Epigenetic patterns in a complete human genome https://www.science.org/doi/10.1126/science.abj5089
Structured Abstract INTRODUCTION The human reference genome has served as the foundation for many large-scale initiatives, including the collective effort to catalog the epigenome, the set of marks and protein interactions that act to control gene activity and cellular function. However, for more than two decades, efforts to construct a complete epigenome have been hampered by an incomplete reference genome. With recent technological advances, we can now study genome structure and function comprehensively across a complete telomere-to-telomere human genome assembly, T2T-CHM13. As a result, we can now broaden the human epigenome to include 225 million base pairs (Mbp) of additional sequence. RATIONALE The epigenome refers to DNA modifications (e.g., CpG methylation), protein-DNA interactions, histone modifications, and chromatin organization that collectively influence gene expression, genome regulation, and genome stability. These epigenetic features are heritable upon cell division but dynamic during development, generating profiles that are unique to different tissues and cell types. Here, we present an epigenetic annotation of the human genome in which we explore previously unresolved regions, including acrocentric chromosome short arms, segmentally duplicated genes, and a diverse collection of repeat classes, including human centromeres. Generating a complete epigenetic annotation of the previously missing 8% of the human genome provides a foundation for elucidating the functional roles of these genomic elements that are critical to our understanding of genome regulation, function, and evolution. RESULTS Completion of the human epigenome required that we develop approaches to profiling the previously unresolved regions. Using the T2T-CHM13 reference with existing short-read epigenetic data, we identified 3 to 19% more enrichment sites for epigenetic markers. However, even with the complete reference, these short-read epigenetic methods cannot correctly resolve regions of the genome of high similarity, including segmental duplications, gene paralogs, or large repeat arrays. On the other hand, long-read epigenetic methods can resolve single-molecule epigenetic patterns within these regions by anchoring to flanking or infrequent unique regions, providing a foundational assessment of these areas. Long-read methylation calls using the T2T-CHM13 assembly increased the number of probeable CpG sites by 10% (3.2 M), revealing epigenetic patterning of genomic regions that were previously intractable. We generated long-read methylomes of distinct developmental time points and surveyed >99% of the genome’s CpGs. We probed highly homologous gene families and observed paralog-specific differences in regulation between disease and nondisease states. In tandem repeats, we identified differences in epigenetic regulation between genetically identical sequences present across different genomic locations, observing locus- and single-molecule-level differences in methylation. Our analysis revealed that these regions vary in epigenetic and transcriptional activity despite high sequence identity, highlighting the importance of the local chromosome environment as a modulator of epigenetics. Finally, the T2T-CHM13 genome assembly has opened exploration of the human centromere, enabling us to probe the epigenetic elements that define centromeric chromatin. The centromere is the site of assembly of the kinetochore complex, an essential complex for eukaryotic cell division. We generated complete epigenetic maps of human centromeres, revealing epigenetic markers of centromere activity that denote active human kinetochores. We predicted kinetochore site localization within active centromeres and report variability of kinetochore localization across individuals representing diverse ancestry. CONCLUSION The improvements in epigenetic profiling using T2T-CHM13 set the foundation for complete assemblies and long-read epigenetics for major biological advancements. Using technological advances in genome resequencing and alignment, we present a comprehensive functional assessment of previously unresolved genomic regions. This study marks the start of exploration into duplicated and repetitive portions of the epigenome, pioneering the exploration of epigenetics in a complete human genome.
Abstract The completion of a telomere-to-telomere human reference genome, T2T-CHM13, has resolved complex regions of the genome, including repetitive and homologous regions. Here, we present a high-resolution epigenetic study of previously unresolved sequences, representing entire acrocentric chromosome short arms, gene family expansions, and a diverse collection of repeat classes. This resource precisely maps CpG methylation (32.28 million CpGs), DNA accessibility, and short-read datasets (166,058 previously unresolved chromatin immunoprecipitation sequencing peaks) to provide evidence of activity across previously unidentified or corrected genes and reveals clinically relevant paralog-specific regulation. Probing CpG methylation across human centromeres from six diverse individuals generated an estimate of variability in kinetochore localization. This analysis provides a framework with which to investigate the most elusive regions of the human genome, granting insights into epigenetic regulation. |
