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Characterizing the Retinoblastoma 1 locus: putative elements for Rb1 regulation by in silico analysis.

ABSTRACT: Limited understanding of the Rb1 locus hinders genetic and epigenetic analyses of Retinoblastoma, a childhood cancer of the nervous systems. In this study, we used in silico tools to investigate and review putative genetic and epigenetic elements of the Rb1 gene. We report transcription start sites, CpG islands, and regulatory moieties that are likely to influence transcriptional states of this gene. These might contribute genetic and epigenetic information modulating tissue-specific transcripts and expression levels of Rb1. The elements we identified include tandem repeats that reside within or next to CpG islands near Rb1's transcriptional start site, and that are likely to be polymorphic among individuals. Our analyses highlight the complexity of this gene and suggest opportunities and limitations for future studies of retinoblastoma, genetic counseling, and the accurate identification of patients at greater risk of developing the malignancy.

SUBMITTER: Hajjari M

PROVIDER: S-EPMC3904107 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Characterizing the Retinoblastoma 1 locus: putative elements for Rb1 regulation by in silico analysis.

Hajjari Mohammadreza M Khoshnevisan Atefeh A Lemos Bernardo B

Frontiers in genetics 20140128

Limited understanding of the Rb1 locus hinders genetic and epigenetic analyses of Retinoblastoma, a childhood cancer of the nervous systems. In this study, we used in silico tools to investigate and review putative genetic and epigenetic elements of the Rb1 gene. We report transcription start sites, CpG islands, and regulatory moieties that are likely to influence transcriptional states of this gene. These might contribute genetic and epigenetic information modulating tissue-specific transcripts ...[more]

PMID: 24478791

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Project description:PurposeRetinoblastoma (RB), an intraocular tumor of childhood, is commonly associated with mutations in the RB1 gene. RB116 is a novel, early passage RB cell line that has not been previously characterized. In this study, we examined RB116 for the expression of RB1 and tested the hypothesis that RB116 cells would express stem cell markers as well as retinal progenitor cell markers. We compared RB116 cells with other well known RB cell lines, including Y79 and WERI-RB27.MethodsWe evaluated expression of RB1 in RB116 cells by sequencing, multiplex ligation-dependent probe amplification, quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), western immunoblot, and immunocytochemistry. Next, RB116 cells, along with Y79 and WERI-RB27 cells, were examined for expression of stem cell markers (ABCG2, Nanog, Oct3/4, ALDH1A1) and retinal progenitor markers (PAX6, CHX10) by quantitative immunocytochemistry. Immunocytochemical findings were accompanied by PCR analysis.ResultsRB116 cells expressed RB1 at the mRNA and protein levels, with no mutations detected by either sequencing analysis, or gene dosage abnormalities detected by multiplex ligation-dependent probe amplification. The RB1 protein was immunoreactive in RB116 cells with an atypical perinuclear localization. RB116 cells also expressed stem cell markers, with 3%-5% of cells immunopositive for ABCG2, Oct3/4 and ALDH1A1, with at least 18% of cells immunoreactive to Nanog. These findings were confirmed by RT-PCR. Small percentages of RB116 cells also exhibited immunoreactivity to retinal progenitor markers PAX6 (9.8%) and CHX10 (1.2%). Expression of mRNAs for these markers was confirmed by qRT-PCR.ConclusionsRB116 cells demonstrate RB1 expression accompanied by atypical perinuclear localization. RB116 cells also express primitive stem cell and retinal progenitor cell markers. Further studies on the phenotypes of both RB1-positive and RB1-negative human RB cells may be important in assessing differentiation potential of these cells, as well as designing targeted differentiation therapies.

Dataset Information

Characterizing the Retinoblastoma 1 locus: putative elements for Rb1 regulation by in silico analysis.

Publications

Characterizing the Retinoblastoma 1 locus: putative elements for Rb1 regulation by in silico analysis.

Similar Datasets

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