ChIP-mini Tech Transforms Bacterial DNA-Protein Study

Abstract

Genome-wide identification of binding profiles for DNA-binding proteins from the limited number of intracellular pathogens in infection studies is crucial for understanding virulence and cellular processes but remains challenging, as the current ChIP-exo is designed for high-input bacterial cells (>1010). Here, we developed an optimized ChIP-mini method, a low-input ChIP-exo utilizing a 5,000-fold reduced number of initial bacterial cells and an analysis pipeline, to identify genome-wide binding dynamics of DNA-binding proteins in host-infected pathogens. Applying ChIP-mini to intracellular Salmonella Typhimurium, we identified 642 and 1,837 binding sites of H-NS and RpoD, respectively, elucidating changes in their binding position and binding intensity during infection. Post-infection, we observed 21 significant reductions in H-NS binding at intergenic regions, exposing the promoter region of virulence genes, such as those in Salmonella pathogenicity islands-2, 3 and effectors. Furthermore, we revealed the crucial phenomenon that novel and significantly increased RpoD bindings were found within regions exhibiting diminished H-NS binding, thereby facilitating substantial upregulation of virulence genes. These findings markedly enhance our understanding of how H-NS and RpoD simultaneously coordinate the transcription initiation of virulence genes within macrophages. Collectively, this work demonstrates a broadly adaptable tool that will enable the elucidation of DNA-binding protein dynamics in diverse intracellular pathogens during infection.

In the quest to understand the pathogenic expression mechanisms of bacteria and the advancements in biofoundry technology, identifying and analyzing protein-DNA binding sites is crucial. Researchers at UNIST and Korea University have developed a novel method known as ChIP-mini, which allows for precise identification of these binding sites using significantly fewer samples than previously possible.

Professor Donghyuk Kim from the School of Energy and Chemical Engineering at UNIST and Dr. Eun-Jin Lee from the Department of Life Sciences at Korea University have collaborated to create the ChIP-mini method, enabling high-resolution analysis of binding sites for specific proteins on DNA-even when using 5,000 times fewer cells than traditional methods.

DNA, which stores genetic information, consists of a long sequence of nucleotide bases. Cells regulate gene expression by allowing transcription factors-specialized proteins-to bind to specific sites on the DNA.

Chromatin Immunoprecipitation (ChIP) is a technique utilized to isolate DNA fragments bound by proteins. The newly-developed ChIP-mini technology achieves base pair-level precision (approximately 0.34 nanometers) by analyzing as few as 4.8 million cells-5,000 times less than the conventional methods. Consequently, even in scenarios where multiple proteins bind nearby, individual binding sites can be accurately identified and analyzed. In contrast, existing ChIP-exo experiments required between 10 billion and 100 billion cells to achieve similar precision.

The research team demonstrated the ChIP-mini technology's capabilities by isolating trace amounts of Salmonella bacteria-the causative agent of infections in hosts-and quantitatively analyzing the changes in DNA binding positions and intensities of two crucial proteins, H-NS and RpoD, within the bacteria. Salmonella restricts pathogenic gene expression by firmly binding H-NS proteins to DNA outside host macrophages. However, upon entering the host cell, it diminishes H-NS binding and selectively activates pathogenic genes through RpoD (RNA polymerase sigma factor 70). This adaptation helps Salmonella evade detection by the host's immune system.

Previous ChIP experiments struggled with insufficient quantities of Salmonella within host cells due to its low abundance at the site of infection. To address this challenge, the research team employed DiffExo, a statistical program developed for quantitative analysis.

Additionally, the cost for individual analyses using the ChIP-mini technology is approximately 20,000 KRW (around $17), representing a twelvefold reduction compared to previous methods. Dr. Jon Young Park, co-first author at UNIST, remarked, "If the developed technology can be integrated with Next Generation Sequencing (NGS) automation platforms, it will facilitate the rapid and cost-effective generation of extensive combined datasets essential for biofoundry applications. We are actively pursuing research to ensure compatibility with NGS automation devices."

Biofoundry technology encompasses the production of high-value-added proteins by engineering microorganisms such as bacteria, akin to semiconductor manufacturing processes. Extensive datasets are crucial for effective microbial gene editing and optimal circuit design.

Professor Kim emphasized, "This research serves as a foundational technology in the biofoundry field, particularly in identifying gene expression networks of infectious microorganisms and discovering novel bio-parts."

The findings of this research have been published in the Nucleic Acids Research on February 10, 2025. This study has been supported by the National Research Foundation of Korea (NRF) and the Ministry of Science and ICT (MSIT).

Journal Reference

Joon Young Park, Minchang Jang, Eunna Choi, et al., "ChIP-mini: a low-input ChIP-exo protocol for elucidating DNA-binding protein dynamics in intracellular pathogens," Nucl. Acids Res., (2025).