Terminal deoxynucleotidyl transferase (TdT) is a template independent DNA polymerase that is the sole activity in the recombinase complex responsible for N region addition during recombination of V, D, and J gene segments and is therefore essential for diversification of the immune system. TdT gene expression is tightly controlled and mRNA transcripts are detected only in pre-B and pre-T cells. TdT is also aberrantly expressed at high levels in certain leukemias and TdT activity measurements can be a determinant of therapy regimens. It is possible that factors controlling expression of TdT are also involved in modifying the expression of genes which are responsible for the leukemic phenotype. In order to study the genetic elements which restrict expression, I have begun to characterize the human TdT initiation complex. The basal promoter has been identified and is unusual in several respects. It exhibits a tripartite structure, but no elements for known transcription factors, such as TATA consensus sequences or CC-rich Spl binding sites are present. A novel role for the TATA- binding protein, TBP, was revealed. In Contrast to other TATA-less promoters which require the complete TFIID complex, activity of the human TdT promoter requires only the 38 kD TBP subunit. This proposal is designed to elucidate the molecular events necessary for transcription initiation at this novel promoter. I have identified by deletion mutagenesis coupled with in vivo and in vitro transcription assays two 5 bp elements in the basal promoter. I hypothesize that two previously unidentified transcription factors or two known transcription factors exhibiting novel interactions participate in the expression of TdT, by binding at these two elements. I further hypothesize that at least one of the TdT promoter-specific factors binds to TBP and thereby initiates recruitment and assembly of a competent RNA polymerase II transcription complex. I propose to isolate these transcription factors, obtain cDNA clones, and use these biological reagents, through functional analyses of mutationally altered variants, to characterize important DNA and protein-binding domains. Knowledge of the protein factors that participate in basal TdT expression, and knowledge of their interactions with their cognate DNA sequences and with other trans-acting elements of the system will ultimately lead to construction of a mechanistic model for the regulation of TdT expression. The results generated in these studies will increase our knowledge of control of gene expression during lymphocyte differentiation.
| Mahajan, K N; Gangi-Peterson, L; Sorscher, D H et al. (1999) Association of terminal deoxynucleotidyl transferase with Ku. Proc Natl Acad Sci U S A 96:13926-31 |
| Gangi-Peterson, L; Sorscher, D H; Reynolds, J W et al. (1999) Nucleotide pool imbalance and adenosine deaminase deficiency induce alterations of N-region insertions during V(D)J recombination. J Clin Invest 103:833-41 |
