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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. © 1996 Wiley‐Liss, Inc.