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1.
Exp Cell Res ; 422(1): 113431, 2023 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-36423660

RESUMO

The genomes of immortalized cell lines (and cancer cells) are characterized by multiple types of aberrations, ranging from single nucleotide polymorphisms (SNPs) to structural rearrangements that have accumulated over time. Consequently, it is difficult to estimate the relative impact of different aberrations, the order of events, and which gene functions were under selective pressure at the early stage towards cellular immortalization. Here, we have established novel cell cultures derived from Drosophila melanogaster embryos that were sampled at multiple time points over a one-year period. Using short-read DNA sequencing, we show that copy-number gain in preferentially stress-related genes were acquired in a dominant fraction of cells in 300-days old cultures. Furthermore, transposable elements were active in cells of all cultures. Only a few (<1%) SNPs could be followed over time, and these showed no trend to increase or decrease. We conclude that the early cellular responses of a novel culture comprise sequence duplication and transposable element activity. During immortalization, positive selection first occurs on genes that are related to stress response before shifting to genes that are related to growth.


Assuntos
Drosophila melanogaster , Duplicação Gênica , Animais , Drosophila melanogaster/genética , Análise de Sequência de DNA , Linhagem Celular , Elementos de DNA Transponíveis/genética
2.
BMC Genomics ; 23(1): 276, 2022 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-35392795

RESUMO

BACKGROUND: Immortalized cell lines are widely used model systems whose genomes are often highly rearranged and polyploid. However, their genome structure is seldom deciphered and is thus not accounted for during analyses. We therefore used linked short- and long-read sequencing to perform haplotype-level reconstruction of the genome of a Drosophila melanogaster cell line (S2-DRSC) with a complex genome structure. RESULTS: Using a custom implementation (that is designed to use ultra-long reads in complex genomes with nested rearrangements) to call structural variants (SVs), we found that the most common SV was repetitive sequence insertion or deletion (> 80% of SVs), with Gypsy retrotransposon insertions dominating. The second most common SV was local sequence duplication. SNPs and other SVs were rarer, but several large chromosomal translocations and mitochondrial genome insertions were observed. Haplotypes were highly similar at the nucleotide level but structurally very different. Insertion SVs existed at various haplotype frequencies and were unlinked on chromosomes, demonstrating that haplotypes have different structures and suggesting the existence of a mechanism that allows SVs to propagate across haplotypes. Finally, using public short-read data, we found that transposable element insertions and local duplications are common in other D. melanogaster cell lines. CONCLUSIONS: The S2-DRSC cell line evolved through retrotransposon activity and vast local sequence duplications, that we hypothesize were the products of DNA re-replication events. Additionally, mutations can propagate across haplotypes (possibly explained by mitotic recombination), which enables fine-tuning of mutational impact and prevents accumulation of deleterious events, an inherent problem of clonal reproduction. We conclude that traditional linear homozygous genome representation conceals the complexity when dealing with rearranged and heterozygous clonal cells.


Assuntos
Drosophila melanogaster , Genoma Mitocondrial , Animais , Linhagem Celular , Drosophila/genética , Drosophila melanogaster/genética , Haplótipos , Reprodução , Retroelementos/genética , Análise de Sequência de DNA
3.
J Biol Chem ; 293(37): 14342-14358, 2018 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-30068546

RESUMO

Polycomb group proteins are essential epigenetic repressors. They form multiple protein complexes of which two kinds, PRC1 and PRC2, are indispensable for repression. Although much is known about their biochemical properties, how mammalian PRC1 and PRC2 are targeted to specific genes is poorly understood. Here, we establish the cyclin D2 (CCND2) oncogene as a simple model to address this question. We provide the evidence that the targeting of PRC1 to CCND2 involves a dedicated PRC1-targeting element (PTE). The PTE appears to act in concert with an adjacent cytosine-phosphate-guanine (CpG) island to arrange for the robust binding of PRC1 and PRC2 to repressed CCND2 Our findings pave the way to identify sequence-specific DNA-binding proteins implicated in the targeting of mammalian PRC1 complexes and provide novel link between polycomb repression and cancer.


Assuntos
Ciclina D2/genética , Ciclina D2/metabolismo , Oncogenes , Proteínas do Grupo Polycomb/metabolismo , Animais , Sítios de Ligação , Inativação Gênica , Humanos , Camundongos , Ligação Proteica , Transcrição Gênica
4.
Nucleic Acids Res ; 45(17): e152, 2017 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-28973466

RESUMO

Hi-C experiments generate data in form of large genome contact maps (Hi-C maps). These show that chromosomes are arranged in a hierarchy of three-dimensional compartments. But to understand how these compartments form and by how much they affect genetic processes such as gene regulation, biologists and bioinformaticians need efficient tools to visualize and analyze Hi-C data. However, this is technically challenging because these maps are big. In this paper, we remedied this problem, partly by implementing an efficient file format and developed the genome contact map explorer platform. Apart from tools to process Hi-C data, such as normalization methods and a programmable interface, we made a graphical interface that let users browse, scroll and zoom Hi-C maps to visually search for patterns in the Hi-C data. In the software, it is also possible to browse several maps simultaneously and plot related genomic data. The software is openly accessible to the scientific community.


Assuntos
Mapeamento Cromossômico/métodos , Marcadores Genéticos , Genoma Humano , Software , Linhagem Celular Tumoral , Mapeamento Cromossômico/estatística & dados numéricos , Gráficos por Computador , Humanos , Armazenamento e Recuperação da Informação , Células K562 , Linfócitos/metabolismo , Linfócitos/patologia
5.
Nucleic Acids Res ; 40(13): 5926-37, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22434883

RESUMO

Variation in the number of individual chromosomes (chromosomal aneuploidy) or chromosome segments (segmental aneuploidy) is associated with developmental abnormalities and reduced fitness in all species examined; it is the leading cause of miscarriages and mental retardation and a hallmark of cancer. However, despite their documented importance in disease, the effects of aneuploidies on the transcriptome remain largely unknown. We have examined the expression effects of seven heterozygous chromosomal deficiencies, both singly and in all pairwise combinations, in Drosophila melanogaster. The results show that genes in one copy are buffered, i.e. expressed more strongly than the expected 50% of wild-type level, the buffering is general and not influenced by other monosomic regions. Furthermore, long genes are significantly more highly buffered than short genes and gene length appears to be the primary determinant of the buffering degree. For short genes the degree of buffering depends on expression level and expression pattern. Furthermore, the results show that in deficiency heterozygotes the expression of genes involved in proteolysis is enhanced and negatively correlates with the degree of buffering. Thus, enhanced proteolysis appears to be a general response to aneuploidy.


Assuntos
Drosophila melanogaster/genética , Monossomia , Proteólise , Aneuploidia , Animais , Drosophila melanogaster/metabolismo , Deleção de Genes , Expressão Gênica , Genes de Insetos
6.
PLoS One ; 6(11): e27942, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22132175

RESUMO

Genome-wide analysis of gene expression or protein binding patterns using different array or sequencing based technologies is now routinely performed to compare different populations, such as treatment and reference groups. It is often necessary to normalize the data obtained to remove technical variation introduced in the course of conducting experimental work, but standard normalization techniques are not capable of eliminating technical bias in cases where the distribution of the truly altered variables is skewed, i.e. when a large fraction of the variables are either positively or negatively affected by the treatment. However, several experiments are likely to generate such skewed distributions, including ChIP-chip experiments for the study of chromatin, gene expression experiments for the study of apoptosis, and SNP-studies of copy number variation in normal and tumour tissues. A preliminary study using spike-in array data established that the capacity of an experiment to identify altered variables and generate unbiased estimates of the fold change decreases as the fraction of altered variables and the skewness increases. We propose the following work-flow for analyzing high-dimensional experiments with regions of altered variables: (1) Pre-process raw data using one of the standard normalization techniques. (2) Investigate if the distribution of the altered variables is skewed. (3) If the distribution is not believed to be skewed, no additional normalization is needed. Otherwise, re-normalize the data using a novel HMM-assisted normalization procedure. (4) Perform downstream analysis. Here, ChIP-chip data and simulated data were used to evaluate the performance of the work-flow. It was found that skewed distributions can be detected by using the novel DSE-test (Detection of Skewed Experiments). Furthermore, applying the HMM-assisted normalization to experiments where the distribution of the truly altered variables is skewed results in considerably higher sensitivity and lower bias than can be attained using standard and invariant normalization methods.


Assuntos
Bases de Dados Genéticas/normas , Genômica/métodos , Viés , Imunoprecipitação da Cromatina , Humanos , Cadeias de Markov , Análise de Sequência com Séries de Oligonucleotídeos , Padrões de Referência
7.
PLoS Genet ; 5(5): e1000465, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19412336

RESUMO

Chromosomal instability, which involves the deletion and duplication of chromosomes or chromosome parts, is a common feature of cancers, and deficiency screens are commonly used to detect genes involved in various biological pathways. However, despite their importance, the effects of deficiencies, duplications, and chromosome losses on the regulation of whole chromosomes and large chromosome domains are largely unknown. Therefore, to explore these effects, we examined expression patterns of genes in several Drosophila deficiency hemizygotes and a duplication hemizygote using microarrays. The results indicate that genes expressed in deficiency hemizygotes are significantly buffered, and that the buffering effect is general rather than being mainly mediated by feedback regulation of individual genes. In addition, differentially expressed genes in haploid condition appear to be generally more strongly buffered than ubiquitously expressed genes in haploid condition, but, among genes present in triploid condition, ubiquitously expressed genes are generally more strongly buffered than differentially expressed genes. Furthermore, we show that the 4th chromosome is compensated in response to dose differences. Our results suggest general mechanisms have evolved that stimulate or repress gene expression of aneuploid regions as appropriate, and on the 4th chromosome of Drosophila this compensation is mediated by Painting of Fourth (POF).


Assuntos
Aneuploidia , Cromossomos/genética , Drosophila melanogaster/genética , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Feminino , Dosagem de Genes , Expressão Gênica , Masculino
8.
Dev Genes Evol ; 217(9): 639-50, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17701050

RESUMO

Thioredoxins are small thiol proteins that have a conserved active site sequence, WCGPC, and reduce disulfide bonds in various proteins using the two active site cysteines, a reaction that oxidizes thioredoxin and renders it inactive. Thioredoxin reductase returns thioredoxin to its reduced, active form in a reaction that converts NADPH to NADP(+). The biological functions of thioredoxins vary widely; they have roles in oxidative stress protection, act as electron donors for ribonucleotide reductase, and form structural components of enzymes. To date, three thioredoxin genes have been characterized in Drosophila melanogaster: the generally expressed Thioredoxin-2 (Trx-2) and the two sex-specific genes ThioredoxinT (TrxT) and deadhead (dhd). The male-specific TrxT and the female-specific dhd are located as a gene pair, transcribed in opposite directions, with only 470 bp between their transcription start points. We show in this study that all three D. melanogaster thioredoxins are conserved in 11 other Drosophilid species, which are believed to have diverged up to 40 Ma ago and that Trx-2 is conserved all the way to Tribolium castaneum. We have found that the intriguing gene organization and regulation of TrxT and dhd is remarkably well conserved and identified potential conserved regulatory sequences. In addition, we show that the 50-70 C terminal amino acids of TrxT constitute a hyper-variable domain, which could play a role in sexual conflict and male-female co-evolution.


Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Genes de Insetos , Tiorredoxinas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sequência Conservada , Primers do DNA/genética , Drosophila/classificação , Drosophila melanogaster/genética , Evolução Molecular , Feminino , Regulação da Expressão Gênica , Genoma de Inseto , Masculino , Modelos Genéticos , Dados de Sequência Molecular , Filogenia , Homologia de Sequência de Aminoácidos , Caracteres Sexuais , Especificidade da Espécie
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