Transcription factors regulating the immunoglobulin heavy chain locus

Detta är en avhandling från Stockholm : Karolinska Institutet, Center for Genomics Research

Sammanfattning: The immunoglobulin heavy (IgH) chain locus is subject to somatic recombination and changes in gene expression in B lineage cells. Enhancers located in the 3' end of the locus may in part regulate these events. This work aimed at increasing the understanding of the function of one of these elements, the HS1,2 enhancer. Analysis of a [beta]-globin promoter/HS 1,2-linked reporter gene demonstrated that CD40 receptor-mediated signalling can activate HS 1,2 in transgenic mice. This activation was concomitant with the recruitment of protein complexes to ETS/AP-1 and octamer DNA motifs, as shown by in vivo footprinting. The CD40 induced complex NFAB-II (nuclear factor of activated B cells) differed in composition to the IgM receptor-induced NFAB-I. While both NFAB complexes contained Elf-1 and JunB, c-Fos in NFAB-I was substituted for a Fos-related protein in NFAB-II. CD40-mediated activation of NFAB-11 proteins potentiated transcription of an ETS/AP-1 linked reporter gene. Thus, two separate signal transduction pathways can not only activate the HS1,2 enhancer, but also appear to converge on a common downstream target, the ETS/AP- I motif, albeit with different AP- I protein complexes. HS1,2 contains a high affinity binding site for the Pax-5-encoded BSAP protein. Pax-5 expression is induced in early B cell development and observed at all stages except the plasma cell stage. When assessing Pax-5 expression following stimulation of a HS 1,2-linked transgene with lipopolysaccharide or via IgM/CD40 receptor-signalling in B lymphocytes, Pax-5 expression remained unaffected until the plasma cell stage. While BSAP repressed a HS1,2-linked reporter gene in a plasma cell line, the same protein could act as an activator when the DNA binding site was multimerised and inserted upstream of a minimal promoter. Thus, BSAP appears to possess seemingly different effector functions depending on the context of the DNA template. A VH promoter-C[my] transgene, potentiated by HS 1,2, demonstrated that this enhancer can interact with an 19 VH promoter in vivo. High levels of transgene expression were observed in spleen and thymus and low levels outside the lymphoid lineage. Moreover, the HS1,2 enhancer directed temporal expression of the VH promoter, since transgene expression was confined to activated splenic B cells and peritoneal B-lymphocytes. This temporal regulation may depend on the lymphoid-restricted cofactor OBF-1, since OBF-/- splenic B cells exhibited a decreased expression of the VH/HS 1,2 transgene upon in vitro stimulation with CD40 and LPS+IL-4. Peritoneal B cells displayed a similar defect in transgene expression in vivo. In contrast, the expression of a [beta]-globin promoter/HS1,2-driven transgene was not affected in the corresponding OBF-/- B cell populations. These data suggest that the IgH locus is a target for OBF- I in late B cell development.

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