Regulation of dihydrofolate reductase gene expression and E2F components in human diploid fibroblasts during growth and senescence.
The induction of dihydrofolate reductase (DHFR), a key enzyme in DNA biosynthesis that is induced just before the onset of S phase, is markedly attenuated in senescent human fibroblasts (Pang and Chen, 1994, J. Cell. Physiol., 160:531-538). Footprinting analysis of the 365 bp promoter region of the human DHFR gene (-381 to -17) indicated that nuclear proteins bind to a cluster of cis-elements, including two overlapping E2F binding sequences, two Sp1 sites, and one Yi sequence. Gel mobility shift assays were performed to assess the role of each cis-element in the regulation of DHFR gene expression. We found that 1) Sp1 binding activity was constitutively expressed throughout the cell cycle in early passage and senescent cells; 2) Yi binding activity was undetectable in both early passage and senescent cells; and 3) E2F binding activity was serum-inducible, senescence-dependent, and prominent in presenescent cells but strikingly diminished in senescent cells. Northern blot analysis of the expression of E2F and DP family members showed that the E2F-1, E2F-4, and E2F-5 mRNA was growth- and senescence-dependent, whereas E2F-3, DP-1, and DP-2 expression was constitutive and senescence-independent. In contrast, E2F-2 mRNA was not detectable in IMR-90 or WI-38 human fibroblasts. Western blot analysis showed that among the E2F-associated proteins, the expression of E2F-1, cyclin A, and cyclin B but not p107 was cell cycle- and senescence-dependent. A nuclear extract mixing experiment suggested that an inhibitory factor may further reduce E2F binding activity in senescent cells.
Good
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Department of Chemistry, Rutgers, State University of New Jersey, Piscataway 08855-0939, USA.
Dimri
G P
GP
Campisi
J
J
Chen
K Y
KY
D001483:Base Sequence; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002478:Cells, Cultured; D016922:Cellular Senescence; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D050703:E2F2 Transcription Factor; D050699:E2F3 Transcription Factor; D050704:E2F4 Transcription Factor; D050700:E2F5 Transcription Factor; D015971:Gene Expression Regulation, Enzymologic; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D056465:Retinoblastoma-Binding Protein 1; D016329:Sp1 Transcription Factor / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000235:genetics
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D050703:E2F2 Transcription Factor; C493761:E2F2 protein, human; D050699:E2F3 Transcription Factor; D050704:E2F4 Transcription Factor; C493762:E2F4 protein, human; D050700:E2F5 Transcription Factor; D056465:Retinoblastoma-Binding Protein 1; D016329:Sp1 Transcription Factor; C493735:TFDP1 protein, human; D050680:Transcription Factor DP1; D014157:Transcription Factors; D013762:Tetrahydrofolate Dehydrogenase