Berardi P, Meyyappan M, Riabowol KTA novel transcriptional inhibitory element differentially regulates the cyclin D1 gene in senescent cells. J Biol Chem 278: 7510-7519

Department of Biochemistry and Molecular Biology, The University of Calgary, Calgary, Alberta, Canada
Journal of Biological Chemistry (Impact Factor: 4.57). 03/2003; 278(9):7510-9. DOI: 10.1074/jbc.M210864200
Source: PubMed


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.

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    • "To examine whether the mRNA levels of the two major ING1 isoforms, ING1a and ING1b (Fig. 1A), are expressed differently in young vs. senescent human diploid fibroblasts, reverse transcription–polymerase chain reaction (RT-PCR) using isoform specific primers was done with GAPDH primers as an internal control for mRNA integrity and amplification efficiency (Wong et al., 1994). Senescent cells used in this study displayed several markers of senescence such as elevated levels of cyclin dependent kinase inhibitors (Hara et al., 1996; Wong & Riabowol, 1996), cyclin Dl (Dulic et al., 1993; Berardi et al., 2003), senescenceassociated β-galactosidase (SA-β-gal) activity (Dimri et al., 1995), and globular actin (Kwak et al., 2004) (Fig. S1). We found that the level of INGlb mRNA decreased in senescent cells as previously reported (Vieyra et al., 2002a). "
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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.
    Full-text · Article · Sep 2008 · Aging cell
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    • "It has been well documented that 5′-UTR plays an important role in the regulation of gene expression, primarily due to its effect on the post-transcriptional process, including the initiation and efficiency of translation, as well as the stability of mRNA (Van der Velden and Thomas 1999; Pickering and Willis 2005; Derrigo et al. 2000; Wilkie et al. 2003); however, since the basal transcription apparatus also contains partial sequence downstream of the transcription start site (TSS), it can be inferred that the 5′-UTR might also affect gene expression via a transcriptional mechanism, as has already been reported for some genes (Berardi et al. 2003; Coppotelli et al. 2006; Minet et al. 1999; Singh et al. 2002). In the present study, the segment +8 to +53 of the TPH2 5′-UTR decreased luciferase activity and mRNA level strikingly, yet had a negligible effect on mRNA stability, suggesting that transcriptional mechanisms may be involved in the repression of gene expression by this region and that negative cis-element(s) should exist in this region. "
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    ABSTRACT: Tryptophan hydroxylase-2 (TPH2) is a recently identified TPH isoform responsible for neuronal serotonin (5-HT) synthesis, and TPH2 polymorphisms are associated with a range of behavioral traits and psychiatric disorders. This study characterized cis-acting elements and three common polymorphisms (-703G/T, -473T/A, and 90A/G) in the 5' regulatory region of human TPH2 by using luciferase reporter assay, quantitative real-time PCR, and electrophoretic mobility shift assay (EMSA). The core promoter of human TPH2 was localized to the region between -107 and +7, and the segment of +8 to +53 within the 5'-UTR was found to exert a potent inhibitory effect on gene expression at both transcriptional and post-transcriptional levels. In both RN46A and HEK-293 cell lines, the TTA (-703T/-473T/90A) haplotype of the three polymorphisms showed the lowest gene expression compared with other haplotypes, and the -703G/T and -473T/A polymorphisms tended to exert a synergic effect on gene expression dependent upon the sequence of the 5'-UTR. In RN46A, the 90A/G polymorphism significantly increased luciferase activity and mRNA level irrespective of the other two polymorphisms, while in HEK-293 cells the effect of 90A/G was dependent on the alleles at loci -703 and -473. EMSA showed that all the three polymorphisms potentially alter DNA-protein interactions, while the 90A/G polymorphism predictably alters the 5'-UTR secondary structure of mRNA and influences RNA-protein interactions. In conclusion, our present study demonstrates that both the 5'-UTR and common polymorphisms (especially the 90A/G) in the 5' regulatory region of human TPH2 have a significant impact on gene expression.
    Full-text · Article · Feb 2008 · Human Genetics
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    • "Variations in the time and method of antigen retrieval can alter the binding characteristics and cellular localisation of this antibody by a considerable margin (720%), while still producing minimal background staining. The apparent reduction in cyclin D1 expression on progression from borderline tumours to serous cystadenocarcinomas may reflect dysregulation of the G1/S checkpoint in the higher grade lesions as cyclin D1 is known to accumulate in the presence of an intact G1/S checkpoint (Berardi et al, 2003). Determination of cyclin A LFs may be of greater value in predicting outcome and/or the likely response to chemotherapy, by indicating the fraction of cycling tumour cells (rather than the total number of tumour cells) that are in S phase. "
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    ABSTRACT: We have investigated whether immunohistochemical markers can identify differences in cell cycle phase distribution in ovarian serous neoplasms, including borderline tumours of different grades. Sections of normal ovary (n=18), serous cystadenoma (n=21), borderline serous tumours (n=21) and serous cystadenocarcinoma (n=15) were analysed by immunohistochemistry using markers of cell cycle entry (Mcm-2) and cell cycle phase, including cyclin D1 (mid-to-late G1), cyclin A (S phase), cyclin B1 (G2 phase) and phosphohistone H3 (mitosis). Double-labelling confocal microscopy confirmed marker phase specificity and phase estimations were corroborated by flow cytometry. On progression from normal ovary through serous cystadenoma and borderline tumours to cystadenocarcinomas, expression of Mcm-2 (P<0.0001), cyclin D1 (P=0.002), cyclin A (P<0.0001), cyclin B1 (P<0.0001) and phosphohistone H3 (P<0.0001) increased, paralleled by an increase in the S-phase fraction (cyclin A : Mcm-2 ratio; P=0.002). Borderline tumours of increasing grade also showed increased Mcm-2 and cyclin A expression, together with an increase in the S-phase fraction. Immunohistochemistry can be used to estimate cell cycle phase distribution in ovarian serous neoplasms, giving results similar to flow cytometric analysis and enabling direct assessment of tumour heterogeneity. Immunohistochemical estimates of the S-phase fraction may identify serous borderline tumours likely to exhibit malignant progression and/or select serous cystadenocarcinomas likely to respond to adjuvant therapy.
    Full-text · Article · Apr 2004 · British Journal of Cancer
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