Major histocompatibility complex class II transcriptional platform: assembly of nuclear factor Y and regulatory factor X (RFX) on DNA requires RFX5 dimers

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.