Transcription imparts architecture, function and logic to enhancer units

Distal enhancers play pivotal roles in development and disease yet remain one of the least understood regulatory elements. We used massively parallel reporter assays to perform functional comparisons of two leading enhancer models and find that gene-distal transcription start sites are robust predic...

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Published inNature genetics Vol. 52; no. 10; pp. 1067 - 1075
Main Authors Tippens, Nathaniel D, Liang, Jin, Leung, Alden King-Yung, Wierbowski, Shayne D, Ozer, Abdullah, Booth, James G, Lis, John T, Yu, Haiyuan
Format Journal Article
LanguageEnglish
Published United States Nature Publishing Group 01.10.2020
Subjects
Analysis
Binding sites
Boundaries
Candidates
Cell Line
Cloning
Clustered Regularly Interspaced Short Palindromic Repeats - genetics
Clusters
CRISPR
Enhancer Elements, Genetic - genetics
Enhancers
Gene expression
Genetic aspects
Genetic transcription
Genomes
Histone Code - genetics
Histones
Humans
Modular units
Multiprocessing
Promoter Regions, Genetic - genetics
Promoters (Genetics)
Protein Processing, Post-Translational - genetics
Regulatory sequences
RNA polymerase
Sequences
Transcription
Transcription Initiation Site
Transcription Initiation, Genetic
Transcription, Genetic
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Summary:Distal enhancers play pivotal roles in development and disease yet remain one of the least understood regulatory elements. We used massively parallel reporter assays to perform functional comparisons of two leading enhancer models and find that gene-distal transcription start sites are robust predictors of active enhancers with higher resolution than histone modifications. We show that active enhancer units are precisely delineated by active transcription start sites, validate that these boundaries are sufficient for capturing enhancer function, and confirm that core promoter sequences are necessary for this activity. We assay adjacent enhancers and find that their joint activity is often driven by the stronger unit within the cluster. Finally, we validate these results through functional dissection of a distal enhancer cluster using CRISPR-Cas9 deletions. In summary, definition of high-resolution enhancer boundaries enables deconvolution of complex regulatory loci into modular units.
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N.D.T., J.L., A.O., J.T.L., and H.Y. conceived of the project and designed the enhancer comparison screen. N.T. conceived of dissecting enhancer cooperativity and mechanisms. J.L. performed cloning, primer design, Cas9 deletions, and all eSTARR- and Clone-seq assays. N.D.T. optimized and prepared enhancer fusions with guidance from A.O., H.Y., and J.T.L.. N.D.T. and A.K.L. performed analysis with feedback from J.G.B., J.L., A.O., J.T.L., and H.Y.. S.D.W. designed sgRNAs. N.T. wrote the manuscript with feedback from all authors.
Author contributions
ISSN:1061-4036
1546-1718
DOI:10.1038/s41588-020-0686-2