Negative regulation contributes to tissue specificity of the immunoglobulin heavy-chain enhancer.

We have identified in and around the immunoglobulin heavy-chain enhancer two apparently distinct negative regulatory elements which repress immunoglobulin H enhancer, simian virus 40 enhancer, and heterologous promoter activity in fibroblasts but not in myeloma cells. We propose that in nonlymphoid cells, negative regulatory elements prevent activation of the immunoglobulin H enhancer by ubiquitous stimulatory trans-acting factors.     

Binding in vitro of multiple cellular proteins to immunoglobulin heavy-chain enhancer DNA.

Seven protein-binding sites on the immunoglobulin heavy-chain (IgH) enhancer element have been identified by exonuclease III protection and gel retardation assays. It appears that the seven sites bind a minimum of four separate proteins. Three of these proteins also bind to other enhancers or promoters, but one protein seems to recognize exclusively IgH enhancer sequences. A complex of four binding sites, recognized by different proteins, is located within one 80-base-pair region of IgH enhancer DNA. Close juxtaposition of enhancer proteins may allow protein-protein interactions or be part of a mechanism for modulating enhancer protein activity. All IgH enhancer-binding proteins identified in this study were found in extracts from nonlymphoid as well as lymphoid cells. These data provide the first direct evidence that multiple proteins bind to enhancer elements and that while some of these proteins recognize common elements of many enhancers, others have more limited specificities.     

Complex protein binding within the mouse immunoglobulin heavy-chain enhancer.

We have begun to purify and characterize several proteins which bind to the mouse immunoglobulin heavy-chain enhancer to understand the molecular interactions important for enhancer activity. Three proteins which bind to different sites on the immunoglobulin heavy-chain enhancer have been chromatographically separated and partially purified. One protein binds a site which has not been reported previously and does not bind to other reported protein-binding sites on the immunoglobulin heavy-chain enhancer. Binding-site boundaries for the three partially purified proteins have been precisely mapped by methylation interference, DNase I footprinting, and orthophenanthroline/copper chemical nuclease footprinting. We have also characterized these three proteins with respect to dissociation rate constants.     

Nuclear factors binding to the human immunoglobulin heavy-chain gene enhancer.

The human immunoglobulin heavy chain (IgH) gene contains at least two tissue-specific regulatory regions, which are similar to the mouse IgH gene. One is the J-C enhancer and another is located in the 5' promoter region. Using an electrophoretic mobility shift assay and DNase I footprint, we have examined the interaction of factors in B cell nuclear extracts with the two regulatory regions of the human IgH gene. We have identified a nuclear factor in mouse B cell nuclear extracts which bound to specific sequence in the human IgH enhancer. This factor is apparently not present in mouse fibroblast nuclear extracts. We also found factor(s) which bound to the highly conserved octanucleotide sequence within the human IgH enhancer and 5' promoter regions.     

Complex regulation of the immunoglobulin mu heavy-chain gene enhancer: microB, a new determinant of enhancer function.

The B-lymphocyte-specific activity of the immunoglobulin mu heavy-chain gene enhancer has been attributed to the octamer motif (ATTTGCAT) present within the enhancer that binds a B-cell-specific factor designated NF-A2/OTF-2. However, significant residual enhancer activity even after deletion of this element has suggested the presence of a second critical functional determinant. We have used deletion and mutational analyses to define an element, microB (TTTGGGGAA), that is essential for B-cell-specific enhancer activity in S194 myeloma cells in the absence of the octamer. Transfection analysis in a panel of lymphoid cell lines suggests that the presence of either microB or octamer leads to considerable enhancer activity in cell lines representing later stages of B-cell differentiation, whereas both elements are needed for function in cell lines representing earlier stages. Furthermore, in contrast to the results in pre-B-cell lines, both microB and octamer elements function independently in certain T-cell lines in which the mu enhancer is active.     

Muscle basic helix-loop-helix proteins and the regulation of myogenesis.

Significant progress has been made in defining the structural motifs that distinguish the muscle-specific from other basic helix-loop-helix proteins. Evidence is accumulating for multiple levels of regulation of the expression and action of the muscle basic helix-loop-helix factors.     

Functional analysis of defined mutations in the immunoglobulin heavy-chain enhancer in transgenic mice.

We have analyzed the effect of defined mutations in the mouse immunoglobulin heavy-chain enhancer after introduction into the germline of transgenic mice. We have tested a mutation of the enhancer octamer motif, a double mutation of the octamer motif and the microB-site, and a triple mutation in the microE2, microE3 and microE4-sites. All constructs are expressed in the spleen of transgenic mice. Furthermore, expression is exclusively detectable in lymphoid organs and not in several nonlymphoid tissues. Whereas mutations in the microE-sites have a more pronounced effect on transgene activity in thymocytes as compared to bone marrow and spleen cells, the octamer/microB double mutation shows significantly reduced expression levels only in B-cells. Finally, our results demonstrate that the intronic heavy-chain enhancer element does not contribute to the increase steady state levels of heavy-chain mRNA after stimulation of spleen cells with LPS.     

B-lymphocyte targeting of gene expression in transgenic mice with the immunoglobulin heavy-chain enhancer.

A hybrid gene containing rabbit beta-globin structural sequences (-9 to +1650), and a chicken conalbumin gene promoter (+62 to -102) in the place of the beta-globin promoter (upstream from -9), was inactive in 5 different transgenic mouse line. Adding the mouse immunoglobulin heavy-chain (IgH) enhancer to this construction specifically stimulated expression in B-cells. These results show that IgH enhancer is specifically active in B-cells. Expression of the hybrid gene was low compared to the endogenous immunoglobulin heavy and light-chain genes. Substituting the mouse immunoglobulin kappa light-chain gene (Ig kappa) promoter (+4 to -800) for the heterologous conalbumin promoter was not sufficient to restore gene expression to level of the endogenous genes. In addition to the reproducible B cell expression, we also found inheritable unexpected expression in certain tissues, which varied from line to line.     

Regeneration of the immunoglobulin heavy-chain repertoire after transient B-cell depletion with an anti-CD20 antibody

B-cell depletive therapies have beneficial effects in patients suffering from rheumatoid arthritis. Nevertheless, the role of B cells in the pathogenesis of the disease is not clear. In particular, it is not known how the regeneration of the B-cell repertoire takes place. Two patients with active rheumatoid arthritis were treated with rituximab, and the rearranged immunoglobulin heavy-chain genes (Ig-V_H) were analysed to follow the B-cell regeneration. Patient A was treated with two courses of rituximab, and B-cell regeneration was followed over 27 months by analysing more than 680 Ig-V_H sequences. Peripheral B-cell depletion lasted 7 months and 10 months, respectively, and each time was accompanied by a clinical improvement. Patient B received one treatment course. B-cell depletion lasted 5 months and was accompanied by a good clinical response. B cells regenerated well in both patients, and the repopulated B-cell repertoire was characterised by a polyclonal and diverse use of Ig-V_H genes, as expected in adult individuals. During the early phase of B-cell regeneration we observed the expansion and recirculation of a highly mutated B-cell population. These cells expressed very different Ig-V_H genes. They were class-switched and could be detected for a short period only. Patient A was followed long term, whereby some characteristic changes in the V_H2 family as well as in specific mini-genes like V_H3–23, V_H 4–34 or V_H 1–69 were observed. In addition, rituximab therapy resulted in the loss of clonal B cells for the whole period.     

Ets proteins: new factors that regulate immunoglobulin heavy-chain gene expression.

We used a DNA-protein interaction screening method to isolate a cDNA, Erg-3, whose product binds to a site, designated pi, present in the immunoglobulin (Ig) heavy-chain gene enhancer. Erg-3 is an alternatively spliced product of the erg gene and contains an Ets DNA-binding domain. Fli-1 and PU.1, related Ets proteins, also bind to the same site. In addition, PU.1 binds to a second site, designated microB, in the Ig heavy-chain enhancer. We demonstrate that the pi binding site is crucial for Ig heavy-chain gene enhancer function. In addition, we show that Erg-3 and Fli.1, but not PU.1, can activate a reporter construct containing a multimer of protein-binding sites, synergistically with helix-loop-helix protein E12. We discuss how combinatorial interactions between members of the helix-loop-helix and Ets families may account for the tissue specificity of these proteins.