Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Identification of Transcription Factor Binding Sites by Cleavage Under Target and Release Using Nuclease in Zebrafish.

ABSTRACT: Cleavage Under Targets and Release Using Nuclease (CUT&RUN) has emerged as a chromatin profiling strategy that excels traditional methods. Although CUT&RUN has been widely utilized in mammalian cells, its use in the zebrafish is at its early stages. In this study, we have developed a protocol to successfully perform CUT&RUN to map transcription factor (TF) binding sites in embryonic, adult tissues, and FACS-sorted zebrafish cells. We also provide a detailed workflow for the identification of predicted TF binding sites that can be utilized in any animal species. Altogether, our strategy will expand this invaluable tool to the zebrafish community, improving the epigenetic resolution that can be achieved in this model organism.

SUBMITTER: Barakat R

PROVIDER: S-EPMC9246268 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Identification of Transcription Factor Binding Sites by Cleavage Under Target and Release Using Nuclease in Zebrafish.

Barakat Radwa R Campbell Clyde A CA Espin-Palazon Raquel R

Zebrafish 20220601 3

Cleavage Under Targets and Release Using Nuclease (CUT&RUN) has emerged as a chromatin profiling strategy that excels traditional methods. Although CUT&RUN has been widely utilized in mammalian cells, its use in the zebrafish is at its early stages. In this study, we have developed a protocol to successfully perform CUT&RUN to map transcription factor (TF) binding sites in embryonic, adult tissues, and FACS-sorted zebrafish cells. We also provide a detailed workflow for the identification of pre ...[more]

PMID: 35704898

Similar Datasets

Single-Cell Factor Localization on Chromatin using Ultra-Low Input Cleavage Under Targets and Release using Nuclease.

Project description:Determining the binding locations of a protein on chromatin is essential for understanding its function and potential regulatory targets. Chromatin Immunoprecipitation (ChIP) has been the gold standard for determining protein localization for over 30 years and is defined by the use of an antibody to pull out the protein of interest from sonicated or enzymatically digested chromatin. More recently, antibody tethering techniques have become popular for assessing protein localization on chromatin due to their increased sensitivity. Cleavage Under Targets & Release Under Nuclease (CUT&RUN) is the genome-wide derivative of Chromatin Immunocleavage (ChIC) and utilizes recombinant Protein A tethered to micrococcal nuclease (pA-MNase) to identify the IgG constant region of the antibody targeting a protein of interest, therefore enabling site-specific cleavage of the DNA flanking the protein of interest. CUT&RUN can be used to profile histone modifications, transcription factors, and other chromatin-binding proteins such as nucleosome remodeling factors. Importantly, CUT&RUN can be used to assess the localization of either euchromatic- or heterochromatic-associated proteins and histone modifications. For these reasons, CUT&RUN is a powerful method for determining the binding profiles of a wide range of proteins. Recently, CUT&RUN has been optimized for transcription factor profiling in low populations of cells and single cells and the optimized protocol has been termed ultra-low input CUT&RUN (uliCUT&RUN). Here, a detailed protocol is presented for single-cell factor profiling using uliCUT&RUN in a manual 96-well format.

| S-EPMC9306226 | biostudies-literature

Transcription factor binding sites are frequently under accelerated evolution in primates.

Project description:Recent comparative genomic studies have identified many human accelerated elements (HARs) with elevated substitution rates in the human lineage. However, it remains unknown to what extent transcription factor binding sites (TFBSs) are under accelerated evolution in humans and other primates. Here, we introduce two pooling-based phylogenetic methods with dramatically enhanced sensitivity to examine accelerated evolution in TFBSs. Using these new methods, we show that more than 6000 TFBSs annotated in the human genome have experienced accelerated evolution in Hominini, apes, and Old World monkeys. Although these TFBSs individually show relatively weak signals of accelerated evolution, they collectively are more abundant than HARs. Also, we show that accelerated evolution in Pol III binding sites may be driven by lineage-specific positive selection, whereas accelerated evolution in other TFBSs might be driven by nonadaptive evolutionary forces. Finally, the accelerated TFBSs are enriched around developmental genes, suggesting that accelerated evolution in TFBSs may drive the divergence of developmental processes between primates.

| S-EPMC9922303 | biostudies-literature

Genomic selection identifies vertebrate transcription factor Fezf2 binding sites and target genes.

Project description:Identification of transcription factor targets is critical to understanding gene regulatory networks. Here, we uncover transcription factor binding sites and target genes employing systematic evolution of ligands by exponential enrichment (SELEX). Instead of selecting randomly synthesized DNA oligonucleotides as in most SELEX studies, we utilized zebrafish genomic DNA to isolate fragments bound by Fezf2, an evolutionarily conserved gene critical for vertebrate forebrain development. This is, to our knowledge, the first time that SELEX is applied to a vertebrate genome. Computational analysis of bound genomic fragments predicted a core consensus binding site, which identified response elements that mediated Fezf2-dependent transcription both in vitro and in vivo. Fezf2-bound fragments were enriched for conserved sequences. Surprisingly, ∼20% of these fragments overlapped well annotated protein-coding exons. Through loss of function, gain of function, and chromatin immunoprecipitation, we further identified and validated eomesa/tbr2 and lhx2b as biologically relevant target genes of Fezf2. Mutations in eomesa/tbr2 cause microcephaly in humans, whereas lhx2b is a critical regulator of cell fate and axonal targeting in the developing forebrain. These results demonstrate the feasibility of employing genomic SELEX to identify vertebrate transcription factor binding sites and target genes and reveal Fezf2 as a transcription activator and a candidate for evaluation in human microcephaly.

| S-EPMC3099680 | biostudies-literature

Interactions between the R2R3-MYB transcription factor, AtMYB61, and target DNA binding sites.

Project description:Despite the prominent roles played by R2R3-MYB transcription factors in the regulation of plant gene expression, little is known about the details of how these proteins interact with their DNA targets. For example, while Arabidopsis thaliana R2R3-MYB protein AtMYB61 is known to alter transcript abundance of a specific set of target genes, little is known about the specific DNA sequences to which AtMYB61 binds. To address this gap in knowledge, DNA sequences bound by AtMYB61 were identified using cyclic amplification and selection of targets (CASTing). The DNA targets identified using this approach corresponded to AC elements, sequences enriched in adenosine and cytosine nucleotides. The preferred target sequence that bound with the greatest affinity to AtMYB61 recombinant protein was ACCTAC, the AC-I element. Mutational analyses based on the AC-I element showed that ACC nucleotides in the AC-I element served as the core recognition motif, critical for AtMYB61 binding. Molecular modelling predicted interactions between AtMYB61 amino acid residues and corresponding nucleotides in the DNA targets. The affinity between AtMYB61 and specific target DNA sequences did not correlate with AtMYB61-driven transcriptional activation with each of the target sequences. CASTing-selected motifs were found in the regulatory regions of genes previously shown to be regulated by AtMYB61. Taken together, these findings are consistent with the hypothesis that AtMYB61 regulates transcription from specific cis-acting AC elements in vivo. The results shed light on the specifics of DNA binding by an important family of plant-specific transcriptional regulators.

| S-EPMC3669277 | biostudies-literature

Pseudocounts for transcription factor binding sites.

Project description:To represent the sequence specificity of transcription factors, the position weight matrix (PWM) is widely used. In most cases, each element is defined as a log likelihood ratio of a base appearing at a certain position, which is estimated from a finite number of known binding sites. To avoid bias due to this small sample size, a certain numeric value, called a pseudocount, is usually allocated for each position, and its fraction according to the background base composition is added to each element. So far, there has been no consensus on the optimal pseudocount value. In this study, we simulated the sampling process by artificially generating binding sites based on observed nucleotide frequencies in a public PWM database, and then the generated matrix with an added pseudocount value was compared to the original frequency matrix using various measures. Although the results were somewhat different between measures, in many cases, we could find an optimal pseudocount value for each matrix. These optimal values are independent of the sample size and are clearly correlated with the entropy of the original matrices, meaning that larger pseudocount vales are preferable for less conserved binding sites. As a simple representative, we suggest the value of 0.8 for practical uses.

| S-EPMC2647310 | biostudies-literature

A strategy for identifying transcription factor binding sites reveals two classes of genomic c-Myc target sites.

Project description:Defining the hardwiring of transcription factors to their cognate genomic binding sites is essential for our understanding of biological processes. We used scanning chromatin immunoprecipitation to identify in vivo binding regions (E boxes) for c-Myc in three target genes as a model system. Along with other c-Myc target genes that have been validated by chromatin immunoprecipitation, we used the publicly available genomic sequences to determine whether experimentally derived in vivo binding sites might be predictable from nonexonic sequence conservation across species. Our studies revealed two classes of target genomic binding sites. Although the majority of target genes studied [class I: B23 (NPM1), CAD, CDK4, cyclin D2, ID2, LDH-A, MNT, PTMa, ODC, NM23B, nucleolin, prohibitin, SHMT1, and SHMT2] demonstrate significant sequence conservation of the E boxes and flanking regions, several genes (cyclin B1, JPO1, and PRDX3) belong to a second class (class II) that does not display sequence conservation at and around the site of c-Myc binding. On the basis of our model, we propose a strategy for predicting transcription factor binding sites using phylogenetic sequence comparisons, which will select potential class I target genes among the many emerging candidates from DNA-microarray studies for experimental validation by chromatin immunoprecipitation.

| S-EPMC154342 | biostudies-literature

Cattle Cleavage Under Targets & Release Using Nuclease (CUT&RUN) for CENP-A locus identification

Project description:CUT&RUN experiment was carried out on bovine semeimbranosous muscle satellite cells to identify the locus of the accumulation of the centromeric protein A (CENP-A) on the cattle Y-chromosome. This was carried out to support the annotation of the DNA signal comprising tandem array of a centromeric satellite signal which characterizes the centromere on the Y-chromosome.

2024-10-02 | GSE262830 | GEO

A regulatory role for repeated decoy transcription factor binding sites in target gene expression.

Project description:Tandem repeats of DNA that contain transcription factor (TF) binding sites could serve as decoys, competitively binding to TFs and affecting target gene expression. Using a synthetic system in budding yeast, we demonstrate that repeated decoy sites inhibit gene expression by sequestering a transcriptional activator and converting the graded dose-response of target promoters to a sharper, sigmoidal-like response. On the basis of both modeling and chromatin immunoprecipitation measurements, we attribute the altered response to TF binding decoy sites more tightly than promoter binding sites. Tight TF binding to arrays of contiguous repeated decoy sites only occurs when the arrays are mostly unoccupied. Finally, we show that the altered sigmoidal-like response can convert the graded response of a transcriptional positive-feedback loop to a bimodal response. Together, these results show how changing numbers of repeated TF binding sites lead to qualitative changes in behavior and raise new questions about the stability of TF/promoter binding.

| S-EPMC3322331 | biostudies-literature

Project description:BackgroundCollections of transcription factor binding profiles (Transfac, Jaspar) are essential to identify regulatory elements in DNA sequences. Subsets of highly similar profiles complicate large scale analysis of transcription factor binding sites.ResultsWe propose to identify and group similar profiles using two independent similarity measures: chi2 distances between position frequency matrices (PFMs) and correlation coefficients between position weight matrices (PWMs) scores.ConclusionWe show that these measures complement each other and allow to associate Jaspar and Transfac matrices. Clusters of highly similar matrices are identified and can be used to optimise the search for regulatory elements. Moreover, the application of the measures is illustrated by assigning E-box matrices of a SELEX experiment and of experimentally characterised binding sites of circadian clock genes to the Myc-Max cluster.

| S-EPMC1261160 | biostudies-literature

Predicting tissue specific transcription factor binding sites.

Project description:BackgroundStudies of gene regulation often utilize genome-wide predictions of transcription factor (TF) binding sites. Most existing prediction methods are based on sequence information alone, ignoring biological contexts such as developmental stages and tissue types. Experimental methods to study in vivo binding, including ChIP-chip and ChIP-seq, can only study one transcription factor in a single cell type and under a specific condition in each experiment, and therefore cannot scale to determine the full set of regulatory interactions in mammalian transcriptional regulatory networks.ResultsWe developed a new computational approach, PIPES, for predicting tissue-specific TF binding. PIPES integrates in vitro protein binding microarrays (PBMs), sequence conservation and tissue-specific epigenetic (DNase I hypersensitivity) information. We demonstrate that PIPES improves over existing methods on distinguishing between in vivo bound and unbound sequences using ChIP-seq data for 11 mouse TFs. In addition, our predictions are in good agreement with current knowledge of tissue-specific TF regulation.ConclusionsWe provide a systematic map of computationally predicted tissue-specific binding targets for 284 mouse TFs across 55 tissue/cell types. Such comprehensive resource is useful for researchers studying gene regulation.

| S-EPMC3898213 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data