RESUMO
The thymidylate synthase (TS) gene, which is induced at the G(1)-S transition in growth-stimulated cells, encodes an enzyme that is essential for DNA replication and cell survival. Here we demonstrate that LSF (LBP-1c, CP2) binds to sites within the TS promoter and intronic regions that are required for this induction. Mutation of the LSF binding sites inhibits G(1)-S induction of mRNA derived from a TS minigene. Furthermore, expression of dominant-negative LSF (LSFdn) prevents the increase in TS enzyme levels during G(1)-S, and induces apoptosis in growth- stimulated mouse and human cell lines. Such apoptosis can be prevented either by circumventing the TS requirement through addition of low concentrations of thymidine, or by coexpression of the TS gene driven by a heterologous promoter. Induction of apoptosis by LSFdn parallels the process known as thymineless death, which is induced by the TS inhibitor and chemotherapeutic drug 5-fluorodeoxyuridine. Thus, LSF is a novel regulatory factor that supports progression through S-phase by targeting a single gene that is critical for cell survival.
Assuntos
Proteínas de Ligação a DNA/antagonistas & inibidores , Regulação para Baixo , Fase S , Timidilato Sintase/genética , Fatores de Transcrição/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , DNA/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Mutagênese , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Especificidade da EspécieRESUMO
Herpesviruses accomplish DNA replication either by expressing their own deoxyribonucleotide biosynthetic genes or by stimulating the expression of the corresponding cellular genes. Cytomegalovirus (CMV) has adopted the latter strategy to allow efficient replication in quiescent cells. In the present report, we show that murine CMV (MCMV) infection of quiescent fibroblasts induces both mRNA and protein corresponding to the cellular thymidylate synthase (TS) gene, which encodes the enzyme that catalyzes the de novo synthesis of thymidylic acid. The increase in TS gene expression was due to an increase in gene transcription, since the activity of a reporter gene driven by the mouse TS promoter was induced following MCMV infection. Mutagenesis of the potential E2F-responsive element immediately upstream from the TS essential promoter region abolished the virus-mediated stimulation of the TS promoter, suggesting that the transactivating activity of MCMV infection was E2F dependent. Cotransfection experiments revealed that expression of the viral immediate-early 1 protein was sufficient to mediate the increase in TS promoter activity. Finally, MCMV replication and viral DNA synthesis were found to be inhibited by ZD1694, a quinazoline-based folate analog that inhibits TS activity. These results demonstrate that upregulation of cellular TS expression is required for efficient MCMV replication in quiescent cells.
Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular , Replicação do DNA , DNA Viral/biossíntese , Proteínas de Ligação a DNA , Regulação Enzimológica da Expressão Gênica , Muromegalovirus/fisiologia , Timidilato Sintase/genética , Ativação Transcricional , Proteínas Virais , Replicação Viral/fisiologia , Células 3T3 , Animais , Sítios de Ligação , Fatores de Transcrição E2F , Inibidores Enzimáticos/farmacologia , Humanos , Proteínas Imediatamente Precoces/metabolismo , Proteínas Imediatamente Precoces/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Muromegalovirus/genética , Muromegalovirus/metabolismo , Regiões Promotoras Genéticas , Quinazolinas/farmacologia , Proteína 1 de Ligação ao Retinoblastoma , Tiofenos/farmacologia , Timidilato Sintase/antagonistas & inibidores , Fator de Transcrição DP1 , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
The mouse thymidylate synthase (TS) promoter is a member of a family of promoters that lack a TATA box as well as an initiator element and that initiate transcription at many sites over a broad initiation window. An element (MED-1) downstream of the initiation window of almost all promoters of this family has been proposed to be important for promoter activity, as well as for multiple start site utilization. Two consensus MED-1 elements are located downstream of the initiation window of the TS promoter. To determine the role of the MED-1 elements in the TS promoter, one or both elements were inactivated by site-directed mutagenesis and the effects on promoter function were determined. We found that inactivation of the MED-1 elements had no measurable effect on promoter strength, the boundaries of the initiation window, or the pattern of transcriptional start sites. Furthermore, inactivation of the elements did not affect the ability of the TS promoter to direct S phase-specific expression of the gene in growth-stimulated cells. We conclude that the MED-1 element does not play a significant role in TS promoter function and therefore is not an essential component of all TATA-less promoters with complex transcriptional initiation patterns.
