Specific complex formation between the type II bare lymphocyte syndrome-associated transactivators CIITA and RFX5 - PubMed (original) (raw)

Specific complex formation between the type II bare lymphocyte syndrome-associated transactivators CIITA and RFX5

T Scholl et al. Proc Natl Acad Sci U S A. 1997.

Abstract

Two of the genes defective in the five complementation groups identified in the class II-negative bare lymphocyte syndrome or corresponding laboratory mutants have been cloned. One gene encodes a protein, RFX5, that is a member of the RFX family of DNA binding proteins. The other, CIITA, encodes a large protein with a defined acidic transcriptional activation domain; this protein does not interact with DNA. Expression plasmids encoding regions of RFX5 fused to the GAL4 DNA binding domain activated transcription from a reporter construct containing GAL4 sites in a cotransfection assay in the Raji human B cell line. However, these plasmids produced transcriptional activity in HeLa cells only in conjunction with interferon gamma stimulation, a condition in which expression of both CIITA and class II major histocompatibility complex surface proteins are induced. Furthermore, these plasmids were not active in RJ2.2.5, an in vitro mutagenized derivative of Raji in which both copies of CIITA are defective. Transcriptional activation by the RFX5 fusion protein could be restored in RJ2.2.5 by cotransfection with a CIITA expression plasmid. Finally, a direct interaction between RFX5 and CIITA was detected with the yeast two-hybrid and far-Western blot assays. Thus, RFX5 can activate transcription only in cooperation with CIITA. RFX5 and CIITA associate to form a complex capable of activating transcription from class II major histocompatibility complex promoters. In this complex, promoter specificity is determined by the DNA binding domain of RFX5 and the general transcription apparatus is recruited by the acidic activation domain of CIITA.

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Figures

Figure 1

Figure 1

Regions of RFX5 that activate transcription in the Raji human B cell line. A series of 5′ deletions of the RFX5 coding region were fused to the GAL4-(1–147) DNA binding domain. These plasmids were designed to sequentially remove putative functional domains detected in the primary structure of RFX5. RFX5-(34–615) is essentially full-length, RFX5-(194–615) removes the DNA-binding domain, RFX5-(318–615) removes the central region, and RFX5-(477–615) removes the proline-rich region. Numbers used are the numbers of amino acid residues deleted from the reported translation initiation codon. pBXGAL-VP expresses the GAL4 DNA binding domain fused to the acidic activation domain of VP16-(413–490). Cells were transfected with 10 μg of pG5EC alone as a control for background activity by this reporter construct. The autoradiograph presents data from independent duplicate transfections of acetylated chloramphenicol species separated by TLC used to measure reporter gene activity.

Figure 2

Figure 2

Transcriptional activity by RFX5–GAL4 fusion proteins is induced by IFN-γ in HeLa cells. Autoradiographs of reporter gene activity from transfected control HeLa cells and HeLa cells treated with IFN-γ. Cells were transfected with 10 μg of the indicated plasmids. Samples transfected with GAL4 fusion proteins were cotransfected with 10 μg of pG5EC.

Figure 3

Figure 3

Transcriptional activity by RFX4–GAL4 fusion proteins in RJ2.2.5 cells required CIITA. Autoradiographs of reporter gene activity from RJ2.2.5 cells cotransfected with the indicated plasmids and 10 μg of pG5EC and cotransfections that also included 10 μg of pCMVCIITA.

Figure 4

Figure 4

Interaction between RFX5 and CIITA detected in yeast. Histograms depict β-galactosidase reporter gene activity in yeast extracts from cells transformed with RFX5–GAL4 fusion protein expression plasmids or cotransformation that included a plasmid for expression of CIITA fused to the GAL4 activation domain. All data represent the mean activity detected in triplicate transformants. Individual measurements were within 5% of each other. pGBT9 encodes the GAL4 DNA binding domain alone. pTD-1 used in the positive control encodes a fusion protein made up of amino acids 84–708 of simian virus 40 large tumor antigen and GAL4 activation domain and pVa3 encodes a fusion protein made up of GAL4 DNA binding domain and amino acids 72–350 of p53 with which the simian virus 40T antigen interacts.

Figure 5

Figure 5

Detection of an interaction between RFX5 and CIITA by far-Western blot analysis. Recombinant proteins (5 μg) were electrophoresed and blotted on quadruplicate gels. Proteins were refolded on the membranes and probed with the indicated radiolabeled _in vitro_-translated proteins.

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