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  1. Introduction
  2. Acetylcholine Receptor Binding Antibody
  3. The GraphPad Guide to Analyzing Radioligand Binding Data

Specifically, a recent study by Rheinbay et al. Their subsequent EMSA and luciferase promoter analysis indicated the functional impact of this mutation on ZNF gene expression, but our genetic base editing data suggest that although expression of ZNF is slightly altered, the major regulatory impact of the mutation is observed at the topologically associated distal genes. These findings further highlight the power of utilizing CRISPR editing approaches in combination with ENCODE-derived topological data in characterizing the functional roles and identifying the potential targets of non-coding mutations in the genome.

It is notable that the two recurrent mutations that we identified and functionally interrogated here are implicated in the expression of multiple distal genes. Such findings demonstrate that it is important to study the regulatory effect of non-coding mutations beyond the most proximal promoter. In addition to the recurrent mutations, it remains to be studied whether non-recurrent mutations also contribute to differential survival or proliferation in cancer cell evolution. Three patient samples have two sections sequenced separately in the original study to detect tumor heterogeneity [ 23 ].

BEDTools multiIntersect [ 54 ] was used to carry out this operation. For all analysis except for Fig. Alignment files bam files for each sample were downloaded for potential somatic mutation identification from GEO. Since DHS in MCF-7 cells with and without hormone treatment were highly correlated with each other minimum pearson correlation coefficient of 0. RSEM normalized results of gene expression were used, which divide the raw counts by the 75th percentile of read counts for each sample and then multiply by For each gene, its median expression value among the samples was calculated for downstream analysis.

We excluded any regions overlapping coding sequences and UCSC Browser blacklisted regions, often misaligned to sites in the reference assembly Duke and DAC , and with low unique mappability of sequencing reads. For analysis centered on ER binding sites, regions that overlap other TF binding sites within flanking regions were also excluded. After the filtering step, mutation data were mapped to ER binding or DHS intervals, and mutation rate at the nucleotide resolution was computed and plotted. For each analyzed interval sets, we calculated the probabilities of occurrence of all possible 96 tri-nucleotide changes similar to computing mutation signatures [ 13 ].

And then the mean expected mutation rate, after times random sampling, based on sequence context tri-nucleotide compositions at each nucleotide position was plotted against the observed rate for comparison. For each time of random sampling, the number of different 96 tri-nucleotide changes was kept the same. Expected mutation rate was not calculated for insertions and deletions, due to unavailability of robust methods to predict their occurrence based on sequence context.

The obtained P values were corrected for multiple testing using the Benjamini-Hochberg procedure [ 56 ]. All the regression models were built using the glm.


The final fitted model was determined by performing ANOVA test for models with different independent variables included. We also compared the final negative binomial model with a corresponding Poisson model. P values for the coefficients of included independent variables were calculated using Wald test. To use any model for prediction, data points for each independent variable were independently simulated.

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Then the model was used to predict values for the response variable. To remove effects of any independent variable on the response variable, residuals function in R was applied to obtain the corrected values of the response variable. Then, we randomly sampled regions from each ERBS group and repeated the sampling for 10 times. Choosing random sites was limited by the number of ERBS with at least 3 mutations. To make sure an equal number of genes were included for each ERBS group, we randomly sampled genes for times and computed the mean expression levels of the genes sampled each time.

The distributions of gene expression levels were compared across ERBS groups with different numbers of mutations. P values were calculated using two-sided t test. Same as above, genes were randomly sampled for times to compute mean expression distributions for different ERBS groups, which avoids bias when comparing with the ChIA-PET based analysis results. Bam files for the 9 ER ChIP-seq samples with good outcome, and the 12 samples with poor or metastasis outcome were merged for mutation discovery [ 23 ]. To increase the credibility of somatic mutations identified from the ChIP-seq data, only the sites covered with at least 10 reads and encompassing both the reference and alternative alleles in BRCA-EU were selected as potential somatic mutations.

In the end, the percentage of outcome-associated ERBS that contain potential somatic mutations was calculated for samples with corresponding outcomes. Motif score ratios between mutant and reference sequences were calculated for the reliably identified motifs.

Motifs with large absolute values of score ratios were presented in Figs. BEDTools utilities [ 54 ] were used to carry out operations such as extensions or overlaps in the various analyses of genomic features. All the statistical tests were performed in the R version 3. Customized bash, R and python scripts were used to perform all the other analysis. Increasing amounts of unlabelled WT or mutant competitor oligonucleotides were used to analyze specificity of mobility shifts.

Competitor probe concentration was 8 pM. Promega FuGene 6 cat. The same molar ratio of plasmids were used for dCas9-target and control sgRNAs. Single colonies were picked up, grown in well plates, and then transferred to well plates for expansion. For each colony, genomic DNA was isolated according to the protocol [ 37 ] and qPCR was performed using the primers that cover the point mutation site of the ZNF gene. Fragmented chromatin was diluted with ChIP-dilution buffer 0. Next, beads were washed well on the magnetic field with each of these buffers two times: low-salt immune complex wash buffer 0.

After reverse cross-linking, proteinase K and RNase digestion, DNA was extracted with ethanol precipitation method and quantified via Qubit Fluorometer. A primer pair for a negative control genomic region was used to calculate fold enrichment. Crosslinking was quenched with ice-cold 0. Sequentially, cell and nuclear membrane lysis reactions were performed with appropriate buffers. DNAs were precipitated by using phenol-chloroform extraction method. Normalized crosslinking frequency was calculated by using Ct value difference between target and genomic control primer pairs.

Wild-type and mutant MCF-7 cells were plated at a density of 1, cells per well in well plates. The plate was inverted overnight and covered to dry before imaging. Evolution of the cancer genome. Nat Rev Genet.

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Nat Genet. International network of cancer genome projects. Landscape of somatic mutations in breast cancer whole-genome sequences. Genome-wide analysis of noncoding regulatory mutations in cancer. Synonymous mutations frequently act as driver mutations in human cancers. Nat Biotechnol. Systematic analysis of noncoding somatic mutations and gene expression alterations across 14 tumor types. Highly recurrent TERT promoter mutations in human melanoma. TERT promoter mutations in familial and sporadic melanoma. Recurrent and functional regulatory mutations in breast cancer.

The large-scale distribution of somatic mutations in cancer genomes. Hum Mutat. Signatures of mutational processes in human cancer. The cancer genome. Nucleotide excision repair is impaired by binding of transcription factors to DNA. Differential DNA repair underlies mutation hotspots at active promoters in cancer genomes.

Supek F, Lehner B. Differential DNA mismatch repair underlies mutation rate variation across the human genome. Cancer statistics, CA Cancer J Clin. Int J Cancer. Quantification of estrogen receptor alpha and beta expression in sporadic breast cancer. Estrogen receptors and human disease. J Clin Invest. Estrogen carcinogenesis in breast cancer. N Engl J Med. Differential oestrogen receptor binding is associated with clinical outcome in breast cancer.

Genome Biol. The effects of chromatin organization on variation in mutation rates in the genome. The topography of mutational processes in breast cancer genomes. Nat Commun. Cell Rep. Consortium EP. An integrated encyclopedia of DNA elements in the human genome. Chromatin interaction analysis reveals changes in small chromosome and telomere clustering between epithelial and breast cancer cells. Extensive promoter-centered chromatin interactions provide a topological basis for transcription regulation. Cook PR. The organization of replication and transcription.

Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. Cancer Genome Atlas Network. Comprehensive molecular portraits of human breast tumours. Exploring the intrinsic differences among breast tumor subtypes defined using immunohistochemistry markers based on the decision tree. Sci Rep.

Mol Oncol. Biophys J. Handl H, Gillies R. Lanthanide-based luminescent assays for ligand-receptor interactions. Life Sci. Rossi A, Taylor C. Analysis of protein-ligand interactions by fluorescence polarization. Nat Protoc. J Vis Exp. Structure and dynamics of the active human parathyroid hormone receptor Labaer J, Ramachandran N. Protein microarrays as tools for functional proteomics. Curr Opin Chem Biol. Screening kinase inhibitors with a microarray-based fluorescent and resonance light scattering assay. Anal Chem. Fluorescence- and bioluminescence-based approaches to study GPCR ligand binding.

Br J Pharmacol. Charest Morin X, Marceau F. Eur J Med Chem. J Med Chem. Selective nonpeptidic fluorescent ligands for oxytocin receptor: design, synthesis, and application to time-resolved FRET binding assay. Nanofluidic Fluorescence Microscopy NFM for real-time monitoring of protein binding kinetics and affinity studies. Biosens Bioelectron. Development of a quantitative fluorescence-based ligand-binding assay.

Sci Rep. Scintillation proximity assays in high-throughput screening. Assay Drug Dev Technol. Hulme E, Trevethick M. Ligand binding assays at equilibrium: validation and interpretation. Radioligand saturation binding for quantitative analysis of ligand-receptor interactions.

Biophys Rep. Catani V, Gasperi V. Assay of CB1 Receptor Binding. Flanagan C. GPCR-radioligand binding assays. Methods Cell Biol. Characterization of monoacylglycerol acyltransferase 2 inhibitors by a novel probe in binding assays. Anal Biochem. Curr Protoc Pharmacol. Chu U, Ruoho A. Sigma Receptor Binding Assays. Use of radiolabeled antagonist assays for assessing agonism at D2 and D3 dopamine receptors: comparison with functional GTP? S assays. J Neurosci Methods. Target engagement and drug residence time can be observed in living cells with BRET.

Nat Commun. Visualizing real-time influenza virus infection, transmission and protection in ferrets. Quantitative measurement of cell membrane receptor internalization by the nanoluciferase reporter: Using the G protein-coupled receptor RXFP3 as a model. Biochim Biophys Acta.

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Application of the novel bioluminescent ligand-receptor binding assay to relaxin-RXFP1 system for interaction studies. Amino Acids. Novel bioluminescent receptor-binding assays for peptide hormones: using ghrelin as a model. Quick preparation of nanoluciferase-based tracers for novel bioluminescent receptor-binding assays of protein hormones: Using erythropoietin as a model.

J Photochem Photobiol B. Daghestani H, Day B. Theory and applications of surface plasmon resonance, resonant mirror, resonant waveguide grating, and dual polarization interferometry biosensors. Sensors Basel. Rich R, Myszka D. Survey of the commercial optical biosensor literature.

J Mol Recognit. Surface plasmon resonance analysis of seven-transmembrane receptors. Methods Enzymol. Del Vecchio K, Stahelin R. Surface plasmon resonance SPR -based biosensor technology for the quantitative characterization of protein-carotenoid interactions. Arch Biochem Biophys. Nikolovska Coleska Z.

Studying protein-protein interactions using surface plasmon resonance. A surface plasmon resonance approach to monitor toxin interactions with an isolated voltage-gated sodium channel paddle motif. J Gen Physiol. Multiplexed immunosensing and kinetics monitoring in nanofluidic devices with highly enhanced target capture efficiency.

Trends Pharmacol Sci. Different structural states of the proteolipid membrane are produced by ligand binding to the human delta-opioid receptor as shown by plasmon-waveguide resonance spectroscopy. Mol Pharmacol. Plasmon-waveguide resonance studies of ligand binding to integral proteins in membrane fragments derived from bacterial and mammalian cells. Surface plasmon resonance imaging for affinity-based biosensors. Quantitative serum proteomics from surface plasmon resonance imaging.

Mol Cell Proteomics. Multiplexed DNA quantification by spectroscopic shift of two microsphere cavities. Quantification of Plasmodium-host protein interactions on intact, unmodified erythrocytes by back-scattering interferometry. Malar J. A fiber-tip label-free biological sensing platform: a practical approach toward in-vivo sensing. High-Q silk fibroin whispering gallery microresonator. Opt Express. Righini G, Soria S. Glial immunoreactivity for metallothionein in the rat brain.

Heeres J, Hergenrother P. High-throughput screening for modulators of protein-protein interactions: use of photonic crystal biosensors and complementary technologies. Chem Soc Rev. Label-free detection of biomolecular interactions using BioLayer interferometry for kinetic characterization. Comb Chem High Throughput Screen. This assay can be used to rapidly ascertain whether an environmental contaminant is capable of acting through the same binding mechanism as endogenous estradiol.

It has been demonstrated that a number of the environmental contaminants are able to function in a manner similar to estradiol Colborn et al. These include such compounds as bisphenol A Krishnan et al. When the ER is bound by its endogenous hormone, subsequent activation of the ER results in conformational changes, protein interactions, and gene transcription Beekman et al. Therefore, xenoestrogen-induced alterations in normal endocrine function can result in adverse effects at the cellular level Roy et al.

The majority of QSAR models developed to date are based on the biological activity of small groups of compounds with similar activity and structural features. However, the structural diversity of estrogenic chemicals is very broad. For the current study, chemicals were selected such that 1 a large data set was generated, 2 a wide diversity of structural features was represented and 3 a wide range of biological activity was measured. To our knowledge, the results presented here represent the largest and most diverse data set publicly available for chemicals binding to the ER.

These data can be utilized to develop a highly robust 3D-QSAR model, as well as separate chemometric models the development and implementation of which will be presented in a separate manuscript. In addition, these data are also useful for comparing ER-binding results from a large number of chemicals to small data sets using different assay conditions. Louis, MO. The source and purity when available for each of the competing test compounds is provided in tabular form with the results from the competitive-binding assays.

Adult retired breeders; Animals received Purina rat chow and filtered tap water ad libitum. Females a mean of 14 rats per cytosol batch were ovariectomized a minimum of 10 days prior to receptor preparation. After sacrifice by CO 2 asphyxiation, uteri were excised, trimmed of excess fat and mesentery, weighed and placed in ice-cold TEDG buffer 10 mM Tris, 1.

The pooled uteri were placed in fresh, ice-cold TEDG buffer at a concentration of 1. These tubes were incubated in an icewater bath for 20 min and vortexed for 10 s at 5-min intervals. The supernatant was discarded and the resulting HAP pellet was resuspended in 2. After 3 washes, the supernatant was discarded and 2. Tubes were incubated on ice for 15 min and vortexed at 5-min intervals.

Acetylcholine Receptor Binding Antibody

The resulting supernatant was decanted into vials containing 10 ml of scintillation cocktail. Radioactivity counts dpm of the NSB tubes were subtracted from all tubes prior to calculation of percent [ 3 H]-E 2 bound. Details and validation of the ER competitive-binding assay will be published in a separate manuscript unpublished. Stock solutions were then subsequently diluted in ethanol for analysis in the ER competitive-binding assay.

Due to the large number of chemicals tested, the ER competitive-binding assays were set up in a tiered design. Unless known to bind to the ER, test chemicals were initially run at only 2 high concentrations spanning 3 log concentrations Tier 1. If necessary, a Tier 3 assay consisting of one-half log molar concentrations which bracketed the IC 50 observed in the Tier 2 assay was run to more accurately determine a competitor's IC In the Tier 1 assay, approximately 36 chemicals could be assayed in replicate per cytosol batch, while only 18 chemicals per cytosol batch could be assayed in replicate in either the Tier 2 or Tier 3 assay.

In the final analysis, it required one rat per chemical assayed in replicate. For purposes of clarity and convenience, test compounds were grouped in tables according to chemical or use classifications. Figure 1 shows representative ER-binding curves over the range of concentrations used for various chemicals in the ER competitive-binding assay. As expected, all of the selected steroidal estrogens Table 1 , synthetic estrogens Table 2 and antiestrogens Table 3 showed affinity for the ER.

With the exception of one chemical in each of these 3 classes, all these chemicals exhibited moderate to strong binding affinity. This was especially evident for the synthetic estrogens, which exhibited strong affinity for the ER in 13 of the 16 chemicals tested. The only other chemical analyzed that exhibited greater affinity than E 2 for the ER was the antiestrogen 4-hydroxytamoxifen. A number of miscellaneous steroids were also analyzed in the ER competitive-binding assay Table 4.

The majority of these chemicals was inactive and did not bind to the ER. Nonetheless, the initial portions of these curves were parallel to the E 2 standard curve. Of the chemicals assayed, 22 20 binders and 2 non-binders demonstrated a U-shaped binding curve identified individually in Tables 1— In 5 of these 22 chemicals, an increase in percent [ 3 H]-E 2 bound was evident for the 2—3 highest concentrations tested. In the remaining 17 chemicals, an increase in the percent [ 3 H]-E 2 binding was evident only at the highest concentration tested. The majority 15 of these 22 chemicals consisted of steroidal estrogens, synthetic estrogens, antiestrogens, or miscellaneous steroids.

Alkylphenolic compounds Table 5 were a major group of chemicals assayed. All but 3 of the alkylphenols exhibited binding to the ER with their binding affinity ranging from moderate to weak. Of concern was the possibility that different sources or different lots of an individual chemical might bind to the ER with substantially different affinities.

Therefore, we tested this possibility on 4-nonylphenol, a compound known to be a mixture of isomers. Nonylphenol was chosen due to its importance in National Toxicology Program studies Chapin et al. In addition, we tested 2 different lots of 4-nonylphenol from Aldrich and 2 different lots from Fluka. Also, there were no substantial differences in ER-binding affinities between different lots of 4-nonylphenol from the same source. It also appeared that length of a chemical's side chain influenced the ER-binding affinity of the alkylphenols. However, there appears to be a limit to the number of side-chain carbons that increases binding to the ER since 4-dodecylphenol 12 carbons exhibited a lower RBA than 4-nonylphenol 9 carbons.

Table 6 provides the binding affinities of another major group of test chemicals, the diphenyl derivatives. Of the diphenyl methane derivatives bisphenol As , only bisphenol B was moderately active at binding the ER, while the remaining chemicals in this group were weak binders or inactive at the concentrations tested. Three of the 5 diphenyl ethanes were moderate ER binders, while the biphenyl compounds were either weak binders or non-binders. In this study, we tested 3 groups of organochlorines Table 7 : DDT isomers, methoxychlor and it's derivatives, and polychlorinated biphenyls PCBs.

Only dihydroxymethoxychlor olefin showed strong affinity for the ER. The remaining chemicals exhibited either moderate or weak RBAs or were inactive at the concentrations tested. A difference in ER binding between methoxychlor with different levels of purity was evident. It has been demonstrated previously that a phenolic contaminant in methoxychlor preparations is estrogenic and that this contaminant may be dihydroxymethoxychlor HPTE , a metabolite of methoxychlor Bulger et al.

Since the pesticides the DDT and methoxychlor isomers within this group of chemicals exhibited affinity for the ER, it was important to determine whether other pesticides could also compete. Of the other pesticides tested Table 8 , only kepone bound with moderate affinity to the ER. None of the remaining pesticides analyzed in the ER competitive-binding assay exhibited any activity at the concentrations tested. Table 9 shows the RBAs for several paraben compounds. All of the parabens examined in this study competed for the ER.

Of the 7 chemicals analyzed, one bound the ER with moderate affinity while the rest exhibited weak binding. The parabens, like the alkylphenolic compounds, demonstrated a positive correlation between binding affinity and chain length. The chemicals with the longer side chains 2-ethylhexyl, heptyl, and benzyl 4-hydroxybenzoates showed greater affinity for the ER compared to the parabens with shorter side chains butyl, propyl, ethyl, and methyl 4-hydroxybenzoates.

Under the conditions of our ER competitive-binding assay, none of the 8 phthalate compounds exhibited an IC 50 Table The results of this assay demonstrated that extending the incubation time had no effect on binding of benzylbutyl phthalate to the ER. The remaining test chemicals consisted of benzophenone compounds Table 11 and several miscellaneous classes of chemicals Table Only 2 of the 5 benzophenone compounds competed for the ER. Of the remaining 39 chemicals assayed Table 12 , 3 showed moderate affinity and 4 exhibited weak affinity for the ER, while the other 32 were inactive at the concentrations tested.

There were also a number of test chemicals that competed for the ER, but did so with such a weak affinity that an IC 50 was not attainable Table While the chemicals that exhibited this slight affinity for the ER were structurally diverse, including 4 organochlorines and 2 phthalates, they were also environmentally important. To our knowledge, this is the first paper to report the ER-binding affinities of such a large number of structurally diverse chemicals assayed under identical conditions.

Other published results Andersen et al. In the current study, the rats utilized as the source of the ER were retired breeders. Although this is not a common practice, ER levels in ovariectomized, retired breeders are comparable to both immature, intact animals Clark et al. Similar binding results were evident between the current study and a previous study using immature rats Perez et al.

The GraphPad Guide to Analyzing Radioligand Binding Data

While ER levels are similar between these rats of different ages, the use of retired breeders has three distinct advantages. First, retired breeders have greater uterine weights, which results in more total ER available for use in binding studies. Second, due to the larger uterine weights, fewer animals are required.

Lastly, the use of retired breeders is essentially an animal-sparing process, since the ability to use these animals, which would be disposed of under normal situations, allows us to conduct these studies without having to purchase and subsequently sacrifice new animals. The reason for the U-shaped binding curves observed in the present study remains unclear. However, it has been shown that high concentrations of steroids and antiestrogens can markedly accelerate the dissociation rate of the ER-[ 3 H]-E 2 complex Borgna and Ladrech, Therefore, the U-shaped binding curves presented here are most likely due to the high doses assayed and the kinetics of the binding assay, and as such they are probably not associated with the U-shaped dose-response curves observed at low doses in vivo for some chemicals vom Saal et al.

Of the variety of chemical classes analyzed in the current study, two were of particular interest. These were the phthalates and the parabens. The phthalic acid esters phthalates are widely used as plasticizing agents; however, since they are not covalently bound within the plastic, the phthalates can be released into the environment Autian, ; Giam et al. Since phthalates are capable of altering reproductive function in rats Ema et al. Previous studies have suggested that di- n -butyl phthalate, benzylbutyl phthalate and BIS 2-ethylhexyl phthalate are weak binders or weakly estrogenic in a variety of systems Bolger et al.

However, we were unable to determine IC 50 values for any of the phthalate compounds analyzed in our ER competitive-binding assay. Nonetheless, two of the phthalates, benzylbutyl phthalate and BIS 2-ethylhexyl phthalate, competed with E 2 for the ER. Utilizing rainbow trout ER, Jobling et al. However, the binding curves were not parallel with that of E 2 , suggesting that the phthalates may be working through or influenced by an alternative mechanism.

Other studies have also reported IC 50 values for benzylbutyl phthalate and di- n -butyl phthalate, using a human recombinant ER hrER Bolger et al. Zacharewski and colleagues also demonstrated that dihexyl phthalate competed with [ 3 H]-E 2 for the ER, though not with strong enough affinity to attain an IC 50 value. However, these authors measured binding to a mouse uterine cytosol ER preparation and differences in binding affinities may be related to variation in species sensitivity.

When comparing the data presented here to data collected from competitive-binding assays that utilized a short-term incubation period at room temperature or above Arcaro et al. In general, RBA values for low affinity xenobiotics were slightly higher in the short term, high temperature assays compared to the assay utilized in the current study, while high affinity chemicals exhibited similar binding affinities. This suggests that differences in assay conditions can lead to the observed differences in binding affinities and that high-temperature assays might be somewhat more sensitive to certain chemicals.

However, high-temperature assays run the risk of ER degradation during the assay.