Since its initial release in 2000, the human reference genome has covered only the euchromatic fraction of the genome, leaving important heterochromatic regions unfinished. Addressing the remaining 8% of the genome, the Telomere-to-Telomere (T2T) Consortium presents a complete 3.055 billion–base pair sequence of a human genome, T2T-CHM13, that includes gapless assemblies for all chromosomes except Y, corrects errors in the prior references, and introduces nearly 200 million base pairs of sequence containing 1956 gene predictions, 99 of which are predicted to be protein coding. The completed regions include all centromeric satellite arrays, recent segmental duplications, and the short arms of all five acrocentric chromosomes, unlocking these complex regions of the genome to variational and functional studies.

Existing human genome assemblies have almost entirely excluded repetitive sequences within and near centromeres, limiting our understanding of their organization, evolution, and functions, which include facilitating proper chromosome segregation. Now, a complete, telomere-to-telomere human genome assembly (T2T-CHM13) has enabled us to comprehensively characterize pericentromeric and centromeric repeats, which constitute 6.2% of the genome (189.9 megabases). Detailed maps of these regions revealed multimegabase structural rearrangements, including in active centromeric repeat arrays. Analysis of centromere-associated sequences uncovered a strong relationship between the position of the centromere and the evolution of the surrounding DNA through layered repeat expansions. Furthermore, comparisons of chromosome X centromeres across a diverse panel of individuals illuminated high degrees of structural, epigenetic, and sequence variation in these complex and rapidly evolving regions.

Microrchidia (MORC) proteins are GHKL (gyrase, heat-shock protein 90, histidine kinase, MutL) ATPases that function in gene regulation in multiple organisms. Animal MORCs also contain CW-type zinc finger domains, which are known to bind to modified histones. We solved the crystal structure of the murine MORC3 ATPase-CW domain bound to the nucleotide analog AMPPNP (phosphoaminophosphonic acidadenylate ester) and in complex with a trimethylated histone H3 lysine 4 (H3K4) peptide (H3K4me3). We observed that the MORC3 N-terminal ATPase domain forms a dimer when bound to AMPPNP. We used native mass spectrometry to show that dimerization is ATPdependent, and that dimer formation is enhanced in the presence of nonhydrolyzable ATP analogs. The CW domain uses an aromatic cage to bind trimethylated Lys4 and forms extensive hydrogen bonds with the H3 tail. We found that MORC3 localizes to promoters marked by H3K4me3 throughout the genome, consistent with its binding to H3K4me3 in vitro. Our work sheds light on aspects of the molecular dynamics and function of MORC3.X-ray crystallography | histone mark reader | ATPase | native mass spectrometry T he Microrchidia (MORC) family of ATPase proteins has been shown to be an important regulator of gene silencing in multiple organisms. This family was first described in mice, when it was discovered that morc1 null males showed arrested spermatogenesis (1). This arrest was later shown to be associated with transposon derepression, implicating murine MORC1 as a crucial mediator of transposon silencing (2). Arabidopsis thaliana MORC1 and MORC6 were shown to mediate silencing of transposons in a manner largely independent of changes in DNA methylation (3)(4)(5). Studies in Caenorhabditis elegans, which lack DNA methylation, also concluded that the single MORC gene in this organism plays a role in transgene silencing (4). Although the biological importance of MORC ATPases in enforcing gene silencing across multiple organisms is clear, how they are targeted and how they function are poorly understood.The MORC ATPases share a similar domain arrangement. The N terminus contains a GHKL (gyrase, heat-shock protein 90, histidine kinase, MutL) type ATPase domain, and at the C terminus is a coiled-coil segment. MORCs have been reported to form functional homomultimers or heteromultimers, where multimerization is likely mediated by the N-and/or the C-terminal domains (4, 6, 7). The coiled-coil region has been proposed to promote constitutive dimerization, whereas N-terminal ATPase head dimerization occurs only on ATP binding (6). This is consistent with other GHKL ATPases described in the literature, many of which have been reported to undergo ATP-dependent dimerization (8-10). Both plant and animal MORCs are capable of forming nuclear bodies, and mutations that impair ATP binding and/or hydrolysis disrupt nuclear body formation of human MORC3 (6).Animal MORCs also carry a CW-type zinc finger domain, which has been proposed to read histone H3 lysine 4 (H3K4) dimethylation and trimethylation mark...

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Rhesus macaque is an Old World monkey that shared a common ancestor with human ∼25 Myr ago and is an important animal model for human disease studies. A deep understanding of its genetics is therefore required for both biomedical and evolutionary studies. Among structural variants, inversions represent a driving force in speciation and play an important role in disease predisposition. Here we generated a genome-wide map of inversions between human and macaque, combining single-cell strand sequencing with cytogenetics. We identified 375 total inversions between 859 bp and 92 Mbp, increasing by eightfold the number of previously reported inversions. Among these, 19 inversions flanked by segmental duplications overlap with recurrent copy number variants associated with neurocognitive disorders. Evolutionary analyses show that in 17 out of 19 cases, the Hominidae orientation of these disease-associated regions is always derived. This suggests that duplicated sequences likely played a fundamental role in generating inversions in humans and great apes, creating architectures that nowadays predispose these regions to disease-associated genetic instability. Finally, we identified 861 genes mapping at 156 inversions breakpoints, with some showing evidence of differential expression in human and macaque cell lines, thus highlighting candidates that might have contributed to the evolution of species-specific features. This study depicts the most accurate fine-scale map of inversions between human and macaque using a two-pronged integrative approach, such as single-cell strand sequencing and cytogenetics, and represents a valuable resource toward understanding of the biology and evolution of primate species.