Methods for high-resolution genome-wide functional dissection of transcriptional regulatory regions
AssigneesMassachusetts Institute of Technology, Beth Israel Deaconess Medical Center, Inc.
InventorsMelina Claussnitzer, Liang He, Manolis Kellis, Xinchen Wang
Life of this patent
Estimating the term…
Abstract
Embodiments disclosed herein provide a general, scalable, high-throughput, and high-resolution approach for experimental dissection of regulatory regions and driver nucleotides in the context of human biology and disease. Applicants present HiDRA, a novel high-resolution global screen for transcriptional regulatory activity in accessible chromatin regions, enabling high-efficiency, high-throughput, and high-resolution inference of regulatory activity.
Claims19
1-24. (canceled)
A method of identifying genomic enhancer regulatory elements comprising:
- fragmenting genomic DNA at accessible chromatin in a population of cells thereby generating genomic DNA fragments, wherein said fragmenting comprises transposition;
- amplifying the genomic DNA fragments;
- enriching the amplified genomic DNA fragments by size;
- integrating the enriched fragments into a vector to obtain a vector library, wherein the vector encodes a reporter gene and the enriched fragments are integrated into an untranslated region (UTR) of the reporter gene, whereby transcription of the reporter gene results in a transcript comprising the integrated enriched fragment sequence;
- transfecting or transducing a cell line with the vector library, wherein the transcript comprising the integrated enriched fragment sequences is expressed in the cell line;
- sequencing the transcript expressed in the cell line, whereby integrated fragments comprising enhancer activity are identified;
- and
- high-resolution mapping of driver elements of enhancer activity within identified enhancer regulatory elements by a method comprising comparing the fragment enrichment enhancer activity of a set of overlapping fragments represented in the vector library, whereby driver elements of enhancer activity are identified for enhancer regulatory elements.
A method of identifying genomic enhancer regulatory elements comprising:
- fragmenting genomic DNA in a population of cells, thereby generating genomic DNA fragments comprising overhanging ends;
- filling in the overhanging ends with at least one labeled nucleotide;
- joining the filled in overhanging ends of the fragmented genomic DNA, wherein the joined fragments comprise contact domains;
- isolating the joined genomic DNA fragments using the labeled nucleotide;
- amplifying the isolated joined genomic DNA fragments;
- integrating the amplified fragments into a vector to obtain a vector library, wherein the vector encodes a reporter gene and the amplified fragments are integrated into an untranslated region (UTR) of the reporter gene, whereby transcription of the reporter gene results in a transcript comprising the integrated fragment sequence;
- transfecting or transducing a cell line with the vector library, wherein the transcript comprising the integrated amplified fragment sequence is expressed in the cell line;
- and
- sequencing the transcripts expressed in the cell line, whereby integrated fragments comprising enhancer activity are identified.
Dependencies are inferred from the claim text.