Mikhail A. Rubtsov

Chromatin structure can strongly facilitate enhancer action over a distance

Numerous DNA transactions in eukaryotic nuclei are regulated by elements (enhancers) that can directly interact with their targets over large regions of DNA organized into chromatin. The mechanisms allowing communication over a distance in chromatin are unknown. We have established an experimental system allowing quantitative analysis of the impact of chromatin structure on distant transcriptional regulation. Assembly of relaxed or linear DNA templates into subsaturated chromatin results in a strong increase of the efficiency of distant enhancer–promoter E–P communication and activation of transcription. The effect is directly proportional to the efficiency of chromatin assembly and cannot be explained only by DNA compaction. Transcription activation on chromatin templates is enhancer- and activator-dependent, and must be accompanied by direct E–P interaction and formation of a chromatin loop. Previously we have shown that DNA supercoiling can strongly facilitate E–P communication on histone-free DNA. The effects of chromatin assembly and DNA supercoiling on the communication are quantitatively similar, but the efficiency of enhancer action in subsaturated chromatin does not depend on the level of unconstrained DNA supercoiling. Thus chromatin structure per se can support highly efficient communication over a distance and functionally mimic the supercoiled state characteristic for prokaryotic DNA.

Dynamics of double strand breaks and chromosomal translocations

Chromosomal translocations are a major cause of cancer. At the same time, the mechanisms that lead to specific chromosomal translocations that associate different gene regions remain largely unknown. Translocations are induced by double strand breaks (DSBs) in DNA. Here we review recent data on the mechanisms of generation, mobility and repair of DSBs and stress the importance of the nuclear organization in this process.

Repositioning of ETO gene in cells treated with VP‐16, an inhibitor of DNA‐Topoisomerase II

The translocation t(8;21)(q22;q22) affecting AML1 and ETO genes is known to be one of the frequent chromosome translocations in acute myeloid leukemia. But no data have been available up to date concerning mutual positioning of these particular genes in the nucleus of a living cell as well as the mechanism of their rapprochement and realignment. Here we show that there is no proximity between these two genes in the primary nuclei of normal human male fibroblasts and moreover that these genes are located in different nuclear layers. But we further show that treatment of cells with VP‐16 (etoposide), an inhibitor of DNA topoisomerase II widely used in anticancer chemotherapy, causes the ETO gene repositioning which allows AML1 and ETO genes to be localized in the same nuclear layer. Inhibitor studies demonstrate that such an effect is likely to be connected with the formation of stalled cleavable complexes on DNA. Finally, inhibition of ETO gene repositioning by 2,3‐butanedione monoxime (BDM) suggests that this process depends on nuclear myosin. Together, our data corroborate the so called “breakage first” model of the origins of recurrent reciprocal translocation. J. Cell. Biochem. 104: 692–699, 2008.

The Broken MLL Gene Is Frequently Located Outside the Inherent Chromosome Territory in Human Lymphoid Cells Treated with DNA Topoisomerase II Poison Etoposide

The mixed lineage leukaemia (MLL) gene is frequently rearranged in secondary leukaemias, in which it could fuse to a variety of different partners. Breakage in the MLL gene preferentially occurs within a ~8 kb region that possesses a strong DNA topoisomerase II cleavage site. It has been proposed that DNA topoisomerase II-mediated DNA cleavage within this and other regions triggers translocations that occur due to incorrect joining of broken DNA ends. To further clarify a possible mechanism for MLL rearrangements, we analysed the frequency of MLL cleavage in cells exposed to etoposide, a DNA topoisomerase II poison commonly used as an anticancer drug, and positioning of the broken 3’-end of the MLL gene in respect to inherent chromosomal territories. It was demonstrated that exposure of human Jurkat cells to etoposide resulted in frequent cleavage of MLL genes. Using MLL-specific break-apart probes we visualised cleaved MLL genes in ~17% of nuclei. Using confocal microscopy and 3D modelling, we demonstrated that in cells treated with etoposide and cultivated for 1 h under normal conditions, ~9% of the broken MLL alleles were present outside the chromosome 11 territory, whereas in both control cells and cells inspected immediately after etoposide treatment, virtually all MLL alleles were present within the chromosomal territory. The data are discussed in the framework of the “breakage first” model of juxtaposing translocation partners. We propose that in the course of repairing DNA topoisomerase II-mediated DNA lesions (removal of stalled DNA topoisomerase II complexes and non-homologous end joining), DNA ends acquire additional mobility, which allows the meeting and incorrect joining of translocation partners.

A One-Step PCR-Based Assay to Evaluate the Efficiency and Precision of Genomic DNA-Editing Tools

Despite rapid progress, many problems and limitations persist and limit the applicability of gene-editing techniques. Making use of meganucleases, TALENs, or CRISPR/Cas9-based tools requires an initial step of pre-screening to determine the efficiency and specificity of the designed tools. This step remains time consuming and material consuming. Here we propose a simple, cheap, reliable, time-saving, and highly sensitive method to evaluate a given gene-editing tool based on its capacity to induce chromosomal translocations when combined with a reference engineered nuclease. In the proposed technique, designated engineered nuclease-induced translocations (ENIT), a plasmid coding for the DNA-editing tool to be tested is co-transfected into carefully chosen target cells along with that for an engineered nuclease of known specificity and efficiency. If the new enzyme efficiently cuts within the desired region, then specific chromosomal translocations will be generated between the two targeted genomic regions and be readily detectable by a one-step PCR or qPCR assay. The PCR product thus obtained can be directly sequenced, thereby determining the exact position of the double-strand breaks induced by the gene-editing tools. As a proof of concept, ENIT was successfully tested in different cell types and with different meganucleases, TALENs, and CRISPR/Cas9-based editing tools.

Expression of Cholesterol Hydroxylase/Lyase System Proteins in Yeast S. cerevisiae Cells as a Self-Processing Polyprotein

Abstract2A peptide discovered in Picornaviridae is capable of self-cleavage providing an opportunity to carry out synthesis of several proteins using one transcript. Dissociation in the 2A sequence during translation leads to the individual proteins formation. We constructed cDNA including genes of the bovine cholesterol hydroxylase/lyase (CHL) system proteins—cytochrome P450scc (CYP11A1), adrenodoxin (Adx) and adrenodoxin reductase (AdR), that are fused into a single ORF using FMDV 2A nucleotide sequences. The constructed vectors direct the expression of cDNA encoding polyprotein P450scc-2A-Adx-2A-AdR (CHL-2A) in Escherichia coli and Saccharomyces cerevisiae. The induced bacterial cells exhibit a high level of CHL-2A expression, but polyprotein is not cleaved at the FMDV sites. In yeast S. cerevisiae, the discrete proteins P450scc-2A, Adx-2A and AdR are expressed. Moreover, a significant proportion of AdR and Adx is present in a fusion Adx-2A-AdR. Thus, the first 2A linker provides an efficient cleavage of the polyprotein, while the second 2A linker demonstrates lower efficiency. Cholesterol hydroxylase/lyase activity registered in the recombinant yeast cell homogenate indicates that the catalytically active CHL system is present in these cells. Consequently, for the first time the mammalian system of cytochrome P450 has been successfully reconstructed in yeast cells through expressing the self-processing polyprotein.

Dual Role of the Extracellular Domain of Human Mucin MUC1 in Metastasis: MUC1 DUAL ROLE IN METASTASIS

Human mucin MUC1 plays an important role in cancer development. The increased level of this molecule expression during cancer cell progression induces metastasis and is associated with poor prognosis for patients. There is a large body of experimental data on the role of various functional domains of human mucin MUC1 in metastasis. While, the cytoplasmic domain determined to play a definitive role, the influence of extracellular domain on cancer cell invasiveness still remains unclear. The present paper reveals that the extracellular domain of MUC1 molecule consists of two functional subdomains—the region of tandem repeats (TR) and the region of irregular repeats (IR). We demonstrate the ability of each of these subdomains to alter the invasiveness of cancer cells. The presence of the MUC1 molecules containing TR subdomain (MUC1‐TR) on the surface of low‐invasive cancer cells leads to the increase in their transendothelial migration potency, while the addition of the IR subdomain to the MUC1‐TR molecule (MUC1‐IR‐TR) restores their natural low invasiveness. J. Cell. Biochem. 118: 4002–4011, 2017.

Rational Design of Recombinant Papain-Like Cysteine Protease: Optimal Domain Structure and Expression Conditions for Wheat-Derived Enzyme Triticain-α

Triticain-α is a papain-like cysteine protease from wheat (Triticum aestivum L.) that possesses activity towards toxic gluten-derived peptides, and was thus proposed as a novel therapeutic tool for celiac disease. We report an original approach employing rational design of domain architecture of Triticain-α and selection of the appropriate expression system for development of cheap and efficient protocol yielding active recombinant enzyme. The segregated catalytic domain of Triticain-α did not adopt native structure in bacteria, neither being expressed as a single protein nor upon conjugation or co-expression with extrinsic chaperones. Meanwhile, its attachment to prodomain of the enzyme resulted in generation of insoluble (inclusion bodies) product that can be transformed into active protease upon refolding in vitro. The estimated yield of the product was affected by affinity six-histidine tag required for its single-step purification with the preferable N-terminal position of the tag. Expression of the two-domain Triticain-α construct in yeast (Pichia pastoris) strain GS115 and bacterial (Escherichia coli) strain Rosetta gami B (DE3) led to the accumulation of a soluble protein, which underwent autocatalytic maturation during expression (in yeast)/purification (in bacteria) procedures and exhibited pronounced protease activity. Furthermore, expression and solubility of such construct in Rosetta gami B (DE3) cells was improved by reducing the temperature of the bacterial growth yielding more active enzyme than yeast counterpart presumably due to facilitated formation of a characteristic disulfide bond critical for maintaining the catalytic site. We suggest that these findings are helpful for obtaining active Triticain-α preparations for scientific or medical applications, and can be employed for the design and production of beneficial recombinant products based on other papain-like cysteine proteases.

Polycistronic expression of the mitochondrial steroidogenic P450scc system in the HEK293T cell line: EFIMOVA et al.

The cholesterol hydroxylase/lyase (CHL) system, consisting of cytochrome P450scc, adrenodoxin (Adx) and adrenodoxin reductase (AdR), initiates mammalian steroidogenesis, converting cholesterol to pregnenolone. The foot‐and‐mouth disease virus 2A‐based method allows to express multiple proteins from a single transcript. We developed a 2A‐based multicistronic system for the coexpression of three bovine CHL system proteins as the self‐processing polyprotein pCoxIV‐P450scc‐2A‐Adx‐2A‐AdR‐GFP (pCoxIV‐CHL‐GFP), with a cleavable N‐terminal mitochondrial targeting presequence. HEK293T cells transfected with plasmid, containing complementary DNA (cDNA) for pCoxIV‐CHL‐GFP, efficiently performed the expression of P450scc‐2A, targeted to mitochondria, and Adx‐2A, AdR‐GFP and the fusion protein Adx‐2A‐AdR‐GFP, which were predominantly localized in the cytosol. Despite the spatial separation of expressed P450scc and redox partners, the transfected HEK293T cells were able to convert the steroid substrates of cytochrome P450scc to pregnenolone, whereas control HEK293T cells were not catalytically active. The presence of 2А peptide residue on the C‐terminus of P450scc did not preclude its enzymatic activity. HEK293T cells transfected with a vector directing the synthesis of only P450scc‐2A demonstrated cytochrome P450scc activity comparable to that of cells expressing all three CHL system components, and to that of nature steroidogenic cells. Thus, the P450scc activity detected in cells transfected with both constructed plasmids was the result of the effective functional coupling of the bovine cytochrome P450scc and endogenous mitochondrial electron transport proteins of HEK293T cells. The produced pregnenolone did not undergo further conversion to progesterone, which indicates the absence of catalytically active 3β‐hydroxysteroid dehydrogenase. Therefore, HEK293T cells may be suitable for the expression of steroidogenic enzymes and the study of their characteristics.

From an increase in the number of tandem repeats through the decrease of sialylation to the downregulation of MUC1 expression level: SYRKINA et al.

Enhanced glucose uptake by cancer cells was demonstrated in many studies in vitro and in vivo. Glycolysis is one of the main ways of obtaining energy in hypoxia conditions. However, in addition to energy exchange, carbohydrates are also necessary for the posttranslational modification of the protein molecules. Cancer cells are often characterized by an enhanced expression of different glycoproteides. Correct glycosylation defines the structure and activity of such molecules. We demonstrated that under the same cultivation conditions, the intensity of glycosylation does not depend on the total number of potential O‐glycosylation sites in one molecule. As a model for the investigation, the tandem repeat region (region with variable number of tandem repeats) of the human mucin MUC1, in which each of the repeats carries four potential O‐glycosylation sites, was used. An increase of the tandem repeat number in the recombinant protein did not lead to a proportional increase in the level of sLea glycosides. A consequence of this was a reduction in the number of recombinant proteins associated with the cytoplasmic membrane at an overall high expression level. Prolongation of the cultivation duration led to a reduction in the expression level of the recombinant proteins by up to 30% of the initial level, and the intensity of this reduction was in a direct ratio to the number of tandem repeats in the protein molecule.