Telomeres are essential for linear genomes, yet their repetitive DNA content and somatic variability has hindered attempts to delineate their chromatin architectures. We performed single-molecule chromatin fiber sequencing (Fiber-seq) on human cells with a fully resolved genome, enabling nucleotide-precise maps of the genetic and chromatin structure of all telomeres. Telomere fibers are predominantly comprised of three distinct chromatin domains that co-occupy individual DNA molecules – multi- kilobase telomeric caps, highly accessible telomeric-subtelomeric boundary elements, and subtelomeric heterochromatin. Extended G-rich telomere variant repeats (TVRs) punctuate nearly all telomeres, and telomere caps imprecisely bridge these degenerate repeats. Telomeres demonstrate pervasive somatic alterations in length, sequence, and chromatin composition, with TVRs and adjacent CTCF-bound promoters impacting their stability and composition. Our results detail the structure and function of human telomeres.One sentence summaryWe use single-molecule chromatin fiber sequencing to detail the structure and function of human telomeric DNA and chromatin.

SummaryThe focal attachment of the kinetochore to the centromere core is essential for genome maintenance, yet the highly repetitive nature of human centromeres limits our understanding of their chromatin organization. We demonstrate that single-molecule chromatin fiber sequencing can uniquely resolve chromatin organization within centromeres at single-molecule and single-nucleotide resolution. We find that the centromere core contains a dichotomous chromatin organization not found elsewhere in the genome, which is characterized by highly accessible chromatin patches heterogeneously punctuated amongst tightly compacted nucleosome arrays. These highly accessible chromatin patches correspond to sites of kinetochore attachment, and clustered CENP-B occupancy within these patches phase nucleosome arrays to the alpha-satellite repeat. This dichotomous chromatin organization is conserved among humans despite the marked divergence of the underlying alpha-satellite organization and is similarly conserved in gibbon centromeres that lack alpha-satellite repeats, indicating that functional conservation within centromeres is mediated at the level of chromatin, not DNA sequence.HighlightsDichotomous accessible and compacted chromatin (dichromatin) marks centromere coresHighly accessible chromatin patches punctuate sites of kinetochore attachmentDichromatin can form irrespective of CENP-B occupancyConservation within centromeres is mediated at the level of chromatin, not DNA

Low-level DNA N6-methyldeoxyadenosine (DNA-m6A) has recently been reported across various eukaryotes. Although anti-m6A antibody-based approaches are commonly used to measure DNA-m6A levels, this approach is known to be confounded by DNA secondary structures, RNA contamination, and bacterial contamination. To evaluate for these confounding features, we introduce an approach for systematically validating the selectivity of antibody-based DNA-m6A methods and use a highly selective anti-DNA-m6A antibody to reexamine patterns of DNA-m6A inC. reinhardtii,A. thaliana, andD. melanogaster. Our findings raise caution about the use of antibody-based methods for endogenous m6A quantification and mapping in eukaryotes.