GSBS Dissertations and Theses

Approval Date

May 1991

Document Type

Doctoral Dissertation

Department

Graduate School of Biomedical Sciences, Immunology & Virology

Subjects

Genes, Immunoglobulin Heavy Chain; Promoter Regions (Genetics); Regulatory Sequences, Ribonucleic Acid; Mice; Germ Cells; Molecular Probes; Academic Dissertations; Dissertations, UMMS

Abstract

The antibody class switch is achieved by DNA recombination between the sequences called switch (S) regions located 5' to immunoglobulin (Ig) heavy chain constant (CH) region genes. This process can be induced in cultured B cells by polyclonal stimulation and switching can be directed to specific antibody classes by certain lymphokines. These stimuli may regulate the accessibility of CH genes and their S regions to a recombinase as indicated by hypomethylation and transcriptional activity. For example, RNAs transcribed from specific unrearranged (germ-line) CH genes are induced prior to switching under conditions that promote subsequent switching to these same CH genes. The function of transcription of these germ-line CH genes is unknown. How stimuli regulate the accessibility of CH genes is also unclear.

I report in this dissertation the structure of the RNA transcribed from the unrearranged Cγ1 gene in mouse spleen cells treated with LPS plus a HeLa cell supernatant containing recombinant interleukin 4 (rIL-4). I will also show that an 150-bp region upstream of the first initiation site of germ-line γ1 RNA contains promoter and enhancer elements responsible for basal level expression and inducibility by phorbol 12-myristate 13-acetate (PMA) and synergy with IL-4 in an IgM+ B cell line, L10A6.2, and an IgG2a+ B cell line, A20.3.

The germ-line γ1 RNA is initiated at multiple start sites 5' to the tandem repeats of the γ1 switch (Sγ1) region. As is true for analogous RNAs transcribed from other unrearranged genes, the germ-line γ1 RNA has an I exon transcribed from the region 5' to the Sγ1 region.. The Iγ1 exon is spliced at a unique site to the Cγ1 gene. The germ-line γ1 RNA has an open-reading frame (ORF) that potentially encodes a small protein 48 amino acids in length.

Elements located within the 150 bp region 5' to the first initiation site of germ-line γ1 RNA are necessary and sufficient to confer inducibility by PMA and synergy with IL-4 to a minimal thymidine kinase (TK) promoter in L10A6.2 cells but are not sufficient to confer this inducibility in A20.3 cells. Linker-scanning mutations demonstrated that these multiple elements function in a mutually dependent manner as indicated by the fact that mutation of any single element will decrease constitutive expression and inducibility by PMA and PMA plus IL-4.

This 150-bp region contains several consensus sequences that bind to known or putative transcription factors, including a C/EBP binding site/IL-4 response element (in the promoter for Ia Aαk gene), four CACCC boxes, a PU box, a TGFβ inhibitory element (TIE), an interferon-αβ response element (αβIRE), and an AP-3 site.

My results begin to provide a description of the mechanism of regulation of the accessibility of unrearranged germ-line Sγ1-Cγ1 gene. By activating the germ-line γ1 promoter, IL-4 induces transcription of germ-line γ1 RNA, thereby inducing accessibility of the Sγ1-Cγ1 gene. By inhibiting expression of the germ-line γ1 promoter, IFNγ and TGFβ down-regulate transcription of germ-line γ1 RNA, thus reducing the accessibility of the Sγ1-Cγ1 gene. My results also suggest that signaling via the antigen receptor on B cells may be involved in induction of switch to IgG1. Furthermore, this is the first case reported in which multiple functionally interdependent elements are needed to respond to PMA.

Comments

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