The structure, function and evolution of a complete human chromosome 8

The complete assembly of each human chromosome is essential for understanding human biology and evolutio

Improved assembly and variant detection of a haploid human genome using single-molecule, high-fidelity long reads

The sequence and assembly of human genomes using long-read sequencing technologies has revolutionized our understanding of structural variation and genome organization. We compared the accuracy, continuity, and gene annotation of genome assemblies generated from either high-fidelity (HiFi) or continuous long-read (CLR) datasets from the same complete hydatidiform mole human genome. We find that the HiFi sequence data assemble an additional 10% of duplicated regions and more accurately represent the structure of tandem repeats, as validated with orthogonal analyses. As a result, an additional 5 Mbp of pericentromeric sequences are recovered in the HiFi assembly, resulting in a 2.5-fold increase in the NG50 within 1 Mbp of the centromere (HiFi 480.6 kbp, CLR 191.5 kbp). Additionally, the HiFi genome assembly was generated in significantly less time with fewer computational resources than the CLR assembly. Although the HiFi assembly has significantly improved continuity and accuracy in many complex regions of the genome, it still falls short of the assembly of centromeric DNA and the largest regions of segmental duplication using existing assemblers. Despite these shortcomings, our results suggest that HiFi may be the most effective standalone technology for de novo assembly of human genomes

Both tails and the centromere targeting domain of CENP-A are required for centromere establishment

The centromere—defined by the presence of nucleosomes containing the histone H3 variant, CENP-A—is the chromosomal locus required for the accurate segregation of chromosomes during cell division. Although the sequence determinants of human CENP-A required to maintain a centromere were reported, those that are required for early steps in establishing a new centromere are unknown. In this paper, we used gain-of-function histone H3 chimeras containing various regions unique to CENP-A to investigate early events in centromere establishment. We targeted histone H3 chimeras to chromosomally integrated Lac operator sequences by fusing each of the chimeras to the Lac repressor. Using this approach, we found surprising contributions from a small portion of the N-terminal tail and the CENP-A targeting domain in the initial recruitment of two essential constitutive centromere proteins, CENP-C and CENP-T. Our results indicate that the regions of CENP-A required for early events in centromere establishment differ from those that are required for maintaining centromere identity

The Dynamic Structure and Rapid Evolution of Human Centromeric Satellite DNA

The complete sequence of a human genome provided our first comprehensive view of the organization of satellite DNA associated with heterochromatin. We review how our understanding of the genetic architecture and epigenetic properties of human centromeric DNA have advanced as a result. Preliminary studies of human and nonhuman ape centromeres reveal complex, saltatory mutational changes organized around distinct evolutionary layers. Pockets of regional hypomethylation within higher-order α-satellite DNA, termed centromere dip regions, appear to define the site of kinetochore attachment in all human chromosomes, although such epigenetic features can vary even within the same chromosome. Sequence resolution of satellite DNA is providing new insights into centromeric function with potential implications for improving our understanding of human biology and health

CENP-A Modifications on Ser68 and Lys124 Are Dispensable for Establishment, Maintenance, and Long-Term Function of Human Centromeres

CENP-A is a histone H3 variant key to epigenetic specification of mammalian centromeres. Using transient overexpression of CENP-A mutants, two recent reports in Developmental Cell proposed essential centromere functions for post-translational modifications of human CENP-A. Phosphorylation at Ser68 was proposed to have an essential role in CENP-A deposition at centromeres. Blockage of ubiquitination at Lys124 was proposed to abrogate localization of CENP-A to the centromere. Following gene inactivation and replacement in human cells, we demonstrate that CENP-A mutants that cannot be phosphorylated at Ser68 or ubiquitinated at Lys124 assemble efficiently at centromeres during G1, mediate early events in centromere establishment at an ectopic chromosomal locus, and maintain centromere function indefinitely. Thus, neither Ser68 nor Lys124 post-translational modification is essential for long-term centromere identity, propagation, cell-cycle-dependent deposition, maintenance, function, or mediation of early steps in centromere establishment

TERRA and the histone methyltransferase Dot1 cooperate to regulate senescence in budding yeast

<div><p>The events underlying senescence induced by critical telomere shortening are not fully understood. Here we provide evidence that TERRA, a non-coding RNA transcribed from subtelomeres, contributes to senescence in yeast lacking telomerase (<i>tlc1Δ</i>). Levels of TERRA expressed from multiple telomere ends appear elevated at senescence, and expression of an artificial RNA complementary to TERRA (anti-TERRA) binds TERRA <i>in vivo</i> and delays senescence. Anti-TERRA acts independently from several other mechanisms known to delay senescence, including those elicited by deletions of <i>EXO1</i>, <i>TEL1</i>, <i>SAS2</i>, and genes encoding RNase H enzymes. Further, it acts independently of the senescence delay provided by <i>RAD52</i>-dependent recombination. However, anti-TERRA delays senescence in a fashion epistatic to inactivation of the conserved histone methyltransferase Dot1. Dot1 associates with TERRA, and anti-TERRA disrupts this interaction <i>in vitro</i> and <i>in vivo</i>. Surprisingly, the anti-TERRA delay is independent of the C-terminal methyltransferase domain of Dot1 and instead requires only its N-terminus, which was previously found to facilitate release of telomeres from the nuclear periphery. Together, these data suggest that TERRA and Dot1 cooperate to drive senescence.</p></div

Human-specific tandem repeat expansion and differential gene expression during primate evolution

Short tandem repeats (STRs) and variable number tandem repeats (VNTRs) are important sources of natural and disease-causing variation, yet they have been problematic to resolve in reference genomes and genotype with short-read technology. We created a framework tomodel the evolution and instability of STRs and VNTRs in apes. We phased and assembled 3 ape genomes (chimpanzee, gorilla, and orangutan) using long-read and 10x Genomics linked-read sequence data for 21,442 human tandem repeats discovered in 6 haplotype-resolved assemblies of Yoruban, Chinese, and Puerto Rican origin. We define a set of 1,584 STRs/VNTRs expanded specifically in humans, including large tandem repeats affecting coding and noncoding portions of genes (e.g., MUC3A, CACNA1C). We show that short interspersed nuclear element-VNTR-Alu (SVA) retrotransposition is the main mechanism for distributing GC-rich human-specific tandem repeat expansions throughout the genome but with a bias against genes. In contrast, we observe that VNTRs not originating from retrotransposons have a propensity to cluster near genes, especially in the subtelomere. Using tissue-specific expression from human and chimpanzee brains, we identify genes where transcript isoform usage differs significantly, likely caused by cryptic splicing variation within VNTRs. Using single-cell expression from cerebral organoids, we observe a strong effect for genes associated with transcription profiles analogous to intermediate progenitor cells. Finally, we compare the sequence composition of some of the largest human-specific repeat expansions and identify 52 STRs/VNTRs with at least 40 uninterrupted pure tracts as candidates for genetically unstable regions associated with disease

Dot1 associates with TERRA and anti-TERRA disrupts this interaction.

<p>(A) TERRA-like RNA oligonucleotides but not anti-TERRA molecules can pull down native Dot1 from yeast whole cell extracts. Yeast whole cell extracts (WCE) were subject to RNA affinity purification using biotinylated TERRA or control (random sequence) RNA oligonucleotides. Bound materials were eluted with 1M NaCl in lanes 2 and 3 and then the beads were boiled in lanes 4 and 5. Lane 1 is 5% of WCE as input. Proteins were visualized by western blot with anti-Dot1 antibody and anti- -actin antibody as a control. Dot1 protein migrates near 65 kD as a doublet. (B) V5-tagged Dot1 binds TERRA and anti-TERRA prevents this interaction <i>in vitro</i>. Nuclear extracts were subject to RNA pulldown using the indicated RNA templates. Bound materials were eluted with 2X Laemmli buffer by boiling and assayed by western blot with antibodies specific to V5 or Actin. Lanes 5 and 6: TERRA oligonucleotides were annealed to anti-TERRA molecules under G-quadruplex permissive or minimizing conditions (NaCl or LiCl, respectively) and then were then transferred in the standard buffer used for RNA pulldown. The LiCl conditions rule out the possibility that folding of TERRA into G-quadruplexes, rather than forming duplexes with anti-TERRA, explains the loss of Dot1 binding. Lane 1 is 10% of input. Marker size in kD are indicated at left. Both isoforms of Dot1 can be seen around 110 kD. (C) V5-tagged Dot1 binds TERRA and anti-TERRA prevents this interaction <i>in vivo</i>. RNA immunoprecipitation was performed with V5-tagged Dot1 on yeast WCE. TERRA levels are quantified by qRT-PCR and displayed as fold change relative to an untagged Dot1 strain and to input (n = 2).</p