Genome-wide single-cell and single-molecule footprinting of transcription factors by FOODIE

Decades of research have established that mammalian transcription factors (TFs) bind to each gene’s regulatory regions and cooperatively control tissue specificity, timing, and intensity of gene transcription. Mapping the combination of TF binding sites genome wide is critically important for understanding functional genomics. We used a double-strand DNA cytosine deaminases to measure TFs’ binding sites on the human genome with a near single-base resolution on a single-molecule basis (Figure1). We term this approach FOOtprinting with DeamInasE (FOODIE).

Fig.1 Genome-wide mapping of TF binding sites by FOODIE. (Left) Workflow of FOODIE; (Right) Highlights of FOODIE

We demonstrated that FOODIE accurately detected TF footprint in human cell line and offered major improvements in sensitivity and resolution over previous TF footprinting methods. We further developed single cell FOODIE (scFOODIE) to enable TF footprint analysis in heterogeneous cell populations (Figure 2).

Fig.2 Cell-type specific TF footprinting in heterogeneous tissues by scFOODIE. (A) Cells from a mixture of four human cell

lines are classified into four different cell types from barcoded scFOODIE data. (B) Cell typing of ~11,200 mouse hippocampus cells based on their scFOODIE data.

We used FOODIE to study the cooperativity between Adjacent TFs on genome DNA (figure 3). An algorithm was devised to quantify TF cooperativity from FOODIE data, and we found that more TFs exhibit positive cooperativity than negative cooperativity. TFs with positive binding cooperativity support concentration dependent DNA binding by multiple factors, whereas negative binding cooperativity would assure fast TF dissociation from the DNA, hence rapid switching of transcription.

Fig.3 Cell-type specific TF footprinting in heterogeneous tissues by scFOODIE. (A-B) Converted cytosines on single-molecule sequencing reads are colored in blue, and the nonconverted cytosines are colored in gray. © Binding cooperativity between a pair of adjacent TFs is measured by the γ = ad/bc, where a is the number of single molecules reads with both TFs bound, b left bound only, c right bound only, and d both unbound.

With FOODIE, we analysis how TFs organize in groups of genes expressed at the same time (correlated gene modules, CGMs). We found that genes carrying out a certain biological function together in a housing-keeping correlated gene module (CGM) or a tissues-specific CGM are coordinated by shared TFs in the gene’s promoters and enhancers, respectively.

Fig.4 Genes in a CGM sharing TF(s) in their promoters and enhancers. Heatmap showing the TF enrichment (-log Pvalue) within the major CGMs. Each row indicates a CGM, and each column indicates TFs assigned by ChIPseq to the measured footprints. Color density denotes the -log P-values calculated by the hypergeometric test.

FOODIE is a high-precision method for genome-wide in situ mapping of TF footprints inside the nucleus with near single-base resolution. FOODIE is experimentally simple, high-throughput, and cost-effective. ScFOODIE allows for cell typing in a heterogeneous tissue sample and TF footprinting in any cell type within a complex tissue. A systematic FOODIE database of different human tissues is underway. We anticipate that such information about TF combinations of different cell types and CGMs will lead to the fundamental understanding of transcriptional regulation and cellular functions in eukaryotic biology, especially for human biology, which might hold the potential for understanding many diseases.

Reference:

He, R., Dong, W., Wang, Z., Xie, C., Gao, L., Ma, W., … & Xie, X. S. (2024). Genome-wide single-cell and single-molecule footprinting of transcription factors with deaminase. Proceedings of the National Academy of Sciences, 121(52), e2423270121.