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Biomedical subjects

B Mach

Publications and source records attributed to B Mach.

At least 19 recordsLinked to original sources
PubMed2001

The bare lymphocyte syndrome and the regulation of MHC expression.

The bare lymphocyte syndrome (BLS) is a hereditary immunodeficiency resulting from the absence of major histocompatibility complex class II (MHCII) expression. Considering the central role of MHCII molecules in the development and activation of CD4(+) T cells, it is not surprising that the immune system of the patients is severely impaired. BLS is the prototype of a "disease of gene regulation." The affected genes encode RFXANK, RFX5, RFXAP, and CIITA, four regulatory factors that are highly specific and essential for MHCII genes. The first three are subunits of RFX, a trimeric complex that binds to all MHCII promoters. CIITA is a non-DNA-binding coactivator that functions as the master control factor for MHCII expression. The study of RFX and CIITA has made major contributions to our comprehension of the molecular mechanisms controlling MHCII genes and has made this system into a textbook model for the regulation of gene expression.

Animals↗
PubMed2000

Structure of the winged-helix protein hRFX1 reveals a new mode of DNA binding.

Regulatory factor X (RFX) proteins are transcriptional activators that recognize X-boxes (DNA of the sequence 5'-GTNRCC(0-3N)RGYAAC-3', where N is any nucleotide, R is a purine and Y is a pyrimidine) using a highly conserved 76-residue DNA-binding domain (DBD). DNA-binding defects in the protein RFX5 cause bare lymphocyte syndrome or major histocompatibility antigen class II deficiency. RFX1, -2 and -3 regulate expression of other medically important gene products (for example, interleukin-5 receptor alpha chain, IL-5R alpha). Fusions of the ligand-binding domain of the oestrogen receptor with the DBD of RFX4 occur in some human breast tumours. Here we present a 1.5 A-resolution structure of two copies of the DBD of human RFX1 (hRFX1) binding cooperatively to a symmetrical X-box. hRFX1 is an unusual member of the winged-helix subfamily of helix-turn-helix proteins because it uses a beta-hairpin (or wing) to recognize DNA instead of the recognition helix typical of helix-turn-helix proteins. A new model for interactions between linker histones and DNA is proposed.

Amino Acid Sequence↗
PubMed2000

Dendritic cells and differential usage of the MHC class II transactivator promoters in the central nervous system in experimental autoimmune encephalitis.

In the normal central nervous system (CNS) expression of MHC class II is minimal, but has been found to be highly up-regulated on microglia cells in experimental autoimmune encephalitis (EAE). Here we used the EAE model to examine the regulation of expression of the class II transactivator (CIITA), which is required for activation of MHC class II genes. EAE was induced in C57BL/6 mice by immunization with myelin oligodendrocyte glycoprotein peptide 35-55. CIITA mRNA form I (specific for dendritic cells) and form IV (IFN-gamma inducible) but not form III (B cell specific) were detected in brain and spinal cord of mice with acute EAE. In unimmunized or mock-immunized mice, none of the three CIITA forms was found to be induced. Dendritic cells (DC) were identified by immunostainings for CD11c in perivascular and meningeal cell infiltrates in EAE spinal cord and brain. Time-course analysis showed (1) the appearance of DC in the CNS shortly before onset of disease, (2) the recruitment of CD11b cells occuring much earlier and (3) the absence of CIITA and MHC class II expression in these CD11b+ cells at preclinical stages.

Animals↗
PubMed1999

CIITA-induced occupation of MHC class II promoters is independent of the cooperative stabilization of the promoter-bound multi-protein complexes.

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.

B-Lymphocytes↗
PubMed1999

Escape of human cytomegalovirus from HLA-DR-restricted CD4(+) T-cell response is mediated by repression of gamma interferon-induced class II transactivator expression.

Human cytomegalovirus (HCMV), a betaherpesvirus, is a pathogen which escapes immune recognition through various mechanisms. In this paper, we show that HCMV down regulates gamma interferon (IFN-gamma)-induced HLA-DR expression in U373 MG astrocytoma cells due to a defect downstream of STAT1 phosphorylation and nuclear translocation. Repression of class II transactivator (CIITA) mRNA expression is detected within the first hours of IFN-gamma-HCMV coincubation and results in the absence of HLA-DR synthesis. This defect leads to the absence of presentation of the major immediate-early protein IE1 to specific CD4(+) T-cell clones when U373 MG cells, used as antigen-presenting cells, are treated with IFN-gamma plus HCMV. However, presentation of endogenously synthesized IE1 can be restored when U373 MG cells are transfected with CIITA prior to infection with HCMV. Altogether, the data indicate that the defect induced by HCMV resides in the activation of the IFN-gamma-responsive promoter of CIITA. This is the first demonstration of a viral inhibition of CIITA expression.

Antiviral Agents↗
PubMed1999

Dimeric RFX proteins contribute to the activity and lineage specificity of the interleukin-5 receptor alpha promoter through activation and repression domains.

Interleukin-5 (IL-5) plays a central role in the differentiation, proliferation, and functional activation of eosinophils. The specific action of IL-5 on eosinophils and hematopoietically related basophils is regulated by the restricted expression of IL-5 receptor alpha (IL-5Ralpha), a subunit of high-affinity IL-5R, on these cells. We have previously identified an enhancer-like cis element in the IL-5Ralpha promoter that is important for both full promoter function and lineage-specific activity. Here, we demonstrate by yeast one-hybrid screening that RFX2 protein specifically binds to this cis element. RFX2 belongs to the RFX DNA-binding protein family, the biological role of which remains obscure. Using an electrophoretic mobility shift assay, we further show that RFX1, RFX2, and RFX3 homodimers and heterodimers specifically bind to the cis element of the IL-5Ralpha promoter. The mRNA expression of RFX1, RFX2, and RFX3 was detected ubiquitously, but in transient-transfection assays, multimerized RFX binding sites in front of a basal promoter efficiently functioned in a tissue- and lineage-specific manner. To further investigate RFX functions on transcription, full-length and deletion mutants of RFX1 were targeted to DNA through fusion to the GAL4 DNA binding domain. Tissue- and lineage-specific transcriptional activation with the full-length RFX1 fusion plasmid on a reporter controlled by GAL4 binding sites was observed. Distinct activation and repression domains within the RFX1 protein were further mapped. Our findings suggest that RFX proteins are transcription factors that contribute to the activity and lineage specificity of the IL-5Ralpha promoter by directly binding to a target cis element and cooperating with other tissue- and lineage-specific cofactors.

Animals↗
PubMed1998

DNA binding properties of a chemically synthesized DNA binding domain of hRFX1.

The RFX DNA binding domain (DBD) is a novel highly conserved motif belonging to a large number of dimeric DNA binding proteins which have diverse regulatory functions in eukaryotic organisms, ranging from yeasts to human. To characterize this novel motif, solid phase synthesis of a 76mer polypeptide corresponding to the DBD of human hRFX1 (hRFX1/DBD), a prototypical member of the RFX family, has been optimized to yield large quantities (approximately 90 mg) of pure compound. Preliminary two-dimensional1H NMR experiments suggested the presence of helical regions in this sequence in agreement with previously reported secondary structure predictions. In gel mobility shift assays, this synthetic peptide was shown to bind in a cooperative manner the 23mer duplex oligodeoxynucleotide corresponding to the binding site of hRFX1, with a 2:1 stoichoimetry due to an inverse repeat present in the 23mer. The stoichiometry of this complex was reduced to 1:1 by decreasing the length of the DNA sequence to a 13mer oligonucleotide containing a single half-site. Surface plasmon resonance measurements were achieved using this 5'-biotylinated 13mer oligonucleotide immobilized on an avidin-coated sensor chip. Using this method an association constant (K a = 4 x 10(5)/M/s), a dissociation constant (K d = 6 x 10(-2)/s) and an equilibrium dissociation constant (K D = 153 nM) were determined for binding of hRFX1/DBD to the double-stranded 13mer oligonucleotide. In the presence of hRFX1/DBD the melting temperature of the 13mer DNA was increased by 16 degreesC, illustrating stabilization of the double-stranded conformation induced by the peptide.

Amino Acid Sequence↗
PubMed1998

Quantitative control of MHC class II expression by the transactivator CIITA.

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.

Animals↗