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Inhibition of the mammalian transcription factor LSF induces S-phase-dependent apoptosis by downregulating
1Committee on Virology and Department of Microbiology and Molecular Genetics, Harvard Medical School and Division of Molecular Genetics, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA.
Abstract:
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.
Insights
LSF is a novel regulatory factor essential for DNA replication and cell survival. It targets the thymidylate synthase (TS) gene, and its inhibition leads to apoptosis, mimicking thymineless death.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- Thymidylate synthase (TS) is crucial for DNA replication and cell survival, induced during the G(1)-S phase transition.
- LSF (LBP-1c, CP2) is a transcription factor implicated in cellular processes.
Purpose of the Study:
- To investigate the role of LSF in regulating thymidylate synthase (TS) gene expression.
- To determine if LSF is a novel regulatory factor involved in cell cycle progression and survival.
Main Methods:
- Analysis of LSF binding sites within the TS promoter and intronic regions.
- Site-directed mutagenesis of LSF binding sites in a TS minigene.
- Expression of dominant-negative LSF (LSFdn) in mouse and human cell lines.
- Assessment of TS enzyme levels, mRNA induction, and apoptosis.
Main Results:
- LSF binds to critical regulatory regions of the TS gene promoter and introns.
- Mutating LSF binding sites abrogates G(1)-S induction of TS mRNA.
- Dominant-negative LSF (LSFdn) inhibits TS enzyme upregulation and induces apoptosis in growth-stimulated cells.
- Apoptosis induced by LSFdn can be rescued by thymidine or TS gene re-expression.
Conclusions:
- LSF is a novel regulatory factor essential for the G(1)-S transition and TS gene induction.
- LSF plays a critical role in supporting S-phase progression by regulating TS.
- LSF activity is vital for cell survival, and its inhibition mimics thymineless death.