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Background: Our previous studies have shown that regulatory factor X5 (RFX5), a classical transcription regulator of MHCII genes, was obviously overexpressed in hepatocellular carcinoma (HCC) tumors. However, the role of RFX5 in the carcinogenesis and progress of HCC remains unknown. This study aimed to reveal its biological significance and the underlying mechanism in HCC. Methods: RFX5 mRNA expression level and copy number variation in HCC tumors and cell lines were determined by analyzing deposited data sets in the Cancer Genome Atlas and Gene Expression Omnibus database. The biological significance of RFX5 in HCC was investigated by monitoring the colony formation and subcutaneous tumor growth capacity when RFX5 was silenced with lentiviral short hairpin RNA and CRISPR/Cas9 system in HCC cell lines. The downstream gene transcriptionally activated by RFX5 in HCC cells was determined by chromatin immunoprecipitation and luciferase reporter assay. The involvement of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein theta (YWHAQ) in HCC development was further determined by performing colony formation rescue assay and subcutaneous tumor growth rescue experiment. The association of YWHAQ with recurrence-free survival of patients with HCC was assessed by Kaplan-Meier analysis. Moreover, apoptosis level and the protein level of p53 pathway were determined to reveal the mechanism of RFX5 in driving HCC development. Results: RFX5 was amplified and highly overexpressed in HCC tumor tissues compared with the corresponding non-tumor tissues. The mRNA expression level of RFX5 was significantly correlated with its DNA copy number (r = 0.4, P < 0.001). Functional study demonstrated that RFX5 was required for both clonogenic forming in vitro and subcutaneous tumor growth in vivo of HCC cells. Further study identified YWHAQ, namely 14-3-3 tau, as a key downstream transcriptional target gene of RFX5, which was tightly regulated by RFX5 in HCC. Moreover, overexpression of YWHAQ largely rescued the clonogenic growth of HCC cells that was suppressed by RFX5 knockdown. In addition, overexpression of YWHAQ in primary tumor was linked to poor prognosis of patients with HCC. These results demonstrated that YWHAQ was a downstream effector of RFX5 in HCC. Notably, RFX5-YWHAQ pathway could protect cells from apoptosis by suppressing the p53 and Bax in HCC. Conclusion: RFX5 is a putative HCC driver gene that plays an important role in the development and progression of HCC by transactivating YWHAQ and suppressing apoptosis. Key words: Hepatocellular carcinoma; Transcription factor; Apoptosis; P53

Background MHC class II deficiency leads to defective CD4+ T-cell function that results from impaired antigen presentation. A genetic disorder in 1 of 4 genes results in this syndrome that is associated with the clinical phenotype of combined immunodeficiency. Objective To describe the clinical, immunological, and molecular characteristics of 10 Egyptian patients from 9 different families having presented with MHC class II deficiency between 2012 and 2017. Methods An initial diagnosis based on the combination of clinical features and low HLA-DR expression by flow cytometry was confirmed by genetic analyses. Results Symptoms included failure to thrive (n = 9), persistent diarrhea (n = 5), and pneumonia (n = 8). Septicemia due to coagulase-negative staphylococci (n = 1) and Candida krusei (n = 1) was diagnosed. Nine patients orally received the live attenuated polio vaccine, of whom 3 developed acute flaccid paralysis thereafter. Nine patients received the BCG vaccine and none developed obvious signs of BCGitis. Four patients carried RFXANK gene mutations, 3 carried RFX5 gene mutations, 1 carried a CIITA gene mutation, and none carried RFXAP gene mutations. Six of the 7 detected mutations were previously unreported mutations: c.431T>C, c.247_250delTCAG, and c.600delG in the RFXANK gene; c.116+1G>A and c.715C>T in the RFX5 gene; and c.929delA in the CIITA gene. Conclusions Given that Egypt is a North African country with a high rate of consanguinity, MHC class II deficiency is not rare. However, the molecular defects differ from those reported in nearby countries. Early diagnosis must be based on suspicious clinical signs and laboratory diagnosis because the defect can be missed by T-cell receptor excision circles based on neonatal screening.

Although immunotherapy is a potential strategy to resist cancers, due to the inadequate acknowledge, this treatment is not always effective for diffuse large B cell lymphoma (DLBCL) patients. Based on the current situation, it is critical to systematically investigate the immune pattern. According to the result of univariate and multivariate cox proportional hazards, LASSO regression and Kaplan-Meier survival analysis on immune-related genes (IRGs), a prognostic signature, containing 14 IRGs (AQP9, LMBR1L, FGF20, TANK, CRP, ORM1, JAK1, BACH2, MTCP1, IFITM1, TNFSF10, FGF12, RFX5, and LAP3), was built. This model was validated by external data, and performed well. DLBCL patients were divided into low- and high-risk groups, according to risk scores from risk formula. The results of CIBERSORT showed that different immune status and infiltration pattern were observed in these two groups. Gene set enrichment analysis (GSEA) indicated 12 signaling pathways were significantly enriched in the high-risk group, such as natural killer cell-mediated cytotoxicity, toll-like receptor signaling pathway, and so on. In summary, 14 clinically significant IRGs were screened to build a risk score formula. This formula was an accurate tool to provide a certain basis for the treatment of DLBCL patients.

EBV-associated gastric adenocarcinomas (EBVaGCs) often exhibit better clinical outcomes than EBV negative gastric cancers (GCs), which could be related to their consistent expression of foreign viral antigens. Antigen-presenting cells (APCs) present peptide antigens in the context of the class-II major histocompatibility complex (MHC-II). During inflammatory conditions, epithelial cells express MHC-II and function as accessory APCs. Utilizing RNA-seq data from nearly 400 GC patients, we determined the impact of EBV-status on expression of MHC-II components, genes involved in their regulation, and T-cell co-stimulation. Virtually all MHC-II genes were significantly upregulated in EBVaGCs compared to normal tissues, or other GC subtypes. Genes involved in antigen presentation were also significantly upregulated in EBVaGCs, as were the key MHC-II transcriptional regulators CIITA and RFX5. This was unexpected as the EBV encoded BZLF1 protein can repress CIITA transcription and is expressed in many EBVaGCs. Furthermore, MHC-II upregulation was strongly correlated with elevated intratumoral levels of interferon-gamma. In addition, expression of co-stimulatory molecules involved in T-cell activation and survival was also significantly increased in EBVaGCs. Thus, gastric adenocarcinoma cells may functionally contribute to the highly immunogenic tumor microenvironment observed in EBVaGCs via a previously unappreciated role in interferon-induced antigen presentation.

Major histocompatibility complex (MHC) II deficiency is a rare primary immunodeficiency disorder that is characterized by the deficiency of MHC class II molecules. The disease is caused by transcription factor mutations including class II transactivator (CIITA), regulatory factor X-5 (RFX5), RFX-associated protein (RFXAP), and RFXAP-containing ankyrin repeat (RFXANK), respectively. Mutations in the RFXANK gene account for >70% of all known patients worldwide. Herein, we reported a 10-month-old boy with MHC II deficiency caused by a novel mutation in the RFXANK gene (c.337 + 1G>C). The boy was admitted to the hospital due to pneumonia and diarrhea at 4 months of age. Genetic analysis revealed a novel homozygous mutation in the RFXANK gene, which derived from the c.337 + 1G>C heterozygous mutations in the RFXANK gene of his parents. The boy died 3 months after diagnosis. More than 200 cases have been reported, and a review of the literature revealed different mutation rates of 4 transcription factors in different countries or regions. This is the first case report of MHC II deficiency from East Asia. We also describe all gene mutations that cause MHC II deficiency and the epidemiology of MHC II deficiency with gene mutations in this paper.

Supplemental Digital Content is available in the text., Major histocompatibility complex (MHC) class II regulatory genes play a paramount role in immune response that can exert a predominant influence on clinical outcome of Epstein–Barr virus infection consistently assumed as the main pathogenetic factor for nasopharyngeal carcinoma. To elucidate the relationship between allelic variants of MHC class II regulatory genes and susceptibility to nasopharyngeal carcinoma, a total of 28 polymorphic loci at MHC class II regulatory genes, involving CIITA, CREB1, RFX family genes (RFX5, RFXAP, and RFXANK), and NFY family genes (NFYA, NFYB, and NFYC), were genotyped by multiplex SNaPshot minisequencing in 137 patients with nasopharyngeal carcinoma and 107 healthy controls from the southern Chinese population. Allelic analysis disclosed that rs7404873, rs6498121, rs6498126, and rs56074043 shared correlations with nasopharyngeal carcinoma (Ptrend < 0.05). Further, rs6498126 on CIITA was independently associated with the risk of developing nasopharyngeal carcinoma (CC vs. GG, odds ratio: 7.386, 95% confidence interval: 1.934–28.207, Ptrend < 0.01). Conversely, rs7404873 on CIITA and rs56074043 on NFYB manifested epistatic interaction to decreased susceptibility of nasopharyngeal carcinoma (rs7404873, TT vs. GG, odds ratio: 0.256, 95% confidence interval: 0.088–0.740, Ptrend < 0.05; rs56074043, AA vs. AG, odds ratio: 0.341, 95% confidence interval: 0.129–0.900, Ptrend < 0.05). Additionally, bioinformatics analysis revealed that the three variants were transcriptional regulatory in function and might impact the expression of nearby genes. The findings suggested genetic variants on MHC class II regulatory genes contributed to nasopharyngeal carcinoma susceptibility and might provide new insights for screening high-risk population.

The MHC II deficiency is a rare autosomal recessive primary immunodeficiency syndrome with increased susceptibility to respiratory and gastrointestinal infections, failure to thrive and early mortality. This syndrome is caused by mutations in transcription regulators of the MHC II gene and results in development of blind lymphocytes due to the lack of indicatory MHC II molecules. Despite homogeneity of clinical manifestations of patients with MHC II deficiency, the genetic defects underlying this disease are heterogeneous. Herein, we report an Iranian patient with MHC II deficiency harbouring a novel mutation in RFXANK and novel misleading clinical features. He had ataxic gait and dysarthria from 30 months of age. Epidemiology, clinical and immunological features, therapeutic options and prognosis of patients with MHC II are reviewed in this paper.

Macrophages are professional antigen-presenting cells relying on the expression of class II major histocompatibility complex (MHC II) genes. Interferon-γ (IFN-γ) activates MHC II transcription via the assembly of an enhanceosome centred on class II trans-activator (CIITA). In the present study, we investigated the role of the forkhead transcription factor FOXO3a in IFN- γ-induced MHC II transcription in macrophages. Knockdown of FOXO3a, but not FOXO1 or FOXO4, diminished IFN-γ-induced MHC II expression in RAW cells. On the contrary, over-expression of FOXO3a, but neither FOXO1 nor FOXO4, enhanced CIITA-mediated trans-activation of the MHC II promoter. IFN-γ treatment promoted the recruitment of FOXO3a to the MHC II promoter. Co-immunoprecipitation and RE-ChIP assays showed that FOXO3a was a component of the MHC II enhanceosome forming interactions with CIITA, RFX5, RFXB and RFXAP. FOXO3a contributed to MHC II transcription by altering histone modifications surrounding the MHC II promoter. Of interest, FOXO3a was recruited to the type IV CIITA promoter and directly activated CIITA transcription by interacting with signal transducer of activation and transcription 1 in response to IFN-γ stimulation. In conclusion, our data unveil a novel role for FOXO3a in the regulation of MHC II transcription in macrophages.

The role of tumor cell expression of major histocompatibility class II (MHCII) has been controversial, with evidence indicating that tumor cell expression of MHCII may lead to an anti-tumor immune response and to tumor cell apoptosis and that MHCII has pro-tumorigenic functions. The cancer genome atlas (TCGA) indicates numerous deleterious mutations for the highly specific, MHCII transcriptional activation proteins, RFX5, RFXAP, RFXANK and CIITA. Also, mutations in the non-polymorphic, human leukocyte antigen (HLA)-DRA gene, which encodes the heavy chain for the most prominent human MHCII molecule, HLA-DR, are common. For many, if not most TCGA cancer datasets, the MHCII specific mutations do not associate with clinical outcomes. However, stomach carcinoma represents an exception, where the data indicate that MHCII-specific mutations are associated with a more favorable outcome. These data raise the question of whether stomach cancer mutations represent effective haploinsufficiency or whether mutations that are associated with a favorable outcome occur with other stomach cancer molecular features that limit the function of the two alleles that represent these MHCII-related proteins.

Background Lung adenocarcinoma (LUAD) is the main subtype of non-small cell lung cancer with a low survival prognosis. We aimed to generate a prognostic model for the postoperative recurrence of LUAD. Methods The methylated DNA data of LUAD patients were downloaded from the Cancer Genome Atlas (TCGA). The differentially methylated genes were identified and protein–protein interacting network was constructed, with which prognostic signature of this cancer was generated. Survival and functional pathways analysis w used to evaluate the clustering ability of the prognostic signature. Results We identified 151 differentially methylated genes related to relapse-free survival of patients with LUAD. Nine hub genes were identified in PPI network, with which 4 gene pair signature was selected as prognostic signature. The potential functions of 6 genes (JDP2, SERPINA5, PLG, SEMG2, RFX5, and POLR3B) in the 4-gene pair signature were enriched in intracellular protein synthesis and transportation. Conclusion The four gene pair signature can predict the prognosis of patients with stage I LUAD. Our study provides a reference for patients with postoperative adjuvant therapy.

Besides being expressed on professional antigen-presenting cells, HLA class II antigens are expressed on various tumors of non-lymphoid origin, including a subset of colorectal cancers (CRC). Information about the regulation of HLA class II antigen expression is important for a better understanding of their role in the interactions between tumor and immune cells. Whether lack of HLA class II antigen expression in tumors reflects the selective immune destruction of HLA class II antigen-expressing tumor cells is unknown. To address this question, we tested whether lack of HLA class II antigen expression in CRC was associated with immune cell infiltration. We selected microsatellite-unstable (MSI-H) CRC, because they show pronounced tumor antigen-specific immune responses and, in a subset of tumors, lack of HLA class II antigen expression due to mutations inactivating HLA class II-regulatory genes. We examined HLA class II antigen expression, mutations in regulatory genes, and CD4-positive T cell infiltration in 69 MSI-H CRC lesions. Mutations in RFX5, CIITA, and RFXAP were found in 13 (28.9 %), 3 (6.7 %), and 1 (2.2 %) out of 45 HLA class II antigen-negative tumors. CD4-positive tumor-infiltrating lymphocyte counts were significantly higher in HLA class II antigen-negative tumors harboring mutations in HLA class II-regulatory genes (107.4 T cells per 0.25 mm(2)) compared to tumors without mutations (55.5 T cells per 0.25 mm(2), p = 0.008). Our results suggest that the outgrowth of tumor cells lacking HLA class II antigen expression due to mutations of regulatory genes is favored in an environment of dense CD4-positive T cell infiltration.

Regulatory factor X-5 (RFX5) was previously characterized as an essential and highly specific regulator of major histocompatibility class II (MHCII) gene expression in the immune system. We found that RFX5 is significantly upregulated in hepatocellular carcinoma (HCC) tumors and cell lines compared with non-tumor tissues in mRNA expression levels, but it fails to induce the expression of MHCII. However, RFX5 can strongly bind to the tripeptidyl peptidase 1 (TPP1) promoter region and then increase its transcriptional activity. We also found that manipulation the expression of RFX5 can significantly affect the expression of TPP1 in HepG2, which suggested that RFX5 can transcriptionally activate TPP1 in HCC. Moreover, TPP1 is overexpressed in HCC tissues and significantly correlated with poor prognosis of HCC patients, suggesting that it may have potential biological implications in HCC.

Major histocompatibility complex (MHC) class II deficiency, also called the bare lymphocyte syndrome, is a rare primary immunodeficiency disease characterized by a virtual absence of MHC class II expression associated with a variable reduction in MHC class I expression. The disease is genetically heterogeneous. It can result from loss-of-function mutations in any one of four genes – CIITA , RFX5 , RFXAP , and RFXANK – encoding regulatory factors required for transcription of class I and/or class II MHC genes. RFX5 , RFXAP , and RFXANK encode subunits of regulatory factor X, a DNA-binding transcription factor required for transcription of both class I and class II MHC genes. Class II transactivator (CIITA) encodes a transcriptional coactivator showing high specificity for MHC class II genes. CIITA is referred to as the ‘master regulator’ of MHC class II genes because its highly regulated pattern of expression dictates most qualitative and quantitative aspects of MHC class II expression. At MHC class I promoters, CIITA is replaced by a related coactivator, NLRC5. Deficient MHC expression leads to the inability to mount antigen-specific adaptive immune responses against infectious agents. Patients consequently suffer from a broad range of severe and recurrent infections, generally resulting in death at a young age, unless they are treated by hematopoietic stem cell transplantation.

Recent reports demonstrate that peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the nuclear receptor superfamily, acts as a repressor of type I collagen synthesis. Our data demonstrate that exogenously expressed PPARgamma down-regulates collagen expression in a dose-responsive manner in human lung fibroblast cells. Silencing PPARgamma using lentiviruses expressing short hairpin RNAs partially reverses interferon-gamma (IFN-gamma)-induced repression and activates collagen mRNA levels. Previous studies indicate that IFN-gamma represses collagen gene expression and induces major histocompatibility complex II (MHC II) expression by activating the formation of a regulatory factor for X-box 5 (RFX5) complex with class II transactivator (CIITA). This report demonstrates that PPARgamma is within the RFX5.CIITA complex as judged by co-immunoprecipitation and DNA affinity precipitation studies. Most importantly, occupancy of PPARgamma on the collagen transcription start site and MHC II promoter increases with IFN-gamma treatment. The PPARgamma agonist, troglitazone, sensitizes the cells to IFN-gamma treatment by increasing recruitment of PPARgamma to collagen gene while repressing collagen expression, and these effects are blocked by the PPARgamma antagonist T0070907. PPARgamma may mediate IFN-gamma-stimulated collagen transcription down-regulation and MHC II up-regulation by interacting with CIITA as well as regulating CIITA expression. Therefore, PPARgamma is a critical target for investigations into therapeutics of diseases involving extracellular matrix remodeling and the immune response.

Collagen type I is composed of three polypeptide chains transcribed from two separate genes (COL1A1 and COL1A2) with different promoters requiring coordinate regulation. Our recent publications, centering on COL1A2 regulation, demonstrate that methylation in the first exon of COL1A2 at a regulatory factor for X box (RFX) site (at -1 to +20) occurs in human cancer cells and correlates with increased RFX1 binding and decreased collagen transcription (Sengupta, P. K., Erhlich, M., and Smith, B. D. (1999) J. Biol. Chem. 274, 36649-36655; Sengupta, S., Smith, E. M., Kim, K., Murnane, M. J., and Smith, B. D. (2003) Cancer Res. 63, 1789-1797). In normal cells, RFX5 complex along with major histocompatibility class II transactivator (CIITA) is induced by interferon-gamma to occupy this site and repress collagen transcription (Xu, Y., Wang, L., Buttice, G., Sengupta, P. K., and Smith, B. D. (2004) J. Biol. Chem. 279, 41319-41332). In this paper, we demonstrate that COL1A1 has an RFX consensus binding site surrounding the transcription start site (-11 to +10) that contains three methylation sites rather than one in the COL1A2 gene RFX binding site. RFX1 interacts weakly with the unmethylated COL1A1 site, and binds with higher affinity to the methylated site. RFX1 represses the unmethylated COL1A1 less efficiently than COL1A2. COL1A1 promoter activity is sensitive to DNA methylation and the COL1A1 gene is methylated in human cancer cells with coordinately decreased collagen expression. The DNA methylation inhibitor, 5-aza-2'-deoxycytidine (aza-dC) increases collagen gene expression with time in human cancer cells. On the other hand, RFX5 interacts with both collagen type I genes with a similar binding affinity and represses both promoters equally in transient transfections. Two dominant negative forms of RFX5 activate both collagen genes coordinately. Finally, CIITA RNA interference experiments indicate that CIITA induction is required for interferon gamma-mediated repression of both collagen type I genes.

The extraordinary homology between major histocompatibility complex class II (MHC II) proteins across species from human to bony fish suggests that transcription factors that regulate these proteins might be conserved as well. Deficiencies in four proteins that regulate MHC II genes in humans (RFX-B, RFX5, RFXAP, and CIITA) cause an inherited immunodeficiency disorder known as the bare lymphocyte syndrome (BLS). To understand the structure and mechanism of function of the BLS transcription factors, we analyzed the evolutionary history of RFX-B, the factor deficient in the majority of patients with BLS. Sequence comparison and analysis of the RFX-B proteins showed that RFX-B and a closely related protein, ANKRA2, are present in humans to bony fish and that specific domains are highly conserved. In addition to sequence conservation, functional conservation exists, as mouse and Xenopus RFX-B orthologues, but not the paralogous protein ANKRA2, were able to complement the MHC II deficiency in a BLS-patient-derived cell line deficient in RFX-B. The remarkable conservation of the RFX-B lineage attests to the conservation of the regulation mechanism for this gene system and its importance to precisely regulate MHC class II molecules in both the developing and active immune response.

Major histocompatibility complex (MHC) class II genes are regulated at the transcriptional level by coordinate action of a limited number of transcription factors that include regulatory factor X (RFX), class II transcriptional activator (CIITA), nuclear factor Y (NF-Y), and cyclic AMP-response element binding protein (CREB). Here, the MHC class-II-specific transcription factors and CREB were expressed in insect cells with recombinant baculoviruses, isolated, and characterized by biochemical and biophysical methods. Analytical ultracentrifugation (AUC) has demonstrated that RFX is a heterotrimer. A heterodimer of RFX5 and RFX-AP was also observed. A high-affinity interaction (K(d) = 25 nM) between RFX5 and RFX-AP was measured by isothermal titration calorimetry (ITC), while the interaction between RFX-AP and RFX-ANK is at least an order of magnitude weaker. The biophysical data show that the interaction between RFX-AP and RFX5 is a key event in the assembly of the heterotrimer. Fluorescence anisotropy was used to determine protein-nucleic acid binding affinities for the RFX subunits and complexes binding to duplex DNA. The RFX5 subunit was found to drive recognition of the promoter, while the auxiliary RFX-AP and RFX-ANK subunits were shown to contribute to the specificity of binding for the overall complex. AUC experiments demonstrate that in the absence of additional subunits, monomeric RFX5 binds to X-box DNA with a 1:1 stoichiometry. Interactions between CREB, CIITA, and RFX in the absence of DNA were demonstrated using bead-based immunoprecipitation assays, confirming that preassociation with DNA is not required for forming the macromolecular assemblies that drive MHC class II gene expression.

We analysed the regulation of human leucocyte antigen (HLA)-E, -F and -G genes, focusing on the SXY module, a promoter region that controls major histocompatibility complex (MHC) class II expression and participates in the expression of classical HLA class I molecules. It comprises the X1, X2 and Y boxes, bound by RFX, X2-BP/ATF/CREB and NFY factors, respectively. The complex recruits the master control factor CIITA. The SXY module is conserved in HLA-E and HLA-F gene promoters, whereas in the HLA-G promoter, the only conserved boxes are S and X1. Chromatin immunoprecipitation assays, performed on HLA-G positive and negative cell lines, demonstrated the in situ binding of RFX5 and CIITA to HLA-E and HLA-F, but not to HLA-G, promoters. In B cells from bare lymphocyte syndrome patients lacking RFX5 or CIITA, we observed lower steady-state levels of HLA-E and HLA-F transcripts but did not find any significant decrease in the cell-surface expression of HLA-E/classical HLA class I. In RFX5-deficient fibroblasts, the cell-surface expression of HLA molecules was decreased. RFX5 and CIITA are thus not involved in HLA-G expression and their importance for the surface expression of HLA-E/classical HLA class I molecules may vary depending on the cell type.

MHC class II deficiency provokes a severe immunodeficiency characterized by a lack of antigen-specific immune response. In the absence of bone marrow transplantation (the only curative treatment), patients affected by this genetic recessive disease die in early childhood. However, others and we have recently described cases of mild or asymptomatic immunodeficiencies with defects in either CIITA (class II transactivator) or RFX5, both proteins required for the transcription of HLA-D genes. We describe in this report the first case of moderate immunodeficiency resulting from a defect in RFXANK, another transcription factor essential for HLA-D expression. The patient did not display any detectable expression of MHC class II molecules on B lymphocytes, monocytes or activated T lymphocytes. Accordingly HLA-D transcription was altered in the corresponding B-lymphoblastoid cell line. The defect in RFXANK was observed both at the transcript and protein level. Indeed a homozygous IVS4+5G>A mutation was evidenced in RFXANK, and shown to hamper the splicing of intron 4. However, we had shown previously that a defect in intron 4 can lead to the skipping of exon 4, and that the resulting truncated protein retains the capacity to activate HLA-DR expression. Therefore, like the two cases of moderate immunodeficiencies described previously, we demonstrate that the RFXANK defect presented here is coherent with a residual activity of the mutant protein. We thus propose that the common feature displayed by mildly immunodeficient patients is the leakiness of the mutations, which might allow a local or temporal expression of MHC class II molecules.

Major Histocompatibility Complex class II (MHCII) molecules direct the development, activation and homeostasis of CD4+ T cells. Given these key functions it is not surprising that the absence of MHCII expression results in a severe primary immunodeficiency disease called MHCII deficiency or the Bare Lymphocyte Syndrome (BLS). The genetic defects responsible for BLS lie in genes encoding transcription factors required for MHCII expression. Four different MHCII regulatory genes encoding RFXANK, RFX5, RFXAP and CIITA have been identified. The first three are subunits of RFX, a ubiquitously expressed factor that binds cooperatively with other proteins to MHCII and related promoters to form a highly stable macromolecular nucleoprotein complex referred to as the MHCII enhanceosome. This enhanceosome serves as a landing pad for the MHCII transactivator CIITA. CIITA is a non-DNA binding coactivator that serves as the master control factor for MHCII expression. The highly regulated expression pattern of CIITA ultimately dictates the cell type specificity, induction and level of MHCII expression. The enhanceosome and CIITA collaborate in activating transcription by promoting histone hyperacetylation and by recruiting components of the general transcription machinery. In this review we summarize what is known about the molecular basis of BLS and what this has taught us about the mechanisms regulating transcription of MHCII and related genes. Particular attention is devoted to the structure, function and mode of action of the MHCII enhanceosome and CIITA. In addition, we focus on the highly regulated and cell type specific expression of CIITA.

MHC class II deficiency is a combined immunodeficiency caused by defects in the four regulatory factors, CIITA, RFXANK, RFX5 and RFXAP, that control MHC II expression at the transcriptional level. The RFXANK gene encodes one subunit of the heterotrimeric RFX complex that is involved in the assembly of several transcription factors on MHC II promoters. Seven different RFXANK mutations have previously been reported in 26 unrelated patients. The most frequent mutation, a 26-bp deletion (752delG-25), has been identified in 21 patients. The other mutations are all nonsense or splice-site mutations, leading to proteins lacking all or part of the RFXANK ankyrin repeat region. We report two novel missense mutations, D121V and R212X, resulting in loss of function of the gene. We investigated the in vivo effects of these mutations and of three other point mutations on the expression of the RFXANK RNA and protein. The number of RFXANK transcripts was severely reduced in all patients except one. The RFXANK protein was barely detected in two cases. In addition, guided by a structural model of RFXANK, we investigated experimental mutants of the C-terminal tyrosine 224. Substitution Y224A, but not Y224F, led to the loss of function of RFXANK. Two null mutants, D121V and Y224A, were tested in protein interaction and DNA binding assays. The D121V mutant was unable to form the RFX complex, indicating that D121 is required for RFXAP binding. The Y224A mutant formed an RFX complex that bound normally to the MHC II promoter, but did not lead to MHC class II expression, whereas Y224F RFXANK retained the wild-type function. This indicates that an aromatic ring, but not the phenyl chain of tyrosine, is necessary at position 224 for normal RFXANK function. Studies on the Y224A mutant suggest that, in addition to the RFX subunits and CIITA, another protein is essential for MHC class II expression. This protein appears to interact with the fourth ankyrin repeat of RFXANK.

Major histocompatibility complex class II (MHCII) deficiency is a primary immunodeficiency resulting from defects in one of four different MHCII-specific transcription factors-CIITA, RFX5, RFXAP, and RFXANK. Despite this genetic heterogeneity, the phenotypical manifestations are homogeneous. It is frequently difficult to establish a definitive diagnosis of the disease on the basis of clinical and immunological criteria. Moreover, the phenotypical homogeneity precludes unambiguous identification of the regulatory gene that is affected. Identification of the four genes mutated in the disease has now allowed us to develop a rapid and straightforward diagnostic test for new MHCII-deficiency patients. This test is based on direct correction of the genetic defect by transduction of cells from patients with lentiviral vectors encoding CIITA, RFXANK, RFX5, or RFXAP. We have validated this approach by defining the molecular defects in two new patients. The RFXANK vector restored MHCII expression in a T cell line from one patient. The RFXAP vector corrected primary cells (PBL) from a second patient. Molecular analysis confirmed the presence of homozygous mutations in the RFXANK and RFXAP genes, respectively. Direct genetic correction represents a valuable tool for the diagnosis and classification of new MHCII-deficiency patients.

Four transacting genes, CIITA, RFXANK, RFX5, and RFXAP, control coordinate MHC II expression. In humans, defects in these genes result in the absence of MHC II expression and thus a combined immunodeficiency. CIITA is considered to be a master MHC II regulator and is responsible for the defect in complementation group A. Eight such affected families have been reported. We investigated the molecular basis of the defect in three patients in these families, all presenting a severe immunodeficiency. CIITA transcripts were detected in all three patients but in one at an abnormally low level. Three novel heterozygous mutations of CIITA were found in patients SP and RC. One SP CIITA allele contained a nonsense mutation, G2178A, leading to a premature stop codon and the other allele in SP was found not to be expressed. In patient RC, two in-frame deletions were detected: one of the nucleotides 3003-3084 corresponding to the exon coding from Leu(964)to Asp(991), in the paternal allele, and a CATdel3193-5 of the isoleucine codon at position 1027, in the maternal allele. Transfection of a CIITA-deficient cell line with the recombinant CATdel3193-5-CIITA cDNA revealed a loss of function for this mutant and retention of the protein in the cytoplasm. No mutations were detected in the 4.5-kb cDNA from patient OK but the level of CIITA transcript was found to be profoundly decreased. However, promoters III and IV were not affected. This last case represents the first described CIITA dysfunction due to putative mutation(s) in cis regulatory sequences of CIITA.

Major histocompatibility complex class II (MHC-II) molecules play a central role in adaptive antiviral immunity by presenting viral peptides to CD4 + T cells. Due to their key role in adaptive immunity, many viruses, including Kaposi's sarcoma-associated herpesvirus (KSHV), have evolved multiple strategies to inhibit the MHC-II antigen presentation pathway. The expression of MHC-II, which is controlled mainly at the level of transcription, is strictly dependent upon the binding of the class II transactivator (CIITA) to the highly conserved promoters of all MHC-II genes. The recruitment of CIITA to MHC-II promoters requires its direct interactions with a preassembled MHC-II enhanceosome consisting of cyclic AMP response element-binding protein (CREB) and nuclear factor Y (NF-Y) complex and regulatory factor X (RFX) complex proteins. Here, we show that KSHV-encoded latency-associated nuclear antigen (LANA) disrupts the association of CIITA with the MHC-II enhanceosome by binding to the components of the RFX complex. Our data show that LANA is capable of binding to all three components of the RFX complex, RFX-associated protein (RFXAP), RFX5, and RFX-associated ankyrin-containing protein (RFXANK), in vivo but binds more strongly with the RFXAP component in in vitro binding assays. Levels of MHC-II proteins were significantly reduced in KSHV-infected as well as LANA-expressing B cells. Additionally, the expression of LANA in a luciferase promoter reporter assay showed reduced HLA-DRA promoter activity in a dose-dependent manner. Chromatin immunoprecipitation assays showed that LANA binds to the MHC-II promoter along with RFX proteins and that the overexpression of LANA disrupts the association of CIITA with the MHC-II promoter. These assays led to the conclusion that the interaction of LANA with RFX proteins interferes with the recruitment of CIITA to MHC-II promoters, resulting in an inhibition of MHC-II gene expression. Thus, the data presented here identify a novel mechanism used by KSHV to downregulate the expressions of MHC-II genes. IMPORTANCE Kaposi's sarcoma-associated herpesvirus is the causative agent of multiple human malignancies. It establishes a lifelong latent infection and persists in infected cells without being detected by the host's immune surveillance system. Only a limited number of viral proteins are expressed during latency, and these proteins play a significant role in suppressing both the innate and adaptive immunities of the host. Latency-associated nuclear antigen (LANA) is one of the major proteins expressed during latent infection. Here, we show that LANA blocks MHC-II gene expression to subvert the host immune system by disrupting the MHC-II enhanceosome through binding with RFX transcription factors. Therefore, this study identifies a novel mechanism utilized by KSHV LANA to deregulate MHC-II gene expression, which is critical for CD4 + T cell responses in order to escape host immune surveillance.

The class II transactivator (CIITA) is a master regulator of major histocompatibility complex class II (MHC-II), Ii, and DM genes (8, 9, 10, 55, 56). CIITA was initially identified by complementation of an HLA-DR− mutant B-cell line, RJ2.2.5 (55), which was created by mutagenesis followed by negative immunoselection for MHC-II-defective cells (1). CIITA is important for the constitutive expression of MHC-II in B cells and dendritic cells and the cytokine induction of these genes in a variety of other cell types. Gamma interferon (IFN-γ) is a prime example of a cytokine which induces CIITA and subsequently MHC-II expression. CIITA is a transcriptional coactivator that does not bind DNA (55) but does interact with RFX (consisting of RFX5-RFXANK-RFXAP), CREB, and NF-Y (NF-YA/B/C) (65), all of which directly bind to the X and Y element of the MHC-II promoters. Specifically, CIITA interacts with both RFX5 and RFXANK and NF-YB and NF-YC but not with NF-YA. In addition, CIITA also interacts with the histone acetyltransferase (HAT) CREB-binding protein (CBP) (20, 30), which can acetylate histone at lysine residues to allow gene activation (13, 19, 31, 57, 59, 62). Thus, CIITA appears to be a focal point of interaction for both DNA-binding proteins specific for the MHC-II promoters and for HATs to allow chromatin opening. CIITA expression is strongly induced by IFN-γ (56). Two of its promoters, promoter III and promoter IV, are responsible for IFN-γ inducibility (39, 42, 43). Promoter III contains a proximal sequence that is responsible for the constitutive expression of CIITA in B cells and a distal sequence containing STAT1-responsive element which confers IFN-γ responsiveness. Promoter IV contains the major IFN-responsive sequence regulated by both STAT1 and IRF-1. An invaluable tool to study IFN-γ pathways has been the use of mutant cell lines. Mutant lines which are defective in the IFN-γ induction of MHC-II genes but not defective in the induction of other IFN-γ functions have been useful in elucidating this pathway (9, 37). The G3A cell line was derived from the commonly used IFN-γ-responsive parental cell line 2fTGH (10). IFN-γ treatment of G3A cells causes the normal induction of most responsive genes but causes a selective failure to induce MHC-II, Ii, and DM. This is caused by a defect in the IFN induction of CIITA and places CIITA downstream of the JAK/STAT pathway. In addition to its well-known function in inducing gene expression, IFN-γ also suppresses the expression of a number of genes. There are several examples of IFN-γ-suppressible promoters or genes, and these can be loosely grouped as the following: genes important for cell proliferation and cell differentiation, such as cyclin (51) and c-myc and N-myc (59); certain cytokine genes expressed by the TH2 subpopulation, such as interleukin 4 (IL-4) (15) and IL-10 (17); and genes coding for matrix proteins, such as the collagen (26, 29, 58) and proteoglycan (16, 50) genes. The mechanism by which IFN-γ causes gene suppression is not well known, and the present study shows that one mechanism involves the CIITA molecule. The rationale for this study stems from a previous report, where we and others detected interactions of CIITA with a number of DNA-binding proteins that bind the MHC-II promoters (65). This prompted us to determine if CIITA can selectively induce the expression of MHC-II but simultaneously sequester DNA-binding proteins from their other gene targets. The squelching of transcription factors has been proposed before (4, 21, 34, 43); however, there are few good physiologic examples of squelching. In this report, we determined that CIITA can squelch the expression of NF-Y-dependent promoters. However, this was not reversed by the addition of NF-Y, and the CIITA domain that is required for this effect does not match the CIITA–NF-Y interaction site. Instead, the region within CIITA that is required for squelching maps to a 36-amino-acid (aa) region which binds the CBP. Significantly, squelching was observed under biologic conditions where the effects of endogenous CIITA on endogenous collagen gene expression were examined. These experiments show that this small peptide domain is a potent inhibitor of CBP function and elucidate a possible mechanism by which IFN-γ suppresses gene expression.

Tight control of MHC expression is essential for the outcome of a successful pregnancy. The lack of MHC class II and class I mediated antigen presentation by fetal trophoblast cells is an important mechanism to evade maternal immune recognition. Interestingly, the deficient expression of MHC class II molecules (HLA-DR, -DQ and -DP) and of the classical MHC class I molecules HLA-A and HLA-B is also noted after IFN-gamma treatment in trophoblast-derived cell lines. Our studies show that in trophoblast cell lines the IFN-gamma induced transactivation of HLA-A and HLA-B promoters is repressed. Furthermore, it was found that trophoblast cells lacked IFN-gamma mediated induction of the class II transactivator (CIITA). This lack of CIITA expression in trophoblast cells is due to CIITA promoter hypermethylation. In addition to lack of CIITA expression, trophoblast cells also displayed a repressed expression of RFX5. Together, these observations reveal a silencing of multiple activation pathways that are critical to the transcriptional control of MHC class II and class I antigen presentation functions by trophoblast cells.

The major histocompatibility complex (MHC) class II transactivator CIITA plays a pivotal role in the control of the cellular immune response through the quantitative regulation of MHC class II expression. We have analyzed a region of CIITA with similarity to leucine-rich repeats (LRRs). CIITA LRR alanine mutations abolish both the transactivation capacity of full-length CIITA and the dominant-negative phenotype of CIITA mutants with N-terminal deletions. We demonstrate direct interaction of CIITA with the MHC class II promoter binding protein RFX5 and could also detect novel interactions with RFXANK, NF-YB, and -YC. However, none of these interactions is influenced by CIITA LRR mutagenesis. On the other hand, chromatin immunoprecipitation shows that in vivo binding of CIITA to the MHC class II promoter is dependent on LRR integrity. LRR mutations lead to an impaired nuclear localization of CIITA, indicating that a major function of the CIITA LRRs is in nucleocytoplasmic translocation. There is, however, evidence that the CIITA LRRs are also involved more directly in MHC class II gene transactivation. CIITA interacts with a novel protein of 33 kDa in a manner sensitive to LRR mutagenesis. CIITA is therefore imported into the nucleus by an LRR-dependent mechanism, where it activates transcription through multiple protein-protein interactions with the MHC class II promoter binding complex.

Major histocompatibility complex (MHC) class II molecules are the predominant presenters of exogenous antigens to T helper cells (reviewed in references 21, 77, and 99). These key molecules are critical for numerous aspects of immune function, including T-cell selection, tolerance induction, antibody production, T-cell-mediated immunity, and the inflammatory response. As principal mediators of transplant rejection, these molecules are often common targets for immune therapies to prevent the rejection of grafted tissues. Class II MHC is implicated as a contributing factor in a host of diseases ranging from rheumatoid arthritis and diabetes to Alzheimer's disease and multiple sclerosis. Constitutive expression of class II MHC is restricted to “professional” antigen-presenting cells but can be induced on various tissues by gamma interferon (IFN-γ). In humans, a congenital lack of both constitutive and inducible class II results in a profound and generally fatal immunodeficiency (type II bare lymphocyte syndrome [BLS]) (7, 29, 45, 61, 67, 75, 112) marked by a significant reduction of CD4+ T cells. Early molecular forays addressing BLS revealed that the genes encoding class II MHC were not defective. Instead, the defect lay in transcription factors controlling class II MHC gene expression. BLS thus became the first disease known to be caused by defective or absent transcription factors. The availability of patient-derived cell lines with class II MHC transcription defects provided a unique tool of nature to identify the requisite transcription factors. Transcriptional regulation of class II MHC expression is complex. Class II MHC and related promoters are characterized by the presence of conserved W (or S), X, and Y boxes (Fig. ​(Fig.1)1) (reviewed in references 10, 67, and 75). The X element is bipartite. The upstream X1 region is recognized by RFX, a trimeric complex of RFX family members including RFX5, RFXANK (RFX-B), and RFXAP (32, 74, 87, 117). The downstream X2 box is bound by X2BP (NF-X2), a complex comprising CREB, and an unidentified 120-kDa protein (83, 84). Another trimeric complex, NF-Y (CBF), which is highly conserved in eukaryotes, binds the Y box (69, 71, 146; reviewed in reference 70). A number of factors interacting with the W box have been described, including the RFX complex (26, 48, 120). The factors involved in X and Y box binding are ubiquitous and expressed constitutively yet fail to account for either constitutive or IFN-γ-inducible class II MHC expression. Somatic cell fusions using BLS patient-derived cells allowed the definition of complementation groups, with each group containing a defect in a single genetic locus. This type of analysis revealed a crucial locus, aIr-1, which in all likelihood encodes the class II transactivator (CIITA), which explained the lack of class II transcription in BLS complementation group A (1, 118). Group A cells express the requisite X and Y binding proteins but fail to transcribe class II. CIITA expression appears to be a nearly absolute requisite for expression of class II MHC, whether constitutive or inducible (17, 19, 23, 47, 85, 103, 114, 118, 119). A number of class II MHC-related genes including genes encoding HLA-DM (H-2M in mice) and invariant chain (Ii), with promoters similar to those for classical class II genes, are also regulated by CIITA (17, 18, 22, 23, 50, 137). CIITA can also upregulate expression of class I MHC genes and beta-2-microglobulin (β2m) through effects at site α in addition to X- and Y-like sequences in the promoters for these genes (40, 72, 104). These initial observations have led to the view that CIITA is a master, or global, regulator for expression of class II MHC and related genes. FIG. 1 Organization of W, X, Y, and other motifs in the promoters of class II MHC and related genes. Genes coding for class II MHC and related proteins contain well conserved W, X, and Y boxes, the presence of which correlates with transcriptional regulation ... Since the discovery of CIITA, numerous primary articles and several reviews on its role in regulating the class II MHC have been published. In this review, we will discuss the molecular structure of this novel protein, its mechanism of function, and its biologic and clinical relevance, which is broad.

The X2 box of MHC class II promoters is homologous to TRE/CRE elements and is required for expression of MHC class II genes. The X2 box-specific DNA binding activity, X2BP, was purified to homogeneity, sequenced, and identified as CREB. Transient transactivation experiments showed that CREB can cooperate with CIITA to enhance activation of transcription from MHC class II promoters in a dose-dependent manner. Binding of CREB to the class II promoter in vivo was demonstrated by a chromatin immunoprecipitation assay. Additionally, ICER, a dominant inhibitor of CREB function, was found to repress class II expression. These results demonstrate that CREB binds to the X2 box in vivo and cooperates with CIITA to direct MHC class II expression.

Major histocompatibility class II (MHC-II) molecules are transmembrane proteins that have a central role in development and control of the immune system. They are encoded by a multigene family and their expression is tightly regulated. MHC-II deficiency (OMIM 209920) is an autosomal recessive immunodeficiency syndrome resulting from defects in trans-acting factors essential for transcription of MHC-II genes. There are four genetic complementation groups (A, B, C and D), reflecting the existence of four MHC-II regulators. The factors defective in groups A (CIITA), C (RFX5) and D (RFXAP) have been identified. CIITA is a non-DNA-binding co-activator that controls the cell-type specificity and inducibility of MHC-II expression. RFX5 and RFXAP are two subunits of RFX, a multi-protein complex that binds the X box motif of MHC-II promoters. Mutations in the genes encoding RFX5 (RFX5) or RFXAP (RFXAP) abolish binding of RFX (refs 7,8,12). Similar to groups C and D, group B is characterized by a defect in RFX binding, and although it accounts for the majority of patients, the factor defective in group B has remained unknown. We report here the isolation of RFX by a novel single-step DNA-affinity purification approach and the identification of RFXANK, the gene encoding a third subunit of RFX. RFXANK restores MHC-II expression in cell lines from patients in group B and is mutated in these patients. RFXANK contains a protein-protein interaction region consisting of three ankyrin repeats. Its interaction with RFX5 and RFXAP is essential for binding of the RFX complex to MHC-II promoters.

Two of the genes defective in the five complementation groups identified in the class II-negative bare lymphocyte syndrome or corresponding laboratory mutants have been cloned. One gene encodes a protein, RFX5, that is a member of the RFX family of DNA binding proteins. The other, CIITA, encodes a large protein with a defined acidic transcriptional activation domain; this protein does not interact with DNA. Expression plasmids encoding regions of RFX5 fused to the GAL4 DNA binding domain activated transcription from a reporter construct containing GAL4 sites in a cotransfection assay in the Raji human B cell line. However, these plasmids produced transcriptional activity in HeLa cells only in conjunction with interferon γ stimulation, a condition in which expression of both CIITA and class II major histocompatibility complex surface proteins are induced. Furthermore, these plasmids were not active in RJ2.2.5, anin vitromutagenized derivative of Raji in which both copies of CIITA are defective. Transcriptional activation by the RFX5 fusion protein could be restored in RJ2.2.5 by cotransfection with a CIITA expression plasmid. Finally, a direct interaction between RFX5 and CIITA was detected with the yeast two-hybrid and far-Western blot assays. Thus, RFX5 can activate transcription only in cooperation with CIITA. RFX5 and CIITA associate to form a complex capable of activating transcription from class II major histocompatibility complex promoters. In this complex, promoter specificity is determined by the DNA binding domain of RFX5 and the general transcription apparatus is recruited by the acidic activation domain of CIITA.

The class II transactivator (CIITA) is a master transcription regulator of gene products involved in the exogenous antigen presentation pathway, including major histocompatibility complex (MHC) class II, invariant chain, and DM. An extensive analysis of the putative functional domains of CIITA is undertaken here to explore the action of CIITA. Antibodies to CIITA protein were produced to verify that these mutant proteins are expressed. Both acidic and proline/serine/threonine-rich domains are essential for class II MHC promoter activation. In addition, three guanine nucleotide-binding motifs are essential for CIITA activity. Of these mutants, two exhibited strong transdominant-negative functions. These two mutants provide a plausible approach to manipulate MHC class II expression and immune responses.