Genome-Wide Analyses of Protein-DNA Interaction Using Long-Read Sequencing
Abstract
Prostate cancer is marked by aberrant gene expression, which is controlled by proteins that bind to the DNA. It is therefore critical to map where on the genome a protein binds. The existing method, called ChIP-seq, requires PCR amplification of enriched DNA, followed by short-read, often 50-300 base pair, sequencing. PCR amplification causes sequence biases, while short reads have a problem mapping to repetitive regions, which accounts for 50% of the human genome. In Aim 1 of this study, we propose a new technology called DiMeLo- seq, which uses an enzyme to catalyze DNA adenine methylation (mA) at the binding sites of the target protein. The mA-modified DNA is then directly sequenced using Nanopore technology in long reads that are of average 50 kilobases. Long reads have no problem mapping to repetitive regions of the human genome. This method does not need PCR amplification. The accumulation of mA indicates protein-DNA binding, and long reads allow for the detection of co-binding on distant elements (e.g., promoters and enhancers), suggesting DNA looping. Further, it can detect endogenous DNA methylation, another major regulator of gene expression, along with exogenous mA on the same DNA molecule. Aim 1 will test the performance of DiMeLo-seq using androgen receptor (AR) as a typical transcription factor in prostate cancer cells. In Aim 2, we will further test the method for detecting histone modifications. We chose histone 3 lysine 9 (H3K9) methylation, which is known to bind at repetitive regions, with which conventional ChIP-seq has a problem achieving accurate mapping. We will compare DiMeLo-seq data with ChIP-seq data and evaluate how DiMeLo-seq outperforms ChIP-seq, especially in repetitive regions. We will further examine the ability of DiMeLo-seq to detect protein co-binding at the promoters and enhancers. We will integrate protein-DNA binding with DNA methylation data and determine whether AR binding at sites with extensive DNA methylation indicates gene repression. Last, we will examine whether H3K9 and DNA methylation are increased in Enzalutamide-resistant prostate cancer. Ultimate Applicability of the Research: Our study will develop innovative technology for the prostate cancer research field. It will dramatically advance the studies of gene regulation, thereby increasing our understanding of the molecular mechanisms underlying prostate cancer progression and drug resistance. It might also identify potential targets that are involved in drug resistance that may be important therapeutic targets. Our study is thus paradigm-shifting. It addresses the PCRP overall challenge in defining the biology of lethal prostate cancer. By understanding the mechanism of Enzalutamide resistance, our study will help eliminate death from prostate cancer.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jan 04, 2024
- Source ID
- HT94252310007
Entities
People
- Changsheng Zhao
Organizations
- Emory University
- United States Army