Interplay between REST and nucleolin transcription factors: a key mechanism in the overexpression of genes upon increased phosphorylation

Non-malignant cells can be transformed via the activation of kinases that control degradation of neural-restrictive silencer factor (REST). Here, we identify a mechanism that contributes to the activation of genes, expression of which is controlled by responsive elements containing overlapping binding sites for REST and nucleolin. We demonstrate that both phosphorylated and non-phosphorylated nucleolin-bound DNA; however, only phosphorylated nucleolin successfully competed with either full-length REST or a REST-derived DNA-binding peptide, REST68, for binding to the overlapping binding sites. We show that this interplay between the two transcription factors regulates the activation of cell survival and immunomodulatory genes in tumors and non-malignant cells with activated protein kinase C, which is accompanied with alterations in cell proliferation and apoptosis. We propose a model for the regulation of these genes, which brings a new insight into the molecular mechanisms that control cellular transformation driven by activation of protein kinases

Tandemly repeated DNA families in the mouse genome

<p>Abstract</p> <p>Background</p> <p>Functional and morphological studies of tandem DNA repeats, that combine high portion of most genomes, are mostly limited due to the incomplete characterization of these genome elements. We report here a genome wide analysis of the large tandem repeats (TR) found in the mouse genome assemblies.</p> <p>Results</p> <p>Using a bioinformatics approach, we identified large TR with array size more than 3 kb in two mouse whole genome shotgun (WGS) assemblies. Large TR were classified based on sequence similarity, chromosome position, monomer length, array variability, and GC content; we identified four superfamilies, eight families, and 62 subfamilies - including 60 not previously described. 1) The superfamily of centromeric minor satellite is only found in the unassembled part of the reference genome. 2) The pericentromeric major satellite is the most abundant superfamily and reveals high order repeat structure. 3) Transposable elements related superfamily contains two families. 4) The superfamily of heterogeneous tandem repeats includes four families. One family is found only in the WGS, while two families represent tandem repeats with either single or multi locus location. Despite multi locus location, TRPC-21A-MM is placed into a separated family due to its abundance, strictly pericentromeric location, and resemblance to big human satellites.</p> <p>To confirm our data, we next performed <it>in situ </it>hybridization with three repeats from distinct families. TRPC-21A-MM probe hybridized to chromosomes 3 and 17, multi locus TR-22A-MM probe hybridized to ten chromosomes, and single locus TR-54B-MM probe hybridized with the long loops that emerge from chromosome ends. In addition to <it>in silico </it>predicted several extra-chromosomes were positive for TR by <it>in situ </it>analysis, potentially indicating inaccurate genome assembly of the heterochromatic genome regions.</p> <p>Conclusions</p> <p>Chromosome-specific TR had been predicted for mouse but no reliable cytogenetic probes were available before. We report new analysis that identified <it>in silico </it>and confirmed <it>in situ </it>3/17 chromosome-specific probe TRPC-21-MM. Thus, the new classification had proven to be useful tool for continuation of genome study, while annotated TR can be the valuable source of cytogenetic probes for chromosome recognition.</p

Pericentromeric satellite lncRNAs are induced in cancer-associated fibroblasts and regulate their functions in lung tumorigenesis

The abnormal tumor microenvironment (TME) often dictates the therapeutic response of cancer to chemo- and immuno-therapy. Aberrant expression of pericentromeric satellite repeats has been reported for epithelial cancers, including lung cancer. However, the transcription of tandemly repetitive elements in stromal cells of the TME has been unappreciated, limiting the optimal use of satellite transcripts as biomarkers or anti-cancer targets. We found that transcription of pericentromeric satellite DNA (satDNA) in mouse and human lung adenocarcinoma was observed in cancer-associated fibroblasts (CAFs). In vivo, lung fibroblasts expressed pericentromeric satellite repeats HS2/HS3 specifically in tumors. In vitro, transcription of satDNA was induced in lung fibroblasts in response to TGFβ, IL1α, matrix stiffness, direct contact with tumor cells and treatment with chemotherapeutic drugs. Single-cell transcriptome analysis of human lung adenocarcinoma confirmed that CAFs were the cell type with the highest number of satellite transcripts. Human HS2/HS3 pericentromeric transcripts were detected in the nucleus, cytoplasm, extracellularly and co-localized with extracellular vesicles in situ in human biopsies and activated fibroblasts in vitro. The transcripts were transmitted into recipient cells and entered their nuclei. Knock-down of satellite transcripts in human lung fibroblasts attenuated cellular senescence and blocked the formation of an inflammatory CAFs phenotype which resulted in the inhibition of their pro-tumorigenic functions. In sum, our data suggest that satellite long non-coding (lnc) RNAs are induced in CAFs, regulate expression of inflammatory genes and can be secreted from the cells, which potentially might present a new element of cell-cell communication in the TME

Overexpression of Pericentromeric HSAT2 DNA Increases Expression of EMT Markers in Human Epithelial Cancer Cell Lines

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

Human chromosome 1 satellite 3 DNA is decondensed, demethylated and transcribed in senescent cells and in A431 epithelial carcinoma cells

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