Human Class II Transactivator Research Articles

IFN-γ up-regulates expression of the MHC class II transactivator (CIITA) type IV promoter in multiple myeloma cells (35.20)

Abstract MHC Class II transactivator (CIITA) is the master transcriptional regulator of MHC class II (MHC II) genes. MHC II expression is central to antigen presentation and the immune recognition of tumor cells. The human CIITA gene, MHC2TA, is regulated in a cell-type specific manner by multiple promoters. Regulation by IFN-γ is generally via the type IV promoter (pIV) and involves the binding of IFN regulatory factor (IRF)-1 and IRF-2. The response of pIV to IFN-γ was examined in myeloma cells. Western blot and RT-PCR show that STAT1 is activated by phosphorylation and IRF-1 RNA expression increases in response to IFN-γ. RT-PCR and functional promoter analyses show that IFN-γ up-regulates the activity of MHC2TA pIV as does ectopic expression of IRF-1 or IRF-2, and the IRF-E site is required. Binding studies confirm IRF-1 and IRF-2 binding to MHC2TA pIV. Myeloma cells express positive regulatory domain I-binding factor 1 (PRDI-BF1), also called B lymphocyte-induced maturation protein-1 (Blimp-1), which represses both MHC2TA pIII and pIV. Importantly, these cells retain the ability to up-regulate CIITA pIV and MHC II in response to IFN-γ. Although PRDI-BF1/Blimp-1 diminishes the ability of myeloma cells to present antigen by limiting CIITA and MHC II expression, these studies indicate that expression can be restored by the use of cytokines like IFN-γ. Research support: RO1CA10220, RO1CA114504, MEDCEN Foundation of Central GA

Not polymorphism but methylation of class II transactivator gene promoter IV associated with persistent HBV infection

Background Class II transactivator (CIITA) is the major rate-limiting regulator for expression of class II major histocompability complex (MHC-II). Human CIITA gene expression is controlled by four distinct promoters (pIto pIV). Objective To evaluate the relationship among polymorphism and methylation status of CIITA gene promoters and persistent hepatitis B virus (HBV) infection. Methods We recruited 21 patients with hepatocellular carcinoma (HCC), 45 liver cirrhosis (LC), 65 chronic hepatitis B (CHB), 26 acute hepatitis B (AHB) and 95 healthy blood donors. Polymorphism of CIITA gene promoters was assayed by PCR–SSCP-sequencing. Bioinformatics analysis was employed to predict the existence of CpG islands. Methylation-specific PCR (MSP) was used to detect the methylation status of CIITA gene pIV. Results No sequence differences were observed at CIITA genes pI, III and IV among HCC, LC, CHB, AHB patients and healthy controls. No CpG islands were found in the pI, pII and pIII sequences, but there was a CpG island in pIV. The frequency of methylated POV was not significantly different within persistent HBV infection groups (patients with HCC, LC or CHB). Significance was found between the persistent infection group and acute HBV infection or healthy controls. Conclusions CIITA gene promoter sequences are conserved. PIV is highly methylated and associated with host susceptibility to HBV persistent infection.

Prolonged MHC class II expression and CIITA transcription in human keratinocytes

A transcriptional cofactor, the MHC class II transactivator (CIITA), is a master regulator of MHC class II gene expression. CIITA expression is restricted in MHC class II-positive cells and is regulated by 4 (human) or 3 (mouse) promoters. To clarify the usage of human CIITA promoters in keratinocytes, we analyzed CIITA mRNA levels in IFN-γ-stimulated normal human keratinocytes (NHK) by real-time PCR using promoter-specific primers. When the amount of total CIITA mRNA in NHK was quantified at 6 h after IFN-γ-stimulation, the amount of CIITA mRNA detected in NHK did not differ from that seen in the B cell line Raji or the IFN-γ-stimulated macrophage cell line THP-1. Quantitative real-time PCR using promoter-specific primers showed that type IV CIITA mRNA was strongly transcribed and that type III CIITA mRNA was weakly transcribed in stimulated NHK, while no transcripts from pI and pII were detected. Although type IV mRNA in THP-1 was transiently transcribed by IFN-γ-stimulation, transcription of type IV in IFN-γ-stimulated keratinocytes was prolonged. This difference subsequently caused significantly higher expression at 72 h of MHC class II on NHK, compared with THP-1. This is the first report to quantitatively analyze each type of CIITA transcript in NHK.

Persistence of MHC DR nonexpression on swine cells by introduction of a mutated MHC class II transactivator gene: a comparison with the effect induced by antisense RNA.

CIITA (class II transactivator) is a coactivator essential for transcription of MHC class II genes. In this study, a construct with a mutated CIITA gene with N-terminal domains depleted was constructed. This mutated CIITA (mCIITA) was able to repress DR and DQ expression in 45.0-60.0% of the mCIITA-transfected clones of swine endothelial cell line PIEC and in B cell line L23, as well as in human cell lines HeLa and Raji. Similarly, 30.0-46.7% of swine cell clones containing the human CIITA antisense RNA also failed to express DR molecules. However, the persistence of the DR repression on the cell lines is quite different. Transfection with mCIITA was persistent for at least 120 days, while with the CIITA antisense RNA, persistence existed for only 35-45 days. To explore the underlying mechanism, Raji cells were transfected with pUHD10-3-mCIITA, a mCIITA-containing, doxycycline-dependent plasmid. The intensity of DR repression is correlated quite well with the efficiencies of the mCIITA expression within the cells in a doxycycline dose-dependent manner. This implicates a competition between the mCIITA and its endogenous full-length counterpart. In addition, we were able to show that purified human CD4 T cells did not respond to the mCIITA-transfected PIECs in xenogeneic mixed lymphocyte endothelial reaction (MLER). The stimulating indices (SI) were only 1.0-1.5, compared with 15.2-18.2 for those transfected with empty vector or an initiation codon-depleted mCIITA that is dysfunctional for protein translation. The results we obtained, especially those for persistent suppression of class II genes, show promise for the possible development of mCIITA-transgenic swine for organ/tissue xenotransplantation.

Class II transactivator promoter activity is suppressed through regulation by a trophoblast noncoding RNA.

Trophoblasts lack expression of all classic major histocompatibility complex (MHC) antigens. Determination of the mechanism involved could provide insight into selective gene suppression and allograft tolerance. Suppression of class II expression in trophoblasts is secondary to dominant negative trans-acting factors that suppress class II transactivator (CIITA) transcription. We recently described a trophoblast-derived noncoding RNA (TncRNA) that suppresses class II expression. We examined the effects of TncRNA on the CIITA promoter, CIITA, and MHC class II expression. HeLa clones stably transfected with TncRNA were analyzed for MHC class II and CIITA expression by fluorescence-activated cell sorting, Northern blots, and quantitative polymerase chain reaction. Activity and functional dissection of CIITA promoter IV (pIV) was assessed by transient co-transfection of promoter-reporter constructs. Methylation of pIV was assessed by Southern blots, fluorescence-activated cell sorting, and quantitative polymerase chain reaction. TncRNA suppressed interferon-gamma-induced human leukocyte antigen-DR and CIITA expression in HeLa cells. The mechanism involves inhibition of CIITA pIV through a defined inhibitory domain on the promoter. The mechanism does not involve methylation of the promoter. A novel method of CIITA suppression is described where a noncoding RNA selectively mediates the suppression of CIITA pIV possibly by complementary RNA-DNA binding to an inhibitory domain on the promoter. Selective suppression of MHC class II could have important implications in allograft tolerance and in developing class II-deficient cells or tissues for the purpose of transplantation or drug delivery systems.

Impaired class II transactivator expression in mice lacking interferon regulatory factor-2.

Class II transactivator (CIITA) is required for both constitutive and inducible expression of MHC class II genes. IFN-gamma induced expression of CIITA in various cell types is directed by CIITA type IV promoter. The two transactivators, STAT1 and IRF-1, mediate the IFN-gamma activation of the type IV promoter by binding to the GAS and IRF-E of the promoter, respectively. In addition to IRF-1, IRF-2, another member of the IRF family, also activates the human CIITA type IV promoter, and IRF-2 cooperates with IRF-1 to activate the promoter in transient transfection assays. IRF-1 and IRF-2 can co-occupy the IRF-E of the human CIITA type IV promoter. To understand the effect of loss of IRF-2 on the endogenous CIITA expression, we assayed for CIITA expression in IRF-2 knock-out mice. Both basal and IFN-gamma induced CIITA expression were reduced in IRF-2 knock-out mice. At least half of the amount of inducible CIITA mRNA depends on IRF-2. The reduction of IFN-gamma induced CIITA mRNA in IRF-2 knock-out mice was due to the reduction of the type IV CIITA mRNA induction. The reduction of basal CIITA mRNA was apparently due to the reduction of CIITA mRNA originating from other promoters. These data indicate that IRF-2, like IRF-1, plays a critical role in the regulation of the endogenous CIITA gene. The implications in understanding the previously described phenotypes of IRF-2 defective mice are discussed.

Genetic susceptibility to multiple sclerosis: detection of polymorphic nucleotides and an intron in the 3′ untranslated region of the major histocompatibility complex class II transactivator gene

The master player in the transcriptional regulation of major histocompatibility (MHC) class II genes is a factor known as the MHC class II transactivator (CIITA). In this study we searched for polymorphisms in the 5′ and 3′ ends of the human CIITA gene to assess whether or not there is an association between alleles of this gene and multiple sclerosis (MS). Polymorphism screening based upon detection of single strand conformational changes (SSCP analysis) followed by sequencing revealed six single nucleotide variations, namely one in the promoter utilized by B cells and five in the 3′ untranslated region (UTR) of the gene. Determination of alleles at these polymorphic sites was facilitated by treatment of amplified DNA fragments with a panel of appropriate restriction enzymes. The distributions of CIITA alleles did not differ between MS patients and control subjects ( p > 0.05). After subgrouping of the patients into relapsing-remitting MS and primary progressive MS we found that the distribution of promoter alleles in the latter of these two patient groups differed from that of healthy control subjects ( p = 0.04). There was no evidence of linkage disequilibrium between the polymorphic site in the B cell specific promoter and those in the 3′ UTR. Based upon the polymorphic sites in the 3′ UTR we identified two common CIITA haplotypes which were present at similar frequencies in patients and control subjects. Assuming that susceptibility to MS depends upon type of MHC class II molecule as well as the amounts of expressed class II molecules we tested for interaction between DR15 status and CIITA alleles. No such interaction was detected. Unexpectedly, we identified an intron in the 3′ UTR of the human as well as the mouse CIITA gene. Due to the proximity of these introns to the termination codon in both the human and mouse CIITA gene, the mechanism for regulation of transcript stability known as nonsense-mediated decay is probably not involved in the posttranscriptional control of the expression of these genes. So far, the function and significance of the intron in the human and mouse CIITA genes are unknown.

Co-occupancy of the interferon regulatory element of the class II transactivator (CIITA) type IV promoter by interferon regulatory factors 1 and 2.

Class II transactivator (CIITA) activates the expression of major histocompatibility class II genes, which encode antigen-presenting molecules recognized by the T-cell receptor of CD4+ T cells. IFN-gamma induced CIITA transcription in many cell types is directed by the CIITA Type IV promoter. Here we report that the human CIITA Type IV promoter IRF-E binds IRF-1 and can be activated by exogenous expression of IRF-1. Surprisingly, the CIITA Type IV promoter IRF-E is also activated by IRF-2, another member of the IRF family that generally acts as a transcriptional repressor. In addition, we found that IRF-1 and IRF-2 synergistically activate the CIITA Type IV promoter. Electrophoretic mobility shift assays revealed that IRF-1 and IRF-2 can simultaneously occupy the IRF-E of the CIITA Type IV promoter, suggesting a novel mechanism for the role of these two proteins in promoter activation. Our results also indicate that IRF-1 and IRF-2 can cooperatively activate and co-occupy the IRF-E of the guanylate binding protein (GBP) promoter. Finally, CIITA induction by IFN-gamma does not occur in a pancreatic tumor cell line that expresses a mutated IRF-2, representing the first IRF-2 mutation identified in a human tumor cell line.

Suppression of human anti-porcine T-cell immune responses by major histocompatibility complex class II transactivator constructs lacking the amino terminal domain.

The class II transactivator (CIITA) is a bi- or multifunctional domain protein that acts as a transcriptional activator and plays a critical role in the expression of MHC class II genes. We have previously demonstrated that a mutated form of the human CIITA gene, coding for a protein lacking the amino terminal 151 amino acids, acts as a potent dominant-negative suppressor of HLA class II expression. Porcine MHC class II antigens are potent stimulators of direct T-cell recognition by human CD4+ T cells and are, therefore, likely to play an important role in the rejection responses to transgenic pig donors in clinical xenotransplantation. We were, therefore, interested in examining mutated CIITA constructs for their effect on porcine MHC class II expression. Stable transfectants of the porcine vascular endothelial cell line PIEC with mutated CIITA constructs were tested for SLA-DR and SLA-DQ induction by recombinant porcine interferon-gamma. Transient transfectants of the porcine B-cell line L23 with the mutated CIITA constructs were tested for the suppression of constitutive SLA-DR and SLA-DQ expression. T-cell proliferation studies were performed using highly purified human CD4+ T cells. In preliminary studies, we demonstrated that transfection of the PIEC line with full-length human CIITA constructs resulted in strong expression of SLA-DR and SLA-DQ antigens, thus establishing the cross-species effectiveness of human CIITA in the pig. The mutated human CIITA constructs were, therefore, tested in the pig. PIEC clones stably transfected with one of these constructs showed up to 99% suppression of SLA-DR and SLA-DQ antigen induction and marked suppression of SLA-DRA mRNA induction. Moreover, transient transfection of the porcine B-cell line L23 showed up to 90% suppression of constitutive SLA-DR and SLA-DQ antigen expression in 5-8 days. In functional studies, interferon-gamma-stimulated PIEC clones transfected with this mutated CIITA construct failed to stimulate purified human CD4+ T lymphocytes. Mutated human CIITA constructs are potent suppressors of porcine MHC class II expression.

Isolation of a B-cell-specific promoter for the human class II transactivator.

The class II transactivator (CIITA) is essential for the expression of major histocompatibility complex (MHC) class II antigens. The tissular patterns of CIITA and MHC class II gene expression are tightly correlated: CIITA mRNA is highly expressed in B cells, and is induced by interferon gamma (IFN-gamma) in macrophage and epithelial cell lines. We first isolated two overlapping cosmids encoding human CIITA which, when co-transfected, are able to restore MHC class II expression in a B-lymphoblastoid cell line (B-LCL) defective for CIITA. Subsequently, a 1.8 kilobase (kb) fragment of the CIITA promoter was isolated and sequenced. A motif presenting a strong similarity to an initiator was detected, as well as putative binding sites for Sp1, GATA-2, LyF-1, ets-1, AP1, and MZF1 transcription factors, and two GAS motifs. When introduced in front of a luciferase reporter gene, this promoter is able to direct a high luciferase activity in a human B-LCL. In contrast, luciferase expression was not stimulated after IFN-gamma treatment when the construct was transfected in macrophage or in epithelial cell lines. However, an induction of the human CIITA gene was observed in mouse macrophage and fibrosarcoma cell lines, when the cells were transfected with a cosmid containing the human CIITA gene, but lacking the 1.8 kb promoter described above. Taken together, these data suggest the existence of an intragenic promoter driving an IFN-gamma-inducible expression of CIITA.

Active suppression of the class II transactivator-encoding AIR-1 locus is responsible for the lack of major histocompatibility complex class II gene expression observed during differentiation from B cells to plasma cells.

In this study the genetic control of major histocompatibility complex (MHC) class II gene expression during the transition from B cell to plasma cell has been analyzed. Class II molecules are not expressed in plasma cells because of an active suppression resulting in the abrogation of class II gene transcription. We show here that the plasma cell-specific repressor function, designated SIR (suppressor of immune response genes), does not act directly on the transcription of class II genes, but instead on the transcription of the AIR-1 gene, whose product, the class II transactivator (CIITA), is fundamental for the regulation of the constitutive and inducible expression of MHC class II genes. This was unambiguously demonstrated by the fact that plasmacytoma x B cell hybrids carrying an AIR-1 locus derived from CIITA-expressing cells do not express CIITA-specific transcripts. Transfection of a cDNA containing the human CIITA coding sequence under the control of an heterologous promoter restores expression of human MHC class II genes in the hybrids and is responsible for de novo expression of mouse MHC class II genes in both the mouse plasmacytoma cell line and the hybrids. These results confirm and extend the notion of the functional conservation of the AIR-1 gene product across species barriers. Interestingly, in CIITA-transfected cell hybrids, cell surface expression of the human HLA-DQ heterodimer was not observed. This result was not attributable to lack of HLA-DQ alpha or -DQ beta transcription, because both transcripts were present in the CIITA-transfected hybrids, although at reduced levels. These findings further support our previous observations on the distinct regulation of expression of the human HLA-DQ class II subset, which may be thus controlled at the posttranscriptional level by a CIITA-independent mechanism.

CIITA activates the expression of MHC class II genes in mouse T cells

It has long been a puzzle that MHC class II molecules are expressed in human T cells after activation but not in mouse T cells; this expression is believed to play a role in the cell mediated immune response. Recently the MHC class II transactivator (CIITA) has been reported to be a major regulatory factor for both the constitutive and IFN inducible expression of MHC class II genes. Here we show that human T cells expressing MHC class II have CIITA transcripts while MHC class II-negative human T cells and mouse T cells do not. The expression of MHC class II genes in mouse T cells can be reconstituted upon transfection with the human CIITA cDNA. These data indicate that the expression of CIITA explains the expression or lack of expression of MHC class II in human and mouse T cells respectively.