The impact of genetic background and cell lineage on the level and pattern of gene expression in position effect variegation

Background Chromatin-based transcriptional silencing is often described as a stochastic process, largely because of the mosaic expression observed in position effect variegation (PEV), where a euchromatic reporter gene is silenced in some cells as a consequence of juxtaposition with heterochromatin. High levels of variation in PEV phenotypes are commonly observed in reporter stocks. To ascertain whether background mutations are the major contributors to this variation, we asked how much of the variation is determined by genetic variants segregating in the population, examining both the level and pattern of expression using the fruit fly, Drosophila melanogaster, as the model. Results Using selective breeding of a fourth chromosome PEV reporter line, 39C-12, we isolated two inbred lines exhibiting contrasting degrees of variegation (A1: low expression, D1: high expression). Within each inbred population, remarkable similarity is observed in the degree of variegation: 90% of the variation between the two inbred lines in the degree of silencing can be explained by genotype. Further analyses suggest that this result reflects the combined effect of multiple independent trans-acting loci. While the initial observations are based on a PEV phenotype scored in the fly eye (hsp70-white reporter), similar degrees of silencing were observed using a beta-gal reporter scored across the whole fly. Further, the pattern of variegation becomes almost identical within each inbred line; significant pigment enrichment in the same quadrant of the eye was found for both A1 and D1 lines despite different degrees of expression. Conclusions The results indicate that background genetic variants play the major role in determining the variable degrees of PEV commonly observed in laboratory stocks. Interestingly, not only does the degree of variegation become consistent in inbred lines, the patterns of variegation also appear similar. Combining these observations with the spreading model for local heterochromatin formation, we propose an augmented stochastic model to describe PEV in which the genetic background drives the overall level of silencing, working with the cell lineage-specific regulatory environment to determine the on/off probability at the reporter locus in each cell. This model acknowledges cell type-specific events in the context of broader genetic impacts on heterochromatin formation.

The Impact of Genetic Background and Cell Lineage on the Level and Pattern of Position Effect Variegation

BackgroundChromatin-based transcriptional silencing is often described as a stochastic process, largely because of the mosaic expression observed in position effect variegation (PEV), where a euchromatic reporter gene is juxtaposed with heterochromatin. Here we closely examine the impact of genetic background on PEV phenotypes in the fruit fly, Drosophila melanogaster.ResultsUsing consecutive generations of selective breeding, we isolated, from a single laboratory population, two inbred lines exhibiting contrasting degrees of variegation (A1: low expression, D1: high expression). Within each inbred population, remarkable similarity is observed in both the degree and the pattern of variegation. 89.63% of the differences between the two inbred lines in the degree of silencing can be explained by genotype, while a modest but significant sex effect is also observed. Further analyses of the PEV phenotype in the progeny of crosses between A1 and D1 suggest that the genotypic effect is the result of the combined effect of multiple independent trans-acting loci. While the initial observations are based on a PEV phenotype scored in the fly eye (hsp70-white reporter), similar degrees of silencing were observed using a beta-gal reporter that can be scored across the whole fly. The pattern of variegating hsp70-white expression among individual flies becomes almost identical after five generations of inbreeding. Using a reporter inserted into the heterochromatic fourth chromosome, image analysis found significant enrichment of pigmentation in the ventral-posterior quadrant in both the A1 and D1 lines, and in the F1 and F2 progeny produced from a cross between A1 and D1, despite different degrees of expression.ConclusionsCombining these results with the spreading model for local heterochromatin formation, we propose an augmented stochastic model to describe PEV. In this model, the genetic background, which determines the overall level of silencing, works with the cell lineage specific regulatory environment to determine the on/ off probability at the reporter locus in each cell. This model acknowledges cell-type specific events, as well as the general impact of heterochromatin formation.

The Crk adapter protein is essential for Drosophila embryogenesis, where it regulates multiple actin-dependent morphogenic events

Small Src homology domain 2 (SH2) and 3 (SH3) adapter proteins regulate cell fate and behavior by mediating interactions between cell surface receptors and downstream signaling effectors in many signal transduction pathways. The CT10 regulator of kinase (Crk) family has tissue-specific roles in phagocytosis, cell migration, and neuronal development and mediates oncogenic signaling in pathways like that of Abelson kinase. However, redundancy among the two mammalian family members and the position of the Drosophila gene on the fourth chromosome precluded assessment of Crk’s full role in embryogenesis. We circumvented these limitations with short hairpin RNA and CRISPR technology to assess Crk’s function in Drosophila morphogenesis. We found that Crk is essential beginning in the first few hours of development, where it ensures accurate mitosis by regulating orchestrated dynamics of the actin cytoskeleton to keep mitotic spindles in syncytial embryos from colliding. In this role, it positively regulates cortical localization of the actin-related protein 2/3 complex (Arp2/3), its regulator suppressor of cAMP receptor (SCAR), and filamentous actin to actin caps and pseudocleavage furrows. Crk loss leads to the loss of nuclei and formation of multinucleate cells. We also found roles for Crk in embryonic wound healing and in axon patterning in the nervous system, where it localizes to the axons and midline glia. Thus, Crk regulates diverse events in embryogenesis that require orchestrated cytoskeletal dynamics.

The Crk adapter protein is essential for Drosophila embryogenesis, where it regulates multiple actin-dependent morphogenic events

Small SH2/SH3 adapter proteins regulate cell fate and behavior by mediating interactions between cell surface receptors and downstream signaling effectors in many signal transduction pathways. The Crk family has tissue-specific roles in phagocytosis, cell migration and neuronal development, and mediates oncogenic signaling in pathways like that of Abelson kinase. However, redundancy among the two mammalian family members and the position of the Drosophila gene on the fourth chromosome precluded assessment of Crk’s full role in embryogenesis. We circumvented these limitations with shRNA and CRISPR technology to assess Crk’s function in Drosophila morphogenesis. We found Crk is essential beginning in the first few hours of development, where it ensures accurate mitosis by regulating orchestrated dynamics of the actin cytoskeleton to keep mitotic spindles in syncytial embryos from colliding. In this role, it positively regulates levels of the Arp2/3 complex, its regulator SCAR, and F-actin in actin caps and pseudocleavage furrows. Crk loss leads to loss of nuclei and formation of multinucleate cells. We also found roles for Crk in embryonic wound healing and in axon patterning in the nervous system, where it localizes to the axons and midline glia. Thus, Crk regulates diverse events in embryogenesis that require orchestrated cytoskeletal dynamics.

Polytene chromosomes reflect functional organization of the Drosophila genome

Polytene chromosomes of Drosophila melanogaster are a convenient model for studying interphase chromosomes of eukaryotes. They are giant in size in comparison with diploid cell chromosomes and have a pattern of cross stripes resulting from the ordered chromatid arrangement. Each region of polytene chromosomes has a unique banding pattern. Using the model of four chromatin types that reveals domains of varying compaction degrees, we were able to correlate the physical and cytological maps of some polytene chromosome regions and to show the main properties of genetic and molecular organization of bands and interbands, that we describe in this review. On the molecular map of the genome, the interbands correspond to decompacted aquamarine chromatin and 5’ ends of ubiquitously active genes. Gray bands contain lazurite and malachite chromatin, intermediate in the level of compaction, and, mainly, coding parts of genes. Dense black transcriptionally inactive bands are enriched in ruby chromatin. Localization of several dozens of interbands on the genome molecular map allowed us to study in detail their architecture according to the data of whole genome projects. The distribution of proteins and regulatory elements of the genome in the promoter regions of genes localized in the interbands shows that these parts of interbands are probably responsible for the formation of open chromatin that is visualized in polytene chromosomes as interbands. Thus, the permanent genetic activity of interbands and gray bands and the inactivity of genes in black bands are the basis of the universal banding pattern in the chromosomes of all Drosophila tissues. The smallest fourth chromosome of Drosophila with an atypical protein composition of chromatin is a special case. Using the model of four chromatin states and fluorescent in situ hybridization, its cytological map was refined and the genomic coordinates of all bands and interbands were determined. It was shown that, in spite of the peculiarities of this chromosome, its band organization in general corresponds to the rest of the genome. Extremely long genes of different Drosophila chromosomes do not fit the common scheme, since they can occupy a series of alternating bands and interbands (up to nine chromosomal structures) formed by parts of these genes.

Genomics of expanded avian sex chromosomes shows that certain chromosomes are predisposed towards sex-linkage in vertebrates

ABSTRACTSex chromosomes have evolved from the same autosomes multiple times across vertebrates, suggesting that certain genomic regions are predisposed towards sex-linkage. However, to test this hypothesis detailed studies of independently originated sex-linked regions and their gene content are needed. Here we address this problem through comparative genomics of birds where multiple chromosomes appear to have formed neo-sex chromosomes: larks (Alaudidae; Sylvioidea). We detected the largest known avian sex chromosome (195.3 Mbp) and show that it originates from fusions between (parts of) four avian chromosomes (Z, 3, 4A and 5). We found evidence of five evolutionary strata where recombination has been suppressed at different time points, and that these time points correlate with the level of Z–W gametolog differentiation. We show that there is extensive homology to sex chromosomes in other vertebrate lineages: three of the fused chromosomes (Z, 4A, 5) have independently evolved into sex chromosomes in fish (Z), turtles (Z, 5), lizards (Z, 4A) and mammals (Z, 4A). Moreover, we found that the fourth chromosome, chromosome 3, was significantly enriched for genes with predicted sex-specific functions. These results support a key role of chromosome content in the evolution of sex chromosomes in vertebrates.

Association Between Chromosome 4Q25 Polymorphism RS2200733 and the Incidence of Atrial Fibrillation in Bulgarian Patients

Summary Atrial fibrillation (AF) is the commonest type of arrhythmia seen in everyday clinical practice, which leads to a significant increase in both morbidity and mortality. Its incidence increases with age and tends to turn into an epidemic. The cause of AF in 10-20% of cases remains unknown. Several mutations and polymorphism that might be responsible for the development of AF have been found, including single nucleotide polymorphisms (SNPs) - rs2200733 and rs10033464 in the long arm of the fourth chromosome. These polymorphisms are selected o the basis of genome- wide association study in Iceland from 2007, the results from which were later confirmed in 4 other large populations. The rs2200733 is a common noncoding polymorphism, described in National Center for Biotechnology Information (NCBI) database dbSNP like NC_000004.12:g.110789013C>T, with a frequency of the less common allele between 0.1 and 0.24. In order to investigate the association between the rs2200733 polymorphism in chromosome 4q25 and the development of AF, we studied the frequency of this polymorphism in patients with heart diseases from the Pleven region, and thus evaluate the relationship between the individual genotype and the clinical condition of the patients. We carried out a case-control study on 80 patients: 40 with AF and 40 without AF- from the Pleven region. None of these had structural heart disease. The study was conducted between November 2015 and November 2017. With deoxyribonucleic acid (DNA) analysis, we determined rs2200733 polymorphism, using a TaqMan-based polymerase chain reaction (PCR). The Cochran-Armitage trend test, the Chi-Squared Pearson correlation, Fisher test we used confirmed the statistically significant association between the rs2200733 polymorphism in chromosome 4q25 and the development of AF. In the population examined, the genotypic frequencies were as follows: CC - 45 (56.2%), CT - 19 (23.8%), TT - 16 (20%), with value of Chi-Square (χ2) 24.496, df=2, p<0.001. Screening for SNPs could be a useful marker for the detection of patients predisposed to AF.

Cytogenetic studies in Trachymyrmex holmgreni Wheeler, 1925 (Formicidae: Myrmicinae) by conventional and molecular methods

Over the past several decades, ant cytogenetic studies have focused on chromosome number and morphology; however, recently, additional information concerning heterochromatin composition and 45S rDNA location has become accessible. The fungus-growing ants are a peculiar ant group that cultivates fungus for food, and Trachymyrmex is suspected to be the sister group of leafcutter ants. Cytogenetic data are so far available for sixn Trachymyrmex species. The present study aimed to increase the knowledge about Trachymyrmex cytogenetics by the chromosomal characterization of Trachymyrmex holmgreni including the karyotyping, fluorochromes staining, 18S rDNA, and microsatellite (GA)15 fluorescence in situ hybridization (FISH). Karyotyped samples from four ant colonies showed 2n = 20 metacentric chromosomes. Centromeric heterochromatin rich in GC base pairs was detected in all chromosomes. FISH revealed the presence of rDNA clusters on the fourth chromosome pair, and an intense spreading of the microsatellite (GA)15 including exclusively euchromatic areas of the chromosomes. The GC-rich heterochromatin observed in different ant species may have a common origin and, thus, phylogenetic implication that needs to be further investigated. To the best of our knowledge, this study is the first report of the use of chromosomal physical location of repetitive DNA sequences by means of microsatellite probes in Formicidae.

Leptin Gene as Potential Gene for Molecular Selection on Cattle in Indonesia

<p>Genetic improvement in beef and dairy cattle which can increase meat and milk production can be conducted through livestock selection based on molecular technique using leptin gene. The leptin gene is located on the fourth chromosome and consists of three exons and two introns. The leptin gene serves to produce the leptin hormone secreted by fat tissue. High concentrations of leptin hormone can increase feed intake and body metabolism thereby increasing livestock productivity. This paper describes the leptin gene including structure, mechanism of leptin hormone and polymorphisms in cows. These polymorphisms occur in the promoter, intron and exon sections associated with body and carcass weight and milk production in some cattle breed. Therefore, leptin gene can be used for selection towards increasing cattle productivity. Utilization of leptin gene can be done by single nucleotide polymorphism (SNP) identification on the whole structure of leptin gene and to observe its effect on characteristics of cattle production in Indonesia.</p>

A versatile two-step CRISPR- and RMCE-based strategy for efficient genome engineering in Drosophila

The development of CRISPR/Cas9 technologies promises a quantum leap in genome-engineering of model organisms. However, CRISPR-mediated gene targeting reports in Drosophila are still restricted to a few genes, use variable experimental conditions and vary in efficiency, questioning the universal applicability of the method. Here, we developed an efficient, two-step strategy to flexibly engineer the fly genome by combining CRISPR with recombinase-mediated cassette exchange (RMCE). In the first step, two sgRNAs, whose activity had been tested in cell culture, were co-injected together with a donor plasmid into transgenic Act5C-Cas9, Ligase4 mutant embryos and the homologous integration events were identified by eye fluorescence. In the second step, the eye marker was replaced with DNA sequences of choice using RMCE enabling flexible gene modification. We applied this strategy to engineer four different loci, including a gene on the fourth chromosome, at comparably high efficiencies, suggesting that any fly lab can engineer their favourite gene for a broad range of applications within about three months.