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

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.

Download Full-text

Rank-statistics based enrichment-site prediction algorithm developed for chromatin immunoprecipitation on chip experiments

BMC Bioinformatics ◽

10.1186/1471-2105-7-434 ◽

2006 ◽

Vol 7 (1) ◽

Cited By ~ 7

Author(s):

Srinka Ghosh ◽

Heather A Hirsch ◽

Edward Sekinger ◽

Kevin Struhl ◽

Thomas R Gingeras

Keyword(s):

Chromatin Immunoprecipitation ◽

Prediction Algorithm ◽

Rank Statistics ◽

Site Prediction ◽

On Chip

Download Full-text

Assessing the Epigenetic Status of Human Telomeres

Cells ◽

10.3390/cells8091050 ◽

2019 ◽

Vol 8 (9) ◽

pp. 1050 ◽

Cited By ~ 2

Author(s):

María I. Vaquero-Sedas ◽

Miguel A. Vega-Palas

Keyword(s):

Cancer Cells ◽

Chromatin Immunoprecipitation ◽

Epigenetic Modifications ◽

Telomeric Sequences ◽

Major Interest ◽

Interstitial Telomeric Sequences ◽

Relevant Role ◽

Cancer Types ◽

On Chip ◽

Microscopy Techniques

The epigenetic modifications of human telomeres play a relevant role in telomere functions and cell proliferation. Therefore, their study is becoming an issue of major interest. These epigenetic modifications are usually analyzed by microscopy or by chromatin immunoprecipitation (ChIP). However, these analyses could be challenged by subtelomeres and/or interstitial telomeric sequences (ITSs). Whereas telomeres and subtelomeres cannot be differentiated by microscopy techniques, telomeres and ITSs might not be differentiated in ChIP analyses. In addition, ChIP analyses of telomeres should be properly controlled. Hence, studies focusing on the epigenetic features of human telomeres have to be carefully designed and interpreted. Here, we present a comprehensive discussion on how subtelomeres and ITSs might influence studies of human telomere epigenetics. We specially focus on the influence of ITSs and some experimental aspects of the ChIP technique on ChIP analyses. In addition, we propose a specific pipeline to accurately perform these studies. This pipeline is very simple and can be applied to a wide variety of cells, including cancer cells. Since the epigenetic status of telomeres could influence cancer cells proliferation, this pipeline might help design precise epigenetic treatments for specific cancer types.

Download Full-text

Principles of ChIP-seq Data Analysis Illustrated with Examples

Genomics and Computational Biology ◽

10.18547/gcb.2015.vol1.iss1.e22 ◽

2015 ◽

Vol 1 (1) ◽

pp. 22 ◽

Cited By ~ 3

Author(s):

Giovanna Ambrosini ◽

René Dreos ◽

Philipp Bucher

Keyword(s):

Data Analysis ◽

Next Generation Sequencing ◽

Chromatin Immunoprecipitation ◽

Powerful Method ◽

Associated Proteins ◽

On Chip ◽

Chromatin Profiling ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing ◽

Regulate Gene

Chromatin immunoprecipitation (ChIP) followed by highthroughput sequencing (ChIP-seq) is a powerful method to determine how transcription factors and other chromatin-associated proteins interact with DNA in order to regulate gene transcription. A single ChIPseq experiment produces large amounts of highly reproducible data. The challenge is to extract knowledge from the data by thoughtful application of appropriate bioinformatics tools. Here we present a concise introduction into ChIP-seq data analysis in the form of a tutorial based on tools developed by our group. We expose biological questions, explain methods and provide guidelines for the interpretation of the results. While this article focuses on ChIP-seq, most of the algorithms and tools we present are applicable to other chromatin profiling assays based on next generation sequencing (NGS) technology as well.

Download Full-text

Targeted discovery tools: proteomics and chromatin immunoprecipitation-on-chip

BJU International ◽

10.1111/j.1464-410x.2005.05942.x ◽

2005 ◽

Vol 96 (s2) ◽

pp. 16-22 ◽

Cited By ~ 5

Author(s):

XAVIER LE GUEZENNEC ◽

ARIE B. BRINKMAN ◽

MICHIEL VERMEULEN ◽

SERGEI G. DENISSOV ◽

CINZIA GAZZIOLA ◽

...

Keyword(s):

Chromatin Immunoprecipitation ◽

On Chip

Download Full-text

BCL6 programs lymphoma cells for survival and differentiation through distinct biochemical mechanisms

Blood ◽

10.1182/blood-2007-01-069575 ◽

2007 ◽

Vol 110 (6) ◽

pp. 2067-2074 ◽

Cited By ~ 93

Author(s):

Samir Parekh ◽

Jose M. Polo ◽

Rita Shaknovich ◽

Przemyslaw Juszczynski ◽

Paola Lev ◽

...

Keyword(s):

B Cells ◽

Germinal Center ◽

Chromatin Immunoprecipitation ◽

Target Gene ◽

Target Genes ◽

Constitutive Expression ◽

Biochemical Mechanisms ◽

On Chip ◽

Survival Effect ◽

Germinal Center B Cells

AbstractThe BCL6 transcriptional repressor is the most commonly involved oncogene in diffuse large B-cell lymphomas (DLBCLs). Constitutive expression of BCL6 mediates lymphomagenesis through aberrant proliferation, survival, and differentiation blockade. Binding of BCL6 to the SMRT/N-CoR corepressors mediates the BCL6 survival effect in DLBCL. Although the basis for differentiation blockade is unknown in DLBCL, recent data suggest that BCL6 binding to the MTA3 corepressor might be involved. We report that BCL6 and MTA3 are coexpressed in normal germinal center B cells and DLBCL. Depletion of MTA3 in DLBCL cells induced a differentiation-related BCL6 target gene (PRDM1), but not target genes involved in survival. Accordingly, MTA3 and PRDM1 expression are mutually exclusive in germinal center B cells. We performed chromatin immunoprecipitation (ChIP)–on-chip mapping of the PRDM1 locus, identifying a novel BCL6 binding site on intron 3 of the PRDM1 gene, and show that BCL6 recruits MTA3 to this site. In DLBCL cells, MTA3 depletion induced plasmacytic differentiation but did not decrease viability of DLBCL cells. However, MTA3 depletion synergized with a specific BCL6 inhibitor that blocks SMRT/N-CoR binding to decrease DLBCL viability. Taken together, these results show that BCL6 regulates distinct transcriptional programs through the SMRT/N-CoR and MTA3 corepressors, respectively, and provides a basis for combinatorial therapeutic targeting of BCL6.

Download Full-text

Gut microbiome a promising target for management of respiratory diseases

Biochemical Journal ◽

10.1042/bcj20200426 ◽

2020 ◽

Vol 477 (14) ◽

pp. 2679-2696

Author(s):

Riddhi Trivedi ◽

Kalyani Barve

Keyword(s):

Respiratory Diseases ◽

New Technology ◽

Bacterial Flora ◽

Systemic Circulation ◽

The Body ◽

Lung Pathology ◽

Promising Target ◽

Current Therapies ◽

Intestinal Microbial Flora ◽

On Chip

The intestinal microbial flora has risen to be one of the important etiological factors in the development of diseases like colorectal cancer, obesity, diabetes, inflammatory bowel disease, anxiety and Parkinson's. The emergence of the association between bacterial flora and lungs led to the discovery of the gut–lung axis. Dysbiosis of several species of colonic bacteria such as Firmicutes and Bacteroidetes and transfer of these bacteria from gut to lungs via lymphatic and systemic circulation are associated with several respiratory diseases such as lung cancer, asthma, tuberculosis, cystic fibrosis, etc. Current therapies for dysbiosis include use of probiotics, prebiotics and synbiotics to restore the balance between various species of beneficial bacteria. Various approaches like nanotechnology and microencapsulation have been explored to increase the permeability and viability of probiotics in the body. The need of the day is comprehensive study of mechanisms behind dysbiosis, translocation of microbiota from gut to lung through various channels and new technology for evaluating treatment to correct this dysbiosis which in turn can be used to manage various respiratory diseases. Microfluidics and organ on chip model are emerging technologies that can satisfy these needs. This review gives an overview of colonic commensals in lung pathology and novel systems that help in alleviating symptoms of lung diseases. We have also hypothesized new models to help in understanding bacterial pathways involved in the gut–lung axis as well as act as a futuristic approach in finding treatment of respiratory diseases caused by dysbiosis.

Download Full-text