Janet F. Piskurich

BLIMP-1 mediates extinction of major histocompatibility class II transactivator expression in plasma cells

Class II transactivator (CIITA), a coactivator required for class II major histocompatibility complex (MHC) transcription, is expressed in B cells but extinguished in plasma cells. This report identifies B lymphocyte–induced maturation protein 1 (BLIMP-1), a transcriptional repressor that is capable of triggering plasma cell differentiation, as a developmentally regulated repressor of CIITA transcription. BLIMP-1 represses the B cell–specific promoter of the human gene that encodes CIITA (MHC2TA) in a binding site–dependent manner. Decreased CIITA correlates with increased BLIMP-1 during plasma cell differentiation in cultured cells. Ectopic expression of BLIMP-1 represses endogenous mRNA for CIITA and the CIITA targets, class II MHC, invariant chain and H2-DM (the murine equivalent of HLA-DM) in primary splenic B cells as well as 18-81 pre-B cells. Thus, the BLIMP-1 program of B cell differentiation includes loss of antigen presentation via extinction of CIITA expression.

Two Distinct Gamma Interferon-Inducible Promoters of the Major Histocompatibility Complex Class II Transactivator Gene Are Differentially Regulated by STAT1, Interferon Regulatory Factor 1, and Transforming Growth Factor β

ABSTRACT The major histocompatibility complex (MHC) class II transactivator (CIITA) is the master regulatory factor required for appropriate expression of class II MHC genes. Understanding the expression of CIITA is key to understanding the regulation of class II MHC genes. This report describes the independent regulation of two distinct CIITA promoters by cytokines with opposing functions, gamma interferon (IFN-γ) and transforming growth factor β (TGF-β). A functional analysis of deletion mutations of the upstream promoter (promoter III) identified an IFN-γ-responsive region located approximately 5 kb from the transcriptional start site. An in vivo DNase I hypersensitivity analysis detected a hypersensitive site in this area which supports the relevance of this region. When the downstream promoter (promoter IV) was studied by in vivo genomic footprinting, IFN-γ-induced changes at putative binding sites for STAT1, interferon regulatory factor 1 (IRF-1), and E-box proteins were seen. Gel shift and supershift analyses for IRF-1 confirmed the in vivo footprint results. The role of the IFN-γ-inducible transcription factor STAT1 was examined functionally. Although both promoters were controlled by STAT1, promoter-specific regulation was exhibited. The IFN-γ response of promoter III was completely dependent on STAT1 and not IRF-1, while promoter IV was partially activated by IRF-1 in the total absence of STAT1 expression. While both promoters were affected by TGF-β, activation of promoter III by IFN-γ was more severely diminished by TGF-β treatment. The differential control of CIITA promoters by TGF-β, IRF-1, and STAT1 may be important in refining regulation of class II MHC genes in different cell types and under different stimulatory conditions.

The role of the MHC class II transactivator in class II expression and antigen presentation by astrocytes and in susceptibility to central nervous system autoimmune disease

The role of the MHC class II transactivator (CIITA) in Ag presentation by astrocytes and susceptibility to experimental autoimmune encephalomyelitis (EAE) was examined using CIITA-deficient mice and newly created transgenic mice that used the glial fibrillary acidic protein promoter to target CIITA expression in astrocytes. CIITA was required for class II expression on astrocytes. Like class II-deficient mice, CIITA-deficient mice were resistant to EAE by immunization with CNS autoantigen, although T cells from immunized CIITA-deficient, but not class II-deficient, mice proliferated and secreted Th1 cytokines. CIITA-deficient splenic APC presented encephalitogenic peptide to purified wild-type encephalitogenic CD4+ T cells, indicating that CIITA-independent mechanisms can be used for class II-restricted Ag presentation in lymphoid tissue. CIITA-deficient mice were also resistant to EAE by adoptive transfer of encephalitogenic class II-restricted CD4+ Th1 cells, indicating that CIITA-dependent class II expression was required for CNS Ag presentation. Despite constitutive CIITA-driven class II expression on astrocytes in vivo, glial fibrillary acidic protein-CIITA transgenic mice were no more susceptible to EAE than controls. CIITA-transfected astrocytes presented peptide Ag, but in contrast to IFN-γ-activated astrocytes, they could not process and present native Ag. CIITA-transfected astrocytes did not express cathepsin S without IFN-γ activation, indicating that CIITA does not regulate other elements that may be required for Ag processing by astrocytes. Although our results demonstrate that CIITA-directed class II expression is required for EAE induction, CIITA-directed class II expression by astrocytes does not appear to increase EAE susceptibility. These results do not support the role of astrocytes as APC for class II-restricted Ag presentation during the induction phase of EAE.

Interferon-γ induces polymeric immunoglobulin receptor mrna in human intestinal epithelial cells by a protein synthesis dependent mechanism

Transport of secretory IgA into external fluids is mediated by the polymeric immunoglobulin receptor (pIgR) on the surface of mucosal epithelial cells. We studied the mechanism by which interferon-gamma (IFN-gamma) induces pIgR expression in HT-29.74 cells, a subclone of the HT-29 cell line selected for high concns of pIgR. Here we report the isolation of genomic DNA and cDNA clones encoding human pIgR and development of a sensitive ribonuclease protection assay for pIgR mRNA. This assay was used to determine if induction of pIgR by IFN-gamma is mediated by accumulation of pIgR mRNA. After an initial lag of 12 hr, pIgR mRNA increased seven-fold in response to IFN-gamma, reaching a plateau at 24 hr. Concentrations of pIgR protein also increased seven-fold, but the increase was delayed until 48 hr following stimulation with IFN-gamma. Cycloheximide treatment abolished the IFN-gamma induced increase in pIgR mRNA, indicating that induction of pIgR mRNA by IFN-gamma requires de novo protein synthesis. These results suggest that induction of pIgR expression by IFN-gamma involves an increase in steady-state concns of pIgR mRNA via a protein synthesis dependent mechanism.

Regulation and Specificity of MHC2TA Promoter Usage in Human Primary T Lymphocytes and Cell Line

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.

Differential selectivity of CIITA promoter activation by IFN-γ and IRF-1 in astrocytes and macrophages: CIITA promoter activation is not affected by TNF-α

Abstract During demyelinating disease of the central nervous system (CNS), locally elevated cytokine levels may induce upregulation of MHC class II molecules on otherwise low expressing or negative cell types such as microglia and astrocytes, since IFN-γ has been shown to induce MHC class II expression on these cell types in vitro. While many transcription factors are involved with MHC class II expression, only the class II transactivator (CIITA) is tightly coordinated with IFN-γ-inducibility. Control of CIITA gene expression is complex, involving four distinct promoters, two of which (promoters III and IV) are IFN-γ-inducible in certain cell types. Here we demonstrate that IFN-γ treatment of rat astrocytes induces only CIITA promoter IV activity in contrast to the murine macrophage cell line RAW 264.7 that uses both IFN-γ-inducible promoters. In contrast to previously published reports, promoter IV activation is completely dependent upon an intact interferon regulatory factor-1 (IRF-1) but not STAT1 binding site using promoter constructs specifically mutated at these positions. Importantly, while TNF-α is able to synergize with IFN-γ to increase astrocyte MHC class II expression in vitro, we show that treatment of rat astrocytes with TNF-α has no effect on CIITA promoter activity. These data demonstrate that TNF-α augments MHC class II expression through a mechanism downstream or independent of CIITA induction.

A Novel Element and a TEF-2-like Element Activate the Major Histocompatibility Complex Class II Transactivator in B-lymphocytes

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.

Adult-derived stem cells and their potential for use in tissue repair and molecular medicine.

This report reviews three categories of precursor cells present within adults. The first category of precursor cell, the epiblast‐like stem cell, has the potential of forming cells from all three embryonic germ layer lineages, e.g., ectoderm, mesoderm, and endoderm. The second category of precursor cell, the germ layer lineage stem cell, consists of three separate cells. Each of the three cells is committed to form cells limited to a specific embryonic germ layer lineage. Thus the second category consists of germ layer lineage ectodermal stem cells, germ layer lineage mesodermal stem cells, and germ layer lineage endodermal stem cells. The third category of precursor cells, progenitor cells, contains a multitude of cells. These cells are committed to form specific cell and tissue types and are the immediate precursors to the differentiated cells and tissues of the adult. The three categories of precursor cells can be readily isolated from adult tissues. They can be distinguished from each other based on their size, growth in cell culture, expressed genes, cell surface markers, and potential for differentiation. This report also discusses new findings. These findings include the karyotypic analysis of germ layer lineage stem cells; the appearance of dopaminergic neurons after implantation of naive adult pluripotent stem cells into a 6‐hydroxydopamine‐lesioned Parkinsons model; and the use of adult stem cells as transport mechanisms for exogenous genetic material. We conclude by discussing the potential roles of adult‐derived precursor cells as building blocks for tissue repair and as delivery vehicles for molecular medicine.

Constitutive Expression of MHC Class II Genes in Melanoma Cell Lines Results from the Transcription of Class II Transactivator Abnormally Initiated from Its B Cell-Specific Promoter

In melanoma cell lines, two different patterns of MHC class II expression have been described, either an IFN γ-inducible expression of HLA-DR and HLA-DP, with a faint or null expression of HLA-DQ, resembling that described for melanocytes, or a constitutive expression, i.e., IFN-γ independent, of all three HLA-D isotypes. As this latter phenotype has been associated with a more rapid progression of melanoma tumors, we have analyzed in different melanoma cell lines the molecular mechanisms leading to this abnormal pattern of MHC class II expression. In agreement with the evidence of a coordinate transcription of the HLA-D genes in these cell lines, we have shown the constitutive expression of CIITA (class II transactivator) transcripts, CIITA being known as the master switch of MHC class II expression. Unexpectedly, these transcripts initiate from promoter III of the CIITA gene, a promoter that is mainly used constitutively in B lymphocytes. This expression was further shown to occur through factor(s) acting on the enhancer located upstream of CIITA promoter III, which was previously described in epithelioid cells as an IFN-γ-response sequence. The hypothesis of a general abnormality of the IFN-γ transduction pathway was dismissed. Constitutive transcription of CIITA from promoter III having been observed in unrelated melanoma cell lines, we propose the hypothesis that this phenomenon might not be a random event, but could be linked to the neoplasic state of the melanoma cells.

Class II transactivator (CIITA) promoter methylation does not correlate with silencing of CIITA transcription in trophoblasts

Abstract Trophoblast cells are unique because they do not express major histocompatibility complex (MHC) class II antigens, either constitutively or after exposure to interferon-γ (IFN-γ). The absence of MHC class II antigens on trophoblasts is thought to play a critical role in preventing rejection of the fetus by the maternal immune system. The inability of trophoblasts to express MHC class II genes is primarily due to lack of the class II transactivator (CIITA), a transacting factor that is required for constitutive and IFN-γ-inducible MHC class II transcription. We, therefore, investigated the silencing of CIITA expression in trophoblasts. In transient transfection assays, transcription from the IFN-γ-responsive CIITA type IV promoter was upregulated by IFN-γ in trophoblasts, which suggests that CIITA is silenced by an epigenetic mechanism in these cells. Polymerase chain reaction analysis demonstrated that the CIITA type IV promoter is methylated in both the human choriocarcinoma cell lines JEG-3 and Jar and in 2fTGH fibrosarcoma cells, which are IFN-γ inducible for CIITA. Conversely, methylation of the CIITA type IV promoter was not observed in human primary cytotrophoblasts isolated from term placentae or in mouse or rat trophoblast cell lines. Simultaneous treatment with IFN-γ and the histone deacetylase inhibitor trichostatin A weakly activated CIITA transcription in mouse trophoblasts. Stable hybrids between human choriocarcinoma and fibrosarcoma cells and between mouse trophoblasts and fibroblasts expressed CIITA following treatment with IFN-γ. These results suggest that silencing of CIITA transcription is recessive in trophoblasts and involves an epigenetic mechanism other than promoter methylation. The fact that CIITA is expressed in the stable hybrids implies that trophoblasts may be missing a factor that regulates chromatin structure at the CIITA promoter.