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Topologically associated domains (TADs) restrict promoter-enhancer interactions, thereby maintaining the spatiotemporal pattern of gene activity. However, rearrangements of the TADs boundaries do not always lead to significant changes in the activity pattern. Here, we investigated the consequences of the TAD boundaries deletion on the expression of developmentally important genes encoding tyrosine kinase receptors:Kit, Kdr, Pdgfra. We used genome editing in mice to delete the TADs boundaries at theKitlocus and characterized chromatin folding and gene expression in pure cultures of fibroblasts, mast cells, and melanocytes. We found that althoughKitis highly active in both mast cells and melanocytes, deletion of the TAD boundary between theKitandKdrgenes results in ectopic activation only in melanocytes. Thus, the epigenetic landscape, namely the mutual arrangement of enhancers and actively transcribing genes, is important for predicting the consequences of the TAD boundaries removal. We also found that mice without a TAD border between theKitandKdrgenes have a phenotypic manifestation of the mutation — a lighter coloration. Thus, the data obtained shed light on the principles of interaction between the 3D chromatin organization and epigenetic marks in the regulation of gene activity.

Chromosome level assemblies are accumulating in various taxonomic groups including mosquitoes. However, even in the few reference-quality mosquito assemblies, a significant portion of the heterochromatic regions including telomeres remain unresolved. Here we produce a de novo assembly of the New World malaria mosquito, Anopheles albimanus by integrating Oxford Nanopore sequencing, Illumina, Hi-C and optical mapping. This 172.6 Mbps female assembly, which we call AalbS3, is obtained by scaffolding polished large contigs (contig N50=13.7 Mbps) into three chromosomes. All chromosome arms end with telomeric repeats, which is the first in mosquito assemblies and represents a significant step towards the completion of a genome assembly. These telomeres consist of tandem repeats of a novel 30-32 bp telomeric repeat unit (TRU) and are confirmed by analysing the termini of long reads and through both chromosomal in situ hybridization and a Bal31 sensitivity assay. The AalbS3 assembly included previously uncharacterized centromeric and rDNA clusters and more than doubled the content of transposable elements and other repetitive sequences. This telomere-to-telomere assembly, although still containing gaps, represents a significant step towards resolving biologically important but previously hidden genomic components. The comparison of different scaffolding methods will also inform future efforts to obtain reference-quality genomes for other mosquito species.100-word Article SummaryWe report AalbS3, a telomere-to-telomere assembly of the Anopheles albimanus genome produced by integrating advancing technologies including Oxford Nanopore and Bionano optical mapping. AalbS3 features much of the difficult-to-assemble genomic ‘dark matters’ including previously missed transposons, centromeres and rDNA clusters. We describe novel telomeric repeats that are confirmed by analysis of long reads and by telomere hybridization assays. This reference-quality assembly represents a significant step towards completing the genomic puzzle pieces and informs efforts to improve the assembly of other mosquito species. Future research into the relationship between telomere and mosquito life span may have significant implications to disease control.

The step-by-step Hi-C protocol is described for mosquito embryos at the 15-18 h developmental stage. In brief, we collect mosquito eggs, fix them with 3% formaldehyde, and prepare Hi-C libraries using nuclei isolated from ~1000-3000 embryos of mixed sexes. We use MboI restriction enzyme and T4-ligase to generate two biological replicates per species. Libraries can be prepared with NEBNext® Ultra™ II DNA Library Prep Kit for Illumina. The protocol was adapted for Anopheles species and successfully verified for Culex and Aedes embryos.

Chromosomes are hierarchically folded within cell nuclei into territories, domains and subdomains, but the functional importance and evolutionary dynamics of these hierarchies are poorly defined. Here, we comprehensively profile genome organizations of five Anopheles mosquito species and show how different levels of chromatin architecture influence each other. Patterns observed on Hi-C maps are associated with known cytological structures, epigenetic profiles, and gene expression levels. Evolutionary analysis reveals conservation of chromatin architecture within synteny blocks for tens of millions of years and enrichment of synteny breakpoints in regions with increased genomic insulation. However, in-depth analysis shows a confounding effect of gene density on both insulation and distribution of synteny breakpoints, suggesting limited causal relationship between breakpoints and regions with increased genomic insulation. At the level of individual loci, we identify specific, extremely long-ranged looping interactions, conserved for similar to 100 million years. We demonstrate that the mechanisms underlying these looping contacts differ from previously described Polycomb-dependent interactions and clustering of active chromatin. NSF [MCB-1715207]; NIH NIAID [R21AI135298, R21AI159382]; USDA National Institute of Food and Agriculture Hatch project [223822]; RFBR [19-34-50051]; University of Lausanne Department of Ecology and Evolution; NIH [AI133571, AI121284]; Novartis Foundation [18B116]; Swiss National Science Foundation [PP00P3_170664, PP00P3_202669]; Fulbright Foreign Student Program [15161026]; Ministry of Education and Science of Russian Federation [2019-0546 (FSUS-2020-0040)]; [121031800061-7] Published version The following reagents were obtained through BEI Resources, NIAID, NIH: An. coluzzii, Strain MOPTI, Eggs, MRA-763, contributed by Gregory C. Lanzaro; An. merus, Strain MAF, MRA-1156, contributed by Maureen Coetzee; An. atroparvus, Strain EBRO, Eggs, MRA-493, contributed by Carlos Aranda and Mark Q. Benedict; An. albimanus, Strain STECLA, Eggs, MRA-126, contributed by Mark Q. Benedict. All computations were performed using nodes of the high-throughput cluster of the Novosibirsk State University, and bioinformatics resource center of the Institute of Cytology and Genetics. We are sincerely grateful to Nariman Battulin for fruitful discussions. This work was supported by the NSF grant MCB-1715207, NIH NIAID grants R21AI135298 and R21AI159382, and the USDA National Institute of Food and Agriculture Hatch project 223822 to IVS. The reported study of An. atroparvus was partly funded by RFBR according to the research project no 19-34-50051 to IVS and VL. PacBio sequencing of An. merus was funded by a grant from the University of Lausanne Department of Ecology and Evolution to RMW and NIH grants AI133571 and AI121284 to Z.T. M.J.M.F.R., L.R., R.F., and R.M.W. were supported by Novartis Foundation for medical-biological research grant #18B116 and Swiss National Science Foundation grants PP00P3_170664 and PP00P3_202669. V.L. was partly supported by the Fulbright Foreign Student Program, Grantee ID: 15161026. Hi-C data analysis was supported by the Ministry of Education and Science of Russian Federation, grant #2019-0546 (FSUS-2020-0040). ChIP-seq data analysis was supported by project 121031800061-7 (Mechanisms of genetic control of development, physiological processes and behavior in animals).

BackgroundAnopheles coluzzii and An. arabiensis belong to the An. gambiae complex and are among the major malaria vectors in Sub-Saharan Africa. However, chromosome-level reference genome assemblies are still lacking for these medically important mosquito species.FindingsIn this study, we produced de novo chromosome-level genome assemblies for An. coluzzii and An. arabiensis using the long-read Oxford Nanopore sequencing technology and the Hi-C scaffolding approach. We obtained 273.4 Mbp and 256.8 Mbp of the total assemblies for An. coluzzii and An. arabiensis, respectively. Each assembly consists of three chromosome-scale scaffolds (X, 2, 3), complete mitochondrion, and unordered contigs identified as autosomal pericentromeric DNA, X pericentromeric DNA, and Y sequences. Comparison of these assemblies with the existing assemblies for these species demonstrated that we obtained improved reference-quality genomes. The new assemblies allowed us to identify genomiccoordinates for the breakpoint regions of fixed and polymorphic chromosomal inversions in An. coluzzii and An. arabiensis.ConclusionThe new chromosome-level assemblies will facilitate functional and population genomic studies in An. coluzzii and An. arabiensis. The presented assembly pipeline will accelerate progress toward creating high-quality genome references for other disease vectors.

Chromosomes are hierarchically folded within cell nuclei into territories, domains and subdomains, but the functional importance and evolutionary dynamics of these hierarchies are poorly defined. Here, we comprehensively profiled genome organizations of five Anopheles mosquito species and showed how different levels of chromatin architecture influence contacts between genomic loci. Patterns observed on Hi-C maps are associated with known cytological structures, epigenetic profiles, and gene expression levels. At the level of individual loci, we identified specific, extremely long-ranged looping interactions, conserved for ~100 million years. We showed that the mechanisms underlying these looping contacts differ from previously described Polycomb-dependent interactions and clustering of active chromatin.