Characterization of the human intestinal CD98 promoter and its regulation by interferon-gamma.

Growing evidence that epithelial CD98 plays an important role in intestinal inflammation focused our interest to investigate the transcriptional regulation of CD98. Our mouse-based in vivo and in vitro experiments revealed that epithelial colonic CD98 mRNA expression was transcriptionally increased in intestinal inflammation. We then isolated and characterized a 5'-flanking fragment containing the promoter region required for CD98 gene transcription. Primer extension and rapid amplification of 5'-cDNA ends were used to map a transcriptional initiation site 129 bp upstream from the translational start codon (ATG). Direct sequencing of the 5'-flanking region revealed the presence of four GC-rich stimulating protein (Sp)1 binding domains, one NF-kappaB binding domain, and no TATA box. Binding of Sp1 [Sp1(-874), SP1(-386), Sp1(-187), and Sp1(-177)] and NF-kappaB [NF-kappaB(-213)] to the promoter was confirmed by EMSA and supershift assays. Furthermore, chromatin immunoprecipitation experiments showed the in vivo DNA-Sp1 and DNA-NF-kappaB interactions under basal and IFN-gamma-stimulated conditions. Reporter genes driven by serially truncated and site-mutated CD98 promoters were used to examine basal and IFN-gamma-responsive transcription in transiently transfected Caco2-BBE cells. Our results revealed that Sp1(-187), Sp1(-177), and the NF-kappaB binding site were essential for basal and IFN-gamma-stimulated CD98 promoter activities, whereas Sp1(-874) and Sp1(-386) were not. The results from additional site-mutated CD98 promoters suggested that Sp1(-187), Sp1(-177), and the NF-kappaB site may cooperate in mediating basal and IFN-gamma-stimulated CD98 promoter activities. Finally, we demonstrated that a reduction of Sp1 or NF-kappaB expression reduced CD98 protein expression in unstimulated and IFN-gamma-stimulated Caco2-BBE cells. Collectively, these findings indicate that the Sp1 and NF-kappaB transcription factors are likely to play a significant role in IFN-gamma-mediated transcriptional regulation of CD98 in the intestinal epithelium, providing new insights into the regulation of CD98 expression in intestinal inflammation.

Yan Y ，Dalmasso G ，Sitaraman S ，Merlin D ... -  《AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY》

Characterization of the infection-responsive bovine lactoferrin promoter.

The concentration of lactoferrin in bovine milk is dramatically increased in response to infection. The high levels of lactoferrin may have a role in the prevention of microbial infection of the mammary gland. However, molecular mechanisms of how the lactoferrin gene is regulated in the mammary gland in response to infection remain unknown. In this study, we isolated and characterized the 5' flanking region of the bovine lactoferrin gene. An 8.2 kilobase (kb) fragment of the bovine lactoferrin gene, containing 4.4 kb of 5' flanking region, exon 1, intron 1, and exon 2, was isolated from a bovine genomic library on two overlapping bacterial artificial chromosome (BAC) clones. Sequence analysis of the isolated lactoferrin gene revealed that the promoter region contains a high GC content, a non-canonical TATA box, multiple stimulating protein 1 (SP1)/GC elements, and other putative binding sites for transcription factors including nuclear factor-kappaB (NF-kappaB), activator protein 1 (AP1), signal transducer and activator of transcriptions 3 and 5 (STAT3 and STAT5), and steroid hormone receptors. To demonstrate that the isolated promoter is functional, 4.4 kb of 5' flanking region was inserted upstream from the firefly luciferase gene and the chimeric construct was transiently transfected into murine mammary epithelial cells. Transfection studies showed that the basal promoter activity is quite potent, being similar in strength to that of the simian virus 40 (SV40) promoter/enhancer. In addition, a 24-h treatment with Escherichia coli lipopolysaccharide (LPS) significantly stimulated its activity up to 2.3-fold in a dose-dependent manner. Furthermore, promoter deletion analysis indicated that the sequence up to -543 was sufficient for basal activity, whereas the sequence up to -1029 was required for maximal basal activity. The basal activity of the promoter is affected by both positive regulatory regions (-2462/-1879 and -1029/-75) and a negative regulatory region (-1407/-1029). LPS-responsive regions of the promoter were localized to the region from -1029 to -543 containing one STAT3 site and two NF-kappaB sites, and the region from -4355 to -2462 containing three AP1 sites and six NF-kappaB sites. Taken together, our findings suggested that the lactoferrin promoter responds to infection via the NF-kappaB pathway.

Zheng J ，Ather JL ，Sonstegard TS ，Kerr DE ... -  《GENE》

Sp1 and AP2 enhance promoter activity of the mouse GM3-synthase gene.

Promoters of the glycosyltransferase genes for ganglioside synthesis are TATA-less and often have multiple binding sites for transcription factors Sp1 and AP2 in their proximal regions. However, the function of Sp1 and AP2 in the promoters has not yet been defined. Here, we cloned 5'-flanking fragments of the mouse GM3-synthase gene and assessed the promoter activity of these fragments in mouse Neuro-2a cells. This promoter is TATA-less and contains a number of potential transcription factor-binding sites. Multiple putative transcriptional initiation sites for this gene were identified, including several downstream initiation sites. We then set out to dissect the regulatory elements important for GM3-synthase promoter function. We found that a 5'-flanking 254-bp DNA fragment of the gene contained regulatory elements including two Sp1-binding and six AP2-binding sites that were essential for the basal activity of the promoter in mouse Neuro-2a cells. The effects of the individual Sp1- and AP2-binding sites on basal activity of the GM3-synthase gene were investigated. Mutations in the juxtaposed Sp1/AP2-binding site and in an AP2-binding site decreased the activity of the proximal promoter to approximately 50%. In vitro and in vivo interactions between transcription factors Sp1 and AP2 and these regulatory elements were confirmed by EMSA and the chromatin immunoprecipitation approach, respectively. Therefore, our results demonstrate that Sp1 and AP2 enhance the basal activity of the TATA-less mouse GM3-synthase promoter.

Xia T ，Zeng G ，Gao L ，Yu RK ... -  《GENE》

In vivo binding of NF-kappaB to the IkappaBbeta promoter is insufficient for transcriptional activation.

Despite certain structural and biochemical similarities, differences exist in the function of the NF-kappaB (nuclear factor kappaB) inhibitory proteins IkappaBalpha (inhibitory kappaBalpha) and IkappaBbeta. The functional disparity arises in part from variance at the level of gene regulation, and in particular from the substantial induction of IkappaBalpha, but not IkappaBbeta, gene expression post-NF-kappaB activation. In the present study, we probe the differential effects of IL (interleukin)-1beta on induction of IkappaBalpha and perform the first characterization of the human IkappaBbeta promoter. A consensus NF-kappaB-binding site, capable of binding NF-kappaB both in vitro and in vivo, is found in the IkappaBbeta gene 5' flanking region. However, the IkappaBbeta promoter was not substantially activated by pro-inflammatory cytokines, such as IL-1beta and tumour necrosis factor alpha, that are known to cause strong activation of NF-kappaB. Furthermore, in contrast with IkappaBalpha, NF-kappaB activation did not increase expression of endogenous IkappaBbeta as assessed by analysis of mRNA and protein levels. Unlike kappaB-responsive promoters, IkappaBbeta promoter-bound p65 inefficiently recruits RNA polymerase II, which stalls at the promoter. We present evidence that this stalling is likely due to the absence of transcription factor IIH engagement, a prerequisite for RNA polymerase II phosphorylation and transcriptional initiation. Differences in the conformation of promoter-bound NF-kappaB may underlie the variation in the ability to engage the basal transcriptional apparatus at the IkappaBbeta and kappaB-responsive promoters. This accounts for the differential expression of IkappaB family members in response to NF-kappaB activation and furthers our understanding of the mechanisms involved in transcription factor activity and IkappaBbeta gene regulation.

Griffin BD ，Moynagh PN 《-》

Regulation of KLF5 involves the Sp1 transcription factor in human epithelial cells.

Human Kruppel-like factor 5 (hKLF5) is a transcription factor with a potential tumor suppressor function in prostate and breast cancers. In the majority of cancer samples examined, a significant loss of expression for KLF5 has been detected. Whereas hemizygous deletion appears to be responsible for KLF5's reduced expression in about half of the cases, the mechanism for reduction is unknown in the remaining half; gene promoter methylation does not appear to be involved. In this report, we studied the regulation of KLF5 and cloned and functionally characterized a 1944-bp fragment of the 5'-flanking region of the hKLF5 gene. Several mitogens as well as global demethylation induced the expression of KLF5, implicating multiple factors in the regulation of KLF5. KLF5's promoter lacks a TATA box and has a GC-rich region. Deletion mapping in combination with promoter activity assay showed that multiple cis-elements are involved in the transcriptional regulation of KLF5, some of which may play a repressor role whereas some others play an enhancer role. The Sp1 site between position -239 and -219 is essential for a basal promoter activity. Deletion or mutations of this Sp1 site significantly reduced promoter activity in several epithelial cell lines. Electrophoretic mobility shift assays (EMSAs) revealed that the Sp1 site binds Sp1 protein in nucleic extracts of different cell lines. In addition, overexpression of Sp1 protein transactivates KLF5 promoter activity. These findings suggest that Sp1 is a key transcription factor in KLF5's dynamic transcriptional regulation.

Chen C ，Zhou Y ，Zhou Z ，Sun X ，Otto KB ，Uht RM ，Dong JT ... -  《GENE》