Single nucleotide polymorphisms and functional analysis of class II transactivator (CIITA) promoter IV in persistent HBV infection.

BACKGROUND/AIMS: CIITA plays a pivotal role in immune response, and the outcome of HBV infection is influenced by immune response. The aims of this study were to analyze the effect of CIITA promoter IV polymorphisms on its activity, and their association with persistent HBV infection. METHODS: The polymorphisms in promoter IV (C-1350T and G-944C) were analyzed by tetra-primer amplification refractory mutation system polymerase chain reaction (ARMS-PCR), and four haplotypes were assigned in 1420 HBV infected subjects. The functional analysis on haplotypes of promoter IV was studied using pGL3-basic and pGL3-promoter vectors. RESULTS: There were significantly decreased-TG and increased-CC haplotype frequencies in persistent HBV infected subjects (34.8% and 41.3%), compared with spontaneously recovered subjects (46.5% and 36.2%). There were significantly higher CC/CC and lower TG/TG genotype frequencies in persistent infected subjects (20.3% and 11.1%) than in spontaneously recovered subjects (10.9% and 23.1%). The mean relative luciferase activity of promoter IV were the highest in TG haplotype (0.558+/-0.023), and the lowest in CC haplotype (0.302+/-0.016). CONCLUSIONS: The polymorphisms of CIITA promoter IV are associated with persistent HBV infection. The CC haplotype with the lowest activity of promoter and CC/CC genotype are responsible for persistent HBV infection.     

Quantitative control of MHC class II expression by the transactivator CIITA

Activation of T lymphocytes is quantitatively controlled by the level of expression of MHC class II molecules. Both constitutive and inducible expression of MHC class II genes is regulated by the transactivator CIITA, which is itself tightly regulated. Since the level of MHC class II molecules expressed is a functionally essential parameter, it was of interest to explore whether MHC class II expression is quantitatively controlled by the level of the transactivator. This report shows that in a variety of experimental conditions one does indeed observe, in both mouse and man, a quantitative control of MHC class II expression by the level of CIITA. This relationship between the regulator gene, which behaves as a rate-limiting factor, and its target genes clarifies our understanding of the quantitative modulation of MHC class II expression, and thus of T lymphocyte activation.     

Revisiting the specificity of the MHC class II transactivator CIITA in vivo

CIITA is a master regulatory factor for the expression of MHC class II (MHC-II) and accessory genes involved in Ag presentation. It has recently been suggested that CIITA also regulates numerous other genes having diverse functions within and outside the immune system. To determine whether these genes are indeed relevant targets of CIITA in vivo, we studied their expression in CIITA-transgenic and CIITA-deficient mice. In contrast to the decisive control of MHC-II and related genes by CIITA, nine putative non-MHC target genes (Eif3s2, Kpna6, Tap1, Yars, Col1a2, Ctse, Ptprr, Tnfsf6 and Plxna1) were found to be CIITA independent in all cell types examined. Two other target genes, encoding IL-4 and IFN-gamma, were indeed found to be up- and down-regulated, respectively, in CIITA-transgenic CD4(+) T cells. However, there was no correlation between MHC-II expression and this Th2 bias at the level of individual transgenic T cells, indicating an indirect control by CIITA. These results show that MHC-II-restricted Ag presentation, and its indirect influences on T cells, remains the only pathway under direct control by CIITA in vivo. They also imply that precisely regulated MHC-II expression is essential for maintaining a proper Th1-Th2 balance.     

MHC class II transactivator (CIITA) expression is upregulated in multiple myeloma cells by IFN-gamma

The MHC class II transactivator (CIITA) acts in the cell nucleus as the master regulator of MHC class II (MHC II) gene expression. It is important to study CIITA regulation in multiple myeloma since MHC expression is central to ability of myeloma cells to present antigen and to the ability of the immune system to recognize and destroy this malignancy. Regulation of CIITA by IFN-gamma in B lymphocytes occurs through the CIITA type IV promoter (pIV), one of the four potential promoters (pI-pIV) of this gene. To investigate regulation of CIITA by IFN-gamma in multiple myeloma cells, first the ability of these cells to respond to IFN-gamma was examined. RT-PCR analyses show that IFN-gammaR1, the IFN-gamma-binding chain of the IFN-gamma receptor, is expressed in myeloma cells and IRF-1 expression increases in response to IFN-gamma treatment. Western blotting demonstrates that STAT1 is activated by phosphorylation in response to IFN-gamma. RT-PCR and functional promoter analyses show that IFN-gamma upregulates the activity of CIITA pIV, as does ectopic expression of IRF-1 or IRF-2. In vivo protein/DNA binding studies demonstrate protein binding at the GAS, E box and IRF-E sites. In vitro studies confirm the binding of IRF-1 and IRF-2 to CIITA pIV. Although multiple myeloma cells express PRDI-BF1/Blimp-1, a factor that represses both the CIITA type III and IV promoters, they retain the capability to upregulate CIITA pIV and MHC II expression in response to IFN-gamma treatment. These findings are the first to demonstrate that although PRDI-BF1/Blimp-1 diminishes the constitutive ability of these cells to present antigen by limiting CIITA and MHC II expression, it is possible to enhance this expression through the use of cytokines, like IFN-gamma.     

Inactivating mutations of class II transactivator (CIITA) gene in gastric and colorectal cancers

Expression of MHC class II, which is important against cancer immunity, depends on the transcactivator CIITA. These data suggest a possibility that CIITA gene might be inactivated in cancers. In this study, we studied inactivating mutation status of CIITA gene in gastric (GC) and colorectal (CRC) cancers by analyzing the C7 repeat in the CIITA (exon 11) gene. We found frameshift mutations in 3 GCs and 6 CRCs in cancers with high microsatellite instability (MSI-H) (9/113), but not in those with microsatellite-stable (MSS) (0/90) (P = 0.004). They were all deletion or duplication of one base in the C7 repeat that would result in truncation of amino acid synthesis. Immune therapy is now a major option in cancer therapy and our results on the genetic alterations of MHC II-related CTIITA in MSH-H GC and CRC might provide useful information for the treatment of MSI-H cancers.     

Invariant chain+ N2a neuroblastoma cells stably expressing the class II MHC transactivator CIITA fail to stimulate anti-tumor immunity

A promising cancer treatment strategy involves stimulation of anti-tumor immune responses. CD4⁺ T cell responses are particularly desirable, as they enhance CD8⁺ T cell activity and provide immune memory. The major histocompatibility complex (MHC) class II transactivator CIITA can be used to stimulate expression of MHC II on tumor cells, thereby promoting CD4⁺ T cell activation. In this study, N2a neuroblastoma cells were stably transfected with CIITA. N2aCIITA cells displayed increased expression of MHC I, MHC II and invariant chain; CD80 and CD86 were expressed by neither the parental N2a cells nor by the N2aCIITA cells. All mice injected with N2aCIITA cells developed tumors. Furthermore, no increase in the numbers of T cells, natural killer cells, macrophages, or eosinophils was observed in the spleens or tumors of mice injected with N2aCIITA cells, compared to tissues from mice injected with the parental N2a cells. This absence of an anti-tumor immune response despite MHC II expression is likely due to the presence of invariant chain, in support of the MHCII⁺/Ii⁻ paradigm.     

Single nucleotide polymorphisms of the resistin (RSTN) gene

Type 2 diabetes mellitus is a complex phenotype that is frequently associated with central obesity and insulin resistance. Recently, a protein named resistin, encoded by RSTN (OMIM #605565), was identified in adipose tissue. Serum resistin was elevated in obese and diabetic mice, and administration of resistin to normal mice was found to interfere with glucose tolerance and insulin action. Because of these functions, resistin is a candidate gene for diabetes and obesity. Through the use of DNA sequencing, we thus developed amplification primers for rapid screening of the RSTN gene that encodes resistin. No putative mutations were found, but two noncoding single-nucleotide polymorphisms (SNPs) were identified, and these were found to vary in frequency across various ethnic groups. The identification of amplification primers and SNPs provides tools to investigate resistin for association with other phenotypes. Received: April 11, 2001 / Accepted: June 11, 2001     

Tumor rejection by gene transfer of the MHC class II transactivator in murine mammary adenocarcinoma cells

The murine mammary adenocarcinoma cell line TS/A is a highly malignant MHC class II-negative tumor. We show that transfection of TS/A cells with the MHC class II transactivator CIITA renders them MHC class II-positive and highly immunogenic in vivo. These cells were fully rejected by 51% of syngeneic recipients and had a significantly lower growth rate in the remaining 49% of animals. This directly correlated to the amount of MHC class II molecules expressed in the transfected tumor. Tumor rejecting animals were protected against rechallenge with the parental TS/A tumor. The rejection required CD4(+) and CD8(+) T cells. CD4(+) T cells were fundamental in the priming phase of the antitumor response. CTL-specific for a peptide of the envelope gp70 of an endogenous ecotropic retrovirus were identified and explained the specificity of the effector mechanism of rejection against the TS/A and the antigenically related C26 carcinoma cells but not against the unrelated gp70-negative syngeneic fibrosarcoma F1F cells. This is the first example of successful tumor vaccination by genetic transfer of CIITA. These results open the way to a possible use of CIITA for increasing both the inducing and the effector phase of the antitumor immune response.     

Single nucleotide polymorphisms of the fukutin gene

Mutations in the LMNA gene, which encodes nuclear lamins A and C, underlie both Emery-Dreifuss muscular dystrophy (EMD2) and Dunnigan-type familial partial lipodystrophy (FPLD). This indicates that one gene can cause different phenotypes characterized by tissue degeneration. The gene for one form of Berardinelli-Seip-type congenital total lipodystrophy (BSCL) has been mapped to chromosome 9q34. Based on the observation that one gene caused both FPLD and EMD2, we considered that a known gene for muscular dystrophy at or near the BSCL locus on chromosome 9q would be an appropriate candidate for BSCL. The gene encoding fukutin, which is mutated in Fukuyama congenital muscular dystrophy has been mapped to 9q31. We thus developed amplification primers for the coding regions of the fukutin gene. We found no putative disease mutations, but through screening of diseased and normal subjects, we identified three novel single nucleotide polymorphisms (SNPs). We conclude that mutations in fukutin are not present in subjects with BSCL. However, the identification of SNPs provides tools to investigate this protein for association with other phenotypes. Received: April 9, 2001 / Accepted: May 1, 2001     

Single nucleotide polymorphisms of the ALDH2 gene in six Indian populations

BACKGROUND: Aldehyde dehydrogenase-2 (ALDH2) degrades acetaldehyde metabolized from ethanol. Its encoding gene ALDH2 has a functional polymorphism, ALDH2 Glu487Lys associated with low enzyme activity. AIM: Since Glu487Lys of this locus is fixed for the functional subunit in all non-East Asian populations, this polymorphism was examined along with G-357A promoter (SacI) and four other intronic loci to identify informative markers to study the role of this gene in Indian populations. SUBJECTS AND METHODS: A total of 397 males belonging to six ethnic populations, from four linguistic groups of India were included in the present study. No test was performed to detect the phenotype of alcoholism. Genotype of ALDH2*E487K and G-357A promoter site along with four non-coding single nucleotide polymorphisms (SNPs) in the upstream of this polymorphism were determined by PCR and sequencing. RESULTS: All of the subjects were found to have the common homozygous genotype (ALDH2*1/ALDH2*1) for the E487K site. Allele frequencies of non-coding SNPs varied among populations but genetic variance (F(st)) indicated little variation among populations. Four major SNP-defined haplotypes accounted for almost all chromosomes in all populations. The ancestral haplotype was found in high frequency in all populations and linkage disequilibrium was strong and highly significant between all sites (p < 0.05). CONCLUSION: The small number of haplotypes in this region is suggesting the strong linkage disequilibrium across the region and confirms the global long-range linkage disequilibrium around the ALDH2 locus. This study provides a baseline for future research into the role of the ALDH2 locus in alcoholism in Indian populations.