Robertsonian Fusion and Centromere Repositioning Contributed to the Formation of Satellite-free Centromeres During the Evolution of Zebras.

Centromeres are epigenetically specified by the histone H3 variant CENP-A and typically associated with highly repetitive satellite DNA. We previously discovered natural satellite-free neocentromeres in Equus caballus and Equus asinus. Here, through ChIP-seq with an anti-CENP-A antibody, we found an extraordinarily high number of centromeres lacking satellite DNA in the zebras Equus burchelli (15 of 22) and Equus grevyi (13 of 23), demonstrating that the absence of satellite DNA at the majority of centromeres is compatible with genome stability and species survival and challenging the role of satellite DNA in centromere function. Nine satellite-free centromeres are shared between the two species in agreement with their recent separation. We assembled all centromeric regions and improved the reference genome of E. burchelli. Sequence analysis of the CENP-A binding domains revealed that they are LINE-1 and AT-rich with four of them showing DNA amplification. In the two zebras, satellite-free centromeres emerged from centromere repositioning or following Robertsonian fusion. In five chromosomes, the centromeric function arose near the fusion points, which are located within regions marked by traces of ancestral pericentromeric sequences. Therefore, besides centromere repositioning, Robertsonian fusions are an important source of satellite-free centromeres during evolution. Finally, in one case, a satellite-free centromere was seeded on an inversion breakpoint. At 11 chromosomes, whose primary constrictions seemed to be associated with satellite repeats by cytogenetic analysis, satellite-free neocentromeres were instead located near the ancestral inactivated satellite-based centromeres; therefore, the centromeric function has shifted away from a satellite repeat containing locus to a satellite-free new position.

Molecular Dynamics and Evolution of Centromeres in the Genus Equus.

The centromere is the chromosomal locus essential for proper chromosome segregation. While the centromeric function is well conserved and epigenetically specified, centromeric DNA sequences are typically composed of satellite DNA and represent the most rapidly evolving sequences in eukaryotic genomes. The presence of satellite sequences at centromeres hampered the comprehensive molecular analysis of these enigmatic loci. The discovery of functional centromeres completely devoid of satellite repetitions and fixed in some animal and plant species represented a turning point in centromere biology, definitively proving the epigenetic nature of the centromere. The first satellite-free centromere, fixed in a vertebrate species, was discovered in the horse. Later, an extraordinary number of satellite-free neocentromeres had been discovered in other species of the genus Equus, which remains the only mammalian genus with numerous satellite-free centromeres described thus far. These neocentromeres arose recently during evolution and are caught in a stage of incomplete maturation. Their presence made the equids a unique model for investigating, at molecular level, the minimal requirements for centromere seeding and evolution. This model system provided new insights on how centromeres are established and transmitted to the progeny and on the role of satellite DNA in different aspects of centromere biology.

Birth, evolution, and transmission of satellite-free mammalian centromeric domains.

Mammalian centromeres are associated with highly repetitive DNA (satellite DNA), which has so far hindered molecular analysis of this chromatin domain. Centromeres are epigenetically specified, and binding of the CENPA protein is their main determinant. In previous work, we described the first example of a natural satellite-free centromere on Equus caballus Chromosome 11. Here, we investigated the satellite-free centromeres of Equus asinus by using ChIP-seq with anti-CENPA antibodies. We identified an extraordinarily high number of centromeres lacking satellite DNA (16 of 31). All of them lay in LINE- and AT-rich regions. A subset of these centromeres is associated with DNA amplification. The location of CENPA binding domains can vary in different individuals, giving rise to epialleles. The analysis of epiallele transmission in hybrids (three mules and one hinny) showed that centromeric domains are inherited as Mendelian traits, but their position can slide in one generation. Conversely, centromere location is stable during mitotic propagation of cultured cells. Our results demonstrate that the presence of more than half of centromeres void of satellite DNA is compatible with genome stability and species survival. The presence of amplified DNA at some centromeres suggests that these arrays may represent an intermediate stage toward satellite DNA formation during evolution. The fact that CENPA binding domains can move within relatively restricted regions (a few hundred kilobases) suggests that the centromeric function is physically limited by epigenetic boundaries.

The Unique DNA Sequences Underlying Equine Centromeres.

Centromeres are highly distinctive genetic loci whose function is specified largely by epigenetic mechanisms. Understanding the role of DNA sequences in centromere function has been a daunting task due to the highly repetitive nature of centromeres in animal chromosomes. The discovery of a centromere devoid of satellite DNA in the domestic horse consolidated observations on the epigenetic nature of centromere identity, showing that entirely natural chromosomes could function without satellite DNA cues. Horses belong to the genus Equus which exhibits a very high degree of evolutionary plasticity in centromere position and DNA sequence composition. Examination of horses has revealed that the position of the satellite-free centromere is variable among individuals. Analysis of centromere location and composition in other Equus species, including domestic donkey and zebras, confirms that the satellite-less configuration of centromeres is common in this group which has undergone particularly rapid karyotype evolution. These features have established the equids as a new mammalian system in which to investigate the molecular organization, dynamics and evolutionary behaviour of centromeres.

Centromere sliding on a mammalian chromosome.

The centromere directs the segregation of chromosomes during mitosis and meiosis. It is a distinct genetic locus whose identity is established through epigenetic mechanisms that depend on the deposition of centromere-specific centromere protein A (CENP-A) nucleosomes. This important chromatin domain has so far escaped comprehensive molecular analysis due to its typical association with highly repetitive satellite DNA. In previous work, we discovered that the centromere of horse chromosome 11 is completely devoid of satellite DNA; this peculiar feature makes it a unique model to dissect the molecular architecture of mammalian centromeres. Here, we exploited this native satellite-free centromere to determine the precise localization of its functional domains in five individuals: We hybridized DNA purified from chromatin immunoprecipitated with an anti CENP-A antibody to a high resolution array (ChIP-on-chip) of the region containing the primary constriction of horse chromosome 11. Strikingly, each individual exhibited a different arrangement of CENP-A binding domains. We then analysed the organization of each domain using a single nucleotide polymorphism (SNP)-based approach and single molecule analysis on chromatin fibres. Examination of the ten instances of chromosome 11 in the five individuals revealed seven distinct 'positional alleles', each one extending for about 80-160 kb, were found across a region of about 500 kb. Our results demonstrate that CENP-A binding domains are autonomous relative to the underlying DNA sequence and are characterized by positional instability causing the sliding of centromere position. We propose that this dynamic behaviour may be common in mammalian centromeres and may determine the establishment of epigenetic alleles.

Genome-wide evolutionary and functional analysis of the Equine Repetitive Element 1: an insertion in the myostatin promoter affects gene expression.

BACKGROUND: In mammals, an important source of genomic variation is insertion polymorphism of retrotransposons. These may acquire a functional role when inserted inside genes or in their proximity. The aim of this work was to carry out a genome wide analysis of ERE1 retrotransposons in the horse and to analyze insertion polymorphism in relation to evolution and function. The effect of an ERE1 insertion in the promoter of the myostatin gene, which is involved in muscle development, was also investigated. RESULTS: In the horse population, the fraction of ERE1 polymorphic loci is related to the degree of similarity to their consensus sequence. Through the analysis of ERE1 conservation in seven equid species, we established that the level of identity to their consensus is indicative of evolutionary age of insertion. The position of ERE1s relative to genes suggests that some elements have acquired a functional role. Reporter gene assays showed that the ERE1 insertion within the horse myostatin promoter affects gene expression. The frequency of this variant promoter correlates with sport aptitude and racing performance. CONCLUSIONS: Sequence conservation and insertion polymorphism of ERE1 elements are related to the time of their appearance in the horse lineage, therefore, ERE1s are a useful tool for evolutionary and population studies. Our results suggest that the ERE1 insertion at the myostatin locus has been unwittingly selected by breeders to obtain horses with specific racing abilities. Although a complex combination of environmental and genetic factors contributes to athletic performance, breeding schemes may take into account ERE1 insertion polymorphism at the myostatin promoter.

Alike but different: the evolution of the Tubifex tubifex species complex (Annelida, Clitellata) through polyploidization.

BACKGROUND: Tubifex tubifex is a widespread annelid characterized by considerable variability in its taxonomic characteristics and by a mixed reproductive strategy, with both parthenogenesis and biparental reproduction. In a molecular phylogenetic analysis, we detected substantial genetic variability among sympatric Tubifex spp. from the Lambro River (Milano, Italy), which we suggested comprise several cryptic species. To gain insights into the evolutionary events that generated this differentiation, we performed a cytogenetic analysis in parallel with a molecular assay. Approximately 80 cocoons of T. tubifex and T. blanchardi were collected and dissected. For each cocoon, we sequenced a fragment of the 16S rRNA from half of the sibling embryos and karyotyped the other half. To generate a robust phylogeny enabling the reconstruction of the evolutionary processes shaping the diversity of these sympatric lineages, we complemented our original 16S rRNA gene sequences with additional COI sequences. RESULTS: The chromosome number distribution was consistent with the presence of at least six sympatric euploid chromosome complements (one diploid, one triploid, three tetraploids and one hexaploid), as confirmed by a FISH assay performed with an homologous 18S rDNA probe. All the worms with 2n = 50 chromosomes belonged to an already identified sibling species of T. tubifex, T. blanchardi. The six euploid sets were coherently arranged in the phylogeny, with each lineage grouping specimens with the same chromosome complement. CONCLUSIONS: These results are compatible with the hypothesis that multiple polyploidization events, possibly enhanced by parthenogenesis, may have driven the evolution of the T. tubifex species complex.

Discovery and comparative analysis of a novel satellite, EC137, in horses and other equids.

Centromeres are the sites of kinetochore assembly and spindle fiber attachment and consist of protein-DNA complexes in which the DNA component is typically characterized by the presence of extended arrays of tandem repeats called satellite DNA. Here, we describe the isolation and characterization of a 137-bp-long new satellite DNA sequence from the horse genome (EC137), which is also present, even if less abundant, in the domestic donkey, the Grevy's zebra and the Burchelli's zebra. We investigated the chromosomal distribution of the EC137 sequence in these 4 species. Moreover, we analyzed its architectural organization by high-resolution FISH. The position of this sequence with respect to the primary constriction and in relation to the 2 major horse satellite tandem repeats (37 cen and 2PI) on horse chromosomes suggests that the new centromeric equine satellite is an accessory DNA element, presumably contributing to the organization of pericentromeric chromatin. FISH on combed DNA fibers reveals that the EC137 satellite is organized in relatively short stretches (2-8 kb) which are strictly intermingled within 37 cen or 2PI arrays. This arrangement suggests that interchanges between satellite families are a frequent occurrence in the horse genome.

Chromosome Transplantation: Opportunities and Limitations.

There are thousands of rare genetic diseases that could be treated with classical gene therapy strategies such as the addition of the defective gene via viral or non-viral delivery or by direct gene editing. However, several genetic defects are too complex for these approaches. These "genomic mutations" include aneuploidies, intra and inter chromosomal rearrangements, large deletions, or inversion and copy number variations. Chromosome transplantation (CT) refers to the precise substitution of an endogenous chromosome with an exogenous one. By the addition of an exogenous chromosome and the concomitant elimination of the endogenous one, every genetic defect, irrespective of its nature, could be resolved. In the current review, we analyze the state of the art of this technique and discuss its possible application to human pathology. CT might not be limited to the treatment of human diseases. By working on sex chromosomes, we showed that female cells can be obtained from male cells, since chromosome-transplanted cells can lose either sex chromosome, giving rise to 46,XY or 46,XX diploid cells, a modification that could be exploited to obtain female gametes from male cells. Moreover, CT could be used in veterinary biology, since entire chromosomes containing an advantageous locus could be transferred to animals of zootechnical interest without altering their specific genetic background and the need for long and complex interbreeding. CT could also be useful to rescue extinct species if only male cells were available. Finally, the generation of "synthetic" cells could be achieved by repeated CT into a recipient cell. CT is an additional tool for genetic modification of mammalian cells.

RNA-Seq analysis discloses early senescence and nucleolar dysfunction triggered by Tdp1α depletion in Medicago truncatula.

An intron-spliced hairpin RNA approach was used for the targeted silencing of the MtTdp1α gene encoding the αisoform of tyrosyl-DNA phosphodiesterase 1 in Medicago truncatula Gaertn. Tyrosyl-DNA phosphodiesterase 1, involved in the repair of DNA topoisomerase I-mediated DNA damage, has been poorly investigated in plants. RNA-Seq analysis, carried out in the MtTdp1α-depleted plants, revealed different levels of transcriptional modulation (up- and down-regulation, alternative splicing, activation of alternative promoter) in genes involved in DNA damage sensing, DNA repair, and chromatin remodelling. It is suggested that the MtTdp1α gene has new, previously undetected roles in maintaining genome integrity. Up-regulation of senescence-associated genes and telomere shortening were observed. Moreover, impaired ribosome biogenesis indicated that the MtTdp1α gene is required for the nucleolar function. In agreement with the RNA-Seq data, transmission electron microscopy detected an altered nucleolar architecture in the MtTdp1α-depleted cells. Based on the reported data, a working hypothesis related to the occurrence of a nucleolar checkpoint in plant cells is proposed.

Uncoupling of satellite DNA and centromeric function in the genus Equus.

In a previous study, we showed that centromere repositioning, that is the shift along the chromosome of the centromeric function without DNA sequence rearrangement, has occurred frequently during the evolution of the genus Equus. In this work, the analysis of the chromosomal distribution of satellite tandem repeats in Equus caballus, E. asinus, E. grevyi, and E. burchelli highlighted two atypical features: 1) several centromeres, including the previously described evolutionary new centromeres (ENCs), seem to be devoid of satellite DNA, and 2) satellite repeats are often present at non-centromeric termini, probably corresponding to relics of ancestral now inactive centromeres. Immuno-FISH experiments using satellite DNA and antibodies against the kinetochore protein CENP-A demonstrated that satellite-less primary constrictions are actually endowed with centromeric function. The phylogenetic reconstruction of centromere repositioning events demonstrates that the acquisition of satellite DNA occurs after the formation of the centromere during evolution and that centromeres can function over millions of years and many generations without detectable satellite DNA. The rapidly evolving Equus species gave us the opportunity to identify different intermediate steps along the full maturation of ENCs.

Cytological, molecular, cytogenetic, and physiological characterization of a novel immortalized human enteric glial cell line.

Enteric glial cells (EGCs), the major components of the enteric nervous system (ENS), are implicated in the maintenance of gut homeostasis, thereby leading to severe pathological conditions when impaired. However, due to technical difficulties associated with EGCs isolation and cell culture maintenance that results in a lack of valuable in vitro models, their roles in physiological and pathological contexts have been poorly investigated so far. To this aim, we developed for the first time, a human immortalized EGC line (referred as ClK clone) through a validated lentiviral transgene protocol. As a result, ClK phenotypic glial features were confirmed by morphological and molecular evaluations, also providing the consensus karyotype and finely mapping the chromosomal rearrangements as well as HLA-related genotypes. Lastly, we investigated the ATP- and acetylcholine, serotonin and glutamate neurotransmitters mediated intracellular Ca2+ signaling activation and the response of EGCs markers (GFAP, SOX10, S100ß, PLP1, and CCL2) upon inflammatory stimuli, further confirming the glial nature of the analyzed cells. Overall, this contribution provided a novel potential in vitro tool to finely characterize the EGCs behavior under physiological and pathological conditions in humans.

Cell death induction and nitric oxide biosynthesis in white poplar (Populus alba) suspension cultures exposed to alfalfa saponins.

The present work reports on the biological activity of alfalfa (Medicago sativa) saponins on white poplar (Populus alba, cultivar 'Villafranca') cell suspension cultures. The extracts from alfalfa roots, aerial parts and seeds were characterized for their saponin content by means of thin layer chromatography (TLC) and electrospray ionisation coupled to mass spectrometry. The quantitative saponin composition from the different plant extracts was determined considering the aglycone moieties and determined by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS) analyses. Only soyasapogenin I was detected in the seed extract while several other saponins were found in the root and leaf extracts. Actively proliferating white poplar cell cultures were challenged with the different saponin extracts. Only alfalfa root saponins, at 50 µg ml⁻¹, induced significant cell death rates (75.00 ± 4.90%). Different cell subpopulations with peculiar cell death morphologies were observed and the programmed cell death (PCD)/necrosis ratio was reduced at increasing saponin concentrations. Enhancement of nitric oxide (NO) production was observed in white poplar cells treated with root saponins (RSs) at 50 µg ml⁻¹ and release of reactive oxygen species (ROS) in the culture medium was also demonstrated. Saponin-induced NO production was sensitive to sodium azide and N(G)-monomethyl-L-arginine, two specific inhibitors of distinct pathways for NO biosynthesis in plant cells.

Polymorphic organization of constitutive heterochromatin in Equus asinus (2n = 62) chromosome 1.

In the karyotype of Equus asinus (domestic donkey, 2n = 62), non-centromeric heterochromatic bands have been described in subcentromeric and telomeric positions. In particular, chromosome 1 is characterised by heterochromatic bands in the proximal region of the long arm and in the short arm; it has been shown that these regions are polymorphic in size. Here we investigated the variation in the intensity and distribution of fluorescence signals observed on donkey chromosome 1 after in situ hybridization with two DNA probes containing fragments from the two major equine satellite DNA families. Our results show that, in Equus asinus chromosome 1, the amount and distribution of large clusters of satellite DNA can define at least nine polymorphic variants of the constitutive heterochromatin that cannot be detected by C-banding alone.

Is cognitive behavioural therapy an effective complement to antidepressants in adolescents? A meta-analysis.

Calati R, Pedrini L, Alighieri S, Alvarez MI, Desideri L, Durante D, Favero F, Iero L, Magnani G, Pericoli V, Polmonari A, Raggini R, Raimondi E, Riboni V, Scaduto MC, Serretti A, De Girolamo G. Is cognitive behavioural therapy an effective complement to antidepressants in adolescents? A meta-analysis. OBJECTIVE: Evidence on effectiveness of combined treatments versus antidepressants alone in adolescents consists on a few studies in both major depressive and anxiety disorders. A meta-analysis of randomised 12-week follow-up studies in which antidepressant treatment was compared to combined treatment consisting of the same antidepressant with cognitive behavioural therapy has been performed. METHODS: Data were entered into the Cochrane Collaboration Review Manager software and were analysed within a random effect framework. A quality assessment has been performed through Jadad Scale. RESULTS: Higher global functioning at the Children's Global Assessment Scale was found in the combined treatment group (p < 0.0001) as well as higher improvement at the Clinical Global Impressions Improvement Scale (p = 0.04). No benefit of combined treatment was found on depressive symptomatology at the Children's Depression Rating Scale - Revised. CONCLUSION: Combined treatment seems to be more effective than antidepressant alone on global functioning and general improvement in adolescents with major depressive and anxiety disorders.

The life cycle of Myxobolus lentisuturalis (Myxozoa: Myxobolidae), from goldfish (Carassius auratus auratus), involves a Raabeia-type actinospore.

We studied a natural infection of the oligochaete Branchiura sowerbyi Beddard, 1892 with the Raabeia-type actinosporean stage of Myxobolus lentisuturalis Dyková, Fiala et Nie, 2002 which infected goldfish Carassius auratus auratus (L.) in Italy, using molecular analysis of the SSU rRNA gene. The existence of intraoligochaete development shows that this parasite follows the life-cycle pattern described by Wolf and Markiw (1984) for Myxobolus cerebralis. Histological examinations of the goldfish infected by M. lentisuturalis showed at low magnification the presence of two bilateral crescent-shaped masses in the dorsal epaxial muscle. These lesions were not circumscribed, presented irregular edges and infiltrated the underlying bundles of skeletal muscle and interstitial tissue. At higher magnification, disappearance of muscle fibres and substitution of the muscle tissue with Myxobolus spores and plasmodia were observed.

Satellite DNA at the Centromere is Dispensable for Segregation Fidelity.

The typical vertebrate centromeres contain long stretches of highly repeated DNA sequences (satellite DNA). We previously demonstrated that the karyotypes of the species belonging to the genus Equus are characterized by the presence of satellite-free and satellite-based centromeres and represent a unique biological model for the study of centromere organization and behavior. Using horse primary fibroblasts cultured in vitro, we compared the segregation fidelity of chromosome 11, whose centromere is satellite-free, with that of chromosome 13, which has similar size and a centromere containing long stretches of satellite DNA. The mitotic stability of the two chromosomes was compared under normal conditions and under mitotic stress induced by the spindle inhibitor, nocodazole. Two independent molecular-cytogenetic approaches were used-the interphase aneuploidy analysis and the cytokinesis-block micronucleus assay. Both assays were coupled to fluorescence in situ hybridization with chromosome specific probes in order to identify chromosome 11 and chromosome 13, respectively. In addition, we tested if the lack of centromeric satellite DNA affected chromatid cohesion under normal and stress conditions. We demonstrated that, in our system, the segregation fidelity of a chromosome is not influenced by the presence of long stretches of tandem repeats at its centromere. To our knowledge, the present study is the first analysis of the mitotic behavior of a natural satellite-free centromere.

Transfer of a human chromosomal vector from a hamster cell line to a mouse embryonic stem cell line.

Two transchromosomic mouse embryonic stem (ES) sublines (ESMClox1.5 and ESMClox2.1) containing a human minichromosome (MC) were established from a sample of hybrid colonies isolated in fusion experiments between a normal diploid mouse ES line and a Chinese hamster ovary line carrying the MC. DNA cytometric and chromosome analyses of ESMClox1.5 and ESMClox2.1 indicated a mouse chromosome complement with a heteroploid constitution in a subtetraploid range; the karyotypes showed various degrees of polysomy for different chromosomes. A single copy of the MC was found in the majority of cells in all the isolated hybrid colonies and was stably maintained in the established sublines for more than 100 cell generations either with or without the selective agent. No significant differences from the ES parental cells were observed in growth characteristics of the transchromosomic ES sublines. ESMClox1.5 cells were unable to grow in soft agar; when cultured in hanging drops, they formed embryoid bodies, and when inoculated in nude mice, they produced teratomas. They were able to express the early development markers Oct4 and Nanog, as demonstrated by reverse transcription-polymerase chain reaction assay. All these features are in common with the ES parental line. Further research using the transchromosomic ES sublines described here may allow gene expression studies on transferred human minichromosomes and could shed light on the relationships among ploidy, pluripotency, cell transformation, and tumorigenesis. Disclosure of potential conflicts of interest is found at the end of this article.

Human chromosomes 9, 12, and 15 contain the nucleation sites of stress-induced nuclear bodies.

We previously reported the identification of a novel nuclear compartment detectable in heat-shocked HeLa cells that we termed stress-induced Src-activated during mitosis nuclear body (SNB). This structure is the recruitment center for heat shock factor 1 and for a number of RNA processing factors, among a subset of Serine-Arginine splicing factors. In this article, we show that stress-induced SNBs are detectable in human but not in hamster cells. By means of hamster>human cell hybrids, we have identified three human chromosomes (9, 12, and 15) that are individually able to direct the formation of stress bodies in hamster cells. Similarly to stress-induced SNB, these bodies are sites of accumulation of hnRNP A1-interacting protein and heat shock factor 1, are usually associated to nucleoli, and consist of clusters of perichromatin granules. We show that the p13-q13 region of human chromosome 9 is sufficient to direct the formation of stress bodies in hamster>human cell hybrids. Fluorescence in situ hybridization experiments demonstrate that the pericentromeric heterochromatic q12 band of chromosome 9 and the centromeric regions of chromosomes 12 and 15 colocalize with stress-induced SNBs in human cells. Our data indicate that human chromosomes 9, 12, and 15 contain the nucleation sites of stress bodies in heat-shocked HeLa cells.