Identification of Class II Transcriptional Activator-Induced Genes by Representational Difference Analysis: Discoordinate Regulation of the DNα/DOβ Heterodimer

Abstract

Class II transcriptional activator (CIITA) is a master regulator of MHC class II genes, including DR, DP, and DQ, and MHC class II-associated genes DM and invariant chain. To determine the repertoire of genes that is regulated by CIITA and to identify uncharacterized CIITA-inducible genes, we used representational difference analysis. Representational difference analysis screens for differentially expressed transcripts. All CIITA-induced genes were MHC class II related. We have identified the α subunit, DNα, of the class II processing factor DO as an additional CIITA-inducible gene. Northern analysis confirmed that DNα is induced by IFN-γ in 2fTGH fibrosarcoma cells, and CIITA is necessary for high-level expression in B cells. The β subunit, DOβ, is not inducible in fibrosarcoma cells by IFN-γ or exogenous CIITA expression. Moreover, in contrast to other class II genes, DOβ expression remains high in the absence of CIITA in B cells. The promoters for DNα and DOβ contain the highly conserved WXY motifs, and, like other class II genes, expression of both DNα and DOβ requires RFX. These findings demonstrate that both DNα and DOβ are regulated by RFX. However, DNα is defined for the first time as a CIITA-inducible gene, and DOβ as a MHC class II gene whose expression is independent of CIITA.