RESUMO
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.
Assuntos
Desoxirribonuclease I/farmacologia , Genes de Imunoglobulinas/efeitos dos fármacos , Cadeias gama de Imunoglobulina/genética , Animais , Antígenos CD40/fisiologia , Éxons , Genes de Imunoglobulinas/genética , Regiões Constantes de Imunoglobulina/genética , Região de Troca de Imunoglobulinas , Interleucina-4/fisiologia , Região de Controle de Locus Gênico/genética , Camundongos , Camundongos Transgênicos , Especificidade da Espécie , Transcrição Gênica , Transgenes/imunologiaRESUMO
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.
Assuntos
Rearranjo Gênico de Cadeia Pesada de Linfócito B/genética , Genes de Imunoglobulinas/genética , Switching de Imunoglobulina/genética , Transcrição Gênica , Transgenes/genética , Animais , Linfócitos B/imunologia , Ligante de CD40 , Citometria de Fluxo , Regulação da Expressão Gênica , Haptenos/imunologia , Hibridomas , Região de Junção de Imunoglobulinas/genética , Região de Troca de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Cadeias delta de Imunoglobulina/análise , Cadeias delta de Imunoglobulina/genética , Cadeias gama de Imunoglobulina/análise , Cadeias gama de Imunoglobulina/sangue , Cadeias gama de Imunoglobulina/genética , Cadeias mu de Imunoglobulina/análise , Cadeias mu de Imunoglobulina/genética , Interleucina-4/farmacologia , Lipopolissacarídeos/farmacologia , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/farmacologia , Camundongos , Camundongos Transgênicos , RNA Mensageiro/metabolismoRESUMO
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.
