Major Histocompatibility Complex Class II Promoters Research Articles

The Atlantic Salmon Mhc Class II α and β Promoters are Active in Mammalian Cell Lines

The major histocompatibility complex class II (MHCII) genes are only constitutively expressed in certain immune response cells such as B cells, macrophages, dendritic cells and other antigen presenting cells. This cell specific expression pattern and the presence of conserved regions such as the X-, X2-, Y-, and W-boxes make the MHCII promoters especially interesting as vector constructs. We tested whether the Atlantic salmon (Salmo salar L.) MHCII promoters can function in cell lines from other organisms. We found that the salmon MHCII α and MHCII β promoters could drive expression of a LacZ reporter gene in adherent lymphoblast cell lines from dog (DH82) and rabbit (HybL-L). This paper shows that the promoters of Atlantic salmon MHCII α and β genes can function in mammalian cell lines.

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

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.

CIITA and the MHCII Enhanceosome in the Regulation of MHCII Expression

Major Histocompatibility Complex class II (MHCII) molecules direct the development, activation and homeostasis of CD4+ T cells. Given these key functions it is not surprising that the absence of MHCII expression results in a severe primary immunodeficiency disease called MHCII deficiency or the Bare Lymphocyte Syndrome (BLS). The genetic defects responsible for BLS lie in genes encoding transcription factors required for MHCII expression. Four different MHCII regulatory genes encoding RFXANK, RFX5, RFXAP and CIITA have been identified. The first three are subunits of RFX, a ubiquitously expressed factor that binds cooperatively with other proteins to MHCII and related promoters to form a highly stable macromolecular nucleoprotein complex referred to as the MHCII enhanceosome. This enhanceosome serves as a landing pad for the MHCII transactivator CIITA. CIITA is a non-DNA binding coactivator that serves as the master control factor for MHCII expression. The highly regulated expression pattern of CIITA ultimately dictates the cell type specificity, induction and level of MHCII expression. The enhanceosome and CIITA collaborate in activating transcription by promoting histone hyperacetylation and by recruiting components of the general transcription machinery. In this review we summarize what is known about the molecular basis of BLS and what this has taught us about the mechanisms regulating transcription of MHCII and related genes. Particular attention is devoted to the structure, function and mode of action of the MHCII enhanceosome and CIITA. In addition, we focus on the highly regulated and cell type specific expression of CIITA.

Phosphorylation of CIITA directs its oligomerization, accumulation and increased activity on MHCII promoters.

The class II transactivator (CIITA) is the master regulator of major histocompatibility complex class II (MHCII) transcription. Its activity is regulated at the post-transcriptional level by phosphorylation and oligomerization. This aggregation mapped to and depended on the phosphorylation of residues between positions 253 and 321 in CIITA, which resulted in a dramatic accumulation of the protein and increased expression of MHCII genes in human promonocytic U937 cells, which represent immature antigen-presenting cells. Thus, the post-transcriptional modification of CIITA plays an important role in the immune response.

A functionally essential domain of RFX5 mediates activation of major histocompatibility complex class II promoters by promoting cooperative binding between RFX and NF-Y.

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.

CIITA is a transcriptional coactivator that is recruited to MHC class II promoters by multiple synergistic interactions with an enhanceosome complex

By virtue of its control over major histocompatibility complex class II (MHC-II) gene expression, CIITA represents a key molecule in the regulation of adaptive immune responses. It was first identified as a factor that is defective in MHC-II deficiency, a hereditary disease characterized by the absence of MHC-II expression. CIITA is a highly regulated transactivator that governs all spatial, temporal, and quantitative aspects of MHC-II expression. It has been proposed to act as a non-DNA-binding transcriptional coactivator, but evidence that it actually functions at the level of MHC-II promoters was lacking. By means of chromatin immunoprecipitation assays, we show here for the first time that CIITA is physically associated with MHC-II, as well as HLA-DM, Ii, MHC-I, and beta(2)m promoters in vivo. To dissect the mechanism by which CIITA is recruited to the promoter, we have developed a DNA-dependent coimmunoprecipitation assay and a pull-down assay using immobilized promoter templates. We demonstrate that CIITA recruitment depends on multiple, synergistic protein-protein interactions with DNA-bound factors constituting the MHC-II enhanceosome. CIITA therefore represents a paradigm for a novel type of regulatory and gene-specific transcriptional cofactor.

RFXAP, a novel subunit of the RFX DNA binding complex is mutated in MHC class II deficiency.

Major Histocompatibility Complex class II (MHC-II) deficiency is a disease of gene regulation that provides a unique opportunity for the genetic dissection of the molecular mechanisms controlling transcription of MHC-II genes. Cell lines from MHC-II deficiency patients have been assigned to three complementation groups (A, B and C) believed to reflect the existence of distinct essential MHC-II regulatory genes. Groups B and C, as well as an in vitro generated regulatory mutant representing a fourth group (D), are characterized by a specific defect in the binding activity of RFX, a multimeric DNA binding complex that is essential for activation of MHC-II promoters. RFX5, a subunit of RFX, was recently shown to be mutated in group C. We have now isolated a novel gene, RFXAP (RFX Associated Protein), that encodes a second subunit of the RFX complex. RFXAP is mutated in the 6.1.6 cell line (group D), as well as in an MHC-II deficiency patient (DA). This establishes that group D is indeed a fourth MHC-II deficiency complementation group. Complementation of the 6.1.6 and DA cell lines by transfection with RFXAP fully restores expression of all endogenous MHC-II genes in vivo, demonstrating that RFXAP is a novel essential MHC-II regulatory gene.