Complement receptor 2 (CR2) is regulated at the transcriptional level, but the promoter elements and the transcription factors that bind to them and contribute to its regulation are unknown. After documenting that PMA and cAMP induced the activity of the CR2 promoter by 10-fold, we conducted promoter truncation and mutagenesis experiments, in conjunction with shift assays, to determine the functionally important regions of the promoter and the proteins that bind to them. We identified two regions, separated by ∼900 nucleotides, which together were responsible for inducible promoter activity. Mutagenesis of single promoter elements demonstrated a functional upstream stimulatory factor/E box in the TATA box-proximal region and three equally important, closely spaced, CREB/AP-1 half-sites in the upstream promoter region. The cAMP response element-binding protein (CREB)/AP-1 half-sites bound in vitro Jun and CREB that are induced by protein kinases A and/or C. The 900-nucleotide segment stretching between the above two regions had no functional impact on the induced transcription, and its deletion increased the promoter activity. Finally, a region upstream of the distal site had a repressor activity on CR2 transcription. Moreover, IL-4 induced binding of CREB and AP-1 to the upstream promoter elements and resulted in increased CR2 surface protein expression. These studies have characterized regions of the CR2 promoter and the transcription factors that bind to them and are crucial to induced CR2 expression. Our studies may provide insights to novel approaches to modulate B cell function by regulating CR2 gene transcription.

CR2 is a key regulator of the B cell response to Ag. Here we show that NF-κB enhances the expression of the human CR2 gene. Promoter truncation, deletion, and mutagenesis studies indicated a functional role for a consensus NF-κB promoter element, as well as a heterogeneous nuclear ribonucleoprotein D element and an overlapping X box/E box. By supershift analysis, the first two elements bound NF-κB p50 and p65 and heterogeneous nuclear ribonucleoprotein RNP D, respectively. The X box/E box bound regulatory factor X5 and, surprisingly, NF-κB p50 and p65. Overexpression of NF-κB p50 enhanced the activity of the CR2 promoter in B cell lines and primary B cells, suggesting a direct role for NF-κB in regulating promoter activity. Importantly, mutation of the NF-κB element or the X box/E box rendered the promoter unresponsive to NF-κB p50. Using chromatin immunoprecipitation in live B cell lines and primary B cells, we found that NF-κB proteins p50, p65, and c-Rel bound to the genomic promoter at two locations that overlap with the consensus NF-κB element or the X box/E box. Finally, stimuli that activate NF-κB enhanced the activity of the CR2 promoter, and LPS rapidly increased the number of CR2 proteins on the surface of primary B cells. We propose that the NF-κB signaling pathway enhances the expression of the CR2 gene, as a result of NF-κB proteins binding to two CR2 promoter elements. Thus, at the onset of an infection, LPS could sensitize the B cell to Ag by enhancing the level of CR2-costimulatory molecules on the cell surface.

Complement receptor 2 (CR2) is important in the regulation of the B lymphocyte response; the regulation of its expression is therefore of central importance. We recently reported that a 42 kDa heterogeneous nuclear ribonucleoprotein (hnRNP) is involved in the transcriptional regulation of the human CR2 gene [Tolnay, Lambris and Tsokos (1997) J. Immunol. 159, 5492-5501]. We cloned the cDNA encoding this protein and found it to be identical with hnRNP D0B, a sequence-specific RNA-binding protein. By using a set of mutated oligonucleotides, we demonstrated that the recombinant hnRNP D0B displays sequence specificity for double-stranded oligonucleotide defined by the CR2 promoter. We conducted electrophoretic mobility-shift assays to estimate the apparent Kd of hnRNP D0B for the double-stranded DNA motif and found it to be 59 nM. Interestingly, hnRNP D0B displayed affinities of 28 and 18 nM for the sense and anti-sense strands of the CR2 promoter-defined oligonucleotide respectively. The significantly greater binding affinity of hnRNP D0B for single-stranded DNA than for double-stranded DNA suggests that the protein might melt the double helix. The intranuclear concentration of sequence-specific protein was estimated to be 250-400 nM, indicating that the protein binds to the CR2 promoter in vivo. Co-precipitation of a complex formed in vivo between hnRNP D0B and the TATA-binding protein demonstrates that hnRNP D0B interacts with the basal transcription apparatus. Our results suggest a new physiological role for hnRNP D0B that involves binding to double- and single-stranded DNA sequences in a specific manner and functioning as a transcription factor.

No abstract

This chapter summarizes the preclinical and clinical development to date of investigational anticancer agents whose mechanism of action is thought to be via direct interaction with tubulin or microtubules. All of the compounds discussed are agents discovered or derived from screening natural materials for anti-cancer activity. The underlying theme for pursuit of these agents is that tubulin is a validated anticancer target and that novel interactions between new chemical entities and tubulin may overcome resistance, increase therapeutic index, or alter the spectrum of clinical utility against different cancer types.