Precision single-cell mapping of chromatin-associated protein activity by molecular footprinting to chart disruptions to gene regulation and dissect tumor heterogeneity in cancer - ABSTRACT Single-cell analysis has transformed biomedical research, revolutionizing our understanding of biological processes while providing invaluable insight into human physiology and disease, including cancer. However, current single-cell methodologies are largely restricted to analyzing DNA or RNA features, with analysis of the crucial regulatory protein activity layer remaining almost completely unexplored from the single-cell perspective, with current technologies only able to profile the most stable chromatin-binding factors such as histones in single cells. This has led to significant gaps in our knowledge about mechanisms of gene regulation in both normal and tumor cells, and has further prevented the development of potential precision therapies that could target pathways that are dysregulated in cancer development or progression. Therefore, the ability to assess the activity of regulatory proteins with different DNA-binding affinities, including weak or transient factors that are critical for transcription regulation, at the single-cell level would open up a new era in the field, similar to the single-cell RNA-seq revolution. To address this challenge, we will pioneer the development of new transformative methods for high-throughput single-cell mapping of DNA-binding proteins across binding affinities to define genome regulation, as well as dysregulation, in human cancer. We present Docking & Deamination plus sequencing (D&D-seq) for direct single-cell DNA footprinting, which tethers a species-specific antibody-binding nanobody to a cytosine base editing enzyme, catalyzing C-to-U edits in proximal genomic regions reflecting target binding to DNA. Combined with single-cell ATAC-seq, this approach enables high-throughput profiling of even weak factor binding to DNA in single-cells using common sequencing platforms. To develop D&D-seq for high-throughput analysis of single-cell gene regulation in cancer, we will optimize profiling across high- and low-affinity binders, and engineer a variety of molecular footprinting base editors to map multiple functional DNA:protein interactions in tumor cells. To capture gene regulation across different layers in the same cell, we will incorporate D&D-seq into common single-cell multi-omics workflows to profile transcription factor (TF) binding and chromatin accessibility together with gene expression and protein levels or histone modifications in single cells for the first time. To better assess the effects of cancer-associated somatic mutations on gene regulatory networks, we will integrate D&D-seq with our single-cell genotyping framework to identify TF binding patterns that are specific to mutant cells. Finally, to support wide adoption by the single-cell cancer genomics community, we will adapt D&D for split-and-pool barcoding, enabling high-thoughput, multimodal analysis of gene regulatory networks in hundreds of thousands of single cells without requiring specific sequencing platforms. Together, these methods will enable the direct single-cell measurement of DNA:protein interactions and TF activity in native chromatin contexts to empower novel discoveries about chromatin dynamics and transcriptional regulation in human tumor cells at unprecedented resolution, opening up entire new fields of inquiry that were unable to be pursued before.
