Differential Binding Of Nuclear Proteins Research Articles

FNTB Promoter Polymorphisms Are Independent Predictors of Survival in Patients with Triple Negative Breast Cancer.

Simple SummaryIn breast cancer, the promising efficacy of farnesyltransferase inhibitors (FTIs) in preclinical studies is in contrast to only limited effects in clinical trials. Therefore, this study focussed on the clinical relevance of polymorphisms in FNTB, the gene encoding the catalytically active β-subunit of farnesyltransferase, in early breast cancer. This is the first study on breast cancer suggesting that FNTB promoter polymorphisms are independent prognostic biomarkers, particularly in patients with early triple negative breast cancer (TNBC), and possibly modulate FNTB transcriptional activity. Taken together, we describe for the first time, a link between FNTB promoter polymorphism and the prognosis of breast cancer patients. We propose that FNTB genotyping, which is easily possible from a single blood drawing, may allow independent prognostic stratification, particularly in TNBC, in order to identify patients with a high risk of recurrence and poor prognosis. Ultimately, our results encourage further prospective evaluations of the role of FNTB promoter polymorphisms in predicting response to FTIs, particularly in TNBC patients.In breast cancer, the promising efficacy of farnesyltransferase inhibitors (FTIs) in preclinical studies is in contrast to only limited effects in clinical Phase II–III trials. The objective of this study was to explore the clinical relevance of farnesyltransferase β-subunit (FNTB) single nucleotide promoter polymorphisms (FNTB-173 6G > 5G (rs3215788), -609 G > C (rs11623866) and -179 T > A (rs192403314)) in early breast cancer. FNTB genotyping was performed by pyrosequencing in 797 patients from a prospective multicentre observational PiA trial (NCT 01592825). In the total cohort, the FNTB-173 6G > 5G polymorphism was an independent predictor of RFI (HR = 0.568; 95% CI = 0.339–0.949, p = 0.031), OS (HR = 0.629; 95% CI = 0.403–0.980, p = 0.040) and BCSS (HR = 0.433; 95% CI = 0.213–0.882; p = 0.021), whereas the FNTB-609 G > C polymorphism was an independent predictor of RFI (HR = 0.453; 95% CI = 0.226–0.910, p = 0.026) and BCSS (HR = 0.227; 95% CI = 0.075–0.687, p = 0.009). Subtype analysis revealed the independent prognostic relevance of FNTB promoter polymorphisms, particularly in TNBC but not in luminal or HER2-positive intrinsic subtypes. Finally, we used electrophoretic mobility shift assays (EMSAs) to confirm in vitro that the polymorphism FNTB-173 6G > 5G resulted in the differential binding of nuclear proteins from five different breast cancer cell lines. This is the first study on breast cancer suggesting that FNTB promoter polymorphisms (i) are independent prognostic biomarkers, particularly in patients with early TNBC, and (ii) could modulate FNTB’s transcriptional activity.

Abstract 2913: The SOX11 transcription factor is critical for basal-like breast cancer growth and migration and is associated with poor survival

Abstract Introduction: Basal-like breast cancers (BLBCs) are aggressive breast cancers that are associated with poor survival and have distinctive mRNA expression profiles compared to non-BLBC tumors. Through an integrated screen of transcription factors (TFs) we identified SOX11 as uniquely critical for growth in BLBC, but not essential in estrogen-receptor (ER)-positive breast cancer cells. In this study we examine the role of SOX11 regulating growth and other BLBC phenotypes, including increased migration, invasion and poor survival. Methods: SOX11 was identified through an integrated primary screen comparing mRNA expression of TFs, DNA sequences of differentially expressed BLBC genes, and differential binding of nuclear proteins to oligonucleotide sequences of transcription factor binding sites, and a secondary siRNA screen of growth in a panel of breast cancer cell lines. Further growth studies were performed using siRNA to SOX11 and measuring 2D-growth, or 3D-growth of cell lines grown in soft-agar. Immuno-fluorescent staining of phosphorylated histone H3 (pH3) was used to measure the effect of SOX11 depletion on proliferation. Migration and invasion assays were performed with membranes coated in Matrigel (invasion) or uncoated (migration). Survival analyses were performed with patients dichotomized by median SOX11 expression level, and log rank statistical comparisons as well as a multivariable Cox Proportional Hazards model to investigate the prognostic value of SOX11. Results: SOX11 depletion resulted in reduced growth of ER-negative breast cancer cell lines, including ER-negative/ Her2-positive cells; whereas, growth of ER-positive breast cancer cells or ER-negative, non-transformed breast cells was not affected. In MDA-MB-468 cells, SOX11 depletion resulted in a 60% reduction of pH3-positive cells. Additionally, depletion of SOX11 in MDA-MB-231 or MDA-MB-468 cells reduced migration and invasion 40-60%, while over-expression of SOX11 in MCF7 cells increased migration nearly 12-fold. High SOX11 is correlated with worse prognosis, whether analyzed for all patients or subsets of patients divided by ER or HER2 status. In the multivariable analysis accounting for tumor size, grade, lymph node status, and PAM50 class, high SOX11 expression was associated with increased risk of breast cancer related death with a hazard ratio of 1.42 and p-value < 0.001. Conclusions: SOX11 is a critical regulator of multiple BLBC phenotypes, including growth, migration, invasion, and expression of signature BLBC genes. Growth reduction of BLBC cells following SOX11 depletion is a result of decreased proliferation. High SOX11 expression was also found to be an independent prognostic indicator of poor survival in women with breast cancer. These results identify SOX11 as a potential target for the treatment of BLBC, the most aggressive form of breast cancer. Citation Format: Jonathan Shepherd, Abhijit Mazumdar, Anna Tsimelzon, Susan G. Hilsenbeck, Powel H. Brown. The SOX11 transcription factor is critical for basal-like breast cancer growth and migration and is associated with poor survival. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2913.

Functional Characterization of Polymorphisms in the Peptidase Inhibitor 3 (Elafin) Gene and Validation of Their Contribution to Risk of Acute Respiratory Distress Syndrome

Elafin (peptidase inhibitor 3 [PI3]) and its biologically active precursor, pre-elafin, are neutrophil serine proteinase inhibitors with an important role in preventing excessive tissue injury during inflammatory events. Recently, we reported an association between single-nucleotide polymorphism (SNP) rs2664581 in the PI3 gene, increased risk of acute respiratory distress syndrome (ARDS) and pre-elafin circulating levels. This study aims to validate the legitimacy of this association by using a cohort of patients who met the criteria for systemic inflammatory response syndrome and were at risk of developing ARDS (n = 840). A comprehensive functional study of SNPs in PI3 gene was also performed. Luciferase assays and electrophoretic mobility shift assays were conducted to determine the functional relevance of promoter region variants. The effect of the coding SNP rs2664581 on the neutrophil elastase inhibitory activity and transglutaminase binding properties of pre-elafin was also investigated. The variant allele of rs2664581 (C) was significantly associated with increased ARDS risk, mainly among subjects with sepsis (odds ratio = 1.44; 95% confidence interval = 1.04-1.99; P = 0.0276, adjusted by age, sex, and Acute Physiology and Chronic Health Evaluation III). Pre-elafin recombinant protein carrying the amino acid change associated with rs2664581 (Thr34Pro, mutant protein [MT]) had greater capacity to undergo transglutaminase-mediated cross-linking to immobilized fibronectin than wild-type protein in vitro (P < 0.003). No differences were observed in the neutrophil elastase inhibitory activities of wild-type versus MT proteins. In addition, the risk allele-promoter construct had significantly lower cytokine-induced transcriptional activity. Electrophoretic mobility shift assay results indicated a differential binding of nuclear proteins to the G and A alleles of SNP -338G > A. Our results confirm the association between SNP rs2664581 and enhanced risk of ARDS, further supporting the role of PI3 in ARDS development. SNPs in the PI3 locus may act synergistically by regulating PI3 gene expression and pre-elafin biological functions.

Identification of aCis-Acting Regulatory Polymorphism in a EucalyptCOBRA-Like Gene Affecting Cellulose Content

Populations with low linkage disequilibrium (LD) offer unique opportunities to study functional variants influencing quantitative traits. We exploited the low LD in forest trees to identify functional polymorphisms in a Eucalyptus nitens COBRA-like gene (EniCOBL4A), whose Arabidopsis homolog has been implicated in cellulose deposition. Linkage analysis in a full-sib family revealed that EniCOBL4A is the most strongly associated marker in a quantitative trait locus (QTL) region for cellulose content. Analysis of LD by genotyping 11 common single-nucleotide polymorphisms (SNPs) and a simple sequence repeat (SSR) in an association population revealed that LD declines within the length of the gene. Using association studies we fine mapped the effect of the gene to SNP7, a synonymous SNP in exon 5, which occurs between two small haplotype blocks. We observed patterns of allelic expression imbalance (AEI) and differential binding of nuclear proteins to the SNP7 region that indicate that SNP7 is a cis-acting regulatory polymorphism affecting allelic expression. We also observed AEI in SNP7 heterozygotes in a full-sib family that is linked to heritable allele-specific methylation near SNP7. This study demonstrates the potential to reveal functional polymorphisms underlying quantitative traits in low LD populations.

Binding of estrogen receptor α promoter to nuclear proteins of mouse cerebral cortex: effect of age, sex, and gonadal steroids

Majority of estrogen actions in the brain are mediated by estrogen receptor (ER) alpha which in turn is regulated by several factors like circulating levels of gonadal steroid hormones 17beta-estradiol and testosterone, sex and age of the organism. The expression of ERalpha is regulated through interaction between cis-elements of its promoter and proteins present in the nuclei. Here, we have used electrophoretic mobility shift assay (EMSA) to analyze the effect of age, sex, 17beta-estradiol, and testosterone on the binding of ERalpha promoter (-91 to +46 bp) to nuclear proteins from the mouse cerebral cortex. EMSA revealed the formation of three specific complexes in all groups. However, the intensity of these complexes varied as a function of age, sex and treatment with 17beta-estradiol and testosterone. Nuclear proteins from the cerebral cortex of both sexes showed reduced binding with promoter fragment in old mice. Further, competition analysis indicated stronger binding in females than males of both ages. The extent of binding was reduced by 17beta-estradiol and testosterone treatment in both ages and sexes. Thus, these findings demonstrate differential binding of nuclear proteins to mouse ERalpha promoter which may account for different functions of estrogen in the brain.

Functional characterization of a -100_-102delAAG deletion-insertion polymorphism in the promoter region of the HTR3B gene

The HTR3B gene encodes the B-subunit of the type 3 serotonin receptor (5-HT3). A -100_-102delAAG deletion in the promoter region has been associated with poor response to antiemetic medication and susceptibility to bipolar affective disorders. The molecular mechanisms underlying these associations, however, remained unclear. We performed electrophoretic mobility shift and luciferase reporter gene assays to elucidate the effect of this polymorphism on the HTR3B promoter activity in PC-12 and HEK293 cells. The reporter constructs carried a 2171 bp fragment of the native HTR3B promoter or 30 bp of the polymorphic locus in tandem triplication upstream of the thymidine kinase minimal promoter. Deletion mapping indicated that the sequence around the -100_-102delAAG polymorphism had significant promoter activity. Electrophoretic mobility shift assays indicated differential binding of nuclear proteins to the polymorphic DNA region with stronger binding to the insertion than to the deletion allele. The activity of the native promoter carrying the deletion allele was 25% higher in PC-12 (P=0.016) and 40% higher in HEK cells (P=0.016) compared with the respective insertion construct. Constructs carrying the deletion allele in tandem triplicates showed 43% (PC-12 cells, P=0.002) and 28% (HEK293 cells, P=0.015) higher activity than those carrying the insertion allele. The polymorphism was not linked with known amino acid substitutions in HTR3A and HTR3B. The -100_-102delAAG 3 bp deletion increases the HTR3B promoter activity in vitro. The consequences of this for the structure and the function of the resulting 5-HT3 receptors remain to be elucidated.

Haplotype-dependent binding of nuclear proteins to the promoter of the neural tube defects-associated platelet-derived growth factor alpha-receptor gene

We have previously shown that polymorphisms in the promoter of the human platelet-derived growth factor alpha-receptor (PDGFRA) gene can be grouped into five distinct haplotypes, designated H1, H 2 alpha, H 2 beta, H 2 gamma and H 2 delta, and that specific combinations of these promoter haplotypes predispose to neural tube defects (NTDs). These promoter haplotypes differ strongly in their ability to drive reporter gene expression in various human cell lines, with highest activity for H 2 alpha and H 2 beta. Here, we show that the haplotype-linked PDGFRA promoter region extends to 3.6 kb upstream from the transcription start site, and contains a total of ten polymorphic sites. For two of these polymorphic sites, i.e. -909 C/A and +68 GAins/del, we observed differential binding of nuclear proteins from human osteosarcoma (HOS) cells. The protein complex binding specifically to -909 C, which is present in all haplotypes except the low activity haplotype H 2 gamma, contained members of the upstream stimulatory factor (USF) family of transcription factors. Furthermore, we identified a protein complex of 125 kDa which bound specifically to the low activity haplotype H1 at position +68 GAdel and may represent an H1-specific PDGFRA transcriptional repressor. The current identification of cis-acting elements in the PDGFRA promoter and the transcription factors that bind them, provides a new strategy for the identification of genes that are potentially involved in neural tube defects.

Genotypic Effect of the −565C&gt;T Polymorphism in the ABCA1 Gene Promoter on ABCA1 Expression and Severity of Atherosclerosis

Loss-of-function mutations of the ATP-binding cassette transporter A1 (ABCA1) gene cause Tangier disease, a rare genetic disorder with accumulation of lipid-laden macrophages and increased risk of atherosclerosis. Common variants of this gene may be a genetic factor for atherosclerosis in the general population. This study was performed to test the reported association between the -565C>T polymorphism and atherosclerosis severity and to investigate whether this variant per se had an effect on promoter activity of the ABCA1 gene. A cohort of patients with coronary atherosclerosis were genotyped for the -565C>T polymorphism. Logistic regression analyses showed that homozygotes of the -565T allele had greatest mean number of diseased coronary arteries, particular in nonsmokers. Real-time reverse-transcriptase polymerase chain reaction showed that in atherosclerotic plaques removed from patients undergoing endarteretomy, ABCA1 expression levels were lowest in those who had the T/T genotype and highest in those of the C/C genotype. Transfection and reporter assays demonstrated that in cultured macrophages, the -565T allelic promoter had a lower activity in driving gene expression than the -565C allelic promoter. Electrophoretic mobility shift assays displayed differential binding of nuclear proteins to the 2 alleles. These results indicate that the -565C>T polymorphism has an allele-specific effect on ABCA1 gene expression and provide further evidence of a genotypic effect on coronary atherosclerosis severity. The study showed that the ABCA1 gene -565C>T polymorphism was associated with severity of coronary atherosclerosis in a cohort of patients from Southern England and that this sequence variant per se had an effect on promoter activity of the ABCA1 gene. The data support the notion that common ABCA1 gene variants can contribute to interindividual variability in atherosclerosis susceptibility and severity.

Variation at the von Willebrand Factor (vWF) Gene Locus Is Associated With Plasma vWF:Ag Levels: Identification of Three Novel Single Nucleotide Polymorphisms in the vWF Gene Promoter

Both genetic and environmental factors contribute to the normal population variability of plasma von Willebrand Factor (vWF) levels, however, regulatory mechanisms at the vWF gene locus itself have not yet been identified. We have investigated the association between polymorphic variation in the 5′-regulatory region of the vWF gene and levels of plasma vWF:Ag in a study of 261 group O blood donors. Three novel single nucleotide polymorphisms (SNPs) were identified in the vWF promoter: C/T at -1234, A/G at -1185, and G/A at -1051. These SNPs had identical allele frequencies of 0.36 for the -1234C, -1185A, and -1051G alleles and 0.64 for the -1234T, -1185G, and -1051A alleles and were in strong linkage disequilibrium. In fact, these polymorphisms segregated as two distinct haplotypes: -1234C/-1185A/-1051G (haplotype 1) and -1234T/-1185G/-1051A (haplotype 2) with 12.6% of subjects homozygous for haplotype 1, 40.6% homozygous for haplotype 2, and 42.5% of subjects heterozygous for both haplotypes. Only 4.3% of individuals had other genotypes. A significant association between promoter genotype and level of plasma vWF:Ag was established (analysis of covariance [ANCOVA], P = .008; Kruskal-Wallis test,P = .006); individuals with the CC/AA/GG genotype had the highest mean vWF:Ag levels (0.962 U/mL), intermediate values of vWF:Ag (0.867 U/mL) were observed for heterozygotes (CT/AG/GA), and those with the TT/GG/AA genotype had the lowest mean plasma vWF:Ag levels (0.776 U/mL). Interestingly, when the sample was subgrouped according to age, the significant association between promoter genotype and plasma vWF:Ag level was accentuated in subjects &gt; 40 years of age (analysis of variance [ANOVA], P = .003; Kruskal-Wallis test, P= .001), but was not maintained for subjects ≤ 40 years of age (ANOVA, P &gt; .4; Kruskal-Wallis test, P &gt; .4). In the former subgroup, mean levels of plasma vWF:Ag for subjects with the CC/AA/GG, CT/AG/GA, and TT/GG/AA genotypes were 1.075, 0.954, and 0.794 U/mL, respectively. By searching a transcription factor binding site profile database, these polymorphic sequences were predicted to interact with several transcription factors expressed in endothelial cells, including Sp1, GATA-2, c-Ets, and NFκB. Furthermore, the binding sites at the -1234 and -1051 SNPs appeared to indicate allelic preferences for some of these proteins. Electrophoretic mobility shift assays (EMSAs) performed with recombinant human NFκB p50 showed preferential binding of the -1234T allele (confirmed by supershift EMSAs), and EMSAs using bovine aortic endothelial cell (BAEC) nuclear extracts produced specific binding of a nuclear protein to the -1051A allele, but not the -1051G allele. These findings suggest that circulating levels of vWF:Ag may be determined, at least in part, by polymorphic variation in the promoter region of the vWF gene, and that this association may be mediated by differential binding of nuclear proteins involved in the regulation of vWF gene expression.

Variation at the von Willebrand Factor (vWF) Gene Locus Is Associated With Plasma vWF:Ag Levels: Identification of Three Novel Single Nucleotide Polymorphisms in the vWF Gene Promoter

AbstractBoth genetic and environmental factors contribute to the normal population variability of plasma von Willebrand Factor (vWF) levels, however, regulatory mechanisms at the vWF gene locus itself have not yet been identified. We have investigated the association between polymorphic variation in the 5′-regulatory region of the vWF gene and levels of plasma vWF:Ag in a study of 261 group O blood donors. Three novel single nucleotide polymorphisms (SNPs) were identified in the vWF promoter: C/T at -1234, A/G at -1185, and G/A at -1051. These SNPs had identical allele frequencies of 0.36 for the -1234C, -1185A, and -1051G alleles and 0.64 for the -1234T, -1185G, and -1051A alleles and were in strong linkage disequilibrium. In fact, these polymorphisms segregated as two distinct haplotypes: -1234C/-1185A/-1051G (haplotype 1) and -1234T/-1185G/-1051A (haplotype 2) with 12.6% of subjects homozygous for haplotype 1, 40.6% homozygous for haplotype 2, and 42.5% of subjects heterozygous for both haplotypes. Only 4.3% of individuals had other genotypes. A significant association between promoter genotype and level of plasma vWF:Ag was established (analysis of covariance [ANCOVA], P = .008; Kruskal-Wallis test,P = .006); individuals with the CC/AA/GG genotype had the highest mean vWF:Ag levels (0.962 U/mL), intermediate values of vWF:Ag (0.867 U/mL) were observed for heterozygotes (CT/AG/GA), and those with the TT/GG/AA genotype had the lowest mean plasma vWF:Ag levels (0.776 U/mL). Interestingly, when the sample was subgrouped according to age, the significant association between promoter genotype and plasma vWF:Ag level was accentuated in subjects > 40 years of age (analysis of variance [ANOVA], P = .003; Kruskal-Wallis test, P= .001), but was not maintained for subjects ≤ 40 years of age (ANOVA, P > .4; Kruskal-Wallis test, P > .4). In the former subgroup, mean levels of plasma vWF:Ag for subjects with the CC/AA/GG, CT/AG/GA, and TT/GG/AA genotypes were 1.075, 0.954, and 0.794 U/mL, respectively. By searching a transcription factor binding site profile database, these polymorphic sequences were predicted to interact with several transcription factors expressed in endothelial cells, including Sp1, GATA-2, c-Ets, and NFκB. Furthermore, the binding sites at the -1234 and -1051 SNPs appeared to indicate allelic preferences for some of these proteins. Electrophoretic mobility shift assays (EMSAs) performed with recombinant human NFκB p50 showed preferential binding of the -1234T allele (confirmed by supershift EMSAs), and EMSAs using bovine aortic endothelial cell (BAEC) nuclear extracts produced specific binding of a nuclear protein to the -1051A allele, but not the -1051G allele. These findings suggest that circulating levels of vWF:Ag may be determined, at least in part, by polymorphic variation in the promoter region of the vWF gene, and that this association may be mediated by differential binding of nuclear proteins involved in the regulation of vWF gene expression.

Differential regulation of transcription termination occurring at two different sites on the micro-delta gene complex.

The progression of polymerases across the micro-delta Ig heavy chain gene complex is characterized by two termination events occurring at different sites on the transcription unit and at different times during B cell differentiation. We have utilized two mouse strains to analyze the regulatory determinants for these events in primary B cells. In the transgenic pmicro.microdeltaRatt strain a 1160 bp intervening DNA segment (the att site) has been inverted. This mutation results in the abrogation of transcription termination that occurs in early B cells. Using a novel method that takes advantage of an internal ribosome entry site we have further restricted the size of the segment that is needed for inducing transcription termination in transfectants. This 200 bp termination-inducing sequence operates in tumor equivalents of early but not mature B cells and the activity is correlated with differential binding of nuclear proteins. To explore the regulatory basis for the change in site of transcription termination upon B cell activation we have examined the microS-/- deletion mutant strain in which the microS poly(A) site has been eliminated. The results suggest that polyadenylation at the microS site plays a dominant but not exclusive role in regulating transcription termination in activated B cells.

Allelic polymorphisms in the transcriptional regulatory region of apolipoprotein E gene

In this work, we explored the existence of genetic variants within the apolipoprotein E gene transcriptional regulatory region, using a denaturing gradient gel electrophoresis screening of a region comprising nucleotides −1017 to +406. Upon a population study, three new polymorphic sites (−491, −427 and −219) and two mutations were found. Functional effects of the polymorphisms, assayed by transient transfection and electrophoretic mobility shift assays in a human hepatoma cell line, showed that polymorphisms at sites −491 and −219 of the APOE promoter produce variations in the transcriptional activity of the gene, most probably through differential binding of nuclear proteins.

Differential binding of nuclear proteins to the TP53 gene promoter in male breast tumour.

It is well established that TP53 regulates the expression of many genes, but the regulation of expression of TP53 itself is poorly understood. Recently, it has been shown that there is a tissue-specific binding of nuclear proteins in the TP53 gene promoter. The aim of this study was to determine the nuclear proteins that bind to the TP53 promoter elements (between -104 and -458) in male breast cancer. The results of our study, using the electrophoretic mobility shift assay (EMSA) and Southwestern analysis, have showed: (1) nuclear proteins or factors other than p53 bind to the TP53 promoter; (2) the levels of at least four nuclear proteins vary between normal and tumour breast tissue; and (3) two newly discovered nuclear proteins bind to the TP53 promoter in tumour tissue but are absent in normal tissue. This differential binding of nuclear proteins to the TP53 gene promoter might play a critical role in TP53 transcription and cancer progression in male breast tumours.

Genomic simple repetitive DNAs are targets for differential binding of nuclear proteins

The biological meaning of abundant simple repetitive DNA sequences in eukaryote genomes is obscure. Therefore, (GAA) n , (GT) n , and composite (GT) n (GA) m blocks were characterized for protein binding in the repeat and flanking sequences of cloned genomic DNA fragments. In gel mobility shift and competition assays the binding of nuclear proteins to the repeats was specific (including some flanking single copy sequences). DNase footprinting revealed the target sequences within and adjacent to the repeats. Chemical modifications (Os0 4, DAPC) DAmonstrated non-B DNA structures in the polypurine blocks. The binding of nuclear proteins in and around simple repeat sequences refute biological insignificance of all of these ubiquitously interspersed elements.