scholarly journals Mutations in the Bare Lymphocyte Syndrome Define Critical Steps in the Assembly of the Regulatory Factor X Complex

2000 ◽  
Vol 20 (12) ◽  
pp. 4455-4461 ◽  
Author(s):  
Nada Nekrep ◽  
Nabila Jabrane-Ferrat ◽  
B. Matija Peterlin
Keyword(s):  
Severe Combined Immunodeficiency ◽  
Factor X ◽  
Regulatory Factor ◽  
Bare Lymphocyte Syndrome ◽  
Mhc Ii ◽  
Histocompatibility Complex ◽  
Class Ii Mhc ◽  
Weak Binding ◽  
Complementation Groups ◽  
Rfx Complex

ABSTRACT The regulatory factor X (RFX) complex, which contains RFXANK(B), RFXAP, and RFX5, binds to X and S boxes in major histocompatibility complex class II (MHC II) promoters. In the bare lymphocyte syndrome (BLS), which is a human severe combined immunodeficiency, MHC II promoters are neither occupied nor transcribed. Thus, the absence of any one subunit prevents the formation of the RFX complex. Nevertheless, except for a weak binding between RFX5 and RFXAP, no other interactions between RFX proteins have been described. In this study, we demonstrate that RFXANK(B) binds to RFXAP to form a scaffold for the assembly of the RFX complex, which then binds to DNA. Moreover, mutant RFXANK(B) and RFXAP proteins from complementation groups B and D of BLS, respectively, cannot support this interaction. Our data elucidate an intriguing medical situation, where a genetic disease targets two different surfaces that are required for the nucleation of a multisubunit DNA-protein complex.

scholarly journals Major Histocompatibility Complex Class II Transcriptional Platform: Assembly of Nuclear Factor Y and Regulatory Factor X (RFX) on DNA Requires RFX5 Dimers

2002 ◽  
Vol 22 (15) ◽  
pp. 5616-5625 ◽  
Author(s):  
Nabila Jabrane-Ferrat ◽  
Nada Nekrep ◽  
Giovanna Tosi ◽  
Laura J. Esserman ◽  
B. Matija Peterlin
Keyword(s):  
Class Ii ◽  
Nuclear Factor ◽  
Factor X ◽  
Complex Class ◽  
Regulatory Factor ◽  
Mhc Ii ◽  
Nuclear Factor Y ◽  
Histocompatibility Complex

ABSTRACT Major histocompatibility complex class II (MHC-II) genes are regulated in a B-cell-specific and gamma interferon-inducible manner. Conserved upstream sequences (CUS) in their compact promoters bind nuclear factor Y (NFY) and regulatory factor X (RFX) complexes. These DNA-bound proteins form a platform that attracts the class II transactivator, which initiates and elongates MHC-II transcription. In this report, we analyzed the complex assembly of these DNA-bound proteins. First, we found that NFY can interact with RFX in cells. In particular, NFYA and NFYC bound RFXANK/B in vitro. Next, RFX5 formed dimers in vivo and in vitro. Within a leucine-rich stretch N-terminal to the DNA-binding domain in RFX5, the leucine at position 66 was found to be critical for this self-association. Mutant RFX5 proteins that could not form dimers also did not support the formation of higher-order DNA-protein complexes on CUS in vitro or MHC-II transcription in vivo. We conclude that the MHC-II transcriptional platform begins to assemble off CUS and then binds DNA via multiple, spatially constrained interactions. These findings offer one explanation of why in the Bare Lymphocyte Syndrome, which is a congenital severe combined immunodeficiency, MHC-II promoters are bare when any subunit of RFX is mutated or missing.

scholarly journals Analysis of Ankyrin Repeats Reveals How a Single Point Mutation in RFXANK Results in Bare Lymphocyte Syndrome

2001 ◽  
Vol 21 (16) ◽  
pp. 5566-5576 ◽  
Author(s):  
Nada Nekrep ◽  
Matthias Geyer ◽  
Nabila Jabrane-Ferrat ◽  
B. Matija Peterlin
Keyword(s):  
Wide Spectrum ◽  
Single Point ◽  
Severe Combined Immunodeficiency ◽  
Single Point Mutation ◽  
Factor X ◽  
Ankyrin Repeats ◽  
Binding Studies ◽  
Bare Lymphocyte Syndrome ◽  
Mhc Ii

ABSTRACT Ankyrin repeats are well-known structural modules that mediate interactions between a wide spectrum of proteins. The regulatory factor X with ankyrin repeats (RFXANK) is a subunit of a tripartite RFX complex that assembles on promoters of major histocompatibility complex class II (MHC II) genes. Although it is known that RFXANK plays a central role in the nucleation of RFX, it was not clear how its ankyrin repeats mediate the interactions within the complex and with other proteins. To answer this question, we modeled the RFXANK protein and determined the variable residues of the ankyrin repeats that should contact other proteins. Site-directed alanine mutagenesis of these residues together with in vitro and in vivo binding studies elucidated how RFXAP and CIITA, which simultaneously interact with RFXANK in vivo, bind to two opposite faces of its ankyrin repeats. Moreover, the binding of RFXAP requires two separate surfaces on RFXANK. One of them, which is located in the ankyrin groove, is severely affected in the FZA patient with the bare lymphocyte syndrome. This genetic disease blocks the expression of MHC II molecules on the surface of B cells. By pinpointing the interacting residues of the ankyrin repeats of RFXANK, the mechanism of this subtype of severe combined immunodeficiency was revealed.

scholarly journals The SA85-1.1 Protein of the Trypanosoma cruzi trans-Sialidase Superfamily Is a Dominant T-Cell Antigen

2000 ◽  
Vol 68 (6) ◽  
pp. 3574-3580 ◽  
Author(s):  
Amanda E. Millar ◽  
Stuart J. Kahn
Keyword(s):  
T Cell ◽  
Trypanosoma Cruzi ◽  
Surface Protein ◽  
T Cell Response ◽  
Cd4 Cells ◽  
Protective Response ◽  
Mhc Ii ◽  
Histocompatibility Complex ◽  
Class Ii Mhc ◽  
Ifn Γ

ABSTRACT Trypanosoma cruzi currently infects 18 million people, and 30% of those infected develop a chronic inflammatory process that causes significant morbidity or mortality. The major histocompatibility complex class II (MHC-II)-restricted T-cell response is critical to the control of the infection and to the ensuing inflammatory pathology. The specific epitopes or major antigens of this response have not been identified. The parasite simultaneously expresses variant members of the trans-sialidase superfamily. To begin to analyze the MHC-II response to these variant proteins, the response to a single surface protein, SA85-1.1, was initiated. These studies have demonstrated that a biased gamma interferon (IFN-γ) response to the SA85-1.1 protein develops during T. cruzi infection. In addition, adoptive transfer of a CD4 clone that recognizes an SA85-1.1 epitope, named epitope 1, and immunization with a peptide encoding epitope 1 were protective and suggested that epitope 1 may be immunodominant. In this report IFN-γ intracellular staining demonstrated that splenocytes from acutely and chronically infected mice, incubated with SA85-1.1 protein or peptides that encode epitope 1, result in IFN-γ synthesis by 4 to 6% of the splenic CD4 cells. These data indicate that during T. cruzi infection epitope 1 is a major epitope and that 4 to 6% of the CD4 cells are stimulated by a single trans-sialidase superfamily epitope and suggest that a combination of trans-sialidase superfamily proteins combines to stimulate a majority of CD4 cells. These data suggest that during T. cruzi infection the CD4 response to thetrans-sialidase superfamily is critical to the protective response and to the ensuing chronic inflammatory pathology.

scholarly journals Ets-1 activates the DRA promoter in B cells.

1994 ◽  
Vol 14 (11) ◽  
pp. 7314-7321 ◽  
Author(s):  
N Jabrane-Ferrat ◽  
B M Peterlin
Keyword(s):  
Major Histocompatibility Complex ◽  
B Cells ◽  
Dna Binding Proteins ◽  
Factor X ◽  
Regulatory Factor ◽  
Mobility Shift ◽  
The Core ◽  
Histocompatibility Complex ◽  
Ets Family ◽  
Electrophoretic Mobility Shift Assays

The X box in promoters of class II major histocompatibility complex genes plays a crucial role in the B-cell-specific and gamma interferon-inducible expression of these genes. The sequence TTCC is located in the pyrimidine tract which extends 5' to and partially overlaps the X box of the DRA promoter. This sequence resembles the core binding site for the Ets family of DNA-binding proteins. In this study, we demonstrate that mutations within the pyrimidine tract which change the TTCC motif, but do not affect the binding of regulatory factor X to the X box, decrease the activity of the DRA promoter in B cells. Furthermore, using electrophoretic mobility shift assays and cotransfection experiments, we demonstrate that Ets-1, but not Ets-2 or PU.1, functionally interacts with the pyrimidine tract and activates the DRA promoter.

Sumoylation of the zinc finger protein ZXDC enhances the function of its transcriptional activation domain

2007 ◽  
Vol 388 (9) ◽  
pp. 965-972 ◽  
Author(s):  
Srikarthika Jambunathan ◽  
Joseph D. Fontes
Keyword(s):  
Zinc Finger ◽  
Transcriptional Activation ◽  
Zinc Finger Protein ◽  
Class Ii ◽  
Activation Domain ◽  
Transcriptional Activation Domain ◽  
Mhc Ii ◽  
Histocompatibility Complex ◽  
Class Ii Mhc ◽  
Important Regulator

Abstract The transcription of major histocompatibility complex class II (MHC II) genes is dependent on the co-activator protein class II trans-activator (CIITA). We have recently identified a protein known as zinc finger X-linked duplicated family member C (ZXDC) that, along with its binding partner ZXDA, forms a complex that interacts with CIITA and regulates MHC II transcription. Western blot analysis with anti-ZXDC antibodies identified two species of the ZXDC protein, one migrating near its predicted molecular mass and one with slower electrophoretic mobility. We report here that the slower migrating form is the result of sumoylation at a single lysine residue within the transcriptional activation domain of ZXDC. Three SUMO proteins (SUMO-1, -2 and -3) can modify the ZXDC protein. Multiple SUMO E3 ligase enzymes and HDAC4 can facilitate ZXDC sumoylation, and one ligase, PIASy, interacts with a specific region of the ZXDC protein. We found that sumoylation does not appear to disrupt or modulate the interaction of ZXDC with its binding partners. Rather, sumoylation of ZXDC is required for full activity of the transcriptional activation domain. Our findings suggest that sumoylation is an important regulator of ZXDC.

scholarly journals A Functionally Essential Domain of RFX5 Mediates Activation of Major Histocompatibility Complex Class II Promoters by Promoting Cooperative Binding between RFX and NF-Y

2000 ◽  
Vol 20 (10) ◽  
pp. 3364-3376 ◽  
Author(s):  
Jean Villard ◽  
Marie Peretti ◽  
Krzysztof Masternak ◽  
Emmanuèle Barras ◽  
Giuseppina Caretti ◽  
...  
Keyword(s):  
Major Histocompatibility Complex ◽  
Major Histocompatibility Complex Class ◽  
Class Ii ◽  
Terminal Region ◽  
Cooperative Binding ◽  
Complex Class ◽  
Major Histocompatibility ◽  
Mhc Ii ◽  
Histocompatibility Complex ◽  
Rfx Complex

ABSTRACT Major histocompatibility complex class II (MHC-II) molecules occupy a pivotal position in the adaptive immune system, and correct regulation of their expression is therefore of critical importance for the control of the immune response. Several regulatory factors essential for the transcription of MHC-II genes have been identified by elucidation of the molecular defects responsible for MHC-II deficiency, a hereditary immunodeficiency disease characterized by regulatory defects abrogating MHC-II expression. Three of these factors, RFX5, RFXAP, and RFXANK, combine to form the RFX complex, a regulatory protein that binds to the X box DNA sequence present in all MHC-II promoters. In this study we have undertaken a dissection of the structure and function of RFX5, the largest subunit of the RFX complex. The results define two distinct domains serving two different essential functions. A highly conserved N-terminal region of RFX5 is required for its association with RFXANK and RFXAP, for assembly of the RFX complex in vivo and in vitro, and for binding of this complex to its X box target site in the MHC-II promoter. This N-terminal region is, however, not sufficient for activation of MHC-II expression. This requires an additional domain within the C-terminal region of RFX5. This C-terminal domain mediates cooperative binding between the RFX complex and NF-Y, a transcription factor binding to the Y box sequence of MHC-II promoters. This provides direct evidence that RFX5-mediated cooperative binding between RFX and NF-Y plays an essential role in the transcriptional activation of MHC-II genes.

scholarly journals New Functions of the Major Histocompatibility Complex Class II-Specific Transcription Factor RFXANK Revealed by a High-Resolution Mutagenesis Study

2005 ◽  
Vol 25 (19) ◽  
pp. 8607-8618 ◽  
Author(s):  
Michal Krawczyk ◽  
Krzysztof Masternak ◽  
Madeleine Zufferey ◽  
Emmanuèle Barras ◽  
Walter Reith
Keyword(s):  
Major Histocompatibility Complex ◽  
High Resolution ◽  
Major Histocompatibility Complex Class ◽  
Class Ii ◽  
Ankyrin Repeat ◽  
Complex Class ◽  
Major Histocompatibility ◽  
Mhc Ii ◽  
Histocompatibility Complex ◽  
Rfx Complex

ABSTRACT The transcription factors RFX and CIITA are major players in regulation of the expression of all classical and nonclassical major histocompatibility complex class II (MHC-II) genes. RFX nucleates the formation of a multiprotein complex, called the MHC-II enhanceosome, on MHC-II promoters. Assembly of this enhanceosome is an obligatory step for recruitment of the coactivator CIITA and thus for activation of MHC-II gene transcription. We have analyzed the function of the ankyrin repeat-containing protein RFXANK, which forms the heterotrimeric RFX complex together with RFX5 and RFXAP. We discovered that ANKRA2, the closest paralogue of RFXANK, can substitute for RFXANK in the activation of MHC-II genes and that this ability is mediated by its ankyrin repeat domain (ARD). This finding provided the basis for a high-resolution structure-function analysis of the ARD of RFXANK, which allowed us to map the RFX5 interaction domain and residues critical for assembly of the RFX complex. We also found that mutations in the fourth ankyrin repeat of RFXANK abolish assembly of the enhanceosome on MHC-II promoters in vivo but not in vitro, suggesting a new role of RFXANK in facilitating promoter occupation in the context of chromatin.

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