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Genes Mar 2020Heterochromatin is identified as a potential factor driving diversification of species. To understand the magnitude of heterochromatin variation within the complex of...
Heterochromatin is identified as a potential factor driving diversification of species. To understand the magnitude of heterochromatin variation within the complex of malaria mosquitoes, we analyzed metaphase chromosomes in , , , , and . Using fluorescence hybridization (FISH) with ribosomal DNA (rDNA), a highly repetitive fraction of DNA, and heterochromatic Bacterial Artificial Chromosome (BAC) clones, we established the correspondence of pericentric heterochromatin between the metaphase and polytene X chromosomes of . We then developed chromosome idiograms and demonstrated that the X chromosomes exhibit qualitative differences in their pattern of heterochromatic bands and position of satellite DNA (satDNA) repeats among the sibling species with postzygotic isolation, , , , and or . The identified differences in the size and structure of the X chromosome heterochromatin point to a possible role of repetitive DNA in speciation of mosquitoes. We found that and , incipient species with prezygotic isolation, share variations in the relative positions of the satDNA repeats and the proximal heterochromatin band on the X chromosomes. This previously unknown genetic polymorphism in malaria mosquitoes may be caused by a differential amplification of DNA repeats or an inversion in the sex chromosome heterochromatin.
Topics: Animals; Anopheles; DNA, Satellite; Genomic Structural Variation; Heterochromatin; Polytene Chromosomes; X Chromosome
PubMed: 32204543
DOI: 10.3390/genes11030327 -
BMC Biology Mar 2020Aedes aegypti is the principal mosquito vector of Zika, dengue, and yellow fever viruses. Two subspecies of Ae. aegypti exhibit phenotypic divergence with regard to...
BACKGROUND
Aedes aegypti is the principal mosquito vector of Zika, dengue, and yellow fever viruses. Two subspecies of Ae. aegypti exhibit phenotypic divergence with regard to habitat, host preference, and vectorial capacity. Chromosomal inversions have been shown to play a major role in adaptation and speciation in dipteran insects and would be of great utility for studies of Ae. aegypti. However, the large and highly repetitive genome of Ae. aegypti makes it difficult to detect inversions with paired-end short-read sequencing data, and polytene chromosome analysis does not provide sufficient resolution to detect chromosome banding patterns indicative of inversions.
RESULTS
To characterize chromosomal diversity in this species, we have carried out deep Illumina sequencing of linked-read (10X Genomics) libraries in order to discover inversion loci as well as SNPs. We analyzed individuals from colonies representing the geographic limits of each subspecies, one contact zone between subspecies, and a closely related sister species. Despite genome-wide SNP divergence and abundant microinversions, we do not find any inversions occurring as fixed differences between subspecies. Many microinversions are found in regions that have introgressed and have captured genes that could impact behavior, such as a cluster of odorant-binding proteins that may play a role in host feeding preference.
CONCLUSIONS
Our study shows that inversions are abundant and widely shared among subspecies of Aedes aegypti and that introgression has occurred in regions of secondary contact. This library of 32 novel chromosomal inversions demonstrates the capacity for linked-read sequencing to identify previously intractable genomic rearrangements and provides a foundation for future population genetics studies in this species.
Topics: Aedes; Animals; Chromosome Inversion; Chromosomes; Genetic Introgression; Genetic Variation; High-Throughput Nucleotide Sequencing; Mosquito Vectors
PubMed: 32164699
DOI: 10.1186/s12915-020-0757-y -
Genes Feb 2020underreplicate the DNA of thoracic nuclei, stalling during S phase at a point that is proportional to the total genome size in each species. In polytene tissues, such...
underreplicate the DNA of thoracic nuclei, stalling during S phase at a point that is proportional to the total genome size in each species. In polytene tissues, such as the salivary glands, all of the nuclei initiate multiple rounds of DNA synthesis and underreplicate. Yet, only half of the nuclei isolated from the thorax stall; the other half do not initiate S phase. Our question was, why half? To address this question, we use flow cytometry to compare underreplication phenotypes between thoracic tissues. When individual thoracic tissues are dissected and the proportion of stalled DNA synthesis is scored in each tissue type, we find that underreplication occurs in the indirect flight muscle, with the majority of underreplicated nuclei in the dorsal longitudinal muscles (DLM). Half of the DNA in the DLM nuclei stall at S phase between the unreplicated G0 and fully replicated G1. The dorsal ventral flight muscle provides the other source of underreplication, and yet, there, the replication stall point is earlier (less DNA replicated), and the endocycle is initiated. The differences in underreplication and ploidy in the indirect flight muscles provide a new tool to study heterochromatin, underreplication and endocycle control.
Topics: Animals; Cell Nucleus; DNA; DNA Replication; Drosophila melanogaster; Flight, Animal; Flow Cytometry; G1 Phase; Muscle, Skeletal; Polytene Chromosomes; Resting Phase, Cell Cycle; S Phase; Salivary Glands; Thorax
PubMed: 32111003
DOI: 10.3390/genes11030246 -
BMC Genomics Feb 2020Proximity ligation based techniques, like Hi-C, involve restriction digestion followed by ligation of formaldehyde cross-linked chromatin. Distinct chromatin states can...
BACKGROUND
Proximity ligation based techniques, like Hi-C, involve restriction digestion followed by ligation of formaldehyde cross-linked chromatin. Distinct chromatin states can impact the restriction digestion, and hence the visibility in the contact maps, of engaged loci. Yet, the extent and the potential impact of digestion bias remain obscure and under-appreciated in the literature.
RESULTS
Through analysis of 45 Hi-C datasets, lamina-associated domains (LADs), inactive X-chromosome in mammals, and polytene bands in fly, we first established that the DNA in condensed chromatin had lesser accessibility to restriction endonucleases used in Hi-C as compared to that in decondensed chromatin. The observed bias was independent of known systematic biases, was not appropriately corrected by existing computational methods, and needed an additional optimization step. We then repurposed this bias to identify novel condensed domains outside LADs, which were bordered by insulators and were dynamically associated with the polycomb mediated epigenetic and transcriptional states during development.
CONCLUSIONS
Our observations suggest that the corrected one-dimensional read counts of existing Hi-C datasets can be reliably repurposed to study the gene-regulatory dynamics associated with chromatin condensation and decondensation, and that the existing Hi-C datasets should be interpreted with cautions.
Topics: Animals; Chromatin; Chromatin Assembly and Disassembly; Chromatin Immunoprecipitation; Chromosome Positioning; Drosophila; Epigenomics; Genomics; Humans; Mice; Polytene Chromosomes; Sequence Analysis, DNA; X Chromosome
PubMed: 32087673
DOI: 10.1186/s12864-020-6580-6 -
Cells Feb 2020Spatial organization of chromosome territories and interactions between interphase chromosomes themselves, as well as with the nuclear periphery, play important roles in...
Spatial organization of chromosome territories and interactions between interphase chromosomes themselves, as well as with the nuclear periphery, play important roles in epigenetic regulation of the genome function. However, the interplay between inter-chromosomal contacts and chromosome-nuclear envelope attachments in an organism's development is not well-understood. To address this question, we conducted microscopic analyses of the three-dimensional chromosome organization in malaria mosquitoes. We employed multi-colored oligonucleotide painting probes, spaced 1 Mb apart along the euchromatin, to quantitatively study chromosome territories in larval salivary gland cells and adult ovarian nurse cells of , , and . We found that the X chromosome territory has a significantly smaller volume and is more compact than the autosomal arm territories. The number of inter-chromosomal, and the percentage of the chromosome-nuclear envelope, contacts were conserved among the species within the same cell type. However, the percentage of chromosome regions located at the nuclear periphery was typically higher, while the number of inter-chromosomal contacts was lower, in salivary gland cells than in ovarian nurse cells. The inverse correlation was considerably stronger for the autosomes. Consistent with previous theoretical arguments, our data indicate that, at the genome-wide level, there is an inverse relationship between chromosome-nuclear envelope attachments and chromosome-chromosome interactions, which is a key feature of the cell type-specific nuclear architecture.
Topics: Animals; Anopheles; Female; Germ Cells; Malaria; Nuclear Envelope; Ovary; Polytene Chromosomes; Salivary Glands; X Chromosome
PubMed: 32024176
DOI: 10.3390/cells9020339 -
Chromosoma Mar 2020Su(var) mutations define epigenetic factors controlling heterochromatin formation and gene silencing in Drosophila. Here, we identify SU(VAR)2-1 as a novel chromatin...
Su(var) mutations define epigenetic factors controlling heterochromatin formation and gene silencing in Drosophila. Here, we identify SU(VAR)2-1 as a novel chromatin regulator that directs global histone deacetylation during the transition of cleavage chromatin into somatic blastoderm chromatin in early embryogenesis. SU(VAR)2-1 is heterochromatin-associated in blastoderm nuclei but not in later stages of development. In larval polytene chromosomes, SU(VAR)2-1 is a band-specific protein. SU(VAR)2-1 directs global histone deacetylation by recruiting the histone deacetylase RPD3. In Su(var)2-1 mutants H3K9, H3K27, H4K8 and H4K16 acetylation shows elevated levels genome-wide and heterochromatin displays aberrant histone hyper-acetylation. Whereas H3K9me2- and HP1a-binding appears unaltered, the heterochromatin-specific H3K9me2S10ph composite mark is impaired in heterochromatic chromocenters of larval salivary polytene chromosomes. SU(VAR)2-1 contains an NRF1/EWG domain and a C2HC zinc-finger motif. Our study identifies SU(VAR)2-1 as a dosage-dependent, heterochromatin-initiating SU(VAR) factor, where the SU(VAR)2-1-mediated control of genome-wide histone deacetylation after cleavage and before mid-blastula transition (pre-MBT) is required to enable heterochromatin formation.
Topics: Animals; Blastula; CRISPR-Cas Systems; Centrosome; Chromatin Assembly and Disassembly; Cloning, Molecular; Drosophila; Drosophila Proteins; Embryonic Development; Female; Gene Expression Regulation, Developmental; Genome-Wide Association Study; Heterochromatin; Histones; Immunohistochemistry; In Situ Hybridization, Fluorescence; Male; Mutation; Phylogeny
PubMed: 31950239
DOI: 10.1007/s00412-020-00732-x -
Genes Dec 2019Notch is a key factor of a signaling cascade which regulates cell differentiation in all multicellular organisms. Numerous investigations have been directed mainly at...
Notch is a key factor of a signaling cascade which regulates cell differentiation in all multicellular organisms. Numerous investigations have been directed mainly at studying the mechanism of Notch protein action; however, very little is known about the regulation of activity of the gene itself. Here, we provide the results of targeted 5'-end editing of the gene in its native environment and genetic and cytological effects of these changes. Using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) system in combination with homologous recombination, we obtained a founder fly stock in which a 4-kb fragment, including the 5' nontranscribed region, the first exon, and a part of the first intron of , was replaced by an attachment Phage (attP) site. Then, fly lines carrying a set of six deletions within the 5'untranscribed region of the gene were obtained by 31-mediated integration of transgenic constructs. Part of these deletions does not affect gene activity, but their combinations with transgenic construct in the first intron of the gene cause defects in the Notch target tissues. At the polytene chromosome level we defined a DNA segment (~250 bp) in the 5'-nontranscribed region which when deleted leads to disappearance of the 3C6/C7 interband and elimination of CTC-Factor (CTCF) and Chromator (CHRIZ) insulator proteins in this region.
Topics: 5' Untranslated Regions; Animals; CRISPR-Cas Systems; Chromosome Structures; Drosophila Proteins; Drosophila melanogaster; Gene Expression Regulation; Homologous Recombination; Polytene Chromosomes; Receptors, Notch; Structure-Activity Relationship
PubMed: 31842424
DOI: 10.3390/genes10121037 -
Genes Nov 2019In the ciliate somatic macronuclei differentiate from germline micronuclei during sexual reproduction, accompanied by developmental sequence reduction. Concomitantly,... (Review)
Review
In the ciliate somatic macronuclei differentiate from germline micronuclei during sexual reproduction, accompanied by developmental sequence reduction. Concomitantly, over 95% of micronuclear sequences adopt a heterochromatin structure characterized by the histone variant H3.4 and H3K27me3. RNAi-related genes and histone variants dominate the list of developmentally expressed genes. Simultaneously, 27nt-ncRNAs that match sequences retained in new macronuclei are synthesized and bound by PIWI1. Recently, we proposed a mechanistic model for 'RNA-induced DNA replication interference' (RIRI): during polytene chromosome formation PIWI1/27nt-RNA-complexes target macronucleus-destined sequences (MDS) by base-pairing and temporarily cause locally stalled replication. At polytene chromosomal segments with ongoing replication, H3.4K27me3-nucleosomes become selectively deposited, thus dictating the prospective heterochromatin structure of these areas. Consequently, these micronucleus-specific sequences become degraded, whereas 27nt-RNA-covered sites remain protected. However, the biogenesis of the 27nt-RNAs remains unclear. It was proposed earlier that in stichotrichous ciliates 27nt-RNA precursors could derive from telomere-primed bidirectional transcription of nanochromosomes and subsequent Dicer-like (DCL) activity. As a minimalistic explanation, we propose here that the 27nt-RNA precursor could rather be mRNA or pre-mRNA and that the transition of coding RNA from parental macronuclei to non-coding RNAs, which act in premature developing macronuclei, could involve RNA-dependent RNA polymerase (RDRP) activity creating dsRNA intermediates prior to a DCL-dependent pathway. Interestingly, by such mechanism the partition of a parental somatic genome and possibly also the specific nanochromosome copy numbers could be vertically transmitted to the differentiating nuclei of the offspring.
Topics: Ciliophora; DNA Replication; Gene Expression Regulation, Developmental; Genome, Protozoan; Histones; Micronucleus, Germline; Nucleosomes; RNA Interference; RNA Precursors; RNA, Messenger; RNA, Small Nuclear; Telomere
PubMed: 31752243
DOI: 10.3390/genes10110940 -
Comparative Cytogenetics 2019The study presents data on the karyotype characteristics, features of chromosomal polymorphism and the gene COI sequences of Wülker, 1996 (Diptera, Chironomidae) from...
The study presents data on the karyotype characteristics, features of chromosomal polymorphism and the gene COI sequences of Wülker, 1996 (Diptera, Chironomidae) from the South Caucasus. We found 8 banding sequences in the Caucasian population. Overall, The Caucasian population of the species can be characterized as having a low level of polymorphism. We found one new banding sequence hpiA2 in the banding sequence pool of . We observed inversion polymorphism only in the arm F. The dendrogram of genetic distances by Nei criteria (1972) shows a clear separation of the Caucasian population from populations of Siberia. At the same time, the distance between populations of Siberia and the population of South Caucasus (0.379-0.445) almost reach the mean distance (0.474 ± 0.314) between subspecies (Gunderina 2001). Due to this, we can assume that the population of South Caucasus separated from Siberian populations at the level of subspecies. Constructed on data for COI gene sequences the phylogenetic tree estimated by the Bayesian inference shows that the sequences of from the South Caucasus form a separate line in the general branch of sequences. At the same time, calculated K2P genetic distances between sequences from Norway and Caucasus (2.0-2.2%) do not exceed the 3% threshold for the genus .
PubMed: 31723376
DOI: 10.3897/CompCytogen.v13i4.35572 -
Ecology and Evolution Jul 2019We tested the Rothfels sympatric speciation model for black flies by comparing all available data for sex-chromosome diversity with the geographic locations of larval...
ABSTRACT
We tested the Rothfels sympatric speciation model for black flies by comparing all available data for sex-chromosome diversity with the geographic locations of larval collection sites within the complex of black flies (Diptera: Simuliidae). Five separate data sets equaling about 20,000 larvae were included from throughout the geographic range of this complex. We record a total of 31 taxa having unique sex chromosomes, all of which demonstrate linkage disequilibrium with most taxa sharing autosomal polymorphisms. All siblings share portions of their distributions with , the presumed oldest member of the complex. Twenty-one of 22 cytotypes have distributions within the ranges of siblings thus supporting the sympatric speciation model of Rothfels. Chromosomally diverse sites may require analysis of as many as 200 larvae to be properly described. There is no effect of any inversions influencing the occurrence of other inversions. Finally, we report a new cytotype, IIL-6, which we originally discovered in Alaska. Aspects of future genomic research are discussed as they relate to the main chromosomal structural/functional tenants of the model.
OPEN RESEARCH BADGE
This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data are available at https://doi.org/10.6084/m9.figshare.7719398.
PubMed: 31380088
DOI: 10.1002/ece3.5402