Biomedical subjects

J M Boss

Publications and source records attributed to J M Boss.

At least 19 recordsLinked to original sources

PubMed1995

Interferon (IFN) beta acts downstream of IFN-gamma-induced class II transactivator messenger RNA accumulation to block major histocompatibility complex class II gene expression and requires the 48-kD DNA-binding protein, ISGF3-gamma.

Interferon (IFN) gamma, a cardinal proinflammatory cytokine, induces expression of the gene products of the class II locus of the major histocompatibility complex (MHC), whereas IFN-alpha or -beta suppresses MHC class II expression. The mechanism of IFN-beta-mediated MHC class II inhibition has been unclear. Recently, a novel factor termed class II transactivator (CIITA) has been identified as essential for IFN-gamma-induced MHC class II transcription. We studied the status of IFN-gamma-induced CIITA messenger RNA (mRNA) accumulation and CIITA-driven transactivation in IFN-beta-treated cells and used cell lines that had defined defects in the type I IFN response pathway to address the roles of IFN signaling components in the inhibition of MHC class II induction. IFN-beta treatment did not suppress IFN-gamma-induced accumulation of CIITA mRNA. After cells were stably transfected with CIITA, endogenous MHC class II genes were constitutively expressed, and MHC class II promoters, delivered by transfection, were actively transcribed in CIITA-expressing cells. Expression of these promoters was significantly impaired by pretreatment with IFN-beta. These results suggest that IFN-beta acts downstream of CIITA mRNA accumulation, and acts in part by reducing the functional competence of CIITA for transactivating MHC class II promoters. IFN stimulated gene factor 3 (ISGF3) gamma was essential for IFN-beta to mediate inhibition of MHC class II induction, regardless of whether MHC class II transcription was stimulated by IFN-gamma or directly by CIITA expression. Results of these experiments suggest that inhibition of MHC class II in IFN-beta-treated cells requires expression of gene(s) directed by the ISGF3-IFN-stimulated response element pathway, and that these gene product(s) may act by blocking CIITA-driven transcription of MHC class II promoters.

DNA-Binding Proteins↗

PubMed1995

Purified X2 binding protein (X2BP) cooperatively binds the class II MHC X box region in the presence of purified RFX, the X box factor deficient in the bare lymphocyte syndrome.

The conserved X2 box sequence of MHC class II promoters is homologous to TRE/CRE elements, and is required for B cell expression and IFN-gamma induction of MHC class II genes. The X2 binding protein (X2BP) was initially identified as a DNA-binding activity that specifically interacts with the conserved X2 box sequence in both the MHC HLA-DRA and HLA-DRB promoters. To begin to demonstrate that X2BP is the X2 box factor responsible for class II expression in B cells, we have purified X2BP to homogeneity from B cell nuclear extracts using DNA-affinity chromatography. X-box DNA-affinity purification indicates that X2BP is most likely composed of two polypeptides of 120 kDa and 46 kDa. The 120-kDa protein was specifically cross-linked to an X-box probe by exposure to UV irradiation. The 46-kDa subunit of X2BP cross-reacted with anti-rat CREB polyclonal Abs but not to anti-human CREB Abs in Western analysis and supershift assays, indicating that it may be a novel member of the ATF/CREB family. Purified X2BP interacted with purified RFX, a factor that binds to the adjacent X1 box and is absent in some cell lines that are mutant for MHC class II transcription. This interaction increases the DNA-binding half-life of RFX from 5 to at least 60 min, suggesting that X2BP functions in class II MHC gene expression by forming a stable complex with RFX.

Base Sequence↗

PubMed1995

Activation of class II MHC genes requires both the X box region and the class II transactivator (CIITA).

CIITA, a gene that can complement a transcriptional mutation of the major histocompatibility complex (MHC) class II genes, was tested for its ability to function as a coactivator, CIITA cDNA clones isolated showed alternative RNA splicing, but only one splice site combination was able to restore class II MHC gene expression. DNA-mediated transfection experiments showed that CIITA directs its activity through the X box element; the presence of CIITA leads to the formation of a higher order complex at the X box region; and CIITA contains a potent activation domain. These findings support the hypothesis that CIITA directly interacts with the MHC class II-specific transcription factors and is required for expression.

Alternative Splicing↗

PubMed1994

Molecular analysis of G1B and G3A IFN gamma mutants reveals that defects in CIITA or RFX result in defective class II MHC and Ii gene induction.

Class II major histocompatibility complex (MHC) genes and the invariant (Ii) gene are inducible by interferon-gamma (IFN gamma) but not by interferon-alpha and interferon-beta. The promoter regions of these genes contain three regulatory elements that mediate constitutive and IFN gamma-induced expressions; however, none of the DNA-binding proteins that interact with these elements are regulated by IFN gamma. Recently, a gene coding for a transactivator (CIITA) of class II MHC genes that complements a HLA-DR-negative immunodeficiency has been isolated. Using one IFN gamma mutant cell line (G3A) that is selectively defective in HLA-DR and Ii induction, four lines of evidence are presented to show that CIITA mediates the IFN gamma induction of HLA-DR and Ii genes. Analysis of another mutant line, G1B, indicates that the lack of DRA and Ii gene induction by IFN gamma is correlated with the lack of RFX DNA binding activity, thus providing the link between RFX and an IFN gamma response.

Antigens, Differentiation, B-Lymphocyte↗

PubMed1992

Tumor necrosis factor induces genes involved in inflammation, cellular and tissue repair, and metabolism in murine fibroblasts.

TNF-alpha is a primary mediator of the inflammatory response and has been ascribed a wide range of biologic activities including the cytolysis of some but not all transformed cell lines in vitro. Since most cells, normal and transformed, will also be lysed if they are concurrently treated with inhibitors of transcription or translation, the ability to resist TNF lysis depends on the de novo induction of specific gene products. To identify genes that might be involved in the ability to resist TNF-mediated cytolysis, cDNA libraries enriched for TNF-induced genes were constructed and screened. Twenty-one genes that are induced by TNF were isolated. Ten of the cDNA clones were identified by DNA sequence analysis and function in inflammatory responses, tissue or cellular repair, or cellular metabolism. Seven genes were not identified and are novel. The remaining four genes are encoded in the mitochondrial genome. Interestingly, not all the mitochondrial transcripts are induced. This may reflect a fine tuning of certain components of mitochondria that may be necessary for survival after TNF treatment.

Animals↗

PubMed1992

Single base pair substitutions within the HLA-DRA gene promoter separate the functions of the X1 and X2 boxes.

The class II MHC genes are expressed on the surfaces of B cells, activated T cells, and macrophages and may be induced in other cell types by IFN-gamma. The control of class II gene expression has been shown to be mediated by a series of conserved cis-acting sequences (W, X1, X2, and Y boxes) located immediately 5' to the genes. Although these sequences are conserved, the bp that are important for transcriptional regulation have yet to be identified. To address this issue with regard to the MHC gene HLA-DRA, a series of single bp substitutions spanning the conserved upstream sequences was created and analyzed for their effects on transcription in both B cells and IFN-gamma-treated fibroblasts. In addition, the effects of X1 and X2 box mutations on DNA/protein interactions were examined and compared to the transcriptional data. The results of these studies show that each of the conserved elements participate in maximal expression in B cells and that W, X1, and X2 boxes are important for IFN-gamma induction and expression in fibroblasts. Interestingly, some of the bp changes that altered B cell expression did not alter expression and IFN-gamma induction in fibroblasts, suggesting that different or altered factors control the expression of these genes in the different cell types. Mutant templates designed to eliminate the binding of X1- and X2-specific DNA binding proteins in vivo suggest that these elements and their factors may interact to promote transcription.

B-Lymphocytes↗

PubMed1991

Two B cell factors bind the HLA-DRA X box region and recognize different subsets of HLA class II promoters.

The class II genes of the human Major Histocompatibility Complex (MHC) encode three isotypes of alpha/beta heterodimeric proteins, HLA-DR, -DQ, and -DP, which are responsible for presenting processed antigens to T helper lymphocytes. These MHC class II genes are expressed in a coordinate manner. The promoter regions of all MHC class II genes share a set of highly conserved elements that mediate different levels of tissue-specific and inducible transcription. One element, the X box, appears to be the major positive element in B cell-specific expression, and nuclear protein binding studies have subdivided this region into the X1 and X2 boxes. Regulatory Factor X (RFX) binds to the X1 box whereas several other factors have been described that bind to the X2 box. In this report, we further characterize the X1 binding protein RFX and show that RFX binds poorly to beta chain gene promoters. In particular, RFX does not bind to the DRB gene, which is expressed at the highest levels of all beta chain genes. In addition, we have identified an X2 box binding activity in human B cell extracts that binds with high affinity to the HLA-DRA promoter. This X2 binding protein, X2BP, binds to a different subset of class II promoters than does RFX. These findings suggest that coordinate regulation of class II expression may involve different combinations or arrangements of transcriptional elements and factors instead of a common set.

Autoradiography↗

PubMed1991

DNA binding properties of YB-1 and dbpA: binding to double-stranded, single-stranded, and abasic site containing DNAs.

A number of eukaryotic DNA binding proteins have been isolated by screening phage expression libraries with DNA probes containing the binding site of the DNA-binding protein. This methodology was employed here to isolate clones of the factor that interacts with the W box element of the human major histocompatibility complex HLA-DQB gene. Surprisingly, several cDNA clones of YB-1, a cDNA clone that was previously isolated with a CCAAT element-containing sequence were found. Independently, the screening of phage expression libraries with depurinated DNA resulted in the isolation of YB-1 and dbpA, a previously isolated cDNA that has homology to YB-1. Additional characterization of YB-1 showed that it bound a wide variety of DNA sequences and suggested that the binding of this protein is promiscuous. Furthermore, we show that both YB-1 and dbpA bind to depurinated DNA better than undamaged DNA and that the extent of specificity of binding is influenced by Mg2+. Due to the lack of sequence specificity and high degree of binding to depurinated DNA, we suggest that these proteins might be involved in chromosome functions such as maintenance of chromatin structure or DNA repair that do not require sequence-specific binding.

Base Sequence↗

PubMed1991

Regulatory factor-X binding to mutant HLA-DRA promoter sequences.

The class II genes of the major histocompatibility complex (MHC) encode a family of cell surface glycoproteins that present processed antigen to the T cell receptor. Class II genes are regulated coordinately, responding to both immunologic and developmental signals. Conserved sequence elements 5' to class II genes have been shown to be important in transcriptional control. One of these sequences, the X box, is a specific target for the binding of the factor RF-X. In the hereditary HLA class II deficiency, a form of primary immunodeficiency, a regulatory defect in expression of class II genes is associated with a defect in the binding of RF-X. To determine the basepairs that are important for this binding interaction, a series of single basepair substitutions spanning the X box motif of the DRA gene was constructed and tested for binding of RF-X by gel electrophoresis mobility shift assays (EMSAs). Several, but not all, of the mutants severely affected binding of RF-X. In addition, the binding of both the natural and the recombinant form of RF-X was affected with the same specificity. A comparison of X box basepair positions important for RF-X binding to DRA with sequences conserved between X boxes of other class II alpha chain genes suggests that high affinity RF-X binding is important for a high level of expression and may explain differences in the levels of class II expression of different class II alpha chains.

Animals↗

PubMed1991

Sensitivity to tumour necrosis factor-mediated cytolysis is unrelated to manganous superoxide dismutase messenger RNA levels among transformed mouse fibroblasts.

The ability of cells to resist the cytolytic actions of tumour necrosis factor (TNF) has been shown to require TNF-induced gene expression. It has been shown in some human cells that the gene encoding manganese superoxide dismutase (MnSOD), a TNF-induced gene, can provide resistance to TNF killing. Variation in the sensitivity to TNF was observed during subcloning of mouse SV40-transformed cell lines. This variation fell into three phenotypic classes. Cells were found that were either always resistant to TNF, always sensitive to TNF, and sensitive to TNF if inhibitors of transcription or translation were present. To determine if the regulation of MnSOD was responsible for the TNF sensitivity, Northern blot analysis was carried out. These experiments showed no correlation between expression and/or induction of the MnSOD mRNA and sensitivity or resistance to TNF. These data suggest that other pathways and gene products must therefore play a role for cells to resist TNF-mediated cellular lysis.

Animals↗

PubMed1989

The neuron-specific protein PGP 9.5 is a ubiquitin carboxyl-terminal hydrolase.

A complementary DNA (cDNA) for ubiquitin carboxyl-terminal hydrolase isozyme L3 was cloned from human B cells. The cDNA encodes a protein of 230 amino acids with a molecular mass of 26.182 daltons. The human protein is very similar to the bovine homolog, with only three amino acids differing in over 100 residues compared. The amino acid sequence deduced from the cDNA was 54% identical to that of the neuron-specific protein PGP 9.5. Purification of bovine PGP 9.5 confirmed that it is also a ubiquitin carboxyl-terminal hydrolase. These results suggest that a family of such related proteins exists and that their expression is tissue-specific.

Amino Acid Sequence↗

PubMed1988

Conserved upstream sequences of human class II major histocompatibility genes enhance expression of class II genes in wild-type but not mutant B-cell lines.

Class II major histocompatibility genes contain a conserved upstream sequence (CUS) that is important in the expression of these genes. This region has been divided into two major elements, the X box and the Y box. The ability of these elements to mediate transcription of a heterologous promoter was assayed upon transfection into a B-cell line (Raji), a class II-specific trans-acting factor-deficient B-cell line (RJ2.2.5 cells), and a T-cell line (Jurkat). The results showed that the X box element was responsible for directing tissue-specific expression when Raji cells were compared to Jurkat cells. The X box could not direct expression of the heterologous promoter in the trans-acting factor-deficient cell line, indicating that the X box is an ultimate target of the missing or defective factor in the RJ2.2.5 cell line. The Y box directed an equal but extremely low level of transcription in this system in both the mutant and wild-type B-cell lines, suggesting that this element is not involved in B-cell expression or as a target of the mutant factor.

B-Lymphocytes↗

PubMed1987

Structure and expression of HLA-DQ alpha and -DX alpha genes: interallelic alternate splicing of the HLA-DQ alpha gene and functional splicing of the HLA-DQ alpha gene using a retroviral vector.

The nucleotide sequences of the two closely related HLA-DQ alpha and HLA-DX alpha genes have been determined. Exons coding for the signal peptide, alpha 2 and transmembrane domains are 94-99% homologous, whereas the alpha 1 exon and the promoter region have diverged as much as or more than introns and the 3' untranslated region. The promoter regions of both genes contain two short sequences thought to be important for regulation of transcription by gamma-interferon. Transfection studies established that the DQ alpha and DQ beta genes encode the HLA-DQ antigen. Transcripts of varying length are produced from different alleles as the result of the use of alternate splice and polyadenylation signals at the 3' end of the DQ alpha gene. Thus typing at the DQ alpha locus can be achieved by Northern blot analysis. No transcript of DX alpha was detected in B lymphocytes. The DX alpha gene was accurately spliced when introduced into a retroviral vector, suggesting that the lack of expression of DX alpha is not due to aberrant splice signals.

Alleles↗

PubMed1987

A highly diverged beta 1 exon in the DR region of the human MHC: sequence and evolutionary implications.

The DR subregion of the human major histocompatibility complex from a DR4 haplotype includes the well-characterized DR alpha, DR4 beta, DR(MT3)beta psi genes. In addition, the region between the DR alpha and the proximal DR(MT3)beta contains several copies of conserved DR beta-related sequences. These repeated elements, numbered II, III, and IV, include the DR beta signal sequence and a region located further upstream. Further examination of these conserved sequences showed that DR beta first intron sequences are present at the 3' ends of these repeats. Progressively longer portions of the DR beta first intron are conserved from repeat II to repeat IV, producing a gradient of conservation. The most complete repeat element of repeats II, III, and IV is associated with a lone beta 1 exon (DR beta 1). Upon sequencing, DR beta 1 was found to contain several deleterious mutations, indicating that it is nonfunctional. DR beta 1 has accumulated a large number of replacement substitutions and mutations at positions which are invariant in beta 1 domains from expressed DR beta genes: 77.8% of the nucleotide substitutions were replacement substitutions, and 41.5% of the amino acids at invariant positions have been altered. Calculations based on these figures suggest that DR beta 1 may have become inactive approximately 25 million years ago. There are, however, two histidine residues within a variable region which are unique to DR beta 1 and the DR4 beta gene, suggesting that they represent a gene pair which probably evolved by duplication of a single DR beta chain gene.

Amino Acid Sequence↗

PubMed1986

Regulation of a transfected human class II major histocompatibility complex gene in human fibroblasts.

To investigate the cis-acting DNA elements that are involved in regulation of class II major histocompatibility complex genes, including gamma-interferon (gamma-IFN) induction, 5' flanking DNA deletions of a DQ beta "minigene" were analyzed in stable transfected cell lines. At least four elements 5' to the gene were found to be involved in DQ beta regulation. Deletion of sequences from -2500 to -159 base pairs (bp) resulted in increased transcription, suggesting that negative regulatory elements resided in the deleted region. These clones were all capable of responding to gamma-IFN. Further deletion of sequences from -159 to -128 bp resulted in constitutive high level transcription and the inability of these constructions to respond to gamma-IFN. A deletion to -107 bp resulted in a decrease in the basal level of expression that was restored by removal of the 5' DNA sequence to -82 bp, suggesting the presence of a second negative element. Finally, deletion to -64 bp caused a marked decrease in expression, suggesting the loss of an element necessary for high levels of transcription. The gamma-IFN control and the transcription control elements contain the conserved upstream sequences found in all class II genes, suggesting a role for these sequences.

Fibroblasts↗

PubMed1986

Cloning and sequence analysis of complementary DNA encoding an aberrantly rearranged human T-cell gamma chain.

Complementary DNA (cDNA) encoding a human T-cell gamma chain has been cloned and sequenced. At the junction of the variable and joining regions, there is an apparent deletion of two nucleotides in the human cDNA sequence relative to the murine gamma-chain cDNA sequence, resulting simultaneously in the generation of an in-frame stop codon and in a translational frameshift. For this reason, the sequence presented here encodes an aberrantly rearranged human T-cell gamma chain. There are several surprising differences between the deduced human and murine gamma-chain amino acid sequences. These include poor homology in the variable region, poor homology in a discrete segment of the constant region precisely bounded by the expected junctions of exon CII, and the presence in the human sequence of five potential sites for N-linked glycosylation.

Amino Acid Sequence↗

PubMed1985

SB subregion of the human major histocompatibility complex: gene organization, allelic polymorphism and expression in transformed cells.

The SB region of the human major histocompatibility complex (MHC) has been cloned from cosmid and lambda phage libraries made from the human B-lymphoblastoid cell line Priess (DR4/4, DC4/4, SB3/4). Two alpha genes and two beta genes are encoded in the 100 kb long SB region in the order SB alpha-SB beta-SX alpha-SX beta. The SB alpha and SB beta genes encode the alpha and beta subunits of the SB subset of class II MHC molecules. Both the SX alpha and the SX beta genes are pseudogenes in the haplotype examined. From the isolated clones, the two haplotypes of the Priess cell line, SB3 and SB4, are distinguished by nucleotide sequencing and blot hybridization analyses. Restriction site polymorphisms between the SB3 and SB4 clones were observed only in relatively small regions of the SB beta and SX beta genes. A mouse macrophage cell line was transfected with one of the cosmid clones containing both SB alpha and SB beta genes. Expression of the alpha and beta genes was detected by fluorescene-activated cell sorting (FACS) and two-dimensional gel electrophoresis using SB-specific monoclonal antibodies.

Alleles↗

PubMed1985

Gene organization of DC and DX subregions of the human major histocompatibility complex.

The DC and DX subregions of the human major histocompatibility complex (MHC) have been cloned from a cosmid library made from a human B-cell line, Priess. The DC subregion, 48 kilobases, includes the DC alpha and DC beta genes. A second DC-like region, the DX subregion, 35 kilobases, contains the DX alpha gene and a newly found beta gene termed DX beta. Since the DC and DX genes are highly homologous in nucleotide sequence, gene size, exon-intron organization, and direction of transcription, the DC and DX subregions were presumably generated by duplication of an ancestral alpha-beta gene pair. Nucleotide sequencing indicates that all four genes have intact coding sequences and promoter regions. Homology between the upstream promoter sequences of these four genes and seven other class II genes at nucleotides -69 to -78 and -98 to -110 highlights these previously described conserved elements. Moreover, a striking conservation of flanking alpha-gene-specific and beta-gene-specific sequences has been observed. Comparison of Southern blots of Priess DNA with DC alpha and DC beta cDNA probes with isolated cosmid clones showed that (i) the human chromosome encodes only two DC alpha-related and two DC beta-related genes, namely, DC alpha, DX alpha, DC beta, and DX beta, and (ii) the DC and DX subregions are homozygous in Priess cells.

Chromosome Mapping↗