Promoter-specific functions of CIITA and the MHC class II enhanceosome in transcriptional activation

Abstract

Transcription of the major histocompatibility complex class II family of genes is regulated by conserved promoter elements and two gene-specific trans-activators, RFX and CIITA. RFX binds DNA and nucleates the assembly of an enhanceosome, which recruits CIITA through protein--protein interactions. Transcriptional activation is a complex, multi-step process involving chromatin modification and recruitment of the transcription apparatus. To examine the roles of the enhanceosome and CIITA in these processes, we analysed the level of promoter-associated hyperacetylated histones H3 and H4, TBP, TFIIB and RNA poly merase II in cells lacking RFX or CIITA. We compared four genes co-regulated by RFX and CIITA (HLA-DRA, HLA-DPB, HLA-DMB and Ii) and found that the enhanceosome and CIITA make variable, promoter-dependent contributions to histone acetylation and transcription apparatus recruitment. CIITA is generally implicated at multiple levels of the activation process, while the enhanceosome contributes in a CIITA-independent manner only at certain promoters. Our results support the general notion that the impact of a particular activator on transcription in vivo may vary depending on the promoter and the chromatin context.

Fig. 1. Regulation of MHCII transcription and molecular defects in BLS. Transcription of MHCII and related genes is directed by a conserved regulatory module consisting of the S, X, X2 and Y ‘boxes’. This module is the target of four essential MHCII-specific trans-activators, RFX5, RFXAP, RFXANK (RFXB) and CIITA, identified by virtue of the fact that they are mutated in BLS. The first three factors assemble into a heterotrimeric complex called RFX. Binding of RFX to the X box nucleates the assembly of an enhanceosome complex containing RFX, X2BP, NF-Y and an as yet unidentified S box-binding factor. The enhanceosome serves as a landing pad for the coactivator CIITA. (A) In cells deficient in one of the RFX subunits (in this study, RFXANK–/– BLS1 cells), the RFX complex cannot assemble and the enhanceosome cannot form. MHCII promoters thus remain unoccupied. (B) In B cells deficient in CIITA (RJ2.2.5), RFX is functional and the promoters are stably occupied by the enhanceosome. However, they remain silent because CIITA is missing. (C) In wild-type cells (Raji and BLS1c), enhanceosome assembly and CIITA recruitment lead to promoter activation.

Fig. 2. MHCII and related genes are co-regulated by RFX and CIITA through the MHCII regulatory module. (A) Schematic representation of the upstream promoter-proximal regions of HLA-DRA (DRA), HLA-DPB (DPB), HLA-DMB (DMB) and Ii. The S–X–X2–Y elements of the MHCII regulatory module are present in all promoters and are shown as filled boxes. The other potential or confirmed regulatory sequences are shown as empty boxes: Oct, octamer-binding site; NF-1, CTF/NF-1-binding site; GC, Sp1-binding site; NFkB, binding site for NFκB/Rel family members; ISRE, interferon-stimulated response element; Y′, an imperfect Y box (NF-Y-binding site). The promoters are drawn to scale and the distance (in bp) from the transcription initiation site (arrow) is shown at the bottom. (B) The effect of a deficiency in CIITA or RFXANK on DRA, DPB, DMB and Ii expression. Steady-state levels of the corresponding mRNAs were compared between isogenic pairs of B-cell lines (wild-type versus mutant). mRNA levels were determined by multiplex real-time RT–PCR and are represented relative to the wild type.

Fig. 3. Occupancy of MHCII and related promoters by the enhanceosome and CIITA. The association of RFX5 with promoter DNA is indicative of MHCII enhanceosome assembly. Antibodies specific for RFX5 or CIITA were used to immunoprecipitate crosslinked chromatin fragments. Immunoprecipitates were then analysed for the abundance of specific promoter sequences by PCR. The inset (top left) shows an example of a classical ChIP analysis; immunoprecipitated fractions (IP, lanes 1–3) and sequential dilutions of input chromatin from wild-type B cells (lanes 4–7) were amplified with DRA-specific primers and PCR products were analysed by gel electrophoresis. Pre-immune serum (p.i.) was used as negative control. In subsequent experiments, the levels of RFX5 and CIITA binding to different promoters in CIITA- and RFXANK-deficient cells and their matching wild-type counterparts were measured by quantitative real-time PCR. Association of RFX5 (A) and CIITA (B) with the DRA promoter. The unrelated CD20, Langerin and HSP70-2 promoters are included as controls. Results are shown relative to the values observed for the DRA promoter in wild-type cells. Association of RFX5 (C) and CIITA (D) with the DPB, DMB and Ii promoters. Results for the mutant cells (RJ2.2.5 and BLS1) are given relative to the values observed in the matching wild-type cells (Raji and BLS1c).

Fig. 4. Contribution of CIITA and the MHCII enhanceosome to histone acetylation at the DRA promoter. Histone acetylation at the DRA and Langerin promoters was analysed by quantitative ChIP using antibodies specific for acetylated histone H3 (A) or acetylated histone H4 (B). Results are normalized with respect to the values obtained with the internal control promoter HSP70-2, and are represented relative to the values observed for the DRA promoter in wild-type cells.

Fig. 5. Contribution of CIITA and the MHCII enhanceosome to histone acetylation at different MHCII and related promoters. Histone acetylation at the DRA, DPB, DMB and Ii promoters was analysed as in Figure 4. Results are normalized and represented relative to the values observed in the matching wild-type cells.

Fig. 6. Contribution of CIITA and the enhanceosome to GTM recruitment at the DRA promoter. GTM recruitment to the DRA and Langerin promoters was analysed by quantitative ChIP experiments using antibodies specific for TBP (A), TFIIB (B) and RNA Pol II (C). Results are represented as in Figure 4.

Fig. 7. Contributions of CIITA and the enhanceosome to GTM recruitment at different MHCII and related promoters. Recruitment of TBP (A), TFIIB (B) and RNA Pol II (C) to the DRA, DPB, DMB and Ii promoters was analysed by quantitative ChIP and is represented as in Figure 5.