Mutant Probes Research Articles

Ehrlichia chaffeensis TRP32 is a Nucleomodulin that Directly Regulates Expression of Host Genes Governing Differentiation and Proliferation.

Ehrlichia chaffeensis is an obligately intracellular bacterium that reprograms the mononuclear phagocyte through diverse effector-host interactions to modulate numerous host cell processes, including transcription. In a previous study, we reported that E. chaffeensis TRP32, a type 1 secreted effector, interacts with multiple host nucleus-associated proteins and also auto-activates reporter gene expression in yeast. In this study, we demonstrate that TRP32 is a nucleomodulin that binds host DNA and alters host gene transcription. TRP32 enters the host cell nucleus via a noncanonical translocation mechanism that involves phosphorylation of Y179 located in a C-terminal tri-tyrosine motif. Both genistein and mutation of Y179 inhibited TRP32 nuclear entry. An electromobility shift assay (EMSA) demonstrated TRP32 host DNA binding via its tandem repeat domain. TRP32 DNA binding and motif preference were further confirmed by supershift assays, as well as competition and mutant probe analyses. Using ChIP-Seq, we determined that TRP32 binds a G-rich motif primarily within ±500 bp of the gene transcription start site. An ontology analysis identified genes involved in processes such as immune cell differentiation, chromatin remodeling, and RNA transcription and processing, as primary TRP32 targets. TRP32 bound genes (n=1223) were distributed on all chromosomes and included several global regulators of proliferation and inflammation such as FOS and JUN, AKT3 and NRAS, and non-coding RNA genes, miRNA 21 and miRNA 142. TRP32 target genes were differentially regulated during infection, the majority of which were repressed, and direct repression/activation of these genes by TRP32 was confirmed in vitro with a cellular luciferase reporter assay.

Effect of a Single Nucleotide Polymorphism in the LAMA1 Promoter Region on Transcriptional Activity: Implication for Pathological Myopia

ABSTRACTPurpose: To elucidate the role of the protein coding laminin α1 (LAMA1) gene in pathological myopia (PM) at the transcriptional level. To achieve this, the binding affinity of single nucleotide polymorphism (SNP) rs2089760—located on the LAMA1 promoter gene—to human fetal scleral fibroblast (HFSF) nucleoprotein was investigated and its effect on LAMA1 transcriptional initiation activity was analyzed.Methods: Binding interactions of the HFSF nucleoprotein and biotin-labeled SNP rs2089760 probe were investigated by amplifying the LAMA1 promoter gene and performing overlap extension polymerase chain reaction (PCR) to obtain the G/A mutation of LAMA1 SNP rs2089760. Recombinant adenovirus vectors, Ad5f11p-pLAMA1SNPa-Luc2, Ad5f11p-pLAMA1SNPg-Luc2, and Ad5f11p-CMV-RLuc, were constructed. Fluorescence intensity ratios of firefly luciferase (FLuc) and renilla luciferase (RLuc) vectors were measured 48 h after HFSF infection.Results: Both specific and mutant probes banded precisely with HFSF nucleoprotein. The intensity value of the mutant probe was significantly lower than that of the specific probe (p < 0.05). HFSFs were successfully infected by the recombinant adenoviruses. The FLuc/RLuc fluorescence intensity ratio of Ad5f11p-pLAMA1SNPa-Luc2 (0.0238 ± 0.0009) was significantly lower than that of Ad5f11p-pLAMA1SNPg-Luc2 (0.0281 ± 0.0015) (p < 0.05).Conclusions: It is highly likely that SNP rs2089760 in the LAMA1 promoter region is located at the transcription factor binding site. The SNP rs2089760 G > A mutation reduces transcription factor binding ability and transcriptional initiation activity, and negatively regulates gene transcription of LAMA1. We suggest that LAMA1 SNP rs2089760 plays an important role in the development of PM.

Alternative method of allelic discrimination.

5' nuclease assays for allelic discrimination use both a wild-type and a mutant probe. Here we present a new method for genotyping by 5' nuclease assays that dispenses with the mutant probe, using the wild-type probe together with a probe for a reference gene known to be present in two copies to determine the copy number of the wild type allele relative to the reference gene. The copy number of the wild-type allele then determines the genotype: two copies indicates homozygous wild-type; one copy indicates heterozygous; and zero copies indicates homozygous mutant. We were able to use our method to correctly genotype three alleles of the thiopurine methyl transferase (TPMT) gene: TPMT *2 (c.G238C), *3B (c.G460A) and *3C (c.A719G). Our approach can be used as an alternate allelic discrimination strategy that is cost effective when multiple TaqMan assays are performed on a sample.

Miz-1 activates gene expression via a novel consensus DNA binding motif.

The transcription factor Miz-1 can either activate or repress gene expression in concert with binding partners including the Myc oncoprotein. The genomic binding of Miz-1 includes both core promoters and more distal sites, but the preferred DNA binding motif of Miz-1 has been unclear. We used a high-throughput in vitro technique, Bind-n-Seq, to identify two Miz-1 consensus DNA binding motif sequences—ATCGGTAATC and ATCGAT (Mizm1 and Mizm2)—bound by full-length Miz-1 and its zinc finger domain, respectively. We validated these sequences directly as high affinity Miz-1 binding motifs. Competition assays using mutant probes indicated that the binding affinity of Miz-1 for Mizm1 and Mizm2 is highly sequence-specific. Miz-1 strongly activates gene expression through the motifs in a Myc-independent manner. MEME-ChIP analysis of Miz-1 ChIP-seq data in two different cell types reveals a long motif with a central core sequence highly similar to the Mizm1 motif identified by Bind-n-Seq, validating the in vivo relevance of the findings. Miz-1 ChIP-seq peaks containing the long motif are predominantly located outside of proximal promoter regions, in contrast to peaks without the motif, which are highly concentrated within 1.5 kb of the nearest transcription start site. Overall, our results indicate that Miz-1 may be directed in vivo to the novel motif sequences we have identified, where it can recruit its specific binding partners to control gene expression and ultimately regulate cell fate.

Abnormal B cell memory subsets dominate HIV-specific responses in infected individuals.

Recently, several neutralizing anti-HIV antibodies have been isolated from memory B cells of HIV-infected individuals. Despite extensive evidence of B cell dysfunction in HIV disease, little is known about the cells from which these rare HIV-specific antibodies originate. Accordingly, we used HIV envelope gp140 and CD4 or coreceptor (CoR) binding site (bs) mutant probes to evaluate HIV-specific responses in peripheral blood B cells of HIV-infected individuals at various stages of infection. In contrast to non-HIV responses, HIV-specific responses against gp140 were enriched within abnormal B cells, namely activated and exhausted memory subsets, which are largely absent in the blood of uninfected individuals. Responses against the CoRbs, which is a poorly neutralizing epitope, arose early, whereas those against the well-characterized neutralizing epitope CD4bs were delayed and infrequent. Enrichment of the HIV-specific response within resting memory B cells, the predominant subset in uninfected individuals, did occur in certain infected individuals who maintained low levels of plasma viremia and immune activation with or without antiretroviral therapy. The distribution of HIV-specific responses among memory B cell subsets was corroborated by transcriptional analyses. Taken together, our findings provide valuable insight into virus-specific B cell responses in HIV infection and demonstrate that memory B cell abnormalities may contribute to the ineffectiveness of the antibody response in infected individuals.

A nucleotide variant in promoter of the human CDH13 gene which affects its transcription activity is associated with colorectal cancer

T-cadherin is frequently down regulated in various cancers, however, the underlying mechanisms responsible have yet to be elucidated. A genome wide association study of a cohort of Korean adults revealed that the T-cadherin rs3865188 single nucleotide polymorphism (SNP) was associated with serum Adiponectin levels and that its genotypic variants were correlated with risk for colorectal cancer (CRC). To test the function of rs12444338, a SNP tightly linked to the rs3865188 SNP, in T-cadherin transcriptional regulation in colorectal cancer, its effect on transcriptional activity and the capacity of binding activity attributable to allelic variation of the rs12444338 SNP was investigated. An electrophoretic-mobility-shift assay (EMSA) revealed a specific nucleoprotein complex unique to the T allele probe, which displayed lower promoter activity when compared to the G allele. Based on the results of the EMSA using mutant probes, the consensus sequence of the putative transcription factor binding site was determined. Additionally, candidates for putative binding factors to the T allele were also identified. Collectively, the study data suggested that the rs12444338 SNP was involved in transcriptional regulation of T-cadherin gene (CDH13) and that the differential binding of transcription factors at the rs12444338 SNP resulted in altered gene expression. These results elucidate, at least in part, the regulation of T-cadherin expression in CRC and provide further information regarding the effect of nucleotide variation in its promoter region.

Molecular patterns of multidrug resistance of Mycobacterium tuberculosis in Georgia

BackgroundTuberculosis (TB) infections caused by multidrug-resistant Mycobacterium tuberculosis (MDR MTB) remain a significant public health concern worldwide. Georgia has a high prevalence of MDR MTB. The genetic mechanisms underlying the emergence of MDR MTB strains in this region are poorly understood and need to be determined for developing better strategies for TB control. This study investigated the frequency of major drug resistance mutations across rpoB, katG and inhA loci of Georgian MDR MTB strains and explored differences between new and previously treated patients. MethodsA total of 634 MTB strains were examined for which an MDR phenotype had been previously determined by the proportions method. The GenoType®MTBDRplus system was applied to screen the strains for the presence of rpoB (S531L, H526D, H526Y, and D516V), katG (S315T) and inhA promoter region (C15T and T8C) mutations. The target loci were amplified by PCR and then hybridized with the respective site-specific and wild type (control) probes. ResultsOut of the 634 isolates tested considered by phenotypic testing to be resistant to RIF and INH, this resistance was confirmed by the GenoType®MTBDRplus assay in 575 (90.7%) isolates. RIF resistance was seen in 589 (92.9%) and INH resistance was seen in 584 (92.1%); 67.2% and 84.3% of MDR strains harbored respectively rpoB S531L and katG S315T mutations (generally known as having low or no fitness cost in MTB). The inhA C15T mutation was detected in 22.6% of the strains, whereas rpoB H526D, rpoB H526Y, rpoB D516V and inhA T8C were revealed at a markedly lower frequency (⩽5.2%). The specific mutations responsible for the RIF resistance of 110 isolates (17.4%) could not be detected as no corresponding mutant probe was indicated in the assay. There was no specific association of the presence of mutations with the gender/age groups. All types of prevailing mutations had higher levels in new cases. ConclusionsA great majority of the Georgian MDR MTB strains have a strong preference for the drug resistance mutations carrying no or low fitness cost. Thus, it can be suggested that MDR MTB strains with such mutations will continue to arise in Georgia at a high frequency even in the absence of antibiotic pressure.

Peroxisome Proliferator-activated Receptor γ Agonists Induce Cell Cycle Arrest through Transcriptional Regulation of Krüppel-like Factor 4 (KLF4)

Peroxisome proliferator-activated receptor γ (PPARγ), a subgroup of ligand-activated nuclear receptors, plays critical roles in cell cycle regulation, differentiation, apoptosis, and invasion. PPARγ is involved in tumorigenesis and is a potent target for cancer therapy. PPARγ transactivation of KLF4 has been demonstrated in various studies; however, how PPARγ regulates KLF4 expression is not clear. In this study, we reveal that PPARγ regulates the expression of KLF4 by binding directly to the PPAR response element (PPRE) within the KLF4 promoter. The PPRE resides at -1657 to -1669 bp upstream of the KLF4 ATG codon, which is essential for the transactivation of troglitazone-induced KLF4 expression. Furthermore, we found that stable silencing of KLF4 obviously suppressed the G(1)/S arrest and anti-proliferation effects induced by PPARγ ligands. Taken together, our data indicate that up-regulation of KLF4 upon PPARγ activation is mediated through the PPRE in the KLF4 promoter, thus providing further insights into the PPARγ signal transduction pathway as well as a novel cancer therapeutic strategy.

Identification of the Novel Erythroid-Specific Enhancer in the First Intron of Human ALAS2 Gene; Tthe Mutation Disrupting GATA Transcription Factor-Binding Site in the Enhancer Causes X-Linked Sideroblastic Anemia

AbstractAbstract 3185Human ALAS2 gene encodes erythroid-specific 5-aminolevulinate synthase, the rate-limiting enzyme of heme biosynthetic pathway in erythroid cells. The mutation of ALAS2 gene causes X-linked sideroblastic anemia (XLSA), and approximate 50 different mutations have been reported in the coding region of ALAS2 gene as disease causative mutations. Here, we report two novel mutations of ALAS2 gene identified in a pedigree with XLSA and one male patient with congenital sideroblastic anemia (CSA). Incidentally, both mutations were predicted to impair or abolish the function of the specific GATA transcription factor-binding motif (GATA element) located at the middle of the first intron of ALAS2 gene (referred as ALAS2int1GATA). In a proband of the pedigree of XLSA, the “GATA” sequence in ALAS2int1GATA, which is the core sequence of consensus for GATA element (WGATAR), was changed to “GGTA”, and the same mutation was identified in two male relatives, his mother’s cousins, both of whom were diagnosed as sideroblastic anemia. Furthermore, a patient with CSA carries a deletion of 35 bps in the first intron of ALAS2 gene, the deleted region of which contains ALAS2int1GATA, although this deletion was not detected in his parents’ ALAS2 gene. No other mutation was detected in the proximal promoter region, the known enhancer region present in eighth intron, and the coding region and exon-intron boundaries of ALAS2 gene. Moreover, no mutation was detected in the coding region and exon-intron boundaries of SLC25A38, ABCB7, GLRX5, PUS1 or SLC19A2 gene, each of which was reported as a responsible gene for CSA. It is of interest that ALAS2int1GATA is present within one of “GATA transcription factor-occupying regions in K562 erythroleukemia cells” identified by genome-wide analysis using chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) (Fujiwara et al., Mol Cell, vol. 36: p667–681, 2009), suggesting that ALAS2int1GATA may act as a cis-regulatory element for ALAS2 transcription in vivo. In fact, our ChIP-PCR analysis in K562 cells, which focused on the proximal promoter (−267 to +29) and the first intron of ALAS2 gene, confirmed that GATA-1 protein selectively bound to the GATA element in the proximal promoter region as well as ALAS2int1GATA out of 17 consensus GATA elements present in the first intron. Then, we examined whether the 467-bp region in the first intron of ALAS2 gene, defined by ChIP-seq analysis (referred as “ALAS2 ChIP-peak”), acts as an enhancer for ALAS2 expression using transient reporter assay, because ALAS2 ChIP-peak contains ALAS2int1GATA. For this assay, we used pGL3-AEpro, which contains the firefly luciferase gene under the control of the ALAS2 proximal promoter, as a parent reporter vector. The presence of ALAS2 ChIP-peak in the downstream region of the luciferase gene increased ALAS2 proximal promoter activity about ten-folds in K562 cells. However, this enhancing activity of ALAS2 ChIP-peak was not observed in non-erythroid HEK293 embryonic kidney cells. Importantly, each newly identified mutation at ALAS2int1GATA diminished the enhancer activity of ALAS2 ChIP-peak on ALAS2 proximal promoter in K562 cells. Moreover, electrophoretic mobility shift assay (EMSA) revealed that GATA-1 protein in nuclear extracts of K562 cells or HEK293 cells overexpressing GATA-1 could bind to a wild-type probe containing ALAS2int1GATA, but GATA-1 failed to bind to a mutant probe, which carries a single base change in ALAS2int1GATA or a deletion of ALAS2int1GATA that was identified in patients with XLSA or CSA, respectively. These results suggest that ALAS2int1GATA plays an essential role for enhancing ALAS2 expression as a core of the ALAS2 ChIP-peak, the function of which may depend on the erythroid-specific transcription factor, GATA-1. Thus, the mutation at or the deletion of ALAS2int1GATA impairs the recruitment of GATA transcription factor(s) to the ALAS2 ChIP-peak, which in turn decreases the transcription of ALAS2 gene, thereby causing XLSA or CSA. In conclusion, we provide the evidence for the existence of an enhancer element in the first intron of ALAS2 gene. Moreover, the loss-of-function mutation at the newly identified enhancer element of ALAS2 gene is associated with XLSA or CSA. Disclosures:No relevant conflicts of interest to declare.

Activation of Transcription Factor MEF2D by Bis(3)-cognitin Protects Dopaminergic Neurons and Ameliorates Parkinsonian Motor Defects

Parkinson disease (PD) is characterized by the selective demise of dopaminergic (DA) neurons in the substantial nigra pars compacta. Dysregulation of transcriptional factor myocyte enhancer factor 2D (MEF2D) has been implicated in the pathogenic process in in vivo and in vitro models of PD. Here, we identified a small molecule bis(3)-cognitin (B3C) as a potent activator of MEF2D. We showed that B3C attenuated the toxic effects of neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) by activating MEF2D via multiple mechanisms. B3C significantly reduced MPP(+)-induced oxidative stress and potentiated Akt to down-regulate the activity of MEF2 inhibitor glycogen synthase kinase 3β (GSK3β) in a DA neuronal cell line SN4741. Furthermore, B3C effectively rescued MEF2D from MPP(+)-induced decline in both nucleic and mitochondrial compartments. B3C offered SN4741 cells potent protection against MPP(+)-induced apoptosis via MEF2D. Interestingly, B3C also protected SN4741 cells from wild type or mutant A53T α-synuclein-induced cytotoxicity. Using the in vivo PD model of C57BL/6 mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP), we showed that B3C maintained redox homeostasis, promoted Akt function activity, and restored MEF2D level in midbrain neurons. Moreover, B3C greatly prevented the loss of tyrosine hydroxylase signal in substantial nigra pars compacta DA neurons and ameliorated behavioral impairments in mice treated with MPTP. Collectedly, our studies identified B3C as a potent neuroprotective agent whose effectiveness relies on its ability to effectively up-regulate MEF2D in DA neurons against toxic stress in models of PD in vitro and in vivo.

Does increasing the JAK2V617F assay sensitivity allow to identify more patients with MPN?

Does increasing the JAK2V617F assay sensitivity allow to identify more patients with MPN?

The role of heat shock factor 1 in transcriptional regulation of high mobility group box

Background: Previous study suggests that high mobility group box 1 (HMGB1) can be a potential late inflammatory mediator. However, whether heat shock factor 1 (HSF1) regulate HMGB1 expression via binding to heat shock element (HSE) is not known. Objective: We investigated the role of HSF1 in the transcriptional regulation of HMGB1 protein. Methods: A probe that included HMGB1 promoter region containing HSE was synthesized for electrophoretic mobility shift assay (EMSA) to determine the binding of HSF1 and HSE in the promoter region of HMGB1 gene. Mutant mouse HMGB1 promoter was prepared by PCR amplification on a template of wild-type plasmid DNA with site-directed mutant primers. The mutant DNA fragments were also inserted into a corresponding plasmid. In addition, luciferase reporter plasmids of HMGB1 promoter were constructed to transfect RAW264.7 cells. After that, luciferase activity was measured to assay the effects of the HSF1 transfection on the promoter activity. Results: EMSA result showed a retardation strap after the coculture of biotin labeled HSF1 binding fragment and nuclear protein extracts. The retardation phenomenon could be competed by unlabeled probe and not by unlabeled mutant probe. A super retardation strap was present after adding HSF1 monoclonal antibody. After the HSE core sites was mutated, the relative luciferase activity of the mutant plasmid decreased by 4.26 folds compared with that in the wild-type (23.54±1.68 vs.100.25±3.26, p Conclusion: HSF1 can bind to the promoter region HSE (-668bp~-651bp) of HMGB1, which down-regulates the expression of HMGB1. Keywords: Heat shock transcription factor 1, high mobility group box 1, mouse, RAW264.7, transcription

The role of heat shock factor 1 in transcriptional regulation of high mobility group box

Background: Previous study suggests that high mobility group box 1 (HMGB1) can be a potential late inflammatory mediator. However, whether heat shock factor 1 (HSF1) regulate HMGB1 expression via binding to heat shock element (HSE) is not known. Objective: We investigated the role of HSF1 in the transcriptional regulation of HMGB1 protein. Methods: A probe that included HMGB1 promoter region containing HSE was synthesized for electrophoretic mobility shift assay (EMSA) to determine the binding of HSF1 and HSE in the promoter region of HMGB1 gene. Mutant mouse HMGB1 promoter was prepared by PCR amplification on a template of wild-type plasmid DNA with site-directed mutant primers. The mutant DNA fragments were also inserted into a corresponding plasmid. In addition, luciferase reporter plasmids of HMGB1 promoter were constructed to transfect RAW264.7 cells. After that, luciferase activity was measured to assay the effects of the HSF1 transfection on the promoter activity. Results: EMSA result showed a retardation strap after the coculture of biotin labeled HSF1 binding fragment and nuclear protein extracts. The retardation phenomenon could be competed by unlabeled probe and not by unlabeled mutant probe. A super retardation strap was present after adding HSF1 monoclonal antibody. After the HSE core sites was mutated, the relative luciferase activity of the mutant plasmid decreased by 4.26 folds compared with that in the wild-type (23.54±1.68 vs.100.25±3.26, p Conclusion: HSF1 can bind to the promoter region HSE (-668bp~-651bp) of HMGB1, which down-regulates the expression of HMGB1. Keywords: Heat shock transcription factor 1, high mobility group box 1, mouse, RAW264.7, transcription

Development of an Allele-Specific Real-Time PCR Assay for Discrimination and Quantification of p63 R279H Mutation in EEC Syndrome

The ectrodactyly-ectodermal dysplasia-clefting syndrome is a rare autosomal dominant disorder caused by heterozygous mutations in the p63 gene, a transcription factor belonging to the p53 family. The majority of cases of ectrodactyly-ectodermal dysplasia syndrome are caused by de novo mutations and are therefore sporadic in approximately 60% of patients. The substitution of arginine to histidine (R279H), due to a c.836G>A mutation in exon 7 of the p63 gene, represents 55% of the identified mutations and is considered a mutational hot spot. A quantitative and sensitive real-time PCR was performed to quantify both wild-type and R279H alleles in DNA extracted from peripheral blood and RNA from cultured epithelial cells. Standard curves were constructed for both wild-type and mutant probes. The sensitivity of the assay was determined by generating serial dilutions of the DNA isolated from heterozygous patients (50% of alleles mutated) with wild-type DNA, thus obtaining decreasing percentages of p63 R279H mutant allele (50%, 37.5%, 25%, 12.5%, 10%, 7.5%, 5%, 2.5%, and 0.0%). The assay detected up to 1% of the mutant p63. The high sensitivity of the assay is of particular relevance to prenatal diagnosis and counseling and to detect therapeutic effects of drug treatment or gene therapy aimed at reducing the amount of mutated p63.

Genetic mutations in non-syndromic deafness patients of uyghur and han chinese ethnicities in xinjiang, China: a comparative study

BackgroundThe deafness-associated gene mutation profile varies greatly among regions and races. Due to the multi-ethnic coalition of over one thousand years, non-syndromic deafness (NSD) patients of Uyghur ethnicity may exhibit a unique deafness-associated gene mutation spectrum as compared to Han Chinese deaf population.MethodsIn order to characterize nine loci of four deafness-associated genes of Uyghur NSD patients in comparison with Chinese Han deaf population, NSD patients (n = 350) were enrolled, including Uyghur (n = 199) and Han Chinese (n = 151). Following the history taking, blood samples were collected for DNA extraction. DNA microarray was performed on nine loci of four deafness-associated genes, including 35delG, 176-191del16, 235delC, 299-300delAT, 538C > T, 1555A > G, 1494C > T, 2168A > G, and IVS7-2A > G. The samples that showed the absence of both wild and mutant probe signals were tested for further DNA sequencing analysis.ResultsThe mutations in the nine loci of prevalent deafness-associated genes were detected in 13.06% of Uyghur NSD patients and 32.45% of Han Chinese patients (P < 0.05), respectively. GJB2 mutation was detected in 9.05% of Uyghur patients and 16.56% of Han Chinese patients (P > 0.05), respectively. 235delC was the hotspot mutation region in NSD patients of the two ethnicities, whereas 35delG was the mutation hotspot in Uyghur patients. 187delG mutation was detected for the first time in Uyghur NSD patients and considered as an unreported pathological variant of GJB2. SLC26A4 mutation was found in 2.01% of Uyghur patients and 14.57% of Han Chinese patients (P < 0.05), respectively. The frequencies of mtDNA 12S rRNA mutation in Uyghur and Han Chinese patients were 2.01% and 2.65% (P > 0.05), respectively. The NSD patients exhibited a low frequency of GJB3 mutation regardless of ethnicity.ConclusionPrevalent deafness-associated gene mutations in the nine loci studied were less frequently detected in Uyghur NSD patients than in Han Chinese patients. GJB2 was the most common mutant gene in the two ethnicities, whilst the two ethnicities differed substantially in hotspot mutations. A low-frequency SLC26A4 mutation was detected in Uyghur NSD patients. Uyghur NSD patients differed significantly from Han Chinese patients in gene mutation profile.

The effects of an insertion in the 5′UTR of the AMCase on gene expression and pulmonary functions

Studies regarding the physiological role of acidic mammalian chitinase (AMCase) and the effects of its genetic variants on asthma have produced conflicting results. We aimed to determine the genetic variants in the AMCase gene that could regulate the gene expression and thus influence disease severity. Genetic variants of the AMCase gene were determined by sequencing of asthmatics and healthy controls in up to -1kb in the promoter region and exon 1 and 2. In an association study, a population of asthmatic (n=504) and healthy Turkish children (n=188) were genotyped for the observed SNPs. A replication study was performed in a North American adult population of patients with mild (n=317) and severe (n=145) asthma. The functional properties of the insertion were determined by promoter reporter assay, electromobility shift assay and transcription factor ELISA experiments. Of the identified SNPs, only a ten base pair insertion (CAATCTAGGC) in the 5'UTR region of exon 2 was significantly associated with lower FEV(1) (β=-14.63 SE=6.241, P=0.019) in Turkish children with asthma. However, in the adult population, the same insertion showed a trend toward higher FEV(1). The insertion was shown to have enhancer activity and the mutant probe possessing the insertion had higher binding affinity for the nuclear extracts. Our study shows that a ten base pair insertion in the 5'UTR region of AMCase gene may modify gene expression and thus may affect the severity of asthma. However, its effects appear to be different in different populations.

Recruitment of SWI/SNF Complex Is Required for Transcriptional Activation of the SLC11A1 Gene during Macrophage Differentiation of HL-60 Cells

The solute carrier family 11 member 1 (SLC11A1) gene is strictly regulated and exclusively expressed in myeloid lineage cells. However, little is known about the transcriptional regulation of the SLC11A1 gene during myeloid development. In this study, we used HL-60 cells as a model to investigate the regulatory elements/factors involved in the transactivation of the SLC11A1 gene during phorbol 12-myristate 13-acetate (PMA)-induced macrophage differentiation of HL-60 cells. Promoter deletion analysis showed that a 7-base AP-1-like element (TGACTCT) was critical for the responsiveness of the SLC11A1 promoter to PMA. Stimulation by PMA induced the binding of ATF-3 and the recruitment of two components of the SWI/SNF complex, BRG1 and β-actin, to this element in an ATF-3-dependent manner. RNAi-mediated depletion of ATF-3 or BRG1 markedly decreased SLC11A1 gene expression and its promoter activity induced by PMA. Luciferase reporter experiments demonstrated that ATF-3 cooperated with BRG1 and β-actin to activate the SLC11A1 promoter. Furthermore, we showed that PMA can induce the proximal (GT/AC)(n) repeat sequence to convert to the Z-DNA structure in the SLC11A1 gene promoter, and depletion of BRG1 resulted in a significant decrease of Z-DNA formation. Our results demonstrated that recruitment of the SWI/SNF complex initiated Z-DNA formation and subsequently helped to transactivate the SLC11A1 gene.

The Role of p38 MAPK/CREB1 Signaling In γ-Globin Gene Regulation

AbstractAbstract 1012The clinical symptoms of sickle cell disease can be ameliorated by increased fetal hemoglobin (HbF) levels. Previous work from our laboratory demonstrated that Trichostatin A (TSA) and sodium butyrate (NaB) activate γ-globin expression via p38 MAPK signaling. In addition, cAMP response element binding protein 1 (CREB1) was shown to trans-activate the -1222 Gγ-globin cAMP response element (G-CRE) in a transient assay system. To study the role of p38 MAPK signaling in γ-globin regulation, loss of function siRNA studies were performed in K562 cells. siRNA-mediated knockdown of p38 MAPK resulted in 72% loss of γ-globin transcription. Furthermore, enforced stable expression of MKK3/6 increased the phosphorylated form of p38 MAPK by 70%, which in turn produced a 2- to 3-fold increase in γ-globin mRNA and HbF levels. Likewise, siCREB1 treatment reduced CREB1 levels by 62% and down regulated γ-globin expression 59%. In the same vein, stable expression of recombinant CREB1 activated HbF by 2-fold. These findings were subsequently confirmed in human primary erythroid cells grown in a two-phase liquid culture system. On day 11, we observed 50–70% γ-globin silencing after CREB1 and p38 MAPK siRNA knockdown with 60% target gene silencing. CREB1 enforced expression trans-activated γ-globin 4.5-fold which was accompanied by a 90% increase in HbF-FITC positive cells and HbF levels. Collectively, these data demonstrate that p38 MAPK and CREB1 are required for steady-state γ-globin gene transcription. To determine the role of the G-CRE in γ-globin regulation, the Gγ-globin promoter (-1500 to +36) was cloned into pGL4.17 Luc2/neo to produce pGγLuc2 (wild type) and mutant plasmids: -1225 G/A (m1), -1227 AC/TG (m2) and a scrambled G-CRE (m3s). Five K562 stable lines including KLuc2 (promoterless), KGγLuc2, KGγLuc2(m1), KGγLuc2(m2) and KGγLuc2(m3s) were established. Luciferase activity in KGγLuc2 was 1000-fold higher than in the control Kluc2 line; all mutations produced >90% loss of luciferase activity and a loss of γ-globin trans-activation by TSA and NaB. Next, siRNA studies were completed to determine if the G-CRE is required for γ-globin activation. A dose-dependent loss of promoter activity was observed after p38 MAPK and CREB1 siRNA knockdown of the KGγLuc2 cell line; however, promoter silencing was not observed in the mutant lines supporting a role for the G-CRE in p38 MAPK/CREB1 mediated γ-globin regulation. To study in vivo binding, chromatin immunoprecipitation (ChIP) assays were performed with CREB1 antibody in the KGγLuc2 stable line. We observed comparable 2- to 3-fold chromatin enrichment with CREB1 compared to the control IgG in the G-CRE regions of the pGγLuc2 plasmid and endogenous Gγ-globin promoter. To determine if an enhanceosome complex is bound to the G-CRE, we performed affinity column pull-down/mass spectrometry analysis. K562 nuclear extract was purified on a Heparin Sepharose column, following which fractions eluting at 0.6M NaCl showing peak gel shift binding activities with the G-CRE oligo were loaded into a size selecting Suprose 6 gel exclusion column. G-CRE eluting fractions were then identified by protein microsequencing (MS/MS). We identified CREB1, ATF2, c-Jun, BRG-1, hnRNPC1/C2, and the TCP-1 complex as major components. To determine protein co-localization, promoter pull-down assays were performed using biotinylated wild type and mutant (AC/TG) G-CRE probes and K562 nuclear extracts. We observed simultaneous CREB1, ATF-2 and cJun binding to the G-CRE which was abolished in the mutant probe. However, Brg1 was bound after NaB (2mM) induction. Subsequent co-IP studies showed interactions between ATF-2 and Brg1, CREB1, cJun, and hnRNPC1C2, which was further confirmed by co-elution profile of these molecules observed by sucrose gradient centrifugation, thus implying association as one complex. These data support complex protein-protein interactions in the G-CRE, which modulate γ-globin gene expression. Additional studies will be performed in primary erythroid cells using siRNA-based gene silencing and ChIP assays to determine novel mechanisms of γ-globin regulation and to define in vivo binding of proteins identified in the G-CRE enhanceosome complex. Disclosures:No relevant conflicts of interest to declare.

Melting temperature of surface-tethered DNA

A method for the accurate determination of the melting temperature ( T m) of surface-immobilized DNA duplexes that exploits the fluorescence-quenching properties of gold is reported. A thiolated single-stranded DNA probe is chemisorbed onto a gold surface and then hybridized to a fluorophore-labeled complementary sequence. On formation of the duplex, the fluorescence of the label is effectively quenched by the gold surface. As the temperature is increased and the duplex denatures, the fluorophore label moves away from the gold surface and the fluorescence signal is again observed. The increase in fluorescence is measured as the temperature is ramped, and using first-derivative plots, the T m is determined. To demonstrate the approach, the T m of the cystic fibrosis DF508 mutation was determined in three different phases: in solution, in suspension immobilized on gold nanoparticles, and immobilized on gold film-coated substrate. The technique was further applied to optimize conditions for differentiation between a surface-immobilized DF508 mutant probe and a mutant/wild-type target exploiting increasing stringency in varying salt and formamide concentrations. The approach has application in optimization of assay conditions for biosensors that use gold substrates as well as in melting curve analysis.

Preaxial polydactyly/triphalangeal thumb is associated with changed transcription factor-binding affinity in a family with a novel point mutation in the long-range cis-regulatory element ZRS

A cis-regulatory sequence also known as zone of polarizing activity (ZPA) regulatory sequence (ZRS) located in intron 5 of LMBR1 is essential for expression of sonic hedgehog (SHH) in the developing posterior limb bud mesenchyme. Even though many point mutations causing preaxial duplication defects have been reported in ZRS, the underlying regulatory mechanism is still unknown. In this study, we analyzed the effect on transcription factor binding of a novel ZRS point mutation (463T>G) in a Pakistani family with preaxial polydactyly and triphalangeal thumb. Electrophoretical mobility shift assay demonstrated a marked difference between wild-type and the mutant probe, which uniquely bound one or several transcription factors extracted from Caco-2 cells. This finding supports a model in which ectopic anterior SHH expression in the developing limb results from abnormal binding of one or more transcription factors to the mutant sequence.