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The formation of heterochromatin begins in the differentiating cells. The aim of this work was to study changes of satellite DNA distribution, transcriptional activity and interaction with certain proteins in mouse embryonic stem cells after induction with retinoic acid. We found that pericentromeric satellites entered chromocenters only some days after induction of E-14 and IOUD2 mouse embryonic stem cells. The redistribution was accompanied by association with HP1a and transcription from pericentromeric (but not centromeric) satellite DNA. RNA was polyadenylated and transcribed from the forward chain. Probes made from the cDNA hybridized to all chromosomes. In differentiating cells, the transcript was found exclusively in chromocenters while in differentiated cultured L929 cells it formed 1–2 large clusters outside chromocenters. Using ChIP and immunostaining, we demonstrated that in induced cells pericentromeric DNA interacted with RNA-helicase p68 that was previously shown to be involved in transcription regulation and to be involved in differentiation processes. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Constitutive heterochromatin mainly consists of different classes of satellite DNAs and is defined as a transcriptionally inactive chromatin that remains compact throughout the cell cycle. The aim of this work was to investigate the level of condensation, methylation and transcriptional status of centromeric (alphoid DNA) and pericentromeric satellites (human satellite 3, HS3) in tissues (lymphocytes, placenta cells) and in cultured primary (MRC5, VH-10, AT2Sp) and malignant (A431) cells. We found that alphoid DNA remained condensed and heavily methylated in all the cell types. The HS3 of chromosome 1 (HS3-1) but not of chromosome 9 (HS3-9) was strongly decondensed and demethylated in A431 cells. The same observation was made for aged embryonic lung (MRC5) and juvenile foreskin (VH-10) fibroblasts obtained at late passages (32nd and 23rd, respectively). Decondensation was also found in ataxia telangiectasia AT2Sp fibroblasts at the 16th passage. One of the manifestations of the disease is premature aging. The level of HS3-1 decondensation was higher in aged primary fibroblasts as compared to A431. The HS3-1 extended into the territory of neighbouring chromosomes. An RT-PCR product was detected in A431 and senescent MRC5 fibroblasts using primers specific for HS3-1. The RNA was polyadenylated and transcribed from the reverse chain. Our results demonstrate the involvement of satellite DNA in associations between human chromosomes and intermingling of chromosome territories. The invading satellite DNA can undergo decondensation to a certain level. This process is accompanied by demethylation and transcription. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) are widely used in therapy, but the differences between MSCs of various origins and their ability to undergo osteogenic differentiation and produce extracellular matrix are not fully understood. To address this, we conducted a comparative analysis of mesenchymal cell primary cultures from 6 human sources, including osteoblast-like cells from the adult femur, adipose-derived stem cells, Wharton's jelly-derived mesenchymal cells, gingival fibroblasts, dental pulp stem cells, and periodontal ligament stem cells. We analyzed these cells' secretome, proteome, and transcriptome under standard and osteogenic cultivation conditions. Despite the overall similarity in osteogenic differentiation, the cells maintain their embryonic specificity after isolation and differentiation in vitro. Furthermore, we propose classifying mesenchymal cells into 3 groups: dental stem cells of neural crest origin, mesenchymal stem cells, and fetal stem cells. Specifically, fetal stem cells have the most promising secretome for various applications, while mesenchymal stem cells have a specialized secretome optimal for extracellular matrix production. Nevertheless, mesenchymal cells from all sources secreted core bone extracellular matrix-associated proteins. In conclusion, our study illuminates the distinctive characteristics of mesenchymal stem cells from various sources, providing insights into their potential applications in regenerative medicine and enhancing our understanding of the inherent diversity of mesenchymal cells in vivo., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Pericentromeric tandemly repeated DNA of human satellites 1, 2, and 3 (HS1, HS2, and HS3) is actively transcribed in some cells. However, the functionality of the transcription remains obscure. Studies in this area have been hampered by the absence of a gapless genome assembly. The aim of our study was to map a transcript that we have previously described as HS2/HS3 on chromosomes using a newly published gapless genome assembly T2T-CHM13, and create a plasmid overexpressing the transcript to assess the influence of HS2/HS3 transcription on cancer cells. We report here that the sequence of the transcript is tandemly repeated on nine chromosomes (1, 2, 7, 9, 10, 16, 17, 22, and Y). A detailed analysis of its genomic localization and annotation in the T2T-CHM13 assembly revealed that the sequence belonged to HSAT2 (HS2) but not to the HS3 family of tandemly repeated DNA. The transcript was found on both strands of HSAT2 arrays. The overexpression of the HSAT2 transcript increased the transcription of the genes encoding the proteins involved in the epithelial-to-mesenchymal transition, EMT ( SNAI1 , ZEB1 , and SNAI2 ), and the genes that mark cancer-associated fibroblasts ( VIM , COL1A1 , COL11A1 , and ACTA2 ) in cancer cell lines A549 and HeLa. Co-transfection of the overexpression plasmid and antisense nucleotides eliminated the transcription of EMT genes observed after HSAT2 overexpression. Antisense oligonucleotides also decreased transcription of the EMT genes induced by tumor growth factor beta 1 (TGFβ1). Thus, our study suggests HSAT2 lncRNA transcribed from the pericentromeric tandemly repeated DNA is involved in EMT regulation in cancer cells.

The culture of osteoblasts (OB) of human origin is a useful experimental model in studying bone biology, osteogenic differentiation, functions of bone proteins, oncological processes in bone tissue, testing drugs against bone desires, and many other fields. The purpose of the present study is to share a workflow that has established the conditions to efficiently isolate and grow OB cells obtained from surgically removed bones from human donors. The protocol described here also shows how to determine cell phenotype. Here we provide characteristics of cells isolated by this protocol that might help researchers to decide if such OB are suitable for the purposes of their study. Osteoblasts isolated from collagenase-treated explants of adult bones are able to proliferate and keep their phenotype in culture. OB cells have high synthetic properties. They express osteomarkers, such as RUNX2, osteocalcin, BMP2, and osteopontin both in control conditions and in an osteogenic medium that could be estimated by qPCR and immunocytochemical staining and by Western blotting. Induction of osteogenic differentiation does not dramatically influence the synthetic properties of OB cells, while the cells gain the ability to extracellular mineralization only in an osteogenic medium., Competing Interests: The authors declare no conflict of interest.

Finding new, safe strategies to prevent and control rheumatoid arthritis is an urgent task. Bioactive peptides and peptide-rich protein hydrolyzate represent a new trend in the development of functional foods and nutraceuticals. The resulting tissue hydrolyzate of the chicken embryo (CETH) has been evaluated for acute toxicity and tested against chronic arthritis induced by Freund's full adjuvant (modified Mycobacterium butyricum) in rats. The antiarthritic effect of CETH was studied on the 28th day of the experiment after 2 weeks of oral administration of CETH at doses of 60 and 120 mg/kg body weight. Arthritis was evaluated on the last day of the experiment on the injected animal paw using X-ray computerized microtomography and histopathology analysis methods. The CETH effect was compared with the non-steroidal anti-inflammatory drug diclofenac sodium (5 mg/kg). Oral administration of CETH was accompanied by effective dose-dependent correction of morphological changes caused by the adjuvant injection. CETH had relatively high recovery effects in terms of parameters for reducing inflammation, inhibition of osteolysis, reduction in the inflammatory reaction of periarticular tissues, and cartilage degeneration. This study presents for the first time that CETH may be a powerful potential nutraceutical agent or bioactive component in the treatment of rheumatoid arthritis., Competing Interests: The authors declare that they have no competing interests., (© 2021 The Authors. Food Science & Nutrition published by Wiley Periodicals LLC.)

The expression of DDX5 protein (RNA-helicase p68) correlates with processes of proliferation and differentiation. However there is no direct evidence of involvement of the protein in these processes. In present work, we studied the influence of DDX5 protein inactivation by si-RNA on the proliferation of Jurkat cells and dynamic of DDX5 expression during differentiation of U-937 cells induced by phorbol 12-myristate-13-acetate (PMA). We showed that the content of DDX5 in Jurkat cells is less in phases G0/G1 as compare to phases G2/M. The treatment of cells with the antisense LV-shDDX5 was followed by the increase of G0/G1 cells. It was also shown that the increase of expression of the DDX5 protein occurred during the initial stages of differentiation, and the peak of expression was registered during the first 2-3 hours after the induction of the cells, later the DDX5 content decreases. The increase of the number of macrophage surface marker CR3 on the membrane of cells occurred only in 24 hours after induction of the cells by PMA. Thus, these data confirm that: (1) the DDX5 protein is essential for normal cell proliferation; (2) the transition from G1 to S/G2 phase is accompanied by an increase of DDX5 protein concentration in the cells; (3) the concentration of the DDX5 protein increases on early stages of U-937 cells differentiation and after decreases.

Physical connections between mitotic chromosomes have been reported previously. It was assumed that the interchromosome connection was based on the DNA-protein thread. However, the data about DNA sequences and protein component in the thread is fragmentary. We demonstrated on the mouse cultured cell line and prematurely condensed chromosomes that: (a) all four mouse satellite DNA fragments (major and minor satellite, mouse satellite 3 (MS3) and mouse satellite 4 (MS4)) were involved in the thread formation; (b) MS4 was involved in the thread to the least extent among all the other fragments; (c) telomere was never a member of the thread; (d) the thread was synthesized at a late G(2) phase; (e) RNA helicase p68 and CENP-B were among the protein components of the interchromosome connection. It was shown by FACS analysis that in mouse and human cell lines: (1) the flow karyotype spectrums were never free from chromosome aggregates; (2) chromosome association did not depend on the chromosome length and each chromosome was free to associate with the other.

We purified a 68-kDa protein from the mouse nuclear matrix using ion exchange and affinity chromatography. Column fractions were tested for specific binding to mouse minor satellite DNA using a gel mobility shift assay. The protein was identified by mass spectrometry as RNA helicase P68. In fixed cells, P68 was found to shuttle in and out of SC35 domains, forming fibres and granules in a cell-cycle dependent manner. Analysis of the P68 sequence revealed a short potential coiled-coil domain that might be involved in the formation of P68 fibres. Contacts between centromeres and P68 granules were observed during all phases of the cycle but they were most prominent in mitosis. At this stage, P68 was found in both the centromeric regions and the connections between chromosomes. Direct interaction of P68/DEAD box RNA helicase with satellite DNAs in vitro has not been demonstrated for any other members of the RNA helicase family.

Any functions of tandem repetitive sequences need proteins that specifically bind to them. Telomere-binding TRF2/MTBP attaches telomeres to the nuclear envelope in interphase due to its rod-domain-like motif. Interphase nuclei organized as a number of sponge-like ruffly round chromosome territories that could be rotated from outside. SAF-A/hnRNP-U and p68-helicase are proteins suitable to do that. Their location in the interchromosome territory space, ATPase domains, and the ability to be bound by satellite DNAs (satDNA) make them part of the wires used to help chromosome territory rotates. In case of active transcription p68-helicase can be involved in the formation of local "gene expression matrices" and due to its satDNA-binding specificity cause the rearrangement of the local chromosome territory. The marks of chromatin rearrangement, which have to be heritable, could be provided by SAF-A/hnRNP-U. During telophase unfolding the proper chromatin arrangement is restored according to these marks. The structural specificity of both proteins to the satDNAs provides a regulative but relatively stable mode of binding. The structural specificity of protein binding could help to find the "magic" centromeric sequence. With future investigations of proteins with the structural specificity of binding during early embryogenesis, when heterochromatin formation goes on, the molecular mechanisms of the "gene gating" hypothesis (Blobel, 1985) will be confirmed.

A complex of three proteins (of 80, 70, 58 kDa-p80, p70, and p58, respectively) with the ability to bind alphoid DNA (alpha-satDNA) was revealed by gel mobility shift assay (GMSA) in human nuclear matrix. The probes of the alpha-satDNA bound in the GMSA with the greatest specificity, but the complex was capable of binding human satellite 3 fragment. According to ion exchange and affinity chromatography, the complex includes two DNA-binding proteins, p70 and p80, and a non-DNA-binding one, p58, which enhances the specificity of binding to the alpha-satDNA. GMSA, SDS-PAGE and immunoblotting showed that the lamins, as well as constitutive centromeric proteins (CENP-A, CENP-B, CENP-C, CENP-G), were not incorporated into the complex. It was demonstrated by immunoprecipitation assay that p70 and, probably p58, share a common antigen determinant with the rod domain of intermediate filaments (IF) proteins. The results obtained indicate that the nuclear matrix contains at least one IF-related protein that is able to bind specifically to alpha-satDNA in vitro and that this protein is distinct from the lamins., (Copyright 2000 Academic Press.)

A nuclear matrix protein complex has been found which specifically binds to alpha-satellite DNA as revealed by gel shift assay. The complex contains three proteins--two DNA-binding proteins with molecular masses of 70 and 80 kD and a 58-kD protein which does not bind DNA but enhances the specificity of binding of the others to alpha-satellite DNA. The complex does not contain CENP-A, CENP-B, CENP-C, CENP-G, or lamins A/C and B.