Conservation of dichromatin organization along regional centromeres

Abstract

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

Centromeric chromatin clearings demarcate the site of kinetochore formation

“…This attribute is also reflected in our tomograms where we observe kinetochores of varied size and with varied distribution of kinetochore complexes. Specifically, the finding that CENP-A occupancy is often split between multiple regions within a single centromere (Altemose et al, 2022a; Dubocanin et al, 2023; Gershman et al, 2022; Logsdon et al, 2024; Sacristan et al, 2022) is consistent with our own observation that some kinetochores are comprised of a single chromatin clearing whereas others are comprised of 2-3 clearings. Each of the clearings likely corresponds to a region on the linear DNA sequence with a high local density of CENP-A nucleosomes.…”

“…The three-dimensional visualization of the kinetochore we present here complements recent one-dimensional DNA maps of the chromatin landscape of the centromere locus (Altemose et al, 2022b; a; Dubocanin et al, 2023; Gershman et al, 2022; Logsdon et al, 2021; Miga et al, 2020). Areas of CENP-A occupancy coincide with regions of reduced CpG methylation termed centromere dip regions (Altemose et al, 2022a; b; Gershman et al, 2022; Logsdon et al, 2021; Miga et al, 2020), and Fiber-Seq of centromeres revealed these same regions to be highly physically accessible, forming the most accessible chromatin domain in the human genome (Dubocanin et al, 2023). These DNA sequencing-based results align remarkably well with our findings where tomograms show kinetochore complexes clustered within a distinct clearing space composed of open chromatin.…”

“…At the level of kinetochore-forming centromeric chromatin, CENP-A-containing nucleosomes are thought to be found in multiple patches along the linear DNA sequence interspersed with canonical nucleosomes (i.e., those harboring conventional histone H3) (Blower et al, 2002; Ribeiro et al, 2010; Zinkowski et al, 1991). These proposals are supported and extended by genomic approaches that build on the recent complete human centromere assemblies (Altemose et al, 2022a; b; Dubocanin et al, 2023; Gershman et al, 2022; Logsdon et al, 2021).…”

Centromere innovations within a mouse species

“…In humans, where the spacing between CENP-B box position is 171 bp in α-satellite DNA, CENP-A also has a specific favored position (relative to the more heterogeneous positioning of conventional nucleosomes harboring histone H3), but it is located between CENP-B boxes (40). Furthermore, recent analysis of single-molecule chromatin fiber sequencing data suggests that the CENP-B occupancy may phase nucleosomes on α-satellite DNA (41). In considering M. musculus centromeres, we considered two possibilities.…”

Synchronized long-read genome, methylome, epigenome, and transcriptome for resolving a Mendelian condition

“…For example, with single-molecule chromatin fiber sequencing (Fiber-seq) prior to DNA extraction permeabilized cells are treated with a non-specific N6-methyladenine methyltransferase (m6A-MTase), which stencils the architecture of each chromatin fiber onto its underlying DNA molecule in the form of m6A-modified bases, which can be directly read out during standard Single Molecule, Real-Time (SMRT) sequencing. Furthermore, recent advances in Fiber-seq enable the co-identification of both accurate chromatin features 16 and CpG methylation along each sequenced read 17 , providing genomic sequence, CpG methylation, and chromatin epigenetic information in the same sequencing run. Finally, long-read full-length transcript sequencing is emerging as a powerful tool for resolving the transcript impact of genetic variants 18, 19 , and recent cDNA concatenation workflows for processing full-length transcript data (MAS-Seq) 20 provide an order-of-magnitude higher throughput identification of full-length transcript data.…”