RESUMO
Molecular genetic analysis of tumor tissues is the most important step towards understanding the mechanisms of cancer development; it is also necessary for the choice of targeted therapy. The Hi-C (high-throughput chromatin conformation capture) technology can be used to detect various types of genomic variants, including balanced chromosomal rearrangements, such as inversions and translocations. We propose a modification of the Hi-C method for the analysis of chromatin contacts in formalin-fixed paraffin-embedded (FFPE) sections of tumor tissues. The developed protocol allows to generate high-quality Hi-C data and detect all types of chromosomal rearrangements. We have analyzed various databases to compile a comprehensive list of translocations that hold clinical importance for the targeted therapy selection. The practical value of molecular genetic testing is its ability to influence the treatment strategies and to provide prognostic insights. Detecting specific chromosomal rearrangements can guide the choice of the targeted therapies, which is a critical aspect of personalized medicine in oncology.
Assuntos
Formaldeído , Neoplasias , Inclusão em Parafina , Humanos , Neoplasias/genética , Neoplasias/patologia , Formaldeído/química , Translocação Genética , Fixação de Tecidos , Cromatina/genética , Cromatina/metabolismo , Cromatina/químicaRESUMO
Chromosome conformation capture techniques have revolutionized our understanding of chromatin architecture and dynamics at the genome-wide scale. In recent years, these methods have been applied to a diverse array of species, revealing fundamental principles of chromosomal organization. However, structural organization of the extrachromosomal entities, like viral genomes or plasmids, and their interactions with the host genome, remain relatively underexplored. In this work, we introduce an enhanced 4C-protocol tailored for probing plasmid DNA interactions. We design specific plasmid vector and optimize protocol to allow high detection rate of contacts between the plasmid and host DNA.
Assuntos
Plasmídeos , Plasmídeos/metabolismo , Plasmídeos/genética , DNA/química , DNA/genética , Cromatina/genética , Cromatina/metabolismo , Cromatina/química , GenomaRESUMO
Copy number variations (CNVs) of the human CNTN6 gene caused by megabase-scale microdeletions or microduplications in the 3p26.3 region are often the cause of neurodevelopmental disorders, including intellectual disability and developmental delay. Surprisingly, patients with different copy numbers of this gene display notable overlapping of neuropsychiatric symptoms. The complexity of the study of human neuropathologies is associated with the inaccessibility of brain material. This problem can be overcome through the use of reprogramming technologies that permit the generation of induced pluripotent stem (iPS) cells from fibroblasts and their subsequent in vitro differentiation into neurons. We obtained a set of iPS cell lines derived from a patient carrier of the CNTN6 gene duplication and from two healthy donors. All iPS cell lines displayed the characteristics of pluripotent cells. Some iPS cell lines derived from the patient and from healthy donors were differentiated in vitro by exogenous expression of the Ngn2 transcription factor or by spontaneous neural differentiation of iPS cells through the neural rosette stage. The obtained neurons showed the characteristics of mature neurons as judged by the presence of neuronal markers and by their electrophysiological characteristics. Analysis of allele-specific expression of the CNTN6 gene in these neuronal cells by droplet digital PCR demonstrated that the level of expression of the duplicated allele was significantly reduced compared to that of the wild-type allele. Importantly, according to the sequencing data, both copies of the CNTN6 gene, which were approximately 1 Mb in size, showed no any additional structural rearrangements.
Assuntos
Alelos , Duplicação Cromossômica/genética , Cromossomos Humanos Par 3/genética , Contactinas/genética , Regulação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/patologia , Deficiência Intelectual/genética , Neurônios/patologia , Adolescente , Adulto , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Deficiência Intelectual/fisiopatologia , Cariotipagem , Camundongos SCID , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismoRESUMO
Immunofluorescent analysis of markers specific for parental genomes was used to study heterokaryons and hybrid cells soon after the fusion of diploid embryonic stem (ES) cells marked with green fluorescent protein and diploid fibroblasts labeled by blue fluorescent beads. Heterokaryons were identified by an analysis of parental mitochondrial DNAs. Within 20 h after fusion, most heterokaryons (up to 80%) had a fibroblast-like phenotype, being positive for typical fibroblast markers (collagen type I, fibronectin, lamin A/C) and for the modification me3H3K27 chromatin marking the inactive X chromosome but being negative for Oct4 and Nanog. Approximately 20% of heterokaryons had an alternative ES-like phenotype being positive for Oct4 and Nanog, with signs of reactivation of the previously inactive X-chromosome but negative for fibroblast markers. Hybrid cells having alternative phenotypes were easily identified from 24-48 h. The level of DNA methylation at the promoter of the fibroblast Oct4 allele in the ES-like hybrid cells at day 4 was similar to that of ES cells but at the same time, both parental Oct4 alleles were heavily methylated in fibroblast-like hybrid cells. Thus, bidirectional reprogramming initiated at the heterokaryon stage seems to lead to the formation of two types of hybrid cells with alternative dominance of the parental genomes. However, the further fates of two types of hybrid cells are different: ES-like hybrid cells form colonies at 4-6 days but no colonies are derived from the fibroblast-like hybrid cells. The latter grow as disconnected single cells and are unable to form colonies, like mouse embryonic fibroblasts.
Assuntos
Fusão Celular , Reprogramação Celular , Células-Tronco Embrionárias/fisiologia , Fibroblastos/fisiologia , Células Híbridas/fisiologia , Animais , Biomarcadores , Linhagem Celular , Cromatina , Metilação de DNA , DNA Mitocondrial/análise , Imunofluorescência , Corantes Fluorescentes , Genoma , Proteínas de Fluorescência Verde , Proteínas de Homeodomínio/biossíntese , Camundongos , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/biossíntese , Fenótipo , Reação em Cadeia da Polimerase , Cromossomo XRESUMO
Two dozen hybrid clones were produced by fusion of diploid embryonic stem (ES) cells positive for green fluorescent protein (GFP) with tetraploid fibroblasts derived from DD/c and C57BL-I(I)1RK mice. Cytogenetic analysis demonstrated that most cells from these hybrid clones contained near-hexaploid chromosome sets. Additionally, the presence of chromosomes derived from both parental cells was confirmed by polymerase chain reaction (PCR) analysis of polymorphic microsatellites. All hybrid cells were positive for GFP and demonstrated growth characteristics and fibroblast-like morphology. In addition, most hybrid cells were positive for collagen type I, fibronectin, and lamin A/C but were negative for Oct4 and Nanog proteins. Methylation status of the Oct4 and Nanog gene promoters was evaluated by bisulfite genomic sequencing analysis. The methylation sites (CpG-sites) of the Oct4 and Nanog gene promoters were highly methylated in hybrid cells, whereas the CpG-sites were unmethylated in the parental ES cells. Thus, the fibroblast genome dominated the ES genome in the diploid ES cell/tetraploid fibroblast hybrid cells. Immunofluorescent analysis of the pluripotent and fibroblast markers demonstrated that establishment of the fibroblast phenotype occurred shortly after fusion and that the fibroblast phenotype was further maintained in the hybrid cells. Fusion of karyoplasts and cytoplast derived from tetraploid fibroblasts with whole ES cells demonstrated that karyoplasts were able to establish the fibroblast phenotype of the reconstructed cells but not fibroblast cytoplasts. Thus, these data suggest that the dominance of parental genomes in hybrid cells of ES cell/somatic cell type depends on the ploidy of the somatic partner.