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Principal Investigator: JOHN T LIS
Organization: CORNELL UNIVERSITY
Fiscal Year: 2024
Award: $680,349
Funding agency: National Human Genome Research Institute
PROJECT SUMMARY/ABSTRACT
Human genome is peppered with an estimated one million enhancers that can regulate their specific target
gene(s) from a distance up to megabases away, and in an orientation-independent manner. The broad goal of
this project is to define the fundamental architecture and function of human enhancers in a in-depth manner
and to better understand the specificity of their interplay with different classes of promoters that use different
transcription factors and/or are regulated at distinct steps in transcription. We have shown that enhancers can
be identified and precisely mapped using our GRO/PRO-cap assay that map transcription start sites of nascent
RNA with highest sensitivity of all available methods. This assay has shown that both enhancers and
promoters share a common architecture whereby both are delimited by two divergent core promoters (CPs)
and a central cluster of transcription factor (TF) binding motifs. The roles and required organization of the
multiple sequence motifs that constitute enhancers and the two CPs need to be fully dissected to understand
how active enhancers function. Additionally, enhancers can interact productively with specific promoters, and
the basis of this specificity especially at long range remains ill-defined. Finally, we know that promoter and
enhancer elements have sequence motifs that can act at distinct regulatory steps of the transcription cycle, but
how these activities coordinate gene regulation has yet to be examined. In Aim 1, we will test the activity of all
PRO-cap identified enhancer candidates in K562 from representative human Chromosomes 8 and 11 with a
carefully chosen set of promoters harboring distinct regulatory features. A set of active enhancer-promoter
combinations will then be subjected to a comprehensive motif mutagenesis of the central clusters of TF binding
motifs and each of the two core promoters. These studies test our fundamental enhancer unit hypothesis and
assess the relationships of enhancers to targeted promoters, and the role of specific motifs and sequence
features in enhancer function. In Aim 2, we examine quantitatively enhancers and key mutants identified in
Aim 1 by barcoding and integrating WT and mutant enhancers 5 kb upstream of their normally responsive
promoter in a chromosomal context at the AAVS1 safe harbor locus. These assays will rigorously test function
of enhancer motifs, core promoters, and the overall architecture of enhancers in a constant chromosomal
background. In addition, we will also test the regulatory code underlying enhancer specificity for promoters and
evaluate effects of mutant TF motifs on TF binding and on nascent transcription using targeted genomic
assays. Finally, in Aim 3, we explore the ability of enhancers to act over long distances, using the NMU
enhancer (eNMU), which resides 94 kb upstream of the NMU promoter and stimulates its transcription by
10,000-fold. We will utilize this robust model enhancer, which is not confounded by redundant/shadow
enhancers at the native locus, to establish a novel long-range, chromosomal, massively-parallel reporter
system using a landing pad at the eNMU locus and characterize functional motifs of eNMU by mutagenesis.
Terms: <4C-seq><Allergy><Architecture><Assay><Bar Codes><Basal Transcription Factor><Basal transcription factor genes><Base Pairing><Binding><Bioassay><Biological Assay><CCRL2><CCRL2 gene><CKRX><CRAM-A><CRAM-B><Capsicum><Chemokine (C-C Motif) Receptor-Like 2 Gene><Chromatin Conformation Capture and Sequencing><Chromosomes><Code><Coding System><Communication><Complex><DNA><DNA Polymerase II><DNA Polymerase epsilon><DNA-Dependent DNA Polymerase II><Deoxyribonucleic Acid><Disease><Disorder><Dissection><Elements><Engineering / Architecture><Enhancer Elements><Enhancers><GRO-seq><GROseq><Gene Action Regulation><Gene Expression><Gene Expression Regulation><Gene Regulation><Gene Regulation Process><Gene Transcription><General Transcription Factor Gene><General Transcription Factors><Genes><Genetic Enhancer Element><Genetic Transcription><Genetics-Mutagenesis><Genomics><Goals><HCR><Histones><Human><Human Chromosomes><Human Genome><Hypersensitivity><K-562><K562><K562 Cells><K562 blasts><Maps><Measures><Messenger RNA><Methods><Modeling><Modern Man><Molecular><Molecular Interaction><Mutagenesis><Mutagenesis Molecular Biology><Mutate><NMU gene><Non-Polyadenylated RNA><Normal Cell><Nucleic Acid Regulator Regions><Nucleic Acid Regulatory Sequences><PRO-seq><Pol II><Precision Run-On Sequencing><Precision nuclear run-on sequencing><Productivity><Property><RNA><RNA Expression><RNA Gene Products><Regulation><Regulator Genes><Regulatory Regions><Reporter><Reporting><Ribonucleic Acid><Role><Site><Specificity><System><Testing><Transcript><Transcription><Transcription Factor Proto-Oncogene><Transcription Initiation><Transcription Initiation Site><Transcription Start Site><Transcription factor genes><Transcriptional Regulatory Elements><Upstream Enhancer><Work><barcode><enhancer sequence><genetic enhancer sequence><genetic regulatory element><global run on sequencing><global run on transcription sequencing><human study><human whole genome><insight><mRNA><mRNA Stability><mutant><novel><pepper><promoter><promotor><regulatory gene><social role><trans acting element><transcription factor>