Regulation of germline transcription in the immunoglobulin heavy chain locus

During an immune response, activated B cells develop into high rate immunoglobulin (Ig) secreting plasma cells. They also switch from production of IgM to IgG, IgA or IgE. Specific isotypes of antibodies have functional and structural features that make them particularly well suited to defend against different types of pathogens. IgM is changed to another isotype via class switch recombination (CSR) - a non-homologous DNA recombination process. CSR is at least in part regulated at the transcriptional level. The external stimuli (cytokines or cell-to-cell contact) induce CSR to a particular constant heavy chain (CH) gene via their ability to modulate germline (GL) transcription of a given CH gene. The GL transcription is driven by GL promoters located upstream of each CH region. The GL promoters contain multiple transcription-factor binding sites and are tightly regulated during B cell differentiation. In addition, several enhancer elements with developmentally regulated activities are located in the IgH locus. The mouse 3' enhancers span a region of more than thirty kilobases and consist of four DNAse hypersensitivity sites - HS3a, HSI,2, HS3b and HS4. One possible function of the 3' enhancers could be regulation of CSR. We aimed to clarify the role of different IgH locus regulatory elements and transcription factors in regulation of GL transcription. Much evidence indicated that T cell-dependent activation of B lymphocytes is generated through the interaction of CD40 (on B cells) with CD40 ligand (on activated T cells). Therefore, using agonistic rat anti-mouse CD40 monoclonal antibody, we investigated the role of CD40 signalling in CSR. We found that stimulation of murine cells through CD40 induced GL y 1, y2b and low levels of e transcripts. However, cells were expressing only IgG2b, but not IgG1 or IgE. CD40, but not lipopolysaccharide (LPS), could induce GL y I transcripts, although both CD40 and LPS signalling involve NF-kB family proteins. We showed that LPS and CD40 stimulation induce different NF-kB complexes binding to GL y I promoter. he increased levels of IgE in atopic individuals could be associated with upregulation of GL e transcription. The transcription factor STAT6 is an important activator of the GL e promoter, induced by interleukin 4 (IL-4). We investigated whether increased levels of IgE in allergic individuals may be associated with alterations in the level or activation of STAT6. Our results demonstrated for the first time that upon IL-4 signalling, STAT6 activation and basal GL E promoter activity differ in B cells from different individuals. Although we did not find any association between STAT6 activation and allergy, we do not exclude the possibility that stronger activation of this transcription factor is associated with the atopic phenotype. Next, we investigated regulation of GL transcription by the IgH locus 3' enhancers. Previous studies have shown that the activity of the HS1,2 enhancer was induced in B cells approximately at the same differentiation stage when GL transcription is…