A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Underwood, Karen; Mochin, Maria T.; Brusgard, Jessica L.; Choe, Mu Hyeon; Gnatt, Avi; Passaniti, Antonino

doi:10.3791/50512

Cited by 12 publications

(22 citation statements)

References 9 publications

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“…Additionally, certain transcription factors, such as eukaryotic nuclear receptors ( 39 ), do not tightly bind to the target DNA sequences in the absence of a ligand. PDI-ELISA could be a great tool to screen and identify small molecule ligands that enable the transcription factor binding to the target DNA sequence ( 40 ) (Figure 6B ). Furthermore, small molecule or protein activators may induce DNA conformational change and therefore greatly enhance the DNA binding of a DNA-binding protein to the target DNA sequence.…”

Section: Resultsmentioning

confidence: 99%

A rapid and sensitive high-throughput screening method to identify compounds targeting protein–nucleic acids interactions

Alonso

Guillen

Chambers³

et al. 2015

Nucleic Acids Research

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DNA-binding and RNA-binding proteins are usually considered ‘undruggable’ partly due to the lack of an efficient method to identify inhibitors from existing small molecule repositories. Here we report a rapid and sensitive high-throughput screening approach to identify compounds targeting protein–nucleic acids interactions based on protein–DNA or protein–RNA interaction enzyme-linked immunosorbent assays (PDI-ELISA or PRI-ELISA). We validated the PDI-ELISA method using the mammalian high-mobility-group protein AT-hook 2 (HMGA2) as the protein of interest and netropsin as the inhibitor of HMGA2–DNA interactions. With this method we successfully identified several inhibitors and an activator for HMGA2–DNA interactions from a collection of 29 DNA-binding compounds. Guided by this screening excise, we showed that netropsin, the specific inhibitor of HMGA2–DNA interactions, strongly inhibited the differentiation of the mouse pre-adipocyte 3T3-L1 cells into adipocytes, most likely through a mechanism by which the inhibition is through preventing the binding of HMGA2 to the target DNA sequences. This method should be broadly applicable to identify compounds or proteins modulating many DNA-binding or RNA-binding proteins.

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Section: Resultsmentioning

confidence: 99%

A rapid and sensitive high-throughput screening method to identify compounds targeting protein–nucleic acids interactions

Alonso

Guillen

Chambers³

et al. 2015

Nucleic Acids Research

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“…A critical Met residue within the Runt domain also mediates association with its essential cofactor Cbfβ, which increases DNA binding 10-fold relative to Runt alone (Tahirov et al, 2001). Using a quantitative DNA-binding assay (D-ELISA) specific for RUNX2 (Underwood et al, 2012; Underwood et al, 2013), we showed that RUNX2 with a Cys mutation in the Runt domain (Cys118Ser) did not bind DNA (Underwood et al, 2012). Furthermore, a GFP-tagged Runt domain vector mutated at position Met-143 of the Runt domain (which inhibits Cbfβ binding) exhibited reduced DNA binding relative to wild type Runt domain (Underwood et al, 2012).…”

Section: Resultsmentioning

confidence: 99%

Hyperglycemia and redox status regulate RUNX2 DNA-binding and an angiogenic phenotype in endothelial cells

Mochin

Underwood

Cooper

et al. 2015

Microvascular Research

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Angiogenesis is regulated by hyperglycemic conditions, which can induce cellular stress responses, reactive oxygen species (ROS), and anti-oxidant defenses that modulate intracellular signaling to prevent oxidative damage. The RUNX2 DNA-binding transcription factor is activated by a glucose-mediated intracellular pathway, plays an important role in endothelial cell (EC) function and angiogenesis, and is a target of oxidative stress. RUNX2 DNA-binding and EC differentiation in response to glucose were conserved in ECs from different tissues and inhibited by hyperglycemia, which stimulated ROS production through the aldose reductase glucose-utilization pathway. Furthermore, the redox status of cysteine and methionine residues regulated RUNX2 DNA-binding and reversal of oxidative inhibition was consistent with an endogenous Methionine sulfoxide reductase-A (MsrA) activity. Low molecular weight MsrA substrates and sulfoxide scavengers were potent inhibitors of RUNX2 DNA binding in the absence of oxidative stress, but acted as antioxidants to increase DNA binding in the presence of oxidants. MsrA was associated with RUNX2:DNA complexes, as measured by a sensitive, quantitative DNA-binding ELISA. The related RUNX2 protein family member, RUNX1, which contains an identical DNA-binding domain, was a catalytic substrate of recombinant MsrA. These findings define novel redox pathways involving aldose reductase and MsrA that regulate RUNX2 transcription factor activity and biological function in ECs. Targeting of these pathways could result in more effective strategies to alleviate the vascular dysfunction associated with diabetes or cancer.

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“…As a first step to determine, which of the five ACGT motifs function as XBP1s and/or 228 ATF6(N) binding sites for promoter activation, we measured the binding of these TFs to 229 portions of the MIEP by using a DNA-protein interaction ELISA (DPI-ELISA, (Brand et al, 230 2010;Underwood et al, 2013). Microtiter plates were coated with double-stranded 231 oligonucleotides encoding three copies (27 nucleotides each) of a putative binding motif with 232 adjacent sequences on either side (Table S3) XBP1s, XBP1u, or ATF6(N) (Fig.…”

Section: Xbp1 -/-mentioning

confidence: 99%

“…The DPI-ELISA was performed essentially as described in detail elsewhere (Brand et al, 415 2010;Underwood et al, 2013). Terminally biotinylated dsDNA oligonucleotides containing 3 416 copies of putative TF binding sites (Table S3) were purchased from Eurofins.…”

Section: Immunoblot Analysis 367mentioning

confidence: 99%

Repression of Viral Gene Expression and Replication by the Unfolded Protein Response Effector XBP1u

Hinte

Anken

Tirosh

et al. 2019

Preprint

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1The unfolded protein response (UPR) is a cellular homeostatic circuit regulating protein 2 synthesis and processing in the ER by three ER-to-nucleus signaling pathways. One 3 pathway is triggered by the inositol-requiring enzyme 1 (IRE1), which splices the X-box 4 binding protein 1 (XBP1) mRNA, thereby enabling expression of XBP1s. Another UPR 5 pathway activates the activating transcription factor 6 (ATF6). Here we show that murine 6 cytomegalovirus (MCMV), a prototypic β-herpesvirus, harnesses the UPR to regulate its own 7 life cycle. MCMV activates the IRE1-XBP1 pathway early post infection to relieve repression 8 by XBP1u, the product of the unspliced XBP1 mRNA. XBP1u inhibits viral gene expression 9 and replication by blocking the activation of the viral major immediate-early promoter by 10 XBP1s and ATF6. These findings reveal a redundant function of XBP1s and ATF6 as 11 activators of the viral life cycle, and an unexpected role of XBP1u as a potent repressor of 12 both XBP1s and ATF6-mediated activation. 13 14 Key words: unfolded protein response / transcription factor / XBP1u / ATF6 / 15 cytomegalovirus. 16 17 18Upon activation by ER stress, ATF6 travels to the Golgi, where it undergoes 38 intramembrane proteolysis. This process liberates its cytosolic N-terminus, the basic leucine 39 zipper (bZIP) transcription factor ATF6(N), and enables it to travel to the nucleus, where it 40 activates the transcription of chaperone genes as well as of the gene encoding XBP1 (Lee et 41 al, 2002). 42The third sensor, IRE1, is an ER transmembrane protein kinase that oligomerizes 43 upon accumulation of unfolded proteins in the ER lumen. Oligomerization and auto-44 transphosphorylation activates the RNase function of IRE1, which mediates an 45 unconventional splicing of the XBP1 mRNA in the cytosol (Calfon et al, 2002; Lee et al, 2002; 46 Yoshida et al, 2001). Removal of the 26-nt intron from the XBP1 mRNA leads to a frame shift 47 99 Results 100 Early activation of IRE1-XBP1 signaling promotes MCMV replication. 101Previous studies have shown that MCMV inhibits IRE1-XBP1 signaling at late times (≥24 h) 102 post infection (Qian et al, 2012; Stahl et al, 2013). However, cellular ER stress response 103 transcripts were shown to be upregulated at 5-6 hours after MCMV infection (Marcinowski et 104 al, 2012), suggesting that UPR signaling is activated at early times post infection. Thus, we 105 decided to analyze whether MCMV activates the IRE1-XPB1 signaling pathway within the 106 first few hours after infection. To do this, we infected mouse embryonic fibroblasts (MEFs) 107 with MCMV and quantified spliced and unspliced XBP1 transcripts by qRT-PCR. We 108 detected a short and transient increase of XBP1 splicing between 5 and 7 hours post 109 infection (hpi) (Fig. 1A). This increase was massively reduced when cells were infected with 110 UV-inactivated MCMV (Fig. 1A), suggesting that XBP1 splicing was not caused by viral 111 Statistical analysis 444All statistical analyses were performed with GraphPad Prism 5.0 soft...

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A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Cited by 12 publications

References 9 publications

A rapid and sensitive high-throughput screening method to identify compounds targeting protein–nucleic acids interactions

A rapid and sensitive high-throughput screening method to identify compounds targeting protein–nucleic acids interactions

Hyperglycemia and redox status regulate RUNX2 DNA-binding and an angiogenic phenotype in endothelial cells

Repression of Viral Gene Expression and Replication by the Unfolded Protein Response Effector XBP1u

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