DiMeLo-cito: a one-tube protocol for mapping protein-DNA interactions reveals CTCF bookmarking in mitosis

Abstract

Genome regulation relies on complex and dynamic interactions between DNA and proteins. Recently, powerful methods have emerged that leverage third-generation sequencing to map protein-DNA interactions genome-wide. For example, Directed Methylation with Long-read sequencing (DiMeLo-seq) enables mapping of protein-DNA interactions along long, single chromatin fibers, including in highly repetitive genomic regions. However, DiMeLo-seq involves lossy centrifugation-based wash steps that limit its applicability to many sample types. To address this, we developed DiMeLo-cito, a single-tube, wash-free protocol that maximizes the yield and quality of genomic DNA obtained for long-read sequencing. This protocol enables the interrogation of genome-wide protein binding with as few as 100,000 cells and without the requirement of a nuclear envelope, enabling confident measurement of protein-DNA interactions during mitosis. Using this protocol, we detected strong binding of CTCF to mitotic chromosomes in diploid human cells, in contrast with earlier studies in karyotypically unstable cancer cell lines, suggesting that CTCF "bookmarks" specific sites critical for maintaining genome architecture across cell divisions. By expanding the capabilities of DiMeLo-seq to a broader range of sample types, DiMeLo-cito can provide new insights into genome regulation and organization.

Figure 1.. Genome-wide mapping with enhanced DNA recovery using DiMeLo-cito.

A. Schematic overview of the DiMeLo-cito workflow. Permeabilized nuclei are incubated with antibodies that bind target molecules in the genome, which in turn are bound by a nanobody-fused Hia5 adenine methyltransferase. Instead of washing, the sample is diluted and incubated with an affinity resin enclosed in a semipermeable membrane in an assembly known as an EMPaNADA to remove free enzyme-antibody complexes. After activation of Hia5 with SAM, nearby adenines are methylated, and DNA is directly extracted and subjected to long-read library preparation and sequencing. B. DNA extraction recoveries from ~1 million digitonin-permeabilized nuclei in the presence of paramagnetic affinity resins directly added to the reaction or enclosed in an EMPaNADA. Samples were eluted from beads with 100 μL buffer. C. DNA extraction recoveries from varying inputs of permeabilized nuclei subjected to the washing protocol of DiMeLo-seq or diluted with an EMPaNADA and kept in a single tube. D. Methyladenine enrichment profiles at bound CTCF sites for DiMeLo-seq and DiMeLo-cito targeting CTCF and sequenced to ~1X coverage genome-wide. Plots were generated with a 50 bp smoothing window. Peaks in methyladenine signal were fit to exponential decays. Fit parameters for the maximal value (a) and the decay constant (lambda) are shown in the figure legends. E. Screenshot from IGV at the H19 imprinting control region with reads aligned to the HG002v1.1 genome assembly. Locations of methyladenine and methylcytosine basecalls with a confidence threshold >0.99 are shown. The regions shown correspond to chr11_MATERNAL:2,065,542–2,078,512 and chr11_PATERNAL:2,057,830–2,069,830. F Methyladenine and methylcytosine distributions at bound CTCF sites with DiMeLo-cito sequenced to approximately 30X coverage. Methyladenine signal was subtracted from nontargeting (IgG isotype) controls.

Figure 2.. DiMeLo-cito reveals CTCF binding in unfixed mitotic GM24385 LCs.

A. Methyladenine and methylcytosine distributions from DiMeLo-cito targeting CTCF in unfixed mitotic GM24385 with nontargeting background subtracted. DNA from 1 million cells was sequenced to ~30X coverage and plots were generated with a 30 bp smoothing window B. Methyladenine distribution from DiMeLo-cito targeting CTCF in unfixed HeLa cells synchronized in mitosis sequenced to ~2X coverage. Plots were generated with a 50 bp smoothing window. C. Quantification of immunofluorescence (IF) signal targeting CTCF in various unfixed cell lines. Asterisks denote p-values <10⁻¹⁰ for a two-sample tailed t-test calculated between the secondary-only control condition and each cell line. D. Plots of single reads from B centered on CTCF sites and separated based on strand and sorted in descending order by the fraction of the read containing methyladenine. E. Comparison of standard DiMeLo-seq targeting CTCF in mitotic cells at ~0.1X coverage and DiMeLo-cito data in B downsampled to 0.1X coverage for comparison. Both plots were smoothed with a 50 bp smoothing window. F. An enrichment profile plot of CTCF signal at M2 motifs within centromeric transition zones during mitosis (blue) or in unsynchronized cells (green).