Although centromeres have conserved function, centromere-specific histone H3 (CenH3) and centromeric DNA evolve rapidly. The centromere drive model explains this phenomenon as a consequence of the conflict between fast-evolving DNA and CenH3, suggesting asymmetry in female meiosis as a crucial factor. We characterized evolution of the CenH3 protein in three closely related, polyploid mitotic parthenogenetic species of the Meloidogyne incognita group, and in the distantly related meiotic parthenogen Meloidogyne hapla. We identified duplication of the CenH3 gene in a putative sexual ancestral Meloidogyne. We found that one CenH3 (αCenH3) remained conserved in all extant species, including in distant Meloidogyne hapla, whereas the other evolved rapidly and under positive selection into four different CenH3 variants. This pattern of CenH3 evolution in Meloidogyne species suggests the subspecialization of CenH3s in ancestral sexual species. Immunofluorescence performed on mitotic Meloidogyne incognita revealed a dominant role of αCenH3 on its centromere, whereas the other CenH3s have lost their function in mitosis. The observed αCenH3 chromosome distribution disclosed cluster-like centromeric organization. The ChIP-Seq analysis revealed that in M. incognita αCenH3-associated DNA dominantly comprises tandem repeats, composed of divergent monomers which share a completely conserved 19-bp long box. Conserved αCenH3-associated DNA is also confirmed in the related mitotic Meloidogyne incognita group species suggesting preservation of both centromere protein and DNA constituents. We hypothesize that the absence of centromere drive in mitosis might allow for CenH3 and its associated DNA to achieve an equilibrium in which they can persist for long periods of time.

Segments of the genome enriched in repetitive sequences still present a challenge and are omitted in genome assemblies. For that reason, the exact composition of DNA sequences underlying the heterochromatic regions and the active centromeres are still unexplored for many organisms. The centromere is a crucial region of eukaryotic chromosomes responsible for the accurate segregation of genetic material. The typical landmark of centromere chromatin is the rapidly-evolving variant of the histone H3, CenH3, while DNA sequences packed in constitutive heterochromatin are associated with H3K9me3-modified histones. In the Pacific oyster Crassostrea gigas we identified its centromere histone variant, Cg-CenH3, that shows stage-specific distribution in gonadal cells. In order to investigate the DNA composition of genomic regions associated with the two specific chromatin types, we employed chromatin immunoprecipitation followed by high-throughput next-generation sequencing of the Cg-CenH3- and H3K9me3-associated sequences. CenH3-associated sequences were assigned to six groups of repetitive elements, while H3K9me3-associated-ones were assigned only to three. Those associated with CenH3 indicate the lack of uniformity in the chromosomal distribution of sequences building the centromeres, being also in the same time dispersed throughout the genome. The heterochromatin of C. gigas exhibited general paucity and limited chromosomal localization as predicted, with H3K9me3-associated sequences being predominantly constituted of DNA transposons.

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Centromeres are chromosomal domains essential for kinetochore assembly and correct chromosome segregation. Inconsistent in their underlying DNA sequences, centromeres are defined epigenetically by the presence of the centromere-specific histone H3 variant CenH3. Most of the analyzed eukaryotes have monocentric chromosomes in which CenH3 proteins deposit into a single, primary constriction visible at metaphase chromosomes. Contrary to monocentrics, evolutionary sporadic holocentric chromosomes lack a primary constriction and have kinetochore activity distributed along the entire chromosome length. In this work, we identified cCENH3 protein, the centromeric H3 histone of the coleopteran model beetle Tribolium castaneum . By ChIP-seq analysis we disclosed that cCENH3 chromatin assembles upon a repertoire of repetitive DNAs. cCENH3 in situ mapping revealed unusually elongated T . castaneum centromeres that comprise approximately 40% of the chromosome length. Being the longest insect regional centromeres evidenced so far, T . castaneum centromeres are characterized by metapolycentric structure composed of several individual cCENH3-containing domains. We suggest that the model beetle T . castaneum with its metapolycentromeres could represent an excellent model for further studies of non-canonical centromeres in insects.

Cartilage tissue is avascular and hypoxic which regulates chondrocyte phenotype via stabilization of HIFs. Here, we investigated the role of hypoxia and HIFs in regulation of Rho and canonical Wnt signaling in chondrocytes. Our data demonstrates that hypoxia controls the expression of RhoA in chondrocytes in a context-dependent manner on the culturing conditions. Within a 3D microenvironment, hypoxia suppresses RhoA on which hypoxia-driven expression of chondrogenic markers depends. Conversely, hypoxia leads to upregulation of RhoA in chondrocytes on 2D with a failure in re-expression of chondrogenic markers. Similarly to RhoA, hypoxic regulation of Wnt/β-catenin signaling depends on the microenvironment. Hypoxia downregulates β-catenin within 3D hydrogels whereas it causes a potent increase on 2D. Hypoxia-induced suppression of canonical Wnt signaling in 3D contributes to the promotion of chondrogenic phenotype as induction of Wnt signaling abrogates the hypoxic re-differentiation of chondrocytes. Inhibiting Wnt/β-catenin signaling via stabilization of Axin2 leads to a synergistic enhancement of hypoxia-induced expression of chondrogenic markers. The effects of hypoxia on Rho and Wnt/β-catenin signaling are HIF-dependent as stabilizing HIFs under normoxia revealed similar effects on chondrocytes. The study reveals important insights on hypoxic signaling of chondrocytes and how hypoxia regulates cellular mechanisms depending on the cellular microenvironment.

The flour beetle Tribolium freemani is a sibling species of the model organism and important pest Tribolium castaneum. The two species are so closely related that they can produce hybrid progeny, but the genetic basis of their differences has not been revealed. In this work, we sequenced the T. freemani genome by applying PacBio HiFi technology. Using the well-assembled T. castaneum genome as a reference, we assembled 262 Mb of the T. freemani genomic sequence and anchored it in 10 linkage groups corresponding to nine autosomes and sex chromosome X. The assembly showed 99.8% completeness of conserved insect genes, indicating a high-quality reference genome. Comparison with the T. castaneum assembly revealed that the main differences in genomic sequence between the two sibling species come from repetitive DNA, including interspersed and tandem repeats. In this work, we also provided the complete assembled mitochondrial genome of T. freemani. Although the genome assembly needs to be ameliorated in tandemly repeated regions, the first version of the T. freemani reference genome and the complete mitogenome presented here represent useful resources for comparative evolutionary studies of related species and for further basic and applied research on different biological aspects of economically important pests.

The red flour beetle Tribolium castaneum is an important pest of stored agricultural products and the first beetle whose genome was sequenced. So far, one high-copy-number and ten moderate-copy-number satellite DNAs (satDNAs) have been described in the assembled part of its genome. In this work, we aimed to catalog the entire collection of T. castaneum satDNAs. We resequenced the genome using Illumina technology and predicted potential satDNAs via graph-based sequence clustering. In this way, we discovered 46 novel satDNAs that occupied a total of 2.1% of the genome and were, therefore, considered low-copy-number satellites. Their repeat units, preferentially 140–180 bp and 300–340 bp long, showed a high A + T composition ranging from 59.2 to 80.1%. In the current assembly, we annotated the majority of the low-copy-number satDNAs on one or a few chromosomes, discovering mainly transposable elements in their vicinity. The current assembly also revealed that many of the in silico predicted satDNAs were organized into short arrays not much longer than five consecutive repeats, and some of them also had numerous repeat units scattered throughout the genome. Although 20% of the unassembled genome sequence masked the genuine state, the predominance of scattered repeats for some low-copy satDNAs raises the question of whether these are essentially interspersed repeats that occur in tandem only sporadically, with the potential to be satDNA “seeds”.

Eukaryotic genomes are replete with satellite DNAs (satDNAs), large stretches of tandemly repeated sequences which are mostly underrepresented in genome assemblies. As a result, we know little about their spread, movement, and rearrangement across the genome or about their potential impact on the genome. Here, we have combined Oxford Nanopore long-read sequencing with a reference-guided assembly approach to generate a new, high-quality TcasONT genome assembly of the model beetle Tribolium castaneum. The new TcasONT is enriched by 50 Mb in the repetitive genome part. Therefore, we used the enhanced assembly to conduct global and in-depth analyses of ten abundant non-(peri)centromeric satDNAs, Cast1-Cast9. We found a large fraction of long arrays embedded in gene-rich regions common for almost all Cast1-Cast9 satDNAs and a negative correlation between the abundance of these satDNAs and transposable elements. Contrary to accepted opinion, our finding clearly speaks to the ability of satDNAs to expand in euchromatic regions and also demonstrate the flexibility of gene-rich regions to tolerate and accommodate satDNA sequences. In addition, we find that suppressed recombination on X chromosomes has less effect on the spread of satDNAs in euchromatin, but rather tolerates amplification of satDNAs into longer arrays. Our phylogenetic analyses show the extensive spread of Cast1-Cast9 satDNAs, characterized by distribution along the entire chromosome length and between (non)homologous chromosomes. This observed pattern points out very efficient mechanisms of propagation satDNAs on the whole genome scale including gene-rich regions. In conclusion, the presence of such a large portion of satDNAs with extensive propagation and amplification potential in gene-rich regions of the T. castaneum genome can strongly influence both gene expression and the dynamics of genome evolution.