based models. With a linear model that combines chromatin annotations and sequence information, we achieve a Pearson''s R2 of 0.347 for predicting the results of chromosomally integrated reporter assays. This is substantially better than with either chromatin annotations or sequence information alone and also outperforms predictive models of episomal assays. Our results have broad implications for how cis-regulatory elements are identified, prioritized and functionally validated. Overall design: We developed and applied a novel lentivirus-based massively parallel reporter assay (lentiMPRA) to directly compare the functional activities of 2,236 candidate liver enhancers and 204 controls in an episomal versus a chromosomally integrated context. By using integration-competent vs. integration-defective components of the lentiviral system, we directly compared the functional activities of 2,236 candidate liver enhancers in a chromosomally integrated versus an episomal context in the human liver hepatocellular carcinoma cell line, HepG2.