Document text
Principal Investigator: Philipp Oberdoerffer
Organization: JOHNS HOPKINS UNIVERSITY
Fiscal Year: 2024
Award: $229,659
Funding agency: National Cancer Institute
Project Summary
Chromatin context defines all DNA transactions, from gene regulation to genome maintenance. Changes in
chromatin composition are hallmarks of cancer cells that contribute to malignant transformation and have been
studied extensively using a wide range of Next Generation Sequencing (NGS) approaches. However,
conventional genome-wide mapping efforts are limited to the detection of individual targets of interest, impeding
the study of their often diverse biological roles. Multi-subunit chromatin-regulatory complexes as well as protein
interactions with histones or noncanonical nucleic acid structures are largely defined through correlative analyses
of separate mapping efforts, which are unable to determine physical interaction, proximity on the same DNA
fragment or even presence in the same cell. These limitations underline an urgent need for improved, context-
dependent chromatin mapping and characterization efforts. In this application, we will establish a versatile and
broadly applicable technique that allows for the genome-scale analysis of two-component interactions on
chromatin. The proposal builds on our extensive experience in the study of genome-wide chromatin responses
to DNA damage, as well as the expertise of co-Investigator Dr. Michael Seidman in the analysis and visualization
of close-proximity molecular interactions in the context of DNA replication stress. Aim 1 will combine this
complementary expertise to develop Proximity-based Chromatin Immunoprecipitation (ProxiChIP), a tool to
characterize functional subsets of a given chromatin feature based on its interacting partners, shifting the current
ChIP paradigm towards combinatorial feature mapping. Using well-characterized, interacting chromatin binding
proteins as a proof of principle, we will convert a method currently restricted to the imaging-based detection of
protein interactions (proximity ligation assay, PLA) to a broadly applicable biochemistry tool suitable for
immunoprecipitation and a diverse set of downstream applications including NGS. In Aim 2, we will apply this
methodology to advance the epigenomic characterization of pathological RNA:DNA hybrids, a complex and
poorly understood feature of many cancer genomes thought to contribute to DNA replication stress, cancer
genome instability and therapy response. RNA:DNA hybrids have been mapped genome-wide using DNA:RNA
immunoprecipitation (DRIP) and related methods. However, existing approaches fail to distinguish between
physiological and pathological RNA:DNA hybrid subsets. ProxiChIP-based mapping of RNA:DNA hybrids in the
context of replication stress is expected to define the genomic features that underly pathological R loop formation,
which presents an essential step towards understanding their impact on genome integrity and malignant
transformation.
Terms: <Abscission><Antibodies><Assay><Basic Cancer Research><Binding><Binding Proteins><Bioassay><Biological><Biological Assay><Biology><Biotin><Cancers><Cell Body><Cell Communication and Signaling><Cell Signaling><Cells><ChIP assay><Chromatin><Closure by Ligation><Complex><DNA><DNA Damage><DNA Injury><DNA Replication><DNA Synthesis><DNA biosynthesis><DNA mapping><DNA replication fork><Data Set><Deoxyribonucleic Acid><Detection><Excision><Extirpation><Foundations><Gene Action Regulation><Gene Expression Regulation><Gene Regulation><Gene Regulation Process><Genes><Genome><Genome Instability><Genome Mappings><Genomic Instability><Genomics><Goals><Histones><Hybrids><Image><Immune Precipitation><Immunoprecipitation><In Situ><Individual><Intracellular Communication and Signaling><Investigators><Label><Ligand Binding Protein><Ligand Binding Protein Gene><Ligation><Maintenance><Malignant><Malignant - descriptor><Malignant Cell><Malignant Neoplasms><Malignant Tumor><Maps><Methodology><Methods><Molecular><Molecular Interaction><NGS Method><NGS system><Non-Polyadenylated RNA><Nucleotides><Pathologic><Physiologic><Physiological><Poison><Position><Positioning Attribute><Protein Binding><Proteins><RNA><RNA Gene Products><RNase H1><RNase HI><Removal><Research Personnel><Researchers><Ribonucleic Acid><Ribonucleoside Phosphates><Ribonucleotides><Role><Signal Transduction><Signal Transduction Systems><Signaling><Single-Stranded DNA><Source><Surgical Removal><Techniques><Toxic Chemical><Toxic Substance><Visualization><Vitamin H><Work><analog><assay development><biochemical tools><biochemistry tools><biologic><biological signal transduction><bound protein><cancer cell><cancer cell genome><cancer genome><cancer type><chromatin immunoprecipitation><coenzyme R><combinatorial><epigenomics><experience><genome integrity><genome scale><genome wide analysis><genome wide studies><genome-wide><genome-wide analysis><genome-wide identification><genomewide><genomic integrity><imaging><imaging detection><imaging-based detection><imaging-based disease detection><improved><interest><malignancy><neoplasm/cancer><next gen sequencing><next generation sequencing><nextgen sequencing><novel><nucleic acid structure><replication fork><replication stress><resection><response><response to therapy><response to treatment><ribonuclease H1><ribonuclease HI><social role><ssDNA><targeted cancer therapy><therapeutic response><therapy response><tool><toxic compound><treatment response><treatment responsiveness><tumor genome>