Kuang Yu Chen

Department of Chemistry and The Cancer Institute of New Jersey, Rutgers - The State University of New Jersey, Piscataway, NJ. 08855-0939

Received 8/19/97 Accepted 8/25/97

2. INTRODUCTION

Normal diploid fibroblasts have a limited doubling potential in tissue culture (1). The remarkable consistency of the life span of these cells in culture, which is inversely related to the age of the donor, and the species specificity of the life span (2) has made them a useful model to study the biochemistry of cellular aging. Since the hallmark of cellular aging is the failure of old cells to initiate DNA synthesis (3), possible alterations of gene regulation at late G1/S boundary during senescence are of interest (4, 5). Many of early and mid-G1 genes are fully induced by serum in senescent cells, indicating that senescent cells still retain the ability to receive growth stimulatory signals, (6, 7). However, it has been reported that many genes at the G1/S boundary are suppressed following serum stimulation in senescent cells (6-9). The attenuation of genes such as TK, DHFR, thymidylate synthase (TS), proliferating cell nuclear antigen (PCNA) and histones could lead to a defect in DNA synthetic machinery and thus renders cells incapable to enter S phase of the cell cycle.

Gene expression is commonly regulated at the level of transcription initiation. This step, in turn, is controlled by general and gene-specific transcription factors which bind to cognate DNA sequences in gene promoter regions. The combined action of activating and repressing transcription factors determines the transcription of target genes, and ultimately specifies physiological phenotypes. We have investigated the mechanism of the age-dependent regulation of TK and DHFR. We have identified the CBP/tk (CCAAT Binding Protein for tk gene) and E2F1 as the key transcription factor, respectively, responsible for the down-regulation of TK and DHFR gene (10. 11). The TK-specific transcription factor, CBP/tk, recognizes either one of the two inverted CCAAT elements embedded in Y-box which has a consensus sequence CTGATTGGYYRR (Y=C or T, R=A or G) (10). DNA affinity purification and gel mobility supershift assay using antisera against NF-YA and NF-YB suggests that CBP/tk is either identical to NF-Y (12) or contains NF-Y like proteins (13). NF-Y is a heteromeric protein with three subunits, NF-YA, NF-YB, and NF-YC (14). The transcription factor, E2F, specific for DHFR regulation, was first identified as a DNA-binding protein for the sequence TTTCGCGC within the adenovirus E2 promoter (15). E2F is a heterodimer, consisting of one E2F family protein and one DP family protein (16, 17). Among the known E2F family and DP family members, the attenuation of E2F1 gene expression at both the mRNA and protein levels appears to be primarily responsible for the down-regulation of DHFR gene in senescent cells (11).

We found that the promoter organizations of most of G1/S genes share features resembling that of either TK or DHFR. Specifically, we found that five G1/S genes contain tandem CCAAT elements arranged in a manner similar to that in TK promoter whereas the other six G1/S genes, including DHFR, contain E2F sequence. Most of the CCAAT element in G1/S gene promoters is present as part of Y-box. The presence of these two prominent motif features in almost all age-dependent G1/S gene promoters suggests that the E2F and Y-box binding proteins such as E2F1 and CBP/tk may be important for the global attenuation of G1/S genes during cell senescence. Interestingly, the promoters of NF-Y subunits, A and B, also contain tandem multiple CCAAT elements, similar to that in TK promoter and the promoter of E2F1 contains E2F sites. Thus, it is likely that these two age-dependent transcription factors, CBP/tk and E2F1, may be subject to autoregulatory control by themselves. Further studies will determine whether the tandem, multiple CCAAT elements and the E2F site, individually or together, may represent unique cis-element motifs important for the age-dependent regulation of G1/S genes.