The vertebrate immune response consists of humoral and cellular immunereactions, which are mediated mainly by immunoglobulins (Ig) and Tcell receptors (TCR)respectively. The organization of Ig and TCR genes has been well established. Each ofthe lg and TCR genes consists of multiple germ line gene segments that rearrange duringlymphocyte development to generate diverse receptor structures expressed on mature Band T cells. The transcriptional regulation of Ig genes has been well studied. Theoctamer motif in the lg gene promoter or enhancer, the E-box and the KB site have beenfunctionally characterized. The regulation of transcription factors that bind to thesesites is well understood. The transcriptional regulation of TCR genes is not as wellstudied as that of lg genes. TCR-α, -β, -γ, -δ gene enhancers and a TCR α genesilencer have been reported. Some of the transcription factors that bind to these ciselementshave been cloned. A T cell-specific transcription factor, GATA-3, may playan important regulatory role on the expression of TCR genes in T cells. The promotersof TCR genes also have been investigated, however, the transcription factors thatinteract with them have not been characterized. The aim of this thesis was to isolateand characterize transcription factors that function in TCR gene transcription.
A eDNA clone htβ, encoding a zinc finger protein that binds to the promoterregion of the human TCR gene Vβ8.1, was cloned from a human peripheral blood T celllibrary. The region of this protein containing four zinc fingers of the class Cys_2-X_(12)-His_2 may be responsible for DNA binding to the TCR Vβ8.1 promoter sequenceGAAGTTGGGGGTGGTG. A putative transcriptional activation domain that is highlynegatively charged has also been found in htβ. Analysis of expression of htβ mRNAreveales similar expression levels in Hela cells, Jurkat T cells, Ramos B cells and U -937monocyte line. In addition to binding to the human TCR Vβ8.1 promoter, htβ also canbind to the mouse TCR gene α silencer. The comparison of htβ binding sites betweenthe human TCR Vβ8.1 promoter and the mouse TCR gene α silencer reveals a coresequence of the CACCC box. Gel-shift assay analysis of five repeats of the CACCCbox with bacterially expressed htβ protein indicates that htβ can bind to the CACCCbox. Gel-shift assays of the CACCC box with nuclear extracts from various cell linesreveal four common bands in T cell, B cell, monocyte and Hela cell lines, and one extraband in Hela cell extracts. CAT assay analysis indicates the CACCC box is essential forefficient transcription of the Vβ8.l promoter. Cotransfection with a htβ expressionplasmid and a reporter plasmid show that htβ can activate human TCR Vβ8.1 genetranscription. Htβ also is able to counteract the silencing effect of the TCR α silencer.Htl3 may have an interaction with the cAMP response element binding protein (CREB)to negatively regulate human Vβ8.1 gene transcription in Hela cells, and that negativeeffect is not significant in Jurkat T cells. The CACCC box has been found in almost allVβ8 subfamily members (4 of 5 Vβ8 members in human, and 2 of 3 Vβ8 members inmouse), and both TCR α and β enhancers in human and mouse. These results suggestthat the CACCC box binding protein may have an important function in the immunesystem.
A murine zinc finger protein (M-zif) has been isolated and characterized. It hasfour fingers in the zinc finger domain, the putative DNA binding domain. Also, aglutamine-rich region was found, which may be involved in transcriptional activation.A previously reported eDNA molecule was shown to contain in opposite orientatioins thecoding regions of both the interleukin-2 receptor α (IL-2Rα.) and M-zif genes. Theresults presented here indicate that the cDNA is a chimeric molecule resulting fromcloning artifact. The zinc finger domain of M-zif is highly homologous to that of htβ, ahuman T cell receptor Vβ8.1 promoter binding protein. They may have a similar DNAbinding site. M-zif is not the mouse equivalent of htβ.
