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Proliferation-dependent positioning of individual centromeres in the interphase nucleus of human lymphoblastoid cell lines.

ABSTRACT: The cell nucleus is a highly organized structure and plays an important role in gene regulation. Understanding the mechanisms that sustain this organization is therefore essential for understanding genome function. Centromeric regions (CRs) of chromosomes have been known for years to adopt specific nuclear positioning patterns, but the significance of this observation is not yet completely understood. Here, using a combination of fluorescence in situ hybridization and immunochemistry on fixed human cells and high-throughput imaging, we directly and quantitatively investigated the nuclear positioning of specific human CRs. We observe differential attraction of individual CRs toward both the nuclear border and the nucleoli, the former being enhanced in nonproliferating cells and the latter being enhanced in proliferating cells. Similar positioning patterns are observed in two different lymphoblastoid cell lines. Moreover, the positioning of CRs differs from that of noncentromeric regions, and CRs display specific orientations within chromosome territories. These results suggest the existence of not-yet-characterized mechanisms that drive the nuclear positioning of CRs and therefore pave the way toward a better understanding of how CRs affect nuclear organization.

SUBMITTER: Ollion J

PROVIDER: S-EPMC4571307 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Proliferation-dependent positioning of individual centromeres in the interphase nucleus of human lymphoblastoid cell lines.

Ollion Jean J Loll François F Cochennec Julien J Boudier Thomas T Escudé Christophe C

Molecular biology of the cell 20150506 13

The cell nucleus is a highly organized structure and plays an important role in gene regulation. Understanding the mechanisms that sustain this organization is therefore essential for understanding genome function. Centromeric regions (CRs) of chromosomes have been known for years to adopt specific nuclear positioning patterns, but the significance of this observation is not yet completely understood. Here, using a combination of fluorescence in situ hybridization and immunochemistry on fixed hu ...[more]

PMID: 25947134

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Project description:BackgroundWhite blood cells have been shown in animal studies to play a central role in the pathogenesis of diabetic retinopathy. Lymphoblastoid cells are immortalized EBV-transformed primary B-cell leukocytes that have been extensively used as a model for conditions in which white blood cells play a primary role. The purpose of this study was to investigate whether lymphoblastoid cell lines, by retaining many of the key features of primary leukocytes, can be induced with glucose to demonstrate relevant biological responses to those found in diabetic retinopathy.MethodsLymphoblastoid cell lines were obtained from twenty-three human subjects. Differences between high and standard glucose conditions were assessed for expression, endothelial adhesion, and reactive oxygen species.ResultsCollectively, stimulation of the lymphoblastoid cell lines with high glucose demonstrated corresponding changes on molecular, cellular and functional levels. Lymphoblastoid cell lines up-regulated expression of a panel of genes associated with the leukocyte-mediated inflammation found in diabetic retinopathy that include: a cytokine (IL-1B fold change = 2.11, p-value = 0.02), an enzyme (PKCB fold change = 2.30, p-value = 0.01), transcription factors (NFKB-p50 fold change = 2.05, p-value = 0.01), (NFKB-p65 fold change = 2.82, p-value = 0.003), and an adhesion molecule (CD18 fold change = 2.59, 0.02). Protein expression of CD18 was also increased (p-value = 2.14x10-5). The lymphoblastoid cell lines demonstrated increased adhesiveness to endothelial cells (p = 1.28x10-5). Reactive oxygen species were increased (p = 2.56x10-6). Significant inter-individual variation among the lymphoblastoid cell lines in these responses was evident (F = 18.70, p < 0.0001).ConclusionsExposure of lymphoblastoid cell lines derived from different human subjects to high glucose demonstrated differential and heterogeneous gene expression, adhesion, and cellular effects that recapitulated features found in the diabetic state. Lymphoblastoid cells may represent a useful tool to guide an individualized understanding of the development and potential treatment of diabetic complications like retinopathy.

Project description:BACKGROUND:Statins are widely prescribed to lower plasma low-density lipoprotein cholesterol levels. Though statins reduce cardiovascular disease risk overall, statin efficacy varies, and some people experience adverse side effects while on statin treatment. Statins also have pleiotropic effects not directly related to their cholesterol-lowering properties, but the mechanisms are not well understood. To identify potential genetic modulators of clinical statin response, we looked for genetic variants associated with statin-induced changes in gene expression (differential eQTLs or deQTLs) in lymphoblastoid cell lines (LCLs) derived from participants of the Cholesterol and Pharmacogenetics (CAP) 40?mg/day 6-week simvastatin clinical trial. We exposed CAP LCLs to 2??M simvastatin or control buffer for 24?h and performed polyA-selected, strand-specific RNA-seq. Statin-induced changes in gene expression from 259 European ancestry or 153 African American ancestry LCLs were adjusted for potential confounders prior to association with genotyped and imputed genetic variants within 1?Mb of each gene's transcription start site. RESULTS:From the deQTL meta-analysis of the two ancestral populations, we identified significant cis-deQTLs for 15 genes (TBC1D4, MDGA1, CHI3L2, OAS1, GATM, ASNSD1, GLUL, TDRD12, PPIP5K2, OAS3, SERPINB1, ANKDD1A, DTD1, CYFIP2, and GSDME), eight of which were significant in at least one of the ancestry subsets alone. We also conducted eQTL analyses of the endogenous (control-treated), statin-treated, and average of endogenous and statin-treated LCL gene expression levels. We identified eQTLs for approximately 6000 genes in each of the three (endogenous, statin-treated, and average) eQTL meta-analyses, with smaller numbers identified in the ancestral subsets alone. CONCLUSIONS:Several of the genes in which we identified deQTLs have functions in human health and disease, such as defense from viruses, glucose regulation, and response to chemotherapy drugs. This suggests that DNA variation may play a role in statin effects on various health outcomes. These findings could prove useful to future studies aiming to assess benefit versus risk of statin treatment using individual genetic profiles.

Dataset Information

Proliferation-dependent positioning of individual centromeres in the interphase nucleus of human lymphoblastoid cell lines.

Publications

Proliferation-dependent positioning of individual centromeres in the interphase nucleus of human lymphoblastoid cell lines.

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