scholarly journals Genome-wide identification of DNA–protein interactions using chromatin immunoprecipitation coupled with flow cell sequencing

2009 ◽  
Vol 201 (1) ◽  
pp. 1-13 ◽  
Author(s):  
Brad G Hoffman ◽  
Steven J M Jones
Keyword(s):  
Protein Interactions ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Cell Types ◽  
Flow Cell ◽  
Transcriptional Networks ◽  
Genome Wide ◽  
Cellular Development ◽  
And Function ◽  
Dna Protein Interactions

The transcriptional networks underlying mammalian cell development and function are largely unknown. The recently described use of flow cell sequencing devices in combination with chromatin immunoprecipitation (ChIP-seq) stands to revolutionize the identification of DNA–protein interactions. As such, ChIP-seq is rapidly becoming the method of choice for the genome-wide localization of histone modifications and transcription factor binding sites. As further studies are performed, the information generated by ChIP-seq is expected to allow the development of a framework for networks describing the transcriptional regulation of cellular development and function. However, to date, this technology has been applied only to a small number of cell types, and even fewer tissues, suggesting a huge potential for novel discovery in this field.

scholarly journals Genome-wide identification of protein binding sites in mammalian cells

2021 ◽  
Author(s):  
Fenglin Liu ◽  
Tianyu Ma ◽  
Yu-Xiang Zhang
Keyword(s):  
Transcription Factors ◽  
Protein Binding ◽  
Protein Interactions ◽  
Binding Sites ◽  
Mammalian Cells ◽  
Protein Binding Sites ◽  
Genome Wide ◽  
Transcription Binding Sites ◽  
Dna Protein Interactions

AbstractWe present GWPBS-Cap, a method to capture genome-wide protein binding sites (PBSs) without using antibodies. Using this technique, we identified many protein binding sites with different binding strengths between proteins and DNA. The PBSs can be useful to predict transcription binding sites and the co-localization of multiple transcription factors in the genome. The results also revealed that active promoters contained more protein binding sites with lower NaCl tolerances. Taken together, GWPBS-Cap can be used to efficiently identify protein binding sites and reveal genome-wide landscape of DNA-protein interactions.

scholarly journals AKT Alters Genome-Wide Estrogen Receptor α Binding and Impacts Estrogen Signaling in Breast Cancer

2008 ◽  
Vol 28 (24) ◽  
pp. 7487-7503 ◽  
Author(s):  
Poornima Bhat-Nakshatri ◽  
Guohua Wang ◽  
Hitesh Appaiah ◽  
Nikhil Luktuke ◽  
Jason S. Carroll ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Estrogen Receptor ◽  
Binding Sites ◽  
Transforming Growth Factor ◽  
Cell Types ◽  
Estrogen Receptor Α ◽  
Estrogen Signaling ◽  
Primary Tumors ◽  
Genome Wide ◽  
Extracellular Signal

ABSTRACT Estrogen regulates several biological processes through estrogen receptor α (ERα) and ERβ. ERα-estrogen signaling is additionally controlled by extracellular signal activated kinases such as AKT. In this study, we analyzed the effect of AKT on genome-wide ERα binding in MCF-7 breast cancer cells. Parental and AKT-overexpressing cells displayed 4,349 and 4,359 ERα binding sites, respectively, with ∼60% overlap. In both cell types, ∼40% of estrogen-regulated genes associate with ERα binding sites; a similar percentage of estrogen-regulated genes are differentially expressed in two cell types. Based on pathway analysis, these differentially estrogen-regulated genes are linked to transforming growth factor β (TGF-β), NF-κB, and E2F pathways. Consistent with this, the two cell types responded differently to TGF-β treatment: parental cells, but not AKT-overexpressing cells, required estrogen to overcome growth inhibition. Combining the ERα DNA-binding pattern with gene expression data from primary tumors revealed specific effects of AKT on ERα binding and estrogen-regulated expression of genes that define prognostic subgroups and tamoxifen sensitivity of ERα-positive breast cancer. These results suggest a unique role of AKT in modulating estrogen signaling in ERα-positive breast cancers and highlights how extracellular signal activated kinases can change the landscape of transcription factor binding to the genome.

scholarly journals Rapid and Low-Input Profiling of Histone Marks in Plants Using Nucleus CUT&Tag

2021 ◽  
Vol 12 ◽  
Author(s):  
Weizhi Ouyang ◽  
Xiwen Zhang ◽  
Yong Peng ◽  
Qing Zhang ◽  
Zhilin Cao ◽  
...  
Keyword(s):  
Histone Modifications ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Protein A ◽  
Transcriptional Factor ◽  
Experimental Procedure ◽  
Histone Marks ◽  
Genome Wide ◽  
Efficient Alternative ◽  
Tn5 Transposase

Characterizing genome-wide histone posttranscriptional modifications and transcriptional factor occupancy is crucial for deciphering their biological functions. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a powerful method for genome-wide profiling of histone modifications and transcriptional factor-binding sites. However, the current ChIP-seq experimental procedure in plants requires significant material and several days for completion. CUT&Tag is an alternative method of ChIP-seq for low-sample and single-cell epigenomic profiling using protein A-Tn5 transposase fusion proteins (PAT). In this study, we developed a nucleus CUT&Tag (nCUT&Tag) protocol based on the live-cell CUT&Tag technology. Our results indicate that nCUT&Tag could be used for histone modifications profiling in both monocot rice and dicot rapeseed using crosslinked or fresh tissues. In addition, both active and repressive histone marks such as H3K4me3 and H3K9me2 can be identified using our nCUT&Tag. More importantly, all the steps in nCUT&Tag can be finished in only 1 day, and the assay can be performed with as little as 0.01 g of plant tissue as starting materials. Therefore, our results demonstrate that nCUT&Tag is an efficient alternative strategy for plant epigenomic studies.

DNA-PROTEIN INTERACTIONS: INTEGRATING STRUCTURE, SEQUENCE, AND FUNCTION – AN INTRODUCTION

2006 ◽  
Author(s):  
MARTHA L. BULYK ◽  
ALEXANDER J. HARTEMINK ◽  
ERNEST FRAENKEL ◽  
GARY STORMO
Keyword(s):  
Protein Interactions ◽  
And Function ◽  
Dna Protein Interactions

scholarly journals The story of the fibrin(ogen) αC-domains: evolution of our view on their structure and interactions

2021 ◽  
Author(s):  
Leonid Medved ◽  
John W Weisel
Keyword(s):  
Binding Sites ◽  
Current Knowledge ◽  
Optical Trap ◽  
Cell Types ◽  
Ordered Structures ◽  
Multiple Binding Sites ◽  
New Information ◽  
Multiple Binding ◽  
And Function ◽  
Fibrinogen Molecule

Although much has been established concerning the overall structure and function of fibrinogen, much less has been known about its two αC regions, each consisting of an αC-connector and αC-domain, but new information has been accumulating. This review summarizes the state of our current knowledge of the structure and interactions of fibrinogen’s αC regions. A series of studies with isolated αC regions and their fragments demonstrated that the αC-domain forms compact ordered structures consisting of N- and C-terminal sub-domains including β sheets and suggested that the αC-connector has a poly(L-proline) type II structure. Functionally, the αC-domains interact intramolecularly with each other and with the central region of the molecule, first demonstrated by electron microscopy and then quantified by optical trap force spectroscopy. Upon conversion of fibrinogen into fibrin, the αC-domains switch from intra- to intermolecular interactions to form ordered αC polymers. The formation of αC polymers occurs mainly through the homophilic interaction between the N-terminal sub-domains; interaction between the C-terminal sub-domains and the αC-connectors also contributes to this process. Considerable evidence supports the idea that the αC-regions accelerate fibrin polymerization and affect the final structure of fibrin clots. The interactions between αC-regions are important for the mechanical properties of clots, increasing their stiffness and extensibility. Conversion of fibrinogen into fibrin results in exposure of multiple binding sites in its αC regions, providing interaction of fibrin with different proteins and cell types during hemostasis and wound healing. This heretofore mysterious part of the fibrinogen molecule is finally giving up its secrets.

scholarly journals RedChIP identifies noncoding RNAs associated with genomic sites occupied by Polycomb and CTCF proteins

2021 ◽  
Vol 119 (1) ◽  
pp. e2116222119
Author(s):  
Alexey A. Gavrilov ◽  
Rinat I. Sultanov ◽  
Mikhail D. Magnitov ◽  
Aleksandra A. Galitsyna ◽  
Erdem B. Dashinimaev ◽  
...  
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Wide Spectrum ◽  
Noncoding Rnas ◽  
Ctcf Binding ◽  
Polycomb Repressive Complex 2 ◽  
Proximity Ligation ◽  
Chromatin Interactions ◽  
Genome Wide ◽  
Architectural Protein

Nuclear noncoding RNAs (ncRNAs) are key regulators of gene expression and chromatin organization. The progress in studying nuclear ncRNAs depends on the ability to identify the genome-wide spectrum of contacts of ncRNAs with chromatin. To address this question, a panel of RNA–DNA proximity ligation techniques has been developed. However, neither of these techniques examines proteins involved in RNA–chromatin interactions. Here, we introduce RedChIP, a technique combining RNA–DNA proximity ligation and chromatin immunoprecipitation for identifying RNA–chromatin interactions mediated by a particular protein. Using antibodies against architectural protein CTCF and the EZH2 subunit of the Polycomb repressive complex 2, we identify a spectrum of cis- and trans-acting ncRNAs enriched at Polycomb- and CTCF-binding sites in human cells, which may be involved in Polycomb-mediated gene repression and CTCF-dependent chromatin looping. By providing a protein-centric view of RNA–DNA interactions, RedChIP represents an important tool for studies of nuclear ncRNAs.

scholarly journals The epigenetic landscape in purified myonuclei from fast and slow muscles

2021 ◽  
Author(s):  
Mads Bengtsen ◽  
Ivan Myhre Winje ◽  
Einar Eftestøl ◽  
Johannes Landskron ◽  
Chengyi Sun ◽  
...  
Keyword(s):  
Binding Sites ◽  
Association Studies ◽  
Cell Types ◽  
Specific Marker ◽  
Fiber Types ◽  
Epigenetic Landscape ◽  
Super Enhancer ◽  
Genome Wide ◽  
A Genome ◽  
Pericentriolar Material

AbstractMuscle cells have different phenotypes adapted to different usage and can be grossly divided into fast/glycolytic and slow/oxidative types. While most muscles contain a mixture of such fiber types, we aimed at providing a genome-wide analysis of chromatin environment by ChIP-Seq in two muscle extremes, the almost completely fast/glycolytic extensor digitorum longus (EDL) and slow/oxidative soleus muscles. Muscle is a heterogeneous tissue where less than 60% of the nuclei are inside muscle fibers. Since cellular homogeneity is critical in epigenome-wide association studies we devised a new method for purifying skeletal muscle nuclei from whole tissue based on the nuclear envelope protein Pericentriolar material 1 (PCM1) being a specific marker for myonuclei. Using antibody labeling and a magnetic-assisted sorting approach we were able to sort out myonuclei with 95% purity. The sorting eliminated influence from other cell types in the tissue and improved the myo-specific signal. A genome-wide comparison of the epigenetic landscape in EDL and soleus reflected the functional properties of the two muscles each with a distinct regulatory program involving distal enhancers, including a glycolytic super-enhancer in the EDL. The two muscles are also regulated by different sets of transcription factors; e.g. in soleus binding sites for MEF2C, NFATC2 and PPARA were enriched, while in EDL MYOD1 and SOX1 binding sites were found to be overrepresented. In addition, novel factors for muscle regulation such as MAF, ZFX and ZBTB14 were identified.

scholarly journals AIControl: Replacing matched control experiments with machine learning improves ChIP-seq peak identification

2018 ◽  
Author(s):  
Nao Hiranuma ◽  
Scott M. Lundberg ◽  
Su-In Lee
Keyword(s):  
Protein Interactions ◽  
Binding Sites ◽  
Current Practice ◽  
Matched Control ◽  
Cell Types ◽  
Data Driven ◽  
Background Signal ◽  
Control Dataset ◽  
The Cost ◽  
Fine Resolution

AbstractChIP-seq is a technique to determine binding locations of transcription factors, which remains a central challenge in molecular biology. Current practice is to use a “control” dataset to remove background signals from a immunoprecipitation (IP) target dataset. We introduce the AlControl framework, which eliminates the need to obtain a control dataset and instead identifies binding peaks by estimating the distributions of background signals from many publicly available control ChIP-seq datasets. We thereby avoid the cost of running control experiments while simultaneously increasing the accuracy of binding location identification. Specifically, AIControl can (1) estimate background signals at fine resolution, (2) systematically weigh the most appropriate control datasets in a data-driven way, (3) capture sources of potential biases that may be missed by one control dataset, and (4) remove the need for costly and time-consuming control experiments. We applied AIControl to 410 IP datasets in the ENCODE ChIP-seq database, using 440 control datasets from 107 cell types to impute background signal. Without using matched control datasets, AIControl identified peaks that were more enriched for putative binding sites than those identified by other popular peak callers that used a matched control dataset. We also demonstrated that our framework identifies binding sites that recover documented protein interactions more accurately.

scholarly journals QstR-dependent regulation of natural competence and type VI secretion in Vibrio cholerae

2018 ◽  
Author(s):  
Milena Jaskólska ◽  
Sandrine Stutzmann ◽  
Candice Stoudmann ◽  
Melanie Blokesch
Keyword(s):  
Vibrio Cholerae ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Dna Binding Protein ◽  
Dependent Manner ◽  
Regulatory Pathway ◽  
Type Vi Secretion System ◽  
Natural Competence ◽  
Type Vi Secretion ◽  
And Function

AbstractDuring growth on chitinous surfaces in its natural aquatic environment Vibrio cholerae develops natural competence for transformation and kills neighboring non-immune bacteria using a type VI secretion system (T6SS). Activation of these two phenotypes requires the chitin-induced regulator TfoX, but also integrates signals from quorum sensing via the intermediate regulator QstR, which belongs to the LuxR-type family of regulators. Here, we define the QstR regulon using RNA sequencing. Moreover, by mapping QstR binding sites using chromatin immunoprecipitation coupled with deep sequencing we demonstrate that QstR is likely a dual transcription factor that binds upstream of the up- and down-regulated genes. Like other LuxR-type family transcriptional regulators we show that QstR function is dependent on dimerization. However, in contrast to the well-studied LuxR-type biofilm regulator VpsT of V. cholerae, which requires the second messenger c-di-GMP, we show that QstR dimerization and function is c-di-GMP independent. Surprisingly, although ComEA, which is a periplasmic DNA-binding protein essential for transformation, is produced in a QstR-dependent manner, QstR-binding was not detected upstream of comEA suggesting the existence of a further regulatory pathway. Overall these results provide detailed insights into the function of a key regulator of natural competence and type VI secretion in V. cholerae.

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