ABSTRACT
The hepatitis B virus X protein (HBx) is essential for establishing natural viral infection and has been implicated in the development of liver cancer associated with chronic infection. The basis for HBx function in either process is not understood. In cell culture, HBx exhibits pleiotropic activities affecting transcription, DNA repair, cell growth, and apoptotic cell death. Numerous cellular proteins including the p127-kDa subunit of UV-damaged DNA-binding activity have been reported to interact with HBx but the functional significance of these interactions remains unclear. Here we show that the binding of HBx to p127 interferes with cell viability. Mutational analysis reveals that HBx contacts p127 via a region to which no function has been assigned previously. An HBx variant bearing a single-charge reversal substitution within this region loses p127 binding and concomitant cytotoxicity. This mutant regains activity when directly fused to p127. These studies confirm that p127 is an important cellular target of HBx, and they indicate that HBx does not exert its effect by sequestering p127, and thereby preventing its normal function, but instead by conferring to p127 a deleterious activity.
Subject(s)
DNA-Binding Proteins/metabolism , Trans-Activators/physiology , Amino Acid Sequence , Cell Death , Cell Survival/physiology , DNA-Binding Proteins/radiation effects , HeLa Cells , Humans , Liver Neoplasms/metabolism , Liver Neoplasms/virology , Molecular Sequence Data , Molecular Weight , Mutagenesis , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/radiation effects , Sequence Homology, Amino Acid , Trans-Activators/metabolism , Transfection , Ultraviolet Rays , Viral Regulatory and Accessory ProteinsABSTRACT
Choline-O-acetyltransferase (ChAT) is the enzyme which catalyses the biosynthesis of the neurotransmitter acetylcholine in cholinergic neurons. Here we show that in mouse cholinergic NS-20Y neuroblastoma cells cultured in the presence of either okadaic acid (serine/threonine phosphatases 1 and 2A inhibitor) or KN-62 (CaM kinase inhibitor) ChAT activity and mRNA either increased or decreased as a function of the drug concentration, respectively. After 24 h exposure, okadaic acid exerted a dramatic effect on cell morphology; cells became round and had no more neurites. On the contrary, KN-62 induced a slight morphological differentiation of the cells. The present results suggest that phosphatases 1 and 2A and CaM kinase could mediate regulation of ChAT gene expression.
Subject(s)
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/analogs & derivatives , Calcium-Calmodulin-Dependent Protein Kinases/physiology , Choline O-Acetyltransferase/biosynthesis , Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Neoplasm Proteins/biosynthesis , Neuroblastoma/pathology , Okadaic Acid/pharmacology , Phosphoprotein Phosphatases/physiology , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/pharmacology , Animals , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Choline O-Acetyltransferase/genetics , Enzyme Induction/drug effects , Mice , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , Neuroblastoma/enzymology , Phosphoprotein Phosphatases/antagonists & inhibitors , Phosphorylation/drug effects , Protein Processing, Post-Translational/drug effects , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , RNA, Neoplasm/biosynthesis , RNA, Neoplasm/genetics , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/enzymologyABSTRACT
Precise regulation of MHC class II expression plays a crucial role in the control of the immune response. The transactivator CIITA behaves as a master controller of constitutive and inducible MHC class II gene activation, but its exact mechanism of action is not known. Activation of MHC class II promoters requires binding of at least three distinct multi-protein complexes (RFX, X2BP and NF-Y). It is known that the stability of this binding results from cooperative interactions between these proteins. We show here that expression of CIITA in MHC class II- cells triggers occupation of the promoters by these complexes. This observation raised the possibility that the effect of CIITA on promoter occupation is mediated by an effect on the cooperative stabilization of the DNA-bound multi-protein complexes. We show, however, that the presence of CIITA does not affect the stability of the higher-order protein complex formed on DNA by RFX, X2BP and NF-Y. This suggests other mechanisms for CIITA-induced promoter occupancy, such as an effect on chromatin structure leading to increased accessibility of MHC class II promoters. This ability of CIITA to facilitate promoter occupation is undissociable from its transactivation potential. Finally, we conclude that this effect of CIITA is cell-type specific, since expression of CIITA is not required for normal occupation of MHC class II promoters in B lymphocytes.
Subject(s)
Genes, MHC Class II , Histocompatibility Antigens Class II/biosynthesis , Nuclear Proteins , Promoter Regions, Genetic , Trans-Activators/metabolism , Transcription Factors/metabolism , B-Lymphocytes/cytology , B-Lymphocytes/immunology , CCAAT-Enhancer-Binding Proteins , Cells, Cultured , DNA Footprinting , DNA-Binding Proteins/metabolism , Gene Expression Regulation , HLA-DR Antigens/biosynthesis , HLA-DR Antigens/genetics , HLA-DR alpha-Chains , Histocompatibility Antigens Class II/genetics , Humans , Models, Genetic , Monocytes/cytology , Monocytes/immunology , Protein Binding , Regulatory Factor X Transcription Factors , Transcriptional Activation , Tumor Cells, CulturedABSTRACT
Activation of T lymphocytes is quantitatively controlled by the level of expression of MHC class II molecules. Both constitutive and inducible expression of MHC class II genes is regulated by the transactivator CIITA, which is itself tightly regulated. Since the level of MHC class II molecules expressed is a functionally essential parameter, it was of interest to explore whether MHC class II expression is quantitatively controlled by the level of the transactivator. This report shows that in a variety of experimental conditions one does indeed observe, in both mouse and man, a quantitative control of MHC class II expression by the level of CIITA. This relationship between the regulator gene, which behaves as a rate-limiting factor, and its target genes clarifies our understanding of the quantitative modulation of MHC class II expression, and thus of T lymphocyte activation.
Subject(s)
Gene Expression Regulation/immunology , Genes, MHC Class II , Nuclear Proteins , Trans-Activators/physiology , Animals , Cell Line , Gene Expression Regulation/drug effects , Genes, MHC Class II/drug effects , HeLa Cells , Histocompatibility Antigens Class II/biosynthesis , Histocompatibility Antigens Class II/genetics , Humans , Interferon-gamma/pharmacology , Mice , Mice, Inbred BALB C , Organ Specificity/genetics , Trans-Activators/biosynthesisABSTRACT
Major histocompatibility complex class II (MHC-II) molecules present peptide antigens to CD4-positive T cells and are of critical importance for the immune response. The MHC-II transactivator CIITA is essential for all aspects of MHC-II gene expression examined so far and thus constitutes a master regulator of MHC-II expression. In this study, we generated and analyzed mutant CIITA molecules which are able to suppress endogenous MHC-II expression in a dominant negative manner for both constitutive and inducible MHC-II expression. Dominant negative CIITA mutants were generated via specific restriction sites and by functional selection from a library of random N-terminal CIITA deletions. This functional selection strategy was very effective, leading to strong dominant negative CIITA mutants in which the N-terminal acidic and proline/serine/threonine-rich regions were completely deleted. Dominant negative activity is dependent on an intact C terminus. Efficient repression of endogenous MHC-II mRNA levels was quantified by RNase protection analysis. The quantitative effects of various dominant negative CIITA mutants on mRNA expression levels of the different MHC-II isotypes are very similar. The optimized dominant negative CIITA mutants isolated by functional selection should be useful for in vivo repression of MHC-II expression.
Subject(s)
Genes, MHC Class II/genetics , Nuclear Proteins , Sequence Deletion , Trans-Activators/genetics , Transcriptional Activation/genetics , Burkitt Lymphoma , Cell Separation , Cloning, Molecular , DNA, Recombinant , Flow Cytometry , Genes, Dominant , HLA-DR Antigens/genetics , HeLa Cells , Humans , Interferon-gamma/pharmacology , RNA, Messenger/biosynthesis , Trans-Activators/analysis , Trans-Activators/metabolism , Tumor Cells, CulturedABSTRACT
Congenital MHC class II deficiency or bare lymphocyte syndrome (BLS; McKusick 209920) is caused by defects in trans-acting regulatory factors that control MHC class II expression and is therefore a disease of gene regulation. There are at least four complementation groups and the genetic and molecular dissection of this rare disease has contributed considerably to our current understanding of the molecular mechanisms governing MHC class II expression. Identification of the gene that is defective in BLS complementation group A, CIITA (MHC class II transactivator), has led to the discovery that CIITA acts as a master control factor of MHC class II expression. We have identified the CIITA mutations in a second patient from BLS group A. Two novel mutations abolish CIITA function, as shown by transfection experiments. Molecular analysis of these two novel mutations, together with the one described earlier in the first patient, is informative in terms of CIITA structure-function relationships.