Expression of the V(D)J recombinase gene RAG-1 is tightly regulated and involves both transcriptional and post-transcriptional controls.
The V(D)J recombinase activating genes, RAG-1 and RAG-2, are coexpressed only in immature lymphocytes, and are sufficient and necessary for V(D)J recombination to occur in non-lymphoid cells. In order to examine control mechanisms operative in the regulation of RAG-1 and RAG-2, we have studied the pattern of expression of these genes in human pre-T cells, pre-B cells, and thymocytes treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA); an agent which mimics some of the lymphocyte maturation changes seen in vivo. The expression of RAG-1 and RAG-2 was tightly controlled in a rapid, yet very complex, manner with both positive and negative control elements operating. Treatment of immature lymphocytes with TPA caused the specific and rapid elimination of steady-state RAG-1 and RAG-2 RNA. Nuclear run-on assays showed that TPA completely repressed the transcription of RAG-1 within 30 min. In addition to repressing the transcription of RAG-1, TPA treatment caused the rapid and specific degradation of RAG-1 transcripts by decreasing the apparent half-life of RAG-1 mRNA more than two-fold. As judged by cycloheximide treatment of cells, the effects of TPA were not dependent on new protein synthesis. A labile transcriptional repressor, separate from the TPA-associated repression of transcription, was also active in cells transcribing RAG-1 and RAG-2 RNA. After depletion of this labile repressor by cycloheximide treatment, steady-state RAG-1 and RAG-2 RNA levels, and their transcription rates, were elevated four- to six-fold; but were still susceptible to elimination by TPA treatment. Treatment of pre-T CEM cells with interleukin-2, or theophylline (an agent that increases intracellular cAMP) resulted in a two-fold increase in RAG-1 RNA suggesting that lymphokines, either independently or through second messengers, may modulate RAG-1 and RAG-2 expression. The complex, rapid and precise regulation of RAG-1 and RAG-2 expression is consistent with the view that it is necessary for the cell to tightly regulate V(D)J recombinase levels; lower expression may result in inefficient recombination of Ig/TCR genes, whereas increased expression may lead to recombination errors that are deleterious to the cell.
Neale
G A
GA
Department of Virology and Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN 38101.
Fitzgerald
T J
TJ
Goorha
R M
RM
D000818:Animals; D015152:Blotting, Northern; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D004254:DNA Nucleotidyltransferases / Q000096:biosynthesis* / Q000235:genetics; D004268:DNA-Binding Proteins; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D018398:Homeodomain Proteins; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology; D008214:Lymphocytes / Q000378:metabolism; D009687:Nuclear Proteins; D014176:Protein Biosynthesis; D012323:RNA Processing, Post-Transcriptional / Q000187:drug effects; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013806:Theophylline / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D045567:VDJ Recombinases
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D007376:Interleukin-2; D009687:Nuclear Proteins; C499311:RAG2 protein, human; D012333:RNA, Messenger; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein; D003513:Cycloheximide; D013806:Theophylline; D004254:DNA Nucleotidyltransferases; D045567:VDJ Recombinases; D013755:Tetradecanoylphorbol Acetate