DNA Binding Activity Of Factors Research Articles

A sticky-end probe biosensor for homogeneous detection of transcription factor binding activity.

Transcription factors are essential regulators of various physiological and pathological processes. However, detecting transcription factor-DNA binding activities is often time-consuming and labor-intensive. Homogeneous biosensors that are compatible with mix-and-measure protocols have the potential to simplify the workflow for therapeutic screening and disease diagnostics. In this study, we apply a combined computational-experimental approach to investigate the design of a sticky-end probe biosensor, where the transcription factor-DNA complex stabilizes the fluorescence resonance energy transfer signal of the donor-acceptor pair. We design a sticky-end biosensor for the SOX9 transcription factor based on the consensus sequence and characterize its sensing performance. A systems biology model is also developed to investigate the reaction kinetics and optimize the operating conditions. Taken together, our study provides a conceptual framework for the design and optimization of sticky-end probe biosensors for homogeneous detection of transcription factor-DNA binding activity.

Novel ZNF414 activity characterized by integrative analysis of ChIP-exo, ATAC-seq and RNA-seq data

Transcription factor binding to DNA is a central mechanism regulating gene expression. Thus, thorough characterization of this process is essential for understanding cellular biology in both health and disease. We combined data from three sequencing-based methods to unravel the DNA binding function of the novel ZNF414 protein in cells representing two tumor types. ChIP-exo served to map protein binding sites, ATAC-seq allowed identification of open chromatin, and RNA-seq examined the transcriptome. We show that ZNF414 is a DNA-binding protein that both induces and represses gene expression. This transcriptional response has an impact on cellular processes related to proliferation and other malignancy-associated functions, such as cell migration and DNA repair. Approximately 20% of the differentially expressed genes harbored ZNF414 binding sites in their promoters in accessible chromatin, likely representing direct targets of ZNF414. De novo motif discovery revealed several putative ZNF414 binding sequences, one of which was validated using EMSA. In conclusion, this study illustrates a highly efficient integrative approach for the characterization of the DNA binding and transcriptional activity of transcription factors.

Abstract 1452: Deciphering mechanisms of Ref-1 signaling and its inhibition in aggressive tumor-stroma PDAC models

Abstract Targeted therapy for cancer using small molecules has progressed exponentially, but agents that can affect cancer cells rather than non-tumorigenic cells are crucial to avoid pernicious side effects. AP endonuclease-1/Redox factor-1 (APE1/Ref-1 or Ref-1) is a multifunctional protein with DNA repair activity and redox signaling activity as major functions. The DNA repair function is indispensable for cell survival. Its role as a redox factor that stimulates the DNA binding activity of numerous transcription factors (TFs) such as HIF-1α, NFΚB, STAT3, and AP-1 tends to be dysregulated in cancer cells. Ref-1 is overexpressed in many cancers including, aggressive and invasive pancreatic ductal adenocarcinoma (PDAC), which is the 3rd leading cause of death in U.S. The dense stroma of PDAC primarily constituting cancer-associated fibroblasts (CAFs) makes it highly hypoxic, nutrient poor, and drug resistant. Current treatment regimens offer only modest improvement in survival. Targeting the PDAC stroma or ECM did not result in clinical benefit thus far presumably due to complex interactions between PDAC and its TME. Better treatments options that selectively target the tumor within its protective stroma are hence needed. Our group was able to generate a Ref-1 redox inhibitor, APX3330, that completed Phase I clinical trial (NCT03375086) with a good safety profile, verified target engagement and a recommended phase II dose. However, its potency in preclinical models is in the high µM range indicating the need for more potent second-generation inhibitors. Based on initial SAR studies, we selected 13 second-generation compounds from > 350 that were further characterized for positive properties including increased efficacy for cell killing, mouse and human S9 fraction metabolic stability, plasma half-life, and in silico ADMET properties. Target engagement studies involving blockade of TF activity via luciferase assay and EMSA as well as validation of direct interaction of these inhibitors with Ref-1 using thermal shift assay are ongoing. In two 3D co-culture models, second-generation Ref-1 redox analogs suppressed tumor survival significantly while sparing the CAFs. These findings were confirmed in vivo with xenografts co-implanted with tumor and CAF lines. Gene expression and mitochondria functional data revealed Ref-1's control of TCA cycle in tumor cells, but not in CAFs. To confirm and compare the effects of Ref-1 redox signaling inhibitors in cells, CRISPR editing was used to generate Ref-1 redox deficient cell lines (Ref-1C65A). As confirmation of significantly reduced Ref-1 redox activity, PDAC cells expressing Ref-1C65A did not induce hypoxia marker (CA9) under hypoxia, similar to when Ref-1 was knocked down or blocked via small molecule inhibitor. Ref-1 redox signaling and validation for selective killing of PDAC cells leaving the stomal cells undisturbed paves the way to improved PDAC treatment. Citation Format: Silpa Gampala, Nayela Chowdhury, Olivia Babb, Rachel A. Caston, Randall S. Wireman, Hye-ran Moon, George Sandusky, Emily Hulsey, Bumsoo Han, Millie M. Georgiadis, Sara K. Quinney, Andi R. Masters, James H. Wikel, Mark R. Kelley, Melissa L. Fishel. Deciphering mechanisms of Ref-1 signaling and its inhibition in aggressive tumor-stroma PDAC models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1452.

Quantitative assessment of NFκB transcription factor activity

The Nuclear Factor Kappa B (NFκB) pathway is an important signalling pathway in the immune system. Single gene defects in the NFκB pathway are described in a number of immunodeficiency diseases. These conditions provide a unique opportunity to investigate the mechanisms of NFκB function and how genetic mutations that disrupt this function lead to human disease. Here we describe a robust method for quantifying small differences in the functional activity of the NFκB pathway.Peripheral blood mononuclear cells from healthy donors were stimulated over several days, with a combination of anti-IgM antibody and multimeric CD40 ligand. Nuclear proteins were thereafter extracted and tested for the ability of activated transcription factors, to bind known NFκB DNA binding motifs.Repeatability experiments showed that the DNA binding Activity can be quantified with an average inter and intra assay coefficient of variation of less than 10% (RelB and p52) and less than 15% (p50 and RelA). In healthy individuals there is a significant increase in the DNA binding activity of NFκB transcription factors in response to stimulation, although the magnitude of this response varies across individuals. The kinetics of the DNA binding activity also differs between the canonical and non-canonical transcription factors. P50 and RelA DNA binding activity responds within hours of stimulation, whilst RelB and p52 response was delayed to more than a day after stimulation.Activation of NFκB signalling in response to B cell specific stimulation, can be precisely measured to distinguish individuals with differences in the functional activity of this pathway. This test may prove to be an important biomarker for investigating the functional impact of genetic variants on NFκB signalling.

The involvement of toll-like receptors 2 and 4 in human platelet signalling pathways

In addition to haemostasis, platelets play an essential role in mechanisms of inflammation and in immunological reactions. Platelets express various toll-like receptors (TLR) on their surface, among them TLR2 and TLR4, which are important for the recognition of bacterial patterns. This study compared TLR2- and TLR4-dependent platelet signalling and their effect on platelet function.Platelet-rich-plasma and washed platelets were prepared from peripheral blood samples of healthy donors. Pam3CSK4 or LPS (lipopolysaccharides from Escherichia coli) were used for stimulation of TLR2 and TLR4. Intracellular signalling pathways were investigated by Western blot. TLR2- and TLR4-mediated specific transcription factor DNA binding activity was measured by the nuclear factor kappa B (NFκB) transcription factor assay kit. Platelet adhesion and glycoprotein Ib function were assessed by immunofluorescence staining and analysis of ristocetin-induced agglutination.Both, Pam3CSK4 and LPS were able to induce NFκB-mediated and classical activating platelet signalling with a higher stimulatory capacity of TLR2. In addition, TLR2 and TLR4 activation led to a similar activation of inhibitory pathways. In contrast to TLR2, stimulation of TLR4 resulted in decreased Akt/protein kinase B phosphorylation conditioned by enhanced protein phosphatase 2A activity. TLR4-mediated signalling induced platelet adhesion and facilitated ristocetin-induced platelet agglutination.In conclusion, Pam3CSK4 directly induces aggregation via classical activation cascades, whereas LPS enhances platelet adhesion and glycoprotein receptor Ib-dependent platelet agglutination.

Three Copies of Four Interferon Receptor Genes Underlie a Mild Type I Interferonopathy in Down Syndrome.

Down syndrome (DS) is characterized by the occurrence of three copies of human chromosome 21 (HSA21). HSA21 contains a cluster of four interferon receptor (IFN-R) genes: IFNAR1, IFNAR2, IFNGR2, and IL10RB. DS patients often develop mucocutaneous infections and autoimmune diseases, mimicking patients with heterozygous gain-of-function (GOF) STAT1 mutations, which enhance cellular responses to three types of interferon (IFN). A gene dosage effect at these four loci may contribute to the infectious and autoimmune manifestations observed in individuals with DS. We report high levels of IFN-αR1, IFN-αR2, and IFN-γR2 expression on the surface of monocytes and EBV-transformed-B (EBV-B) cells from studying 45 DS patients. Total and phosphorylated STAT1 (STAT1 and pSTAT1) levels were constitutively high in unstimulated and IFN-α- and IFN-γ-stimulated monocytes from DS patients but lower than those in patients with GOF STAT1 mutations. Following stimulation with IFN-α or -γ, but not with IL-6 or IL-21, pSTAT1 and IFN-γ activation factor (GAF) DNA-binding activities were significantly higher in the EBV-B cells of DS patients than in controls. These responses resemble the dysregulated responses observed in patients with STAT1 GOF mutations. Concentrations of plasma type I IFNs were high in 12% of the DS patients tested (1.8% in the healthy controls). Levels of type I IFNs, IFN-Rs, and STAT1 were similar in DS patients with and without recurrent skin infections. We performed a genome-wide transcriptomic analysis based on principal component analysis and interferon modules on circulating monocytes. We found that DS monocytes had levels of both IFN-α- and IFN-γ-inducible ISGs intermediate to those of monocytes from healthy controls and from patients with GOF STAT1 mutations. Unlike patients with GOF STAT1 mutations, patients with DS had normal circulating Th17 counts and a high proportion of terminally differentiated CD8+ T cells with low levels of STAT1 expression. We conclude a mild interferonopathy in Down syndrome leads to an incomplete penetrance at both cellular and clinical level, which is not correlate with recurrent skin bacterial or fungal infections. The constitutive upregulation of type I and type II IFN-R, at least in monocytes of DS patients, may contribute to the autoimmune diseases observed in these individuals.

Methyl 3,4,5-trimethoxycinnamate suppresses inflammation in RAW264.7 macrophages and blocks macrophage\u2013adipocyte interaction

Methyl 3,4,5-trimethoxycinnamate (MTC) is a bioactive natural phenylpropanoid. We evaluated anti-inflammatory effects of synthetic MTC in RAW264.7 macrophages and RAW264.7–3T3-L1 adipocytes co-culture. Levels of cytokines and chemokines, as well as NO and PGE2 in cell supernatants were analysed using ELISAs, Griess assay and enzyme immunoassays, respectively. In-cell cytoblot was used to assess levels of proteins; while DNA binding and reporter gene assays were used to measure transcription factor DNA binding and transcriptional activities, respectively. Glucose uptake in adipocytes was evaluated with 2‐deoxy‐2‐[(7‐nitro‐2, 1, 3‐benzoxadiazol‐4‐yl) amino]‐d‐glucose uptake. MTC (5–20 µM) suppressed LPS + IFNγ-induced release of TNFα, IL-6 and IL-1β, as well as NO/iNOS and PGE2/COX-2 levels in RAW264.7 cells. Furthermore, there was a reduction in phospho-IκB and phospho-p65 proteins, accompanied by a reduction in total IκB in RAW264.7 cells. Further studies showed that MTC also produced a reduction in NF-κB DNA binding and luciferase activity. Treatment of RAW264.7 cells with MTC (5–20 µM) resulted in enhanced DNA binding of Nrf2 and an increase in ARE-luciferase activity. In a macrophage–adipocyte co-culture, the compound reduced the release of TNFα, IL-6, IL-1β, MCP-1 and RANTES, while enhancing glucose uptake and activation of AMPKα. Our results suggest that MTC produced anti-inflammatory and antioxidant activities in macrophages. MTC also prevented inflammation in macrophage–adipocyte co-culture. The effect of MTC on glucose uptake in adipocytes is proposed to be linked to activation of AMPK.

The C-terminal tails of the mitochondrial transcription factors Mtf1 and TFB2M are part of an autoinhibitory mechanism that regulates DNA binding

The structurally homologous Mtf1 and TFB2M proteins serve as transcription initiation factors of mitochondrial RNA polymerases in Saccharomyces cerevisiae and humans, respectively. These transcription factors directly interact with the nontemplate strand of the transcription bubble to drive promoter melting. Given the key roles of Mtf1 and TFB2M in promoter-specific transcription initiation, it can be expected that the DNA binding activity of the mitochondrial transcription factors is regulated to prevent DNA binding at inappropriate times. However, little information is available on how mitochondrial DNA transcription is regulated. While studying C-terminal (C-tail) deletion mutants of Mtf1 and TFB2M, we stumbled upon a finding that suggested that the flexible C-tail region of these factors autoregulates their DNA binding activity. Quantitative DNA binding studies with fluorescence anisotropy-based titrations revealed that Mtf1 with an intact C-tail has no affinity for DNA but deletion of the C-tail greatly increases Mtf1's DNA binding affinity. Similar observations were made with TFB2M, although autoinhibition by the C-tail of TFB2M was not as complete as in Mtf1. Analysis of available TFB2M structures disclosed that the C-tail engages in intramolecular interactions with the DNA binding groove in the free factor, which, we propose, inhibits its DNA binding activity. Further experiments showed that RNA polymerase relieves this autoinhibition by interacting with the C-tail and engaging it in complex formation. In conclusion, our biochemical and structural analyses reveal autoinhibitory and activation mechanisms of mitochondrial transcription factors that regulate their DNA binding activities and aid in specific assembly of transcription initiation complexes.

A time-resolved multi-omic atlas of the developing mouse liver.

Liver organogenesis and development are composed of a series of complex, well-orchestrated events. Identifying key factors and pathways governing liver development will help elucidate the physiological and pathological processes including those of cancer. We conducted multidimensional omics measurements including protein, mRNA, and transcription factor (TF) DNA-binding activity for mouse liver tissues collected from embryonic day 12.5 (E12.5) to postnatal week 8 (W8), encompassing major developmental stages. These data sets reveal dynamic changes of core liver functions and canonical signaling pathways governing development at both mRNA and protein levels. The TF DNA-binding activity data set highlights the importance of TF activity in early embryonic development. A comparison between mouse liver development and human hepatocellular carcinoma (HCC) proteomic profiles reveal that more aggressive tumors are characterized with the activation of early embryonic development pathways, whereas less aggressive ones maintain liver function–related pathways that are elevated in the mature liver. This work offers a panoramic view of mouse liver development and provides a rich resource to explore in-depth functional characterization.

Salinomycin inhibits breast cancer progression via targeting HIF-1α/VEGF mediated tumor angiogenesis in vitro and in vivo

Cancer is a complex disease wherein cells begin to divideabnormally and spread into surrounding tissues. Angiogenesis plays a crucial role in tumor progression as it is required for sustained growth and metastasis, therefore targeting angiogenesis is a promising therapeutic approach for breast cancer management. Salinomycin (SAL) has been reported to exhibit anticancer response on various types of cancer. In the present study, we explored the antiangiogenic and anticancer efficacy of the polyether ionophore SAL in the breast cancer model. It effectively inhibited cell proliferation, invasion, and migration. It also inhibited the expression of pro-angiogenic cell surface marker CD31 in HUVEC, thereby interrupting the endothelial tubulogenesis. It decreased the HIF-1α transcription factor DNA binding activity to HRE sequence in HUVEC and human breast cancer cells. Further, corresponding to our in vitro findings, SAL suppressed neovascularization in the chick chorioallantoic membrane and the Matrigel plug implanted mice model. Bioluminescence and immunofluorescence imaging revealed that SAL treatment in mice inhibits breast cancer growth and tumor angiogenesis. SAL also suppressed the serum VEGFA level in tumor-bearing mice and induced caspase-dependent apoptosis in breast cancer cells. Taken together our findings suggested that SAL inhibits VEGF induced angiogenesis and breast cancer growth via interrupting HIF-1α/VEGF signalling and could be used as a promising antiangiogenic agent for breast cancer treatment.

Blockade of platelet-activating factor receptor attenuates abnormal behaviors induced by phencyclidine in mice through down-regulation of NF-κB

Accumulating evidence suggests that neuroinflammation is one of the important etiologic factors of abusive and neuropsychiatric disorders. Platelet-activating factor (PAF) is potent proinflammatory lipid mediat1or and plays a pivotal role in neuroinflammatory disorders through the specific PAF receptor (PAF-R). Phencyclidine (PCP) induces a psychotomimetic state that closely resembles schizophrenia. Here, we investigated the role of PAF-R in the abnormal behaviors induced by PCP in mice. Repeated treatment with PCP resulted in a significant increase in PAF-R gene expression in the prefrontal cortex (PFC) and in the hippocampus. This increase was more pronounced in the PFC than hippocampus. Treatment with PCP resulted in a significant increase in nuclear translocation of the nuclear factor kappa beta (NF-κB) p65 and DNA binding activity, indicating that the proinflammatory molecule NF-κB was increased through up-regulation of PAF-R. Consistently, NF-κB activation was significantly protected by the PAF-R antagonist, ginkgolide B (Gink B), in PAF-R knockout mice and by the NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC). In addition, PCP-induced abnormal behaviors (i.e., reduced sociability, depression, cognitive impairment, and behavioral sensitization) were significantly attenuated by Gink B, in PAF-R knockout mice, and by PDTC. Importantly, PDTC did not significantly alter the attenuations observed in Gink B-treated mice or PAF-R knockout mice, indicating that NF-κB is a critical target for neuropsychotoxic modulation of PAF-R. Therefore, the results suggest that PAF-R mediates PCP-induced neuropsychotoxicity via a NF-κB-dependent mechanism, and that up-regulation of PAF-R may be associated with schizophrenia-like behavior in animal models.

Chondroitin sulphate inhibits NF-κB activity induced by interaction of pathogenic and damage associated molecules

To evaluate the anti-inflammatory mechanism of action of Chondroitin Sulphate (CS). THP-1 macrophages were cultured with a range of sizes and concentrations of HA fragments with TLR4 (LPS in a physiologically relevant concentration determined by analyses of sera of a community clinic ascertained knee osteoarthritis (OA) cohort) or TLR2 (heat killed listeria bacteria) agonists and varying concentrations of CS in a physiologically relevant range (10-200μg/ml). We measured IL-1β release, intracellular IL-1β, proIL-1β, caspase-1 and NF-κB activity and DNA binding activity of NF-κB transcription factors from nuclear and cytoplasmic extracts. Serum LPS was significantly associated with radiographic knee joint space narrowing (JSN) (P=0.02) in the OA cohort (n=40). The priming dose of LPS used for these experiments (10ng/ml) was below the lowest serum concentration of the OA cohort (median 47.09, range 14.43-81.36ng/ml). Priming doses of LPS and HA fragments alone did not elicit an inflammatory response. However, primed with LPS, HA fragments produced large dose-dependent increases in IL-1β that were inhibitable by CS. CS did not inhibit caspase-1 activity but in physiologically achievable concentrations, attenuated NF-κB activity induced by either the TLR4 (LPS 1000ng/ml) or TLR2 agonists alone or in combination with HA fragments. LPS induced and CS significantly reduced activity of canonical NF-κB transcription factors, p65, p50, c-Rel and RelB. Subinflammatory concentrations of pathogenic (LPS, listeria) and damage associated (HA) molecules interact to induce macrophage-related inflammation. CS works upstream of the inflammasome by inhibiting activation of NF-κB transcription factors.

Modulatory effects of resveratrol on endoplasmic reticulum stress-associated apoptosis and oxido-inflammatory markers in a rat model of rotenone-induced Parkinson's disease

The mechanisms leading to neuronal death in Parkinson's disease (PD) are not fully elucidated; however, mounting evidence implicates endoplasmic reticulum (ER) stress, oxidative damage, and inflammatory changes are the crucial factors in its pathogenesis. This study was undertaken to investigate the modulatory effects of resveratrol on ER stress-mediated apoptosis, inflammatory and oxidative stress markers in a rat model of rotenone-induced PD. mRNA expression levels of ER stress markers; C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP78), were estimated in the rat brain using quantitative real-time PCR. Caspase-3 activity, IL-1β levels and Nuclear Factor Erythroid 2-related factor (Nrf2) DNA-binding activity were estimated by ELISA, while glutathione peroxidase and Xanthine oxidase activities, as well as protein carbonyl contents in the rat brain were evaluated spectrophotometrically. Our data revealed that Resveratrol ameliorated rotenone-induced ER stress by downregulating CHOP and GRP78 genes expression and hampered caspase-3 activity in the brain of rotenone exposed rats. It also restored redox balance as evident by suppressing Xanthine oxidase activity and protein carbonyls formation; in addition to preservation of intracellular antioxidants status via activating glutathione peroxidase and Nrf2 signaling pathway. In conclusion; our study launched promising avenues for the potential use of resveratrol as a neuroprotective therapeutic agent in Parkinson's disease.

A feedback inhibition between nitric oxide and hydrogen peroxide in the heat shock pathway in Arabidopsis seedlings

We even reported that hydrogen peroxide (H2O2) acts upstream of nitric oxide (NO) in the heat shock (HS) pathway in Arabidopsis (Arabidopsis thaliana) seedlings. In this work, we found a strange phenomenon that exogenous application of high concentration of H2O2 could not elevate internal H2O2 level under HS. NO donors sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) intensified this trend, whereas a special NO scavenger, 2-(4-phenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxyde (cPTIO) counteracted it, suggesting NO effects on reducing H2O2 accumulation. Western blotting and real-time reverse transcription-polymerase chain reaction demonstrated that NO eliminated H2O2 effects on the DNA-binding activity of HS factors and the accumulation of HS proteins. Subsequent experiments revealed that under HS, SNP and SNAP enhanced the activities of antioxidant enzymes catalase, ascorbate peroxidase and glutathione reductase, whereas c-PTIO inhibited them. Collectively, H2O2-induced NO stimulated the activities of antioxidant enzymes so as to eliminate excessive H2O2 in plant and then inhibit HS factor DNA-binding activity and HS protein accumulation, indicating a feedback inhibition between NO and H2O2 in thermotolerance.

Moderate alcohol induces stress proteins HSF1 and hsp70 and inhibits proinflammatory cytokines resulting in endotoxin tolerance.

Binge or moderate alcohol exposure impairs host defense and increases susceptibility to infection because of compromised innate immune responses. However, there is a lack of consensus on the molecular mechanism by which alcohol mediates this immunosuppression. In this study, we show that cellular stress proteins HSF1 and hsp70 play a mechanistic role in alcohol-mediated inhibition of the TLR4/MyD88 pathway. Alcohol exposure induced transcription factor HSF1 mRNA expression and DNA binding activity in primary human monocytes and murine macrophages. Furthermore, HSF1 target gene hsp70 mRNA and protein are upregulated by alcohol in monocytes. In vitro pre-exposure to moderate alcohol reduced subsequent LPS-induced NF-κB promoter activity and downstream TNF-α, IL-6 and IL-1β production in monocytes and macrophages, exhibiting endotoxin tolerance. Mechanistic analysis demonstrates that alcohol-induced HSF1 binds to the TNF-α promoter in macrophages at early time points, exerting transrepression and decreased TNF-α expression. Furthermore, association of hsp70 with NF-κB subunit p50 in alcohol-treated macrophages correlates with reduced NF-κB activation at later time points. Hsp70 overexpression in macrophages was sufficient to block LPS-induced NF-κB promoter activity, suggesting alcohol-mediated immunosuppression by hsp70. The direct crosstalk of hsp70 and HSF1 was further confirmed by the loss of alcohol-mediated endotoxin tolerance in hsp70- and HSF1-silenced macrophages. Our data suggest that alcohol-mediated activation of HSF1 and induction of hsp70 inhibit TLR4-MyD88 signaling and are required for alcohol-induced endotoxin tolerance. Using stress proteins as direct drug targets would be clinically relevant in alcohol abuse treatment and may serve to provide a better understanding of alcohol-mediated immunosuppression.

Effects of Echinomycin on PCNA-Dependent Follicular Development in PMSG-Induced Sprague-Dawley Rats

Echinomycin (Ech) is a small-molecule inhibitor of hypoxia-inducible factor-1 DNA-binding activity, which plays a crucial role in the regulation of ovarian functions in mammals. The present study was designed to test the hypothesis that hypoxia-inducible factor (HIF)-1alpha-mediated proliferation cell nuclear antigen (PCNA) expressions contributed to the follicular development in the rat ovary primed by pregnant mare serum gonadotropin (PMSG). Through the histological examination, the decrease of growing and antral follicle numbers was found after Ech treatment both in control and PMSG treated groups. And then PCNA mRNA and protein expressions were found to significantly increase in the ovaries treated with PMSG, and the similar changes were found in HIF-1alpha mRNA and protein expressions, indicating PMSG-induced follicular development may be through HIF-1alpha/PCNA signaling. Furthermore, PCNA expression was found to significantly decrease in the ovaries after Ech treatment, while HIF-1alpha mRNA and protein expression was no obviously changes. Further analysis found the changes of PCNA expression were consistent with HIF-1 activity in the ovaries, further suggesting the regulatory roles in the follicular development. Taken together, these results demonstrated this HIF-1alpha-mediated PCNA expression is one of the important mechanisms regulating the ovarian follicular development in mammals. Keywords: HIF-1alpha; PCNA; echinomycin; HIF prolyl hyodroxylase acitvity; follicular development

Hydrogen peroxide acts upstream of nitric oxide in the heat shock pathway in Arabidopsis seedlings.

We previously reported that nitric oxide (NO) functions as a signal in thermotolerance. To illustrate its relationship with hydrogen peroxide (H₂O₂) in the tolerance of Arabidopsis (Arabidopsis thaliana) to heat shock (HS), we investigated the effects of heat on Arabidopsis seedlings of the following types: the wild type; three NADPH oxidase-defective mutants that exhibit reduced endogenous H₂O₂ levels (atrbohB, atrbohD, and atrbohB/D); and a mutant that is resistant to inhibition by fosmidomycin (noa1, for nitric oxide-associated protein1). After HS, the NO levels in atrbohB, atrbohD, and atrbohB/D seedlings were lower than that in wild-type seedlings. Treatment of the seedlings with sodium nitroprusside or S-nitroso-N-acetylpenicillamine partially rescued their heat sensitivity, suggesting that NO is involved in H₂O₂ signaling as a downstream factor. This point was verified by phenotypic analyses and thermotolerance testing of transgenic seedlings that overexpressed Nitrate reductase2 and NOA1, respectively, in an atrbohB/D background. Electrophoretic mobility shift assays, western blotting, and real-time reverse transcription-polymerase chain reaction demonstrated that NO stimulated the DNA-binding activity of HS factors and the accumulation of HS proteins through H₂O₂. These data indicate that H₂O₂ acts upstream of NO in thermotolerance, which requires increased HS factor DNA-binding activity and HS protein accumulation.

Alteration of DNA binding activity of transcription factors in NRK-52E rat proximal tubular cells treated with cadmium.

Cadmium is a toxic heavy metal that causes severe clinical symptoms in various tissues including the kidney. In this study, activities of transcription factors were measured to identify what type of transcription factor was affected by cadmium in rat proximal tubular cells (NRK-52E cells) using the protein/DNA binding assay. After treatment of NRK-52E cells with 5 µM CdCl2 for 3 hr, nuclear extracts were used for the protein/DNA binding assay. Among 65 transcription factors, cadmium increased the activities of 6 transcription factors by more than 2.0-fold and decreased those of 15 transcription factors by less than 0.5-fold. These findings may provide new information about novel transcription factors associated with the mechanism of cadmium toxicity.

Exploring the utility of organo-polyoxometalate hybrids to inhibit SOX transcription factors.

BackgroundSOX transcription factors constitute an attractive target class for intervention with small molecules as they play a prominent role in the field of regenerative biomedicine and cancer biology. However, rationally engineering specific inhibitors that interfere with transcription factor DNA interfaces continues to be a monumental challenge in the field of transcription factor chemical biology. Polyoxometalates (POMs) are inorganic compounds that were previously shown to target the high-mobility group (HMG) of SOX proteins at nanomolar concentrations. In continuation of this work, we carried out an assessment of the selectivity of a panel of newly synthesized organo-polyoxometalate hybrids in targeting different transcription factor families to enable the usage of polyoxometalates as specific SOX transcription factor drugs.ResultsThe residual DNA-binding activities of 15 different transcription factors were measured after treatment with a panel of diverse polyoxometalates. Polyoxometalates belonging to the Dawson structural class were found to be more potent inhibitors than the Keggin class. Further, organically modified Dawson polyoxometalates were found to be the most potent in inhibiting transcription factor DNA binding activity. The size of the polyoxometalates and its derivitization were found to be the key determinants of their potency.ConclusionPolyoxometalates are highly potent, nanomolar range inhibitors of the DNA binding activity of the Sox-HMG family. However, binding assays involving a limited subset of structurally diverse polyoxometalates revealed a low selectivity profile against different transcription factor families. Further progress in achieving selectivity and deciphering structure-activity relationship of POMs require the identification of POM binding sites on transcription factors using elaborate approaches like X-ray crystallography and multidimensional NMR. In summary, our report reaffirms that transcription factors are challenging molecular architectures and that future polyoxometalate chemistry must consider further modification strategies, to address the substantial challenges involved in achieving target selectivity.

APE1/Ref-1 enhances DNA binding activity of mutant p53 in a redox-dependent manner

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a dual function protein; in addition to its DNA repair activity, it can stimulate DNA binding activity of numerous transcription factors as a reduction-oxidation (redox) factor. APE1/Ref-1 has been found to be a potent activator of wild-type p53 (wtp53) DNA binding in vitro and in vivo. Although p53 is mutated in most types of human cancer including hepatocellular carcinoma (HCC), little is known about whether APE1/Ref-1 can regulate mutant p53 (mutp53). Herein, we reported the increased APE1/Ref-1 protein and accumulation of mutp53 in HCC by immunohistochemistry. Of note, it was observed that APE1/Ref-1 high-expression and mutp53 expression were associated with carcinogenesis and progression of HCC. To determine whether APE1/Ref-1 regulates DNA binding of mutp53, we performed electromobility shift assays (EMSAs) and quantitative chromatin immunoprecipitation (ChIP) assays in HCC cell lines. In contrast to sequence-specific and DNA structure-dependent binding of wtp53, reduced mutp53 efficiently bound to nonlinear DNA, but not to linear DNA. Notably, overexpression of APE1/Ref-1 resulted in increased DNA binding activity of mutp53, while downregulation of APE1/Ref-1 caused a marked decrease of mutp53 DNA binding. In addition, APE1/Ref-1 could not potentiate the accumulation of p21 mRNA and protein in mutp53 cells. These data indicate that APE1/Ref-1 can stimulate mutp53 DNA binding in a redox-dependent manner.