Aleksandar Vojta

Repurposing the CRISPR-Cas9 system for targeted DNA methylation

Epigenetic studies relied so far on correlations between epigenetic marks and gene expression pattern. Technologies developed for epigenome editing now enable direct study of functional relevance of precise epigenetic modifications and gene regulation. The reversible nature of epigenetic modifications, including DNA methylation, has been already exploited in cancer therapy for remodeling the aberrant epigenetic landscape. However, this was achieved non-selectively using epigenetic inhibitors. Epigenetic editing at specific loci represents a novel approach that might selectively and heritably alter gene expression. Here, we developed a CRISPR-Cas9-based tool for specific DNA methylation consisting of deactivated Cas9 (dCas9) nuclease and catalytic domain of the DNA methyltransferase DNMT3A targeted by co–expression of a guide RNA to any 20 bp DNA sequence followed by the NGG trinucleotide. We demonstrated targeted CpG methylation in a ∼35 bp wide region by the fusion protein. We also showed that multiple guide RNAs could target the dCas9-DNMT3A construct to multiple adjacent sites, which enabled methylation of a larger part of the promoter. DNA methylation activity was specific for the targeted region and heritable across mitotic divisions. Finally, we demonstrated that directed DNA methylation of a wider promoter region of the target loci IL6ST and BACH2 decreased their expression.

Preprotein recognition by the Toc complex

The Toc core complex consists of the pore‐forming Toc75 and the GTPases Toc159 and Toc34. We confirm that the receptor form of Toc159 is integrated into the membrane. The association of Toc34 to Toc75/Toc159 is GTP dependent and enhanced by preprotein interaction. The N‐terminal half of the pSSU transit peptide interacts with high affinity with Toc159, whereas the C‐terminal part stimulates its GTP hydrolysis. The phosphorylated C‐terminal peptide of pSSU interacts strongly with Toc34 and therefore inhibits binding and translocation of pSSU into Toc proteoliposomes. In contrast, Toc159 recognises only the dephosphorylated forms. The N‐terminal part of the pSSU presequence does not influence binding to the Toc complex, but is able to block import into proteoliposomes through its interaction with Toc159. We developed a model of differential presequence recognition by Toc34 and Toc159.

Functional and Phylogenetic Properties of the Pore-forming β-Barrel Transporters of the Omp85 Family

β-Barrel-shaped channels of the Omp85 family are involved in the translocation or assembly of proteins of bacterial, mitochondrial, and plastidic outer membranes. We have compared these proteins to understand the evolutionary development of the translocators. We have demonstrated that the proteins from proteobacteria and mitochondria have a pore diameter that is at least five times smaller than found for the Omp85 in cyanobacteria and plastids. This finding can explain why Omp85 from cyanobacteria (but not the homologous protein from proteobacteria) was remodeled to become the protein translocation pore after endosymbiosis. Further, the pore-forming region of the Omp85 proteins is restricted to the C terminus. Based on a phylogenetic analysis we have shown that the pore-forming domain displays a different evolutionary relationship than the N-terminal domain. In line with this, the affinity of the N-terminal domain to the C-terminal region of the Omp85 from plastids and cyanobacteria differs, even though the N-terminal domain is involved in gating of the pore in both groups. We have further shown that the N-terminal domain of nOmp85 takes part in homo-oligomerization. Thereby, the differences in the phylogeny of the two domains are explained by different functional constraints acting on the regions. The pore-forming domain, however, is further divided into two functional regions, where the distal C terminus itself forms a dimeric pore. Based on functional and phylogenetic analysis, we suggest an evolutionary scenario that explains the origin of the contemporary translocon.

Intracellular localization of VDAC proteins in plants

Voltage-dependent anion channels (VDACs) are porin-type β-barrel diffusion pores. They are prominent in the outer membrane of mitochondria and facilitate metabolite exchange between the organelle and the cytosol. Here we studied the subcellular distribution of a plant VDAC-like protein between plastids and mitochondria in green and non-green tissue. Using in vitro studies of dual-import into mitochondria and chloroplasts as well as transient expression of fluorescence-labeled polypeptides, it could be clearly demonstrated that this VDAC isoform targets exclusively to mitochondria and not to plastids. Our results support the idea that plastids evolved a concept of solute exchange with the cytosol different from that of mitochondria.

IgG Glycome in Colorectal Cancer

Purpose: Alternative glycosylation has significant structural and functional consequences on IgG and consequently also on cancer immunosurveillance. Because of technological limitations, the effects of highly heritable individual variations and the differences in the dynamics of changes in IgG glycosylation on colorectal cancer were never investigated before. Experimental Design: Using recently developed high-throughput UPLC technology for IgG glycosylation analysis, we analyzed IgG glycome composition in 760 patients with colorectal cancer and 538 matching controls. Effects of surgery were evaluated in 28 patients sampled before and three times after surgery. A predictive model was built using regularized logistic regression and evaluated using a 10-cross validation procedure. Furthermore, IgG glycome composition was analyzed in 39 plasma samples collected before initial diagnosis of colorectal cancer. Results: We have found that colorectal cancer associates with decrease in IgG galactosylation, IgG sialylation and increase in core-fucosylation of neutral glycans with concurrent decrease of core-fucosylation of sialylated glycans. Although a model based on age and sex did not show discriminative power (AUC = 0.499), the addition of glycan variables into the model considerably increased the discriminative power of the model (AUC = 0.755). However, none of these differences were significant in the small set of samples collected before the initial diagnosis. Conclusions: Considering the functional relevance of IgG glycosylation for both tumor immunosurveillance and clinical efficacy of therapy with mAbs, individual variation in IgG glycosylation may turn out to be important for prediction of disease course or the choice of therapy, thus warranting further, more detailed studies of IgG glycosylation in colorectal cancer. Clin Cancer Res; 22(12); 3078–86. ©2016 AACR.

Phospho‐mimicry mutant of atToc33 affects early development of Arabidopsis thaliana

The precursor protein receptor at the chloroplast outer membrane atToc33 is a GTPase, which can be inactivated by phosphorylation in vitro, being arrested in the GDP loaded state. To assess the physiological function of phosphorylation, attoc33 knock out mutants were complemented with a mutated construct mimicking the constitutively phosphorylated state. Our data suggest that the reduced functionality of the mutant protein can be compensated by its upregulation. Chloroplast biogenesis and photosynthetic activity are impaired in the mutants during the early developmental stage, which is consistent with the requirement of atToc33 in young photosynthetic tissues.

DNA hypomethylation upregulates expression of the MGAT3 gene in HepG2 cells and leads to changes in N-glycosylation of secreted glycoproteins

Changes in N-glycosylation of plasma proteins are observed in many types of cancer, nevertheless, few studies suggest the exact mechanism involved in aberrant protein glycosylation. Here we studied the impact of DNA methylation on the N-glycome in the secretome of the HepG2 cell line derived from hepatocellular carcinoma (HCC). Since the majority of plasma glycoproteins originate from the liver, the HepG2 cells represent a good model for glycosylation changes in HCC that are detectable in blood, which is an easily accessible analytic material in a clinical setting. Two different concentrations of 5-aza-2′-deoxycytidine (5-aza-2dC) differentially affected global genome methylation and induced different glycan changes. Around twenty percent of 84 glyco-genes analysed changed expression level after the 5-aza-2dC treatment as a result of global genome hypomethylation. A correlation study between the changes in glyco-gene expression and the HepG2 glycosylation profile suggests that the MGAT3 gene might be responsible for the glycan changes consistently induced by both doses of 5-aza-2dC. Core-fucosylated tetra-antennary structures were decreased in quantity likely as a result of hypomethylated MGAT3 gene promoter followed by increased expression of this gene.

Adaptation or malignant transformation: the two faces of epigenetically mediated response to stress.

Adaptive response to stress is a fundamental property of living systems. At the cellular level, many different types of stress elicit an essentially limited repertoire of adaptive responses. Epigenetic changes are the main mechanism for medium- to long-term adaptation to accumulated (intense, long-term, or repeated) stress. We propose the adaptive deregulation of the epigenome in response to stress (ADERS) hypothesis which assumes that the unspecific adaptive stress response grows stronger with the increasing stress level, epigenetically activating response gene clusters while progressively deregulating other cellular processes. The balance between the unspecific adaptive response and the general epigenetic deregulation is critical because a strong response can lead to pathology, particularly to malignant transformation. The main idea of our hypothesis is the continuum traversed by a cell subjected to accumulated stress, which lies between an unspecific adaptive response and pathological deregulation—the two extremes sharing the same underlying cause, which is a manifestation of a unified epigenetically mediated adaptive response to stress. The evolutionary potential of epigenetic regulation in multigenerational adaptation is speculatively discussed in the light of neo-Lamarckism. Finally, an approach to testing the proposed hypothesis is presented, relying on either the publicly available datasets or on conducting new experiments.

Inhibition of DNA Methylation Alters Chromatin Organization, Nuclear Positioning and Activity of 45S rDNA Loci in Cycling Cells of Q. robur

Around 2200 copies of genes encoding ribosomal RNA (rRNA) in pedunculate oak, Quercus robur, are organized into two rDNA loci, the major (NOR-1) and the minor (NOR-2) locus. We present the first cytogenetic evidence indicating that the NOR-1 represents the active nucleolar organizer responsible for rRNA synthesis, while the NOR-2 probably stays transcriptionally silent and does not participate in the formation of the nucleolus in Q. robur, which is a situation resembling the well-known phenomenon of nucleolar dominance. rDNA chromatin topology analyses in cycling root tip cells by light and electron microscopy revealed the minor locus to be highly condensed and located away from the nucleolus, while the major locus was consistently associated with the nucleolus and often exhibited different levels of condensation. In addition, silver precipitation was confined exclusively to the NOR-1 locus. Also, NOR-2 was highly methylated at cytosines and rDNA chromatin was marked with histone modifications characteristic for repressive state. After treatment of the root cells with the methylation inhibitor 5-aza-2′-deoxycytidine, we observed an increase in the total level of rRNA transcripts and a decrease in DNA methylation level at the NOR-2 locus. Also, NOR-2 sites relocalized with respect to the nuclear periphery/nucleolus, however, the relocation did not affect the contribution of this locus to nucleolar formation, nor did it affect rDNA chromatin decondensation, strongly suggesting that NOR-2 has lost the function of rRNA synthesis and nucleolar organization.

Detection and characterisation of hepatitis E virus in naturally infected swine in Croatia.

Hepatitis E is a viral zoonotic disease infecting swine worldwide. Since pigs represent a likely animal reservoir for the hepatitis E virus, the epidemiology of naturally occurring hepatitis E was investigated in Croatian swine herds. Nearly all tested animals were seropositive for antibodies against the hepatitis E virus (55/60, 91.7%). Active infection was detected in all age groups by RT-PCR of viral RNA in serum (8/60, 13.3%) and bile samples (3/37, 8.1%), which was further confirmed by histopathological findings of characteristic lesions in the livers of the infected animals. Three new strains of hepatitis E virus were isolated from Croatian pig herds. Phylogenetic analysis using median-joining networks clustered those Croatian strains with isolates from various parts of the world, indicating their likely origin in international trade. Similarity to human isolates implies a zoonotic potential of Croatian strains, which raises a public health concern, especially in the light of the high prevalence of hepatitis E in the herds studied.