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

Candidate enhancers can be identified on the basis of chromatin modifications, the binding of chromatin modifiers and transcription factors and cofactors, or chromatin accessibility. However, validating such candidates as bona fide enhancers requires functional characterization, typically achieved t...

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Autores principales: Inoue, Fumitaka, Kircher, Martin, Martin, Beth, Cooper, Gregory M., Witten, Daniela M., McManus, Michael T., Ahituv, Nadav, Shendure, Jay
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory Press 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5204343/
https://www.ncbi.nlm.nih.gov/pubmed/27831498
http://dx.doi.org/10.1101/gr.212092.116
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author Inoue, Fumitaka
Kircher, Martin
Martin, Beth
Cooper, Gregory M.
Witten, Daniela M.
McManus, Michael T.
Ahituv, Nadav
Shendure, Jay
author_facet Inoue, Fumitaka
Kircher, Martin
Martin, Beth
Cooper, Gregory M.
Witten, Daniela M.
McManus, Michael T.
Ahituv, Nadav
Shendure, Jay
author_sort Inoue, Fumitaka
collection PubMed
description Candidate enhancers can be identified on the basis of chromatin modifications, the binding of chromatin modifiers and transcription factors and cofactors, or chromatin accessibility. However, validating such candidates as bona fide enhancers requires functional characterization, typically achieved through reporter assays that test whether a sequence can increase expression of a transcriptional reporter via a minimal promoter. A longstanding concern is that reporter assays are mainly implemented on episomes, which are thought to lack physiological chromatin. However, the magnitude and determinants of differences in cis-regulation for regulatory sequences residing in episomes versus chromosomes remain almost completely unknown. To address this systematically, we developed and applied a novel lentivirus-based massively parallel reporter assay (lentiMPRA) to directly compare the functional activities of 2236 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 R(2) of 0.362 for predicting the results of chromosomally integrated reporter assays. This level of prediction is 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.
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spelling pubmed-52043432017-07-01 A systematic comparison reveals substantial differences in chromosomal versus episomal encoding of enhancer activity Inoue, Fumitaka Kircher, Martin Martin, Beth Cooper, Gregory M. Witten, Daniela M. McManus, Michael T. Ahituv, Nadav Shendure, Jay Genome Res Research Candidate enhancers can be identified on the basis of chromatin modifications, the binding of chromatin modifiers and transcription factors and cofactors, or chromatin accessibility. However, validating such candidates as bona fide enhancers requires functional characterization, typically achieved through reporter assays that test whether a sequence can increase expression of a transcriptional reporter via a minimal promoter. A longstanding concern is that reporter assays are mainly implemented on episomes, which are thought to lack physiological chromatin. However, the magnitude and determinants of differences in cis-regulation for regulatory sequences residing in episomes versus chromosomes remain almost completely unknown. To address this systematically, we developed and applied a novel lentivirus-based massively parallel reporter assay (lentiMPRA) to directly compare the functional activities of 2236 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 R(2) of 0.362 for predicting the results of chromosomally integrated reporter assays. This level of prediction is 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. Cold Spring Harbor Laboratory Press 2017-01 /pmc/articles/PMC5204343/ /pubmed/27831498 http://dx.doi.org/10.1101/gr.212092.116 Text en © 2017 Inoue et al.; Published by Cold Spring Harbor Laboratory Press http://creativecommons.org/licenses/by-nc/4.0/ This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
spellingShingle Research
Inoue, Fumitaka
Kircher, Martin
Martin, Beth
Cooper, Gregory M.
Witten, Daniela M.
McManus, Michael T.
Ahituv, Nadav
Shendure, Jay
A systematic comparison reveals substantial differences in chromosomal versus episomal encoding of enhancer activity
title A systematic comparison reveals substantial differences in chromosomal versus episomal encoding of enhancer activity
title_full A systematic comparison reveals substantial differences in chromosomal versus episomal encoding of enhancer activity
title_fullStr A systematic comparison reveals substantial differences in chromosomal versus episomal encoding of enhancer activity
title_full_unstemmed A systematic comparison reveals substantial differences in chromosomal versus episomal encoding of enhancer activity
title_short A systematic comparison reveals substantial differences in chromosomal versus episomal encoding of enhancer activity
title_sort systematic comparison reveals substantial differences in chromosomal versus episomal encoding of enhancer activity
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5204343/
https://www.ncbi.nlm.nih.gov/pubmed/27831498
http://dx.doi.org/10.1101/gr.212092.116
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