Ig Heavy Chain Intron Enhancer Research Articles

Elucidation of IgH intronic enhancer functions via germ-line deletion

Studies of chimeric mice demonstrated that the core Ig heavy chain (IgH) intronic enhancer (iEmu) functions in V(D)J and class switch recombination at the IgH locus. To more fully evaluate the role of this element in these and other processes, we generated mice homozygous for germ-line mutations in which the core sequences of iEmu (cEmu) were either deleted (cEmu(Delta/Delta) mice) or replaced with a pgk-Neo(R) cassette (cEmu(N/N) mice). The cEmu(Delta/Delta) mice had reduced B cell numbers, in association with impaired D to J(H) and V(H) to DJ(H) rearrangement, whereas cEmu(N/N) mice had a complete block in IgH V(D)J(H) recombination, confirming that additional cis elements cooperate with iEmu to enforce D to J(H) recombination. In addition, developing cEmu(Delta/Delta) and cEmu(N/N) B lineage cells had correspondingly decreased levels of germ-line transcripts from the J(H) region of the IgH locus (mu0 and Imu transcripts); although both had normal levels of germ-line V(H) transcripts, suggesting that cEmu may influence IgH locus V(D)J recombination by influencing accessibility of J(H) proximal regions of the locus. Consistent with chimera studies, peripheral cEmu(Delta/Delta) B cells had normal surface Ig and relatively normal class switch recombination. However, cEmu(Delta/Delta) B cells also had relatively normal somatic hypermutation of their IgH variable region genes, showing unexpectedly that the cEmu is not required for this process. The availability of mice with the iEmu mutation in their germ line will facilitate future studies to elucidate the roles of iEmu in V(H)(D)J(H) recombination in the context of IgH chromatin structure and germ-line transcription.

T cell receptor (TCR) alpha/delta locus enhancer identity and position are critical for the assembly of TCR delta and alpha variable region genes.

T cell receptor (TCR) delta and alpha variable region genes are assembled from germ-line gene segments located in a single chromosomal locus in which TCR delta segments are situated between TCR alpha segments. The TCR alpha enhancer (E alpha) located at the 3' end of the TCR alpha/delta locus functions over a long chromosomal distance to promote TCR alpha rearrangement and maximal TCR delta expression; whereas the TCR delta enhancer (E delta) is located among the TCR delta segments and functions with additional element(s) to mediate TCR delta rearrangement. We used gene-targeted mutation to evaluate whether the identity of E alpha and the position of E delta are critical for the developmental stage-specific assembly of TCR delta and alpha variable region genes. Specific replacement of E alpha with E delta, the core E alpha element (E alpha C), or the Ig heavy chain intronic enhancer (iE mu), all of which promote accessibility in the context of transgenic V(D)J recombination substrates, did not promote a significant level of TCR alpha rearrangement beyond that observed in the absence of E alpha. Therefore, the identity and full complement of E alpha-binding sites are critical for promoting accessibility within the TCR alpha locus. In the absence of the endogenous E delta element, specific replacement of E alpha with E delta also did not promote TCR delta rearrangement. However, deletion of intervening TCR alpha/delta locus sequences to restore the inserted E delta to its normal chromosomal position relative to 5' sequences rescued TCR delta rearrangement. Therefore, unlike E alpha, E delta lacks ability to function over the large intervening TCR alpha locus and or E delta function requires proximity to additional upstream element(s) to promote TCR delta accessibility.

Novel control motif cluster in the IgH delta-gamma 3 interval exhibits B cell-specific enhancer function in early development.

The majority of the human Ig heavy chain (IgH) constant (C) region locus has been cloned and mapped. An exception is the region between C delta and C gamma 3, which is unstable and may be a recombination hot spot. We isolated a pBAC clone (pHuIgH3'delta-gamma 3) that established a 52-kb distance between C delta and C gamma 3. Sequence analysis identified a high number of repeat elements, explaining the instability of the region, and an unusually large accumulation of transcription factor-binding motifs, for both lymphocyte-specific and ubiquitous transcription activators (IKAROS, E47, Oct-1, USF, Myc/Max), and for factors that may repress transcription (Delta EF1, Gfi-1, E4BP4, C/EBP beta). Functional analysis in reporter gene assays revealed the importance of the C delta-C gamma 3 interval in lymphocyte differentiation and identified independent regions capable of either enhancement or silencing of reporter gene expression and interaction with the IgH intron enhancer E mu. In transgenic mice, carrying a construct that links the beta-globin reporter to the novel delta-gamma 3 intron enhancer (E delta-gamma 3), transgene transcription is exclusively found in bone marrow B cells from the early stage when IgH rearrangement is initiated up to the successful completion of H and L locus recombination, resulting in Ab expression. These findings suggest that the C delta-C gamma 3 interval exerts regulatory control on Ig gene activation and expression during early lymphoid development.

The Ig heavy chain intronic enhancer core region is necessary and sufficient to promote efficient class switch recombination.

The intronic IgH enhancer E(mu), which consists of the core enhancer (cE(mu) flanked by 5' and 3' matrix attachment regions (MAR), has been implicated in the control of IgH locus recombination and transcription. Both cE(mu) and the MAR are required to enhance transcription of an IgH transgene. To elucidate the regulatory functions of cE(mu) versus its associated MAR in IgH class switch recombination (CSR), we have assayed ES cell lines which have targeted deletions of these elements, both individually and in combination, by the Rag-2-deficient blastocyst complementation method. Mutant B cells from chimeric mice were activated in culture and the influence of the mutations on CSR was assessed by analysis of B cell hybridomas. We find that the cE(mu) is necessary and sufficient for providing the functions of E(mu) required for efficient CSR at the IgH locus. However, the 5' and 3' MAR sequences, as well as the known I(mu) transcription start sites and the bulk of I(mu) coding sequences, were dispensable for the process.

Recombination and transcription of the endogenous Ig heavy chain locus is effected by the Ig heavy chain intronic enhancer core region in the absence of the matrix attachment regions.

The intronic Ig heavy chain (IgH) enhancer, which consists of the core enhancer flanked by 5' and 3' matrix attachment regions, has been implicated in control of IgH locus recombination and transcription. To elucidate the regulatory functions of the core enhancer and its associated matrix attachment regions in the endogenous IgH locus, we have introduced targeted deletions of these elements, both individually and in combination, into an IgHa/b-heterozygous embryonic stem cell line. These embryonic stem cells were used to generate chimeric mice by recombination activating gene-2 (Rag-2)-deficient blastocyst complementation, and the effects of the introduced mutations were assayed in mutant B cells. We find that the core enhancer is necessary and sufficient to promote normal variable (V), diversity (D), and joining (J) segment recombination in developing B lineage cells and IgH locus transcription in mature B cells. Surprisingly, the 5' and 3' matrix attachment regions were dispensable for these processes.

Functional motifs in the IgH enhancer of the channel catfish.

The transcriptional enhancer (Emu3') within the Ig heavy chain (IgH) locus of the channel catfish differs from those found in mammalian IgH loci in both its location and structure. However, upon transfection into fish or mouse lymphocytes, it activates transcription to an extent equivalent to that of the mouse IgH intronic enhancer (Emu). Potential transcription factor binding motifs in Emu3' are more numerous than in mammalian IgH enhancers, and are dispersed over 1.6 kilobases. We transfected catfish and mouse lymphoid cells with reporters under the control of artificial promoters containing motifs from the catfish enhancer. We demonstrate that 9 of 11 octamer motifs identified in the catfish enhancer, representing five variations of the consensus octamer (ATGCAAAT), are functional in both a catfish B-cell line (1B10) and the mouse plasmacytoma J558L. Only those octamer variants in which one of the first four bases is altered are active. Clear species differences in the strengths of the variant octamer motifs were evident, and in catfish B cells the ATGtAAAT motif was over threefold more active than the consensus octamer. The one muA and two muB motifs in Emu3' do not contribute to transcriptional activation. These results suggest that the relative functional contributions of IgH enhancer motifs has changed significantly during vertebrate evolution.

Rapid induction of B-cell lymphomas in mice carrying a human IgH/c-mycYAC.

Activation of the c-myc proto-oncogene by one of the immunoglobulin (Ig) loci after chromosomal translocation is a consistent feature of Burkitt's lymphoma. Different subtypes of this tumor vary in the molecular architecture of the translocation region. In most cases there are no known regulatory elements of the Ig locus neighboring the oncogene and this considerably obscures the mechanism of its deregulation. In order to assess possible oncogene activation signals, we produced an experimental translocation region by insertion of a c-myc gene about 50 kb from the IgH intron enhancer in a yeast artificial chromosome (YAC) containing a 220 kb region of the human Ig heavy chain (IgH) locus. Single copy integration of this YAC into the genome of mouse embryonic stem (ES) cells was achieved by spheroplast fusion. Chimeric mice derived from these ES cells developed monoclonal B-cell lymphomas expressing surface IgM by 8-16 weeks of age. The IgH/c-myc translocus showed different V(h)DJ(H) rearrangement in almost all tumors without any alterations of the distance between c-myc and the IgH intron enhancer. This mouse model can be used for the in vivo analysis of c-myc deregulation and the tumor formation capacity of the IgH locus in aberrant rearrangements.

Expression of Epstein-Barr virus nuclear antigen-1 induces B cell neoplasia in transgenic mice.

The Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA-1) is a pleiotropic protein which has been characterized extensively both biochemically and functionally. It is the only one of the identified latent protein-encoding genes to be consistently expressed in viral-associated endemic Burkitt's lymphoma cells. As such, it is the only candidate viral protein to possibly perform a maintenance function in the tumour pathology. Despite this, no oncogenic activity has been attributed to the protein in tissue culture assays. The experiments described here were initiated to explore the activity of the protein in B cells in vivo. EBNA-1 transgenic mice were generated with transgene expression directed to the B cell compartment using the mouse Ig heavy chain intron enhancer. Transgene expression was demonstrated in the lymphoid tissues of mice of two independent lines. Transgenic positive mice of both lines succumb to B cell lymphoma. The B cell tumours are monoclonal, frequently of follicular centre cell origin and remarkably similar to those induced by transgenic c-myc expression. These results demonstrate that EBNA-1 is oncogenic in vivo and suggest that the gene product may play a direct role in the pathogenesis of Burkitt's lymphoma and possibly other EBV-associated malignancies.

An Ig heavy chain enhancer of the channel catfish Ictalurus punctatus: evolutionary conservation of function but not structure.

The teleost fishes are among the earliest evolutionary lineages to have an Ig heavy chain (IgH) locus whose organization approximates that of mammals. To understand transcriptional control of the IgH locus in a teleost fish and to gain insight into the evolution of the control elements, the enhancer activity in the IgH locus of the channel catfish, Ictalurus punctatus, was investigated. Segments of the locus extending from upstream of the proximal JH gene to 2.5 kb downstream of the second transmembrane (TM2) exon of the mu gene were tested in transient transfection expression assays in murine myeloma and T cell lines, and in catfish B lymphoblastoid, monocyte-like, and putative T cell lines. In marked contrast to mammals, no enhancer activity was observed in the catfish JH to C mu intron, but strong enhancer activity (approaching that of the murine IgH intronic enhancer) was identified in a 1.8-kb segment that included the TM2 exon. This catfish enhancer was active in a B lineage-specific manner in both catfish and murine cells. It was not localized in a small core region, but appeared to contain multiple, dispersed cooperative elements rich in octamer- and mu E5-related motifs. Although the catfish IgH enhancer shares functional characteristics with the mammalian IgH intronic and 3' enhancers, its unusual organization does not permit any obvious inferences concerning evolutionary relationships between the catfish enhancer and any one of the murine IgH enhancers.

Transcriptional regulation of immunoglobulin gene expression by anti-Ig.

When transfected into mouse splenic B cells stimulated with lipopolysaccharide (LPS) the expression of DNA vectors containing the chloramphenicol acetyl transferase gene under the control of a SP6 kappa promoter and the Ig heavy chain intron enhancer could be down-regulated 5- to 10-fold by treatment of the cells with anti-Ig prior to transfection. Exchanging the SP6 kappa promoter by minimal promoters consisting of an octamer or a SP1 motif linked to TATA box did not impair the anti-Ig induced down-regulation while inserting a rabbit beta-globin promoter did. The transcriptional regulation could be observed after replacing the Ig heavy chain intron enhancer with a SV40 enhancer, or duplicated minimal Ig heavy chain enhancers containing or lacking the octamer element. The down-regulation was not dependent on the level of transcriptional stimulation observed. A difference in Oct2 expression could neither be detected at the RNA nor protein level after treatment of LPS stimulated B cells with anti-Ig or phorbol-dibutyrate. Anti-Ig treatment, but not phorbol-di-butyrate treatment, induced increased levels of AP1 and NF kappa B transcription factors. Thus, either differentiation specific transcriptional control of Ig genes is exerted via transcription factors common to several distinct enhancers or via transcriptional adaptor molecules that can interact with several distinct DNA binding proteins.

A survey of protein-DNA interaction sites within the murine immunoglobulin heavy chain locus reveals a particularly complex pattern around the DQ52 element.

The expression of immunoglobulin (Ig) genes is regulated at two levels: rearrangement of individual gene segments and transcription of continuous genes. To find transacting factors involved in mediating locus- and segment specific gene activation and expression, we surveyed a 3600-bp genomic region of the murine Ig heavy chain locus, spanning from the DQ52 element to the Ig heavy chain intron enhancer. We discovered nine, previously undescribed, protein-DNA complexes and estimated their individual binding-affinity preferences (Kr) by quantitative gel shift measurements. We observed one novel protein DNA interaction at the enhancer, two in the JH1 region and six within a 300-bp region immediately 5' to the DQ52 locus. The latter show a complex and specific binding pattern when comparing nuclear extracts derived from pre-B cells and fibroblasts. Further characterization of the interactions at the DQ52 locus by electron microscopy revealed the preferential formation of a protein complex binding to the DQ52 locus with pre B cell extracts. This behavior and the clustering of interaction sites 5' to the DQ52 element suggest that this region is involved in the regulation of heavy chain gene assembly.

‘E‐Boxes’ as Promoter Elements in B Cell Lines and Untransformed B Lymphocytes

The penta-deca (pd) promoter element from the SP6 kappa promoter and the muE2 box from the Ig heavy chain intron enhancer were analysed in an electrophoretic mobility shift assay (EMSA), utilising cell extracts from total mouse splenic B cells before and after stimulation with lipopolysaccharide. With both probes a changed EMSA pattern was observed after stimulation of the cells, where higher molecular weight DNA/protein complexes became dominant. When the pd and muE2 sequence elements were cloned in front of a TATA box and analysed for their transcriptional promoting activity in lipopolysaccharide stimulated B cells, they were inactive. On the contrary, the same constructs were transcriptionally active in some but not all B cell lines. Thus, E-boxes display functional heterogeneity as transcriptional activators depending on host cell.