Regulation of the murine Immunoglobulin GL γ 1 and ε promoters

The immune system takes advantage of different genetic alterations to amplify its diversity. First at early stages of B cell differentiation when the antigen (ag) receptors are assembled from different gene segments. Secondly during the proliferative stage induced by ag challenge, when the Ig genes undergo two types of modifications: somatic hypermutation and class switch recombination (CSR). During CSR the effector function of an Ig molecule is altered, while the antigen specificity is remained, resulting in expression of IgG, IgA or IgE instead of the initial IgM. Switching to a given isotype requires induction of transcription over the targeted CH-gene, while it is still in germline (GL) configuration. However, the specific function of GL transcripts in CSR is still unknown and regulation of GL transcription over the Cγ1 and Cε regions has been the main focus of this thesis. Initially, we explored whether the CD40/CD40L interaction was sufficient for B cells to undergo CSR. In vivo distribution of an agonistic monoclonal rat anti-mouse CD40-antibody (ab) induced upregulation of all Ig isotypes in mice completely devoid of T cells. This induction was to some extent independent of IL-4, since IgE synthesis was only partially inhibited by co-injection of anti-IL4. AntiCD40 also induced GL ε transcripts and expression of IgE in B cells from IL-4 mice in vitro. We then investigated the molecular mechanisms of CD40 signalling in induction of GL transcripts and CSR. We found that signalling through CD40 stimulated GL γ1, γ2b and low levels of ε transcripts. In B cells activated by LPS or anti-CD40, we could detect CSR to Cγ2b. In addition, anti-CD40 together with IL4 or IL-5 induced CSR to the γ1 region. We showed that NF-κB activated both by LPS and anti-CD40 bound to the γ1 promoter. However the total concentration of bound NF-κB varied, in addition to the subunit composition induced by the two stimuli. Furthermore, we have investigated the dependency of proliferation for CSR and synthesis of GL transcripts. We examined what effect inhibition of DNA synthesis had on CSR. Cell cycle arrest was induced in cells stimulated with LPS+IL-4 by addition of the DNA synthesis inhibitors Hydroxyurea (HU) and Aphidicholin (AC). We found that CSR was completely abrogated. Addition of HU also reduced GL γ1 and ε transcripts in LPS plus IL-4 activated B cells. To enrich for B cell blasts in different cell cycle phases we used elutriator centrifugation and found that GL γ1 transcripts were expressed in G1 and S phases, at lower levels in G2/M but not in G0. When investigating binding patterns of nuclear proteins to the GL γ1 promoter, we found two major LPS-induced DNA binding protein complexes. These complexes were shown by elutriation experiments to be expressed in G1 and presumably S, but not G0 or G2/M. Furthermore, the complexes bound to an Ets consensus element. We concluded that there exists a relation between proliferation, Ig class switching and GL transcription. The Ets consensus binding site in the γ1 promoter overlaps with a binding site for Ikaros, a protein important for development of all lymphoid cells, which was initially defined as a transcriptional activator but later has been suggested to act as a repressor. Ikaros forms spectacular clusters in the nuclei of activated B cells at loci of heterochromatin. There are a number of possible Ikaros binding sites in the γ1 and ε GL promoters. We showed that Ikaros could bind to several of the possible binding sites in the GL γ1 and ε promoters, presumably in a cooperative manner. Co-transfection of dominant negative Ikaros, unable to bind DNA, with γ1 and ε promoter-luciferase reporter constructs to a B cell line, led to modestly increased basal transcription levels from these promoters. However, retroviral transduction of wildtype or a dominant negative mutation of Ikaros into primary LPS activated B cells, had no effect on Ig class switching or GL transcription, even though the endogenous clusters were disrupted by introduction of the mutated Ikaros. Thus, Ikaros might not be directly involved in transcriptional regulation of specific genes but might exert its function at other levels. Colocalisation of Ikaros with methyltransferase, which serves to direct replication-coupled DNA methylation, might suggest a role in inheritable silencing of genetic material somehow selected for methylation. ©Lena Ström, 2002 ISBN 91-7349-178-0