Electrophoretic Mobility Shift Assay Assays Research Articles

Baohuoside I inhibits virulence of multidrug-resistant Staphylococcus aureus by targeting the transcription Staphylococcus accessory regulator factor SarZ

BackgroundStaphylococcus aureus is a versatile pathogen that can cause a wide range of infections in humans. Biofilms play a crucial role in the pathogenicity of S. aureus and contribute to its ability to cause persistent and chronic infections. Baohuoside I has garnered increasing recognition as a natural flavonol glycoside with a wide spectrum of health-related activities. PurposeThe antibacterial and anti-biofilm properties of Baohuoside I have not been extensively investigated. Our study aimed to assess its inhibitory effects and the underlying mechanisms on biofilm formation and hemolytic capacity in S. aureus. Study Design/MethodsThe impact of Baohuoside I on the biofilm and virulence of S. aureus was evaluated through in vitro experiments and Galleria mellonella as an in vivo infection model. The mechanisms were explored by Drug affinity responsive target stability (DARTS) and validated in genetic knockout strain and through molecular biological experiments using DARTS, molecular docking, electrophoretic mobility shift assay (EMSA), and bio-layer interferometry (BLI). ResultsBaohuoside I significantly inhibits the formation of S. aureus biofilms and hemolytic activity at 6.25 µM. Proteomics analysis revealed that treatment with Baohuoside I led to a reduction in the expression of quorum-sensing system agr-regulated genes. DARTS analysis identified Staphylococcus accessory regulator factor (SarZ), a key regulator involved in the expression of virulence factors in S. aureus by acting as activator of the agr quorum-sensing system, was the direct target of Baohuoside I. Molecular docking, DARTS, BLI and EMSA assays collectively confirmed the direct binding of Baohuoside I to SarZ, inhibiting its binding to downstream promoters. Furthermore, it is found through site-directed protein mutagenesis that the Tyr27 and Phe117 residues are key for Baohuoside I binding to SarZ. Additionally, the knockout of SarZ significantly diminished the hemolytic ability of S. aureus, underscoring its crucial role as a pivotal regulator of virulence. Lastly, in vivo tests utilizing the G. mellonella infection model demonstrated the efficacy of Baohuoside I. ConclusionThis study provides valuable insights into the mechanism by which Baohuoside I inhibits the virulence of S. aureus through its interaction with SarZ. These findings highlight the significance of SarZ as an effective target against the virulence of S. aureus.

Genetic and functional variants of the TBX20 gene promoter in dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a major cause of heart failure and sudden cardiac death. As DCM is a genetically heterogeneous disease, genetic variants of cardiac transcription factor genes may play an important role. Transcription factor TBX20, an indispensable factor in normal heart development, is involved in the regulation of cardiac structure and function. Although the TBX20 gene is associated with the occurrence and development of DCM, the influence of genetic variants of the TBX20 gene promoter region on DCM has not been reported. We conducted a case-control study consisting of 107 DCM patients and 210 healthy controls. Genetic variants within TBX20 gene promoter region were identified using sequencing techniques and were functionally analyzed by dual-luciferase reporting assay. Electrophoretic mobility shift assay (EMSA) was used to investigate DNA-protein interactions. In this study cohort (n = 317), we identified eight variants within TBX20 gene promoter. One novel DNA sequence variants (DSV) (g.4275G>T) and four single-nucleotide polymorphisms (SNPs) [g.4169G>A (rs1263874255), g.4949C>T (rs1191745927), g.5114G>A (rs112076877), g.5252C>T (rs1356932911)] were identified in DCM patients, but in none of controls. Among them, the DSV (g.4275G>T) and three SNPs [g.4949C>T (rs1191745927), g.5114G>A (rs112076877) and g.5252C>T (rs1356932911)] significantly altered the transcription activity of TBX20 gene promoter by dual-luciferase reporting assay (p < 0.05). Further, EMSA assay indicated that the DSV (g.4275G>T) and three SNPs [g.4949C>T (rs1191745927), g.5114G>A (rs112076877) and g.5252C>T (rs1356932911)] affected the binding of transcription factors. These data indicate that the DSV (g.4275G>T) and three SNPs [g.4949C>T (rs1191745927), g.5114G>A (rs112076877) and g.5252C>T (rs1356932911)] increase transcription activity of TBX20 gene promoter in both HEK-293 and neonatal rat cardiomyocytes (NRCMs) cell lines by affecting the binding of transcription factors. But the mechanism remains to be verified invivo.

Non-Canonical Transcription Activator Activity of RNA-Binding Protein MBNL1 Uniquely Controls FLT3 expression in MLL-Rearranged Leukemias

MLL-rearranged (MLL-r) leukemias count for more than 80% of infant leukemia, ~5-10% of B-cell acute lymphoblastic leukemia (B-ALL), and ~10% of acute myeloid leukemia (AML) cases, where they confer a particularly poor outcome. Despite treatment with intensive multi-agent chemotherapy, most MLL-r patients ultimately relapsed after an initial remission. So far, the molecular mechanism by which MLL-r leukemia maintains progression and prevents differentiation remains largely unclear. Observations in the clinic support aberrant expression and concomitant activating driver mutations in the gene encoding the tyrosine kinase FLT3 that occur in leukemia, including the MLL-r subtype. To interrogate the novel MLL-r vulnerable genes regulating FLT3, we developed an algorithm so-called “context-dependent association analysis” (CDAA) to explore potential MLL-r dependent FLT3 regulators using the DepMap dataset. The RNA-binding protein MBNL1 was identified as the top candidate. MBNL1 has been recognized as an RNA-binding protein (RBP) involved in splicing, RNA export, and stability. However, our study suggested its non-canonical transcriptional activator function by uniquely controlling FLT3 expression in MLL-r leukemias. CRISPR/Cas9 disruption of the coding region of MBNL1 or CRISPR-interference (CRISPRi) against the MBNL1-bound site in the FLT3 distal enhancer notably decreased FLT3 mRNA expression in MLL-r leukemia cell lines. However, the FLT3 gene splicing defect was not observed upon MBNL1 loss, suggesting MBNL1 could be affecting FLT3 expression through a transcriptional regulation mechanism. Genome-wide ChIP-seq assays performed in MLL-r SEM and MOLM13 cells identified only two reproducible MBNL1-bound peaks at a genome-wide scale, one of which was located in the distal enhancer of FLT3, ~170kb away from the FLT3 promoter. CRISPR-interference (CRISPRi) against the MBNL1-bound site in the FLT3 distal enhancer notably decreased FLT3 mRNA expression in MLL-r leukemia cell lines without affecting enhancer-promoter looping. Using in vivo assay, SEM cells targeted with sgRNA against the MBNL1-bound site in the distal enhancer of FLT3 transplanted into NSG mice exhibited significant growth retardation. Furthermore, the impaired cell fitness crisis phenotype could be entirely rescued by overexpression of FLT3 cDNA. To identify the protein domains of MBNL1 required for its oncogenic function in MLL-r leukemia cells, an MBNL1 domain CRISPR screen was performed on the SEM cell line, stably expressing Cas9 and a pooled sgRNA library targeting the coding region of MBNL1. Essential domains observed from the screen included MBNL1's zinc finger domains. A biochemistry electrophoretic mobility shift assay (EMSA) was conducted using purified recombinant MBNL1 protein and a 207 bp DNA oligo synthesized based on the MBNL1 ChIP-seq peak at the FLT3 enhancer. The result clearly demonstrated that MBNL1 could directly bind to the enhancer DNA of FLT3 through its zinc fingers 1 and 2 domains, supporting its role as a novel transcriptional regulator of FLT3. Using state-of-art research tools, including genome editing, ChIP-seq, and EMSA assays, we have systematically interrogated an undocumented transcriptional regulation axis of MBNL1/FLT3 in MLL-r leukemias. This work will significantly advance our understanding of how transcription programs and novel factors reconstruct the regulatory network specifying MLL-r dependency, promoting the future development of alternative therapeutic targets for MLL-r leukemia in patients.

SALL4 promotes angiogenesis in gastric cancer by regulating VEGF expression and targeting SALL4/VEGF pathway inhibits cancer progression

BackgroundSpalt-like protein 4 (SALL4) is a stemness-related transcription factor whose abnormal re-expression contributes to cancer initiation and progression. However, the role of SALL4 in cancer angiogenesis remains unknown.MethodsAnalyses of clinical specimens via TCGA datasets were performed to determine the expression level and clinical significance of SALL4 in STAD (Stomach Adenocarcinoma). SALL4 knockdown, knockout, and overexpression were achieved by siRNA, CRISPR/Cas9, and plasmid transfection. The effects of conditioned medium (CM) from SALL4 knockdown or overexpression of gastric cancer cells on endothelial cell proliferation, migration, and tube formation were investigated by CCK-8 assay, transwell migration assay, and tube formation assay. The regulation of VEGF gene expression by SALL4 was studied by qRT-PCR, western blot, chromatin immunoprecipitation (ChIP) assay, and electrophoretic mobility shift assay (EMSA). Engineered exosomes from 293T cells loaded with si-SALL4-B and thalidomide were produced to test their therapeutic effect on gastric cancer progression.ResultsSALL4 expression was increased in STAD and positively correlated with tumor progression and poor prognosis. SALL4-B knockdown or knockout decreased while over-expression increased the promotion of human umbilical vein endothelial cells (HUVEC) cell proliferation, migration, and tube formation by gastric cancer cell-derived CM. Further investigation revealed a widespread association of SALL4 with angiogenic gene transcription through the TCGA datasets. Additionally, SALL4-B knockdown reduced, while over-expression enhanced the expression levels of VEGF-A, B, and C genes. The results of ChIP and EMSA assays indicated that SALL4 could directly bind to the promoters of VEGF-A, B, and C genes and activate their transcription, which may be associated with increased histone H3-K79 and H3-K4 modifications in their promoter regions. Furthermore, si-SALL4-B and thalidomide-loaded exosomes could be efficiently uptaken by gastric cancer cells and significantly reduced SALL4-B and Vascular Endothelial Growth Factor (VEGF) expression levels in gastric cancer cells, thus inhibiting the pro-angiogenic role of their derived CM.ConclusionThese findings suggest that SALL4 plays an important role in angiogenesis by transcriptionally regulating VEGF expression. Co-delivery of the functional siRNA and anticancer drug via exosomes represents a useful approach to inhibiting cancer angiogenesis by targeting SALL4/VEGF pathway.

NF45/NF90-mediated rDNA transcription provides a novel target for immunosuppressant development.

Herein, we demonstrate that NFAT, a key regulator of the immune response, translocates from cytoplasm to nucleolus and interacts with NF45/NF90 complex to collaboratively promote rDNA transcription via triggering the directly binding of NF45/NF90 to the ARRE2‐like sequences in rDNA promoter upon T‐cell activation in vitro. The elevated pre‐rRNA level of T cells is also observed in both mouse heart or skin transplantation models and in kidney transplanted patients. Importantly, T‐cell activation can be significantly suppressed by inhibiting NF45/NF90‐dependent rDNA transcription. Amazingly, CX5461, a rDNA transcription‐specific inhibitor, outperformed FK506, the most commonly used immunosuppressant, both in terms of potency and off‐target activity (i.e., toxicity), as demonstrated by a series of skin and heart allograft models. Collectively, this reveals NF45/NF90‐mediated rDNA transcription as a novel signaling pathway essential for T‐cell activation and as a new target for the development of safe and effective immunosuppressants.

PML-RAR Binds to the +7kb Enhancer of CEBPE and Inhibits Its Expression

Acute promyelocytic leukemia (APL) is a unique subtype of acute myeloid leukemia (AML). The disease is identified by distinctive morphology and is distinguished by a balanced reciprocal translocation between chromosomes 15 and 17. This aberration leads to the fusion between promyelocytic leukemia (PML) gene located on chromosome 15q21, and retinoic acid receptor α (RARA) gene from chromosome 17q21, leading to the resultant chimeric onco-fusion protein PML-RARA, which is detectable in more than 95% patients and disturbs proper promyelocytic differentiation. All-trans retinoic acid (ATRA) can induce granulocytic differentiation in APL and is used to treat APL patients. Genes containing PML-RARA-targeted promoters are transcriptionally suppressed in APL and most likely constitute a major mechanism of transcriptional repression occurring in APL. A growing body of evidence points to the role of distal regulatory elements, including enhancers, in the control of gene expression. In order to understand the unique sets of enhancers that might be under the control of PML-RAR and crucial for granulocytic differentiation of NB4 cells, we analysed the enhancer landscape of control and ATRA treated NB4 cells. H3K9Ac mapping identified a repertoire of enhancers that were gained in NB4 cells treated with ATRA. Closer investigation of these enhancer elements revealed enrichment of H3K9Ac signals around major drivers of myeloid differentiation. Of note, we identified a gain in enhancer signature for a region about 7kb downstream of the CEBPE gene. Our previous studies identified a novel enhancer for CEBPE in murine hematopoietic cells, which was 6 downstream of CEBPE core promoter. It appears that the +7kb region we identified in human APL cells may be analogous to the murine enhancer. We also observed that PML-RAR binds this +7kb region and ATRA treatment of NB4 cells displaced binding of PML-RAR from the + 7kb region, suggestive of a transcriptional repressive effect of PML-RAR at such enhancer elements. To test the transcription regulating potential of this +7kb region, we used catalytically inactive Cas9 fused to Krüppel associated box (KRAB) domain (dCas9-KRAB). We designed three guide RNAs covering this regulatory region. The sgRNAs effectively repressed expression of CEBPE accompanied by lowered granulocytic differentiation of these guide RNA targeted NB4 cells after ATRA treatment. To explore transcription factor (TF) occupancy at this +7 kb region, we analysed public available ChIP-seq datasets for hematopoiesis-specific factors. Analysis revealed that the +7kb region was marked by an open chromatin signature, accompanied by binding of a majority of hematopoietic TFs around this putative regulatory element with concurrent binding of EP300. Strikingly we noticed binding of CEBPA, CEBPB and CEBPE at this regulatory element. To assess whether binding of these members of the CEBP family of TFs is functionally relevant, luciferase reporter and electrophoretic mobility shift assays (EMSA) were performed. Co expression of the CEBP TFs led to significant induction of luciferase expression, and this data was further confirmed using EMSA assays. Based on these observations, we propose that PML-RAR blocks granulocytic differentiation by occupying this +7kb enhancer of CEBPE, hinders binding of other cell type/lineage specific TFs, and blocks CEBPE expression. When cells are stimulated with ATRA, PML-RAR is displaced from the CEBPE enhancer, allowing for efficient binding of myeloid-specific TFs. This results in increased CEBPE expression, which in turn promotes efficient granulocytic differentiation. The findings from our study expands our current understanding of the mechanism of differentiation therapy, the role of onco-fusion proteins in inhibiting myeloid differentiation, and may provide new therapeutic approaches to many acute myeloid leukemias. Disclosures Ong: National University of Singapore: Other: Royalties.

QseBC is involved in the biofilm formation and antibiotic resistance in Escherichia coli isolated from bovine mastitis.

BackgroundMastitis is one of the most common infectious diseases in dairy cattle and causes significant financial losses in the dairy industry worldwide. Antibiotic therapy has been used as the most effective strategy for clinical mastitis treatment. However, due to the extensive use of antibacterial agents, antimicrobial resistance (AMR) is considered to be one of the reasons for low cure rates in bovine mastitis. In addition, biofilms could protect bacteria by restricting antibiotic access and shielding the bacterial pathogen from mammary gland immune defences. The functional mechanisms of quorum sensing E. coli regulators B an d C (QseBC) have been well studied in E. coli model strains; however, whether QseBC regulates antibiotic susceptibility and biofilm formation in clinical E. coli strain has not been reported.MethodsIn this study, we performed construction of the qseBC gene mutant, complementation of the qseBC mutant, antimicrobial susceptibility testing, antibacterial activity assays, biofilm formation assays, real-time reverse transcription PCR (RT-PCR) experiments and electrophoretic mobility shift assays (EMSAs) to investigate the role of qseBC in regulating biofilm formation and antibiotic susceptibility in the clinical E. coli strain ECDCM2.ResultsWe reported that inactivation of QseBC led to a decrease in biofilm formation capacity and an increase in antibiotic susceptibility of an E. coli strain isolated from a dairy cow that suffered from mastitis. In addition, this study indicated that QseBC increased biofilm formation by upregulating the transcription of the biofilm-associated genes bcsA, csgA, fliC, motA, wcaF and fimA and decreased antibiotic susceptibility by upregulating the transcription of the efflux-pump-associated genes marA, acrA, acrB, acrD, emrD and mdtH. We also performed EMSA assays, and the results showed that QseB can directly bind to the marA promoter.ConclusionsThe QseBC two-component system affects antibiotic sensitivity by regulating the transcription of efflux-pump-associated genes. Further, biofilm-formation-associated genes were also regulated by QseBC TCS in E. coli ECDCM2. Hence, this study might provide new clues to the prevention and treatment of infections caused by the clinical E. coli strains.

RNA sequencing reveals an additional Crz1-binding motif in promoters of its target genes in the human fungal pathogen Candida albicans

BackgroundThe calcium/calcineurin signaling pathway is mediated by the transcription factors NFAT (nuclear factor of activated T cells) in mammals and Crz1 (calcineurin-responsive zinc finger 1) in yeasts and other lower eukaryotes. A previous microarray analysis identified a putative Crz1-binding motif in promoters of its target genes in Candida albicans, but it has not been experimentally demonstrated.MethodsAn inactivation mutant for CaCRZ1 was generated through CRISPR/Cas9 approach. Transcript profiling was carried out by RNA sequencing of the wild type and the inactivation mutant for CaCRZ1 in response to 0.2 M CaCl2. Gene promoters were scanned by the online MEME (Multiple Em for Motif Elicitation) software. Gel electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) analysis were used for in vitro and in vivo CaCrz1-binding experiments, respectively.ResultsRNA sequencing reveals that expression of 219 genes is positively, and expression of 59 genes is negatively, controlled by CaCrz1 in response to calcium stress. These genes function in metabolism, cell cycling, protein fate, cellular transport, signal transduction, transcription, and cell wall biogenesis. Forty of these positively regulated 219 genes have previously been identified by DNA microarray analysis. Promoter analysis of these common 40 genes reveals a consensus motif [5′-GGAGGC(G/A)C(T/A)G-3′], which is different from the putative CaCrz1-binding motif [5′-G(C/T)GGT-3′] identified in the previous study, but similar to Saccharomyces cerevisiae ScCrz1-binding motif [5′-GNGGC(G/T)CA-3′]. EMSA and ChIP assays indicate that CaCrz1 binds in vitro and in vivo to both motifs in the promoter of its target gene CaUTR2. Promoter mutagenesis demonstrates that these two CaCrz1-binding motifs play additive roles in the regulation of CaUTR2 expression. In addition, the CaCRZ1 gene is positively regulated by CaCrz1. CaCrz1 can bind in vitro and in vivo to its own promoter, suggesting an autoregulatory mechanism for CaCRZ1 expression.ConclusionsCaCrz1 differentially binds to promoters of its target genes to regulate their expression in response to calcium stress. CaCrz1 also regulates its own expression through the 5′-TGAGGGACTG-3′ site in its promoter.3QEHhwZ2kG3j3zrcs7CSeNVideo abstract

Identification of a novel melatonin-binding nuclear receptor: Vitamin D receptor.

Previous studies confirmed that melatonin regulates Runx2 expression but the mechanism is unclear. There is a direct interaction between Runx2 and the vitamin D receptor (VDR). Herein, we observed a direct interaction between melatonin and the VDR but not Runx2 using isothermal titration calorimetry. Furthermore, this direct binding was detected only in the C-terminal ligand binding domain (LBD) of the VDR but not in the N-terminal DNA-binding domain (DBD) or the hinge region. Spectrophotometry indicated that melatonin and vitamin D3 (VD3) had similar uptake rates, but melatonin's uptake was significantly inhibited by VD3 until the concentration of melatonin was obviously higher than that of VD3 in a preosteoblastic cell line MC3T3-E1. GST pull-down and yeast two-hybrid assay showed that the interactive smallest fragments were on the 319-379 position of Runx2 and the N-terminus 110-amino acid DBD of the VDR. Electrophoretic mobility shift assay (EMSA) demonstrated that Runx2 facilitated the affinity between the VDR and its specific DNA substrate, which was further documented by a fluorescent EMSA assay where Cy3 labeled Runx2 co-localized with the VDR-DNA complex. Another fluorescent EMSA assay confirmed that the binding of the VDR to Runx2 was significantly enhanced with an increasing concentrations of the VDR, especially in the presence of melatonin; it was further documented using a co-immunoprecipitation assay that this direct interaction was markedly enhanced by melatonin treatment in the MC3T3-E1 cells. Thus, the VDR is a novel melatonin-binding nuclear receptor, and melatonin indirectly regulates Runx2 when it directly binds to the LBD and the DBD of the VDR, respectively.

Association between polymorphism in the promoter region of lncRNA GAS5 and the risk of colorectal cancer.

The growth arrest special 5 (GAS5), as a research hotspot of long noncoding RNAs (lncRNAs), has been reported to be associated with colorectal cancer (CRC). However, the association between polymorphisms in GAS5 and the risk of CRC was not clear. In the present study, a case–control study in 1078 CRC patients and 1175 matched healthy controls was performed to evaluate the association between the potential functional genetic variants in GAS5 and the risk of CRC. PCR-TaqMan, qPCR, dual-luciferase assay, electrophoretic mobility shift assay (EMSA), flow cytometry, migration and invasion assays were performed to evaluate the function of polymorphism. Results showed that subjects carrying the rs55829688 CT/TT genotypes had a significantly higher risk of CRC when compared with the CC genotype. Further qPCR assay confirmed that the CRC tissues with rs55829688 CT/TT genotypes had a higher GAS5 mRNA expression level. The dual-luciferase assay, qPCR and EMSA assay revealed that rs55829688 T>C polymorphism could decrease the expression level of GAS5 by impacting the binding ability of the transcription factor Yin Yang-1 (YY1) to the GAS5 promoter region. The expression of apoptosis-related proteins were detected by Western blot. Further, flow cytometry, migration, and invasion experiments showed that GAS5 repressed apoptosis and increased invasion and migration capability of CRC cells. Taken together, our findings provided evidence that the rs55829688 variant in the GAS5 promoter was associated with the risk of CRC and decreased expression of GAS5 by affecting the binding affinity of the transcription factors YY1 to GAS5.

Electrophoretic Mobility Shift Assay and Dimethyl Sulfate Footprinting for Characterization of G-Quadruplexes and G-Quadruplex-Protein Complexes.

DNA G-quadruplexes are globular nucleic acid secondary structures which occur throughout the human genome under physiological conditions. There is accumulating evidence supporting G-quadruplex involvement in a number of important aspects of genome functions, including transcription, replication, and genomic stability, and that protein and enzyme recognition of G-quadruplexes may represent a key event to regulate physiological or pathological pathways. Two important techniques to study G-quadruplexes and their protein interactions are the electrophoretic mobility shift assay (EMSA) and dimethyl sulfate (DMS) footprinting assay. EMSA, one of the most sensitive and robust methods for studying the DNA-protein interactions, can be used to determine the binding parameters and relative affinities of a protein for the G-quadruplex. DMS footprinting is a powerful assay for the initial characterization of G-quadruplexes, which can be used to deduce the guanine bases involved in the formation of G-tetrads under physiological salt conditions. DMS footprinting can also reveal important information in G-quadruplex-protein complexes on protein contacts and regional changes in DNA G-quadruplex upon protein binding. In this paper, we will provide a detailed protocol for the EMSA and DMS footprinting assays for characterization of G-quadruplexes and G-quadruplex-protein complexes. Expected outcomes and references to extensions of the method will be further discussed.

Association Between a CCL17 Genetic Variant and Risk of Coronary Artery Disease in a Chinese Han Population.

In the present study we investigated the effects of genetic variations in the C-C motif chemokine ligand 17 (CCL17) gene on serum CCL17 levels and risk of coronary artery disease (CAD).Methods and Results:A case-control study was conducted to determine causal inferences amongCCL17single-nucleotide polymorphisms (SNPs), serum CCL17 levels, and risk of CAD. Luciferase assays, electrophoretic mobility shift assays (EMSA), and allele-specific quantitative chromatin immunoprecipitation (ChIP) assays were used to assess the function of the SNPs. In all, 947 participants (794 with CAD, 153 without CAD) were included in the study. The T allele in rs223828, located in intron of theCCL17gene, was associated with increased serum CCL17 levels as well as increased CAD risk. A causal inference test using mediation analysis suggested that rs223828 had a significant indirect casual effect on the increased risk of CAD mediated via serum CCL17 levels. Luciferase assays confirmed that the rs223828T allele enhancesCCL17promoter activity. Protein-DNA binding studies using EMSA and allele-specific quantitative ChIP assays indicated preferential activator protein-1 (AP-1) complex formation and recruitment with the rs223828 T allele compared with the C allele. We propose that theCCL17SNP rs223828 is associated with increased risk of CAD, and that this site may be a potential AP-1 binding site.

NF-κB-vimentin is involved in steroidogenesis stimulated by mono-butyl phthalate in primary cultured ovarian granulosa cells

Di-n-butyl phthalate (DBP) and its active metabolite, monobutyl phthalate (MBP) are the most common endocrine disrupting chemicals. Many studies indicated the effects of MBP on male steroidogenesis, however, little attention have been paid on the effects of low levels of MBP on female steroidogenesis. This study was aimed to assess steroidogenesis stimulated by low-dose MBP on primary cultured ovarian granulosa cells (mGCs). Ovarian granulosa cells were isolated from ICR female mice. Hormone levels in medium were detected by ELISA, mRNA and protein expressions of vimentin, NF-κB p65 and phosphorylation of NF-κB p65 (p-p65) were assayed by qRT-PCR, western blot and immunohistochemistry, respectively. Besides, confocal immunofluorescence and electrophoretic mobility shift assay (EMSA) were used for detecting vimentin expression and activity of NF-κB p65 binding to the promoter of vimentin, respectively. Progesterone levels, mRNA and protein levels of vimentin and p-p65 in cells were increased significantly in mGCs treated by MBP at 10−10M. Additionally, MBP-induced steroidogenesis was blocked when vimentin protein was knocked down or activity of NF-κB was inhibited. EMSA assay showed that binding activity of NF-κB to the promoter regions of vimentin was boosted after MBP exposure. Accordingly, the results suggested that MBP could up-regulated steroidogenesis through NF-κB-vimentin signal in mGCs.

Transcriptional Regulation of SLC26A3 (DRA) by CDX2

BackgroundSLC26A3 (Down Regulated in Adenoma, DRA) plays a key role in mammalian intestinal NaCl absorption via mediating apical membrane Cl−/HCO3− exchange. DRA function and expression is significantly decreased in diarrheal diseases associated with infections and inflammatory bowel diseases (IBD). Since, DRA is now being touted as an important therapeutic target for infectious and IBD associated diarrhea, it is critical to understand the detailed mechanisms of regulation of DRA expression at the molecular and cellular level in health and disease. Earlier studies showed that, the intestinal epithelial caudal‐related homeobox transcription factor 2 (CDX2) is an essential regulator of intestinal epithelial homeostasis and is also involved in differentiation. Interestingly, DRA expression is higher in differentiated cells and in silico analysis of DRA promoter showed 2 consensus sites for CDX2 binding.AimCurrent studies were, therefore, undertaken to test the hypothesis that CDX2 regulates DRA transcription.MethodsCdx2Fl/Fl;Villin‐CreERT2 mice, [where tamoxifen (TAM) deletes Cdx2 in intestine], were used for these studies and Caco2 cells were used as an in vitro model to study the overexpression and silencing effects of CDX2 by transfecting the cDNA and siRNA, respectively. mRNA and protein levels were assessed by real time RT‐PCR and immunoblotting/immuno‐staining, respectively. DRA promoter activity was determined by luciferase assay. Direct binding of CDX2 to DRA promoter region was assessed by chromatin immuno‐precipitation (CHIP) and electrophoretic mobility shift assay (EMSA).ResultsCDX2− /− mice showed marked reduction in DRA mRNA in ileum (Arbitrary units: WT=2.73 ± 1.04, KO=0.029 ± 0.02 (P&lt;0.05), proximal colon (PC) WT=1.87 ± 0.53, KO=0.51 ± 0.08; P &lt;0.05) and distal colon (DC) (WT=1.75 ± 0.46, KO=0.02 ±0.02, P&lt;0.05) as well as in protein levels as compared to wild type mice. Similarly, silencing of CDX2 in Caco2 cells resulted in a marked (~ 50%) decrease in DRA mRNA and protein levels, whereas, ectopic over‐expression of CDX2 significantly up‐regulated DRA expression. CDX2 overexpression also stimulated DRA promoter activity by ~10‐fold, indicating its role in transcriptional regulation. We next utilized EMSA and ChIP assays to demonstrate direct binding of CDX2 to one putative cis elements identified in the 645 to 663 (+) region of the DRA promoter.ConclusionOur studies, for the first time, demonstrate a direct transcriptional regulation of DRA by CDX2. Given that CDX2 has been shown to be downregulated in UC (ulcerative colitis) and CD (crohn's disease) patients, CDX2 may at least partly be responsible for the observed decrease in DRA expression in IBD.Support or Funding InformationSupported by NIDDK/Department of Veteran Affairs

Identification of Thyroid Hormones and Functional Characterization of Thyroid Hormone Receptor in the Pacific Oyster Crassostrea gigas Provide Insight into Evolution of the Thyroid Hormone System.

Thyroid hormones (THs) play important roles in development, metamorphosis, and metabolism in vertebrates. During the past century, TH functions were regarded as a synapomorphy of vertebrates. More recently, accumulating evidence has gradually convinced us that TH functions also occur in invertebrate chordates. To date, however, TH-related studies in non-chordate invertebrates have been limited. In this study, THs were qualitatively detected by two reliable methods (HPLC and LC/MS) in a well-studied molluscan species, the Pacific oyster Crassostrea gigas. Quantitative measurement of THs during the development of C. gigas showed high TH contents during embryogenesis and that oyster embryos may synthesize THs endogenously. As a first step in elucidating the TH signaling cascade, an ortholog of vertebrate TH receptor (TR), the most critical gene mediating TH effects, was cloned in C. gigas. The sequence of CgTR has conserved DNA-binding and ligand-binding domains that normally characterize these receptors. Experimental results demonstrated that CgTR can repress gene expression through binding to promoters of target genes and can interact with oyster retinoid X receptor. Moreover, CgTR mRNA expression was activated by T4 and the transcriptional activity of CgTR promoter was repressed by unliganded CgTR protein. An atypical thyroid hormone response element (CgDR5) was found in the promoter of CgTR, which was verified by electrophoretic mobility shift assay (EMSA). These results indicated that some of the CgTR function is conserved. However, the EMSA assay showed that DNA binding specificity of CgTR was different from that of the vertebrate TR and experiments with two dual-luciferase reporter systems indicated that l-thyroxine, 3,3′,5-triiodothyronine, and triiodothyroacetic acid failed to activate the transcriptional activity of CgTR. This is the first study to functionally characterize TR in mollusks. The presence of THs and the functions of CgTR in mollusks contribute to better understanding of the evolution of the TH system.

Abstract 4610: Functional characterization of a multicancer risk locus on chr5p15.33 reveals regulation of TERT by ZNF148

Abstract Genome wide association studies (GWAS) have mapped multiple independent cancer risk loci (n = 6) to a small region on chr5p15.33 for at least ten distinct cancers, including bladder, breast, glioma, lung, melanoma, non-melanoma skin, ovarian, pancreas, prostate, and testicular germ cell cancer. This region harbors two plausible target genes, TERT which encodes the catalytic subunit of telomerase reverse transcriptase which maintains chromosome ends by adding telomeres repeats, and CLPTM1L which encodes the cleft lip and palate transmembrane protein 1-like protein which promotes cancer cell growth, protect cells from apoptosis, and can induce abnormal cytokinesis. The most significant pancreatic cancer GWAS SNP on chr5p15.33 was rs401681 (P = 3.7×10−7, OR = 1.19), located in the 13th intron of the CLPTM1L gene and within the independent multi-cancer risk locus in this region most distant from TERT. Imputation and fine mapping in pancreatic cancer resolved this signal by three orders of magnitude (P = 1.4×10−10, OR = 1.30) to a set of seven highly correlated SNPs. Electrophoretic mobility shift assays (EMSA) and luciferase assays across these SNPs led us to a single SNP, rs36115365, that showed allele-specific effects in both assays and overlapped with prominent ENCODE marks indicating regulatory potential. Examining this SNP across cell lines derived from multiple types of cancer linked to this locus, specifically pancreatic, testicular, lung, and melanoma, established a consistent pattern of a specific protein binding and enhanced regulatory activity for the risk allele across eight cancer cell lines (two cell lines per cancer type). siRNA-based targeting of the putative gene regulatory-region harboring rs36115365 resulted in a strong inhibition of TERT expression (average 60%, range 45-85%) but no effect on CLPTM1L expression. This inhibition of TERT expression was stronger from the pancreatic cancer risk as compared to the protective allele. Quantitative mass spectrometry identified Zinc finger protein 148 (ZNF148) as the protein specifically binding the risk allele, further supported by direct and allele-specific binding of purified recombinant ZNF148 in EMSA assays. Lastly, siRNA-mediated knockdown of ZNF148 resulted in significant reduction of TERT but not CLPTM1L expression across multiple cancer cell lines. Our results indicate that the pancreatic cancer GWAS signal at the single independent chr5p15.33 multi-cancer risk locus most distant from TERT may be explained by a single SNP that influences TERT expression via ZNF148. Citation Format: Kevin M. Brown, Jun Fang, Jinping Jia, Zhaoming Wang, Matthew Makowski, Tongwu Zhang, Jason Hoskins, Jiyeon Choi, Younghun Han, Mingfeng Zhang, Mai Xu, Peter Kanetsky, Andresson Thorkell, Gloria M. Petersen, Katherine L. Nathanson, Christopher I. Amos, Maria T. Landi, Stephen J. Chanock, Michiel Vermeulen, Laufey T. Amundadottir. Functional characterization of a multicancer risk locus on chr5p15.33 reveals regulation of TERT by ZNF148. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4610. doi:10.1158/1538-7445.AM2015-4610

EIN3 and ORE1 Accelerate Degreening during Ethylene-Mediated Leaf Senescence by Directly Activating Chlorophyll Catabolic Genes in Arabidopsis.

Degreening, caused by chlorophyll degradation, is the most obvious symptom of senescing leaves. Chlorophyll degradation can be triggered by endogenous and environmental cues, and ethylene is one of the major inducers. ETHYLENE INSENSITIVE3 (EIN3) is a key transcription factor in the ethylene signaling pathway. It was previously reported that EIN3, miR164, and a NAC (NAM, ATAF, and CUC) transcription factor ORE1/NAC2 constitute a regulatory network mediating leaf senescence. However, how this network regulates chlorophyll degradation at molecular level is not yet elucidated. Here we report a feed-forward regulation of chlorophyll degradation that involves EIN3, ORE1, and chlorophyll catabolic genes (CCGs). Gene expression analysis showed that the induction of three major CCGs, NYE1, NYC1 and PAO, by ethylene was largely repressed in ein3 eil1 double mutant. Dual-luciferase assay revealed that EIN3 significantly enhanced the promoter activity of NYE1, NYC1 and PAO in Arabidopsis protoplasts. Furthermore, Electrophoretic mobility shift assay (EMSA) indicated that EIN3 could directly bind to NYE1, NYC1 and PAO promoters. These results reveal that EIN3 functions as a positive regulator of CCG expression during ethylene-mediated chlorophyll degradation. Interestingly, ORE1, a senescence regulator which is a downstream target of EIN3, could also activate the expression of NYE1, NYC1 and PAO by directly binding to their promoters in EMSA and chromatin immunoprecipitation (ChIP) assays. In addition, EIN3 and ORE1 promoted NYE1 and NYC1 transcriptions in an additive manner. These results suggest that ORE1 is also involved in the direct regulation of CCG transcription. Moreover, ORE1 activated the expression of ACS2, a major ethylene biosynthesis gene, and subsequently promoted ethylene production. Collectively, our work reveals that EIN3, ORE1 and CCGs constitute a coherent feed-forward loop involving in the robust regulation of ethylene-mediated chlorophyll degradation during leaf senescence in Arabidopsis.

Investigation of the molecular mechanism how genistein combined with 5-fluorouracil inhibits proliferation of human colon cancer cell line SW480

Objective To investigate the molecular mechanism how genistein increased the sensitivity of colon cancer cell to 5-fluorouracil(FU) and promoted colon cancer cell apoptosis. Methods SW480 colon cancer cell line was chosen as experimental object. Genistein 80 μmol/L and 5-FU 30 μg/mL used separated or combined for 48 hours were set as drug experiment group. There were four experiment groups in this study: control group (without any drug), 5-FU group, genistein group, 5-FU and genistein combined group. The expression of survivin, Bcl-2, p21, caspase3 and caspase 9 in the tumor cells of each group at mRNA and protein level was deteced by real-time quantitative polymerase chain reaction (PCR) and Western blotting. The relative expression quantity of genes was determined by comparative threshold method (2–ΔΔCT) and β-actin was taken as an internal reference. Semi quantitative analysis was performed for protein relative expression quantity. The DNA combination activity of nuclear factor(NF)-κB of each group was measured by electrophoretic mobility shift assay (EMSA). Single factor analysis of variance was used for mean comparison among multiple groups and LSD test was for mean comparison between two groups. Results The expression of caspase3, caspase 9 and p21 of 5-FU and genistein combination group at mRNA (1.903±0.122, 2.726±0.050 and 2.541±0.393) and protein level (0.534±0.077, 1.161±0.172 and 0.463±0.016) were all higher than those of control group (1.001±0.052, 1.000±0.014 and 1.001±0.037; 0.080±0.043, 0.248±0.059 and 0.139±0.021), genistein group (1.559±0.038, 2.394±0.095 and 1.686±0.061; 0.335±0.052, 0.478±0.059 and 0.304±0.018) and 5-FU group (1.198±0.063, 1.051±0.043 and 1.399±0.055; 0.194±0.015, 0.337±0.036 and 0.231±0.011); the expression of survivin of 5-FU and genistein combined group at mRNA and protein level (0.165±0.018 and 0.216±0.014) were all lower than those of control group, genistein group and 5-FU group (1.001±0.033, 0.775±0.044 and 0.395±0.030; 0.594±0.079, 0.375±0.014 and 0.295±0.014), and all the differences were statistically significant (gene, F=802.865, 52.760, 39.992, 187.288, 37.435; protein, F=10.466, 44.483, 19.490, 200.011, 45.238; all P<0.01); the difference was also statistically significant in the expression of caspase3 and p21 of 5-FU group and genistein group compared with those of control group (LSD test, P<0.05). The results of EMSA assay showed that the DNA binding activity of NF-κB protein of genistein group (461.64±15.41) and combined group (585.28±7.82) significantly decreased compared with that of control group (1 067.97±36.01) and 5-FU group (718.83±23.18, LSD-test, P<0.05). Conclusions Genistein combined with 5-FU seemed to have synergistic effects on apoptosis of colon cancer cell. The mechanism was that genistein inhibited the DNA binding activity of NF-κB mediated by genistein, further up-regulated caspase 3, caspase 9, p21 and down regulated survivin. Therefore, genistein may provide assistance in colon cancer chemotherapy. Key words: Genistein; Fluorouracil; Colonic neoplasms; Apoptosis; Mechanism

Abstract 346: 9p21.3 Coronary Artery Disease Risk Variants Disrupt Tead Transcription Factor Binding and Tead-dependent Tgfβ Induction of P16 Expression

Background: The mechanism by which 9p21.3 locus increases the risk of CAD is elusive. The 9p21.3 risk locus is associated with reduced expression of the cell cycle suppressor genes CDKN2A (p16) and CDKN2B (p15) and increased vascular smooth muscle cell proliferation. We asked whether risk alleles disrupt transcription factor binding that could account for this effect. Methods and Results: A bioinformatic screen predicted which of 59 single nucleotide polymorphisms (SNPs) disrupt or create transcription factor binding sites at the 9p21.3 risk locus. Electrophoretic mobility shift assay (EMSA) and luciferase reporter assays examined the binding and functionality of the predicted regulatory sequences. Primary human aortic smooth muscle cells (HAoSMCs) genotyped for the 9p21.3 risk allele were used in the functional studies. HAoSMCs homozygous for the risk allele showed reduced expression of p15 and p16 protein levels and increased cellular proliferation compared to the other genotypes. Although 14 out of the 59 SNPs were predicted to have an effect on transcription factor binding,. only rs10811656 and rs4977757 disrupted functional binding sites for TEAD transcription factors by EMSA and luciferase reporter assays. TEAD3 and TEAD4 over-expression induced p16 but not p15 in HAoSMCs homozygous for the non-risk allele, but not for the risk allele. TGFβ is known to activate p16 expression and also to functionally interact with TEAD3 and TEAD4. In HAoSMCs homozygous for the risk allele, TGFβ failed to induce p16 or to inhibit cell proliferation. Knockdown of TEAD3 blocked TGFβ-induced p16 expression and dual knockdown of TEAD3 and TEAD4 markedly reduced p16 expression in heterozygous HAoSMCs. Conclusion: We show for the first time a novel mechanism whereby p16 expression is perturbed by the 9p21.3 risk locus through the disruption of TEAD3 and TEAD4 binding. Furthermore, we show that TGFβ induction of p16 is TEAD3-dependent in HAoSMCs. This mechanism accounts, in part, for the effect of the 9p21.3 risk alleles on CAD. Emerging therapies that activate TEAD factors to inhibit cellular proliferation may also be useful to limit atherosclerosis progression in individuals heterozygous or homozygous for the non-risk allele of 9p21.3.

An E2F1-HOXB9 transcriptional circuit is associated with breast cancer progression.

Homeobox B9 (HOXB9), a member of the homeobox gene family, is overexpressed in breast cancer and promotes tumor progression and metastasis by stimulating epithelial-to-mesenchymal transition and angiogenesis within the tumor microenvironment. HOXB9 activates the TGFβ-ATM axis, leading to checkpoint activation and DNA repair, which engenders radioresistance in breast cancer cells. Despite detailed reports of the role of HOXB9 in breast cancer, the factors that regulate HOXB9 transcription have not been extensively examined. Here we uncover an underlying mechanism that may suggest novel targeting strategies for breast cancer treatment. To identify a transcription factor binding site (TFBS) in the HOXB9 promoter region, a dual luciferase reporter assay was conducted. Protein candidates that may directly attach to a TFBS of HOXB9 were examined by Q-PCR, electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), and mutation analysis. A HOXB9 promoter region from −404 to −392 was identified as TFBS, and E2F1 was a potential binding candidate in this region. The induction of HOXB9 expression by E2F1 was observed by Q-PCR in several breast cancer cell lines overexpressing E2F1. The stimulatory effect of E2F1 on HOXB9 transcription and its ability to bind the TFBS were confirmed by luciferase, EMSA and ChIP assay. Immunohistochemical analysis of 139 breast cancer tissue samples revealed a significant correlation between E2F1 and HOXB9 expression (p<0.001). Furthermore, a CDK4/6 inhibitor suppressed E2F1 expression and also reduced expression of HOXB9 and its downstream target genes. Our in vitro analysis identified the TFBS of the HOXB9 promoter region and suggested that E2F1 is a direct regulator of HOXB9 expression; these data support the strong correlation we found between E2F1 and HOXB9 in clinical breast cancer samples. These results suggest that targeting the E2F1/HOXB9 axis may be a novel strategy for the control or prevention of cancer progression and metastasis.