Positive Regulatory Domain I (PRDM1) and IRF8/PU.1 Counter-regulate MHC Class II Transactivator (CIITA) Expression during Dendritic Cell Maturation

Matthew A Smith,Gabriela Wright,Jian Wu,Prafullakumar Tailor,Keiko Ozato,Xianghong Chen,Sheng Wei,Janet F Piskurich,Jenny P.-Y Ting,Kenneth L Wright

doi:10.1074/jbc.m110.165431

Abstract

Dendritic cells (DCs) are key mediators of immune function through robust and tightly regulated presentation of antigen in the context of the MHC Class II. MHC Class II expression is controlled by the transactivator CIITA. CIITA expression in conventional DCs is uniquely dependent on an uncharacterized myeloid cell-specific promoter, CIITApI. We now identify in vivo the promoter structure and factors regulating CIITApI. In immature DCs transcription requires binding of PU.1, IRF8, NFκB, and Sp1 to the promoter. PU.1 binds independently at one site and in a required heterodimer with IRF8 at a composite element. DCs from IRF8-null mice have an unoccupied CIITApI promoter that can be rescued by reconstitution with IRF8 in vitro. Furthermore, mutation of either PU.1 site or the IFR8 site inhibits transcriptional activation. In vivo footprinting and chromatin immunoprecipitation reveals that DC maturation induces complete disassociation of the bound activators paralleled by recruitment of PRDM1/Blimp-1 to the promoter. PRDM1 is a transcriptional repressor with essential roles in B cells, T cells, NK cells, and DCs. We show that PRDM1 co-repressors, G9a and HDAC2, are recruited to CIITApI, leading to a loss of histone acetylation and acquisition of histone H3K9 dimethylation and heterochromatin protein 1γ (HP1γ). PRDM1 binding also blocks IRF8-mediated activation dependent on the PU.1/IRF composite element. Together these findings reveal the mechanisms regulating CIITA and, thus, antigen presentation in DCs, demonstrating that PRDM1 and IRF8/PU.1 counter-regulate expression. The activity of PRDM1 in silencing all three cell type-specific CIITA promoters places it as a central regulator of antigen presentation.

Highlights

In Vivo Genomic Footprinting Analysis Detects Multiple Protein/DNA Interactions over the CIITA from promoter I (CIITApI) Promoter in Dendritic cells (DCs)— MHC-II expression is critical for the antigen presentation function of DCs, and the cell surface MHC-II levels are significantly increased during DC maturation
We examined the CIITApIII promoter as mRNA from this promoter was detected in monocyte-derived DC, and previous in vivo footprinting studies showed that it was occupied by transcription factors [5]
In this report we have provided evidence for counter regulation of CIITA by IRF8/PU.1 and PRDM1 during the maturation of human monocyte-derived DCs

Summary

Introduction

In Vivo Genomic Footprinting Analysis Detects Multiple Protein/DNA Interactions over the CIITApI Promoter in DCs— MHC-II expression is critical for the antigen presentation function of DCs, and the cell surface MHC-II levels are significantly increased during DC maturation. PU.1, IRF8, NF␬B, and Sp1 Associate with the CIITApI in Vitro—To examine whether the candidate transcription factors implicated by the in vivo footprinting directly interact with their specific binding motif in vitro, we performed EMSAs using nuclear extracts prepared from primary cultured human immature monocyte-derived DCs. A probe spanning the tandem PU.1 sites at Ϫ53 forms two fast migrating complexes that are both diminished by an antibody to PU.1 but not to Sp1 (Fig. 4A).

Results

Conclusion