Abstract
Thymidine kinase (TK) is a growth factor-inducible enzyme that is highly expressed in proliferating mammalian cells. Expression of mouse TK mRNA is controlled by transcriptional and posttranscriptional mechanisms including antisense transcription. Here we report the identification of a novel gene that is divergently transcribed from the bidirectional TK promoter. This gene encodes kynurenine formamidase (KF), an enzyme of the tryptophan metabolism. Whereas the TK gene is induced upon interleukin-2-mediated activation of resting T cells, the KF gene becomes simultaneously repressed. The TK promoter is regulated by E2F, SP1, histone acetyltransferases, and deacetylases. The binding site for the growth-regulated transcription factor E2F is beneficial for TK promoter activity but not required for KF expression. In contrast, the SP1 binding site is crucial for transcription in both directions. Inhibition of histone deacetylases by trichostatin A leads to increased histone acetylation at the TK/KF promoter and thereby to selective activation of the TK promoter and simultaneous shut-off of KF expression. Similarly, TK gene activation by interleukin-2 is linked to histone hyperacetylation, whereas KF expression correlates with reduced histone acetylation. The KF gene is the rare example of a mammalian gene whose expression is linked to histone hypoacetylation at its promoter.
Highlights
Thymidine kinase (TK)1 belongs to a group of enzymes including dihydrofolate reductase, thymidylate synthase, DNA polymerase ␣, and proliferating cell nuclear antigen that are involved in DNA synthesis and precursor production
Primer extension and 5Ј-rapid amplification of cDNA ends (5Ј-RACE) analysis showed that transcription of the kynurenine formamidase (KF) gene was mainly initiated within the bidirectional promoter region and revealed a minor transcription start site within intron 2 of the TK gene, which was infrequently used in liver
We detected a significant homology between a sequence within the murine TK promoter and an expressed sequence tag (EST) clone derived from mouse liver (GenBankTM accession number AA245789)
Summary
Cell Culture and Transfection—Cell lines Swiss 3T3, HeLa, and TIB73 were cultured in Dulbecco’s modified Eagle’s medium containing 10% (v/v) fetal calf serum. A series of 5Ј deletion constructs were generated spanning from Ϫ1 to Ϫ1800 bp of the translational start codon using PCR primers containing XhoI and KpnI restriction sites and cloned into the pGL2neo vector. After a 20-min incubation at room temperature, the transfection mix was added to the cells, which, prior to the transfection, had their growth medium replaced by 800 l of serum-free medium. 5Ј-RACE—The 5Ј-RACE procedure was carried out on 1 g of poly(A)ϩ RNA isolated from a mouse liver using the Marathon cDNA amplification kit (Clontech) according to the manufacturer’s instructions. Southern Blot Analysis—Analysis of genomic DNA was performed as described [16]. The following day, chromatin-antibody complexes were isolated from the solution by incubation with 30 l of protein A-Sepharose beads (50% slurry, 100 g/ml salmon sperm DNA, 500 g/ml bovine serum albumin) while rocking at 4 °C for 2 h. PCR products were resolved on 2% agarose-TAE gels and quantified using the ImageQuant program (Amersham Biosciences)
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