Communicated by: Shunsuke Ishii
Transgenic over-expression of MafK suppresses T cell proliferation and function in vivo
Article first published online: 20 DEC 2001
Genes to Cells
Volume 6, Issue 12, pages 1055–1066, December 2001
How to Cite
Yoh, K., Sugawara, T., Motohashi, H., Takahama, Y., Koyama, A., Yamamoto, M. and Takahashi, S. (2001), Transgenic over-expression of MafK suppresses T cell proliferation and function in vivo. Genes to Cells, 6: 1055–1066. doi: 10.1046/j.1365-2443.2001.00489.x
- Issue published online: 20 DEC 2001
- Article first published online: 20 DEC 2001
- Received: 9 July 2001Accepted: 27 September 2001
Background The small Maf proteins regulate gene transcription from Maf recognition elements (MARE). These proteins do not contain a canonical transactivation domain. Depending upon the ratio of small Maf proteins to their partner proteins, which either possess a transactivation domain or not, transcription can be switched on or off.
Results Transgenic mice were generated which over-express the small Maf family member MafK, specifically in the T cell lineage. It was our expectation that the high level of MafK would shift the balance to the formation of MafK homodimer and thereby repress MARE-dependent transcription. The transgenic mice had a shortened life span because of Pneumocystis carinii pneumonia and displayed a decrease in thymocytes and lower IL-2 and IL-4 mRNA expression levels. Analyses by electrophoretic gel mobility shift assay revealed that over-expressed MafK could interact with the proximal AP-1 sequence of IL-2 and the MARE in the IL-4 promoter region.
Conclusion These results indicate that when over-expressed, MafK binds to a MARE-like sequence and represses MARE-dependent transcription. Consequently, T cell proliferation and cytokine secretion are affected. The MafK homodimer serves as an important molecular probe for evaluating the role played by cis-acting MAREs in the proliferation and function of T cells.
The maf oncogene was originally identified within the genome of the avian musculoaponeurotic fibrosarcoma virus, AS42 (Nishizawa et al. 1989). The Maf proteins are characterized by a basic region linked to a leucine zipper domain (b-Zip domain) which mediates DNA binding and subunit dimerization, respectively. To date, eight members of the Maf family have been identified and have been divided into two subgroups, the large Maf proteins and the small Maf proteins. The large Maf proteins differ from the small Maf proteins in that they contain a transactivation domain. Members of this family include c-Maf (Kataoka et al. 1993), MafB (Kataoka et al. 1994a), NRL (Swaroop et al. 1992), L-Maf (Ogino & Yasuda 1998) and SMaf2 (Kajihara et al. 2001). The small Maf proteins are MafK (Fujiwara et al. 1993), MafF (Fujiwara et al. 1993) and MafG (Kataoka et al. 1995). It has been shown that the small Maf proteins can form homodimers, and since the small Maf proteins do not possess any canonical transactivation domains, homodimers of these proteins act as transcriptional repressors. Alternatively, the small Maf proteins can form various heterodimers with Cap'n'collar (CNC) family proteins and also with c-Fos (reviewed in Motohashi et al. 1997). It has been demonstrated experimentally that gene activation though MARE can be turned on or off in living cells and also in vivo, depending on the balance of the small Maf proteins and their partner molecules containing transactivation domains (Igarashi et al. 1994; Motohashi et al. 2000).
Two types of MARE have been reported: T-MARE, a 13 bp palindromic sequence, and C-MARE, a 14 bp palindromic sequence. The T-MARE consists of a TPA-responsive element (TRE) which substantially overlaps with a nuclear factor erythroid 2 (NF-E2) binding site, while C-MARE contains a cAMP-responsive element (Kataoka et al. 1994b). Both sequences contain a consensus activated protein-1 (AP-1) binding site, which is frequently seen in the regulatory elements of many cytokine genes, including the interleukin-2 (IL-2) gene (Fuse et al. 1984; Serfling et al. 1989; Jain et al. 1992). A further link between Maf proteins and cytokine gene expression comes from studies on c-Maf, a large Maf protein, which has been shown to promote IL-4 gene transcription through binding to the MARE in the IL-4 gene promoter, thereby playing an indispensable role in immune system function (Ho et al. 1996).
The mechanisms which regulate the development and activity of the immune systems are diverse and complicated. The b-Zip transcription factors, including AP-1 and c-Maf, have been shown to play important roles for T cell development and cell-type-specific gene expression (Foletta et al. 1998; Ho et al. 1996, 1998). For example, AP-1 sites in the IL-2 gene regulatory region are essential for IL-2 gene expression (Jain et al. 1992; Foletta et al. 1998). c-Maf acts as an up-regulating factor of IL-4 gene expression (Ho et al. 1996), and c-Maf promotes Th2 cell differentiation through an IL-4-dependent mechanism (Ho et al. 1998). However, the importance of MARE motifs in T cell development and cytokine gene expression in vivo still remains to be elucidated.
The redundancy of the MARE and AP-1 sequences has hampered the precise identification of effector molecules that interact with these sequences in T cells. To address this issue, we made use of the dominant negative activity of the MafK homodimer, since the MafK homodimer suppresses gene activation from MARE. To this end, we made transgenic mice over-expressing MafK in T cells, and analysed T cell development and cytokine gene expression in them. Since, theoretically, the over-expression of MafK causes the inactivation of every MARE-containing gene regulatory region in T cells, we called this method a ‘cis-element targeting’ strategy.
We found that the MafK transgenic mice develop characteristic Pneumocystis carinii pneumonia due to immune deficiency, and have a short life span. T cell proliferation and cytokine secretion are affected by MafK over-expression. These results indicate that MARE-mediated regulation of T cell gene expression is indeed an important element in the proliferation and cytokine secretion of T cells. This strategy may be applicable for alteration of immune system functions and also for the development of new therapeutic approaches to immune diseases.
Generation of transgenic mouse lines over-expressing MafK in T cells
To generate transgenic mouse lines that specifically over-express MafK in the T cell lineage, the mouse MafK cDNA and FLAG sequence was inserted into the VA vector, which contains the human CD2 gene regulatory region and locus control region (LCR) (Fig. 1A). The mafK transgenic construct was injected into fertilized eggs to make transgenic mice. Of the 91 independent offspring analysed, three founders had incorporated the mafK transgene, as identified by PCR analysis (data not shown).
A genomic Southern blot analysis was performed to ensure the integrity and copy number of each transgenic mouse line. The length of the XhoI/NcoI fragment containing the mafK transgene was 0.5 kb, while the corresponding fragment for the endogenous mafK gene was 1.1 kb (Fig. 1A). The mafK transgene was detected in line 194, line 784 and line 792 mice (Fig. 1B). Through densitometric analysis, line 194 appeared to contain two copies of the transgene, while the other two lines each contained approximately six copies. These transgenes showed stable transmission to the next generation.
Expression of MafK mRNA and protein from the transgene
To confirm expression from the transgene, reverse transcription PCR (RT-PCR) analysis was performed with RNA samples taken from the thymocytes of the four transgenic mouse lines. The VA vector contains the human CD2 gene LCR, in addition to its upstream gene regulatory regions, so that the vector regulates the expression of the transgene mainly in T cells (Zhumabekov et al. 1995). The primer set used in this analysis was able to amplify only the transgene-derived MafK mRNA, as a 484 bp product, but not the endogenous MafK mRNA. Transgenic lines 194 and 792 expressed the MafK mRNA, but line 784 did not (Fig. 2A). In the latter case, the mafK transgene seems to be affected by a severe position effect. Thus, transgenic lines 194 and 792 were selected for further analysis.
We then carried out an immunoblot analysis to monitor MafK protein expression from the transgene. MafK protein and FLAG peptide were detected in lines 194 and 792 (Fig. 2B). The amount of MafK protein in line 792 was several fold higher than that in line 194, indicating that expression levels of the protein are copy number dependent.
Mice which over-express MafK have a short life span
During the initial analysis of the transgenic mouse cohort we found that the transgenic mice had a shortened life span. Transgenic mice of lines 194 and 792 developed normally, but after 6 weeks they started dying. Figure 3 shows the survival rate in the MafK transgenic mouse lines. To our surprise, 50% of the mortality occurred at 38.6 weeks and 25.1 weeks for lines 194 and 792, respectively. Transgenic mouse line 792 displayed a more severe phenotype than did line 194, perhaps as a consequence of the higher expression of MafK in line 792 relative to line 194. The transgene-negative littermates did not show any mortality during the experimental period.
MafK transgenic mice develop severe pneumonia
To identify the cause of death in the transgenic mice, histological analyses were performed. Notably, signs of severe pneumonia were found in all the dead mice (25 dead mice were examined). A comparison to lung sections from 16-week-old negative littermates (Fig. 4A) revealed that the alveolar spaces of the lung (line 792, Fig. 4B) were filled with a pink frothy honeycombed material. In addition, an examination the lung of a 50-week-old mouse (line 194), found that the lung disease was exacerbated with age (Fig. 4C). Significantly, a common histological feature seen in all the dead mice was an eosinophilic substance in the alveoli of the lung. Giemsa staining revealed the presence of Trophozoites (Fig. 4D). Consequently, these results established that pneumonia was caused by P. carinii. In general, P. carinii is a very weak, widespread pathogen that is unable to induce disease in normal mice.
FACS analysis of thymocytes in the transgenic mouse
Since MafK protein was over-expressed exclusively in the T cell lineage, a disturbance in T cell function must constitute the basis of the immune deficiency and the susceptibility to P. carinii pneumonia. As expected, the total number of thymocytes in the MafK transgenic mice was reduced to almost a half of that in the negative littermates (Fig. 5A), indicating that the development or proliferation of T cells is affected by the over-expression of MafK. Surface marker analysis revealed that the numbers of thymus CD4–CD8–, CD4+ CD8– and CD4–CD8+ cells were also significantly decreased in the MafK transgenic mice as compared to transgene-negative littermates (Fig. 5A). A decrease in T cell number was also seen in the spleen of transgenic mice. The number of splenic CD19–CD3+, CD4+ CD8– and CD4–CD8+ cells was identifiably lower than that of negative littermates (Fig. 5B). These results therefore indicate that over-expression of MafK protein impedes T cell proliferation.
Due to the possibility that the proliferation of T cells may be affected during the embryonic stage of the MafK transgenic mouse, we examined foetal thymocytes on days post-coitum 16 and 18 (Table 1). Although the number of thymocytes in the transgenic mice was lower than that in negative littermates, the difference was not thought to be sufficiently significant as to explain the decrease of T cells at the adult stage. Similarly, the numbers of CD3+, CD4+, CD8+, CD44–CD25–, CD44+CD25+, CD44+CD25– and CD44–CD25+ T cells were not significantly reduced in the transgenic mouse embryos (data not shown).
|16.5 dpc||18.5 dpc|
|Tg(–)||2.3 × 105± 0.8 × 105 (n = 6)||4.2 × 106± 2.9 × 106 (n = 10)|
|Tg(+)||2.1 × 105± 0.6 × 105 (n = 6)||3.0 × 106± 1.3 × 106 (n = 6)|
RT-PCR analysis of cytokine expression in MafK transgenic mouse
To establish a possible molecular mechanism that may lead to the immune deficiency, the expression of various cytokines were analysed by RT-PCR using primers in Table 2. Thymocytes isolated from two mice of line 194, two mice of line 792, and two negative littermates were used for this analysis. Ten-week-old mice were used for this analysis. Importantly, the expression of IL-2 mRNA was markedly decreased in all four MafK transgenic mice (Fig. 6A). Densitometric analysis revealed that the IL-2 expression in these MafK transgenic mice was reduced, on average, more than 20-fold relative to that of the negative littermates. We also examined the mRNA expression levels of other cytokines, including IL-4, IL-7 and IFN-γ, using RT-PCR. The levels of IL-4 mRNA appeared to be decreased in two out of four MafK transgenic mice, whereas mRNA levels of IL-7 and IFN-γ were expressed at wild-type level (Fig. 6A). These results indicated that the mRNA levels of IL-2 and IL-4 were specifically affected in the thymocytes of MafK over-expressing mice. To extend these observations to the peripheral immune system, we measured the serum levels of IL-4 and IFN-γ in the MafK transgenic mice by ELISA. Firstly we measured the serum levels of IL-4 in the MafK transgenic mice within 10 weeks of age. No significant difference was observed between the MafK transgenic mice and negative littermates (data not shown). Therefore, 35-week-old mice were used for this purpose in the hope of a clearer phenotype. As expected, the IL-4 level in the MafK transgenic mouse was suppressed when compared to that of negative littermates (Fig. 6B). In contrast, the level of serum IFN-γ in the transgenic mice was higher than that of the negative littermates (Fig. 6C).
|Primer name||Sequence (5′ to 3′)|
|FLAG sense||ACT ACA AGG ACG ACG ATG AC|
|MafK anti-sense||GCG GCT GAG AAG GGT ACA GA|
|mouse HPRT sense||GCT GGT GAA AAG GAC CTC T|
|mouse HPRT anti-sense||CAC AGG ACT AGA ACA CCT GC|
|mouse IFN-γ sense||TGA ACG CTA CAC ACT GCA TCT TGG|
|mouse IFN-γ anti-sense||CGA CTC CTT TTC CGC TTC CTG AG|
|mouse IL-2 sense||AGG ATG GAG AAT TAC AG|
|mouse IL-2 anti-sense||TGC TGA CTC ATC ATC GA|
|mouse IL-4 sense||GTC TCT CGT CAC TGA CGG C|
|mouse IL-4 anti-sense||CAT GGT GGC TCA GTA CTA|
|mouse IL-7 sense||CTG CCT GTC ACA TCA TCT GA|
|mouse IL-7 anti-sense||TCT CTC AGT AGT CTC TTT AG|
Decrease of serum IgG in MafK transgenic mice
All the data obtained thus far have focused upon the disturbance of T cell function in the MafK transgenic mice. Therefore, we next examined the serum immunoglobulin levels in these mice. The MafK transgenic mice showed significantly lower serum levels of IgG when compared to negative littermates, whereas there was no significant difference in the IgM levels between the two groups (Figs 7A, B). To identify which subclass of IgG was decreased, we analysed the amount of IgG1 and IgG2a in these mice (Figs 7C, D). The levels of IgG1 and IgG2a in transgenic mice were significantly lower than those of negative littermates. Since IL-4 is known to be a cytokine that stimulates IgG1 production, the ratio of IgG1 to IgG2a was also studied. The ratio of IgG1 to IgG2a in transgenic mice was also lower than that in negative littermates, but not significantly (Fig. 7E).
Over-expressed MafK protein interacted with proximal AP-1 binding sequence in IL-2 and MARE in IL-4 promoter regions
The possibility that over-expressed MafK protein interacted with a MARE-like sequence in gene regulatory regions of IL-2 and IL-4 in transgenic mice was investigated by EMSA analysis using thymocyte nuclear extracts isolated from transgenic mice and negative littermates (Fig. 8). The MARE consensus sequence, #25, was used as a probe (Kataoka et al. 1994b). An intense shifted band was detected in the nuclear extract from the thymocytes of transgenic mice (lane 3 in Fig. 8A). The band was not as apparent in the nuclear extract from negative littermates (lane 2 in Fig. 8A) and completely undetectable in the presence of anti-MafK antibody (lane 4 in Fig. 8A), indicating that the shifted-band contained MafK. The presence of the shifted band was completely eliminated by cold #25 probe (lane 5 in Fig. 8A), but not competed by #18, which did not bind Maf protein (lane 6 in Fig. 8A, Kataoka et al. 1994b). Competition analysis (lanes 5 and 6 in Fig. 8A) revealed that the band was MARE specific. Next, we performed a competition assay using MARE-like sequences found in the gene regulatory regions of IL-2, IL-4 and IFN-γ (see Table 3). We observed binding inhibition in lanes 8 and 10 which contained cold oligonucleotides identical in sequence to the proximal AP-1 binding sequence of IL-2 or to the MARE sequence of the IL-4 promoter region. No significant inhibition was observed using cold oligonucleotides identical in sequence to the proximal or distal AP-1 binding sequence from the IFN-γ promoter region (lanes 11 or 12 in Fig. 8A). The anti-FLAG antibody was also used in this study (Fig. 8B). The band which was generated with endogenous or/and exogenous MafK protein (shown by arrow (a)) was shifted in lane 6 (arrow (b)), but not in lane 3 in Fig. 8B. These results clearly indicated that the intense band seen in lane 5 was generated with MafK protein from the transgene.
|no. 25||5′- TCGAGCTCGGAATTGCTGACTCATCATTACTC-3′|
|no. 18||5′- TCGAGCTCGGAATTGAGGACGTCCTCATTACTC-3′|
|IL-2 dAP-1||5′- TCACCTAAATCCATTCAGTCAGTATATGGGGT-3′|
|IL-2 pAP-1||5′- AAACAAATTCCAGAGAGTCATCAGAAGAGGAA-3′|
|IL-4 AP-1||5′- ACTGACAATCTGGTGTAATAAAATTTTCCAAT-3′|
|IL-4 MARE||5′- TCATTTTCCCTTGGTTTCAGCAACTTTAACTCTA-3′|
|IFN-γ dAP-1||5′- GAGTCGAAAGGAAACTCTAACATGCCACAAAACC-3′|
|IFN-γ pAP-1||5′- AAAAAACTTGTGAAAATACCTAATCCCGAGGA-3′|
A comprehensive understanding of transcription factor activity requires in vivo study, in that a gain-of-function or loss-of-function mutant phenotype can be considered to be a reflection of transcription factor function. However, these efforts are often hampered by the presence of related transcription factors, which, for instance, compensate the loss-of-function phenotype. For this reason, many single gene targeting experiments have yielded mice with no or minimal phenotype.
In this study, we examined the contribution of the MARE-mediated transcriptional activation process to the development and function of T cells in vivo through a cis-element targeting approach. Since the homodimer of MafK is known to act as a transcriptional repressor, we exploited this fact to repress en bloc the MARE-mediated transactivation signals, expecting a wide-range targeting of the MARE motifs in the T cell genome. While it is well known that some cytokines produced in T cells are under the regulation of an AP-1/TRE, we suspect that AP-1 regulation may also include incidentally a MARE-mediated regulation, since the 13 bp T-MARE motif completely overlaps the 7 bp AP-1 motif. To address this hypothesis, we made transgenic mouse lines that expressed MafK specifically in the T cell lineage.
As expected, over-expression of MafK protein impedes T cell proliferation in the MafK transgenic mice. The MafK transgenic mice also showed a short life span and all dead mice suffered from a typical P. carinii pneumonia. The severe immune deficiency in these transgenic mice appears to result from a decrease in the numbers of thymocytes and a lower expression of IL-2 and IL-4. The amounts of IgG, IgG1 and IgG2a in these transgenic mice were also significantly decreased and the ratio of IgG1 to IgG2a was also lower than that of negative littermates (Fig. 7E). These results are consistent with the decrease in the levels of IL-4 in the MafK transgenic mouse, since AP-1 binding site and MARE in the mouse IL-4 promoter region is reported to be important for the gene expression (Ho et al. 1996) and IL-4 is known to stimulate IgG1 production (Snapper et al. 1988). Furthermore, the results unequivocally point to the importance of MARE and AP-1 binding sequences in T cell proliferation and function.
It is well known that the activation of naive T cells requires two signalling events (Bretscher 1992). Signal 1 is delivered after antigen binding to the T cell receptor, and signal 2 is subsequently required to stimulate the T cell to produce IL-2 and other cytokines to allow proliferation and differentiation into an effector cell (Linsley & Ledbetter 1993). The production of IL-2 by an activated T cell is critical for the proliferation and differentiation of the T cell, as well as for the development of a T cell-dependent immune response (Arai et al. 1990). Importantly, the IL-2 gene was one of the first cellular genes which was shown to contain AP-1 binding sites within its promoter region (Fuse et al. 1984; Serfling et al. 1989; Jain et al. 1992). AP-1 binding sites have been shown to play an integral role in the regulation of the gene, both alone and in combination with other transcription factors. The sequences of the distal and proximal AP-1 binding sites in the IL-2 gene are CATTCAGTCAGTA and AGAGTCATCAGAA, respectively (the underlined bases denote sequences which match the canonical MARE). Since EMSA analyses revealed that the proximal AP-1 binding site interfered with MafK binding to the canonical MARE, over-expressed MafK binds to the proximal AP-1 site in the IL-2 promoter region and suppresses the transcription of IL-2.
In addition to the IL-2 gene, AP-1 binding sites have been identified in the regulatory regions of IL-4 and IFN-γ genes (reviewed in Foletta et al. 1998). In this study we examined the expression of IL-4 and found that the mRNA levels in thymocytes and the serum IL-4 protein levels were significantly decreased in transgenic mice. There are two possible explanations for the suppression of IL-4 gene expression by transgenic MafK expression. The suppression may be caused by a dominant negative effect of MafK through binding the AP-1 binding site of the gene. Alternatively, suppression may have occurred via a MARE in the gene promoter region, since c-Maf has been shown to bind to the IL-4 gene promoter and thereby activate IL-4 gene expression (Ho et al. 1996) through a consensus MARE sequence (TCAGCA), albeit this MARE is a half site. EMSA analyses clearly demonstrated that the MARE, but not the AP-1 binding site in the IL-4 promoter region, interfered with MafK binding to the consensus MARE. This observation indicates that the observed decrease of IL-4 production in the transgenic mouse is mainly because of MafK binding to the MARE of the IL-4 promoter region. The expression of IFN-γ was also analysed and although there was no observed difference in mRNA levels from thymocytes of 10-week-old mice, there were significantly higher levels of serum IFN-γ in 35-week-old transgenic mice over controls. A functional AP-1 binding site, of sequence TTGTGAAAATACG, was reported to exist in the proximal promoter region of the IFN-γ gene (Penix et al. 1996). However, this sequence does not conform to the canonical MARE sequence, making it unlikely that MafK would bind the proximal AP-1 binding site of the IFN-γ gene. Our EMSA results support this idea. In contrast to the results from thymocytes, the IFN-γ levels in the serum from the transgenic mice were higher than the negative littermates. This may be because the 35-week-old transgenic mice suffered from P. carinii pneumonia, since IFN-γ has been described as playing a role in P. carinii infection (Ishimine et al. 1995; Kolls et al. 1999).
It is also interesting to note that these transgenic mice develop severe P. carinii pneumonia under normal conditions. It is well established that CD4+ T cells are the cornerstones in the defence against P. carinii (Beck et al. 1991) and that this disease is capable of inducing a pathological state in normal mice with depleted CD4+ cell levels. It has also been reported that the CD4+ cell count is a useful clinical marker in humans for the identification of specific individuals who are at a particularly high clinical risk of developing P. carinii pneumonia (Mansharamani et al. 2000). Based on these observations, a decrease in the number of CD4+ cells may be the main cause for the increased susceptibility of the MafK over-expressing mice to this pathogen. Recently, Shellito et al. (2000) reported that the Th2 response is dominant during the course of disease in the murine model of P. carinii pneumonia. Impairment of IL-4 production may contribute to the increased susceptibility of the MafK transgenic mice to the disease. Although detailed analyses are needed for the developmental mechanisms of P. carinii pneumonia, MafK over-expressing mice seem to be a good animal model for P. carinii pneumonia.
Since the MafK transgenic mouse displayed severe immune suppression, it is possible that the over-expression of MafK may affect expression of many other genes having a MARE in their gene regulatory regions. It would be of great interest to know which target genes are affected in the MafK transgenic mouse, and at present we are exploiting DNA array technology to identify target genes containing functional MAREs in their regulatory regions. Since MafK over-expression mimics the targeted disruption of the majority of MAREs in the T cell gene regulatory regions, to which the MafK protein can bind, this approach would allow us to evaluate the contribution of a cis-acting element MARE to the development and maintenance of a specific cell lineage. Therefore, we think that the application for this ‘cis-element targeting’ strategy will be wide, including the alteration of the immune system and therapeutics for immune diseases.
Generation of MafK transgenic mice
A 0.8 kb full-length cDNA encoding the murine MafK protein, including a FLAG peptide at the 5′ end, was inserted into a VA CD2 transgene cassette containing the upstream gene regulatory region and LCR of the human CD2 gene. The VA vector has been reported to direct the expression of the inserted cDNA in all single positive mature T lymphocytes of transgenic mice, and the expression is linearly proportional to the transgene copy number. Expression of the transgene is also detected at low levels in double negative CD4–CD8– peripheral lymphocytes (Zhumabekov et al. 1995). Mice were maintained in specific pathogen-free conditions in a Laboratory Animal Resource Center. All experiments were performed according to the Guide for the Care and Use of Laboratory Animals in University of Tsukuba.
Analysis of genomic DNA by Southern hybridization
High molecular weight DNA was prepared from the tail of each mouse, and 10 µg of each DNA was digested with XhoI and NcoI and then electrophoresed on a 0.8% agarose gel. After electrophoresis, the DNA was transferred to a nylon membrane (Zeta-probe, Bio-Rad, Hercules, CA). Southern hybridization was performed using a [32P]-labelled XhoI–SmaI fragment (473 bp) of the mafK gene as probe. Transgene copy number was determined from the blot with a BAS 1500 Mac image analyser.
Western blot analysis
Thymocyte nuclear extracts were prepared from 10-week-old MafK transgenic mice or negative littermates. The extracts were size-fractionated on a 15% SDS/polyacrylamide gel, transferred to a polyvinylidene difluoride membrane (Fluorotrans) and reacted with primary and secondary antibodies. To detect the MafK protein, anti-NF-E2 p18 rabbit polyclonal IgG (Santa Cruz Biotechnology Inc., Santa Cruz, CA) was used as the primary antibody and peroxidase-conjugated goat anti-rabbit IgG (Zymed Laboratories Inc., San Francisco, CA) was used as the secondary antibody. For detection of the FLAG peptide, anti-FLAG M2 monoclonal antibody (Sigma, St. Louis, MO) was used as the primary antibody and peroxidase-conjugated goat anti-mouse IgG (Zymed Laboratories) as the secondary antibody. To check the amount of extract in all samples, anti-lamin B antibody (Santa Cruz Biotechnology) was used as a control, with peroxidase-conjugated goat anti-goat IgG (Sigma) as the secondary antibody.
Each mouse was bled while under ether anaesthesia. Organs were fixed in 10% buffered formalin and then embedded in paraffin for histological analysis. Sections were stained with haematoxylin and eosin. Giemsa staining was used to detect cysts of P. carinii.
Multi-colour flow cytometric analysis
Single-cell suspensions were prepared from each mouse thymus and spleen, and incubated with anti-FcγR antibody (2.4G2) for 10 min on ice to block the interaction of staining reagents with FcR-bearing cells. Multi-colour flow cytometry analysis was performed using FACS-Vantage and Cellquest software (BD Bioscience, San Jose, CA) on viable cells, as determined by forward light scatter intensity and propidium iodide exclusion. The following phycoerythrin (PE)- and allophycocyanin (APC)-labelled monoclonal antibodies were used: anti-CD8-PE, anti-CD19-PE, anti-CD3-APC, and anti-CD4-APC. Fluorescein isothiocyanate (FITC)-labelled anti-normal rat immunoglobulin monoclonal antibodies were used as controls (BD PharMingen, Franklin Lakes, NJ).
Reverse transcription PCR for transgene and cytokine expression analyses
Total RNA was prepared from the thymus of adult MafK transgenic mice or their negative littermates using Trizol reagent according to the manufacturer's instructions (Gibco BRL, Life Technologies Inc., Rockville, MD). First-strand cDNA was synthesized at 37 °C for 1 h using the SUPERSCRIPT™ II RNase H− Reverse Transcriptase kit (Gibco BRL), and 1 µL of this 20 µL reaction mixture was used for the PCR. Amplified products were analysed on 2% agarose gels. The primer sets used are shown in Table 2. The expression of the following genes were analysed: mouse HPRT (Hypoxanthine guanine phosphoribosyl transferase), FLAG, MafK, mouse IFN-γ, mouse IL-2, mouse IL-4, and mouse IL-7. After 1 min denaturation at 94 °C, 45 amplification cycles of 30 s at 94 °C, 30 s at 55 °C and 30 s at 72 °C were performed. PCR products were digested with restriction endonucleases to confirm specific amplification of the target genes.
Measurement of serum cytokine levels
Serum cytokine levels were measured for 35-week-old mice using a Genzyme Techne AN'ALYSA™ Immunoassay System kit for mouse IL-4 and IFN-γ (Techne Co., Minneapolis, MN).
Serum levels of immunoglobulin were determined by enzyme-linked immunosorbent assay (ELISA) as previously described (Takahashi et al. 1991, 1996). Briefly, 96-well plates (Nunc A/S, Roskilde, Denmark) were coated with goat anti-mouse antibody, washed three times and blocked with 0.5% BSA-PBS. The plates were kept at room temperature for 1 h and then washed with 0.1 m phosphate buffered saline (PBS). After washing, the plates were blocked with 0.5% bovine serum albumin (BSA) in PBS solution. Then diluted serum samples were added and incubated for 1 h at room temperature. After five washes, alkaline phosphatase-conjugated goat anti-mouse subclass-specific antibody was added at room temperature for 1 h. Finally, the plates were washed five times, and the alkaline phosphatase substrate (Sigma) was incubated on the plates. After the substrate developed, absorption was measured at 405 nm on an Immuno-plate reader (BenchMark, Bio-Rad).
Electrophoretic gel mobility shift assay (EMSA)
Thymocyte nuclear extracts from 10-week-old MafK transgenic line 792 mouse and negative littermates were incubated with the MARE probe: the oligonucleotide containing the MARE consensus sequence described in Table 3 (no. 25; Kataoka et al. 1994b) was labelled with [γ-32P] ATP. The conditions for binding and electrophoresis were as previously described (Kataoka et al. 1994b). A 1000-fold molar excess of unlabelled oligonucleotide was added to the reactions for specific competitors. The anti-MafK antibody was a gift from Dr K. Igarashi (Igarashi et al. 1995). Anti-FLAG M2 monoclonal antibody (Sigma) was also used to detect MafK protein from transgene. Oligonucleotide no. 18 is the mutant MARE which did not bind for Maf protein (Kataoka et al. 1994b). Two binding sites in mouse IL-2 promoter (Fuse et al. 1984; Serfling et al. 1989; Jain et al. 1992) were used for the IL-2 distal AP-1 (dAP-1) and proximal AP-1 (pAP-1) oligonucleotides in Table 3, respectively. The AP-1 binding site and MARE in mouse IL-4 described in Ho et al. (Ho et al. 1996) were used. The mouse IFN-γ dAP-1 binding sequence is the homology matched sequence to human IFN-γ NFAT/AP-1 binding site (Sweetser et al. 1998), and mouse IFN-γ pAP-1 sequence is the homology matched sequence to human IFN-γ proximal element, which is the AP-1 binding site (Penix et al. 1996).
We would like to thank Drs K. Igarashi, A. Shibuya, K. Shibuya, K.C. Lim, N. Kajiwara, F. Sugiyama, N. Kaneko, V. Kelly and K. Yagami for help and discussion. This work was supported by the Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan, ISPS RFTF and PROBRAIN.
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