Determinant differences between the rabbit and mouse immunoglobulin kappa enhancers impair the activity of the rabbit enhancer in mouse myeloma cells.

Enhancer activity of the rabbit immunoglobulin kappa light chain gene intron conserved region (KICR) was examined in mouse myeloma cells using transient expression experiments. Compared to the homologous region of the mouse kappa light chain gene, the rabbit KICR shows nearly no stimulatory effect on expression of the indicator gene, cat. Experiments with mouse-rabbit chimeric KICRs indicated that differences in the region around the NF-kappa B binding site are responsible for the impaired activity of the rabbit KICR whereas mouse sequences covering the kappa E2 and kappa E3 motifs can be replaced by the equivalent rabbit fragment without affecting enhancer function. Creation of a perfect mouse NF-kappa B target sequence in the rabbit gene only partially restores enhancer activity. Furthermore, mouse and rabbit DNA fragments encompassing the NF-kappa B target sequence behave in an identical manner in an electrophoretic mobility shift assay. The results indicate species-related functional differences in the immunoglobulin kappa light chain gene enhancer and suggest that although the NF-kappa B binding site plays a crucial role in enhancer activity surrounding gene elements are also necessary for full enhancer effect.     

Kappa immunoglobulin promoters and enhancers display developmentally controlled interactions.

We have investigated the interaction of the kappa immunoglobulin light chain intron and 3' enhancers with two different kappa promoters at distinct stages of B-cell development. We find that transiently transfected reporter gene constructs driven by either the kappa V-region promoter, or the kappa germline promoter, are controlled by the known enhancers of the locus in a developmentally regulated fashion. We have, however, observed differences in promoter activation by each enhancer. Moreover, constructs controlled by a combination of both enhancers are synergistically activated at the B-cell and plasma cell stages as compared with constructs containing either enhancer alone. This synergy is not observed early in development, at the pre-B cell stage. The pattern of enhancer and promoter interactions is discussed in the context of the known developmental regulation of the locus.     

Structural analysis of a rabbit immunoglobulin kappa 2 J-C locus reveals multiple deletions.

We previously reported that domestic rabbits harbor at least two DNA sequences that hybridize strongly to immunoglobulin kappa C region probes in Southern blots. One of these was cloned from a domestic b4 rabbit and identified as the gene for the nominal b4 allotype kappa chain which is expressed at high levels. We now have cloned (from a b4 rabbit) the other homologous sequence and find that it encodes a kappa chain nearly identical to the kappa 2 chain of "bas" rabbits, which is not normally expressed at detectable levels in domestic rabbits. Sequence analysis of this kappa 2 chain reveals a J kappa -C kappa locus with no obvious coding sequence defects that could explain its low expression. However, several base changes in a putative enhancer region as well as deletions (totalling about 1.5 kb) in the J-C intron might be related to low expression. The comparison between these two kappa genes raises questions about the selective pressures operating during the evolution of this gene system.     

An enhancer at the 3'' end of the mouse immunoglobulin heavy chain locus.

A tissue-specific enhancer (E mu) lies between the joining (JH) and mu constant region (C mu) gene segments of the immunoglobulin heavy chain (IgH) locus. Since mouse endogenous IgH genes are efficiently transcribed in its absence, the normal function of this enhancer remains ill-defined. Recently, another lymphoid-specific enhancer of equal strength has been identified 3' of the rat IgH locus. We have isolated an analogous sequence from mouse and have mapped it 12.5 kb 3' of the 3'-most constant region gene (C alpha-membrane) of the BALB/c mouse locus. The mouse and rat sequences are 82% homologous and share with other enhancers several DNA sequence motifs capable of binding protein. However, in transient transfection assays, the mouse sequence behaves as a weaker enhancer. The role of this distant element in the expression of endogenous IgH genes, both in E mu-deficient, Ig-producing cell lines and during normal B cell development, is discussed.     

Deletion of the immunoglobulin kappa chain intron enhancer abolishes kappa chain gene rearrangement in cis but not lambda chain gene rearrangement in trans.

Immunoglobulins (Ig) secreted from a plasma cell contain either kappa or lambda light chains, but not both. This phenomenon is termed isotypic kappa-lambda exclusion. While kappa-producing cells have their lambda chain genes in germline configuration, in most lambda-producing cells the kappa chain genes are either non-productively rearranged or deleted. To investigate the molecular mechanism for isotypic kappa-lambda exclusion, in particular the role of the Ig kappa intron enhancer, we replaced this enhancer by a neomycin resistance (neoR) gene in embryonic stem (ES) cells. B cells heterozygous for the mutation undergo V kappa-J kappa recombination exclusively in the intact Ig kappa locus but not in the mutated Ig kappa locus. Homozygous mutant mice exhibited no rearrangements in their Ig kappa loci. However, splenic B cell numbers were only slightly reduced as compared with the wild-type, and all B cells expressed lambda chain bearing surface Ig. These findings demonstrate that rearrangement in the Ig kappa locus is not essential for lambda gene rearrangement. We also generated homozygous mutant mice in which the neoR gene was inserted at the 3' end of the Ig kappa intron enhancer. Unexpectedly, mere insertion of the neoR gene showed some suppressive effect on V kappa-J kappa recombination. However, the much more pronounced inhibition of V kappa-J kappa recombination by the replacement of the Ig kappa intron enhancer suggests that this enhancer is essential for V kappa-J kappa recombination.     

Regulation of human Ig lambda light chain gene expression by NF-kappa B.

The human Iglambda enhancer consists of three separated sequence elements that we identified previously by mapping DNase I-hypersensitive regions (HSS) downstream of the C region of the Iglambda L chain genes (HSS-1, HSS-2, and HSS-3). It has been shown by several laboratories that expression of the H chain genes as well as the kappa genes, but not the lambda genes, is dependent on constitutive NF-kappaB proteins present in the nucleus. In this study we show by band-shift experiments, in vivo footprinting, and transient transfection assays that all three hypersensitive sites of the human Iglambda enhancer contain functional NF-kappaB sites that act synergistically on expression. We further show that the chicken lambda enhancer also contains a functional NF-kappaB site but the mouse lambda enhancer contains a mutated, nonfunctional NF-kappaB site that is responsible for its low enhancer activity. It is possible that the inactivating mutation in the mouse Iglambda enhancer was compensated for by an expansion of the Igkappa L chain locus, followed by a contraction of the Iglambda locus in this species.     

Characterization of a cardiac-specific enhancer, which directs {alpha}-cardiac actin gene transcription in the mouse adult heart

Expression of the mouse alpha-cardiac actin gene in skeletal and cardiac muscle is regulated by enhancers lying 5' to the proximal promoter. Here we report the characterization of a cardiac-specific enhancer located within -2.354/-1.36 kbp of the gene, which is active in cardiocytes but not in C2 skeletal muscle cells. In vivo it directs reporter gene expression to the adult heart, where the proximal promoter alone is inactive. An 85-bp region within the enhancer is highly conserved between human and mouse and contains a central AT-rich site, which is essential for enhancer activity. This site binds myocyte enhancer factor (MEF)2 factors, principally MEF2D and MEF2A in cardiocyte nuclear extracts. These results are discussed in the context of MEF2 activity and of the regulation of the alpha-cardiac actin locus.