TAD border deletion at the Kit locus causes tissue-specific ectopic activation of a neighboring gene.

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 the Kit locus and characterized chromatin folding and gene expression in pure cultures of fibroblasts, mast cells, and melanocytes. We found that although Kit is highly active in both mast cells and melanocytes, deletion of the TAD boundary between the Kit and Kdr genes 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 the Kit and Kdr genes 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.

Ultrastructural Abnormalities in Induced Pluripotent Stem Cell-Derived Neural Stem Cells and Neurons of Two Cohen Syndrome Patients.

Cohen syndrome is an autosomal recessive disorder caused by VPS13B (COH1) gene mutations. This syndrome is significantly underdiagnosed and is characterized by intellectual disability, microcephaly, autistic symptoms, hypotension, myopia, retinal dystrophy, neutropenia, and obesity. VPS13B regulates intracellular membrane transport and supports the Golgi apparatus structure, which is critical for neuron formation. We generated induced pluripotent stem cells from two patients with pronounced manifestations of Cohen syndrome and differentiated them into neural stem cells and neurons. Using transmission electron microscopy, we documented multiple new ultrastructural changes associated with Cohen syndrome in the neuronal cells. We discovered considerable disturbances in the structure of some organelles: Golgi apparatus fragmentation and swelling, endoplasmic reticulum structural reorganization, mitochondrial defects, and the accumulation of large autophagosomes with undigested contents. These abnormalities underline the ultrastructural similarity of Cohen syndrome to many neurodegenerative diseases. The cell models that we developed based on patient-specific induced pluripotent stem cells can serve to uncover not only neurodegenerative processes, but the causes of intellectual disability in general.

A Cre-LoxP-based approach for combinatorial chromosome rearrangements in human HAP1 cells.

Alterations of human karyotype caused by chromosomal rearrangements are often associated with considerable phenotypic effects. Studying molecular mechanisms underlying these effects requires an efficient and scalable experimental model. Here, we propose a Cre-LoxP-based approach for the generation of combinatorial diversity of chromosomal rearrangements. We demonstrate that using the developed system, both intra- and inter-chromosomal rearrangements can be induced in the human haploid HAP1 cells, although the latter is significantly less effective. The obtained genetically modified HAP1 cell line can be used to dissect genomic effects associated with intra-chromosomal structural variations.

The human EF1a promoter does not provide expression of the transgene in mice.

In this work, we set out to create mice susceptible to the SARS-CoV-2 coronavirus. To ensure the ubiquitous expression of the human ACE2 gene we used the human EF1a promoter. Using pronuclear microinjection of the transgene construct, we obtained six founders with the insertion of the EF1a-hACE2 transgene, from which four independent mouse lines were established. Unfortunately, only one line had low levels of hACE2 expression in some organs. In addition, we did not detect the hACE2 protein in primary lung fibroblasts from any of the transgenic lines. Bisulfite sequencing analysis revealed that the EF1a promoter was hypermethylated in the genomes of transgenic animals. Extensive analysis of published works about transgenic animals indicated that EF1a transgenic constructs are frequently inactive. Thus, our case cautions against using the EF1a promoter to generate transgenic animals, as it is prone to epigenetic silencing.

Erythrocytes 3D genome organization in vertebrates.

Generation of mature red blood cells, consisting mainly of hemoglobin, is a remarkable example of coordinated action of various signaling networks. Chromatin condensation is an essential step for terminal erythroid differentiation and subsequent nuclear expulsion in mammals. Here, we profiled 3D genome organization in the blood cells from ten species belonging to different vertebrate classes. Our analysis of contact maps revealed a striking absence of such 3D interaction patterns as loops or TADs in blood cells of all analyzed representatives. We also detect large-scale chromatin rearrangements in blood cells from mammals, birds, reptiles and amphibians: their contact maps display strong second diagonal pattern, representing an increased frequency of long-range contacts, unrelated to TADs or compartments. This pattern is completely atypical for interphase chromosome structure. We confirm that these principles of genome organization are conservative in vertebrate erythroid cells.

Comparison of American mink embryonic stem and induced pluripotent stem cell transcriptomes.

BACKGROUND: Recently fibroblasts of many mammalian species have been reprogrammed to pluripotent state using overexpression of several transcription factors. This technology allows production of induced pluripotent stem (iPS) cells with properties similar to embryonic stem (ES) cells. The completeness of reprogramming process is well studied in such species as mouse and human but there is not enough data on other species. We produced American mink (Neovison vison) ES and iPS cells and compared these cells using transcriptome analysis. RESULTS: We report the generation of 10 mink ES and 22 iPS cell lines. The majority of the analyzed cell lines had normal diploid chromosome number. The only ES cell line with XX chromosome set had both X-chromosomes in active state that is characteristic of pluripotent cells. The pluripotency of ES and iPS cell lines was confirmed by formation of teratomas with cell types representing all three germ layers. Transcriptome analysis of mink embryonic fibroblasts (EF), two ES and two iPS cell lines allowed us to identify 11831 assembled contigs which were annotated. These led to a number of 6891 unique genes. Of these 3201 were differentially expressed between mink EF and ES cells. We analyzed expression levels of these genes in iPS cell lines. This allowed us to show that 80% of genes were correctly reprogrammed in iPS cells, whereas approximately 6% had an intermediate expression pattern, about 7% were not reprogrammed and about 5% had a "novel" expression pattern. We observed expression of pluripotency marker genes such as Oct4, Sox2 and Rex1 in ES and iPS cell lines with notable exception of Nanog. CONCLUSIONS: We had produced and characterized American mink ES and iPS cells. These cells were pluripotent by a number of criteria and iPS cells exhibited effective reprogramming. Interestingly, we had showed lack of Nanog expression and consider it as a species-specific feature.

Comparison of the three-dimensional organization of sperm and fibroblast genomes using the Hi-C approach.

BACKGROUND: The three-dimensional organization of the genome is tightly connected to its biological function. The Hi-C approach was recently introduced as a method that can be used to identify higher-order chromatin interactions genome-wide. The aim of this study was to determine genome-wide chromatin interaction frequencies using the Hi-C approach in mouse sperm cells and embryonic fibroblasts. RESULTS: The obtained data demonstrate that the three-dimensional genome organizations of sperm and fibroblast cells show a high degree of similarity both with each other and with the previously described mouse embryonic stem cells. Both A- and B-compartments and topologically associated domains are present in spermatozoa and fibroblasts. Nevertheless, sperm cells and fibroblasts exhibit statistically significant differences between each other in the contact probabilities of defined loci. Tight packaging of the sperm genome results in an enrichment of long-range contacts compared with the fibroblasts. However, only 30% of the differences in the number of contacts are based on differences in the densities of their genome packages; the main source of the differences is the gain or loss of contacts that are specific for defined genome regions. We find that the dependence of the contact probability on genomic distance for sperm is close to the dependence predicted for the fractal globular folding of chromatin. CONCLUSIONS: Overall, we can conclude that the three-dimensional structure of the genome is passed through generations without being dramatically changed in sperm cells.

Reprogramming somatic cells by fusion with embryonic stem cells does not cause silencing of the Dlk1-Dio3 region in mice.

AIM: To examine the imprinted Dlk1-Dio3 locus in pluripotent embryonic stem (ES) cell/fibroblast hybrid cells. METHODS: Gtl2, Rian, and Mirg mRNA expression in mouse pluripotent ES cell/fibroblast hybrid cells was examined by real-time reverse transcription-polymerase chain reaction. Pyrosequencing and bisulfate sequencing were used to determine the DNA methylation level of the Dlk1-Dio3 locus imprinting control region. RESULTS: The selected hybrid clones had a near-tetraploid karyotype and were highly pluripotent judging from their capacity to generate chimeric embryos and adult chimeras. Our data clearly demonstrate that Gtl2, Rian, and Mirg, which are imprinted genes within the Dlk1-Dio3 locus, are active in all examined ES cell/fibroblast hybrid clones. In spite of interclonal variability, the expression of the imprinted genes is comparable to that of ES cells and fibroblasts. Quantitative analysis of the DNA methylation status of the intergenic differentially methylated region (IG DMR) within the Dlk1-Dio3 locus by pyrosequencing and bisulfite sequencing clearly showed that the DNA methylation status of the imprinted region in the tested hybrid clones was comparable to that of both ES cells and fibroblasts. CONCLUSION: Reprogramming process in a hybrid cell system is achieved without marked alteration of the imprinted Dlk1-Dio3 locus.

Reprogramming mediated by cell fusion technology.

This review is focused on recent advances in fusion-based reprogramming of cells of different pluripotent statuses or lineage origins. Recent findings are discussed from standpoints of both the developmental potency of hybrid cells generated by fusion of pluripotent embryonic stem (ES) cells, embryonal carcinoma (EC) cells, and somatic cells and epigenetic mechanisms and other aspects involved in the reprogramming process. Complete reprogramming occurs at least 5-7 days after fusion and includes at least two steps. (i) initiation at the heterokaryon stage and choice of the direction of reprogramming using an "all-or-none principle" to establish the dominance of one parental genome and (ii) "fixation" of the newly acquired expression profile by epigenetic mechanisms. The first step is realized without cell division, whereas the second requires cell proliferation. Reprogramming in hybrid cells is rapid and complete. Thus, cell fusion is a powerful tool for reprogramming.