Chromatin Immunoprecipitation Assays for Myc and N-Myc

Abstract Cancer-associated fibroblasts (CAFs) tend to have tumor-promoting capacity, and can provide therapeutic targets. Even without cancer cells, CAF phenotypes are stably maintained, and DNA methylation and H3K27me3 changes have been shown to be involved. Here, we searched for a potential therapeutic target in primary CAFs from gastric cancer and a mechanism for its dysregulation. Expression microarray using eight CAFs and seven non-CAFs (NCAFs) revealed that serum amyloid A1 (SAA1), which encodes an acute phase secreted protein, was second most upregulated in CAFs, following IGF2. Conditioned medium (CM) derived from SAA1-overexpressing NCAFs was shown to increase migration of gastric cancer cells compared to that from control NCAFs, and its tumor-promoting effect was comparable to that of CM from CAFs. In addition, increased migration of cancer cells by CM from CAFs was mostly canceled with CM from CAFs with SAA1 knockdown. Chromatin immunoprecipitation (ChIP)-quantitative PCR showed that CAFs had higher levels of H3K27ac, an active enhancer mark, in the promoter and the two far upstream regions of SAA1 than NCAFs. Also, BET bromodomain inhibitors, JQ1 and mivebresib, decreased SAA1 expression and tumor-promoting effects in CAFs, suggesting SAA1 upregulation by enhancer activation in CAFs. Our present data showed that SAA1 is a candidate therapeutic target from gastric CAFs and indicated that increased enhancer acetylation is important for its overexpression.

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Using chromatin immunoprecipitation to monitor 1α,25-dihydroxyvitamin D3-dependent chromatin activity on the human CYP24 promoter

The Journal of Steroid Biochemistry and Molecular Biology ◽

10.1016/j.jsbmb.2004.03.075 ◽

2004 ◽

Vol 89-90 ◽

pp. 277-279 ◽

Cited By ~ 11

Author(s):

Sami Väisänen ◽

Thomas W. Dunlop ◽

Christian Frank ◽

Carsten Carlberg

Keyword(s):

Chromatin Immunoprecipitation ◽

Dihydroxyvitamin D3

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Binding of an X-Specific Condensin Correlates with a Reduction in Active Histone Modifications at Gene Regulatory Elements

Genetics ◽

10.1534/genetics.119.302254 ◽

2019 ◽

Vol 212 (3) ◽

pp. 729-742 ◽

Cited By ~ 2

Author(s):

Lena Annika Street ◽

Ana Karina Morao ◽

Lara Heermans Winterkorn ◽

Chen-Yu Jiao ◽

Sarah Elizabeth Albritton ◽

...

Keyword(s):

Gene Expression ◽

Histone Modifications ◽

Chromatin Immunoprecipitation ◽

Protein Complexes ◽

Regulatory Elements ◽

Evolutionarily Conserved ◽

Chromatin Immunoprecipitation Sequencing ◽

Gene Regulatory Elements ◽

Gene Regulatory ◽

Regulatory Sites

Condensins are evolutionarily conserved protein complexes that are required for chromosome segregation during cell division and genome organization during interphase. In Caenorhabditis elegans, a specialized condensin, which forms the core of the dosage compensation complex (DCC), binds to and represses X chromosome transcription. Here, we analyzed DCC localization and the effect of DCC depletion on histone modifications, transcription factor binding, and gene expression using chromatin immunoprecipitation sequencing and mRNA sequencing. Across the X, the DCC accumulates at accessible gene regulatory sites in active chromatin and not heterochromatin. The DCC is required for reducing the levels of activating histone modifications, including H3K4me3 and H3K27ac, but not repressive modification H3K9me3. In X-to-autosome fusion chromosomes, DCC spreading into the autosomal sequences locally reduces gene expression, thus establishing a direct link between DCC binding and repression. Together, our results indicate that DCC-mediated transcription repression is associated with a reduction in the activity of X chromosomal gene regulatory elements.

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Two Sides of the Same Coin: TFIIH Complexes in Transcription and DNA Repair

The Scientific World JOURNAL ◽

10.1100/tsw.2010.46 ◽

2010 ◽

Vol 10 ◽

pp. 633-643 ◽

Cited By ~ 12

Author(s):

Alexander Zhovmer ◽

Valentyn Oksenych ◽

Frédéric Coin

Keyword(s):

Dna Repair ◽

Western Blotting ◽

Chromatin Immunoprecipitation ◽

Transcription Initiation ◽

Dna Lesions ◽

Dynamic Composition ◽

The Core ◽

Repair Factor ◽

A New Technique ◽

Two Sides

TFIIH is organized into a seven-subunit core associated with a three-subunit Cdk-activating kinase (CAK) module. TFIIH has roles in both transcription initiation and DNA repair. During the last 15 years, several studies have been conducted to identify the composition of the TFIIH complex involved in DNA repair. Recently, a new technique combining chromatin immunoprecipitation and western blotting resolved the hidden nature of the TFIIH complex participating in DNA repair. Following the recruitment of TFIIH to the damaged site, the CAK module is released from the core TFIIH, and the core subsequently associates with DNA repair factors. The release of the CAK is specifically driven by the recruitment of the DNA repair factor XPA and is required to promote the incision/excision of the damaged DNA. Once the DNA lesions have been repaired, the CAK module returns to the core TFIIH on the chromatin, together with the release of the repair factors. These data highlight the dynamic composition of a fundamental cellular factor that adapts its subunit composition to the cell needs.

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