A DNase I hypersensitive site near the murine gamma1 switch region contributes to insertion site independence of transgenes and modulates the amount of transcripts induced by CD40 ligation.

Several cis-acting elements regulate the expression of germline transcripts of heavy chain constant region genes and their subsequent switch recombination. To study such elements in the murine gamma1 gene, we have utilized a transgenic approach. In this study we focused on a DNase I hypersensitive site (termed 'Site II') that lies about 2 kb 3' of the gamma1 promoter region and I exon, just 5' to the gamma1 switch region. We have reported that gamma1 transgenes with Site II display the characteristics of a locus control region (LCR) in that they are insertion site independent and copy number dependent. For the present study we prepared six lines of transgenic mice that have the promoter region and I exon, but lack Site II. Expression of RNA from gamma1 transgenes that lack Site II is not correlated with transgene copy number; expression is insertion site dependent. This result indicates that DNase hypersensitive Site II is an important part of the LCR-like elements in the murine gamma1 gene. RNA expression from the gamma1 transgenes that lack Site II is inducible by IL-4 and by CD40 ligation. However, the induction of transgenic RNA expression by CD40 ligation is greater than expected, suggesting that elements within Site II participate in negative regulation of the amount of germline transcripts after CD40 ligation.

Cutting edge: IFN-gamma regulated germline transcripts are expressed from gamma2a transgenes independently of the heavy chain 3' enhancers.

Several results indicate that transcriptional enhancers lying 3' of the Calpha gene regulate RNA expression and switch recombination of heavy chain genes. To investigate this regulation we prepared transgenic mice with a 10.5-kb transgene that included the germline form of the murine gamma2alpha gene, including promoter, I, S, and C regions. RNA was expressed from these gamma2a transgenes with correct IFN-gamma regulation, in spite of the fact that they lacked the 3' enhancers. This RNA expression was independent of insertion site and dependent on copy number, indicating that the gamma2a gene includes locus control region-like elements. Addition of either a cassette containing 3' enhancer DNase I hypersensitive sites 1, 2, 3B, and 4 or the intronic micro enhancer increased transcription from the gamma2a transgene by approximately 75-fold in B cells. However, this increased transcription was not responsive to IFN-gamma treatment of the transgenic B cells.

Germline transcription and recombination of a murine VDJmudeltagamma1 transgene.

To investigate the regulation of Ig switch recombination, we have constructed mice with a 56 kb VDJmudeltagamma1 transgene. This transgene included an anti-nitrophenyl VDJ segment, Smu, Cmu, Cdelta, Igamma1, Sgamma1, Cgamma1 and the Cgamma1 membrane exons from the murine Igh(a) haplotype. Two founder lines were produced, with very similar characteristics. Transgenic B cells expressed normal amounts of Cmu (which is >95% transgenic), Cdelta and other cell surface markers, and normal amounts of VDJ and Cmu RNA. Gamma1 germline transcription of the transgenes is properly regulated since stable transcripts were not expressed in B cells treated with lipopolysaccharide (LPS) alone, nor in thymus or non-lymphoid tissues, but were expressed after treatment of B cells with LPS + IL-4 or CD40L + IL-4. B cells from both lines of transgenic mice expressed transgenic gamma1a after in vitro culture with CD40L + IL-4, but not after culture with CD40L alone. However, the CD40L + IL-4 induced IgG1 precursor frequency is much lower for VDJmudeltagamma1 transgenic B cells (1:240-760) than for non-transgenic B cells (1:9). Analysis of DNA from transgenic hybridomas indicated that switch recombination can take place in switch (S) regions, but can also take place outside S regions. These results indicate that targeting of switch recombinase to S regions must include regulation beyond the S regions themselves and correct germline transcription. This additional regulation might include cis-acting elements or appropriate spacing or arrangement of the recombining elements.

Potential regulatory elements for germline transcription in or near murine Sgamma1.

We were interested in identifying cis-acting elements that regulate germline transcription and switch recombination of heavy chain genes. The murine gamma1 heavy chain gene includes two DNase I hypersensitive sites, which may represent protein:DNA interactions important for germline transcription and switch recombination. One DNase hypersensitive site is at the promoter/I exon boundary (termed 'Site I'); we localized a second pair of DNase hypersensitive sites to just 5' of the Sgamma1 region (termed 'Site II'). The DNA region of hypersensitive Site II includes a NF-kappaB/Rel binding site and a STAT6 binding site. It is noteworthy that NF-kappaB and STAT6 are induced by the same agents (CD40 ligation and IL-4 respectively) that stimulate germline transcription and switch recombination of the murine gamma1 gene. Transgenes with the gamma1 promoter region (DNase hypersensitive Site I), Igamma1 and DNase I hypersensitive Site II expressed germline transcripts with correct regulation, including IL-4 inducibility. However, the level of stable transcripts produced by the transgenes was much lower than that of the endogenous gamma1 gene, a complete 17 kb gamma1 transgene or a derivative of the 17 kb gamma1 transgene that lacked most of Cgamma1. The promoter/Igamma1/Site II transgenes lacked Sgamma1 and we found that gamma1 transgenes that lacked only Sgamma1 also expressed germline transcripts with proper regulation, but at a low level. This suggested that the Sgamma1 region includes positive elements for regulation of the amount of germline transcripts.

Interleukin-12 acts as an adjuvant for humoral immunity through interferon-gamma-dependent and -independent mechanisms.

Interleukin-12 (IL-12) is a pivotal cytokine that has dramatic effects on cell-mediated immunity. It is now becoming increasingly recognized that IL-12 also strongly controls humoral immunity. We have investigated the mechanism by which IL-12 induces alterations in antibody isotype expression by determining the influence of IL-12 on in vitro immunoglobulin (Ig) production in polyclonally activated murine spleen cell cultures. Cells exposed to IL-12 plus lipopolysaccharide or anti-CD40 monoclonal antibody showed dramatically elevated IgG2a and suppressed IgG1 production compared to cells cultured in the absence of IL-12. IL-12 treatment of spleen cell cultures induced expression of gamma2a germ-line transcripts, consistent with initiation of switch recombination to IgG2a. In addition, exposure of limiting dilution cultures to IL-12 increased IgG2a+ cell precursor frequency. All of the above results were dependent on interferon-gamma (IFN-gamma). However, in the absence of IFN-gamma, IL-12 still had significant effects on Ig secretion. Specifically, IL-12 enhanced IgG1 and IgG2b anti-DNP antibody levels in mice containing specific disruptions in the IFN-gamma gene. Our results suggest that IL-12 induces T helper type 1 and natural killer cells to secrete large amounts of IFN-gamma which then causes B cells to switch to IgG2a and IgG3 production. In addition, IL-12 has direct or indirect effects on B cells that are independent of IFN-gamma. The IFN-gamma-independent effects may include enhancement of Ig expression by post-switched cells.

gamma1 heavy chain transgenes are responsive to IFN-gamma repression and CD40 ligation.

The cis-acting elements that regulate production of germ-line transcripts from Ig heavy chain genes and subsequent switch recombination to those genes are poorly defined. We reported that a 17-kb transgene that includes Igamma1, Sgamma1, and Cgamma1 is regulated for germ-line transcription like the endogenous gamma1 gene. Transcripts from such transgenes are expressed only in B cells treated with both LPS and IL-4, and not in B cells treated with LPS alone or in thymocytes or nonlymphoid tissues. We have now found that transcripts from these transgenes are induced by treatment of transgenic B cells by IL-4 alone. As reported by others, IFN-gamma acts to inhibit the IL-4-mediated induction of germ-line transcripts of the endogenous gamma1 gene. We have found that LPS-plus IL-4-induced germ-line transcription of gamma1 transgenes is likewise inhibited by treatment of B cells with IFN-gamma, so the gamma1 gene must include the cis-acting element(s) that confers this inhibition. It is also known that CD40 ligation induces a modest amount of germ-line transcripts from the endogenous gamma1 gene and synergizes with IL-4 to induce large amounts of germ-line transcripts. The gamma1 transgenes are likewise induced by CD40 ligation, suggesting that the response element(s) for CD40 ligation can be found in the gamma1 gene. The promoter region for the germ-line transcripts and the I exon are likely to include some of these cis-acting elements. A series of transgenic mice with the promoter/Igamma1 region conferred low level, lymphoid-specific, RNA expression to a reporter gene, including significant expression in thymocytes. However, the promoter/Igamma1 transgenes were not regulated like the endogenous gamma1 gene, in that transcription in splenic B cells was not increased by LPS plus IL-4.

Enhancement of humoral immunity by interleukin-12.

We have found that IL-12 treatment of mice leads to long-lasting enhancement in production of most antibody isotypes in conventional B-cell responses. Initial recruitment of new B-cell clones into the response is mediated by IFN-gamma, but subsequent enhancement of Ig secretion appears to be IFN-gamma-independent. We have further found that activated B cells can directly bind IL-12. Taken together, our results suggest a two-step model for the role of IL-12 in enhancement of humoral immunity. Initially, IL-12 induces production of IFN-gamma from Th1 and NK cells. Enough cytokine can be produced from either cell type to then mediate gamma 2a heavy chain isotype switching as well as temporary suppression of IgG1 production. IL-12 further stimulates post-switched cells, including cells producing IgG1, to secrete greatly increased amounts of antibody. This step is not mediated by IFN-gamma but might be due to direct IL-12 binding to activated B lymphocytes. Depletion of B1 cells by IL-12 may further enhance antibody responsiveness since B1 cells are known to competitively inhibit Ig secretion by conventional B cells. The end result is that IL-12 causes a generalized upregulation in production of all antibodies and therefore acts as a strong adjuvant for humoral as well as cellular immunity.

The gamma 1 heavy chain gene includes all of the cis-acting elements necessary for expression of properly regulated germ-line transcripts.

The regulation of heavy chain switch recombination and the production of germ-line transcripts are highly correlated. IL-4 induces the production of murine gamma 1 germ-line transcripts, and much, if not all, of the regulation is transcriptional. We have investigated the cis-acting elements involved in the regulation of expression of germ-line transcripts by preparing transgenes with the gamma 1 locus. A construct that includes 5' flanking regions, I gamma 1, S gamma 1, and C gamma 1, is regulated like the endogenous gene. Deletion of either most of S gamma 1 or most of C gamma 1 does not alter the correctly regulated expression of the transgenes. An element common to these three different gamma 1 transgenes confers insertion-site independence and copy-number dependence on the transgenes. Finally, the absolute amount of gamma 1-line transcripts is regulated, as transgenic mice with more than 30 gamma 1 genes express no more germ-line transcripts than nontransgenic mice.

Antigen receptor cross-linking differentially regulates germ-line CH ribonucleic acid expression in murine B cells.

Cytokines are believed to regulate Ig class switching, in part, through selective modulation of germ-line constant heavy (CH) gene transcription. B cell activators such as LPS or activated T cell membranes also influence germ-line CH RNA expression in the absence of exogenous cytokines. In this report we determined whether multivalent Ag receptor cross-linking, utilizing dextran-conjugated anti-IgD Abs (alpha delta-dex), could also regulate germ-line CH RNA expression. We demonstrated that alpha delta-dex markedly inhibited germ-line epsilon RNA expression, but strongly augmented germ-line gamma 1 RNA, in LPS + IL-4-stimulated cultures. This was correlated with > 90% alpha delta-dex-mediated suppression in the secretion of IgE and generation of membrane (m)IgE+ cells, and a more modest 50% reduction in IgG1 synthesis and mIgG1+ cells. Furthermore, alpha delta-dex inhibited the LPS induction of both gamma 3 and gamma 2b germ-line RNA and the associated secretion of IgG3 and IgG2b. A similar alpha delta-dex-mediated suppression of germ-line gamma 2a RNA and IgG2a secretion in LPS + IFN-gamma-stimulated cultures was observed. By contrast, activation of resting B cells with alpha delta-dex alone led to induction of germ-line gamma 3, gamma 1, and gamma 2b RNA but did not stimulate detectable expression of RNA specific for gamma 2a or epsilon. These studies demonstrate that: 1) germ-line gamma 1 gene expression is regulated uniquely, 2) germ-line transcription and switch recombination can be dissociated, 3) the germ-line transcription of each IgG isotype has an independent pattern of regulation, and 4) cross-linking of the Ag receptor, by itself, can stimulate small amounts of germ-line CH RNA.

Germline transcripts of the murine immunoglobulin gamma 2a gene: structure and induction by IFN-gamma.

The switch in expression by B cells from IgM to IgG, IgE, or IgA is accomplished by a DNA deletion. The deletion event is regulated, in that specific cytokines direct the B cell to switch to one, or sometimes two, of the six possible murine heavy chain genes. Prior to switch recombination, cytokine treatment also induces the transcription of the constant, switch, and upstream regions of the targeted heavy chain. Much evidence indicates that IFN-gamma directs switch recombination to the murine gamma 2a gene. By developing probes specific for the gamma 2a gene, we demonstrate that IFN-gamma increases germline transcription of this gene in both normal B cells and in the 18.81.A20 pre-B lymphoma. We have obtained cloned copies of three different germline gamma 2a transcripts, each with a different donor splice site. We have also located the 5' ends of these transcripts. The vast majority of the germline gamma 2a transcripts have a long first exon (> 700 bp), consistent with observations by Severinson and her colleagues.

mnd2: a new mouse model of inherited motor neuron disease.

The autosomal recessive mutation mnd2 results in early onset motor neuron disease with rapidly progressive paralysis, severe muscle wasting, regression of thymus and spleen, and death before 40 days of age. mnd2 has been mapped to mouse chromosome 6 with the gene order: centromere-Tcrb-Ly-2-Sftp-3-D6Mit4-mnd2-D6Mit 6, D6Mit9-D6Rck132-Raf-1, D6Mit11-D6Mit12-D6Mit14, mnd2 is located within a conserved linkage group with homologs on human chromosome 2p12-p13. Spinal motor neurons of homozygous affected animals are swollen and stain weakly, and electromyography revealed spontaneous activity characteristic of muscle denervation. Myelin staining was normal throughout the neuraxis. The clinical observations are consistent with a primary abnormality of lower motor neuron function. This new animal model will be of value for identification of a genetic defect responsible for motor neuron disease and for evaluation of new therapies.

DNA sequences at immunoglobulin switch region recombination sites.

The immunoglobulin heavy chain switch from synthesis of IgM to IgG, IgA or IgE is mediated by a DNA recombination event. Recombination occurs within switch regions, 2-10 kb segments of DNA that lie upstream of heavy chain constant region genes. A compilation of DNA sequences at more than 150 recombination sites within heavy chain switch regions is presented. Switch recombination does not appear to occur by homologous recombination. An extensive search for a recognition motif failed to find such a sequence, implying that switch recombination is not a site-specific event. A model for switch recombination that involves illegitimate priming of one switch region on another, followed by error-prone DNA synthesis, is proposed.

Nuclear protein binding to octamer motifs in the immunoglobulin gamma 1 switch region.

Initiation of the immunoglobulin heavy chain switch DNA rearrangement event is thought to involve conversion of the target switch region DNA to an accessible state. Accessibility is likely to be mediated by the binding of regulatory proteins to sequences in or near switch regions. A DNase hypersensitivity assay was used to recognize possible regions of protein binding in the gamma 1 switch region of the B cell hybridoma 470.25. A strong DNase hypersensitive site was identified 5' of the tandemly repeated S gamma 1 sequences. Data from other laboratories suggest that this hypersensitive site is associated with switch recombination to gamma 1. However, the 470.25 cell does hypersensitive sites within the repetitive portion of the gamma 1 switch region was also identified. A gel retardation assay for protein--DNA interaction revealed a sequence present in several copies in the gamma 1 switch region that specifically binds nuclear proteins. This binding sequence, SG1BS, contains the octanucleotide sequence ATGCAAAA, a 7/8 match to the transcriptional enhancer octamer motif found in immunoglobulin promoters and the heavy chain enhancer. Binding competition studies of SG1BS demonstrate that both the octamer and flanking sequences are critical for binding. By size- and tissue-distribution, the factors that bind SG1BS are not distinguishable from the previously identified octamer-binding factors OTF-1 and OTF-2. The ability of proteins to bind the S gamma 1 octamer motif is increased 2.3-fold upon IL-4 induction of lipopolysaccharide-stimulated B cells.

Two subfamilies of murine retrotransposon ETn sequences.

Early transposon (ETn) elements are 5.7-kb retrotransposons found in the murine genome. We have sequenced large portions of two ETn elements that have apparently transposed within the DNA of a murine myeloma cell line, P3.26Bu4. One of the transposed ETn elements has 5' and 3' long terminal repeats (LTRs) that are exact duplicates of each other and has a 6-bp target site duplication. These results suggest that this element, which inserted into an immunoglobulin gamma 1 switch region, moved by a retrotransposition process. Our nucleotide sequences confirm that individual ETn elements are very similar to one another and lack open reading frames. However, the ETn sequences reported here and those previously described differ significantly near their 5' LTRs, including 200 bp of weak similarity and 240 bp of complete disparity. Southern hybridization analysis suggests that both subfamilies of ETn sequences are represented many times in the mouse genome. The possibility that the disparate sequences have a role in transposition by ETn elements is discussed.

Copy choice mechanism of immunoglobulin heavy-chain switch recombination.

The immunoglobulin heavy-chain switch is mediated by a recombination event between DNA switch regions associated with donor and recipient constant-region genes. We have determined that the mutations which can be found in some switch regions after recombination appear to arise on only one strand of DNA. This result suggests that switch recombination involves error-prone synthesis of one DNA strand and ligation of the other strand from preexisting DNA.

Patterns and extent of isotype-specificity in the murine H chain switch DNA rearrangement.

We have analyzed the configuration of the H chain locus of 41 hybridomas by Southern blot analysis. Each H chain switch region was determined to be germ line, rearranged, or deleted. Including 13 previously analyzed hybridomas, 60% of those with rearrangements on both alleles showed a correlation of the two alleles, i.e., both the expressed and the nonexpressed alleles have rearranged to the same H chain constant region gene segment. When the two H chain alleles did not rearrange to the same gene, they often rearranged to neighboring H chain genes. These results support a role for isotype-specific factors in H chain switch recombination. The action of these isotype-specific factors may be propagated to some extent along the chromosome, which would lead to rearrangements to neighboring genes.