Mechanical Tensions Regulate Gene Expression in the Xenopus laevis Axial Tissues.

During gastrulation and neurulation, the chordamesoderm and overlying neuroectoderm of vertebrate embryos converge under the control of a specific genetic programme to the dorsal midline, simultaneously extending along it. However, whether mechanical tensions resulting from these morphogenetic movements play a role in long-range feedback signaling that in turn regulates gene expression in the chordamesoderm and neuroectoderm is unclear. In the present work, by using a model of artificially stretched explants of Xenopus midgastrula embryos and full-transcriptome sequencing, we identified genes with altered expression in response to external mechanical stretching. Importantly, mechanically activated genes appeared to be expressed during normal development in the trunk, i.e., in the stretched region only. By contrast, genes inhibited by mechanical stretching were normally expressed in the anterior neuroectoderm, where mechanical stress is low. These results indicate that mechanical tensions may play the role of a long-range signaling factor that regulates patterning of the embryo, serving as a link coupling morphogenesis and cell differentiation.

An IDH-independent mechanism of DNA hypermethylation upon VHL inactivation in cancer.

Hypermethylation of tumour suppressors and other aberrations of DNA methylation in tumours play a significant role in cancer progression. DNA methylation can be affected by various environmental conditions, including hypoxia. The response to hypoxia is mainly achieved through activation of the transcriptional program associated with HIF1A transcription factor. Inactivation of Von Hippel-Lindau Tumour Suppressor gene (VHL) by genetic or epigenetic events, which also induces aberrant activation of HIF1A, is the most common driver event for renal cancer. With whole-genome bisulphite sequencing and LC-MS, we demonstrated that VHL inactivation induced global genome hypermethylation in human kidney cancer cells under normoxic conditions. This effect was reverted by exogenous expression of wild-type VHL. We showed that global genome hypermethylation in VHL mutants can be explained by transcriptional changes in MDH and L2HGDH genes that cause the accumulation of 2-hydroxyglutarate - a metabolite that inhibits DNA demethylation by TET enzymes. Unlike the known cases of DNA hypermethylation in cancer, 2-hydroxyglutarate was accumulated in the cells with the wild-type isocitrate dehydrogenases.

Progress and Prospects in Epigenetic Studies of Ancient DNA.

Development of technologies for high-throughput whole-genome sequencing and improvement of sample preparation techniques made it possible to study ancient DNA (aDNA) from archaeological samples over a million year old. The studies of aDNA have shed light on the history of human migration, replacement of populations, interbreeding of Cro-Magnons with Neanderthals and Denisovans, evolution of human pathogens, etc. Equally important is the possibility to investigate epigenetic modifications of ancient genomes, which has allowed to obtain previously inaccessible information on gene expression, nucleosome positioning, and DNA methylation. Analysis of methylation status of certain genomic sites can predict an individual's age at death and reconstruct some phenotypic features, as it was done for the Denisovan genome, and even to elucidate unfavorable environmental factors that had affected this archaic individual. In this review, we discuss current progress in epigenetic studies of aDNA, including methodological approaches and promising research directions in this field.

Comparative Analysis of Genome Editors Efficiency on a Model of Mice Zygotes Microinjection.

Genome editing is an indispensable tool for functional genomics. The caveat of the genome-editing pipeline is a prevalence of error-prone non-homologous end joining over homologous recombination, while only the latter is suitable to introduce particularly desired genetic variants. To overcome this problem, a toolbox of genome engineering was appended by a variety of improved instruments. In this work, we compared the efficiency of a number of recently suggested improved systems for genome editing applied to the same genome regions on a murine zygote model via microinjection. As a result, we observed that homologous recombination utilizing an ssDNA template following sgRNA directed Cas9 cleavage is still the method of choice for the creation of animals with precise genome alterations.

Kaiso Regulates DNA Methylation Homeostasis.

Gain and loss of DNA methylation in cells is a dynamic process that tends to achieve an equilibrium. Many factors are involved in maintaining the balance between DNA methylation and demethylation. Previously, it was shown that methyl-DNA protein Kaiso may attract NCoR, SMRT repressive complexes affecting histone modifications. On the other hand, the deficiency of Kaiso resulted in reduced methylation of ICR in H19/Igf2 locus and Oct4 promoter in mouse embryonic fibroblasts. However, nothing is known about how Kaiso influences DNA methylation at the genome level. Here we show that deficiency of Kaiso led to whole-genome hypermethylation, using Kaiso deficient human renal cancer cell line obtained via CRISPR/CAS9 genome editing. However, Kaiso serves to protect genic regions, enhancers, and regions with a low level of histone modifications from demethylation. We detected hypomethylation of binding sites for Oct4 and Nanog in Kaiso deficient cells. Kaiso immunoprecipitated with de novo DNA methyltransferases DNMT3a/3b, but not with maintenance methyltransferase DNMT1. Thus, Kaiso may attract methyltransferases to surrounding regions and modulate genome methylation in renal cancer cells apart from being methyl DNA binding protein.

Cytoskeletal Protein Zyxin Inhibits the Activity of Genes Responsible for Embryonic Stem Cell Status.

Zyxin is a cytoskeletal LIM-domain protein that regulates actin cytoskeleton assembly and gene expression. In the present work, we find that zyxin downregulation in Xenopus laevis embryos reduces the expression of numerous genes that regulate cell differentiation, but it enhances that of several genes responsible for embryonic stem cell status, specifically klf4, pou5f3.1, pou5f3.2, pou5f3.3, and vent2.1/2. For pou5f3 family genes (mammalian POU5F1/OCT4 homologs), we show that this effect is the result of mRNA stabilization due to complex formation with the Y-box protein Ybx1. When bound to Ybx1, zyxin interferes with the formation of these complexes, thereby stimulating pou5f3 mRNA degradation. In addition, in zebrafish embryos and human HEK293 cells, zyxin downregulation increases mRNA levels of the pluripotency genes KLF4, NANOG, and POU5F1/OCT4. Our findings indicate that zyxin may play a role as a switch among morphogenetic cell movement, differentiation, and embryonic stem cell status.

Isolation and phylogenetic analysis of SARS-CoV-2 variants collected in Russia during the COVID-19 outbreak.

OBJECTIVES: The outbreak of coronavirus disease 2019 (COVID-19) started in December 2019 in China and then spread worldwide over the following months, involving 188 countries. The objective of this study was to determine the molecular epidemiology of the COVID-19 outbreak in Russia. METHODS: In this study, two severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains were isolated and genetically characterized. A phylogenetic analysis of all available Russian sequences was then performed and these were compared to the epidemiological data on COVID-19 incidence to evaluate the molecular epidemiology and pattern of virus spread in the territory of Russia. RESULTS AND CONCLUSIONS: Whole genome analysis of the isolates obtained in this study and 216 others isolated in Russia revealed a set of seven common mutations when compared to the original Wuhan virus, including amino acid substitutions in spike protein S and nucleoprotein N, possibly affecting their properties. Phylogenetic analysis of all Russian sequences and 8717 sequences from other countries showed multiple importations of the virus into Russia, local circulation, and several patterns of virus spread.

A partial genome assembly of the miniature parasitoid wasp, Megaphragma amalphitanum.

Body size reduction, also known as miniaturization, is an important evolutionary process that affects a number of physiological and phenotypic traits and helps animals conquer new ecological niches. However, this process is poorly understood at the molecular level. Here, we report genomic and transcriptomic features of arguably the smallest known insect-the parasitoid wasp, Megaphragma amalphitanum (Hymenoptera: Trichogrammatidae). In contrast to expectations, we find that the genome and transcriptome sizes of this parasitoid wasp are comparable to other members of the Chalcidoidea superfamily. Moreover, compared to other chalcid wasps the gene content of M. amalphitanum is remarkably conserved. Intriguingly, we observed significant changes in M. amalphitanum transposable element dynamics over time, in which an initial burst was followed by suppression of activity, possibly due to a recent reinforcement of the genome defense machinery. Overall, while the M. amalphitanum genomic data reveal certain features that may be linked to the unusual biological properties of this organism, miniaturization is not associated with a large decrease in genome complexity.

Sex specific associations in genome wide association analysis of renal cell carcinoma.

Renal cell carcinoma (RCC) has an undisputed genetic component and a stable 2:1 male to female sex ratio in its incidence across populations, suggesting possible sexual dimorphism in its genetic susceptibility. We conducted the first sex-specific genome-wide association analysis of RCC for men (3227 cases, 4916 controls) and women (1992 cases, 3095 controls) of European ancestry from two RCC genome-wide scans and replicated the top findings using an additional series of men (2261 cases, 5852 controls) and women (1399 cases, 1575 controls) from two independent cohorts of European origin. Our study confirmed sex-specific associations for two known RCC risk loci at 14q24.2 (DPF3) and 2p21(EPAS1). We also identified two additional suggestive male-specific loci at 6q24.3 (SAMD5, male odds ratio (ORmale) = 0.83 [95% CI = 0.78-0.89], Pmale = 1.71 × 10-8 compared with female odds ratio (ORfemale) = 0.98 [95% CI = 0.90-1.07], Pfemale = 0.68) and 12q23.3 (intergenic, ORmale = 0.75 [95% CI = 0.68-0.83], Pmale = 1.59 × 10-8 compared with ORfemale = 0.93 [95% CI = 0.82-1.06], Pfemale = 0.21) that attained genome-wide significance in the joint meta-analysis. Herein, we provide evidence of sex-specific associations in RCC genetic susceptibility and advocate the necessity of larger genetic and genomic studies to unravel the endogenous causes of sex bias in sexually dimorphic traits and diseases like RCC.

New insights from Opisthorchis felineus genome: update on genomics of the epidemiologically important liver flukes.

BACKGROUND: The three epidemiologically important Opisthorchiidae liver flukes Opisthorchis felineus, O. viverrini, and Clonorchis sinensis, are believed to harbour similar potencies to provoke hepatobiliary diseases in their definitive hosts, although their populations have substantially different ecogeographical aspects including habitat, preferred hosts, population structure. Lack of O. felineus genomic data is an obstacle to the development of comparative molecular biological approaches necessary to obtain new knowledge about the biology of Opisthorchiidae trematodes, to identify essential pathways linked to parasite-host interaction, to predict genes that contribute to liver fluke pathogenesis and for the effective prevention and control of the disease. RESULTS: Here we present the first draft genome assembly of O. felineus and its gene repertoire accompanied by a comparative analysis with that of O. viverrini and Clonorchis sinensis. We observed both noticeably high heterozygosity of the sequenced individual and substantial genetic diversity in a pooled sample. This indicates that potency of O. felineus population for rapid adaptive response to control and preventive measures of opisthorchiasis is higher than in O. viverrini and C. sinensis. We also have found that all three species are characterized by more intensive involvement of trans-splicing in RNA processing compared to other trematodes. CONCLUSION: All revealed peculiarities of structural organization of genomes are of extreme importance for a proper description of genes and their products in these parasitic species. This should be taken into account both in academic and applied research of epidemiologically important liver flukes. Further comparative genomics studies of liver flukes and non-carcinogenic flatworms allow for generation of well-grounded hypotheses on the mechanisms underlying development of cholangiocarcinoma associated with opisthorchiasis and clonorchiasis as well as species-specific mechanisms of these diseases.

Correction: Kaiso is required for MTG16-dependent effects on colitis-associated carcinoma.

In the original version of this article the authors noted that the GEO accession number for the relevant dataset was listed incorrectly as GSE12454.

Kaiso is required for MTG16-dependent effects on colitis-associated carcinoma.

The myeloid translocation gene family member MTG16 is a transcriptional corepressor that relies on the DNA-binding ability of other proteins to determine specificity. One such protein is the ZBTB family member Kaiso, and the MTG16:Kaiso interaction is necessary for repression of Kaiso target genes, such as matrix metalloproteinase-7. Using the azoxymethane and dextran sodium sulfate (AOM/DSS) murine model of colitis-associated carcinoma, we previously determined that MTG16 loss accelerates tumorigenesis and inflammation. However, it was unknown whether this effect was modified by Kaiso-dependent transcriptional repression. To test for a genetic interaction between MTG16 and Kaiso in inflammatory carcinogenesis, we subjected single and double knockout (DKO) mice to the AOM/DSS protocol. Mtg16-/- mice demonstrated increased colitis and tumor burden; in contrast, disease severity in Kaiso-/- mice was equivalent to wild-type controls. Surprisingly, Kaiso deficiency in the context of MTG16 loss reversed injury and pro-tumorigenic responses in the intestinal epithelium following AOM/DSS treatment, and tumor numbers were returned to near to wild-type levels. Transcriptomic analysis of non-tumor colon tissue demonstrated that changes induced by MTG16 loss were widely mitigated by concurrent Kaiso loss, and DKO mice demonstrated downregulation of metabolism and cytokine-associated gene sets with concurrent activation of DNA damage checkpoint pathways as compared with Mtg16-/-. Further, Kaiso knockdown in intestinal enteroids reduced stem- and WNT-associated phenotypes, thus abrogating the induction of these pathways observed in Mtg16-/- samples. Together, these data suggest that Kaiso modifies MTG16-driven inflammation and tumorigenesis and suggests that Kaiso deregulation contributes to MTG16-dependent colitis and CAC phenotypes.

The influence of obesity-related factors in the etiology of renal cell carcinoma-A mendelian randomization study.

BACKGROUND: Several obesity-related factors have been associated with renal cell carcinoma (RCC), but it is unclear which individual factors directly influence risk. We addressed this question using genetic markers as proxies for putative risk factors and evaluated their relation to RCC risk in a mendelian randomization (MR) framework. This methodology limits bias due to confounding and is not affected by reverse causation. METHODS AND FINDINGS: Genetic markers associated with obesity measures, blood pressure, lipids, type 2 diabetes, insulin, and glucose were initially identified as instrumental variables, and their association with RCC risk was subsequently evaluated in a genome-wide association study (GWAS) of 10,784 RCC patients and 20,406 control participants in a 2-sample MR framework. The effect on RCC risk was estimated by calculating odds ratios (ORSD) for a standard deviation (SD) increment in each risk factor. The MR analysis indicated that higher body mass index increases the risk of RCC (ORSD: 1.56, 95% confidence interval [CI] 1.44-1.70), with comparable results for waist-to-hip ratio (ORSD: 1.63, 95% CI 1.40-1.90) and body fat percentage (ORSD: 1.66, 95% CI 1.44-1.90). This analysis further indicated that higher fasting insulin (ORSD: 1.82, 95% CI 1.30-2.55) and diastolic blood pressure (DBP; ORSD: 1.28, 95% CI 1.11-1.47), but not systolic blood pressure (ORSD: 0.98, 95% CI 0.84-1.14), increase the risk for RCC. No association with RCC risk was seen for lipids, overall type 2 diabetes, or fasting glucose. CONCLUSIONS: This study provides novel evidence for an etiological role of insulin in RCC, as well as confirmatory evidence that obesity and DBP influence RCC risk.

VHL inactivation without hypoxia is sufficient to achieve genome hypermethylation.

VHL inactivation is a key oncogenic event for renal carcinomas. In normoxia, VHL suppresses HIF1a-mediated transcriptional response, which is characteristic to hypoxia. It has previously been shown that hypoxic conditions inhibit TET-dependent hydroxymethylation of cytosines and cause DNA hypermethylation at gene promoters. In this work, we performed VHL inactivation by CRISPR/Cas9 and studied its effects on gene expression and DNA methylation. We showed that even without hypoxia, VHL inactivation leads to hypermethylation of the genome. Hypermethylated cytosines were evenly distributed throughout the genome with a slight preference for AP-1 (JUN and FOS) binding sites. Hypermethylated cytosines tended to be enriched within the binding sites of transcription factors that showed increased gene expression after VHL inactivation. We also observed promoter hypermethylation associated with decreased gene expression for several regulators of transcription and DNA methylation including SALL3.

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.

Genome-Wide DNA Methylation Profiling Reveals Epigenetic Adaptation of Stickleback to Marine and Freshwater Conditions.

The three-spined stickleback (Gasterosteus aculeatus) represents a convenient model to study microevolution-adaptation to a freshwater environment. Although genetic adaptations to freshwater environments are well-studied, epigenetic adaptations have attracted little attention. In this work, we investigated the role of DNA methylation in the adaptation of the marine stickleback population to freshwater conditions. DNA methylation profiling was performed in marine and freshwater populations of sticklebacks, as well as in marine sticklebacks placed into a freshwater environment and freshwater sticklebacks placed into seawater. We showed that the DNA methylation profile after placing a marine stickleback into fresh water partially converged to that of a freshwater stickleback. For six genes including ATP4A ion pump and NELL1, believed to be involved in skeletal ossification, we demonstrated similar changes in DNA methylation in both evolutionary and short-term adaptation. This suggested that an immediate epigenetic response to freshwater conditions can be maintained in freshwater population. Interestingly, we observed enhanced epigenetic plasticity in freshwater sticklebacks that may serve as a compensatory regulatory mechanism for the lack of genetic variation in the freshwater population. For the first time, we demonstrated that genes encoding ion channels KCND3, CACNA1FB, and ATP4A were differentially methylated between the marine and the freshwater populations. Other genes encoding ion channels were previously reported to be under selection in freshwater populations. Nevertheless, the genes that harbor genetic and epigenetic changes were not the same, suggesting that epigenetic adaptation is a complementary mechanism to selection of genetic variants favorable for freshwater environment.

Kaiso differentially regulates components of the Notch signaling pathway in intestinal cells.

BACKGROUND: In mammalian intestines, Notch signaling plays a critical role in mediating cell fate decisions; it promotes the absorptive (or enterocyte) cell fate, while concomitantly inhibiting the secretory cell fate (i.e. goblet, Paneth and enteroendocrine cells). We recently reported that intestinal-specific Kaiso overexpressing mice (Kaiso Tg ) exhibited chronic intestinal inflammation and had increased numbers of all three secretory cell types, hinting that Kaiso might regulate Notch signaling in the gut. However, Kaiso's precise role in Notch signaling and whether the Kaiso Tg secretory cell fate phenotype was linked to Kaiso-induced inflammation had yet to be elucidated. METHODS: Intestines from 3-month old Non-transgenic and Kaiso Tg mice were "Swiss" rolled and analysed for the expression of Notch1, Dll-1, Jagged-1, and secretory cell markers by immunohistochemistry and immunofluorescence. To evaluate inflammation, morphological analyses and myeloperoxidase assays were performed on intestines from 3-month old Kaiso Tg and control mice. Notch1, Dll-1 and Jagged-1 expression were also assessed in stable Kaiso-depleted colon cancer cells and isolated intestinal epithelial cells using real time PCR and western blotting. To assess Kaiso binding to the DLL1, JAG1 and NOTCH1 promoter regions, chromatin immunoprecipitation was performed on three colon cancer cell lines. RESULTS: Here we demonstrate that Kaiso promotes secretory cell hyperplasia independently of Kaiso-induced inflammation. Moreover, Kaiso regulates several components of the Notch signaling pathway in intestinal cells, namely, Dll-1, Jagged-1 and Notch1. Notably, we found that in Kaiso Tg mice intestines, Notch1 and Dll-1 expression are significantly reduced while Jagged-1 expression is increased. Chromatin immunoprecipitation experiments revealed that Kaiso associates with the DLL1 and JAG1 promoter regions in a methylation-dependent manner in colon carcinoma cell lines, suggesting that these Notch ligands are putative Kaiso target genes. CONCLUSION: Here, we provide evidence that Kaiso's effects on intestinal secretory cell fates precede the development of intestinal inflammation in Kaiso Tg mice. We also demonstrate that Kaiso inhibits the expression of Dll-1, which likely contributes to the secretory cell phenotype observed in our transgenic mice. In contrast, Kaiso promotes Jagged-1 expression, which may have implications in Notch-mediated colon cancer progression.

Genome-wide association study identifies multiple risk loci for renal cell carcinoma.

Previous genome-wide association studies (GWAS) have identified six risk loci for renal cell carcinoma (RCC). We conducted a meta-analysis of two new scans of 5,198 cases and 7,331 controls together with four existing scans, totalling 10,784 cases and 20,406 controls of European ancestry. Twenty-four loci were tested in an additional 3,182 cases and 6,301 controls. We confirm the six known RCC risk loci and identify seven new loci at 1p32.3 (rs4381241, P=3.1 × 10-10), 3p22.1 (rs67311347, P=2.5 × 10-8), 3q26.2 (rs10936602, P=8.8 × 10-9), 8p21.3 (rs2241261, P=5.8 × 10-9), 10q24.33-q25.1 (rs11813268, P=3.9 × 10-8), 11q22.3 (rs74911261, P=2.1 × 10-10) and 14q24.2 (rs4903064, P=2.2 × 10-24). Expression quantitative trait analyses suggest plausible candidate genes at these regions that may contribute to RCC susceptibility.

De novo transcriptome assembly of the mycoheterotrophic plant Monotropa hypopitys.

Monotropa hypopitys (pinesap) is a non-photosynthetic obligately mycoheterotrophic plant of the family Ericaceae. It obtains the carbon and other nutrients from the roots of surrounding autotrophic trees through the associated mycorrhizal fungi. In order to understand the evolutionary changes in the plant genome associated with transition to a heterotrophic lifestyle, we performed de novo transcriptomic analysis of M. hypopitys using next-generation sequencing. We obtained the RNA-Seq data from flowers, flower bracts and roots with haustoria using Illumina HiSeq2500 platform. The raw data obtained in this study can be available in NCBI SRA database with accession number of SRP069226. A total of 10.3 GB raw sequence data were obtained, corresponding to 103,357,809 raw reads. A total of 103,025,683 reads were filtered after removing low-quality reads and trimming the adapter sequences. The Trinity program was used to de novo assemble 98,349 unigens with an N50 of 1342 bp. Using the TransDecoder program, we predicted 43,505 putative proteins. 38,416 unigenes were annotated in the Swiss-Prot protein sequence database using BLASTX. The obtained transcriptomic data will be useful for further studies of the evolution of plant genomes upon transition to a non-photosynthetic lifestyle and the loss of photosynthesis-related functions.