Characterization of the RFX complex and the RFX5(L66A) mutant: implications for the regulation of MHC class II gene expression.
Major histocompatability complex class II (MHCII) molecules are an essential component of the mammalian adaptive immune response. The expression of MHCII genes is regulated by a cell-specific multiprotein complex, termed the MHCII enhanceosome. The heterotrimeric RFX complex is the key DNA-binding component of the MHCII enhanceosome. The RFX complex is comprised of three proteins, RFXB, RFXAP, and RFX5, all of which are required for DNA binding and activation of MHCII gene expression. Static light scattering and chemical cross-linking of the three RFX proteins show that RFXB and RFXAP are monomers and that RFX5 dimerizes through two separate domains. One of these domains, the oligomerization domain, promotes formation of a dimer of dimers of RFX5. In addition, we show that the RFX complex forms a 2:1:1 complex of RFX5.RFXAP.RFXB, which can associate with a further dimer of RFX5 to form a 4:1:1 complex through the oligomerization domain of RFX5. On the basis of these studies, we propose DNA-binding models for the interaction between the RFX complex and the MHCII promoter including a DNA looping model. We also provide direct evidence that the RFX5(L66A) point mutation prevents dimerization of the RFX complexes and propose a model for how this results in a loss of MHCII gene expression.
Garvie
Colin W
CW
Department of Chemistry and Biochemistry, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250, USA. garvie@umbc.edu
Stagno
Jason R
JR
Reid
Sarah
S
Singh
Ashina
A
Harrington
Erik
E
Boss
Jeremy M
JM
D002234:Carbodiimides / Q000737:chemistry; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics*; D005786:Gene Expression Regulation; D005802:Genes, MHC Class II / Q000502:physiology*; D006801:Humans; D008954:Models, Biological; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics; D020836:Protein Structure, Quaternary; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics; D014461:Ultracentrifugation
C518491:1-ethyl-3-(3-(diethylamino)propyl)carbodiimide; D002234:Carbodiimides; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D010446:Peptide Fragments; C093524:RFX5 protein, human; C117892:RFXANK protein, human; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; D004247:DNA