The MHC class II transactivator (CIITA), the master regulator of MHC class II (MHC II) expression, is a co-activator that controls MHC II transcription. Human B lymphocytes express MHC II constitutively due to persistent activity of CIITA promoter III (pIII), one of the four potential promoters (pI-pIV) of this gene. Although increases in MHC II expression in B cells in response to cytokines have been observed and induction of MHC II and CIITA by IFN-gamma has been studied in a number of different cell types, the specific effects of IFN-gamma on CIITA expression in B cells have not been studied. To investigate the regulation of CIITA expression by IFN-gamma in B cells, RT-PCR, in vivo and in vitro protein/DNA binding studies, and functional promoter analyses were performed. Both MHC II and CIITA type IV-specific RNAs increased in human B lymphocytes in response to IFN-gamma treatment. CIITA promoter analysis confirmed that pIV is IFN-gamma inducible in B cells and that the GAS and IRF-E sites are necessary for full induction. DNA binding of IRF-1 and IRF-2, members of the IFN regulatory factor family, was up-regulated in B cells in response to IFN-gamma and increased the activity of CIITA pIV. In vivo genomic footprint analysis demonstrated proteins binding at the GAS, IRF-E and E box sites of CIITA pIV. Although CIITA pIII is considered to be the hematopoietic-specific promoter of CIITA, these findings demonstrate that pIV is active in B lymphocytes and potentially contributes to the expression of CIITA and MHC II in these cells.

B cell differentiation into a plasma cell requires expression of the positive regulatory domain zinc finger protein 1 gene (PRDM1) that encodes the positive regulatory domain I binding factor 1 (PRDI-BF1 or Blimp-1) protein. It represses the transcription of specific target genes, including c-myc, the MHC class II trans-activator, Pax-5, and CD23b. In this study we demonstrate the presence of an alternative protein product of the PRDM1 gene. The new protein, PRDI-BF1 beta, has a disrupted PR domain and lacks the amino-terminal 101 aa of the originally described protein. PRDI-BF1 beta has a dramatic loss of repressive function on multiple target genes, but maintains normal DNA-binding activity, nuclear localization, and association with histone deacetylases and deacetylase activity. Myeloma cell lines express the highest levels of PRDM1 beta mRNA relative to the full-length form, while primary cells and several other cell lines have very low, but detectable, levels of PRDM1 beta. RNA analysis and analysis of the PRDM1 promoters demonstrate that PRDI-BF1 beta is generated from the same gene by alternative transcription initiation using an internal promoter. These newly described features of the PRDM1 gene are highly analogous to the PRDM2 (RIZ) and PRDM3 (MDS1-EVI1) genes, in which each express a truncated protein missing the PR domain. The expression of each of the truncated proteins is elevated in cancerous cells and may play an important role in the disease.

Although activated human T cells express MHC class II antigens, the regulation of these antigens in T cells is poorly understood. This study focuses on the control of the MHC2TA gene in these cells. MHC2TA encodes the transcriptional master regulator of MHC class II, the class II trans-activator (CIITA). It has at least three distinct promoters (PI, PIII, and PIV), each active in an overlapping subset of cell types and directing a slightly different product. This report used highly purified blood T cells prepared by negative immunoselection to analyze CIITA. Real-time PCR analysis indicates that resting T cells do not express detectable CIITA transcript, while activated T cells express the PIII CIITA form. Transient transfection of activated blood T cells using wild-type and mutant PIII promoter-reporter constructs shows that two promoter elements, activation response element-1 (ARE-1) and ARE-2, are important for PIII function. cAMP response element binding protein, a known activator of gene expression in activated T cells, activates PIII in primary T cells. However, an intact ARE-2 site is not required for this activation, indicating that cAMP response element binding protein does not activate via this site. EMSAs indicate that an activating transcription factor/cAMP response element binding protein/cAMP response element modulator family member, but not phosphorylated cAMP response element binding protein-1, binds to ARE-2. ARE-2 also forms a complex with an unidentified protein. The ARE-2 binding protein is constitutively expressed in a DR(+) T cell line, reflecting differences between the DR(+) cell line and primary blood lymphocytes. These results show that MHC2TA PIII is induced in activated T lymphocytes, and that the induced binding of ARE-2 is a crucial step in this process.

Melanoma cells commonly express MHC class II molecules constitutively. This is a rare, or possibly unique, phenotype for a nonprofessional antigen-presenting cell, where MHC class II expression ordinarily occurs only after IFN-gamma treatment. Despite the fact that constitutive expression of MHC class II on melanoma cells has been observed for decades and that the regulation of the MHC class II genes is well understood for many different cell types, there is no data regarding the basis for constitutive MHC class II expression in melanoma cells. Here we report that MHC class II expression in melanoma cells can be traced to constitutive expression of the class II transactivator protein (CIITA), which mediates both IFN-gamma-inducible and -constitutive MHC class II expression in all other cell types. In addition, we determined that constitutive CIITA expression is the result of the activation of both the B cell-specific CIITA promoter III and the IFN-gamma-inducible CIITA promoter IV, the latter of which previously has never been known to function as a constitutive promoter in any cell type. The recently described B cell-related ARE-1 activity is important for promoter III activation in the melanoma cells. Constitutive promoter IV activation involves the IFN regulatory factor element (IRF-E), which binds members of the IRF family of proteins, although the major, IFN-gamma inducible member of this family, IRF-1, is not constitutively expressed in these cells. In cells with constitutively active promoter IV, the promoter IV IRF-E is most likely activated by IRF-2. The relevance of these results to the pathway of melanoma development is discussed.

The major histocompatibility complex (MHC) class II transactivator (CIITA) acts as a master switch to activate expression of the genes required for MHC-II antigen presentation. During B-cell to plasma cell differentiation, MHC-II expression is actively silenced, but the mechanism has been unknown. In plasma cell tumors such as multiple myeloma the repression of MHC-II is associated with the loss of CIITA. We have identified that positive regulatory domain I binding factor 1 (PRDI-BF1), a transcriptional repressor, inhibits CIITA expression in multiple myeloma cell lines. Repression of CIITA depends on the DNA binding activity of PRDI-BF1 and its specific binding site in the CIITA promoter. Deletion of a histone deacetylase recruitment domain in PRDI-BF1 does not inhibit repression of CIITA nor does blocking histone deacetylase activity. This is in contrast to PRDI-BF1 repression of the c-myc promoter. Repression of CIITA requires either the N-terminal acidic and conserved PR motif or the proline-rich domain. PRDI-BF1 has been shown to be a key regulator of B-cell and macrophage differentiation. These findings now indicate that PRDI-BF1 has at least two mechanisms of repression whose function is dependent on the nature of the target promoter. Importantly, PRDI-BF1 is defined as the key molecule in silencing CIITA and thus MHC-II in multiple myeloma cells.

The inadequate ability of cancer cells to present antigen on the cell surface via MHC class I molecules is one mechanism by which tumor cells evade antitumor-associated antigen immunity. In many cases, such as in renal cell carcinoma (RCC), the lack of MHC class I antigen presentation can be attributed to the down-regulation of genes needed for antigen processing, such as the transporters associated with antigen processing (TAP)1 and TAP2, and the proteasomal components low molecular weight proteins (LMP)2 and LMP7. The TAP1 and LMP2 genes are transcribed from a shared bidirectional promoter containing an IFN response factor element that confers IFN-gamma inducibility. Here, we investigate the differential responsiveness to IFN-gamma of RCC cell lines, Caki-1 and Caki-2, which have been reported to have abnormally low expressions of TAP1 and LMP2. We now demonstrate that the Caki-2 cell line is defective in the IFN-gamma signaling pathway. The effects of IFN-gamma on TAP1 and LMP2 expression revealed a loss of up-regulation in Caki-2 cells, but not in Caki-1 cells. In vivo DNA footprinting shows a specific loss of occupancy at the IFN response factor element site in Caki-2 cells, whereas Caki-1 cells show full promoter occupancy. Furthermore, in vitro DNA-binding studies indicated that Caki-2 cells do not have IFN-regulatory factor 1- or signal transducer and activator of transcription 1 (Stat1)-binding activity after IFN-gamma stimulation. Examination of Stat1, Jak1, and Jak2 proteins demonstrated that the proteins were expressed, however, not phosphorylated, upon IFN-gamma treatment in Caki-2 cells. Also, this cell line expressed both IFN-gamma receptor chains. IFN-gamma inducibility could not be rescued by introduction of normal Jak1 and/or Jak2 proteins. However, overexpression of Jak1 did increase TAP1 and LMP2 expression independent of IFN-gamma, indicating that the Stat1 and IFN-regulatory factor 1 proteins present in Caki-2 can be activated. These findings suggest that the loss of TAP1 and LMP2 induction is a defect in the earliest steps of the IFN-gamma signaling pathway resulting in the inability of Caki-2 cells to up-regulate the MHC class I antigen-processing pathway. Because immunotherapy may be one of the most promising approaches for treating RCC, understanding the mechanisms by which these tumors circumvent cytokine signaling, thereby evading antitumor-specific-antigen immunity, would greatly aid the efficacy of such therapy.

Analysis of mRNA levels in cells that express or lack signal transducers and activators of transcription 1 (Stat1) reveals that Stat1 mediates the constitutive transcription of many genes. Expression of the low molecular mass polypeptide 2 (LMP2), which requires Stat1, has been studied in detail. The overlapping interferon consensus sequence 2/gamma-interferon-activated sequence (ICS-2/GAS) elements in the LMP2 promoter bind to interferon regulatory factor 1 (IRF1) and Stat1 and are occupied constitutively in vivo. The point mutant of Stat1, Y701F, which does not form dimers involving SH2-phosphotyrosine interactions, binds to the GAS element and supports LMP2 expression. Unphosphorylated Stat1 binds to IRF1 directly and we conclude that this complex uses the ICS-2/GAS element to mediate constitutive LMP2 transcription in vivo. The promoter of the IRF1 gene, which also contains a GAS site but not an adjacent ICS-2 site, is not activated by Stat1 Y701F. The promoters of other genes whose constitutive expression requires Stat1 may also utilize complexes of unphosphorylated Stat1 with IRF1 or other transcription factors.

Major histocompatibility complex (MHC) class II molecules play a central role in immune responses, and transcription of this family of genes requires the MHC class II transactivator (CIITA). CIITA has four promoters, which are transcribed in a tissue-specific manner. CIITA promoter III is constitutively active in mature B-lymphocytes. This report now describes the minimal 319-base pair promoter region necessary for maximal transcriptional activity in B-lymphocytes. Ultraviolet light and dimethylsulfate in vivo genomic footprinting analyses reveal five occupied DNA sequence elements present in intact B-lymphocytes. Functional analysis of these elements using promoter deletions and site-specific mutations demonstrates that at least two of the sites occupied in vivo are critical for transcriptional activity. In vitro protein/DNA analysis suggests that one of the sites is a TEF-2-like element and the other is occupied by a novel transcription activator. In addition, nuclear factor-1 associates with the promoter both in vivo and in vitro. In myeloma cell lines, loss of CIITA transcription correlates with a completely unoccupied CIITA promoter III. These findings suggest that CIITA transcription in B-lymphocytes is activated through at least two strong promoter elements, while loss of expression in myeloma cells is mediated through changes in promoter assembly.

MHC class II deficiency found in bare lymphocyte syndrome patients results from the absence or dysfunction of MHC class II transcriptional regulators, such as regulatory factor X (RFX) and class II transactivator (CIITA). Understanding the roles of these factors has been greatly facilitated by the study of genetic defects in cell lines of bare lymphocyte syndrome patients, as well as in cell lines that have been generated by chemical mutagenesis in vitro. The latter group includes MHC class II-deficient lines that are no longer responsive to induction by IFN-gamma. Here, we show that the defect in G1B, one such cell line, is attributed to the lack of functional RFX5, the largest subunit of RFX. The RFX5 gene isolated from G1B cells contains two separate single-base pair mutations. One alteration does not exhibit a phenotype, whereas a leucine-to-histidine mutation eliminates DNA-binding and transactivating functions. This mutation lies outside of previously defined functional domains of RFX5 but within an unusual, leucine-rich region (62-LYLYLQL-68). To further investigate the significance of the leucine-rich region, we targeted all neighboring leucine residues for mutagenesis. These mutants were also unable to transactivate a MHC class II reporter gene, confirming that these leucine residues play an essential role in RFX activity and characterize a novel leucine-rich motif.

Resistance to the growth inhibitory effects of transforming growth factor β (TGFβ) has been associated with decreased levels of the TGFβ type II receptor (TβR-II) and has been correlated with tumorigenicity. Previously, we reported an A → G mutation at position −364 in the TβR-II promoter in A431 tumor cells which results in reduced TβR-II promoter activity. In this study, we show that the CDP/Cut (CCAAT displacement protein) transcription factor, a transcriptional repressor, binds both the wild type and the mutant TβR-II promoter. We also demonstrate that the A → G mutation increases CDP/Cut binding affinity, and that overexpression of CDP/Cut reduces transcription from TβR-II promoter reporter constructs. Increased binding of the CDP/Cut repressor protein, as a result of a mutation at position −364, represents a novel mechanism of regulation in a neoplastic cell of the promoter of a tumor suppressor gene, TβR-II.

CIITA is a master transactivator of the major histocompatibility complex class II genes, which are involved in antigen presentation. Defects in CIITA result in fatal immunodeficiencies. CIITA activation is also the control point for the induction of major histocompatibility complex class II and associated genes by interferon-gamma, but CIITA does not bind directly to DNA. Expression of CIITA in G3A cells, which lack endogenous CIITA, followed by in vivo genomic footprinting, now reveals that CIITA is required for the assembly of transcription factor complexes on the promoters of this gene family, including DRA, Ii, and DMB. CIITA-dependent promoter assembly occurs in interferon-gamma-inducible cell types, but not in B lymphocytes. Dissection of the CIITA protein indicates that transactivation and promoter loading are inseparable and reveal a requirement for a GTP binding motif. These findings suggest that CIITA may be a new class of transactivator.

The events that lead to promoter accessibility within chromatin are not completely understood. The invariant chain (Ii) promoter was used as a model to determine the contribution of different DNA-binding factors in establishing occupancy of a complex promoter. Gamma interferon induction of the Ii promoter requires the cooperation of multiple cis elements including distal S, X, and Y/CCAAT elements along with proximal GC and Y/CCAAT elements. The heteromeric transcription factor NF-Y binds to both Y/CCAAT elements, Genomic footprinting was used to analyze in vivo protein-DNA contacts for integrated Ii promoters bearing mutations in each element. The results reveal a hierarchy of transcription factor loading with NF-Y binding to the distal Y/CCAAT element being required for establishing protein-DNA interactions over the entire 250 bp analyzed, Mutation of the X box disrupts binding primarily at the adjacent Y/CCAAT element along with a lesser effect on GC box binding. Importantly, this finding is verified with a cell line which lacks a functional X-box-binding factor, RFX, providing physiological validity for the strategy described here. Mutation of both the S element and the GC box results in either no or little effect on transcription factor binding. However, mutation of the proximal Y/CCAAT element disrupts binding to the adjacent GC box and partially reduces binding in the distal S/X/Y domain. The crucial role for NF-Y in establishing promoter occupancy may be related to its histone fold motif, the essential component for assembling nucleosome-like structures.