The S box of major histocompatibility complex class II promoters is a key determinant for recruitment of the transcriptional co-activator CIITA.
Tightly regulated expression of major histocompatibility complex (MHC) class II genes is critical for the immune system. A conserved regulatory module consisting of four cis-acting elements, the W, X, X2 and Y boxes, controls transcription of MHC class II genes. The X, X2, and Y boxes are bound, respectively, by RFX, CREB, and NF-Y to form a MHC class II-specific enhanceosome complex. The latter constitutes a landing pad for recruitment of the transcriptional co-activator CIITA. In contrast to the well defined roles of the X, X2, and Y boxes, the role of the W region has remained controversial. In vitro binding studies have suggested that it might contain a second RFX-binding site. We demonstrate here by means of promoter pull-down assays that the most conserved subsequence within the W region, called the S box, is a critical determinant for tethering of CIITA to the enhanceosome complex. Binding of CIITA to the enhanceosome requires both integrity of the S box and a remarkably stringent spacing between the S and X boxes. Even a 1-2-base pair change in the native S-X distance is detrimental for CIITA recruitment and promoter function. In contrast to current models, binding of RFX to a putative duplicated binding site in the W box is thus not required for either CIITA recruitment or promoter activity. This paves the way for the identification of novel factors mediating the contribution of the S box to the activation of MHC class II promoters.
University of Geneva Medical School, Department of Pathology and Immunology, Centre Médical Universitaire, 1 rue Michel-Servet, CH-1211, Geneva, Switzerland.
Annick
A
Muhlethaler-Mottet
Michal
M
Krawczyk
Krzysztof
K
Masternak
Charalambos
C
Spilianakis
Androniki
A
Kretsovali
Joseph
J
Papamatheakis
Walter
W
Reith
D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D005802:Genes, MHC Class II; D006801:Humans; D015151:Immunoblotting; D008156:Luciferases / Q000378:metabolism; D008285:Major Histocompatibility Complex; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D015534:Trans-Activators / Q000235:genetics*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
C083728:MHC class II transactivator protein; D009687:Nuclear Proteins; D015534:Trans-Activators; D008156:Luciferases