Abstract
MHC class II (MHCII) genes are transactivated by the NOD-like receptor (NLR) family member CIITA, which is recruited to SXY enhancers of MHCII promoters via a DNA-binding “enhanceosome” complex. NLRC5, another NLR protein, was recently found to control transcription of MHC class I (MHCI) genes. However, detailed understanding of NLRC5’s target gene specificity and mechanism of action remained lacking. We performed ChIP-sequencing experiments to gain comprehensive information on NLRC5-regulated genes. In addition to classical MHCI genes, we exclusively identified novel targets encoding non-classical MHCI molecules having important functions in immunity and tolerance. ChIP-sequencing performed with Rfx5−/− cells, which lack the pivotal enhanceosome factor RFX5, demonstrated its strict requirement for NLRC5 recruitment. Accordingly, Rfx5-knockout mice phenocopy Nlrc5 deficiency with respect to defective MHCI expression. Analysis of B cell lines lacking RFX5, RFXAP, or RFXANK further corroborated the importance of the enhanceosome for MHCI expression. Although recruited by common DNA-binding factors, CIITA and NLRC5 exhibit non-redundant functions, shown here using double-deficient Nlrc5−/−CIIta−/− mice. These paradoxical findings were resolved by using a “de novo” motif-discovery approach showing that the SXY consensus sequence occupied by NLRC5 in vivo diverges significantly from that occupied by CIITA. These sequence differences were sufficient to determine preferential occupation and transactivation by NLRC5 or CIITA, respectively, and the S box was found to be the essential feature conferring NLRC5 specificity. These results broaden our knowledge on the transcriptional activities of NLRC5 and CIITA, revealing their dependence on shared enhanceosome factors but their recruitment to distinct enhancer motifs in vivo. Furthermore, we demonstrated selectivity of NLRC5 for genes encoding MHCI or related proteins, rendering it an attractive target for therapeutic intervention. NLRC5 and CIITA thus emerge as paradigms for a novel class of transcriptional regulators dedicated for transactivating extremely few, phylogenetically related genes.
Highlights
Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) constitute a family of innate immune receptors involved mainly in inflammatory responses and cell death
Our work addresses these questions, delineating the unique consensus sequence required for NLR caspase recruitment domain containing protein 5 (NLRC5) recruitment and pinpointing conserved features conferring its specificity
Through genome-wide analyses, we confirm that NLRC5 regulates classical major histocompatibility complex (MHC) class I (MHCI) genes and identify novel target genes, all encoding non-classical MHCI molecules exerting an array of functions in immunity and tolerance
Summary
Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) constitute a family of innate immune receptors involved mainly in inflammatory responses and cell death. The NLR family member CIITA instead functions as the master transcriptional regulator of major histocompatibility complex (MHC) class II (MHCII) genes, and mutations in the CIITA gene lead to severe immunodeficiency [1]. NLR caspase recruitment domain containing protein 5 (NLRC5) was shown to regulate transcription of MHC class I (MHCI) genes, primarily in lymphocytes, where it is highly expressed [2,3,4,5,6]. Overexpression of NLRC5 was initially found to increase mRNA levels for genes encoding human MHCI molecules and proteins functioning in the MHCI-mediated antigen presentation pathway, including beta-2-microglobulin (B2M), transporter associated with antigen processing 1 (TAP1) and the proteasome subunit beta type-9 (PSMB9) [3]. In vivo promoter occupancy by NLRC5 was demonstrated only for human HLA-A and HLA-B, and mouse H2-K, H2-D, and B2m [2,3]
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