Transcriptional Control of D-beta-2

Abstract

ABSTRACT McMILLAN, RUTH ERICA. Transcriptional Control of D?2. (Under the direction of Michael L. Sikes). The immune system?s response to invading pathogens depends on the ability of T and B-lymphocytes to generate a diverse repertoire of antigen receptors. This diversity is created during lymphocyte development by somatic recombination of variable (V), diversity (D) and joining (J) gene segments. Transcription of unrearranged gene segments precedes their recombination. Given the correlation of transcription and recombination, it has been suggested that enhancers and promoters within each locus may regulate the recombinational accessibility of nearby gene segments. For example, in developing T cells, two stages of recombination (first D to J, then V to DJ) are seen in the T cell receptor beta gene (TCR?). Both steps require the TCR? enhancer, E?, while a promoter associated with the first D segment (PD?1) only controls its recombination. In mutant cells that lack PD?1, the second D? rearranges normally. To investigate the regulation of D?2 recombination, we have characterized the transcriptional profile of DJC?2 gene cassette and identified separate promoter activities positioned 5? and 3? of D?2. The relatively simple downstream promoter (3?PD?2) contains no discernable initiator or TATA elements and is located 400 bases downstream of D?2 and is driven by two NFkB binding sites. Ribonuclease protection assay and 5? RACE PCR revealed diffused transcription start sites throughout the entire 150 bp promoter region. Transcription from 3?PD?2 occurs only when the TCR? locus is in its germline configuration. In-vivo germline transcription of the upstream promoter (5?PD?2) is detected in DNA with D?J?2 joints. When luciferase reporter plasmid bearing E? and sequential truncations of the DJ?2 sequence was transiently transfected into a RAG1 deficient cell line in-vitro, transcription from 5?PD?2 was detected when a 290 bp region immediately downstream of D?2 was deleted. Therefore, germline transcription from 5?PD?2 is held in repression while 3?PD?2 is active. Our report indicates that 3?PD?2 is positioned downstream of its associated D segment; a position that does not efficiently promote recombination. We hypothesize that this unique positioning plays an essential role in delaying D?2 recombination, thereby providing the locus with a second recombination opportunity should the recombination with the upstream D?1 fail.