Epigenetic Patterns in a Complete Human Genome

Abstract

The completion of the first telomere-to-telomere human genome, T2T-CHM13, enables exploration of the full epigenome, removing limitations previously imposed by the missing reference sequence. Existing epigenetic studies omit unassembled and unmappable genomic regions (e.g. centromeres, pericentromeres, acrocentric chromosome arms, subtelomeres, segmental duplications, tandem repeats). Leveraging the new assembly, we were able to measure enrichment of epigenetic marks with short reads using k-mer assisted mapping methods. This granted array-level enrichment information to characterize the epigenetic regulation of these satellite repeats. Using nanopore sequencing data, we generated base level maps of the most complete human methylome ever produced. We examined methylation patterns in satellite DNA and revealed organized patterns of methylation along individual molecules. When exploring the centromeric epigenome, we discovered a distinctive dip in centromere methylation consistent with active sites of kinetochore assembly. Through long-read chromatin accessibility measurements (nanoNOMe) paired to CUT&RUN data, we found the hypomethylated region was extremely inaccessible and paired to CENP-A/B binding. With long-reads we interrogated allele-specific, long-range epigenetic patterns in complex macro-satellite arrays such as those involved in X chromosome inactivation. Using the single molecule measurements we can clustered reads based on methylation status alone distinguishing epigenetically heterogeneous and homogeneous areas. The analysis provides a framework to investigate the most elusive regions of the human genome, applying both long and short-read technology to grant new insights into epigenetic regulation.

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

“…CENP-A-directed mA was found to be higher within both large and small CDRs compared to their adjacent CpG methylated regions, consistent with short-read data for this cell line (Fig. 6d-f) (Altemose et al 2021;Gershman et al 2021). Furthermore, we found that the number of CENP-A nucleosomes detected per kilobase on single-molecule reads increased over 4-fold within ChrX CDRs compared to neighboring regions (Methods, Fig.…”

“…To test DiMeLo-seq's ability to measure protein occupancy in heterochromatic, repetitive regions of the genome we targeted H3K9me3, which is abundant in pericentric heterochromatin. We chose to target H3K9me3 in HG002 cells because the chromosome X centromere has been completely assembled for this male-derived lymphoblast line (Nurk et al 2021), and many different sequencing data types are available for it (Gershman et al 2021). To validate the specificity of targeted methylation, we calculated the fraction of adenines methylated within HG002 CUT&RUN H3K9me3 peaks (Altemose et al 2021) compared to the fraction of adenines methylated outside of broadly defined peaks (Methods).…”

“…g, Average number of nucleosomes estimated per read in sliding 5 kb windows for the portion of reads that align within the window (from reads that align to at least 1 kb within the window), to provide a measure of the density of CENP-A nucleosomes within single DNA molecules across the region. ______________________________________________________________________________ To examine the positions of CENP-A nucleosomes within centromeric repeat arrays, we aligned our reads to a hybrid complete human assembly containing a fully assembled chromosome X from HG002 (Methods) (Nurk et al 2021;Gershman et al 2021). We investigated the recently described chromosome X centromere dip region (CDR), a hypomethylated region in the centromeric alpha HOR array where short-read CENP-A datasets align (Gershman et al 2021;Logsdon et al 2021;Karen H. Miga et al 2020;Altemose et al 2021).…”

“…______________________________________________________________________________ To examine the positions of CENP-A nucleosomes within centromeric repeat arrays, we aligned our reads to a hybrid complete human assembly containing a fully assembled chromosome X from HG002 (Methods) (Nurk et al 2021;Gershman et al 2021). We investigated the recently described chromosome X centromere dip region (CDR), a hypomethylated region in the centromeric alpha HOR array where short-read CENP-A datasets align (Gershman et al 2021;Logsdon et al 2021;Karen H. Miga et al 2020;Altemose et al 2021). Aggregating the methylation signal across multiple reads, we confirmed low endogenous CpG methylation within that region as expected (Fig.…”

Chasing perfection: validation and polishing strategies for telomere-to-telomere genome assemblies

“…Genome assembly is a foundational practice of quantitative biological research with increasing utility. By representing the genomic sequence of a sample of interest, genome assemblies enable researchers to annotate important features, quantify functional data, and discover/genotype genetic variants in a population [1][2][3][4][5][6] . Modern draft eukaryotic genome assembly graphs are typically built from a subset of four Whole Genome Shotgun (WGS) sequencing data types: Illumina short reads 7,8 , Oxford Nanopore Technologies (ONT) long reads 9 , PacBio Continuous Long Reads (CLR), and PacBio High-Fidelity (HiFi) long reads 9,10 , all of which have been extensively described [7][8][9][10] .…”