Transforming growth factor beta 1 repression of the HLA-DR alpha gene is mediated by conserved proximal promoter elements.

Transforming growth factor beta 1 (TGF-beta 1) is a pleiotropic cytokine that decreases the expression of class II MHC Ag in the melanoma cell line Hs294T(c). To investigate the mechanism of this repression, we have examined the effect of TGF-beta 1 on expression of the HLA-DR alpha gene. Both the constitutive level of HLA-DR protein and DR alpha mRNA were repressed by treatment with TGF-beta 1. The proximal 176 bp of the DR alpha promoter were sufficient to confer TGF-beta 1 repression on a reporter gene. Deletional and mutational analysis of the DR alpha promoter revealed that the conserved S and X1 promoter elements were important for basal expression of DR alpha and also mediated the down-regulation by TGF-beta 1. Mobility shift assays and in vivo footprinting showed no change in occupancy of the proximal DR alpha promoter after TGF-beta 1 treatment. These results identify the DNA elements that mediate repression of the HLA-DR alpha gene by TGF-beta 1 and suggest that TGF-beta 1 acts at these sites without causing a change in promoter occupancy.

Developmental and cytokine-mediated regulation of MHC class II gene promoter occupancy in vivo.

The class II genes of the major histocompatibility complex are a family of genes whose expression is regulated developmentally in cells of the B lineage and by IFN-gamma in many other cell types. Using the approach of in vivo footprinting, which allows for the examination of protein-promoter interactions within intact cells, we demonstrated a transition from unoccupied to occupied to once again unoccupied class II promoters in cell lines representing the developmental pathway of B cells. IFN-gamma treatment of HeLa cells led to increased promoter occupancy of the DR alpha and DR beta promoters at the same sites that are constitutively bound in mature B cells. No IFN-gamma-specific binding site was induced. Additionally, an octamer element in the DR alpha gene displayed preferential binding in B cells. These results demonstrate that changes in the transcription of the class II genes are associated with changes in factor binding at the promoter in vivo. Moreover, given the ubiquity of class II promoter binding proteins, these results suggest that throughout B cell development and upon IFN-gamma stimulation, the accessibility of class II promoter DNA is subject to regulation.

Promoter accessibility within the environment of the MHC is affected in class II-deficient combined immunodeficiency.

Class II-deficient combined immunodeficiency (CID) is a hereditary disease resulting in abrogation of transcription of the class II genes of the major histocompatibility complex, due to a defect in a trans-acting regulatory factor. Cell lines from certain CID patients lack factor binding at multiple sites in class II promoters in vivo. A mutation in one of the promoter binding proteins could explain this 'bare' phenotype only if these factors bind cooperatively or in a temporal hierarchy. Alternatively, the mutation could affect the configuration of the promoter within the MHC locus. Here, we provide evidence that the factor(s) defective in class II-deficient CID controls the accessibility of class II promoters within the environment of the MHC. The in vivo occupancy of wild type and mutated class II promoter constructs was examined in stable transfectants of normal and CID-derived cell lines. The CID promoter phenotype could not be reproduced in a normal cell line by eliminating binding at any one promoter element, suggesting that these factors bind independently, both spatially and temporally. In contrast, promoter occupancy was partially restored in two CID lines at a randomly integrated wild type promoter, implying that the promoter is inaccessible to factors in its native environment, but accessible when moved to another location in the genome.

Sequences and factors: a guide to MHC class-II transcription.

The expression of the class-II antigens of the major histocompatibility complex is tissue-specific, developmentally regulated in cells of the B lineage, and inducible by cytokine. Some of the molecular mechanisms that account for this regulation have recently been elucidated. DNA sequences upstream of human and mouse class-II genes that are critical for the transcription of these genes have been identified through transfection studies and transgenic analysis. Furthermore, a number of transcription factors that bind to important promoter motifs have recently been isolated. Examination of the class-II promoter in vivo both in the normal state and in patients with bare lymphocyte syndrome has further increased our understanding of class-II regulation.

Upstream elements of the MHC class II E beta gene active in B cells.

The MHC class II (Ia) molecules are heterodimeric cell surface glycoproteins that have a limited tissue distribution and are essential for an Ir. These proteins are known to both display a limited tissue distribution and be responsive to up-regulation by cytokines such as IL-4. To begin to identify the molecular basis for both of these phenomena in B cells, we performed a deletional analysis of the upstream region of the murine E beta gene. We show that there are several cooperating positive as well as negative control elements in the E beta upstream region. The sequence between -2679 and -66 is a strong transcriptional element containing several sequences that act synergistically as positive elements. The negative elements are located more proximal to the transcription start site. Furthermore, two consensus motifs, termed X and Y, which are located in the proximal upstream region of all class II genes, are necessary for the transcriptional activity of more distal elements. Additionally, we identify an IL-4-responsive element located within 666 bp upstream of the transcription initiation site.

In vivo footprinting of MHC class II genes: bare promoters in the bare lymphocyte syndrome.

Major histocompatibility complex (MHC) class II genes are coordinately regulated and show tissue-specific expression. With the use of in vivo footprinting, common promoter sites in these genes were found to be occupied only in cells that expressed the genes, in spite of the presence of the promoter binding proteins. In vivo analysis of mutant cell lines that exhibited coordinate loss of class II MHC expression, including several from individuals with bare lymphocyte syndrome, revealed two in vivo phenotypes. One suggests a defect in gene activation, whereas the other suggests a defect in promoter accessibility.

Regulation of MHC class II gene transcription.

The transcriptional regulation of MHC class II genes involves the interaction of DNA-binding proteins with specific DNA sequences. The cis elements of the promoter region have now been well delineated functionally, while the proteins that interact with these elements are just beginning to be identified and their functional importance assessed.

The presence of a DNA binding complex correlates with E beta class II MHC gene expression.

The class II major histocompatibility complex (MHC, Ia) antigens are a family of membrane proteins whose expression is strictly regulated. They have a limited tissue distribution and their expression is regulated both developmentally and in response to external stimuli. Here we report the identification of a DNA binding protein complex (termed complex A) within the murine E beta MHC gene, which is restricted to cells that express Ia antigens. Complex A binding activity is developmentally regulated in cells of the B lineage in accordance with class II expression and is responsive to two different Ia-inducing lymphokines, interferon-gamma in macrophages and interleukin-4 in pre-B cells. The DNA target sequence in complex A includes three previously defined transcriptional motifs W, X and Y, and acts as a cis-acting transcription element. Complex A is present both in cells that are constitutive for class II MHC expression and in cells that have been induced for class II MHC expression. These results suggest that complex A may play a critical role in the regulation of class II MHC gene expression.

A cDNA for a human cyclic AMP response element-binding protein which is distinct from CREB and expressed preferentially in brain.

The cyclic AMP response element (CRE) is found in many cellular genes regulated by cyclic AMP, and similar elements are present in the early genes of adenovirus that are activated by E1A. The transcription factor CREB has previously been shown to bind this site, and cDNAs for CREB have recently been characterized. We report here the isolation of a cDNA encoding a human DNA-binding protein that also recognizes this motif in cellular and viral promoters. This protein, HB16, displays structural similarity to CREB and to c-Jun and c-Fos, which bind the related 12-O-tetradecanoylphorbol-13-acetate response element (TRE). HB16 contains a highly basic, putative DNA-binding domain and a leucine zipper structure thought to be involved in dimerization. Deletional analysis of HB16 demonstrated that the leucine zipper is required for its interaction with DNA. In addition, HB16 could form a complex with c-Jun but not with c-Fos. Despite its structural similarity to c-Jun and c-Fos and its interaction with c-Jun, HB16 had approximately a 10-fold-lower affinity for the TRE sequence than for the CRE sequence. Although HB16 and CREB both recognized the CRE motif, an extensive binding analysis of HB16 revealed differences in the fine specificity of binding of the two proteins. HB16 mRNA was found at various levels in many human tissues but was most abundant in brain, where its expression was widespread. The existence of more than one CRE-binding protein suggests that the CRE motif could serve multiple regulatory functions.

mXBP/CRE-BP2 and c-Jun form a complex which binds to the cyclic AMP, but not to the 12-O-tetradecanoylphorbol-13-acetate, response element.

Proto-oncogene products c-Fos and c-Jun form a complex which binds with high affinity to the 12-O-tetradecanoylphorbol-13-acetate (TPA) response DNA element and which stimulates transcription of phorbol ester- inducible genes. We have previously identified, by screening a lambda gt11 expression library, murine protein mXBP, which binds to a sequence which overlaps the 3' end of the murine class II major histocompatibility complex A alpha gene X box, a conserved transcription element found upstream of all class II genes. Here, we demonstrate that the target sequence for mXBP is a consensus cyclic AMP response element (CRE). mXBP is a member of the leucine zipper family of DNA-binding proteins and has significant homology to oncoproteins c-Fos and c-Jun. The inferred amino acid sequence of mXBP shows near identity to human CRE-BP1, except it does not contain an internal proline-rich domain. Immunoprecipitation and glutaraldehyde cross-linking studies show that mXBP/CRE-BP2 can form a complex with c-Jun. Complex formation is dependent on intact leucine zipper domains in both proteins. mXBP-c-Jun complexes can coexist with c-Fos-c-Jun complexes and can bind with high affinity to CRE, but not to TPA response DNA element, sequences. These results suggest that changes in the expression of mXBP/CRE-BP2, c-Fos, and c-Jun, which alter the ratio of mXBP-c-Jun to c-Fos-c-Jun complexes, would affect the relative expression of cyclic AMP and phorbol ester-responsive genes. This provides support for a combinatorial model of gene regulation, whereby protein-protein interactions which alter the DNA binding specificity of protein complexes can expand the flexibility of cellular transcriptional responses.

Interferon gamma regulates binding of two nuclear protein complexes in a macrophage cell line.

Interferon gamma (IFN-gamma) is a potent inducer of major histocompatibility complex (MHC) antigens during normal immune responses and in abnormal responses in autoimmune disease. In this report we identify two nuclear factors whose binding to the murine E beta class II MHC beta-chain gene is regulated by this cytokine. IFN-gamma stimulation of murine macrophages results in the appearance of increased binding of one protein complex, complex A, and decreased binding of a second, faster migrating protein complex, complex B. Although the contact residues for both of these proteins lie within the highly conserved Y-box transcriptional element, their binding specificity differs. The protein in complex B is a CCAAT-box-binding protein that may be similar or identical to NF-Y or YB1, previously identified class II Y-box-binding proteins. The DNA sequence requirements for the binding of the slower migrating complex, complex A, are not limited to CCAAT-box sequences but include sequences upstream of the Y box. These upstream sequences are required both for IFN-gamma-induced gene transcription and for IFN-gamma-induced modulation of binding activity. These data suggest a model in which upstream sequences contribute to formation of a lymphokine-regulated complex downstream. The IFN-gamma-induced binding protein described as complex A in this report differs from the IFN-gamma, -alpha, or -beta-induced nuclear factors previously identified.

Functional analysis of a cytoplasmic domain-deleted mutant of the CD4 molecule.

The CD4 molecule is a receptor found on a subset of T lymphocytes. It has been proposed that, upon binding MHC class II molecules expressed on APC, the CD4 molecule enhances the responsiveness of the T cell by increasing intercellular avidity and/or by transducing an intracellular signal. We have analyzed the effect of removing the cytoplasmic domain of the CD4 molecule on the ability of the CD4 molecule to enhance T cell responsiveness. The cytoplasmic domain-deleted mutant of the CD4 molecule (CD4 delta) was found to be as efficient as the CD4 molecule at enhancing responsiveness to cells bearing the appropriate Ag. If subcellular Ag in the form of purified Ag incorporated into liposomes was used, the CD4 molecule was found to be much more efficient than the CD4 delta molecule at enhancing responsiveness. However, the defect in the ability of the CD4 delta molecule to enhance responsiveness could be compensated for by increasing the level of expression of the CD4 delta molecule.