Activation of MHC class I, II, and CD40 gene expression by histone deacetylase inhibitors.

Epigenetic mechanisms are involved in regulating chromatin structure and gene expression through repression. In this study, we show that histone deacetylase inhibitors (DAIs) that alter the acetylation of histones in chromatin enhance the expression of several genes on tumor cells including: MHC class I, II, and the costimulatory molecule CD40. Enhanced transcription results in a significant increase in protein expression on the tumor cell surface, and expression can be elicited on some tumors that are unresponsive to IFN-gamma. The magnitude of induction of these genes cannot be explained by the effect of DAIs on the cell cycle or enhanced apoptosis. Induction of class II genes by DAIs was accompanied by activation of a repressed class II transactivator gene in a plasma cell tumor but, in several other tumor cell lines, class II was induced in the apparent absence of class II transactivator transcripts. These findings also suggest that the abnormalities observed in some tumors in the expression of genes critical to tumor immunity may result from epigenetic alterations in chromatin and gene regulation in addition to well-established mutational mechanisms.

Aberrant development of thymocytes in mice lacking laminin-2.

In previous in vitro studies, we proposed a role for the extracellular matrix component, laminin-2, and its integrin receptor, VLA-6, in thymocyte development. The characterization of two dystrophic mouse strains with different defects in laminin-2 allowed us to examine this proposal in vivo. Mice deficient in laminin-2, dy/dy, show a significant reduction in thymus size and number of thymocytes compared to normal littermates. These mice also exhibited apparent alterations of thymic architecture. Examination of the CD4/CD8 populations in dy/dy thymi showed large relative increases in the DN (CD4- CD8-) and SP (CD4+ CD8-, CD4- CD8+) populations and a significant decrease in the DP (CD4+ CD8+) population. Further examination of the DN population for CD44 and CD25 expression showed a remarkable decrease in the more mature pre-T cell populations. Analysis of apoptosis in situ, and by flow cytometry, in dy/dy thymi revealed a significant increase in apoptotic DN thymocytes in the capsule and subcapsular regions. Interestingly, thymocyte development appeared to proceed normally in dystrophic mice expressing a mutant form of laminin-2, dy2J, as well as, in fetal and neonatal dy/dy mice. We propose that laminin-2 plays an active role in thymocyte development by delivering cell survival and differentiation signals at specific stages of development in young adult mice.

Chromatin-immune connections: regulation of MHC and other genes.

In cells, genes are contained within chromatin - a highly structured array of DNA wrapped around core histone proteins. Packaged genes are subject to a variety of regulatory modifications including, CpG methylation, histone acetylation and phosphorylation. These epigenetic mechanisms of gene regulation involve higher ordered protein complexes possessing enzymatic activities such as ATP hydrolysis and acetylation that are targeted to specific genes by transcription factors, coactivatorsand coreptessors. In this article, we endeavor to providean overview of current research on mechanisms of transcriptional regulation by chromatin remodeling of MHC and other genes that are of interest in reproductive immunology.

Purification and characterization of the recombinant 5 S subunit of transcarboxylase from Escherichia coli.

Transcarboxylase from Propionibacterium shermanii is a biotin-containing enzyme which catalyzes the reversible transfer of a carboxyl group from methylmalonyl-CoA to pyruvate. It is composed of a central, hexameric 12 S subunit, 6 outer dimeric 5 S subunits which are held in a complex by 12 1.3 S biotinyl subunits. The transcarboxylase reaction requires two partial reactions, one of which is specific to 5 S. The cloning and expression of each of these subunits in Escherichia coli have been reported. We have designed a method for the purification of the 5 S subunit from an E. coli expression system. Protein purified to homogeneity by this method was shown to be active in the 5 S partial reaction, but unable to catalyze the overall transcarboxylase reaction. This protein was characterized as to its ability to form stable dimers, associate with the 1.3 S subunit in stable complexes referred to as 6 S, and assemble whole TC. The latter activity was shown to be lacking. The purified protein has a native molecular weight of 120 kDa and a subunit molecular weight of 60 kDa, consistent with the 5 S dimer. Plasma emission analysis of the metal content of the recombinant protein demonstrated the presence of both Co and Zn, comparable to the authentic protein. Fluorescence analysis verified the ability of the purified protein to bind substrates and 1.3 S subunits appropriately. Sequencing of the amino terminus and determination of the amino acid composition of the recombinant protein relative to that of the authentic subunit further verified the identity of the purified protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Primary structure of the 5 S subunit of transcarboxylase as deduced from the genomic DNA sequence.

Transcarboxylase from Propionibacterium shermanii is a complex biotin-containing enzyme composed of 30 polypeptides of three different types. It is composed of six dimeric outer subunits associated with a central cylindrical hexameric subunit through 12 biotinyl subunits; three outer subunits on each face of the central hexamer. Each outer dimer is termed a 5 S subunit which associates with two biotinyl subunits. The enzyme catalyzes a two-step reaction in which methylmalonyl-CoA and pyruvate form propionyl-CoA and oxalacetate, the 5 S subunit specifically catalyzing one of these reactions. We report here the cloning, sequencing and expression of the monomer of the 5 S subunit. The gene was identified by matching amino acid sequences derived from isolated authentic 5 S peptides with the deduced sequence of an open reading frame present on a cloned P. shermanii genomic fragment known to contain the gene encoding the 1.3 S biotinyl subunit. The cloned 5 S gene encodes a protein of 519 amino acids, M(r) 57,793. The deduced sequence shows regions of extensive homology with that of pyruvate carboxylase and oxalacetate decarboxylase, two enzymes which catalyze the same or reverse reaction. A fragment was subcloned into pUC19 in an orientation such that the 5 S open reading frame could be expressed from the lac promoter of the vector. Crude extracts prepared from these cells contained an immunoreactive band on Western blots which co-migrated with authentic 5 S and were fully active in catalyzing the 5 S partial reaction. We conclude that we have cloned, sequenced and expressed the monomer of the 5 S subunit and that the expressed product is catalytically active.

Primary structure of the monomer of the 12S subunit of transcarboxylase as deduced from DNA and characterization of the product expressed in Escherichia coli.

Transcarboxylase from Propionibacterium shermanii is a complex biotin-containing enzyme composed of 30 polypeptides of three different types: a hexameric central 12S subunit to which 6 outer 5S subunits are attached through 12 1.3S biotinyl subunits. The enzyme catalyzes a two-step reaction in which methylmalonyl coenzyme A and pyruvate serve as substrates to form propionyl coenzyme A (propionyl-CoA) and oxalacetate, the 12S subunit specifically catalyzing one of the two reactions. We report here the cloning, sequencing, and expression of the 12S subunit. The gene was identified by matching amino acid sequences derived from isolated authentic 12S peptides with the deduced sequence of an open reading frame present in a cloned P. shermanii genomic fragment known to contain the gene encoding the 1.3S biotinyl subunit. The cloned 12S gene encodes a protein of 604 amino acids and of M(r) 65,545. The deduced sequence shows regions of extensive homology with the beta subunit of mammalian propionyl-CoA carboxylase as well as regions of homology with acetyl-CoA carboxylase from several species. Two genomic fragments were subcloned into pUC19 in an orientation such that the 12S open reading frame could be expressed from the lac promoter of the vector. Crude extracts prepared from these cells contained an immunoreactive band on Western blots (immunoblots) which comigrated with authentic 12S. The Escherichia coli-expressed 12S was purified to apparent homogeneity by a three-step procedure and compared with authentic 12S from P. shermanii. Their quaternary structures were identical by electron microscopy, and the E. coli 12S preparation was fully active in the reactions catalyzed by this subunit. We conclude that we have cloned, sequenced, and expressed the 12S subunit which exists in a hexameric active form in E.coli.

Effect of deletion from the carboxyl terminus of the 12 S subunit on activity of transcarboxylase.

Transcarboxylase from Propionibacterium shermanii is a biotin-containing enzyme which catalyzes the reversible transfer of a carboxyl group from methylmalonyl-CoA to pyruvate. The central hexameric 12 S subunit of the enzyme associates with six 6 S subunits in the complete enzyme complex. We have constructed a series of cloned genes which encode COOH-terminal truncations of the 12 S subunit. Five of these subunits, which remained soluble following expression in Escherichia coli and were missing from 39 to 97 COOH-terminal amino acids, were purified and compared to the full-length subunit after enzyme complexes were assembled in vitro. All of the truncated subunits were 90% as active in the transcarboxylase reaction as wild type except the reaction containing the shortest complex, TC-12 S (1-507), which had 54% of the wild type activity (TC-12 S-WT). The reduced activity was not due to a lack of CoA ester binding sites or the Km for substrate. However, TC-12 S (1-507) was slower to form than TC-12 S-WT and had more incomplete complexes as judged by high performance liquid chromatography gel filtration profiles and electron microscopy. Isolated TC-12 S (1-507) was 70-80% as active as TC-12 S-WT. We also noted that the truncated form was heat-labile compared to wild type. We conclude that the COOH-terminal region of the 12 S subunit plays a role in assembly and stability of the hexamer and also affects the binding of 6 S subunits to form enzyme complexes. Once complexes do form, the catalytic capacity of TC-12 S (1-507) is almost the same as TC-12 S-WT.

The nonbiotinylated form of the 1.3 s subunit of transcarboxylase binds to avidin (monomeric)-agarose: purification and separation from the biotinylated 1.3 S subunit.

Avidin-biotin technology is used routinely to purify biotin-containing carboxylases and also proteins that have been chemically coupled to biotin. The 1.3 S subunit of transcarboxylase (TC) studied here is the biotin-containing subunit of TC which not only acts as a carboxyl carrier between the CoA ester sites on the central 12 S subunit of TC and keto acid sites on the outer 5 S subunit of TC but also links the 12 S and 5 S subunits together to form a 26 S multisubunit TC complex. The 1.3 S subunit has been cloned, sequenced, and expressed in Escherichia coli. A method for purifying recombinant 1.3 S subunits from E. coli using avidin (monomeric)-agarose column chromatography has been developed. This affinity-purified 1.3 S was found to be homogeneous by SDS-PAGE, amino acid composition, and N-terminal sequence analysis but had a biotin content of only 28% based on moles of biotin per mole of 1.3 S. This lack of stoichiometry was found to be due to copurification of apo-1.3 S as evidenced by the holocarboxylase synthetase reaction. A procedure for separating the apo- and biotinylated 1.3 S forms using hydrophobic interaction chromatography on an Ether 5 PW column is described. The method is based on the difference in hydrophobicity between apo and biotinylated 1.3 S forms. The copurification of apo and biotinylated forms of 1.3 S on the avidin (monomeric)-agarose column was found to be due to specific interaction with avidin rather than to interaction between apo- and biotinylated 1.3 S forms as demonstrated by the fluorescence quenching studies. The results suggest that the avidin-biotin system by itself may not be sufficient to obtain homogeneous biotinyl proteins as nonbiotinyl protein can also bind avidly to such columns.