DiMeLo-seq: a long-read, single-molecule method for mapping protein–DNA interactions genome wide

Abstract

Molecular studies of genome regulation often rely on the ability to map where specific proteins interact with genomic DNA. Existing techniques for mapping protein-DNA interactions genomewide rely on DNA amplification methods followed by sequencing with short reads, which dissociates joint binding information at neighboring sites, removes endogenous DNA methylation information, and precludes the ability to reliably map interactions in repetitive regions of the genome. To address these limitations, we created a new protein-DNA mapping method, called Directed Methylation with Long-read sequencing (DiMeLo-seq), which methylates DNA near each target protein's DNA binding site in situ, then leverages the ability to distinguish methylated and unmethylated bases on long, native DNA molecules using long-read, single-molecule sequencing technologies. We demonstrate the optimization and utility of this method by mapping the interaction sites of a variety of different proteins and histone modifications across the human genome, achieving a single-molecule binding site resolution of less than 200 bp. Furthermore, we mapped the positions of the centromeric histone H3 variant CENP-A in repetitive regions that are unmappable with short reads, while simultaneously analyzing endogenous CpG methylation and joint binding events on single molecules. DiMeLo-seq is a versatile method that can provide multimodal and truly genome-wide information for investigating protein-DNA interactions..

Centromere Identity and the Regulation of Chromosome Segregation

“…Efforts to map centromeric nucleosomes using either CENP-A ChIP-seq or chromatin fiber spreading combined with chromosome 8 specific FISH show that CENP-A nucleosomes are clustered within a small portion of the alpha satellite DNA ( Logsdon et al, 2021 ). Dense clustering of CENP-A nucleosomes within a subdomain of alpha satellite DNA has been observed on other chromosomes as well ( Altemose et al, 2022a , 2022b ; Gershman et al, 2022 ; Nurk et al, 2022 ). Because CENP-A nucleosomes reside on only a small fraction of the HOR sequences, there does not appear to be a strict relationship between CENP-A levels at a given centromere and the size of that centromere’s HOR.…”

“…How cells determine and maintain the position of CENP-A (and therefore, the site of microtubule attachment) within the centromere is a key question in centromere biology. The answers to these questions are within our reach now thanks to new experimental methods and the completion of the human genome assembly that includes high confidence and contiguous centromere sequences ( Altemose et al, 2022a , 2022b ; Gershman et al, 2022 ; Hoyt et al, 2022 ; Nurk et al, 2022 ).…”

“…The HOR containing the CDR is enriched for CENP-A ChIP-seq reads suggesting a correlation between DNA methylation levels and the site of CENP-A assembly. Even at the level of individual chromatin fibers (representing individual chromosomes), CENP-A nucleosomes are ∼5 fold enriched within a 100 kb region spanning the CDR compared to adjacent regions ( Altemose et al, 2022b ). The likelihood of a CENP-A nucleosome increases from 1 in 25 across the HOR (on average) ( Bodor et al, 2014 ) to 1 in 4 within the CDR ( Altemose et al, 2022b ).…”

“…The next challenge is to identify CENP-A positions on these DNA sequences. A newly developed technique, DiMeLo-seq relies on antibody-directed localization of an exogenous adenine DNA methyltransferase Hia5 to CENP-A followed by long read sequencing to simultaneously read out DNA sequence and adenine methylation ( Altemose et al, 2022b ). Using DiMeLo-seq one can now map CENP-A nucleosomes confidently onto homogeneously repetitive DNA sequences.…”

“…Using DiMeLo-seq one can now map CENP-A nucleosomes confidently onto homogeneously repetitive DNA sequences. Challenges of low centromeric coverage due to the low representation of centromere sequences in the genome can be overcome by restriction enzyme based digestion of non-centromeric DNA sequences and selective sequencing of centromeric DNA ( Gamba et al, 2021 ; Altemose et al, 2022b ). The next frontier in understanding centromere inheritance and maintenance at the single molecule level is to be able to track CENP-A position using DiMeLo-seq or other approaches before and after CENP-A deposition (in G1-phase) and before and after CENP-A distribution (in S-phase) on the same chromatin fiber.…”