Transcriptional regulation of T cell receptor genes by a novel CACCC box binding protein

Citation

Wang, Yukang (1993) Transcriptional regulation of T cell receptor genes by a novel CACCC box binding protein. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/x9cm-6a16. https://resolver.caltech.edu/CaltechTHESIS:01102013-132313493

Abstract

The vertebrate immune response consists of humoral and cellular immune reactions, which are mediated mainly by immunoglobulins (Ig) and Tcell receptors (TCR) respectively. The organization of Ig and TCR genes has been well established. Each of the lg and TCR genes consists of multiple germ line gene segments that rearrange during lymphocyte development to generate diverse receptor structures expressed on mature B and T cells. The transcriptional regulation of Ig genes has been well studied. The octamer motif in the lg gene promoter or enhancer, the E-box and the KB site have been functionally characterized. The regulation of transcription factors that bind to these sites is well understood. The transcriptional regulation of TCR genes is not as well studied as that of lg genes. TCR-α, -β, -γ, -δ gene enhancers and a TCR α gene silencer have been reported. Some of the transcription factors that bind to these ciselements have been cloned. A T cell-specific transcription factor, GATA-3, may play an important regulatory role on the expression of TCR genes in T cells. The promoters of TCR genes also have been investigated, however, the transcription factors that interact with them have not been characterized. The aim of this thesis was to isolate and characterize transcription factors that function in TCR gene transcription.

A eDNA clone htβ, encoding a zinc finger protein that binds to the promoter region of the human TCR gene Vβ8.1, was cloned from a human peripheral blood T cell library. 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 sequence GAAGTTGGGGGTGGTG. A putative transcriptional activation domain that is highly negatively charged has also been found in htβ. Analysis of expression of htβ mRNA reveales similar expression levels in Hela cells, Jurkat T cells, Ramos B cells and U -937 monocyte line. In addition to binding to the human TCR Vβ8.1 promoter, htβ also can bind to the mouse TCR gene α silencer. The comparison of htβ binding sites between the human TCR Vβ8.1 promoter and the mouse TCR gene α silencer reveals a core sequence of the CACCC box. Gel-shift assay analysis of five repeats of the CACCC box with bacterially expressed htβ protein indicates that htβ can bind to the CACCC box. Gel-shift assays of the CACCC box with nuclear extracts from various cell lines reveal four common bands in T cell, B cell, monocyte and Hela cell lines, and one extra band in Hela cell extracts. CAT assay analysis indicates the CACCC box is essential for efficient transcription of the Vβ8.l promoter. Cotransfection with a htβ expression plasmid and a reporter plasmid show that htβ can activate human TCR Vβ8.1 gene transcription. 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 negative effect is not significant in Jurkat T cells. The CACCC box has been found in almost all Vβ8 subfamily members (4 of 5 Vβ8 members in human, and 2 of 3 Vβ8 members in mouse), and both TCR α and β enhancers in human and mouse. These results suggest that the CACCC box binding protein may have an important function in the immune system.

A murine zinc finger protein (M-zif) has been isolated and characterized. It has four fingers in the zinc finger domain, the putative DNA binding domain. Also, a glutamine-rich region was found, which may be involved in transcriptional activation. A previously reported eDNA molecule was shown to contain in opposite orientatioins the coding regions of both the interleukin-2 receptor α (IL-2Rα.) and M-zif genes. The results presented here indicate that the cDNA is a chimeric molecule resulting from cloning artifact. The zinc finger domain of M-zif is highly homologous to that of htβ, a human T cell receptor Vβ8.1 promoter binding protein. They may have a similar DNA binding site. M-zif is not the mouse equivalent of htβ.

Thesis Files