The Organization of Pericentromeric Heterochromatin in Polytene Chromosome 3 of the Drosophila melanogaster Line with the Rif11; SuURES Su(var)3-906 Mutations Suppressing Underreplication

Although heterochromatin makes up 40% of the Drosophila melanogaster genome, its organization remains little explored, especially in polytene chromosomes, as it is virtually not represented in them due to underreplication. Two all-new approaches were used in this work: (i) with the use of a newly synthesized Drosophila line that carries three mutations, Rif11, SuURESand Su(var)3-906, suppressing the underreplication of heterochromatic regions, we obtained their fullest representation in polytene chromosomes and described their structure; (ii) 20 DNA fragments with known positions on the physical map as well as molecular genetic features of the genome (gene density, histone marks, heterochromatin proteins, origin recognition complex proteins, replication timing sites and satellite DNAs) were mapped in the newly polytenized heterochromatin using FISH and bioinformatics data. The borders of the heterochromatic regions and variations in their positions on arm 3L have been determined for the first time. The newly polytenized heterochromatic material exhibits two main types of morphology: a banding pattern (locations of genes and short satellites) and reticular chromatin (locations of large blocks of satellite DNA). The locations of the banding and reticular polytene heterochromatin was determined on the physical map.

Genomic Differences Between the Sexes in a Fish Species Seen Through Satellite DNAs

Neotropical fishes have highly diversified karyotypic and genomic characteristics and present many diverse sex chromosome systems, with various degrees of sex chromosome differentiation. Knowledge on their sex-specific composition and evolution, however, is still limited. Satellite DNAs (satDNAs) are tandemly repeated sequences with pervasive genomic distribution and distinctive evolutionary pathways, and investigating satDNA content might shed light into how genome architecture is organized in fishes and in their sex chromosomes. The present study investigated the satellitome of Megaleporinus elongatus, a freshwater fish with a proposed Z1Z1Z2Z2/Z1W1Z2W2 multiple sex chromosome system that encompasses a highly heterochromatic and differentiated W1 chromosome. The species satellitome comprises of 140 different satDNA families, including previously isolated sequences and new families found in this study. This diversity is remarkable considering the relatively low proportion that satDNAs generally account for the M. elongatus genome (around only 5%). Differences between the sexes in regards of satDNA content were also evidenced, as these sequences are 14% more abundant in the female genome. The occurrence of sex-biased signatures of satDNA evolution in the species is tightly linked to satellite enrichment associated with W1 in females. Although both sexes share practically all satDNAs, the overall massive amplification of only a few of them accompanied the W1 differentiation. We also investigated the expansion and diversification of the two most abundant satDNAs of M. elongatus, MelSat01-36 and MelSat02-26, both highly amplified sequences in W1 and, in MelSat02-26’s case, also harbored by Z2 and W2 chromosomes. We compared their occurrences in M. elongatus and the sister species M. macrocephalus (with a standard ZW sex chromosome system) and concluded that both satDNAs have led to the formation of highly amplified arrays in both species; however, they formed species-specific organization on female-restricted sex chromosomes. Our results show how satDNA composition is highly diversified in M. elongatus, in which their accumulation is significantly contributing to W1 differentiation and not satDNA diversity per se. Also, the evolutionary behavior of these repeats may be associated with genome plasticity and satDNA variability between the sexes and between closely related species, influencing how seemingly homeologous heteromorphic sex chromosomes undergo independent satDNA evolution.

Satellite-like W Elements: repetitive, transcribed, and putative mobile genetic factors with potential roles for biology and evolution of Schistosoma mansoni

A large portion of animal and plant genomes consists of non-coding DNA. This part includes tandemly repeated sequences and gained attention because it offers exciting insights into genome biology. We investigated satellite-DNA elements of the platyhelminth Schistosoma mansoni, a parasite with remarkable biological features. S. mansoni lives in the vasculature of humans causing schistosomiasis, a disease of worldwide importance. Schistosomes are the only trematodes that have evolved separate sexes, and the sexual maturation of the female depends on constant pairing with the male. The schistosome karyotype comprises eight chromosome pairs, males are homogametic (ZZ), females heterogametic (ZW). Part of the repetitive DNA of S. mansoni are W-elements (WEs), originally discovered as female-specific satellite DNAs in the heterochromatic block of the W-chromosome. Based on new genome and transcriptome data, we performed a reanalysis of the W-element families (WEFs). Besides a new classification of 19 WEFs, we provide first evidence for stage-, sex-, pairing-, gonad-, and strain-specific/preferential transcription of WEs as well as their mobile nature, deduced from autosomal copies of full-length and partial WEs. Structural analyses suggested roles as sources of non-coding RNA like hammerhead ribozymes (HHRs), for which we obtained functional evidence. Finally, the variable WEF occurrence in different schistosome species revealed remarkable divergence. From these results we propose that WEs potentially exert enduring influence on the biology of S. mansoni. Their variable occurrence in different strains, isolates, and species suggests that schistosome WEs may represent genetic factors taking effect on variability and evolution of the family Schistosomatidae.

First Description of a Satellite DNA in Manatees’ Centromeric Regions

Trichechus manatus and Trichechus inunguis are the two Sirenia species that occur in the Americas. Despite their increasing extinction risk, many aspects of their biology remain understudied, including the repetitive DNA fraction of their genomes. Here we used the sequenced genome of T. manatus and TAREAN to identify satellite DNAs (satDNAs) in this species. We report the first description of TMAsat, a satDNA comprising ~0.87% of the genome, with ~684bp monomers and centromeric localization. In T. inunguis, TMAsat showed similar monomer length, chromosome localization and conserved CENP-B box-like motifs as in T. manatus. We also detected this satDNA in the Dugong dugon and in the now extinct Hydrodamalis gigas genomes. The neighbor-joining tree shows that TMAsat sequences from T. manatus, T. inunguis, D. dugon, and H. gigas lack species-specific clusters, which disagrees with the predictions of concerted evolution. We detected a divergent TMAsat-like homologous sequence in elephants and hyraxes, but not in other mammals, suggesting this sequence was already present in the common ancestor of Paenungulata, and later became a satDNA in the Sirenians. This is the first description of a centromeric satDNA in manatees and will facilitate the inclusion of Sirenia in future studies of centromeres and satDNA biology.

Karyotype asymmetry in Cuscuta L. subgenus Pachystigma reflects its repeat DNA composition

Cuscuta is a cytogenetically diverse genus, with karyotypes varying 18-fold in chromosome number and 89-fold in genome size. Each of its four subgenera also presents particular chromosomal features, such as bimodal karyotypes in Pachystigma. We used low coverage sequencing of the Cuscuta nitida genome (subgenus Pachystigma), as well as chromosome banding and molecular cytogenetics of three subgenus representatives, to understand the origin of bimodal karyotypes. All three species, C. nitida, C. africana (2n = 28) and C. angulata (2n = 30), showed heterochromatic bands mainly in the largest chromosome pairs. Eighteen satellite DNAs were identified in C. nitida genome, two showing similarity to mobile elements. The most abundant were present at the largest pairs, as well as the highly abundant ribosomal DNAs. The most abundant Ty1/Copia and Ty3/Gypsy elements were also highly enriched in the largest pairs, except for the Ty3/Gypsy CRM, which also labelled the pericentromeric regions of the smallest chromosomes. This accumulation of repetitive DNA in the larger pairs indicates that these sequences are largely responsible for the formation of bimodal karyotypes in the subgenus Pachystigma. The repetitive DNA fraction is directly linked to karyotype evolution in Cuscuta.

Heterochromatin-dependent transcription of satellite DNAs in the Drosophila melanogaster female germline

Large blocks of tandemly repeated DNAs-satellite DNAs (satDNAs)-play important roles in heterochromatin formation and chromosome segregation. We know little about how satDNAs are regulated, however their misregulation is associated with genomic instability and human diseases. We use the Drosophila melanogaster germline as a model to study the regulation of satDNA transcription and chromatin. Here we show that complex satDNAs (>100-bp repeat units) are transcribed into long noncoding RNAs and processed into piRNAs (PIWI interacting RNAs). This satDNA piRNA production depends on the Rhino-Deadlock-Cutoff complex and the transcription factor Moonshiner—a previously-described non-canonical pathway that licenses heterochromatin-dependent transcription of dual-strand piRNA clusters. We show that this pathway is important for establishing heterochromatin at satDNAs. Therefore, satDNAs are regulated by piRNAs originating from their own genomic loci. This novel mechanism of satDNA regulation provides insight into the role of piRNA pathways in heterochromatin formation and genome stability.

Comparative Repeat Profiling of Two Closely Related Conifers (Larix decidua and Larix kaempferi) Reveals High Genome Similarity With Only Few Fast-Evolving Satellite DNAs

In eukaryotic genomes, cycles of repeat expansion and removal lead to large-scale genomic changes and propel organisms forward in evolution. However, in conifers, active repeat removal is thought to be limited, leading to expansions of their genomes, mostly exceeding 10 giga base pairs. As a result, conifer genomes are largely littered with fragmented and decayed repeats. Here, we aim to investigate how the repeat landscapes of two related conifers have diverged, given the conifers’ accumulative genome evolution mode. For this, we applied low-coverage sequencing and read clustering to the genomes of European and Japanese larch, Larix decidua (Lamb.) Carrière and Larix kaempferi (Mill.), that arose from a common ancestor, but are now geographically isolated. We found that both Larix species harbored largely similar repeat landscapes, especially regarding the transposable element content. To pin down possible genomic changes, we focused on the repeat class with the fastest sequence turnover: satellite DNAs (satDNAs). Using comparative bioinformatics, Southern, and fluorescent in situ hybridization, we reveal the satDNAs’ organizational patterns, their abundances, and chromosomal locations. Four out of the five identified satDNAs are widespread in the Larix genus, with two even present in the more distantly related Pseudotsuga and Abies genera. Unexpectedly, the EulaSat3 family was restricted to L. decidua and absent from L. kaempferi, indicating its evolutionarily young age. Taken together, our results exemplify how the accumulative genome evolution of conifers may limit the overall divergence of repeats after speciation, producing only few repeat-induced genomic novelties.

Satellitome Analysis of the Pacific Oyster Crassostrea gigas Reveals New Pattern of Satellite DNA Organization, Highly Scattered across the Genome

Several features already qualified the invasive bivalve species Crassostrea gigas as a valuable non-standard model organism in genome research. C. gigas is characterized by the low contribution of satellite DNAs (satDNAs) vs. mobile elements and has an extremely low amount of heterochromatin, predominantly built of DNA transposons. In this work, we have identified 52 satDNAs composing the satellitome of C. gigas and constituting about 6.33% of the genome. Satellitome analysis reveals unusual, highly scattered organization of relatively short satDNA arrays across the whole genome. However, peculiar chromosomal distribution and densities are specific for each satDNA. The inspection of the organizational forms of the 11 most abundant satDNAs shows association with constitutive parts of Helitron mobile elements. Nine of the inspected satDNAs are dominantly found in mobile element-associated form, two mostly appear standalone, and only one is present exclusively as Helitron-associated sequence. The Helitron-related satDNAs appear in more chromosomes than other satDNAs, indicating that these mobile elements could be leading satDNA propagation in C. gigas. No significant accumulation of satDNAs on certain chromosomal positions was detected in C. gigas, thus establishing a novel pattern of satDNA organization on the genome level.

Linker histone dH1K27 dimethylation marks Drosophila heterochromatin independently of H3K9 methylation

Post-translational modifications (PTMs) of histones are important epigenetic determinants and specific core histones PTMs correlate with functional chromatin states. However, despite linker histone H1s are heavily post-translationally modified, little is known about the genomic distribution of H1s PTMs and their association with epigenetic chromatin states. Here, we address this question in Drosophila that encodes a single somatic linker histone, dH1. We previously reported that dH1 is dimethylated at K27 (dH1K27me2). Here, we show that dH1K27me2 is a major PTM of Drosophila heterochromatin. At mitosis, dH1K27me2 accumulates at pericentromeric heterochromatin, while, in interphase cells, it is also detected at intercalary heterochromatin. ChIPseq experiments show that dH1K27me2 enriched regions cluster at both the assembled and unassembled heterochromatin regions of all four Drosophila chromosomes. More than 98% of the dH1K27me2 enriched regions map to heterochromatic repetitive DNA elements, including transposable elements, simple DNA repeats and satellite DNAs. We also show that dH1K27me2 is independent of H3K9 methylation, as it is equally detected in flies carrying a H3K9R mutation. Moreover, dH1K27me2 is not affected by depletion of Su(var)3-9, HP1a and Su(var)4-20. Altogether these results suggest that dH1K27me2 is a novel epigenetic mark of Drosophila heterochromatin that acts upstream of the major Su(var)3- 9/HP1a pathway of heterochromatin formation.

The B Chromosomes of Prochilodus lineatus (Teleostei, Characiformes) Are Highly Enriched in Satellite DNAs

B or supernumerary chromosomes are dispensable elements that are widely present in numerous eukaryotes. Due to their non-recombining nature, there is an evident tendency for repetitive DNA accumulation in these elements. Thus, satellite DNA plays an important role in the evolution and diversification of B chromosomes and can provide clues regarding their origin. The characiform Prochilodus lineatus was one of the first discovered fish species bearing B chromosomes, with all populations analyzed so far showing one to nine micro-B chromosomes and exhibiting at least three morphological variants (Ba, Bsm, and Bm). To date, a single satellite DNA is known to be located on the B chromosomes of this species, but no information regarding the differentiation of the proposed B-types is available. Here, we characterized the satellitome of P. lineatus and mapped 35 satellite DNAs against the chromosomes of P. lineatus, of which six were equally located on all B-types and this indicates a similar genomic content. In addition, we describe, for the first time, an entire population without B chromosomes.