Alexey Sokolov

Pros and cons of methylation-based enrichment methods for ancient DNA

The recent discovery that DNA methylation survives in fossil material provides an opportunity for novel molecular approaches in palaeogenomics. Here, we apply to ancient DNA extracts the probe-independent Methylated Binding Domains (MBD)-based enrichment method, which targets DNA molecules containing methylated CpGs. Using remains of a Palaeo-Eskimo Saqqaq individual, woolly mammoths, polar bears and two equine species, we confirm that DNA methylation survives in a variety of tissues, environmental contexts and over a large temporal range (4,000 to over 45,000 years before present). MBD enrichment, however, appears principally biased towards the recovery of CpG-rich and long DNA templates and is limited by the fast post-mortem cytosine deamination rates of methylated epialleles. This method, thus, appears only appropriate for the analysis of ancient methylomes from very well preserved samples, where both DNA fragmentation and deamination have been limited. This work represents an essential step toward the characterization of ancient methylation signatures, which will help understanding the role of epigenetic changes in past environmental and cultural transitions.

Identification of novel microRNA genes in freshwater and marine ecotypes of the three-spined stickleback (Gasterosteus aculeatus)

The three‐spined stickleback (Gasterosteus aculeatus L.) is an important model organism for studying the molecular mechanisms of speciation and adaptation to salinity. Despite increased interest to microRNA discovery and recent publication on microRNA prediction in the three‐spined stickleback using bioinformatics approaches, there is still a lack of experimental support for these data. In this paper, high‐throughput sequencing technology was applied to identify microRNA genes in gills of the three‐spined stickleback. In total, 595 miRNA genes were discovered; half of them were predicted in previous computational studies and were confirmed here as microRNAs expressed in gill tissue. Moreover, 298 novel microRNA genes were identified. The presence of miRNA genes in selected ‘divergence islands’ was analysed and 10 miRNA genes were identified as not randomly located in ‘divergence islands’. Regulatory regions of miRNA genes were found enriched with selective SNPs that may play a role in freshwater adaptation.

Mitochondrial genome of Megaphragma amalphitanum (Hymenoptera: Trichogrammatidae)

Abstract The mitochondrial genome of the parasitic wasp Megaphragma amalphitanum is published in this paper. The mitochondrial DNA (mtDNA) is 15 041 base pairs (bp) in length and contained 13 protein-coding genes, 2 rRNA genes and 22 tRNA genes. The overall base composition of the genome in descending order was 45.67% A, 8.71% C, 39.63% T and 5.99% G, with a significant AT bias of 85.30%.

Profiling of microRNAs in wild type and early flowering transgenic Chrysanthemum morifolium by deep sequencing

Here, we performed comparative miRNA profiling in wild type and early flowering transgenic Chrysanthemum morifolium with constitutive expression of APETALA1 (AP1)-like gene, HAM92 (Helianthus annuus). Six sRNA libraries constructed from leaves and shoot apexes after the short day photoperiod initiation, as well as from opened inflorescence after anthesis were sequenced and analyzed. A total of 324 members (163 families) of putative conserved miRNAs and 30 candidate novel miRNAs specific for C. morifolium (cmo-miRNAs) were identified. Bioinformatic analysis revealed 427 and 138 potential mRNA targets for conserved and novel cmo-miRNAs, respectively. These genes were described in Gene Ontology terms and found to be implicated in a broad range of signaling pathways. Plant- and tissue-specific expression of 9 highly conserved cmo-miRNAs was compared between wild type and transgenic chrysanthemum lines with ectopic expression of AP1-like genes HAM92 and CDM111 (C. morifolium), using RT-qPCR and cmo-miR162a as a reference miRNA. The results of our study provide a framework for further investigation of miRNA evolution and functions in higher plants, as well as their roles in flowering control.

S100A3 Is a Novel Target Gene of Kaiso in Mouse Skin

322 Kaiso is a protein that binds methylated DNA and belongs to the BTB/POZ family of transcription facc tors. Kaiso contains two functional domains, includd ing a BTB/POZ domain in the NNterminus and a C 2 H 2 type CCterminal domain containing three zinc fingers [1]. This bimodal DNAAbinding protein spee cifically recognizes both methylated mCpGmCpG and nonmethylated CTGCNA sequences (below, the Kaisoobinding site is referred to as KBS) [2–4]. The nonmethylated CTGCNA sequence is present in proo moter regions of Wnt11, matrilysinn7, and Siamois genes in Xenopus laevis frogs. It was shown that the presence of 55hydroxymethylcytosine within the KBS inhibits KaisooDNA binding [5]. However, it should be noted that there is no agreement within the scienn tific community regarding which of the DNAAbinding activities of Kaiso (against methylated regions, nonmm ethylated sequences or both) represents its main activv ity in vivo. It has been demonstrated that Kaiso is essential for amphibian development. A reduction in Kaiso levels in Xenopus laevis frogs and Danio rerio fish results in embryonic lethality due to premature gene activation in blastulae and increased expression of the Wnt11 gene [6–8]. On the other hand, Kaisonull mice show no phenotype. However, when Kaiso–/y mice were crossed with APC/Min mice, the progeny acquired resistance to intestinal tumorigenesis. The direct targets of Kaiso have not yet been discovered [9]. The goal of this work was to identify novel target genes of the Kaiso protein. To do this, the gene express sion analysis of total RNA isolated from the skin of C57BL/6 wilddtype and Kaisonull (Kaiso–/y) mice was carried out using MouseReff8 micromatrices (Illumina, United States). A number of genes were identified (1599 genes, p value < 0.01), the expression levels of which were different in the skin of Kaiso–/y mice compared to the C57BL/6 control mice (Fig. 1a). Gene ontology analysis was used to identify genes with increased levels of expression in Kaiso–/y mice. The genes were classified to be involved in epidermal diff ferentiation, morphogenesis, keratinocyte maturaa tion, cytoskeleton formation, and ectoderm developp ment. Also, a group of genes, including S100A3, Sprr, and Padi3, Padi3 which are localized in the central domain of the epidermis differentiation complex (EDC), which plays an important role in the epiderr mis barrier function, were identified (Fig. 1b) [10]. The data obtained in RNAAmicromatrix hybridizaa tion experiments were verified using the S100A3 gene, the expression level of which was significantly increased …

DeSUMOylation switches Kaiso from activator to repressor upon hyperosmotic stress

Kaiso is a member of the BTB/POZ zinc finger family, which is involved in cancer progression, cell cycle control, apoptosis, and WNT signaling. Depending on promoter context, it may function as either a transcriptional repressor or activator. Previous studies found that Kaiso might be SUMOylated due to heat shock, but the biological significance of Kaiso SUMOylation is unclear. Here, we find that K42 is the only amino acid within Kaiso that is modified with SUMO. Kaiso is monoSUMOylated at lysine 42 in cell lines of kidney origin under normal physiological conditions. SUMOylated Kaiso can activate transcription from exogenous methylated promoters, wherein the deSUMOylated form of the protein kept the ability to be a repressor. Rapid Kaiso deSUMOylation occurs in response to hyperosmotic stress and is reversible upon return to an isotonic environment. DeSUMOylation occurs within minutes in HEK293 cells treated with 100 mm NaCl and relaxes in 3 h even in a salt-containing medium. Genomic editing of Kaiso by conversion of K42 into R42 (K42R) in HEK293 cells that resulted in fully deSUMOylated endogenous protein led to misregulation of genes associated with ion transport, blood pressure, and the immune response. TRIM25 was significantly repressed in two K42R HEK293 clones. By a series of rescue experiments with K42R and KO HEK293 cells, we show that TRIM25 is a direct transcriptional target for Kaiso. In the absence of Kaiso, the level of TRIM25 is insensitive to hyperosmotic stress. Extending our observations to animal models, we show that in response to a high salt diet, Kaiso knockout mice are characterized by significantly higher blood pressure increases when compared to wild-type animals. Thus, we propose a novel biological role for Kaiso in the regulation of homeostasis.

Metagenomic analysis of microbial community of a parasitoid wasp Megaphragma amalphitanum

The vast majority of multicellular organisms coexist with bacterial symbionts that may play various roles during their life cycle. Parasitoid wasp Megaphragma amalphitanum (Hymenoptera: Trichogrammatidae) belongs to the smallest known insects whose size is comparable with some bacteria. Using 16S rRNA gene sequencing and Whole Genome Sequencing (WGS), we described microbiota diversity for this arthropod and its potential impact on their lifecycle. Metagenomic sequences were deposited to SRA database which is available at NCBI with accession number SRX2363723 and SRX2363724. We found that small body size and limited lifespan do not lead to a significant reduction of bacterial symbionts diversity. At the same time, we show here a specific feature of microbiota composition in M. amalphitanum – the absence of the Rickettsiaceae family representatives that are known to cause sex-ratio distortion in arthropods and well represented in other populations of parasitoid wasps.

The complete mitochondrial genome of the smallest known free-living insect Scydosella musawasensis

Abstract The mitochondrial genome of the smallest known free-living insect Scydosella musawasensis (Polilov, 2015) is published in this paper. The mitochondrial DNA (mtDNA) is 14 719 base pairs (bp) in length and contained 13 protein-coding genes, 2 rRNA genes and 21 tRNA genes. The overall base composition of the genome in descending order was 40.59% – A, 13.85% – C, 36.82% – T and 8.73% – G, with a significant AT bias of 77.41%.