The ArgR regulatory protein, a helper to the anaerobic regulator ANR during transcriptional activation of the arcD promoter in Pseudomonas aeruginosa.

Pseudomonas aeruginosa, when deprived of oxygen, generates ATP from arginine catabolism by enzymes of the arginine deiminase pathway, encoded by the arcDABC operon. Under conditions of low oxygen tension, the transcriptional activator ANR binds to a site centered 41.5 bp upstream of the arcD transcriptional start. ANR-mediated anaerobic induction was enhanced two- to threefold by extracellular arginine. This arginine effect depended, in trans, on the transcriptional regulator ArgR and, in cis, on an ArgR binding site centered at -73.5 bp in the arcD promoter. Binding of purified ArgR protein to this site was demonstrated by electrophoretic mobility shift assays and DNase I footprinting. This ArgR recognition site contained a sequence, 5'-TGACGC-3', which deviated in only 1 base from the common sequence motif 5'-TGTCGC-3' found in other ArgR binding sites of P. aeruginosa. Furthermore, an alignment of all known ArgR binding sites confirmed that they consist of two directly repeated half-sites. In the absence of ANR, arginine did not induce the arc operon, suggesting that ArgR alone does not activate the arcD promoter. According to a model proposed, ArgR makes physical contact with ANR and thereby facilitates initiation of arc transcription.

A nuclear factor I-like activity and a liver-specific repressor govern estrogen-regulated in vitro transcription from the Xenopus laevis vitellogenin B1 promoter.

A hormone-controlled in vitro transcription system derived from Xenopus liver nuclear extracts was exploited to identify novel cis-acting elements within the vitellogenin gene B1 promoter region. In addition to the already well-documented estrogen-responsive element (ERE), two elements were found within the 140 base pairs upstream of the transcription initiation site. One of them, a negative regulatory element, is responsible for the lack of promoter activity in the absence of the hormone and, as demonstrated by DNA-binding assays, interacts with a liver-specific transcription factor. The second is required in association with the estrogen-responsive element to mediate hormonal induction and is recognized by the Xenopus liver homolog of nuclear factor I.

Vitamin D deficiency and a CYP27B1-1260 promoter polymorphism are associated with chronic hepatitis C and poor response to interferon-alfa based therapy.

BACKGROUND & AIMS: Vitamin D is an important immune modulator and preliminary data indicated an association between vitamin D deficiency and sustained virologic response (SVR) rates in hepatitis C virus (HCV) genotype 1 patients. We, therefore, performed a comprehensive analysis on the impact of vitamin D serum levels and of genetic polymorphisms with functional relevance within the vitamin D cascade on chronic hepatitis C and its treatment. METHODS: Vitamin D serum levels, genetic polymorphisms within the vitamin D receptor and 1α-hydroxylase were determined in a cohort of 468 HCV genotype 1, 2, and 3 infected patients who were treated with interferon-alfa based regimens. RESULTS: Chronic hepatitis C was associated with a high incidence of severe vitamin D deficiency compared to controls (25(OH)D(3)<10 ng/ml in 25% versus 12%, p<0.00001). 25(OH)D(3) deficiency correlated with SVR in HCV genotype 2 and 3 patients (50% and 81% SVR for patients with and without severe vitamin D deficiency, respectively, p<0.0001). In addition, the CYP27B1-1260 promoter polymorphism rs10877012 had substantial impact on 1,25-dihydroxyvitamin D serum levels (72, 61, and 60 pmol/ml for rs10877012 AA, AC, and CC, respectively, p=0.04) and on SVR rates in HCV genotype 1, 2, and 3 infected patients (77% and 65% versus 42% for rs10877012 AA, AC, and CC, respectively, p=0.02). CONCLUSIONS: Chronic hepatitis C virus infection is associated with vitamin D deficiency. Reduced 25-hydroxyvitamin D levels and CYP27B1-1260 promoter polymorphism leading to reduced 1,25-dihydroxyvitamin D levels are associated with failure to achieve SVR in HCV genotype 1, 2, and 3 infected patients.

Identification of HMX1 target genes: a predictive promoter model approach.

PURPOSE: A homozygous mutation in the H6 family homeobox 1 (HMX1) gene is responsible for a new oculoauricular defect leading to eye and auricular developmental abnormalities as well as early retinal degeneration (MIM 612109). However, the HMX1 pathway remains poorly understood, and in the first approach to better understand the pathway's function, we sought to identify the target genes. METHODS: We developed a predictive promoter model (PPM) approach using a comparative transcriptomic analysis in the retina at P15 of a mouse model lacking functional Hmx1 (dmbo mouse) and its respective wild-type. This PPM was based on the hypothesis that HMX1 binding site (HMX1-BS) clusters should be more represented in promoters of HMX1 target genes. The most differentially expressed genes in the microarray experiment that contained HMX1-BS clusters were used to generate the PPM, which was then statistically validated. Finally, we developed two genome-wide target prediction methods: one that focused on conserving PPM features in human and mouse and one that was based on the co-occurrence of HMX1-BS pairs fitting the PPM, in human or in mouse, independently. RESULTS: The PPM construction revealed that sarcoglycan, gamma (35kDa dystrophin-associated glycoprotein) (Sgcg), teashirt zinc finger homeobox 2 (Tshz2), and solute carrier family 6 (neurotransmitter transporter, glycine) (Slc6a9) genes represented Hmx1 targets in the mouse retina at P15. Moreover, the genome-wide target prediction revealed that mouse genes belonging to the retinal axon guidance pathway were targeted by Hmx1. Expression of these three genes was experimentally validated using a quantitative reverse transcription PCR approach. The inhibitory activity of Hmx1 on Sgcg, as well as protein tyrosine phosphatase, receptor type, O (Ptpro) and Sema3f, two targets identified by the PPM, were validated with luciferase assay. CONCLUSIONS: Gene expression analysis between wild-type and dmbo mice allowed us to develop a PPM that identified the first target genes of Hmx1.

Molecular interactions involved in the transactivation of the human T-cell leukemia virus type 1 promoter mediated by Tax and CREB-2 (ATF-4).

The human T-cell leukemia virus type 1 (HTLV-1) Tax protein activates viral transcription through three 21-bp repeats located in the U3 region of the HTLV-1 long terminal repeat and called Tax-responsive elements (TxREs). Each TxRE contains nucleotide sequences corresponding to imperfect cyclic AMP response elements (CRE). In this study, we demonstrate that the bZIP transcriptional factor CREB-2 is able to bind in vitro to the TxREs and that CREB-2 binding to each of the 21-bp motifs is enhanced by Tax. We also demonstrate that Tax can weakly interact with CREB-2 bound to a cellular palindromic CRE motif such as that found in the somatostatin promoter. Mutagenesis of Tax and CREB-2 demonstrates that both N- and C-terminal domains of Tax and the C-terminal region of CREB-2 are required for direct interaction between the two proteins. In addition, the Tax mutant M47, defective for HTLV-1 activation, is unable to form in vitro a ternary complex with CREB-2 and TxRE. In agreement with recent results suggesting that Tax can recruit the coactivator CREB-binding protein (CBP) on the HTLV-1 promoter, we provide evidence that Tax, CREB-2, and CBP are capable of cooperating to stimulate viral transcription. Taken together, our data highlight the major role played by CREB-2 in Tax-mediated transactivation.

Identification of the minimal promoter region of the mouse NKX5-3, a transcription factor implicated in eye development.

Early ocular development is controlled by a complex network of transcription factors, cell cycle regulators, and diffusible signalling molecules. Together, these molecules regulate cell proliferation and apoptosis, and specify retinal fate. NKX5-3 is a homeobox transcription factor implicated in eye development. The analysis of the 5'-flanking region of the mouse Nkx5-3 gene revealed a predicted TATA-less promoter sequence between -416 and -166 of the translation start site. To functionally characterise Nkx5-3 promoter activity, serial deletions of the promoter sequence were introduced in pGL-3 basic vector and promoter activity of these 5'- and 3'-deleted constructions was tested in HeLa and CHO cells. Transactivation assays identified a region between -350 and -296 exhibiting promoter-like activity. Combined analysis by deletions and point mutations showed that this sequence, containing multiple Sp1 binding sites was necessary to promote transcriptional activity. Binding of Sp1 to this region was confirmed by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation, using an antibody specific for Sp1. Altogether, these results demonstrated that the immediate upstream region of Nkx5-3 gene possessed a strong intrinsic promoter activity in vitro, suggesting a potential role in Nkx5-3 transcription in vivo.

Functional analysis of cis- and trans-acting elements of the Candida albicans CDR2 promoter with a novel promoter reporter system.

Azole resistance in Candida albicans can be mediated by the upregulation of the ATP binding cassette transporter genes CDR1 and CDR2. Both genes are regulated by a cis-acting element called the drug-responsive element (DRE), with the consensus sequence 5'-CGGAWATCGGATATTTTTTT-3', and the transcription factor Tac1p. In order to analyze in detail the DRE sequence necessary for the regulation of CDR1 and CDR2 and properties of TAC1 alleles, a one-hybrid system was designed. This system is based on a P((CDR2))-HIS3 reporter system in which complementation of histidine auxotrophy can be monitored by activation of the reporter system by CDR2-inducing drugs such as estradiol. Our results show that most of the modifications within the DRE, but especially at the level of CGG triplets, strongly reduce CDR2 expression. The CDR2 DRE was replaced by putative DREs deduced from promoters of coregulated genes (CDR1, RTA3, and IFU5). Surprisingly, even if Tac1p was able to bind these putative DREs, as shown by chromatin immunoprecipitation, those from RTA3 and IFU5 did not functionally replace the CDR2 DRE. The one-hybrid system was also used for the identification of gain-of-function (GOF) mutations either in TAC1 alleles from clinical C. albicans isolates or inserted in TAC1 wild-type alleles by random mutagenesis. In all, 17 different GOF mutations were identified at 13 distinct positions. Five of them (G980E, N972D, A736V, T225A, and N977D) have already been described in clinical isolates, and four others (G980W, A736T, N972S, and N972I) occurred at already-described positions, thus suggesting that GOF mutations can occur in a limited number of positions in Tac1p. In conclusion, the one-hybrid system developed here is rapid and powerful and can be used for characterization of cis- and trans-acting elements in C. albicans.

Epigenetic alteration of the Wnt inhibitory factor-1 promoter occurs early in the carcinogenesis of Barrett's esophagus.

The role of Wnt antagonists in the carcinogenesis of esophageal adenocarcinoma (EAC) remains unclear. We hypothesized that downregulation of the Wnt inhibitory factor-1 (WIF-1) might be involved in the neoplastic progression of Barrett's esophagus (BE). We analyzed the DNA methylation status of the WIF-1 promoter in normal, preneoplastic, and neoplastic samples from BE patients and in EAC cell lines. We investigated the role of WIF-1 on EAC cell growth and the chemosensitization of the cells to cisplatin. We found that silencing of WIF-1 correlated with promoter hypermethylation. EAC tissue samples showed higher levels of WIF-1 methylation compared to the matched normal epithelium. In addition, we found that WIF-1 hypermethylation was more frequent in BE samples from patients with EAC than in BE samples from patients who had not progressed to EAC. Restoration of WIF-1 in cell lines where WIF-1 was methylation-silenced resulted in growth suppression. Restoration of WIF-1 could sensitize the EAC cells to the chemotherapy drug cisplatin. Our results suggest that silencing of WIF-1 through promoter hypermethylation is an early and common event in the carcinogenesis of BE. Restoring functional WIF-1 might be used as a new targeted therapy for the treatment of this malignancy.

Regulation of the vitellogenin gene B1 promoter after transfer into hepatocytes in primary cultures.

The estrogen-dependent and tissue-specific regulation of the Xenopus laevis vitellogenin gene B1 promoter has been studied by lipid-mediated DNA transfer into Xenopus hepatocytes in primary culture. Hepatocytes achieve an efficient hormonal control of this promoter through a functional interaction between the estrogen responsive elements and a promoter proximal region upstream of the TATA box, which is characterized by a high density of binding sites for the transcription factors CTF/NF-1, C/EBP and HNF3. DNA accessibility to restriction enzymes within the chromosomal copy of the vitellogenin gene B1 promoter shows that the estrogen responsive unit and the promoter proximal region are sensitive to digestion in uninduced and estrogen-induced hepatocytes but not in erythrocyte nuclei. Together, these findings support the notion that chromatin configuration as well as the interplay of promoter elements mediate proper hormone-dependent and tissue-specific expression of the B1 vitellogenin gene.

BDNF promoter I methylation correlates between post-mortem human peripheral and brain tissues.

Several psychiatric disorders have been associated with CpG methylation changes in CG rich promoters of the brain-derived neurotrophic factor (BDNF) mainly by extracting DNA from peripheral blood cells. Whether changes in peripheral DNA methylation can be used as a proxy for brain-specific alterations remains an open question. In this study we aimed to compare DNA methylation levels in BDNF promoter regions in human blood cells, muscle and brain regions using bisulfite-pyrosequencing. We found a significant correlation between the levels of BDNF promoter I methylation measured in quadriceps and vPFC tissues extracted from the same individuals (n = 98, Pearson, r = 0.48, p = 4.5 × 10(-7)). In the hippocampus, BDNF promoter I and IV methylation levels were strongly correlated (Pearson, n = 37, r = 0.74, p = 1.4 × 10(-7)). We found evidence for sex-dependent effect on BDNF promoter methylation levels in the various tissues and blood samples. Taken together, these data indicate a strong intra-individual correlation between peripheral and brain tissue. They also suggest that sex determines methylation patterns in BDNF promoter region across different types of tissue, including muscle, brain, and blood.

MGMT Promoter Methylation Is a Strong Prognostic Biomarker for Benefit from Dose-Intensified Temozolomide Rechallenge in Progressive Glioblastoma: The DIRECTOR Trial.

PURPOSE: Rechallenge with temozolomide (TMZ) at first progression of glioblastoma after temozolomide chemoradiotherapy (TMZ/RT→TMZ) has been studied in retrospective and single-arm prospective studies, applying temozolomide continuously or using 7/14 or 21/28 days schedules. The DIRECTOR trial sought to show superiority of the 7/14 regimen. EXPERIMENTAL DESIGN: Patients with glioblastoma at first progression after TMZ/RT→TMZ and at least two maintenance temozolomide cycles were randomized to Arm A [one week on (120 mg/m(2) per day)/one week off] or Arm B [3 weeks on (80 mg/m(2) per day)/one week off]. The primary endpoint was median time-to-treatment failure (TTF) defined as progression, premature temozolomide discontinuation for toxicity, or death from any cause. O(6)-methylguanine DNA methyltransferase (MGMT) promoter methylation was prospectively assessed by methylation-specific PCR. RESULTS: Because of withdrawal of support, the trial was prematurely closed to accrual after 105 patients. There was a similar outcome in both arms for median TTF [A: 1.8 months; 95% confidence intervals (CI), 1.8-3.2 vs. B: 2.0 months; 95% CI, 1.8-3.5] and overall survival [A: 9.8 months (95% CI, 6.7-13.0) vs. B: 10.6 months (95% CI, 8.1-11.6)]. Median TTF in patients with MGMT-methylated tumors was 3.2 months (95% CI, 1.8-7.4) versus 1.8 months (95% CI, 1.8-2) in MGMT-unmethylated glioblastoma. Progression-free survival rates at 6 months (PFS-6) were 39.7% with versus 6.9% without MGMT promoter methylation. CONCLUSIONS: Temozolomide rechallenge is a treatment option for MGMT promoter-methylated recurrent glioblastoma. Alternative strategies need to be considered for patients with progressive glioblastoma without MGMT promoter methylation.

Promoter region sequence differences in the A and G gamma globin genes of Brazilian sickle cell anemia patients

Fetal hemoglobin (HbF), encoded by the HBG2 and HBG1 genes, is the best-known genetic modulator of sickle cell anemia, varying dramatically in concentration in the blood of these patients. This variation is partially associated with polymorphisms located in the promoter region of the HBG2 and HBG1 genes. In order to explore known and unknown polymorphisms in these genes, the sequences of their promoter regions were screened in sickle cell anemia patients and correlated with both their HbF levels and their βS-globin haplotypes. Additionally, the sequences were compared with genes from 2 healthy groups, a reference one (N = 104) and an Afro-descendant one (N = 98), to identify polymorphisms linked to the ethnic background.The reference group was composed by healthy individuals from the general population. Four polymorphisms were identified in the promoter region of HBG2 and 8 in the promoter region of HBG1 among the studied groups. Four novel single nucleotide polymorphisms (SNP) located at positions -324, -317, -309 and -307 were identified in the reference group. A deletion located between -396 and -391 in the HBG2 promoter region and the SNP -271 C→T in the HBG1 promoter region were associated with the Central African Republic βS-globin haplotype. In contrast, the -369 C→G and 309 A→G SNPs in the HBG2 promoter region were correlated to the Benin haplotype. The polymorphisms -396_-391 del HBG2, -369 SNP HBG2 and -271 SNP HBG1 correlated with HbF levels. Hence, we suggest an important role of HBG2 and HBG1 gene polymorphisms on the HbF synthesis.

Novel regulatory SNPs in the promoter region of the TNFRSF18 gene in a Gabonese population

Parasites are accountable for driving diversity within immune gene families. We identified and investigated regulatory single nucleotide polymorphisms (SNPs) in the promoter regions of the tumor necrosis factor receptor superfamily member 18 (TNFRSF18) gene by direct sequencing in a group of male Gabonese individuals exposed to a wide array of parasitic diseases such as malaria, filariasis and schistosomiasis. Two new promoter variants were identified in 40 individuals. Both novel variants were heterozygous and were linked to SNP #rs3753344 (C/T), which has been described. One of the SNP variants (ss2080581728) was close to the general transcription factor site, the TATA box. We further validated these new promoter variants for their allelic gene expression using transient transfection assays. One new promoter variant with two base changes (C/T - ss2080581728/rs3753344) displayed an altered expression of the marker gene. Both novel variants remained less active at the non-induced state in comparison to the major allele. The allele frequencies observed in this study were consistent with data for other African populations. The detection and analysis of these human immune gene polymorphisms contribute to a better understanding of the interaction between host-parasite and expression of Treg activity.

The Streptococcus mutans GlnR protein exhibits an increased affinity for the glnRA operon promoter when bound to GlnK

The control of nitrogen metabolism in pathogenic Gram-positive bacteria has been studied in a variety of species and is involved with the expression of virulence factors. To date, no data have been reported regarding nitrogen metabolism in the odontopathogenic species Streptococcus mutans. GlnR, which controls nitrogen assimilation in the related bacterial species, Bacillus subtilis, was assessed in S. mutans for its DNA and protein binding activity. Electrophoretic mobility shift assay of the S. mutans GlnR protein indicated that GlnR binds to promoter regions of the glnRA and amtB-glnK operons. Cross-linking and pull-down assays demonstrated that GlnR interacts with GlnK, a signal transduction protein that coordinates the regulation of nitrogen metabolism. Upon formation of this stable complex, GlnK enhances the affinity of GlnR for the glnRA operon promoter. These results support an involvement of GlnR in transcriptional regulation of nitrogen metabolism-related genes and indicate that GlnK relays information regarding ammonium availability to GlnR.

A family of promoter probe vectors incorporating autofluorescent and chromogenic reporter proteins for studying gene expression in Gram-negative bacteria

A series of promoter probe vectors for use in Gram-negative bacteria has been made in two broad-host-range vectors, pOT (pBBR replicon) and pJP2 (incP replicon). Reporter fusions can be made to gfpUV, gfprnut3.1, unstable gfpmut3.1 variants (LAA, LVA, AAV and ASV), gfp+, dsRed2, dsRedT3, dsRedT4, mRFP1, gusA or lacZ. The two vector families, pOT and pJP2, are compatible with one another and share the same polylinker for facile interchange of promoter regions. Vectors based on pJP2 have the advantage of being ultra-stable in the environment due to the presence of the parABCDE genes. As a confirmation of their usefulness, the dicarboxylic acid transport system promoter (dctA(p)) was cloned into a pOT (pRU1097)- and a pJP2 (pRU1156)-based vector and shown to be expressed by Rhizobium leguminosarum in infection threads of vetch. This indicates the presence of dicarboxylates at the earliest stages of nodule formation.