A novel transcriptional inhibitory element differentially regulates the cyclin D1 gene in senescent cells.
ABSTRACT Senescent human diploid fibroblasts are unable to initiate DNA synthesis following mitogenic stimulation and adopt a unique gene expression profile distinct from young or quiescent cells. In this study, a novel transcriptional regulatory element was identified in the 5'-untranslated region of the cyclin D1 gene. We show that this element differentially suppresses cyclin D1 expression in young versus senescent fibroblasts. Electrophoretic mobility shift assays revealed abundant complexes forming with young cell nuclear extracts compared with senescent cell nuclear extracts. Binding was maintained in young quiescent cells, showing that loss of this activity was specific to senescent cells and not an effect of cell cycle arrest. Site-directed mutagenesis within this cyclin D1 inhibitory element (DIE) abolished binding activity and selectively increased cyclin D1 promoter activity in young but not in senescent cells. Sequences with homology to the DIE were found in the 5'-untranslated regions of other genes known to be up-regulated during cellular aging, suggesting that protein(s) that bind the DIE might be responsible for the coordinate increase in transcription of many genes during cellular aging. This study provides evidence that loss of transcriptional repressor activity contributes to the up-regulation of cyclin D1, and possibly additional age-regulated genes, during cellular senescence.
- SourceAvailable from: Radhika Kandaswamy[Show abstract] [Hide abstract]
ABSTRACT: IMPORTANCE Genetic markers at the gene encoding the metabotropic glutamate receptor 3 (GRM3) showed allelic association with bipolar disorder. OBJECTIVE To screen the GRM3 gene and adjacent control regions of genomic DNA in volunteers with bipolar affective disorder for mutations increasing susceptibility to bipolar disorder. DESIGN Sequencing and high-resolution melting curve analysis of DNA followed by genotyping was carried out in 1099 patients with bipolar affective disorder and 1152 healthy comparator individuals. SETTING Participants with bipolar disorder were recruited from National Health Service psychiatric services and from patient organizations. PARTICIPANTS Individuals were included if they had Research Diagnostic Criteria diagnoses of bipolar I and bipolar II disorder and were of British or Irish ancestry. MAIN OUTCOMES AND MEASURES Identification of base pair changes in the GRM3 gene that affected expression or function of the GRM3 receptor that also showed an allelic association with bipolar disorder. RESULTS A base pair variant (rs148754219) was found in the Kozak sequence of exon 1 of the GRM3 gene, 2 bases before the translation start codon of one of the receptor isoforms, in 23 of 2251 people who were screened and genotyped. Nineteen of the 1099 bipolar cases (1.7%) were mutation carriers compared with 4 of 1152 healthy comparators (0.3%). The variant was associated with bipolar disorder (P = .005; odds ratio, 4.20). Bioinformatic, electrophoretic mobility shift assay, and gene expression analysis found that the variant created a new transcription factor protein binding site and had a strong effect on gene transcription and translation. CONCLUSIONS AND RELEVANCE Confirmation of these findings is needed before the Kozak sequence variant can be accepted as a potential marker for personalized treatment of affective disorders with drugs targeting the metabotropic glutamate receptor 3.JAMA Psychiatry 04/2013; · 12.01 Impact Factor
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ABSTRACT: Treatment of IMR-90 human diploid fibroblasts with a sublethal concentration of H(2)O(2) induces premature senescence. We investigated the protein abundance, subcellular localization and involvement of caveolin 1 in premature senescence. Caveolin 1 is a scaffolding protein able to concentrate and organize signaling molecules within the caveolae membrane domains. We report the first evidence of increased nuclear and cytoplasmic localization of caveolin 1 during establishment of H(2)O(2)-induced premature senescence. Moreover, we demonstrate that phosphorylation of caveolin 1 during treatment with H(2)O(2) is dependent on p38alpha mitogen-activated protein kinase.FEBS Letters 06/2008; 582(12):1685-92. · 3.58 Impact Factor
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ABSTRACT: The ING family of tumor suppressor proteins affects cell growth, apoptosis and response to DNA damage by modulating chromatin structure through association with different HAT and HDAC complexes. The major splicing isoforms of the ING1 locus are ING1a and INGlb. While INGlb plays a role in inducing apoptosis, the function of ING1a is currently unknown. Here we show that alternative splicing of the ING1 message alters the INGla:INGlb ratio by approximately 30-fold in senescent compared to low passage primary fibroblasts. INGla antagonizes INGlb function in apoptosis, induces the formation of structures resembling senescence-associated heterochromatic foci containing heterochromatin protein 1 gamma, the accumulation of senescence-associated beta-galactosidase activity and promotes senescent cell morphology and cell cycle arrest. Phenotypic effects may result from differential effects on gene expression since ING1a increases levels of both retinoblastoma and the p16 cyclin-dependent kinase inhibitor and ING1a and ING1b have opposite effects on the expression of proliferating nuclear cell antigen (PCNA), which is required for cell growth. Gene expression appears to be altered by targeting of HDAC complexes to gene promoters since INGla associates with several-fold higher levels of HDAC1 in senescent, compared to replication-competent cells and ING1 is found on the PCNA promoter by chromatin immunoprecipitation analysis. These data demonstrate a novel role for the ING1 proteins in differentially regulating senescence-associated chromatin remodeling vs. apoptosis and support the idea that altered ratios of the ING1 splicing isoforms may contribute to establishing the senescent phenotype through HDAC and HAT complex-mediated effects on chromatin structure.Aging cell 09/2008; 7(6):783-94. · 7.55 Impact Factor