Cyclooxygenase-2 gene transcription in a macrophage model of inflammation.

Infections involving LPS-bearing, Gram-negative bacteria can lead to acute inflammation and septic shock. Cyclooxygenase-2 (COX-2), the target of nonsteroidal anti-inflammatory drugs and selective COX-2 inhibitors, is importantly involved in these responses. We examined the dynamics of COX-2 gene expression in RAW264.7 murine macrophages treated with LPS as a model for COX-2 gene expression during inflammation. We established, using Northern blotting, nuclear run-on assays, and RT-PCR, that COX-2 transcriptional activation continues for at least 12 h after LPS treatment and involves at least three phases. Previous studies with murine macrophages identified an NF-kappaB site, a C/EBP site, and a cAMP response element-1 (CRE-1) as cis-acting elements in the COX-2 promoter. We identified three additional functional elements including a second CRE (CRE-2), an AP-1 site, and an E-box that overlaps CRE-1. The E-box mediates transcriptional repression whereas the other cis-elements are activating. Using electrophoretic mobility supershift and chromatin immunoprecipitation assays, we cataloged binding to each functional cis element and found them occupied to varying extents and by different transcription factors during the 12 h following LPS treatment. This suggests that the cis elements and their cognate transcription factors participate in a sequential, coordinated regulation of COX-2 gene expression during an inflammatory response. In support of this concept, we found, using inhibitors of Jun kinase and NF-kappaB p50 nuclear localization, that COX-2 gene transcription was completely dependent on phospho-c-Jun plus p50 at 6 h after LPS treatment but was only partially dependent on the combination of these factors at later treatment times.

Important roles for E protein binding sites within the immunoglobulin kappa chain intronic enhancer in activating Vkappa Jkappa rearrangement.

The immunoglobulin kappa light chain intronic enhancer (iEkappa) activates kappa rearrangement and is required to maintain the earlier or more efficient rearrangement of kappa versus lambda (lambda). To understand the mechanism of how iEkappa regulates kappa rearrangement, we employed homologous recombination to mutate individual functional motifs within iE(kappa) in the endogenous kappa locus, including the NF-kappaB binding site (kappaB), as well as kappaE1, kappaE2, and kappaE3 E boxes. Analysis of the impacts of these mutations revealed that kappaE2 and to a lesser extent kappaE1, but not kappaE3, were important for activating kappa rearrangement. Surprisingly, mutation of the kappaB site had no apparent effect on kappa rearrangement. Comparable to the deletion of the entire iEkappa, simultaneous mutation of kappaE1 and kappaE2 reduces the efficiency of kappa rearrangement much more dramatically than either kappaE1 or kappaE2 mutation alone. Because E2A family proteins are the only known factors that bind to these E boxes, these findings provide unambiguous evidence that E2A is a key regulator of kappa rearrangement.

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.

Cloning and characterization of a promoter flanking the early B cell factor (EBF) gene indicates roles for E-proteins and autoregulation in the control of EBF expression.

The early B cell factor (EBF) is a transcription factor shown crucial for the development of B lymphocytes. The protein is expressed from the earliest stages of B cell development until the mature B cell stage, but the control elements responsible for the regulation of the gene are unknown. In this study, we report of the identification of a promoter region flanking the EBF gene. Several transcription start sites were identified by primer extension analysis in a region approximately 3.1 kb from the predicted ATG. Transient transfections revealed that this region was able to stimulate transcription of a reporter gene in B lymphoid and to a lesser extent, myeloid cells, but not in a pre-T cell line. The promoter was also able to functionally interact with E47, suggesting that the EBF gene may be a direct target for activation by E-proteins. In addition, functional binding of EBF to its own promoter was confirmed by EMSA and transfection assays indicating that the EBF protein may be involved in an autoregulatory loop. Finally, a tissue-restricted factor was able to bind an upstream regulatory region in B-lineage cells, further supporting the idea that the cloned promoter participates in the regulation of stage and lineage specific expression of the EBF gene.

E2A and HEB activate the pre-TCR alpha promoter during immature T cell development.

The pre-TCRalpha (pTalpha) is exclusively expressed in immature thymocytes and constitutes the pre-TCR complex with TCRbeta, which regulates early T cell differentiation. Despite the recent identification of the pTalpha enhancer, the contribution of the promoter region, the direct DNA-protein interaction, and the regulation of such interaction along with T cell development have not been investigated. We analyzed the pTalpha promoter region and identified the critical elements for transcription of the pTalpha gene. The pTalpha promoter was found to contain two consecutive E-box elements that are critical for pTalpha transcription. The E-box elements in the promoter region formed the specific DNA-protein complex that was exclusively observed in immature thymocytes, not in mature thymocytes and T cells. The E proteins in this complex were identified as E2A and HeLa E-box binding protein (HEB), and overexpression of E2A and HEB resulted in activation of the pTalpha promoter. The binding complex in the consecutive E-boxes in the pTalpha promoter changed along with T cell development, as a distinct DNA-binding complex was observed in mature T cells. Comparing the E-box regions in the enhancer and the promoter, those in the promoter appear to make a greater contribution to pTalpha gene transcription.