Analysis of Protein–DNA Interaction by Chromatin Immunoprecipitation and DNA Tiling Microarray (ChIP-on-chip)

2017 ◽  
pp. 43-51
Author(s):  
Hui Gao ◽  
Chunyan Zhao
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Dna Interaction ◽  
Microarray Chip ◽  
Tiling Microarray ◽  
Dna Tiling ◽  
On Chip

scholarly journals Chromatin Immunoprecipitation–on-Chip Reveals Stress-Dependent p53 Occupancy in Primary Normal Cells but Not in Established Cell Lines

2008 ◽  
Vol 68 (23) ◽  
pp. 9671-9677 ◽  
Author(s):  
Helena Shaked ◽  
Idit Shiff ◽  
Miriam Kott-Gutkowski ◽  
Zahava Siegfried ◽  
Ygal Haupt ◽  
...  
Keyword(s):  
Cell Lines ◽  
Chromatin Immunoprecipitation ◽  
Normal Cells ◽  
Established Cell Lines ◽  
Established Cell ◽  
On Chip ◽  
Stress Dependent

Chromatin Immunoprecipitation Assays: Application of ChIP-on-Chip for Defining Dynamic Transcriptional Mechanisms in Bone Cells

2008 ◽  
pp. 165-176 ◽  
Author(s):  
Margaretha Deen ◽  
Mohammad Q. Hassan ◽  
Jitesh Pratap ◽  
Nadiya M. Teplyuk ◽  
Daniel W. Young ◽  
...  
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Bone Cells ◽  
On Chip

A chromatin immunoprecipitation (ChIP) protocol for use in whole human adipose tissue

2013 ◽  
Vol 305 (9) ◽  
pp. E1172-E1177 ◽  
Author(s):  
Yulia Haim ◽  
Tanya Tarnovscki ◽  
Dana Bashari ◽  
Assaf Rudich
Keyword(s):  
Adipose Tissue ◽  
Chromatin Immunoprecipitation ◽  
High Sensitivity ◽  
Human Adipose Tissue ◽  
Cell Types ◽  
Dna Interaction ◽  
Specific Cell ◽  
Fresh Tissue ◽  
Subcutaneous Adipose

Chromatin immunoprecipitation (ChIP) has become a central method when studying in vivo protein-DNA interactions, with the major challenge being the hope to capture “authentic” interactions. While ChIP protocols have been optimized for use with specific cell types and tissues including adipose tissue-derived cells, a working ChIP protocol addressing the challenges imposed by fresh whole human adipose tissue has not been described. Utilizing human paired omental and subcutaneous adipose tissue obtained during elective abdominal surgeries, we have carefully identified and optimized individual steps in the ChIP protocol employed directly on fresh tissue fragments. We describe a complete working protocol for using ChIP on whole adipose tissue fragments. Specific steps required adaptation of the ChIP protocol to human whole adipose tissue. In particular, a cross-linking step was performed directly on fresh small tissue fragments. Nuclei were isolated before releasing chromatin, allowing better management of fat content; a sonication protocol to obtain fragmented chromatin was optimized. We also demonstrate the high sensitivity of immunoprecipitated chromatin from adipose tissue to freezing. In conclusion, we describe the development of a ChIP protocol optimized for use in studying whole human adipose tissue, providing solutions for the unique challenges imposed by this tissue. Unraveling protein-DNA interaction in whole human adipose tissue will likely contribute to elucidating molecular pathways contributing to common human diseases such as obesity and type 2 diabetes.

scholarly journals Comparative analysis of ChIP-exo peak-callers: impact of data quality, read duplication and binding subtypes

2019 ◽  
Author(s):  
Vasudha Sharma ◽  
Sharmistha MAJUMDAR
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Binding Mode ◽  
Dna Interaction ◽  
Binding Mode Prediction ◽  
Direct Binding ◽  
Single Base Pair ◽  
Self Learning ◽  
Peak Caller ◽  
Better Than

Abstract Background: ChIP (Chromatin immunoprecipitation)-exo has emerged as an important and versatile improvement over conventional ChIP-seq as it reduces the level of noise, maps the transcription factor (TF) binding location in a very precise manner, upto single base-pair resolution, and enables binding mode prediction. Availability of numerous peak-callers for analyzing ChIP-exo reads has motivated the need to assess their performance and report which tool executes reasonably well for the task. Results: This study has focussed on comparing peak-callers that report direct binding events with those that report indirect binding events. The effect of strandedness of reads and duplication of data on the performance of peak-callers has been investigated. The number of peaks reported by each peak-caller is compared followed by a comparison of the annotated motifs present in the reported peaks. The significance of peaks is assessed based on the presence of a motif in top peaks. Indirect binding tools have been compared on the basis of their ability to identify annotated motifs and predict mode of protein-DNA interaction. Conclusion: By studying the output of the peak-callers investigated in this study, it is concluded that the tools that use self-learning algorithms, i.e. the tools that estimate all the essential parameters from the aligned reads, perform better than the algorithms which require formation of peak-pairs. The latest tools that account for indirect binding of TFs appear to be an upgrade over the available tools, as they are able to reveal valuable information about the mode of binding in addition to direct binding. Furthermore, the quality of ChIP-exo reads have important consequences on the output of data analysis.

scholarly journals Comparative analysis of ChIP-exo peak-callers: impact of data quality, read duplication and binding subtypes

2019 ◽  
Author(s):  
Vasudha Sharma ◽  
Sharmistha Majumdar
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Binding Mode ◽  
Dna Interaction ◽  
Binding Mode Prediction ◽  
Direct Binding ◽  
Single Base Pair ◽  
Self Learning ◽  
Peak Caller ◽  
Better Than

AbstractBackgroundChIP (Chromatin immunoprecipitation)-exo has emerged as an important and versatile improvement over conventional ChIP-seq as it reduces the level of noise, maps the transcription factor (TF) binding location in a very precise manner, upto single base-pair resolution, and enables binding mode prediction. Availability of numerous peak-callers for analyzing ChIP-exo reads has motivated the need to assess their performance and report which tool executes reasonably well for the task.ResultsThis study has focussed on comparing peak-callers that report direct binding events with those that report indirect binding events. The effect of strandedness of reads and duplication of data on the performance of peak-callers has been investigated. The number of peaks reported by each peak-caller is compared followed by a comparison of the annotated motifs present in the reported peaks. The significance of peaks is assessed based on the presence of a motif in top peaks. Indirect binding tools have been compared on the basis of their ability to identify annotated motifs and predict mode of protein-DNA interaction.ConclusionBy studying the output of the peak-callers investigated in this study, it is concluded that the tools that use self-learning algorithms, i.e. the tools that estimate all the essential parameters from the aligned reads, perform better than the algorithms which require formation of peak-pairs. The latest tools that account for indirect binding of TFs appear to be an upgrade over the available tools, as they are able to reveal valuable information about the mode of binding in addition to direct binding. Furthermore, the quality of ChIP-exo reads have important consequences on the output of data analysis.

scholarly journals Chromatin Immunoprecipitation (ChIP) on Chip Experiments Uncover a Widespread Distribution of NF-Y Binding CCAAT Sites Outside of Core Promoters

2005 ◽  
Vol 280 (14) ◽  
pp. 13606-13615 ◽  
Author(s):  
Anna Testa ◽  
Giacomo Donati ◽  
Pearlly Yan ◽  
Francesca Romani ◽  
Tim H.-M. Huang ◽  
...  
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Widespread Distribution ◽  
Core Promoters ◽  
On Chip

scholarly journals Deconvolution of Chromatin Immunoprecipitation-Microarray (ChIP-chip) Analysis of MBF Occupancies Reveals the Temporal Recruitment of Rep2 at the MBF Target Genes

2010 ◽  
Vol 10 (1) ◽  
pp. 130-141 ◽  
Author(s):  
Majid Eshaghi ◽  
Lei Zhu ◽  
Zhaoqing Chu ◽  
Juntao Li ◽  
Chee Seng Chan ◽  
...  
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Time Course ◽  
Target Genes ◽  
S Phase ◽  
Deconvolution Analysis ◽  
Microarray Chip ◽  
Rnap Ii ◽  
Chip Chip ◽  
Chip Analysis

ABSTRACT MBF (or DSC1) is known to regulate transcription of a set of G 1 /S-phase genes encoding proteins involved in regulation of DNA replication. Previous studies have shown that MBF binds not only the promoter of G 1 /S-phase genes, but also the constitutive genes; however, it was unclear if the MBF bindings at the G 1 /S-phase and constitutive genes were mechanistically distinguishable. Here, we report a chromatin immunoprecipitation-microarray (ChIP-chip) analysis of MBF binding in the Schizosaccharomyces pombe genome using high-resolution genome tiling microarrays. ChIP-chip analysis indicates that the majority of the MBF occupancies are located at the intragenic regions. Deconvolution analysis using Rpb1 ChIP-chip results distinguishes the Cdc10 bindings at the Rpb1-poor loci (promoters) from those at the Rpb1-rich loci (intragenic sequences). Importantly, Res1 binding at the Rpb1-poor loci, but not at the Rpb1-rich loci, is dependent on the Cdc10 function, suggesting a distinct binding mechanism. Most Cdc10 promoter bindings at the Rpb1-poor loci are associated with the G 1 /S-phase genes. While Res1 or Res2 is found at both the Cdc10 promoter and intragenic binding sites, Rep2 appears to be absent at the Cdc10 promoter binding sites but present at the intragenic sites. Time course ChIP-chip analysis demonstrates that Rep2 is temporally accumulated at the coding region of the MBF target genes, resembling the RNAP-II occupancies. Taken together, our results show that deconvolution analysis of Cdc10 occupancies refines the functional subset of genomic binding sites. We propose that the MBF activator Rep2 plays a role in mediating the cell cycle-specific transcription through the recruitment of RNAP-II to the MBF-bound G 1 /S-phase genes.

THE TRANSCRIPTION FACTOR Y-BOX BINDING PROTEIN-1 (YB-1) INDUCES EXPRESSION OF THE PIK3CA ONCOGENE LEADING TO INCREASED INVASION OFBASAL-LIKE BREAST CARCINOMA CELLS

2008 ◽  
Vol 31 (4) ◽  
pp. 3
Author(s):  
Arezoo Astanehe ◽  
Melanie Finkbeiner ◽  
Karen To ◽  
Sandra E Dunn
Keyword(s):  
Breast Cancer ◽  
Breast Carcinoma ◽  
Chromatin Immunoprecipitation ◽  
Copy Number ◽  
Pik3ca Mutation ◽  
Comparative Genomic ◽  
Over Expression ◽  
Protein Levels ◽  
A Genome ◽  
On Chip

Background: Basal-like breast carcinoma (BLBC) is the mostaggressive subtype of breast cancer. 73% of BLBC over-express YB-1, anoncogenic transcription/translation factor. PIK3CA, which codes for the p110? catalytic subunit ofphosphatidylinositol-3-kinase (PI3K), is another oncogene. The PI3K signalling pathway is fundamental in the regulation of many cellular functions and isoften deregulated in cancer. Despite its importance, the knowledge on the transcriptional regulation of PIK3CA is limited. Indeed, we have recently published the first report on the PIK3CA promoter. Methods and Results: A genome-wide chromatin immunoprecipitation on chip (ChIP-on-chip) analysis of a BLBC cell-line(SUM149) suggested binding of YB-1 to the PIK3CA promoter. This binding was verified using traditional chromatin immunoprecipitation (ChIP). Furthermore, electrophoretic mobility shift assay (EMSA) using oligonucleotides with eitherwild-type or mutated YB-1 responsive elements mapped YB-1 binding to three sites on the PIK3CA promoter. Silencing YB-1 in BLBC cell-lines (SUM149, HCC1937, andMDA-MB-231) decreased, while over-expression of YB-1 increased the PIK3CA promoter activity, transcript, and protein levels. Interestingly, array comparative genomic hybridization(aCGH) and quantitative PCR demonstrated PIK3CA copy number gains in HCC1937 andMDA-MB-231 cells. Although PIK3CA amplifications are overall uncommon (9%) in breast cancer, we demonstrated here that low level gains in PIK3CA copy number are present in 30%of primary BLBC cases. Furthermore, it has previously been demonstrated that mutations of PIK3CA are the most common genetic aberration (27%) found in breast cancer. These mutations lead to constitutive activation of p110? and are highly oncogenic. Over-expression of YB-1in MCF-7 cells, which harbour an activating PIK3CA mutation, increased PIK3CA transcript and protein levels. Furthermore, induction of PIK3CA by YB-1 leads to increased levels of urokinase plasminogenactivator (uPA) and invasion. Conclusions: Our data demonstrates that YB-1 binds to the PIK3CA promoter and induces itsexpression whether the gene is wild-type or amplified. Moreover, since YB-1induces expression of the active mutant p110?, then therapeutic inhibition of YB-1 may lead to decreased p110? and interference with the constitutively activated PI3K pathway in cancers. In addition, the YB-1/PIK3CA/uPA network provides information regarding the possible therapeutic targets for prevention of breast cancer invasion and metastasis. A.A. is supported by a Child and Family–CIHR–UBC MD/PhD Studentship Award.

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