Regulation of the murine immunoglobulin GL gamma-1 and epsilon 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 lg genes undergo two types of modifications: somatic hypermutation and class switch recombination (CSR). During CSR the effector function of an lg molecule is altered, while the antigen specificity is remained, resulting in expression of IgG, lgA or lgE 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 C-gamma-1 and C-epsilon regions has been the main focus of this thesis.

Initially, we explored whether the CD40/CD4OL interaction was sufficient for B cells to undergo CSR. In vivo distribution of an agonistic monoclonal rat anti-mouse CD40-antibody (ab) induced up- regulation of all lg isotypes in mice completely devoid of T cells. This induction was to some extent independent of IL-4, since lgE synthesis was only partially inhibited by co-injection of anti-IL-4. Anti-CD40 also induced GL c transcripts and expression of lgE 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 gamma-1, gamma-2-b and low levels of E transcripts. In B cells activated by LPS or anti-CD40, we could detect CSR to C-gamma-2-b. In addition, antiCD40 together with IL-4 or IL-5 induced CSR to the gamma-1 region. We showed that NF-kappaB activated both by LPS and anti-CD40 bound to the gamma-1 promoter. However the total concentration of bound NF-kappaB 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 gamma-1 and c 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 gamma-1 transcripts were expressed in G1, and S phases, at lower levels in G2/M but not in G. When investigating binding patterns of nuclear proteins to the GL gamma-1 promoter, we found two major LPS-induced DNA binding protein complexes. These complexes were shown by elutriation experiments to be expressed in G, 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 gamma-1 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 gamma-1 and epsilon GL promoters. We showed that Ikaros could bind to several of the possible binding sites in the GL gamma-1 and epsilon promoters, presumably in a cooperative manner. Co-transfection of dominant negative Ikaros, unable to bind DNA, with gamma-1 and epsilon 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 lg 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.

List of scientific papers

I. Ferlin WG, Severinson E, Strom L, Heath AW, Coffman RL, Ferrick DA, Howard MC (1996). CD40 signaling induces interleukin-4-independent IgE switching in vivo. Eur J Immunol. 26(12): 2911-5.
https://doi.org/10.1002/eji.1830261216

II. Strom L, Laurencikiene J, Miskiniene A, Severinson E (1999). Characterization of CD40-dependent immunoglobulin class switching. Scand J Immunol. 49(5): 523-32.
https://doi.org/10.1046/j.1365-3083.1999.00539.

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III. Lundgren M, Strom L, Bergquist LO, Skog S, Heiden T, Stavnezer J, Severinson E (1995). Cell cycle regulation of immunoglobulin class switch recombination and germ-line transcription: potential role of Ets family members. Eur J Immunol. 25(7): 2042-51.
https://doi.org/10.1002/eji.1830250736

IV. Strom L, Lundgren M, Severinson E (2002). The function of Ikaros as a regulator of Ig class switching. [Submitted]