RESUMO
The positions of enhancers and promoters on genomic DNA remain poorly understood. Chromosomes cannot be observed during the cell division cycle because the genome forms a chromatin structure and spreads within the nucleus. However, high-throughput chromosome conformation capture (Hi-C) measures the physical interactions of genomes. In previous studies, DNA extrusion loops were directly derived from Hi-C heat maps. Multidimensional Scaling (MDS) is used in this assessment to more precisely locate enhancers and promoters. MDS is a multivariate analysis method that reproduces the original coordinates from the distance matrix between elements. We used Hi-C data of cultured osteosarcoma cells and applied MDS as the distance matrix of the genome. In addition, we selected columns 2 and 3 of the orthogonal matrix U as the desired structure. Overall, the DNA loops from the reconstructed genome structure contained bioprocesses involved in transcription, such as the pre-transcriptional initiation complex and RNA polymerase II initiation complex, and transcription factors involved in cancer, such as Foxm1 and CREB3. Therefore, our results are consistent with the biological findings. Our method is suitable for identifying enhancers and promoters in the genome.
Assuntos
Neoplasias Ósseas/genética , Cromossomos Humanos/química , Elementos Facilitadores Genéticos , Osteossarcoma/genética , Regiões Promotoras Genéticas , Algoritmos , Bases de Dados Genéticas , Genômica/métodos , Humanos , Análise de Escalonamento Multidimensional , Conformação de Ácido Nucleico , Células Tumorais CultivadasRESUMO
We investigated genome folding across the eukaryotic tree of life. We find two types of three-dimensional (3D) genome architectures at the chromosome scale. Each type appears and disappears repeatedly during eukaryotic evolution. The type of genome architecture that an organism exhibits correlates with the absence of condensin II subunits. Moreover, condensin II depletion converts the architecture of the human genome to a state resembling that seen in organisms such as fungi or mosquitoes. In this state, centromeres cluster together at nucleoli, and heterochromatin domains merge. We propose a physical model in which lengthwise compaction of chromosomes by condensin II during mitosis determines chromosome-scale genome architecture, with effects that are retained during the subsequent interphase. This mechanism likely has been conserved since the last common ancestor of all eukaryotes.
Assuntos
Adenosina Trifosfatases/genética , Adenosina Trifosfatases/fisiologia , Evolução Biológica , Cromossomos/ultraestrutura , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Eucariotos/genética , Genoma , Complexos Multiproteicos/genética , Complexos Multiproteicos/fisiologia , Adenosina Trifosfatases/química , Algoritmos , Animais , Nucléolo Celular/ultraestrutura , Núcleo Celular/ultraestrutura , Centrômero/ultraestrutura , Cromossomos/química , Cromossomos Humanos/química , Cromossomos Humanos/ultraestrutura , Proteínas de Ligação a DNA/química , Genoma Humano , Genômica , Heterocromatina/ultraestrutura , Humanos , Interfase , Mitose , Modelos Biológicos , Complexos Multiproteicos/química , Telômero/ultraestruturaRESUMO
Human chromosomes are large spatially and hierarchically structured entities, the integrity of which needs to be preserved throughout the lifespan of the cell and in conjunction with cell cycle progression. Preservation of chromosomal structure is important for proper deployment of cell type-specific gene expression programs. Thus, aberrations in the integrity and structure of chromosomes will predictably lead to disease, including cancer. Here, we provide an updated standpoint with respect to chromatin misfolding and the emergence of various cancer types. We discuss recent studies implicating the disruption of topologically associating domains, switching between active and inactive compartments, rewiring of promoter-enhancer interactions in malignancy as well as the effects of single nucleotide polymorphisms in non-coding regions involved in long-range regulatory interactions. In light of these findings, we argue that chromosome conformation studies may now also be useful for patient diagnosis and drug target discovery.
Assuntos
Montagem e Desmontagem da Cromatina , Cromossomos Humanos/química , Neoplasias/patologia , Regiões Promotoras Genéticas , Cromossomos Humanos/genética , Genoma Humano , Humanos , Neoplasias/etiologiaRESUMO
Many studies have shown that the spatial distribution of genes within a single chromosome exhibits distinct patterns. However, little is known about the characteristics of inter-chromosomal distribution of genes (including protein-coding genes, processed transcripts and pseudogenes) in different genomes. In this study, we explored these issues using the available genomic data of both human and model organisms. Moreover, we also analyzed the distribution pattern of protein-coding genes that have been associated with 14 common diseases and the insert/deletion mutations and single nucleotide polymorphisms detected by whole genome sequencing in an acute promyelocyte leukemia patient. We obtained the following novel findings. Firstly, inter-chromosomal distribution of genes displays a nonstochastic pattern and the gene densities in different chromosomes are heterogeneous. This kind of heterogeneity is observed in genomes of both lower and higher species. Secondly, protein-coding genes involved in certain biological processes tend to be enriched in one or a few chromosomes. Our findings have added new insights into our understanding of the spatial distribution of genome and disease- related genes across chromosomes. These results could be useful in improving the efficiency of disease-associated gene screening studies by targeting specific chromosomes.
Assuntos
Doença das Coronárias/genética , Epistasia Genética , Lúpus Eritematoso Sistêmico/genética , Neoplasias/genética , Doenças Neurodegenerativas/genética , Acidente Vascular Cerebral/genética , Animais , Composição de Bases , Caenorhabditis elegans/genética , Mapeamento Cromossômico/estatística & dados numéricos , Cromossomos Humanos/química , Doença das Coronárias/diagnóstico , Doença das Coronárias/patologia , Drosophila melanogaster/genética , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos , Lúpus Eritematoso Sistêmico/diagnóstico , Lúpus Eritematoso Sistêmico/patologia , Camundongos , Neoplasias/classificação , Neoplasias/diagnóstico , Neoplasias/patologia , Doenças Neurodegenerativas/classificação , Doenças Neurodegenerativas/diagnóstico , Doenças Neurodegenerativas/patologia , Fases de Leitura Aberta , Acidente Vascular Cerebral/diagnóstico , Acidente Vascular Cerebral/patologia , Peixe-Zebra/genéticaRESUMO
BACKGROUND: Three-dimensional spatial organization of chromosomes is defined by highly self-interacting regions 0.1-1 Mb in size termed Topological Associating Domains (TADs). Genetic factors that explain dynamic variation in TAD structure are not understood. We hypothesize that common structural variation (SV) in the human population can disrupt regulatory sequences and thereby influence TAD formation. To determine the effects of SVs on 3D chromatin organization, we performed chromosome conformation capture sequencing (Hi-C) of lymphoblastoid cell lines from 19 subjects for which SVs had been previously characterized in the 1000 genomes project. We tested the effects of common deletion polymorphisms on TAD structure by linear regression analysis of nearby quantitative chromatin interactions (contacts) within 240 kb of the deletion, and we specifically tested the hypothesis that deletions at TAD boundaries (TBs) could result in large-scale alterations in chromatin conformation. RESULTS: Large (> 10 kb) deletions had significant effects on long-range chromatin interactions. Deletions were associated with increased contacts that span the deleted region and this effect was driven by large deletions that were not located within a TAD boundary (nonTB). Some deletions at TBs, including a 80 kb deletion of the genes CFHR1 and CFHR3, had detectable effects on chromatin contacts. However for TB deletions overall, we did not detect a pattern of effects that was consistent in magnitude or direction. Large inversions in the population had a distinguishable signature characterized by a rearrangement of contacts that span its breakpoints. CONCLUSIONS: Our study demonstrates that common SVs in the population impact long-range chromatin structure, and deletions and inversions have distinct signatures. However, the effects that we observe are subtle and variable between loci. Genome-wide analysis of chromatin conformation in large cohorts will be needed to quantify the influence of common SVs on chromatin structure.
Assuntos
Cromatina/química , Cromossomos Humanos/genética , Variação Estrutural do Genoma , Linhagem Celular Tumoral , Cromatina/genética , Montagem e Desmontagem da Cromatina , Cromossomos Humanos/química , Elementos Facilitadores Genéticos , Humanos , Modelos Lineares , Deleção de Sequência , Inversão de SequênciaRESUMO
Carcinogenesis is caused by genome instability, one of the major causes of which is double-strand DNA breaks (DSBs). Interestingly, infection by particular species of bacteria can induce DSBs in host cells. For example, several reports suggest an association between periodontal disease and oral cancer. Aggregatibacter actinomycetemcomitans, a common periodontal pathogen, causes DSBs in the host cell. Pulsed-field gel electrophoresis (PFGE) is often used to identify DSBs in host cells. However, as established during investigation of A. actinomycetemcomitans infection, it is often difficult to determine whether broken DNA fragments are indeed from human chromosomes or whether they are bacterial in origin using PFGE-based methods. Because the method involves the coculture of human cells with bacteria, both bacterial and human DNA fragments may be present in the broken DNA fraction. To address this problem, we have developed a method to detect only human chromosomal DNA upon PFGE analysis. Human chromosomes were prelabeled with halogenated deoxyuridine (e.g., BrdU and IdU) before being fractionated by PFGE and visualized by immunoblotting. As proof of concept, we successfully used this method to investigate the mechanism of DSB formation in host chromosomes following infection with genotoxic bacterial species.
Assuntos
Aggregatibacter actinomycetemcomitans , Cromossomos Humanos/metabolismo , Quebras de DNA de Cadeia Dupla , Eletroforese em Gel de Campo Pulsado , Infecções por Pasteurellaceae/metabolismo , Linhagem Celular , Cromossomos Humanos/química , Humanos , Infecções por Pasteurellaceae/patologiaRESUMO
Human herpesviruses 6A and 6B (HHV-6A and HHV-6B) are human viruses capable of chromosomal integration. Approximately 1% of the human population carries one copy of HHV-6A/B integrated into every cell in their body, referred to as inherited chromosomally integrated human herpesvirus 6A/B (iciHHV-6A/B). Whether iciHHV-6A/B is transcriptionally active in vivo and how it shapes the immunological response are still unclear. In this study, we screened DNA sequencing (DNA-seq) and transcriptome sequencing (RNA-seq) data for 650 individuals available through the Genotype-Tissue Expression (GTEx) project and identified 2 iciHHV-6A- and 4 iciHHV-6B-positive candidates. When corresponding tissue-specific gene expression signatures were analyzed, low levels HHV-6A/B gene expression was found across multiple tissues, with the highest levels of gene expression in the brain (specifically for HHV-6A), testis, esophagus, and adrenal gland. U90 and U100 were the most highly expressed HHV-6 genes in both iciHHV-6A- and iciHHV-6B-positive individuals. To assess whether tissue-specific gene expression from iciHHV-6A/B influences the immune response, a cohort of 15,498 subjects was screened and 85 iciHHV-6A/B+ subjects were identified. Plasma samples from iciHHV-6A/B+ and age- and sex-matched controls were analyzed for antibodies to control antigens (cytomegalovirus [CMV], Epstein-Barr virus [EBV], and influenza virus [FLU]) or HHV-6A/B antigens. Our results indicate that iciHHV-6A/B+ subjects have significantly more antibodies against the U90 gene product (IE1) than do non-iciHHV-6-positive individuals. Antibody responses against EBV and FLU antigens or HHV-6A/B gene products either not expressed or expressed at low levels, such as U47, U57, and U72, were identical between controls and iciHHV-6A/B+ subjects. CMV-seropositive individuals with iciHHV-6A/B+ have more antibodies against CMV pp150 than do CMV-seropositive controls. These results argue that spontaneous gene expression from integrated HHV-6A/B leads to an increase in antigenic burden that translates into a more robust HHV-6A/B-specific antibody response.IMPORTANCE HHV-6A and -6B are human herpesviruses that have the unique property of being able to integrate into the telomeric regions of human chromosomes. Approximately 1% of the world's population carries integrated HHV-6A/B genome in every cell of their body. Whether viral genes are transcriptionally active in these individuals is unclear. By taking advantage of a unique tissue-specific gene expression data set, we showed that the majority of tissues from iciHHV-6 individuals do not show HHV-6 gene expression. Brain and testes showed the highest tissue-specific expression of HHV-6 genes in two separate data sets. Two HHV-6 genes, U90 (immediate early 1 protein) and U100 (glycoproteins Q1 and Q2), were found to be selectively and consistently expressed across several human tissues. Expression of U90 translates into an increase in antigen-specific antibody response in iciHHV-6A/B+ subjects relative to controls. Future studies will be needed to determine the mechanism of gene expression, the effects of these genes on human gene transcription networks, and the pathophysiological impact of having increased viral protein expression in tissue in conjunction with increased antigen-specific antibody production.
Assuntos
Anticorpos Antivirais/sangue , Cromossomos Humanos/química , Herpesvirus Humano 6/genética , RNA Viral/genética , Infecções por Roseolovirus/virologia , Glândulas Suprarrenais/imunologia , Glândulas Suprarrenais/virologia , Idoso , Encéfalo/imunologia , Encéfalo/virologia , Estudos de Coortes , Citomegalovirus/imunologia , Esôfago/imunologia , Esôfago/virologia , Feminino , Herpesvirus Humano 4/imunologia , Herpesvirus Humano 6/classificação , Herpesvirus Humano 6/imunologia , Humanos , Padrões de Herança , Masculino , Pessoa de Meia-Idade , Especificidade de Órgãos , Orthomyxoviridae/imunologia , Filogenia , RNA Viral/imunologia , Infecções por Roseolovirus/genética , Infecções por Roseolovirus/imunologia , Testículo/imunologia , Testículo/virologia , Integração Viral , Sequenciamento Completo do GenomaRESUMO
Fluorescence in situ hybridization (FISH) is used to examine chromosomal abnormalities and DNA damage. Developed in the early 1980s, this technique remains an important tool for understanding chromosome biology and diagnosing genetic disease and cancer. Use of FISH on metaphase chromosomes allows the visualization of chromosomal abnormalities at specific loci. Here, we describe methods for creating metaphase chromosome spreads and the use of telomere FISH probes to detect chromosome ends.
Assuntos
Cromossomos Humanos/genética , Hibridização in Situ Fluorescente/métodos , Metáfase , Imagem Molecular/métodos , Telômero/genética , Linhagem Celular , Cromossomos Humanos/química , DNA/genética , Humanos , Microscopia de Fluorescência/métodos , Sondas Moleculares/química , Telômero/químicaRESUMO
Complexity in genome architecture determines how gene expression programs are established, maintained, and modified from early developmental stages to normal adult phenotypes. Large scale and hierarchical organization of the genome impacts various aspects of cell functions, ranging from X-chromosome inactivation, stem-cell fate determination to transcription, DNA replication, and cellular repair. While chromatin loops and topologically-associated domains represent a basic structural or fundamental unit of chromatin organization, spatio-temporal organization of the genome further creates a complex network of interacting genome patterns, forming chromosomal compartments and chromosome territories. The understanding of human diseases, including cancers, auto-immune disorders, Alzheimer's, and cardiovascular diseases, relies on the associated molecular and epigenetic mechanisms. There is a growing interest in the impact of three-dimensional chromatin folding upon the genome structure and function, which gives rise to the question "What's in the fold?" and is the main focus of this review. Here we discuss the principles determining the spatial and regulatory relationships between gene regulation and three-dimensional chromatin landscapes, and how changes in chromatin-folding could influence the outcome of genome function in healthy and disease states.
Assuntos
Cromatina/química , Cromossomos Humanos/química , Cromossomos Humanos/genética , Doença/genética , Regulação da Expressão Gênica , Genoma Humano , HumanosRESUMO
Efforts are being directed to systematically analyze the non-coding regions of the genome for cancer-driving mutations1-6. cis-regulatory elements (CREs) represent a highly enriched subset of the non-coding regions of the genome in which to search for such mutations. Here we use high-throughput chromosome conformation capture techniques (Hi-C) for 19,023 promoter fragments to catalog the regulatory landscape of colorectal cancer in cell lines, mapping CREs and integrating these with whole-genome sequence and expression data from The Cancer Genome Atlas7,8. We identify a recurrently mutated CRE interacting with the ETV1 promoter affecting gene expression. ETV1 expression influences cell viability and is associated with patient survival. We further refine our understanding of the regulatory effects of copy-number variations, showing that RASL11A is targeted by a previously identified enhancer amplification1. This study reveals new insights into the complex genetic alterations driving tumor development, providing a paradigm for employing chromosome conformation capture to decipher non-coding CREs relevant to cancer biology.
Assuntos
Transformação Celular Neoplásica/genética , Cromossomos Humanos/química , Códon sem Sentido , Neoplasias Colorretais/genética , Biologia Computacional/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Células CACO-2 , Neoplasias Colorretais/epidemiologia , DNA de Neoplasias/química , Bases de Dados Genéticas , Frequência do Gene , Células HT29 , Células HeLa , Células Hep G2 , Humanos , Células K562 , Células MCF-7 , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Células Tumorais CultivadasRESUMO
While the vast majority of cellular DNA in eukaryotes is contained in long linear strands in chromosomes, we have long recognized some exceptions like mitochondrial DNA, plasmids in yeasts, and double minutes (DMs) in cancer cells where the DNA is present in extrachromosomal circles. In addition, specialized extrachromosomal circles of DNA (eccDNA) have been noted to arise from repetitive genomic sequences like telomeric DNA or rDNA. Recently eccDNA arising from unique (nonrepetitive) DNA have been discovered in normal and malignant cells, raising interesting questions about their biogenesis, function and clinical utility. Here, we review recent results and future directions of inquiry on these new forms of eccDNA.
Assuntos
DNA Circular/genética , DNA Mitocondrial/genética , DNA de Neoplasias/genética , Neoplasias/genética , Células Neoplásicas Circulantes/química , Animais , Cromossomos Humanos/química , Cromossomos Humanos/metabolismo , DNA de Cloroplastos/química , DNA de Cloroplastos/genética , DNA de Cloroplastos/metabolismo , DNA Circular/química , DNA Circular/metabolismo , DNA de Cinetoplasto/química , DNA de Cinetoplasto/genética , DNA de Cinetoplasto/metabolismo , DNA Mitocondrial/química , DNA Mitocondrial/metabolismo , DNA de Neoplasias/química , DNA de Neoplasias/metabolismo , Células Eucarióticas/química , Células Eucarióticas/metabolismo , Humanos , Kinetoplastida/genética , Kinetoplastida/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Células Neoplásicas Circulantes/metabolismo , Plantas/genética , Plantas/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Telômero/química , Telômero/metabolismoRESUMO
DNA double-strand breaks (DSBs) occurring within fragile zones of less than 200 base pairs account for the formation of the most common human chromosomal translocations in lymphoid malignancies, yet the mechanism of how breaks occur remains unknown. Here, we have transferred human fragile zones into S. cerevisiae in the context of a genetic assay to understand the mechanism leading to DSBs at these sites. Our findings indicate that a combination of factors is required to sensitize these regions. Foremost, DNA strand separation by transcription or increased torsional stress can expose these DNA regions to damage from either the expression of human AID or increased oxidative stress. This damage causes DNA lesions that, if not repaired quickly, are prone to nuclease cleavage, resulting in DSBs. Our results provide mechanistic insight into why human neoplastic translocation fragile DNA sequences are more prone to enzymes or agents that cause longer-lived DNA lesions.
Assuntos
Cromossomos Humanos/genética , Citidina Desaminase/genética , Quebras de DNA de Cadeia Dupla , DNA Fúngico/genética , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/genética , Translocação Genética , Cromossomos Humanos/química , Cromossomos Humanos/metabolismo , Citidina Desaminase/metabolismo , DNA Fúngico/química , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA , Endonucleases/genética , Endonucleases/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Humanos , Conformação de Ácido Nucleico , Peroxidases/genética , Peroxidases/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade , Transcrição Gênica , Uracila-DNA Glicosidase/genética , Uracila-DNA Glicosidase/metabolismoRESUMO
Exploring gene function is critical for understanding the complexity of life. DNA sequences and the three-dimensional organization of chromatin (chromosomal interactions) are considered enigmatic factors underlying gene function, and interactions between two distant fragments can regulate transactivation activity via mediator proteins. Thus, a series of chromosome conformation capture techniques have been developed, including chromosome conformation capture (3C), circular chromosome conformation capture (4C), chromosome conformation capture carbon copy (5C), and high-resolution chromosome conformation capture (Hi-C). The application of these techniques has expanded to various fields, but cancer remains one of the major topics. Interactions mediated by proteins or long noncoding RNAs (lncRNAs) are typically found using 4C-sequencing and chromatin interaction analysis by paired-end tag sequencing (ChIA-PET). Currently, Hi-C is used to identify chromatin loops between cancer risk-associated single-nucleotide polymorphisms (SNPs) found by genome-wide association studies (GWAS) and their target genes. Chromosomal conformations are responsible for altered gene regulation through several typical mechanisms and contribute to the biological behavior and malignancy of different tumors, particularly prostate cancer, breast cancer and hematologic neoplasms. Moreover, different subtypes may exhibit different 3D-chromosomal conformations. Thus, C-tech can be used to help diagnose cancer subtypes and alleviate cancer progression by destroying specific chromosomal conformations. Here, we review the fundamentals and improvements in chromosome conformation capture techniques and their clinical applications in cancer to provide insight for future research.
Assuntos
Cromatina/genética , Cromossomos Humanos/química , Neoplasias/genética , Cromatina/química , Mapeamento Cromossômico , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Humanos , Conformação de Ácido NucleicoRESUMO
Condensin complexes play crucial roles in chromosome condensation that is a fundamental process to establish the "rod-like" shape of chromosome structure in mitosis. Failure of the chromosome assembly causes chromosome segregation errors and subsequent genomic instability. However, a molecular mechanism that controls condensin function for the chromosomal organization has not been fully understood. Here, we show that the abundance of CAP-H2, one of the condensin II subunits, is fluctuated during the cell cycle in accordance with Plk1 kinase activity. Inhibition of Plk1 leads to Cdc20-mediated degradation of CAP-H2 in mitosis. Plk1 phosphorylation of CAP-H2 at Ser288 is required for the accumulation of CAP-H2 and accurate chromosomal condensation during prophase. These findings suggest that Plk1 phosphorylation regulates condensin II function by modulating CAP-H2 expression levels to facilitate proper mitotic chromosome organization.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromossomos Humanos/química , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Adenosina Trifosfatases/genética , Proteínas Cdc20/metabolismo , Ciclo Celular , Linhagem Celular , Cromossomos Humanos/metabolismo , Proteínas de Ligação a DNA/genética , Células HEK293 , Células HeLa , Humanos , Mitose , Complexos Multiproteicos/genética , Fosforilação , Quinase 1 Polo-LikeRESUMO
STUDY QUESTION: Can simultaneous comprehensive chromosome screening (CCS) and gene expression analysis be performed on the same biopsy of preimplantation human embryos? SUMMARY ANSWER: For the first time, CCS and reliable gene expression analysis have been performed on the same human preimplantation embryo biopsy. WHAT IS KNOWN ALREADY: A single trophectoderm (TE) biopsy is routinely used for many IVF programs offering CCS for selection of only chromosomally normal embryos for transfer. Although the gene expression profiling of human preimplantation embryos has been described, to date no protocol allows for simultaneous CCS and gene expression profiling from a single TE biopsy. STUDY DESIGN, SIZE AND DURATION: This is a proof of concept and validation study structured in two phases. In Phase 1, cell lines were subjected to a novel protocol for combined CCS and gene expression analysis so as to validate the accuracy and reliability of the proposed protocol. In Phase 2, 20 donated human blastocysts were biopsied and processed with the proposed protocol in order to obtain an accurate CCS result and characterize their gene expression profiles using the same starting material. PARTICIPANTS/MATERIALS, SETTING AND METHOD: A novel protocol coupling quantitative real-time PCR-based CCS and gene expression analysis using RT-PCR was designed for this study. Phase 1: six-cell aliquots of well-characterized fibroblast cell lines (GM00323, 46,XY and GM04435, 48,XY,+16,+21) were subjected to the proposed protocol. CCS results were compared with the known karyotypes for consistency, and gene expression levels were compared with levels of purified RNA from same cell lines for validation of reliable gene expression profiling. Phase 2: four biopsies were performed on 20 frozen human blastocysts previously diagnosed as trisomy 21 (10 embryos) and monosomy 21 (10 embryos) by CCS. All samples were processed with the proposed protocol and re-evaluated for concordance with the original CCS result. Their gene expression profiles were characterized and differential gene expression among embryos and early embryonic cell lineages was also evaluated. MAIN RESULTS AND THE ROLE OF CHANCE: CCS results from cell lines showed 100% consistency with their known karyotypes. ΔΔCt values of differential gene expression of four selected target genes from the cell lines GM4435 and GM0323 were comparable between six-cell aliquots and purified RNA (Collagen type I alpha-1 (COL1A1), P = 0.54; Fibroblast growth factor-5 (FGF5), P = 0.11; Laminin subunit beta-1 (LAMB1), P = 1.00 and Atlastin-1 (ATL1), P = 0.23). With respect to human blastocysts, 92% consistency was reported after comparing embryonic CCS results with previous diagnosis. A total of 30 genes from a human stem cell pluripotency panel were selected to evaluate gene expression in human embryos. Correlation coefficients of expression profiles from biopsies of the same embryo (r = 0.96 ± 0.03 (standard deviation), n = 45) were significantly higher than when biopsies from unrelated embryos were evaluated (r = 0.93 ± 0.03, n = 945) (P < 0.0001). Growth differentiation factor 3 (GDF3) was found to be significantly up-regulated in the inner cell mass (ICM), whereas Caudal type homebox protein-2 (CDX2), Laminin subunit alpha-1 (LAMA1) and DNA methyltransferase 3-beta (DNMT3B) showed down-regulation in ICM compared with TE. Trisomy 21 embryos showed significant up-regulation of markers of cell differentiation (Cadherin-5 (CDH5) and Laminin subunit gamma-1 (LAMC1)), whereas monosomy 21 blastocysts showed higher expression of genes reported to be expressed in undifferentiated cells (Gamma-Aminobutyric Acid Type-A Receptor Beta3 Subunit (GABRB3) and GDF3). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Gene expression profiles of chromosomally normal embryos were not assessed due to restrictive access to euploid embryos for research. Nonetheless, the profile of blastocysts with single aneuploidies was characterized and compared. Only 30 target genes were analyzed for gene expression in this study. Increasing the number of target genes will provide a more comprehensive transcriptomic signature and reveal potential pathways paramount for embryonic competence and correct development. WIDER IMPLICATIONS OF THE FINDINGS: This is the first time that CCS and gene expression analysis have been performed on the same human preimplantation embryo biopsy. Further optimization of this protocol with other CCS platforms and inclusion of more target genes will provide innumerable research and clinical applications, such as discovery of biomarkers for embryonic reproductive potential and characterization of the transcriptomic signatures of embryos, potentially allowing for further embryo selection prior to embryo transfer and therefore improving outcomes. STUDY FUNDING AND COMPETING INTERESTS: This study was funded by the Foundation for Embryonic Competence, Basking Ridge, NJ, USA. No conflicts of interests declared.
Assuntos
Cromossomos Humanos/química , Desenvolvimento Embrionário/genética , Perfilação da Expressão Gênica/métodos , Testes Genéticos/métodos , Diagnóstico Pré-Implantação/métodos , Biópsia , Blastocisto , Linhagem Celular , Cromossomos Humanos Par 21/genética , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Síndrome de Down/diagnóstico , Síndrome de Down/genética , Síndrome de Down/patologia , Técnicas de Cultura Embrionária , Embrião de Mamíferos , Feminino , Fator 5 de Crescimento de Fibroblastos/genética , Fator 5 de Crescimento de Fibroblastos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Humanos , Cariotipagem , Laminina/genética , Laminina/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Monossomia/diagnóstico , Monossomia/genética , Monossomia/patologia , Gravidez , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We developed a targeted chromosome conformation capture (4C) approach that uses unique molecular identifiers (UMIs) to derive high-complexity quantitative chromosome contact profiles with controlled signal-to-noise ratios. UMI-4C detects chromosomal interactions with improved sensitivity and specificity, and it can easily be multiplexed to allow robust comparison of contact distributions between loci and conditions. This approach may open the way to the incorporation of contact distributions into quantitative models of gene regulation.
Assuntos
Mapeamento Cromossômico/métodos , Cromossomos Humanos/química , Cromossomos Humanos/metabolismo , Regulação da Expressão Gênica , Genômica/métodos , Leucemia/genética , Cromatina/genética , Interpretação Estatística de Dados , Loci Gênicos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Conformação de Ácido Nucleico , Razão Sinal-Ruído , Software , Células Tumorais Cultivadas , Globinas beta/genéticaRESUMO
We have found that reagents that reduce oxidized cysteines lead to destabilization of metaphase chromosome folding, suggesting that chemically linked cysteine residues may play a structural role in mitotic chromosome organization, in accord with classical studies by Dounce et al. (J Theor Biol 42:275-285, 1973) and Sumner (J Cell Sci 70:177-188, 1984a). Human chromosomes isolated into buffer unfold when exposed to dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP). In micromanipulation experiments which allow us to examine the mechanics of individual metaphase chromosomes, we have found that the gel-like elastic stiffness of native metaphase chromosomes is dramatically suppressed by DTT and TCEP, even before the chromosomes become appreciably unfolded. We also report protein labeling experiments on human metaphase chromosomes which allow us to tag oxidized and reduction-sensitive cysteine residues. PAGE analysis using fluorescent labels shows a small number of labeled bands. Mass spectrometry analysis of similarly labeled proteins provides a list of candidates for proteins with oxidized cysteines involved in chromosome organization, notably including components of condensin I, cohesin, the nucleosome-interacting proteins RCC1 and RCC2, as well as the RNA/DNA-binding protein NONO/p54NRB.
Assuntos
Proteínas Cromossômicas não Histona/química , Cromossomos Humanos/química , Cromossomos Humanos/ultraestrutura , Cisteína/química , Metáfase/genética , Adenosina Trifosfatases/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ditiotreitol/química , Eletroforese em Gel Bidimensional , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , Humanos , Cariótipo , Espectrometria de Massas , Micromanipulação , Complexos Multiproteicos/metabolismo , Notophthalmus viridescens/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição de Octâmero/metabolismo , Oxirredução , Fosfinas/química , Proteínas de Ligação a RNA/metabolismo , CoesinasRESUMO
Chromothripsis is a recently observed phenomenon in cancer cells in which one or several chromosomes shatter into pieces with subsequent inaccurate reassembly and clonal propagation. This type of event generates a potentially vast number of mutations within a relatively short-time period, and has been considered as a new paradigm in cancer development. Despite recent advances, much work is still required to better understand the molecular mechanisms of this phenomenon, and thus an easy-to-use tool is in urgent need for automatically detecting and annotating chromothripsis. Here we present CTLPScanner, a web server for detection of chromothripsis-like pattern (CTLP) in genomic array data. The output interface presents intuitive graphical representations of detected chromosome pulverization region, as well as detailed results in table format. CTLPScanner also provides additional information for associated genes in chromothripsis region to help identify the potential candidates involved in tumorigenesis. To assist in performing meta-data analysis, we integrated over 50 000 pre-processed genomic arrays from The Cancer Genome Atlas and Gene Expression Omnibus into CTLPScanner. The server allows users to explore the presence of chromothripsis signatures from public data resources, without carrying out any local data processing. CTLPScanner is freely available at http://cgma.scu.edu.cn/CTLPScanner/.
Assuntos
Transformação Celular Neoplásica/genética , Cromossomos Humanos/química , Cromotripsia , Proteínas de Neoplasias/genética , Neoplasias/genética , Interface Usuário-Computador , Atlas como Assunto , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Mapeamento Cromossômico , Gráficos por Computador , Bases de Dados Genéticas , Humanos , Armazenamento e Recuperação da Informação , Internet , Taxa de Mutação , Proteínas de Neoplasias/metabolismo , Neoplasias/classificação , Neoplasias/patologia , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Mammalian chromosome ends are protected by nucleoprotein structures called telomeres. Telomeres ensure genome stability by preventing chromosome termini from being recognized as DNA damage. Telomere length homeostasis is inevitable for telomere maintenance because critical shortening or over-lengthening of telomeres may lead to DNA damage response or delay in DNA replication, and hence genome instability. Due to their repetitive DNA sequence, unique architecture, bound shelterin proteins, and high propensity to form alternate/secondary DNA structures, telomeres are like common fragile sites and pose an inherent challenge to the progression of DNA replication, repair, and recombination apparatus. It is conceivable that longer the telomeres are, greater is the severity of such challenges. Recent studies have linked excessively long telomeres with increased tumorigenesis. Here we discuss telomere abnormalities in a rare recessive chromosomal instability disorder called Fanconi Anemia and the role of the Fanconi Anemia pathway in telomere biology. Reports suggest that Fanconi Anemia proteins play a role in maintaining long telomeres, including processing telomeric joint molecule intermediates. We speculate that ablation of the Fanconi Anemia pathway would lead to inadequate aberrant structural barrier resolution at excessively long telomeres, thereby causing replicative burden on the cell.