scholarly journals ChIPulate : A comprehensive ChIP-seq simulation pipeline

2018 ◽  
Author(s):  
Vishaka Datta ◽  
Sridhar Hannenhalli ◽  
Rahul Siddharthan
Keyword(s):  
Dna Binding ◽  
Pcr Amplification ◽  
Amplification Efficiency ◽  
Binding Motif ◽  
Binding Strength ◽  
Sources Of Variation ◽  
The Mean ◽  
Genomic Regions ◽  
The Impact

AbstractChIP-seq (Chromatin Immunoprecipitation followed by sequencing) is a high-throughput technique to identify genomic regions that are bound in vivo by a particular protein, e.g., a transcription factor (TF). Biological factors, such as chromatin state, indirect and cooperative binding, as well as experimental factors, such as antibody quality, cross-linking, and PCR biases, are known to affect the outcome of ChIP-seq experiments. However, the relative impact of these factors on inferences made from ChIP-seq data is not entirely clear. Here, via a detailed ChIP-seq simulation pipeline, ChIPulate, we assess the impact of various biological and experimental sources of variation on several outcomes of a ChIP-seq experiment, viz., the recoverability of the TF binding motif, accuracy of TF-DNA binding detection, the sensitivity of inferred TF-DNA binding strength, and number of replicates needed to confidently infer binding strength. We find that the TF motif can be recovered despite poor and non-uniform extraction and PCR amplification efficiencies. The recovery of the motif is however affected to a larger extent by the fraction of sites that are either cooperatively or indirectly bound. Importantly, our simulations reveal that the number of ChIP-seq replicates needed to accurately measure in vivo occupancy at high-affinity sites is larger than the recommended community standards. Our results establish statistical limits on the accuracy of inferences of protein-DNA binding from ChIP-seq and suggest that increasing the mean extraction efficiency, rather than amplification efficiency, would better improve sensitivity. The source code and instructions for running ChIPulate can be found at https://github.com/vishakad/chipulate.

scholarly journals Genome-Wide Binding Analyses of HOXB1 Revealed a Novel DNA Binding Motif Associated with Gene Repression

2021 ◽  
Vol 9 (1) ◽  
pp. 6
Author(s):  
Narendra Pratap Singh ◽  
Bony De Kumar ◽  
Ariel Paulson ◽  
Mark E. Parrish ◽  
Carrie Scott ◽  
...  
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Embryonic Stem ◽  
Binding Motif ◽  
Binding Assays ◽  
Histone Marks ◽  
Genome Wide ◽  
Hox Cofactors

Knowledge of the diverse DNA binding specificities of transcription factors is important for understanding their specific regulatory functions in animal development and evolution. We have examined the genome-wide binding properties of the mouse HOXB1 protein in embryonic stem cells differentiated into neural fates. Unexpectedly, only a small number of HOXB1 bound regions (7%) correlate with binding of the known HOX cofactors PBX and MEIS. In contrast, 22% of the HOXB1 binding peaks display co-occupancy with the transcriptional repressor REST. Analyses revealed that co-binding of HOXB1 with PBX correlates with active histone marks and high levels of expression, while co-occupancy with REST correlates with repressive histone marks and repression of the target genes. Analysis of HOXB1 bound regions uncovered enrichment of a novel 15 base pair HOXB1 binding motif HB1RE (HOXB1 response element). In vitro template binding assays showed that HOXB1, PBX1, and MEIS can bind to this motif. In vivo, this motif is sufficient for direct expression of a reporter gene and over-expression of HOXB1 selectively represses this activity. Our analyses suggest that HOXB1 has evolved an association with REST in gene regulation and the novel HB1RE motif contributes to HOXB1 function in part through a repressive role in gene expression.

scholarly journals Relative Rates of Gluten Digestion by Nine Commercial Dietary Digestive Supplements

2021 ◽  
Vol 8 ◽  
Author(s):  
Gregory John Tanner
Keyword(s):  
Amino Acids ◽  
Celiac Disease ◽  
Initial Rate ◽  
Relative Rate ◽  
Elisa Method ◽  
Fasting Stomach ◽  
The Mean ◽  
Stomach Volume ◽  
The Impact

Endopeptidases containing supplements may digest gluten and reduce the impact on celiac and gluten-sensitive subjects who inadvertently consume gluten. We investigated the relative rate of disappearance of coeliac relevant epitopes in extracts of nine commercial supplements, using two competitive enzyme-linked immunosorbent assays (ELISAs)—Ridascreen (detects QQPFP, QQQFP, LQPFP, and QLPFP) and Gluten-Tec (detects Glia-α20 and PFRPQQPYPQ). All epitopes are destroyed by cleavage after P and Q amino acids. Rates at pH 3.5 and pH 7.0 were measured. These experiments were designed to measure relative rates of epitope digestion not to mimic in vivo digestion. The supplements were: 1 GluteGuard, 2 GlutenBlock, 3 GliadinX, 4 GlutnGo, 5 GlutenRescue, 6 Eat E-Z Gluten+, 7 Glutenease, 8 Glutezyme, and 9 Gluten Digest. The mean initial rate and half-lives of epitope digestion were deduced and extrapolated to rates at the recommended dose of one supplement in a fasting stomach volume. At pH 7, supplement 1 was the fastest acting of the supplements, with Ridascreen ELISA, more than twice as fast as the next fastest supplements, 5, 6, 7, and 8. Supplements 2, 3, and 4 showed little activity at pH 7.0. Supplement 1 was also the fastest acting at pH 7 with Gluten-Tec ELISA, more than three times the rate for supplements 2 and 3, with supplements 4–9 showing minimal activity. At pH 3.5, supplement 1 acted more than five times as fast as the next fastest supplements, 2 and 3, when measured by Ridascreen, but supplements 2 and 3 were over two times faster than supplement 1 when measured by Gluten-Tec. Supplements 4–9 demonstrated minimal activity at pH 3.5 with either ELISA. Supplement 1 most rapidly digested the key immuno-reactive gluten epitopes identified by the R5 antibody in the Codex-approved competitive Ridascreen ELISA method and associated with the pathology of celiac disease.

scholarly journals Sequence and functional analysis of the positively acting regulatory gene amdR from Aspergillus nidulans.

1990 ◽  
Vol 10 (6) ◽  
pp. 3194-3203 ◽  
Author(s):  
A Andrianopoulos ◽  
M J Hynes
Keyword(s):  
Dna Binding ◽  
Aspergillus Nidulans ◽  
Nuclear Localization ◽  
Regulatory Gene ◽  
Transcription Activation ◽  
Binding Motif ◽  
Wild Type ◽  
Amino Terminal ◽  
Carboxyl Terminal

The positively acting regulatory gene amdR of Aspergillus nidulans coordinately regulates the expression of five structural genes involved in the catabolism of certain amides (amdS), omega amino acids (gatA and gabA), and lactams (lamA and lamB) in the presence of omega amino acid inducers. Analysis of the amdR gene showed that it contains three small introns, heterogeneous 5' and 3' transcription sites, and multiple AUG codons prior to the major AUG initiator. The predicted amdR protein sequence has a cysteine-rich "zinc finger" DNA-binding motif at the amino-terminal end, four putative acidic transcription activation motifs in the carboxyl-terminal half, and two sequences homologous to the simian virus 40 large T antigen nuclear localization motif. These nuclear localization sequences overlap the cysteine-rich DNA-binding motif. A series of 5', 3', and internal deletions were examined in vivo for transcription activator function and showed that the amdR product contains at least two activation regions in the carboxyl-terminal half. Each of these activator amdR product contains at least two activation regions in the carboxyl-terminal half. Each of these activator regions may function independently, but both are required for wild-type levels of transcription activation. A number of the amdR deletion products were found to compete with the wild-type amdR product in vivo. Development of a rapid method for the localization of amdR mutations is presented, and using this technique, we localized and sequenced the mutation in the semiconstitutive amdR6c allele. The amdR6c missense mutation occurs in the middle of the gene, and it is suggested that it results in an altered protein which activates gene expression efficiently in the absence of an inducer.

scholarly journals Identification and Prevention of Genotyping Errors Caused by G-Quadruplex– and i-Motif–Like Sequences

2009 ◽  
Vol 55 (7) ◽  
pp. 1361-1371 ◽  
Author(s):  
Jürgen J Wenzel ◽  
Heidi Rossmann ◽  
Christian Fottner ◽  
Stefan Neuwirth ◽  
Carolin Neukirch ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Mendelian Inheritance ◽  
Magnesium Concentration ◽  
Amplification Efficiency ◽  
Nucleotide Polymorphisms ◽  
Genotyping Errors ◽  
Genetic Analyses ◽  
Intronic Polymorphism ◽  
G Quadruplex ◽  
The Impact

Abstract Background: Reliable PCR amplification of DNA fragments is the prerequisite for most genetic assays. We investigated the impact of G-quadruplex– or i-motif–like sequences on the reliability of PCR-based genetic analyses. Methods: We found the sequence context of a common intronic polymorphism in the MEN1 gene (multiple endocrine neoplasia I) to be the cause of systematic genotyping errors by inducing preferential amplification of one allelic variant [allele dropout (ADO)]. Bioinformatic analyses and pyrosequencing-based allele quantification enabled the identification of the underlying DNA structures. Results: We showed that G-quadruplex– or i-motif–like sequences can reproducibly cause ADO. In these cases, amplification efficiency strongly depends on the PCR enzyme and buffer conditions, the magnesium concentration in particular. In a randomly chosen subset of candidate single-nucleotide polymorphisms (SNPs) defined by properties deduced from 2 originally identified ADO cases, we confirmed preferential PCR amplification in up to 50% of the SNPs. We subsequently identified G-quadruplex and i-motifs harboring a SNP that alters the typical motif as the cause of this phenomenon, and a genomewide search based on the respective motifs predicted 0.5% of all SNPs listed by dbSNP and Online Mendelian Inheritance in Man to be potentially affected. Conclusions: Undetected, the described phenomenon produces systematic errors in genetic analyses that may lead to misdiagnoses in clinical settings. PCR products should be checked for G-quadruplex and i-motifs to avoid the formation of ADO-causing secondary structures. Truly affected assays can then be identified by a simple experimental procedure, which simultaneously provides the solution to the problem. .

scholarly journals A novel k-mer set memory (KSM) motif representation improves regulatory variant prediction

2017 ◽  
Author(s):  
Yuchun Guo ◽  
Kevin Tian ◽  
Haoyang Zeng ◽  
Xiaoyun Guo ◽  
David Kenneth Gifford
Keyword(s):  
Binding Sites ◽  
Regulatory Networks ◽  
Motif Discovery ◽  
De Novo ◽  
Binding Motif ◽  
Large Set ◽  
Public Resource ◽  
Regulatory Variant ◽  
The Impact

ABSTRACTThe representation and discovery of transcription factor (TF) sequence binding specificities is critical for understanding gene regulatory networks and interpreting the impact of disease-associated non-coding genetic variants. We present a novel TF binding motif representation, the K-mer Set Memory (KSM), which consists of a set of aligned k-mers that are over-represented at TF binding sites, and a new method called KMAC for de novo discovery of KSMs. We find that KSMs more accurately predict in vivo binding sites than position weight matrix models (PWMs) and other more complex motif models across a large set of ChIP-seq experiments. KMAC also identifies correct motifs in more experiments than four state-of-the-art motif discovery methods. In addition, KSM derived features outperform both PWM and deep learning model derived sequence features in predicting differential regulatory activities of expression quantitative trait loci (eQTL) alleles. Finally, we have applied KMAC to 1488 ENCODE TF ChIP-seq datasets and created a public resource of KSM and PWM motifs. We expect that the KSM representation and KMAC method will be valuable in characterizing TF binding specificities and in interpreting the effects of non-coding genetic variations.

scholarly journals Functional analyses of the Neurospora crassa MT a-1 mating type polypeptide.

Genetics ◽  
1994 ◽  
Vol 137 (3) ◽  
pp. 715-722 ◽  
Author(s):  
M L Philley ◽  
C Staben
Keyword(s):  
Dna Binding ◽  
Neurospora Crassa ◽  
Mating Type ◽  
Dna Sequences ◽  
High Mobility ◽  
Binding Motif ◽  
Vegetative Incompatibility ◽  
Hmg Box

Abstract The Neurospora crassa mt a-1 gene, encoding the MT a-1 polypeptide, determines a mating type properties: sexual compatibility and vegetative incompatibility with A mating type. We characterized in vivo and in vitro functions of the MT a-1 polypeptide and specific mutant derivatives. MT a-1 polypeptide produced in Escherichia coli bound to specific DNA sequences whose core was 5'-CTTTG-3'. DNA binding was a function of the MT a-1 HMG box domain (a DNA binding motif found in high mobility group proteins and a diverse set of regulatory proteins). Mutation within the HMG box eliminated DNA binding in vitro and eliminated mating in vivo, but did not interfere with vegetative incompatibility function in vivo. Conversely, deletion of amino acids 216-220 of MT a-1 eliminated vegetative incompatibility, but did not affect mating or DNA binding. Deletion of the carboxyl terminal half of MT a-1 eliminated both mating and vegetative incompatibility in vivo, but not DNA binding in vitro. These results suggest that mating depends upon the ability of MT a-1 polypeptide to bind to, and presumably to regulate the activity of, specific DNA sequences. However, the separation of vegetative incompatibility from both mating and DNA binding indicates that vegetative incompatibility functions by a biochemically distinct mechanism.

scholarly journals Cellular crowding guides and debundles the microtubule cytoskeleton

2019 ◽  
Author(s):  
A. Z. Płochocka ◽  
N. A. Bulgakova ◽  
L. Chumakova
Keyword(s):  
Mathematical Modelling ◽  
Biological Systems ◽  
Follicular Epithelium ◽  
Microtubule Cytoskeleton ◽  
Cytoskeleton Organization ◽  
Cell Organization ◽  
The Mean ◽  
Cellular Components ◽  
The Impact

Cytoplasm is densely packed with macromolecules causing cellular crowding, which alters interactions inside cells and differs between biological systems. Here we investigate the impact of crowding on microtubule cytoskeleton organization. Using mathematical modelling, we find that only anisotropic crowding affects the mean microtubule direction, but any crowding reduces the number of microtubules that form bundles. We validate these predictions in vivo using Drosophila follicular epithelium. Since cellular components are transported along microtubules, our results identify cellular crowding as a novel regulator of this transport and cell organization.

scholarly journals Identification of the promoter region involved in the autoregulation of the transcriptional activator ALCR in Aspergillus nidulans

1992 ◽  
Vol 12 (5) ◽  
pp. 1932-1939
Author(s):  
P Kulmburg ◽  
D Sequeval ◽  
F Lenouvel ◽  
M Mathieu ◽  
B Felenbok
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Aspergillus Nidulans ◽  
Binding Protein ◽  
Transcriptional Activator ◽  
Binding Domain ◽  
Binding Motif ◽  
Affinity Binding ◽  
Coding Region

The ALCR protein is the transcriptional activator of the ethanol utilization pathway in the filamentous fungus Aspergillus nidulans. This activator belongs to a family of fungal proteins having a conserved DNA-binding domain containing six cysteines (C6 class) with some striking features. At variance with other motifs of this class, the binding domain of ALCR is strongly asymmetrical in relation to the central cysteines and moreover was predicted to adopt a helix-turn-helix structure. This domain of ALCR was synthesized in Escherichia coli and purified as a glutathione-S-transferase fusion protein. Our results show that the transcriptional activator ALCR is a DNA-binding protein. The DNA-binding motif contains zinc that is necessary for the specific DNA binding. The ALCR peptide binds upstream of the coding region of alcR to two specific targets with different affinities that are characterized by a conserved 5-nucleotide core, 5'-CCGCA-3' (or its reverse). One site, the lower-affinity binding site, is a direct repeat, and the other, the higher-affinity binding site, is a palindromic sequence with dyad symmetry. Therefore, the ALCR binding protein is able to recognize one DNA sequence in two different configurations. An alcR mutant obtained by deletion of the two specific targets in the cis-acting region of the alcR gene is unable to grow on ethanol and does not express any alcohol dehydrogenase activity. These results demonstrate that the binding sites are in vivo functional targets (UASalc) for the ALCR protein in A. nidulans. They corroborate prior evidence that alcR is autoregulated.

scholarly journals Leakage of Microbial Endotoxin through the Implant-Abutment Interface in Oral Implants: An In Vitro Study

2016 ◽  
Vol 2016 ◽  
pp. 1-6 ◽  
Author(s):  
Rhoodie Garrana ◽  
Govindrau Mohangi ◽  
Paulo Malo ◽  
Miguel Nobre
Keyword(s):  
In Vitro Study ◽  
Endotoxin Level ◽  
Oral Implants ◽  
E Coli ◽  
Implant Abutment ◽  
The Mean ◽  
The Impact ◽  
External Connections

Background. Endotoxin initiates osteoclastic activity resulting in bone loss. Endotoxin leakage through implant abutment connections negatively influences peri-implant bone levels.Objectives. (i) To determine if endotoxin can traverse different implant-abutment connection (IAC) designs; (ii) to quantify the amount of endotoxins traversing the IAC; (iii) to compare the in vitro comportments of different IACs.Materials and Methods. Twenty-seven IACs were inoculated withE. coliendotoxin. Six of the twenty-seven IACs were external connections from one system (Southern Implants) and the remaining twenty-one IACs were made up of seven internal IAC types from four different implant companies (Straumann, Ankylos, and Neodent, Southern Implants).Results. Of the 27 IACs tested, all 6 external IACs leaked measurable amounts of endotoxin. Of the remaining 21 internal IACs, 9 IACs did not show measurable leakage whilst the remaining 12 IACs leaked varying amounts. The mean log endotoxin level was significantly higher for the external compared to internal types (p=0.015).Conclusion. Within the parameters of this study, we can conclude that endotoxin leakage is dependent on the design of the IAC. Straumann Synocta, Straumann Cross-fit, and Ankylos displayed the best performances of all IACs tested with undetectable leakage after 7 days. Each of these IACs incorporated a morse-like component in their design. Speculation still exists over the impact of IAC endotoxin leakage on peri-implant tissues in vivo; hence, further investigations are required to further explore this.

scholarly journals Single position substitution of hairpin pyrrole-imidazole polyamides imparts distinct DNA-binding profiles across the human genome

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0243905
Author(s):  
Paul B. Finn ◽  
Devesh Bhimsaria ◽  
Asfa Ali ◽  
Asuka Eguchi ◽  
Aseem Z. Ansari ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Dna Interaction ◽  
Sequence Specificity ◽  
Entire Genome ◽  
Dna Sequence Specificity ◽  
Interaction Sites ◽  
The Impact

Pyrrole–imidazole (Py–Im) polyamides are synthetic molecules that can be rationally designed to target specific DNA sequences to both disrupt and recruit transcriptional machinery. While in vitro binding has been extensively studied, in vivo effects are often difficult to predict using current models of DNA binding. Determining the impact of genomic architecture and the local chromatin landscape on polyamide-DNA sequence specificity remains an unresolved question that impedes their effective deployment in vivo. In this report we identified polyamide–DNA interaction sites across the entire genome, by covalently crosslinking and capturing these events in the nuclei of human LNCaP cells. This technique confirms the ability of two eight ring hairpin-polyamides, with similar architectures but differing at a single ring position (Py to Im), to retain in vitro specificities and display distinct genome-wide binding profiles.

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