A Novel Element and a TEF-2-like Element Activate the Major Histocompatibility Complex Class II Transactivator in B-lymphocytes

Abstract

Major histocompatibility complex (MHC) class II molecules play a central role in immune responses, and transcription of this family of genes requires the MHC class II transactivator (CIITA). CIITA has four promoters, which are transcribed in a tissue-specific manner. CIITA promoter III is constitutively active in mature B-lymphocytes. This report now describes the minimal 319-base pair promoter region necessary for maximal transcriptional activity in B-lymphocytes. Ultraviolet light and dimethylsulfate in vivo genomic footprinting analyses reveal five occupied DNA sequence elements present in intact B-lymphocytes. Functional analysis of these elements using promoter deletions and site-specific mutations demonstrates that at least two of the sites occupied in vivo are critical for transcriptional activity. In vitro protein/DNA analysis suggests that one of the sites is a TEF-2-like element and the other is occupied by a novel transcription activator. In addition, nuclear factor-1 associates with the promoter both in vivo and in vitro. In myeloma cell lines, loss of CIITA transcription correlates with a completely unoccupied CIITA promoter III. These findings suggest that CIITA transcription in B-lymphocytes is activated through at least two strong promoter elements, while loss of expression in myeloma cells is mediated through changes in promoter assembly.Major histocompatibility complex (MHC) 1 class II molecules play a fundamental role in presenting exogenous antigenic peptides to CD4 ϩ helper T lymphocytes (1). These activated CD4 ϩ T cells release stimulatory cytokines that augment the response of CD8 ϩ cytotoxic T lymphocytes (2). Constitutive expression of MHC class II molecules is restricted to professional antigen-presenting cells, such as B lymphocytes and dendritic cells, and can be induced by interferon-␥ (IFN-␥) in macrophages, endothelial cells, and fibroblasts (3, 4). The MHC class II family of genes are coordinately regulated at the level of transcription through a conserved trimeric promoter motif (3, 5). A major component of this transcription complex was cloned by genetic complementation of an in vitro mutagenized MHC class II negative B cell line, RJ2.2.5. This component was named the MHC class II transactivator (CIITA) and restored MHC class II cell surface expression in one subgroup of bare lymphocyte syndrome patient cells (6). CIITA has since been shown to be a key regulatory molecule in the expression of all the known genes involved in the MHC class II antigen-presenting pathway (6 -9). CIITA is required for constitutive expression of MHC class II on B-lymphocytes (10) and with a few exceptions, such as in respiratory epithelial cells and dendritic cells (11,12), is required for cytokine-induced expression in other cell types. Mice in which functional CIITA protein was ablated clearly demonstrated an absolute requirement for CI-ITA in B cell expression of MHC class II (13). It has been shown that the CIITA gene has four distinct promoters each transcribing a unique first exon, which are lo...

Regulation of the Class II MHC Pathway in Primary Human Monocytes by Granulocyte-Macrophage Colony-Stimulating Factor

“…However, candidate intermediates can be predicted based on what is known of GM-CSF signaling and CIITA promoter regulation. The cis regions important for activation of PIII have been identified in B and T cells (15,35,36,39). In addition, a region upstream of PIII responsible for CIITA expression in response to IFN-␥ has been identified and involves binding of STAT-1, but not IRF-1 (40).…”

Methylation of Class IItrans-Activator Promoter IV: A Novel Mechanism of MHC Class II Gene Control

“…In addition, sites were found in promoter III that showed differential occupation in CIITA-expressing cells and in Jar cells. A recent report by Ghosh et al (50) showed that two sites in promoter III, which they designated ARE-1 and ARE-2, were critical for transcriptional activity. They also found several other sites of protein/DNA interaction in pIII, a region designated site A (Ϫ27 to Ϫ18).…”

“…However, these same regions remain unoccupied in Jar cells treated with IFN-␥, suggesting that the factors that bind these sites are either absent or unable to access the promoter in this trophoblast cell line. Recently, Ghosh et al (50) identified several regions of in vivo occupancy within pIII that were important for B cell expression. Three of these regions, Ϫ142 to Ϫ133, Ϫ66 to Ϫ56, and Ϫ27 to Ϫ18, correspond to the sites we have identified.…”

DNA Alkylating Agents Alleviate Silencing of Class II Transactivator Gene Expression in L1210 Lymphoma Cells

“…Characterization of the CIITA type III promoter by Ghosh et al (43) revealed that the sequences between Ϫ319 and ϩ1 (relative to the start site of transcription) are sufficient for optimal transcriptional activity in the human B lymphoma line Raji. To study CIITA type III promoter activity in L1210 cells, a DNA fragment spanning the region of the human type III promoter from Ϫ322 to ϩ1 was generated by PCR amplification of Raji genomic DNA and cloned into the firefly luciferase vector pGL3-basic (Promega) to generate pCIITA proIII (322)luc.…”