Hayashi R et al. (2006), A combined computational and experimental study...

XB-ART-34932

Genomics Proteomics Bioinformatics 2006 Aug 01;43:156-64. doi: 10.1016/S1672-0229(06)60028-4.

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A combined computational and experimental study on the structure-regulation relationships of putative mammalian DNA replication initiator GINS.

Hayashi R , Arauchi T , Tategu M , Goto Y , Yoshida K .

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GINS, a heterotetramer of SLD5, PSF1, PSF2, and PSF3 proteins, is an emerging chromatin factor recognized to be involved in the initiation and elongation step of DNA replication. Although the yeast and Xenopus GINS genes are well documented, their orthologous genes in higher eukaryotes are not fully characterized. In this study, we report the genomic structure and transcriptional regulation of mammalian GINS genes. Serum stimulation increased the GINS mRNA levels in human cells. Reporter gene assay using putative GINS promoter sequences revealed that the expression of mammalian GINS is regulated by 17beta-Estradiol-stimulated estrogen receptor alpha, and human PSF3 acts as a gene responsive to transcription factor E2F1. The goal of this study is to present the current data so as to encourage further work in the field of GINS gene regulation and functions in mammalian cells.

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Species referenced: Xenopus
Genes referenced: e2f1 gins2 gins3 gins4 tap1

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Fig. 1. Expression profiles of GINS genes. A search was conducted based on the EST counts in human/mouse tissues (A) and during the developmental stages (B) in the UniGene database (EST Profile Viewer, NCBI). The datasets used for PSF1, PSF2, Psf3, and SLD5 genes were Hs. 360033, Hs. 433180, Mm. 35546, and Hs. 521557, respectively. The number of transcripts per million was calculated from the gene EST/total EST in the pool. C. Semi-quantitative RT-PCR analysis of the transcripts of GINS genes in Saos-2 cells after serum stimulation for 24 h. The number of PCR cycles is shown in parentheses. The housekeeping gene, GAPDH, was used as the control.

Fig. 2. Transcriptional regulation of the human and mouse GINS genes. A. Schematic representation of the vicinity of the transcription start sites of the human and mouse GINS genes. The putative transcription factor E2F- (closed isosceles triangles) and NF-Y-binding sites (open isosceles triangles) are indicated with their scores (maximum score 100) calculated by the Transfac program. Exon 1 (open boxes) is indicated. Scale bar equals 500 bp. B. Human and mouse GINS promoter activities in asynchronously growing human cells. HeLa cells were transfected with 200 ng of reporter constructs and 400 ng of the expression vector for E2F1, together with 0.6 ng of pRL-TK. The pcDNA3 vector was used as the negative control. At 48 h after the transfection, the cells were harvested, and extracts were prepared to measure the firefly and Renilla luciferase activities. Values are represented as relative luciferase activities, with that of pGL3-Basic being taken as 1. C. The E2F-binding motif of the PSF3 promoter is sufficient to confer responses to ectopic E2F expression. The experiment was performed as described in panel B. Values are represented as relative luciferase activities, with that of the control vector pcDNA3 being taken as 1. Statistically significant (P < 0.05) induction is indicated by an asterisk. D. E2-induced activation of GINS promoter constructs in HeLa cells. Cells were transfected with the indicated reporter plasmids and pcDNA3 or pSG5-ERα expression vector, and the effects of E2 or DMSO on the luciferase activities were determined. Results are expressed as mean±S.D. for at least three triplicate determinations for each treatment group. Statistically significant (P < 0.05) induction is indicated by an asterisk.

References [+] :

Blow, Preventing re-replication of chromosomal DNA. 2005, Pubmed