Silvia Bongiorni

Heterochromatin, HP1 and methylation at lysine 9 of histone H3 in animals

Abstract. We show that methylated lysine 9 of histone H3 (Me9H3) is a marker of heterochromatin in divergent animal species. It localises to both constitutive and facultative heterochromatin and replicates late in S-phase of the cell cycle. Significantly, Me9H3 is enriched in the inactive mammalian X chromosome (Xi) in female cells, as well as in the XY body during meiosis in the male, and forms a G-band pattern along the arms of the autosomes. Me9H3 is a constituent of imprinted chromosomes that are repressed. The paternal and maternal pronuclei in one-cell mouse embryos show a striking non-equivalence in Me9H3: the paternal pronucleus contains no immunocytologically detectable Me9H3. The levels of Me9H3 on the parental chromosomes only become equivalent after the two-cell stage. Finally, we provide evidence that Me9H3 is neither necessary nor sufficient for localisation of heterochromatin protein 1 (HP1) to chromosomal DNA.

Inverted meiosis and meiotic drive in mealybugs

In the males of lecanoid coccids, or mealybugs, an entire, paternally derived, haploid chromosome set becomes heterochromatic after the seventh embryonic mitotic cycle. In females, both haploid sets are euchromatic throughout the life cycle. In mealybugs, as in all homopteran species, chromosomes are holocentric. Holocentric chromosomes are characterized by the lack of a localized centromere and consequently of a localized kinetic activity. In monocentric species, sister chromatid cohesion and monopolar attachment play a pivotal role in regulating chromosome behavior during the two meiotic divisions. Both these processes rely upon the presence of a single, localized centromere and as such cannot be properly executed by holocentric chromosomes. Here we furnish further evidence that meiosis is inverted in both sexes of mealybugs and we suggest how this might represent an adaptation to chromosome holocentrism. Moreover, we reveal that at the second meiotic division in males a monopolar spindle is formed, to which only euchromatic chromosomes become attached. By this mechanism the paternally derived, heterochromatic, haploid chromosome set strictly segregates from the euchromatic one, and it is then excluded from the genetic continuum as a result of meiotic drive.

Imprinted Facultative Heterochromatization in Mealybugs

In lecanoid Coccids, or mealybugs, the male development is accompanied by the facultative heterochromatization of the entire, paternally derived, haploid chromosome set. This epigenetic phenomenon occurs in all the cells of mid-cleavage male embryos. Consequently, the Coccid chromosome system offers a powerful tool for gaining insights into the structure of facultative heterochromatin, and into the epigenetic mechanisms of its imprinted, developmentally regulated formation. This paper will present new data and summarize recent studies on genomic imprinting and facultative heterochromatization in mealybugs. First, the existence and the possible role of DNA methylation as an epigenetic modification that fulfills the requisites of the imprinting process in mealybugs will be considered. The second part of this paper will focus on proteins involved in the facultative heterochromatization process. In particular, the involvement of an HP-1-like protein in the silencing of the paternally derived haploid chromosome set and its interaction with the lysine 9 methylated isoform of histone H3 will be discussed.

Microarrays and high-throughput transcriptomic analysis in species with incomplete availability of genomic sequences

Microarrays produce a measurement of gene expression based on the relative measures of dye intensities that correspond to the amount of target RNA. This technology is fast developing and its application is expanding from Homo sapiens to a wide number of species, where enough information on sequences and annotations exist. Anyway, the number of species for which a dedicated platform exists is not high. The use of heterologous array hybridization, screening for gene expression in one species using an array developed for another one, is still quite frequent, even though cross-species microarray hybridization has raised many arguments. Some methods which are high throughput and do not rely on knowledge of the DNA/RNA sequence exist, namely serial analysis of gene expression (SAGE), Massively Parallel Signature Sequencing (MPSS) and deep sequencing of full transcriptome. Although very powerful, particularly the latter, they are still quite costly and cumbersome methods. In some species where genome sequences are largely unknown, several anonymous sequences are deposited in gene banks as a result of Expressed Sequence Tags (ESTs) sequencing projects. The ESTs databases represent a valuable knowledge that can be exploited with some bioinformatic effort to build species-specific microarrays. We present here a method of high-density in situ synthesized microarrays starting from available EST sequences in, Ovis aries. Our data indicate that the method is very efficient and can be easily extended to other species of which genetic sequences are present in public databases, but neglected so far with advanced devices like microarrays. As a perspective, the approach can be applied also to species of which no sequences are available to date, thanks to high-throughput deep sequencing methods.

Identification of a Short Region on Chromosome 6 Affecting Direct Calving Ease in Piedmontese Cattle Breed

Calving in cattle is affected by calf morphology and by dam characteristics. It is described by two different traits: maternal calving ease, which is the ability to generate dams with good physiological predisposition to calving, and direct calving ease, which is the ability to generate calves that are easily born. The aim of this study was to identify regions of cattle genome harboring genes possibly affecting direct calving ease in the Piedmontese cattle breed. A population of 323 bulls scored for direct calving ease (EBV) was analyzed by a medium-density SNP marker panel (54,001 SNPs) to perform a genome-wide scan. The strongest signal was detected on chromosome 6 between 37.8 and 38.7 Mb where 13 SNPs associated to direct calving ease were found. Three genes are located in this region: LAP3, encoding for a leucine aminopeptidase involved in the oxytocin hydrolysis; NCAPG, encoding for a non-SMC condensin I complex, which has been associated in cattle with fetal growth and carcass size; and LCORL, which has been associated to height in humans and cattle. To further confirm the results of the genome-wide scan we genotyped additional SNPs within these genes and analyzed their association with direct calving ease. The results of this additional analysis fully confirmed the findings of the GWAS and particularly indicated LAP3 as the most probable gene involved. Linkage Disequilibrium (LD) analysis showed high correlation between SNPs located within LAP3 and LCORL indicating a possible selection signature due either to increased fitness or breeders’ selection for the trait.

Comparison of Milk Fat Globule Membrane (MFGM) Proteins of Chianina and Holstein Cattle Breed Milk Samples Through Proteomics Methods

Identification of proteins involved in milk production is important to understand the biology of lactation. Many studies have advanced the understanding of mammary function and milk secretion, but the critical molecular mechanisms implicated in milk fat secretion is still incomplete. Milk Fat Globules are secreted from the apical surface of the mammary cells, surrounded by a thin membrane bilayer, the Milk Fat Globule Membrane (MFGM), formed by proteins which have been suggested to be cholesterolemia-lowering factors, inhibitors of cancer cell growth, vitamin binders, bactericidal, suppressors of multiple sclerosis. Using a proteomic approach, we compared MFGM from milk samples of individuals belonging to two different cattle breeds, Chianina and Holstein, representative of selection for milk and meat traits, respectively. We were able to isolate some of the major MFGM proteins in the examined samples and to identify differences between the protein fractions of the two breeds. We detected differences in the amount of proteins linked to mammary gland development and lipid droplets formation, as well as host defence mechanisms. We have shown that proteomics is a suitable, unbiased method for the study of milk fractions proteins and a powerful tool in nutritional genomics.

Epigenetic marks for chromosome imprinting during spermatogenesis in coccids

The establishment of sex-specific epigenetic marks during gametogenesis is one of the key feature of genomic imprinting. By immunocytological analysis, we thoroughly characterized the chromatin remodeling events that take place during gametogenesis in the mealybug Planococcus citri, in which an entire haploid set of (imprinted) chromosomes undergoes facultative heterochromatinization in male embryos. Building on our previous work, we have investigated the interplay of several epigenetic marks in the regulation of this genome-wide phenomenon. We characterized the germline patterns of histone modifications, Me(3)K9H3, Me(2)K9H3, and Me(3)K20H4, and of heterochromatic proteins, PCHET2 (HP1-like) and HP2-like during male and female gametogenesis. We found that at all stages in oogenesis chromatin is devoid of any detectable epigenetic marks. On the other hand, spermatogenesis is accompanied by a complex pattern of redistribution of epigenetic marks from euchromatin to heterochromatin, and vice versa. At the end of spermatogenesis, sperm heads are decorated by all the molecules we tested, except for PCHET2. However, only Me(3)K9H3 and Me(2)K9H3 are still detectable in the male pronucleus. We suggest that the histone H3 lysine 9 methylation is the signal used to establish the male-specific imprinting on the paternal genome, thus allowing it to be distinguished from the maternal genome in the developing embryo.

Drosophila rae1 is required for male meiosis and spermatogenesis

Summary Previous studies of RAE1, a conserved WD40 protein, in Schizosaccharomyces pombe and mouse revealed a role in mRNA export and cell cycle progression in mitotic cells. Studies of RAE1 in Drosophila showed that the protein localizes to the nuclear envelope and is required for progression through the G1 phase of the cell cycle but not RNA export in tissue culture cells. Drosophila RAE1 also plays an essential developmental role, as it is required for viability and synaptic growth regulation as a component of an E3 ubiquitin ligase complex. Here we describe characterization of a new Drosophila rae1 mutant that is viable but results in male sterility. The mutant showed striking defects in primary spermatocyte nuclear integrity, meiotic chromosome condensation, segregation, and spindle morphology. These defects led to a failure to complete meiosis but allowed several aspects of spermatid differentiation to proceed, including axoneme formation and elongation. A GFP–RAE1 fusion protein that rescued most of the cytological defects showed a dynamic localization to the nuclear envelope, chromatin and other structures depending on the stage of spermatogenesis. A role for RAE1 in male meiosis, as well as mitotic cells, was also indicated by the defects induced by expression of rae1-RNAi. These studies in Drosophila provide the first evidence for an essential meiotic role of RAE1, and thus define RAE1 as a protein required for both meiotic and mitotic cell cycles.

HP2-like protein: a new piece of the facultative heterochromatin puzzle

In Drosophila melanogaster, the two chromosomal proteins HP1 and HP2 colocalize on heterochromatic and euchromatic sites in polytene chromosomes. Mutations in the HP2 gene act as dominant suppressors of position effect variegation, demonstrating a role for HP2 in the formation or maintenance of heterochromatin. In this paper, we investigated whether a putative homolog of the D. melanogaster HP2 is involved in the facultative heterochromatinization process in mealybugs. Using an antibody raised against the Drosophila HP2, we identified in the mealybug Planococcus citri a cross-reactive epitope, which we refer to as HP2-like. We investigated the HP2-like pattern during the male embryo development where the entire paternal haploid chromosome set becomes heterochromatic. The HP2 antibody heavily decorates the chromocenters, where it localizes with HP1, and marks the chromatin before it acquires the full cytological characteristics of the male-specific heterochromatin. In euchromatic chromosomes, HP2-like is mainly concentrated at telomeric sites. The interplay between HP2-like and HP1-like was studied by dsRNA interference experiments. Extinguishing HP1-like expression by RNAi does not prevent the association of HP2-like with facultative heterochromatin, implying that HP2-like binds to chromatin in a HP1-independent manner. Our results confirm and extend the structural and functional conservation of proteins involved in heterochromatin assembly.

Cytogenetic characterization of the genome of mealybug Planococcus citri (Homoptera, Coccoidea)

Summary Mealybugs although being agriculturally harmful insects have been very poorly studied by modern cytogenetics techniques, and no cytotaxonomic criteria to distinguish between closely related species is available yet. In the mealybug Planococcus citri (2n=10) male and female individuals are both diploid, however in males, at the stage of blastula, the haploid chromosome set of paternal origin becomes heterochromatic, even though its complete inertia has been considered questionable. Here we present data on the cytogenetic characterization of the chromosomes of Planococcus citri. We report on (i) the fluorescence karyotype (D287/170), which to our knowledge is the first banded karyotype of a mealybug to be described; (ii) the chromosome localization of constitutive heterochromatin; (iii) the chromosome localization of rDNA sites; (iv) NORs activity. Our data also show, for the first time, that in the heterochromatic chromosome set ribosomal genes are still active.