Chromatin diminution in nematodes

The process of chromatin diminution in Parascaris and Ascaris is a developmentally controlled genome rearrangement, which results in quantitative and qualitative differences in DNA content between germ line and somatic cells. Chromatin diminution involves chromosomal breakage, new telomere formation and DNA degradation. The programmed elimination of chromatin in presomatic cells might serve as an alternative way of gene regulation. We put forward a new hypothesis of how an ancient partial genome duplication and chromatin diminution may have served to maintain the genetic balance in somatic cells and simultaneously endowed the germ line cells with a selective advantage.     

Chromatin diminution in Ascaris suum: nucleotide sequence of the eliminated satellite DNA.

We have determined the prototype sequence of the DNA which is eliminated in the course of chromatin diminution in Ascaris suum. This DNA which is virtually absent from somatic cells but retained in the germ line consists predominantly of highly repetitive sequences which are variants of an AT rich 123 base pair repeat unit. Both major and minor variants have been sequenced. The overall structure of this germ line limited DNA corresponds to the segmental organization characteristic of satellite DNAs. Possible correlations between the mechanism of chromatin diminution and some properties of the satellite sequence are discussed.     

Genome size and metabolic intensity in tetrapods: a tale of two lines

We show the negative link between genome size and metabolic intensity in tetrapods, using the heart index (relative heart mass) as a unified indicator of metabolic intensity in poikilothermal and homeothermal animals. We found two separate regression lines of heart index on genome size for reptiles-birds and amphibians-mammals (the slope of regression is steeper in reptiles-birds). We also show a negative correlation between GC content and nucleosome formation potential in vertebrate DNA, and, consistent with this relationship, a positive correlation between genome GC content and nuclear size (independent of genome size). It is known that there are two separate regression lines of genome GC content on genome size for reptiles-birds and amphibians-mammals: reptiles-birds have the relatively higher GC content (for their genome sizes) compared to amphibians-mammals. Our results suggest uniting all these data into one concept. The slope of negative regression between GC content and nucleosome formation potential is steeper in exons than in non-coding DNA (where nucleosome formation potential is generally higher), which indicates a special role of non-coding DNA for orderly chromatin organization. The chromatin condensation and nuclear size are supposed to be key parameters that accommodate the effects of both genome size and GC content and connect them with metabolic intensity. Our data suggest that the reptilian-birds clade evolved special relationships among these parameters, whereas mammals preserved the amphibian-like relationships. Surprisingly, mammals, although acquiring a more complex general organization, seem to retain certain genome-related properties that are similar to amphibians. At the same time, the slope of regression between nucleosome formation potential and GC content is steeper in poikilothermal than in homeothermal genomes, which suggests that mammals and birds acquired certain common features of genomic organization.     

Chromatin diminution in the copepod Mesocyclops edax: diminution of tandemly repeated DNA families from somatic cells.

Chromatin diminution, i.e., the loss of selected chromosomal regions during the differentiation of early embryonic cells into somatic cells, has been described in taxa as varied as ciliates, copepods, insects, nematodes, and hagfish. The nature of the eliminated DNA has been extensively studied in ciliate, nematode, and hagfish species. However, the small size of copepods, which makes it difficult to obtain enough DNA from early embryonic cells for cloning and sequencing, has limited such studies. Here, to identify the sequences eliminated from the somatic cells of a copepod species that undergoes chromatin diminution, we randomly amplified DNA fragments from germ line and somatic line cells of Mesocyclops edax, a freshwater cyclopoid copepod. Of 47 randomly amplified germ line clones, 45 (96%) contained short, tandemly repeated sequences composed of either 2 bp CA-repeats, 8 bp CAAATAGA-repeats, or 9 bp CAAATTAAA-repeats. In contrast, of 83 randomly amplified somatic line clones, only 47 (57%) contained such short, tandemly repeated sequences. As previously observed in some nematode species, our results therefore show that there is partial elimination of chromosomal regions containing (CAAATAGA and CAAATTAAA) repeated sequences during the chromatin diminution observed in the somatic cells of M. edax. We speculate that chromatin diminution might have evolved repeatedly by recruitment of RNAi-related mechanisms to eliminate nonfunctional tandemly repeated DNA sequences from the somatic genome of some species.     

Chromatin diminution is a key process explaining the eukaryotic genome size paradox and some mechanisms of genetic isolation

The functions of redundant (junk, selfish, parasitic, etc.) DNA in eukaryotes can be reliably inferred from chromatin diminution (programmed elimination of up to 94% of the genome from somatic germ cells in Ascaris and Cyclops). These functions should be sought in germ cells, where this DNA is preserved during the entire life time of the species. A possible biological role of redundant DNA as a factor disrupting meiotic chromosome synapsis is suggested. At the same time, chromatin diminution itself can act as a mechanism of postzygotic isolation. All stage of the complex diminution mechanism could not be fixed in the genetic program of the species via gradual accumulation of mutations. The "program" of diminution must have appeared at once and in the completed form.     

The occurrence, role and evolution of chromatin diminution in nematodes

Chromatin diminution takes place in presomatic cells of some parasitic nematodes during early development. This phenomenon may play an important role in somatic cell differentiation, since the somatic cells of these species undergo an extensive genome reorganization during development via chromatin diminution and polyploidization, as explained here by Clara Goday and Sergio Pimpinelli.     

DNA-Feulgen cytophotometric determination of genome size for the freshwater-invading copepod Eurytemora affinis.

Variation in nuclear DNA content within some eukaryotic species is well documented, but causes and consequences of such variation remain unclear. Here we report genome size of an estuarine and salt-marsh calanoid copepod, Eurytemora affinis, which has recently invaded inland freshwater habitats independently and repeatedly in North America, Europe, and Asia. Adults and embryos of E. affinis from the St. Lawrence River drainage were examined for somatic cell DNA content and the presence or absence of embryonic chromatin diminution, using Feulgen-DNA cytophotometry to determine a diploid or 2C genome size of 0.6-0.7 pg DNA/cell. The majority of somatic cell nuclei, however, have twice this DNA content (1.3 pg/nucleus) in all of the adults examined and possibly represent a population of cells arrested at the G2 stage of the cell cycle or associated with some degree of endopolyploidy. Both suggestions contradict assumptions that DNA replication does not occur in adult tissues during the determinate growth characteristic of copepods. Absence of germ cell nuclei with markedly elevated DNA values, commonly found for species of cyclopoid copepods that show chromatin diminution, indicates that E. affinis lacks this trait. The small genome size and presumed absence of chromatin diminution increase the potential utility of E. affinis as a model for genomic studies on mechanisms of adaptation during freshwater invasions.     

Cytophotometric determination of genome size in two species of Cyclops of Lake Baikal (Crustacea: Copepoda, Cyclopoida) in ontogenetic development

The genome size of Cyclops in cells at early stages of cleavage (up to the fifth division) and in somatic cells was estimated by static digital Feulgen cytophotometry in order to study quantitative changes in DNA content during chromatin diminution. Described here cytophotometric method was approbated on five different digital-imaging systems in blood cells of four vertebrate species. In all cases, we observed a direct correlation between the data obtained with known from the literature on genome size and high reproducibility, which will allow these systems to be used in future work. We also optimized the conditions for DNA hydrolysis of both blood smears and for two species of Cyclops from the Moscow population as 30 min in 5 N HCl at 24°C. Here, we first revealed chromatin diminution in two endemic Baikal species of Cyclopoida: Acanthocyclops incolotaenia and Diacyclops galbinus. We estimated the extent of chromatin diminution in Diacyclops galbinus as 95.5–96.2%. Cytometric analysis of the third species, Mesocyclops leuckarti, did not reveal obvious chromatin diminution.     

Sex-specific dynamics of global chromatin changes in fetal mouse germ cells

Mammalian germ cells undergo global reprogramming of DNA methylation during their development. Global DNA demethylation occurs around the time when the primordial germ cells colonize the embryonic gonads and this coincides with dynamic changes in chromatin composition. Global de novo DNA methylation takes place with remarkably different dynamics between the two sexes, prospermatogonia attaining methylation during fetal stages and oocytes attaining methylation postnatally. Our hypothesis was that dynamic changes in chromatin composition may precede or accompany the wave of global DNA de novo methylation as well. We used immunocytochemistry to measure global DNA methylation and chromatin components in male and female mouse fetal germ cells compared to control somatic cells of the gonad. We found that global DNA methylation levels sharply increased in male germ cells at 17.5 days post coitum, but remained low in female germ cells at all fetal stages. Global changes in chromatin composition: i, preceded global DNA methylation in fetal germ cells; ii, sex specifically occurred in male but not in female germ cells; iii, affected active and repressive histone marks and iv, included histone tail and histone globular domain modifications. Our data suggest that dynamic changes of chromatin composition may provide a framework for the pattern of male-specific de novo DNA methylation in prospermatogonia.     

A novel UV-damaged DNA binding protein emerges during the chromatin-eliminating cleavage period in Ascaris suum.

During the early cleavage period of Ascaris suum , chromatin diminution takes place in the somatic founder cells. In the process of chromatin diminution numerous heterochromatic blocks, consisting predominantly of highly repeated DNA, are discarded during mitotic anaphase and are later on digested in the cytoplasm. Very little is known about proteins that are involved in chromatin diminution. We have detected a nuclear protein and purified it to near homogeneity by its preferential binding to UV-damaged DNA. We termed this protein chromatin diminution associated factor 1 (CDAF1), because maximum binding activity per nucleus was observed to develop in 4-8-cell stages, when chromatin diminution occurs for the first time. CDAF1 recognizes cyclobutane pyrimidine dimers in UV-damaged double-stranded DNA. Its binding properties identify CDAF1 as a novel kind of damaged-DNA binding protein. CDAF1 activity is almost not detectable in 1-celled embryos. It increases dramatically during formation of somatic founder cells and persists up to the first larval stage. However, CDAF1 is absent in tissues of adults. These findings led us to suggest that CDAF1 plays a dual role: during the early segregative cleavage period it might be involved in chromatin diminution as a transfactor and act in nucleotide excision repair as an accessory factor throughout embryogenesis.     

aniFOUND: analysing the associated proteome and genomic landscape of the repaired nascent non-replicative chromatin

Specific capture of chromatin fractions with distinct and well-defined features has emerged as both challenging and a key strategy towards a comprehensive understanding of genome biology. In this context, we developed aniFOUND (accelerated native isolation of factors on unscheduled nascent DNA), an antibody-free method, which can label, capture, map and characterise nascent chromatin fragments that are synthesized in response to specific cues outside S-phase. We used the ‘unscheduled’ DNA synthesis (UDS) that takes place during the repair of UV-induced DNA lesions and coupled the captured chromatin to high-throughput analytical technologies. By mass-spectrometry we identified several factors with no previously known role in UVC-DNA damage response (DDR) as well as known DDR proteins. We experimentally validated the repair-dependent recruitment of the chromatin remodeller RSF1 and the cohesin-loader NIPBL at sites of UVC-induced photolesions. Developing aniFOUND-seq, a protocol for mapping UDS activity with high resolution, allowed us to monitor the landscape of UVC repair-synthesis events genome wide. We further resolved repair efficacy of the rather unexplored repeated genome, in particular rDNA and telomeres. In summary, aniFOUND delineates the proteome composition and genomic landscape of chromatin loci with specific features by integrating state-of-the-art ‘omics’ technologies to promote a comprehensive view of their function.     

Heterochromatin endoreduplication prior to gametogenesis and chromatin diminution during early embryogenesis in Mesocyclops edax (Copepoda: Crustacea)

The segregation of progenitor somatic cells from those of the primordial germ cells that sequester and retain elevated levels of DNA during subsequent developmental events, poses an interesting, alternative pathway of chromosome behavior during the reproductive cycle of certain species of cyclopoid copepods and several other organisms. Separation of maternal and paternal chromosome sets during very early cleavages (gonomery) is often a feature following marked elevations of DNA levels in germ cells for some of these species. Here, we report on the accumulation of large amounts of DNA in germ line nuclei of both female and male juveniles and adults of a freshwater copepod, Mesocyclops edax (Forbes, 1890). We also report the robust uptake of 3H-thymidine by germ cells prior to gametogenesis in this species. By using cytophotometric analysis of the DNA levels in both germ line cells and somatic cells from the same specimens we demonstrate that germ cell nuclei accumulate high levels of DNA prior to the onset of gametogenesis. These elevated amounts coincide with the levels of heterochromatic DNA discarded during chromatin diminution. A new model is proposed of major cytological events accompanying the process of chromatin diminution in M. edax.     

Some conclusions on the role of redundant DNA and the mechanisms of eukaryotic genome evolution inferred from studies of chromatin diminution in Cyclopoida

The absence of progress in understanding the problem of redundant eukaryotic DNA is stated. This is caused primarily by the attempts to solve this problem either in terms of the traditional approaches (the general phenotypic parameters such as developmental rate, body size, etc. depend on the genome size) or by introducing such vague terms as egoistic, parasitic, or junk DNA. Studying chromatin diminution (CD) in copepods yielded two important conclusions. First, part of the genome of a certain size (94% in Cyclops kolensis first described by the authors) is not needed for somatic functions as it is eliminated during the early (third to seventh) cleavage divisions from the presumptive somatic cells. Second, this DNA is not redundant, let alone selfish or junk, relative to the germline cells. In this sense, it can be regarded as invariant (monomorphic) trait that characterizes the species. Analysis of cloned and sequenced DNA regions eliminated from the somatic cell genome by CD (i.e., confined to the germline), which was first carried out for C. kolensis, showed that the molecular structure of this DNA has at least two features of regular organization: a mosaic structure of repetitive sequences and high (sometimes up to 100%) homology between different repeats and subrepeats. We have suggested that the germline-restricted DNA forms a unique molecular portrait of the species genome, thus acting as a significant factor of genetic isolation. Yet, the phenomenon of CD proper as it occurs in Cyclopoida without disintegration of the chromosome structure) may be regarded as a model of reductional genome evolution, which has repeatedly occurred in the history of eukaryotes.     

Evidence for endoreduplication: germ cell DNA levels prior to chromatin diminution in Mesocyclops edax.

We studied the functional significance of marked differences in the DNA content of somatic cells and germ line nuclei by static Feulgen-DNA cytophotometry for several species of microcrustaceans that exhibit chromatin diminution during very early stages of embryogenesis. Mature females and males showed many gonadal nuclei with elevated amounts of DNA that persist until dispersal of this "extra" DNA throughout the cytoplasm as fragments and coalescing droplets of chromatin during anaphase of the diminution division.     

Chromatin diminution in the parasitic nematodes ascaris suum and parascaris univalens

Chromatin diminution in Parascaris univalens and Ascaris suum undoubtedly represents an interesting case of developmentally programmed DNA rearrangement in higher eukaryotes. It is a complex mechanism involving chromosomal breakage, new telomere addition and DNA degradation, and occurs in all presomatic cells. The process is rather specific with respect to its developmental timing and the chromosomal regions that are eliminated. The functional significance of chromatin diminution still remains an enigma. The fact, however, that single-copy, protein-coding genes are contained in the eliminated DNA demonstrates that in P. univalens and A. suum, there is a qualitative difference between germ-line and somatic genomes, and suggests that chromatin diminution may be used as a "throw-away" approach to gene regulation. We present a hypothesis as to how, during evolution, a partial genome duplication might have been linked to the process of chromatin diminution, in order to provide a selective advantage to parasitic DNA-eliminating nematodes.     

Neither a germ line-specific nor several somatically expressed genes are lost or rearranged during embryonic chromatin diminution in the nematode Ascaris lumbricoides var. suum

Ascaris lumbricoides var. suum is a parasitic nematode of pigs. Its embryos undergo chromatin diminution between the third and fifth cleavages, resulting in the loss of about 30% of the DNA from all somatic precursor cells while the germ line DNA stays intact. Most of the eliminated DNA has been shown to be satellite sequences. Theodor Boveri [(1910) In "Festschrift fur R. Hertwig, III," Vol. 3, pp. 131-214, Fischer] proposed that functions essential only to the germ line might be lost from the soma. We have examined this proposal by cloning a gene encoding the major sperm protein (MSP) using a cloned MSP gene from Caenorhabditis elegans as a probe. The MSP appears to be expressed only in the testis of Ascaris, as it is in Caenorhabditis. Actin and alpha tubulin were also cloned to serve as somatically expressed gene controls. By probing Southern blots of somatic and germ line DNA with these cloned genes, it was found that none of them was lost or rearranged during chromatin diminution. Thus at least one germ line-specific gene is neither lost nor rearranged during chromatin diminution. We also found that the two nematode species differ widely in their numbers of both MSP and actin genes. Caenorhabditis has greater than 30 MSP genes, but Ascaris has no more than three; whereas Ascaris has many more actin genes than Caenorhabditis.     

The regulatory content of intergenic DNA shapes genome architecture

Background  

Factors affecting the organization and spacing of functionally unrelated genes in metazoan genomes are not well understood. Because of the vast size of a typical metazoan genome compared to known regulatory and protein-coding regions, functional DNA is generally considered to have a negligible impact on gene spacing and genome organization. In particular, it has been impossible to estimate the global impact, if any, of regulatory elements on genome architecture.     

New telomere formation after developmentally regulated chromosomal breakage during the process of chromatin diminution in Ascaris lumbricoides

During the process of chromatin diminution, which takes place in all presomatic cells of the early Ascaris embryo, the heterochromatic termini of the chromosomes are lost. Here we show that the newly formed ends of the reduced somatic chromosomes carry tandem repeats of the telomeric sequence TTAGGC. Comparison of a cloned somatic telomere with the corresponding germline chromosomal region revealed that these telomeric repeats are not present at or near the chromosomal breakage site. They are most likely added by a telomerase-mediated event. Chromosomal breakage, which precedes the telomere addition process, takes place within a short, specific chromosomal region (CBR); however, it does not occur at a single locus, but rather at many different sites. Altogether, our data show that chromatin diminution in Ascaris is a complex molecular process that includes site-specific chromosomal breakage, new telomere formation, and DNA degradation.     

Amitotic division of the macronucleus in Tetrahymena thermophila: DNA distribution by genomic unit

The macronucleus of the ciliate Tetrahymena cell contains euchromatin and numerous heterochromatins called chromatin bodies. During cell division, a chromatin aggregate larger than chromatin body appears in the macronucleus. We observed chromatin aggregates in the dividing macronucleus in a living T. thermophila cell, and found that these were globular in morphology and homogeneous in size. To observe globular chromatin clearly, optimal conditions for making it compact were studied. Addition of Mg ion, benomyl and oryzalin, microtubule inhibitors, to cell suspension was effective. Globular chromatin appeared when the micronuclear anaphase began at the cell cortex, and disappeared long after cell separation. Using living cells with a small macronucleus at early log phase, we counted the number of globular chromatin per nucleus and measured the DNA content of globular chromatin in the macronucleus which was stained with Hoechst 33342 by using ImageJ. The number of globular chromatin per nucleus was reduced by half after division, indicating the globular chromatin is a distribution unit of DNA. A globular chromatin contained similar DNA content as that of the macronuclear genome. We developed methods for inducing and isolating a cell with an extremely small macronucleus with a DNA amount of one globular chromatin. These cells grew, divided, and give clones, suggesting that the macronuclear genome is not dispersed within the macronucleus and the globular chromatin may be a macronuclear genome. We named this globular chromatin "macronuclear genome unit" (MGU).