Single-nucleotide polymorphisms and activity analysis of the promoter and enhancer of the pig lactase gene

Lactose intolerance in northern Europeans is strongly associated with a single-nucleotide polymorphism (SNP) located 14 kb upstream of the human lactase gene: − 13,910*C/T. We examined whether SNPs in the 5′ flanking region of the pig lactase gene are similar to those in the human gene and whether these polymorphisms play a functional role in regulating pig lactase gene expression. The 5′ flanking region of the lactase gene from several different breeds of pigs was cloned and analyzed for gene regulatory activity of a luciferase reporter gene. One SNP was found in the enhancer region (− 797*G/A) and two were found in the promoter region (− 308*G/C and − 301*A/G). The promoter C_− 308,G_− 301(Pro-CG) strongly promotes the expression of the lactase gene, but the promoter G_− 308,A_− 301(Pro-GA) does not. The enhancer A_− 797(Enh-A) genotype for Pro-GA can significantly enhance promoter activity, but has an inhibitory effect on Pro-CG. The Enhancer G_− 797(Enh-G) has a significant inhibitory effect on both promoters. In conclusion, the order of effectiveness on the pig lactase gene is Enh-A + Pro-GA > Enh-A/G + Pro-CG > Enh-G + Pro-GA.     

Destruction of a distal hypoxia response element abolishes trans-activation of the PAG1 gene mediated by HIF-independent chromatin looping

A crucial step in the cellular adaptation to oxygen deficiency is the binding of hypoxia-inducible factors (HIFs) to hypoxia response elements (HREs) of oxygen-regulated genes. Genome-wide HIF-1α/2α/β DNA-binding studies revealed that the majority of HREs reside distant to the promoter regions, but the function of these distal HREs has only been marginally studied in the genomic context. We used chromatin immunoprecipitation (ChIP), gene editing (TALEN) and chromosome conformation capture (3C) to localize and functionally characterize a 82 kb upstream HRE that solely drives oxygen-regulated expression of the newly identified HIF target gene PAG1. PAG1, a transmembrane adaptor protein involved in Src signalling, was hypoxically induced in various cell lines and mouse tissues. ChIP and reporter gene assays demonstrated that the −82 kb HRE regulates PAG1, but not an equally distant gene further upstream, by direct interaction with HIF. Ablation of the consensus HRE motif abolished the hypoxic induction of PAG1 but not general oxygen signalling. 3C assays revealed that the −82 kb HRE physically associates with the PAG1 promoter region, independent of HIF-DNA interaction. These results demonstrate a constitutive interaction between the −82 kb HRE and the PAG1 promoter, suggesting a physiologically important rapid response to hypoxia.     

Coordinate regulation of bovine prion protein gene promoter activity by two Sp1 binding site polymorphisms

Relationships between insertion/deletion (Ins/Del) polymorphisms of the bovine prion protein gene (PRNP) promoter and bovine spongiform encephalopathy (BSE) susceptibility have been reported. Our previous study has shown that polymorphisms of −6C → T included in the specific protein 1 (Sp1) site in the 5′-flanking region of bovine PRNP influence the promoter activity of bovine PRNP. The present study shows that 12 and 23 bp Ins/Del polymorphisms in the upstream region and an additional polymorphism (−47C → A) in the Sp1 binding site coordinately affect the promoter activity. Reporter gene assays demonstrated that the bovine PRNP promoter containing −47A and 23 bp Del/12 bp Ins or 23 bp Ins/12 bp Ins showed lower promoter activity compared with other haplotypes (23 bp Del/12 bp Ins or 23 bp Ins/12 bp Del with −47C) or the wild-type haplotype (23 bp Del/12 bp Del with −47C). Furthermore, gel shift assays showed that the binding activity of Sp1 to the PRNP promoter was influenced by both polymorphisms with corresponding effects on the promoter activity. The coordinate regulation of the bovine PRNP promoter suggests the two Sp1 binding site polymorphisms control Sp1 binding to the PRNP promoter and its activity.     

Matrix attachment region (MAR) properties and abnormal expansion of AT island minisatellites in FRA16B fragile sites in leukemic CEM cells

AT-rich minisatellites (AT islands) are sites of genomic instability in cancer cells and targets for extremely lethal AT-specific drugs, such as bizelesin. Here we investigated the AT islands in the FRA16B fragile site region for their possible roles in the organization of DNA on the nuclear matrix. The FRA16B AT island nominally spans ~3 kb of mostly >90% A/T DNA. In silico analysis indicates that this domain exhibits characteristics of nuclear matrix attachment regions (MARs): an exceptionally intense computed ‘MAR potential’ and profound duplex destabilization and flexibility. FRA16B repeats specifically bind to isolated nuclear matrices, which indicates their in vitro MAR function. This binding is several-fold greater than that of a known MAR in the c-myc gene. AT islands in fragile sites FRA16B and FRA16D are significantly more abundant in CEM cells that are hypersensitive to bizelesin compared to normal WI-38 cells. FRA16B overabundance in CEM is due to an ~10-fold expansion of FRA16B repeats. The expanded FRA16B minisatellites in CEM cells preferentially localize to the nuclear matrix-associated DNA indicating their in vivo MAR function. The unexpanded repeats in WI-38 cells localize to the loop DNA. The c-myc MAR is also matrix-associated in CEM cells while localizing to loop DNA in WI-38 cells. These results are the first to demonstrate that AT islands in fragile sites can function as MARs both in vitro and in vivo. The ability of FRA16B-mediated MAR sites to rearrange depending on the repeat expansion status could be relevant to both genomic instability of cancer cells and their sensitivity to AT-island targeting drugs.     

C/EBPε participates in all-trans retinoic acid induction of PI3Kγ in U937 cells via an intronic matrix attachment region sequence

ATRA (all-trans retinoic acid) regulates gene expression by binding as a ligand to its specific receptors like C/EBPε which is directly induced. In the U937 cell line, PI3Kγ is selectively induced over other PI3Ks by ATRA, although the mechanism is still unclear. Here, we show that C/EBPε and PI3Kγ are induced in U937 cells by ATRA both in levels of mRNA and protein. Reporter gene assay revealed that C/EBPε is able to interact with a previously identified 2 kb MAR (matrix attachment region) sequence in the last intron of PI3Kγ gene, and increases its linked heterogenous reporter gene expression. ChIP assay showed that induction of endogenous PI3Kγ is at least partially caused by enhanced, direct C/EBPε binding to a 15 bp sequence at nucleotides 1428–1442 within this MAR sequence, and EMSA analysis confirmed this binding in vitro. The results above collectively show that C/EBPε participates in ATRA induction of PI3Kγ.     

Targeted disruption of the mouse Lipoma Preferred Partner gene

LPP (Lipoma Preferred Partner) is a zyxin-related cell adhesion protein that is involved in the regulation of cell migration. We generated mice with a targeted disruption of the Lpp gene and analysed the importance of Lpp for embryonic development and adult functions. Aberrant Mendelian inheritance in heterozygous crosses suggested partial embryonic lethality of Lpp^−/− females. Fertility of Lpp^−/− males was proven to be normal, however, females from Lpp^−/− × Lpp^−/− crosses produced a strongly reduced number of offspring, probably due to a combination of female embryonic lethality and aberrant pregnancies. Apart from these developmental and reproductive abnormalities, Lpp^−/− mice that were born reached adulthood without displaying any additional macroscopic defects. On the other hand, Lpp^−/− mouse embryonic fibroblasts exhibited reduced migration capacity, reduced viability, and reduced expression of some Lpp interaction partners. Finally, we discovered a short nuclear form of Lpp, expressed mainly in testis via an alternative promoter.