title: Genome-scale high-resolution mapping of activating and repressive nucleotides in regulatory regions.
abstract: Massively parallel reporter assays (MPRAs) enable nucleotide-resolution dissection of transcriptional regulatory regions, such as enhancers, but only few regions at a time. Here we present a combined experimental and computational approach, Systematic high-resolution activation and repression profiling with reporter tiling using MPRA (Sharpr-MPRA), that allows high-resolution analysis of thousands of regions simultaneously. Sharpr-MPRA combines dense tiling of overlapping MPRA constructs with a probabilistic graphical model to recognize functional regulatory nucleotides, and to distinguish activating and repressive nucleotides, using their inferred contribution to reporter gene expression. We used Sharpr-MPRA to test 4.6 million nucleotides spanning 15,000 putative regulatory regions tiled at 5-nucleotide resolution in two human cell types. Our results recovered known cell-type-specific regulatory motifs and evolutionarily conserved nucleotides, and distinguished known activating and repressive motifs. Our results also showed that endogenous chromatin state and DNA accessibility are both predictive of regulatory function in reporter assays, identified retroviral elements with activating roles, and uncovered 'attenuator' motifs with repressive roles in active chromatin.
authors.affiliation: Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, California, USA.|Computer Science Department, University of California, Los Angeles, Los Angeles, California, USA.|Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at University of California, Los Angeles, Los Angeles, California, USA.|Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, California, USA.|Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California, USA.
authors.forename: Jason
authors.initials: J
authors.lastname: Ernst
authors.affiliation: Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, Massachusetts, USA.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Melnikov
authors.affiliation: Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, Massachusetts, USA.
authors.forename: Xiaolan
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, Massachusetts, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Wang
authors.affiliation: Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, Massachusetts, USA.
authors.forename: Peter
authors.initials: P
authors.lastname: Rogov
authors.affiliation: Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, Massachusetts, USA.
authors.forename: Tarjei S
authors.identifier: http://orcid.org/0000-0002-8133-3135
authors.initials: TS
authors.lastname: Mikkelsen
authors.affiliation: Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, Massachusetts, USA.|Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: Manolis
authors.initials: M
authors.lastname: Kellis
mesh_terms: D002874:Chromosome Mapping / Q000379:methods*; D017124:Conserved Sequence / Q000235:genetics; D063185:Epigenetic Repression / Q000235:genetics*; D015894:Genome, Human / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D009711:Nucleotides / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D009711:Nucleotides