MYC directly transactivates CR2/CD21, the receptor of the Epstein-Barr virus, enhancing the viral infection of Burkitt lymphoma cells.

MYC is an oncogenic transcription factor dysregulated in about half of total human tumors. While transcriptomic studies reveal more than 1000 genes regulated by MYC, a much smaller fraction of genes is directly transactivated by MYC. Virtually all Burkitt lymphoma (BL) carry chromosomal translocations involving MYC oncogene. Most endemic BL and a fraction of sporadic BL are associated with Epstein-Barr virus (EBV) infection. The currently accepted mechanism is that EBV is the BL-causing agent inducing MYC translocation. Herein we show that the EBV receptor, CR2 (also called CD21), is a direct MYC target gene. This is based on several pieces of evidence: MYC induces CR2 expression in both proliferating and arrested cells and in the absence of protein synthesis, binds the CR2 promoter and transactivates CR2 in an E-box-dependent manner. Moreover, using mice with conditional MYC ablation we show that MYC induces CR2 in primary B cells. Importantly, modulation of MYC levels directly correlates with EBV's ability of infection in BL cells. Altogether, in contrast to the widely accepted hypothesis for the correlation between EBV and BL, we propose an alternative hypothesis in which MYC dysregulation could be the first event leading to the subsequent EBV infection.

To Be, or Notch to Be: Mediating Cell Fate from Embryogenesis to Lymphopoiesis.

Notch signaling forms an evolutionarily conserved juxtacrine pathway crucial for cellular development. Initially identified in Drosophila wing morphogenesis, Notch signaling has since been demonstrated to play pivotal roles in governing mammalian cellular development in a large variety of cell types. Indeed, abolishing Notch constituents in mouse models result in embryonic lethality, demonstrating that Notch signaling is critical for development and differentiation. In this review, we focus on the crucial role of Notch signaling in governing embryogenesis and differentiation of multiple progenitor cell types. Using hematopoiesis as a diverse cellular model, we highlight the role of Notch in regulating the cell fate of common lymphoid progenitors. Additionally, the influence of Notch through microenvironment interplay with lymphoid cells and how dysregulation influences disease processes is explored. Furthermore, bi-directional and lateral Notch signaling between ligand expressing source cells and target cells are investigated, indicating potentially novel therapeutic options for treatment of Notch-mediated diseases. Finally, we discuss the role of cis-inhibition in regulating Notch signaling in mammalian development.

Notch signaling induces a transcriptionally permissive state at the Complement C3d Receptor 2 (CR2) promoter in a pre-B cell model.

During mammalian lymphoid development, Notch signaling is necessary at multiple stages of T lymphopoiesis, including lineage commitment, and later stages of T cell effector differentiation. In contrast, outside of a defined role in the development of splenic marginal zone B cells, there is conflicting evidence regarding whether Notch signaling plays functional roles in other B cell sub-populations. Complement receptor 2 (CR2) modulates BCR-signaling and is tightly regulated throughout differentiation. During B lymphopoiesis, CR2 is detected on immature and mature B cells with high surface expression on marginal zone B cells. Here, we have explored the possibility that Notch regulates human CR2 transcriptional activity using in vitro models including a co-culture system, co-transfection gene reporters and chromatin accessibility assays. We provide evidence that Notch signaling regulates CR2 promoter activity in a mature B cell line, as well as the induction of endogenous CR2 mRNA in a non-expressing pre-B cell line. The dynamics of endogenous gene activation suggests additional unidentified factors are required to mediate surface CR2 expression on immature and mature B lineage cells.

Preferential association of a functional variant in complement receptor 2 with antibodies to double-stranded DNA.

OBJECTIVES: Systemic lupus erythematosus (SLE; OMIM 152700) is characterised by the production of antibodies to nuclear antigens. We previously identified variants in complement receptor 2 (CR2/CD21) that were associated with decreased risk of SLE. This study aimed to identify the causal variant for this association. METHODS: Genotyped and imputed genetic variants spanning CR2 were assessed for association with SLE in 15 750 case-control subjects from four ancestral groups. Allele-specific functional effects of associated variants were determined using quantitative real-time PCR, quantitative flow cytometry, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP)-PCR. RESULTS: The strongest association signal was detected at rs1876453 in intron 1 of CR2 (pmeta=4.2×10(-4), OR 0.85), specifically when subjects were stratified based on the presence of dsDNA autoantibodies (case-control pmeta=7.6×10(-7), OR 0.71; case-only pmeta=1.9×10(-4), OR 0.75). Although allele-specific effects on B cell CR2 mRNA or protein levels were not identified, levels of complement receptor 1 (CR1/CD35) mRNA and protein were significantly higher on B cells of subjects harbouring the minor allele (p=0.0248 and p=0.0006, respectively). The minor allele altered the formation of several DNA protein complexes by EMSA, including one containing CCCTC-binding factor (CTCF), an effect that was confirmed by ChIP-PCR. CONCLUSIONS: These data suggest that rs1876453 in CR2 has long-range effects on gene regulation that decrease susceptibility to lupus. Since the minor allele at rs1876453 is preferentially associated with reduced risk of the highly specific dsDNA autoantibodies that are present in preclinical, active and severe lupus, understanding its mechanisms will have important therapeutic implications.

Focused transcription from the human CR2/CD21 core promoter is regulated by synergistic activity of TATA and Initiator elements in mature B cells.

Complement receptor 2 (CR2/CD21) is predominantly expressed on the surface of mature B cells where it forms part of a coreceptor complex that functions, in part, to modulate B-cell receptor signal strength. CR2/CD21 expression is tightly regulated throughout B-cell development such that CR2/CD21 cannot be detected on pre-B or terminally differentiated plasma cells. CR2/CD21 expression is upregulated at B-cell maturation and can be induced by IL-4 and CD40 signaling pathways. We have previously characterized elements in the proximal promoter and first intron of CR2/CD21 that are involved in regulating basal and tissue-specific expression. We now extend these analyses to the CR2/CD21 core promoter. We show that in mature B cells, CR2/CD21 transcription proceeds from a focused TSS regulated by a non-consensus TATA box, an initiator element and a downstream promoter element. Furthermore, occupancy of the general transcriptional machinery in pre-B versus mature B-cell lines correlate with CR2/CD21 expression level and indicate that promoter accessibility must switch from inactive to active during the transitional B-cell window.

TNF-inducible expression of lymphotoxin-ß in hepatic cells: an essential role for NF-κB and Ets1 transcription factors.

As TNF is one of the earliest signals that can be detected in the leukocyte-derived inflammatory cascade which drives subsequent cytokine production, we are interested in determining whether TNF is one of the initiating factors controlling liver remodeling and regeneration following chronic liver damage. One of the early responses is the expression of lymphotoxin-ß by hepatic progenitor oval cells. The aim of this study was to determine whether hepatic expression of LT-ß was controlled by TNF and to understand the basis of this regulation. We previously showed that LT-ß expression is transcriptionally controlled via the TNF-induced, inflammatory NF-κB pathway in T lymphocytes. Here we show that TNF is able to upregulate LT-ß expression in hepatic cells at the transcriptional level by the binding of NF-κB p50/p65 heterodimers and Ets1 to their respective sites in the LT-ß promoter.

Transcriptional effects of a lupus-associated polymorphism in the 5' untranslated region (UTR) of human complement receptor 2 (CR2/CD21).

Systemic lupus erythematosus (SLE) is a complex autoimmune disease with a strong genetic component that determines risk. A common three single-nucleotide polymorphism (SNP) haplotype of the complement receptor 2 (CR2) gene has been associated with increased risk of SLE (Wu et al., 2007; Douglas et al., 2009), and a less common haplotype consisting of the major allele at SNP1 and minor alleles at SNP2 and 3 confers protection (Douglas et al., 2009). SNP1 (rs3813946), which is located in the 5' untranslated region (UTR) of the CR2 gene, altered transcriptional activity of a CR2 promoter-luciferase reporter gene construct transiently transfected into a B cell line (Wu et al., 2007) and had an independent effect in the protective haplotype (Douglas et al., 2009). In this study, we show that this SNP alters transcriptional activity in a transiently transfected non B-cell line as well as in stably transfected cell lines, supporting its relevance in vivo. Furthermore, the allele at this SNP affects chromatin accessibility of the surrounding sequence and transcription factor binding. These data confirm the effects of rs3813946 on CR2 transcription, identifying the 5' UTR to be a novel regulatory element for the CR2 gene in which variation may alter gene function and modify the development of lupus.

Cell- and stage-specific chromatin structure across the Complement receptor 2 (CR2/CD21) promoter coincide with CBF1 and C/EBP-beta binding in B cells.

Stringent developmental transcription requires multiple transcription factor (TF) binding sites, cell-specific expression of signaling molecules, TFs and co-regulators and appropriate chromatin structure. During B-lymphopoiesis, human Complement receptor 2 (CR2/CD21) is detected on immature and mature B cells but not on B cell precursors and plasma cells. We examined cell- and stage-specific human CR2 gene regulation using cell lines modeling B-lymphopoiesis. Chromatin accessibility assays revealed a region between -409 and -262 with enhanced accessibility in mature B cells and pre-B cells, compared to either non-lymphoid or plasma cell-types, however, accessibility near the transcription start site (TSS) was elevated only in CR2-expressing B cells. A correlation between histone acetylation and CR2 expression was observed, while histone H3K4 dimethylation was enriched near the TSS in both CR2-expressing B cells and non-expressing pre-B cells. Candidate sites within the CR2 promoter were identified which could regulate chromatin, including a matrix attachment region associated with CDP, SATB1/BRIGHT and CEBP-beta sites as well as two CBF1 sites. ChIP assays verified that both CBF1 and C/EBP-beta bind the CR2 promoter in B cells raising the possibility that these factors facilitate or respond to alterations in chromatin structure to control the timing and/or level of CR2 transcription.

TNF and phorbol esters induce lymphotoxin-beta expression through distinct pathways involving Ets and NF-kappa B family members.

Lymphotoxin-beta (LT-beta) is a transmembrane protein expressed mainly on cells of the lymphoid lineage. It associates with LT-alpha on the cell surface to form the heterotrimeric LTalpha1,beta2 complex, which binds the LT-beta receptor. Membrane lymphotoxin is a crucial signal for the appropriate development of lymph nodes and Peyer's patches, and in the formation of B and T cell compartments in the spleen. In this study we report the characterization of mechanisms governing both basal as well as PMA- and TNF-inducible regulation of the human LT-beta promoter. Using a Jurkat T cell line, induction with either PMA or TNF resulted in an increase in mRNA levels compared with uninduced values. This induction corresponded to an increase in transcriptional activity of the human LT-beta promoter. Mutational and deletion analysis demonstrated the importance of Ets and NF-kappaB motifs in the regulation of basal transcription. Furthermore, the ability of PMA to induce activity was lost in the Ets mutant constructs. Interestingly, the same mutation had little effect on the ability of TNF to induce transcription of the LT-beta promoter. TNF inducibility was localized to the NF-kappaB site positioned at -83 of the promoter sequence. Thus, it appears that the Ets site, although playing a major role in PMA induction, did not mediate TNF inducibility. Therefore, our study suggests that alternative signaling pathways may be present to induce the expression of LT-beta in response to different immunological or inflammatory stimuli.

Functional analysis of the human complement receptor 2 (CR2/CD21) promoter: characterization of basal transcriptional mechanisms.

Human complement receptor (CR) type 2 (CR2/CD21) is a 145-kDa membrane protein encoded within the regulators of complement activation gene cluster localized on human chromosome 1q32. Understanding the mechanisms that regulate CR2 expression is important because CR2 is expressed during specific stages of B cell development, and several lines of evidence suggest a role for altered CR2 function or expression in a number of autoimmune diseases. Additionally, even modest changes in CR2 expression are likely to affect relative B cell responses. In this study we have delineated the transcriptional requirements of the human CR2 gene. We have studied the human CR2 proximal promoter and identified sites important for controlling the level of transcription in CR2-expressing cells. We have determined that four functionally relevant sites lie within very close proximity to the transcriptional initiation site. These sites bind the transcription factors USF1, an AP-2-like transcription factor, and Sp1.