Gamma interferon modulates myogenesis through the major histocompatibility complex class II transactivator, CIITA

Gamma interferon (IFN-γ) is an inflammatory cytokine that has complex effects on myogenesis. Here, we show that the IFN-γ-induced inhibition of myogenesis is mediated by the major histocompatibility complex (MHC) class II transactivator, CIITA, which binds to myogenin and inhibits its activity. In IFN-γ-treated myoblasts, the inhibition of muscle-specific genes includes the expression of myogenin itself, while in myotubes, myogenin expression is unaffected. Thus, CIITA appears to act by both repressing the expression and inhibiting the activity of myogenin at different stages of myogenesis. Stimulation by IFN-γ in skeletal muscle cells induces CIITA expression as well as MHC class II gene expression. The IFN-γ-mediated repression is reversible, with myogenesis proceeding normally upon removal of IFN-γ. Through overexpression studies, we confirm that the expression of CIITA, independent of IFN-γ, is sufficient to inhibit myogenesis. Through knockdown studies, we also demonstrate that CIITA is necessary for the IFN-γ-mediated inhibition of myogenesis. Finally, we show that CIITA, which lacks DNA binding activity, is recruited to muscle-specific promoters coincident with reductions in RNA polymerase II recruitment. Thus, this work reveals how IFN-γ modulates myogenesis and demonstrates a key role for CIITA in this process.     

Polymorphism at the ovine major histocompatibility complex class II loci

Southern hybridization analysis of the ovine major histocompatibility complex (MHC) ( MhcOvar ) class II region, using sheep-specific probes for the DQA1, DQA2, DQB and DRA loci, has revealed extensive polymorphism. DQA1 and DQAP had eight and 16 alleles respectively, DQB had six and DRA had three alleles. Little information was derived from the DRB locus owing to extensive cross-hybridization between the DRB probe and the DQB locus. Differences in allele frequency between breeds were revealed. At the DQA1 locus a null allele (DQA1-N) was observed with a frequency of between 27% and 45%, making this the most common DQA1 allele in all breeds examined. The frequency of DQA1-N homozygotes was between 11% and 18%, raising questions as to the functional significance of the DQA1 gene. Linkage analysis between the DQA1, DQA2, DQB and DRA loci did not reveal any recombination.     

Cloning of the bovine major histocompatibility complex class II genes

Summary. Class II genes of the bovine major histocompatibility complex (MHC) have been cloned from a genomic library. The library was constructed in the bacteriophage Λ vector EMBL3 and comprises approximately 10 times the equivalent of the haploid genome. Half the library was screened with the human DQA, DQB, DRA and DRB cDNA probes. Of the 100 positively hybridizing phage clones, 37 were eventually fully characterized and mapped by means of Southern blot analysis. The exons encoding the first, second and transmembrane domain of all different A and B genes were subcloned and mapped in more detail. These analyses showed that these 37 clones were derived from five different A and 10 different B genes. The hybridization studies indicate that we have cloned and mapped two DQA genes, one DRA gene, two other A genes, four DQB genes, three DRB genes and three other B genes. Since the library was made from a heterozygous animal, this would suggest that there are at least one DQA, one DRA one other undefined A, two DQB, two DRB and one or two other undefined B genes in the haploid genome of Holstein Friesian cattle.     

Molecular mapping class II polymorphisms in the human major histocompatibility complex. II. DQ beta

The restriction fragment length polymorphisms have been determined for six restriction enzymes (Bam HI, Bg1 II, Eco RI, Hinc II, Hind III, and Pvu II) and a DQ beta probe on 25 cell lines that are homozygous by consanguinuity at the MHC. These patterns reflect both DR haplotypes and DQ types of the cells tested. At least one non-polymorphic band is present in all the cell lines with every restriction enzyme except Hinc II. This band most probably represents DX beta hybridization. The polymorphic bands indicate that more polymorphism exists in the DQ subregion than is predicted serologically. Each DR haplotype is associated with a unique set of restriction fragments except for DR2 and DR6. The patterns are largely consistent within each DR haplotype. In addition, some bands reflect the established DQ specificities DQw1 and DQw2. Individual bands can be identified that are unique to the haplotypes DR1, DR4, DR5, and DR6 and the DQw1- and DQw2-associated haplotypes. Subdivisions of haplotypes can be identified with this probe. In particular, MVL (DR1), Akiba (DR2), QBL (DR3), FPF (DR5), and APD (DR6) have polymorphisms that distinguish them from other members of their DR haplotype.     

Cloning of the bovine major histocompatibility complex class II genes

Class II genes of the bovine major histocompatibility complex (MHC) have been cloned from a genomic library. The library was constructed in the bacteriophage lambda vector EMBL3 and comprises approximately 10 times the equivalent of the haploid genome. Half the library was screened with the human DQA, DQB, DRA and DRB cDNA probes. Of the 100 positively hybridizing phage clones, 37 were eventually fully characterized and mapped by means of Southern blot analysis. The exons encoding the first, second and transmembrane domain of all different A and B genes were subcloned and mapped in more detail. These analyses showed that these 37 clones were derived from five different A and 10 different B genes. The hybridization studies indicate that we have cloned and mapped two DQA genes, one DRA gene, two other A genes, four DQB genes, three DRB genes and three other B genes. Since the library was made from a heterozygous animal, this would suggest that there are at least one DQA, one DRA one other undefined A, two DQB, two DRB and one or two other undefined B genes in the haploid genome of Holstein Friesian cattle.     

Modulation of the peptide-binding specificity of a single-chain class II major histocompatibility complex

We designed and expressed a single-chain class II major histocompatibility complex molecule capable of forming a stable complex with an antigenic peptide. The peptide-binding preference of the single-chain (sc) human leukocyte antigen derived from DRB5(*)0101 (DR51) was determined to be similar to that of the authentic one, which requires a bulky hydrophobic residue at position-1 (P1) as a primary anchor. For modulation of the peptide-binding affinity, we modified binding pocket 1 of sc DR51 by site-directed mutagenesis. The relative binding affinity of the engineered sc DR51 for several P1-substituted peptides was measured by competition assaying with a fluorescence labeled peptide. The sc DR51 molecule showed high affinity to the self-peptide derived from myelin basic protein, 87-98 with Phe as the P1 residue (F90F). While reduction of pocket 1 volume (betaG86V) decreased the affinity of F90F, it rather increased the affinity of the Ala-substituted peptide as to the P1 residue (F90A). Through more extensive engineering in the peptide-binding groove of the sc DR51 molecule, it is expected that we can construct sc DR51 variants with various peptide ligand motifs.     

Characterization of the major histocompatibility complex class II genes in miiuy croaker

Major histocompatibility complex (MHC) has a central role in the adaptive immune system by presenting foreign peptide to the T-cell receptor. In order to study the molecular function and genomic characteristic of class II genes in teleost, the full lengths of MHC class IIA and IIB cDNA and genomic sequence were cloned from miiuy croaker (Miichthys miiuy). As in other teleost, four exons and three introns were identified in miiuy croaker class IIA gene; but the difference is that six exons and five introns were identified in the miiuy croaker class IIB gene. The deduced amino acid sequence of class IIA and class IIB had 26.3-85.7% and 11.0-88.8% identity with those of mammal and teleost, respectively. Real-time quantitative RT-PCR demonstrated that the MHC class IIA and IIB were ubiquitously expressed in ten normal tissues; expression levels of MHC genes were found first upregulated and then downregulated, and finally by a recovery to normal level throughout the pathogenic bacteria infection process. In addition, we report on the underlying mechanism that maintains sequences diversity among many fish species. A series of site-model tests implemented in the CODEML program revealed that positive Darwinian selection is likely the cause of the molecular evolution in the fish MHC class II genes.     

Partial nucleotide sequence of a bovine major histocompatibility class II DRβ-like gene

Summary. A genomic clone containing a bovine DRβ-like gene, BoDRβ II , was isolated from a bovine genomic library and characterized by restriction enzyme mapping and nucleotide sequencing of exon regions. Alignment of this sequence with the human DRβ cDNA sequence allowed identification of exon/intron boundaries. The clone contains a 13.3-kilobase (kb) insert, and includes 1.3kb 5' of the β₁ exon and 6.7kb 3' of the transmembrane (TM) exon. Open reading frames were present in the BoDRβ exons sequenced. Nucleotide identities of the bovine β₁, β₂ and TM exons with the corresponding human DRβ exons were 73, 91 and 83%, respectively. Nucleotide identities of these exons with those of a previously described bovine DRβ-like pseudogene, BoDRβ I , were 69, 95 and 81%, respectively. Although a limited amount of sequence data was obtained for the intron regions, a 71% identity was found within a 514-nucleotide region immediately 3' to the β₂ exons in BoDRβ I and BoDRβ II . A series of GT residues followed by a longer series of GA residues began about 35 nucleotides 3' of the β₁ exon in both BoDRβ I and BoDRβ II .     

Survey of major histocompatibility complex class II diversity in pig-tailed macaques

Pig-tailed macaques (Macaca nemestrina) serve as important models for human infectious disease research. Major histocompatibility complex (MHC) class II molecules are important to this research since they present peptides to CD4+ T cells. Despite the importance of characterizing the MHC-II alleles expressed in model species like pig-tailed macaques, to date, less than 150 MHC-II alleles have been named for the six most common classical class II loci (DRA, DRB, DQA, DQB, DPA, and DPB) in this population. Additionally, only a small percentage of these alleles are full-length, making it impossible to use the known sequence for reagent development. To address this, we developed a fast, high-throughput method to discover full-length MHC-II alleles and used it to characterize alleles in 32 pig-tailed macaques. By this method, we identified 128 total alleles across all six loci. We also performed an exon 2-based genotyping assay to validate the full-length sequencing results; this genotyping assay could be optimized for use in determining MHC-II allele frequencies in large cohorts of pig-tailed macaques.     

Varying functions of specific major histocompatibility class II transactivator promoter III and IV elements in melanoma cell lines.

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.