Chromatin Immunoprecipitation Assay as a Tool for Analyzing Transcription Factor Activity

Gade, Padmaja; Kalvakolanu, Dhan V.

doi:10.1007/978-1-61779-376-9_6

Cited by 69 publications

(38 citation statements)

References 40 publications

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“…The ChIP assay was performed using established protocols as previously described and is detailed in the Supporting Information. Primers for the real‐time PCR analysis of the interested DNA fragments in the immunoprecipitated DNA fragments are listed in Supporting Table .…”

Section: Methodsmentioning

confidence: 99%

Hepatic IRF2BP2 Mitigates Nonalcoholic Fatty Liver Disease by Directly Repressing the Transcription of ATF3

Fang

Zhang

et al. 2020

Hepatology

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Background and Aims Although knowledge regarding the pathogenesis of nonalcoholic fatty liver disease (NAFLD) has profoundly grown in recent decades, the internal restrictive mechanisms remain largely unknown. We have recently reported that the transcription repressor interferon regulatory factor‐2 binding protein 2 (IRF2BP2) is enriched in cardiomyocytes and inhibits pathological cardiac hypertrophy in mice. Notably, IRF2BP2 is abundantly expressed in hepatocytes and dramatically down‐regulated in steatotic livers, whereas the role of IRF2BP2 in NAFLD is unknown. Approach and Results Herein, using gain‐of‐function and loss‐of‐function approaches in mice, we demonstrated that while hepatocyte‐specific Irf2bp2 knockout exacerbated high‐fat diet–induced hepatic steatosis, insulin resistance and inflammation, hepatic Irf2bp2 overexpression protected mice from these metabolic disorders. Moreover, the inhibitory role of IRF2BP2 on hepatosteatosis is conserved in a human hepatic cell line in vitro. Combinational analysis of digital gene expression and chromatin immunoprecipitation sequencing identified activating transcription factor 3 (ATF3) to be negatively regulated by IRF2BP2 in NAFLD. Chromatin immunoprecipitation and luciferase assay substantiated the fact that IRF2BP2 is a bona fide transcription repressor of ATF3 gene expression via binding to its promoter region. Functional studies revealed that ATF3 knockdown significantly relieved IRF2BP2 knockout‐exaggerated hepatosteatosis in vitro. Conclusion IRF2BP2 is an integrative restrainer in controlling hepatic steatosis, insulin resistance, and inflammation in NAFLD through transcriptionally repressing ATF3 gene expression.

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Section: Methodsmentioning

confidence: 99%

Hepatic IRF2BP2 Mitigates Nonalcoholic Fatty Liver Disease by Directly Repressing the Transcription of ATF3

Fang

Zhang

et al. 2020

Hepatology

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“…Chromatin immunoprecipitation assay. Chromatin immunoprecipitation assay (ChIP) was performed as described previously with modifications (11). MLg cells were serum starved overnight and then exposed to 10 ng/ml TGF-␤1 1h.…”

Section: Methodsmentioning

confidence: 99%

TGF-β1 induces Fstl1 via the Smad3-c-Jun pathway in lung fibroblasts

Zheng

Zhang

et al. 2017

American Journal of Physiology-Lung Cellular and Molecular Phys

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Transforming growth factor (TGF)-β1 has long been regarded as a central mediator of tissue fibrosis. Follistatin-like 1 (Fstl1) is a crucial profibrotic glycoprotein that is upregulated in fibrotic lung tissues, and it promotes fibrogenesis via facilitating TGF-β signaling. Here we examined the signaling pathway by which TGF-β1 upregulates Fstl1 expression in mouse pulmonary fibroblasts. TGF-β1 regulated Fstl1 expression at both the transcriptional and translational levels. Although TGF-β1 rapidly activated the Smad, MAPK, and Akt pathways in lung fibroblasts, only Smad2/3 inhibition eliminated TGF-β1-induced Fstl1 expression. Analysis of the luciferase reporter activity identified a functional c-Jun transcription site in the promoter. Our results suggested a critical role for the Smad3-c-Jun pathway in the regulation of Fstl1 expression by TGF-β1 during fibrogenesis.

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“…However, because most genomic instances of the motif are not actually bound, having the recognition motif is insufficient. Protein-DNA interactions can be measured genome wide using chromatin immunoprecipitation followed by sequencing (ChIP-seq)[23–25]. Unfortunately, numerous lines of evidence indicate that not all binding events influence transcription[26–28].…”

Section: Introductionmentioning

confidence: 99%

Detecting differential transcription factor activity from ATAC-seq data

Tripodi

Allen

Dowell

2018

Preprint

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Transcription factors are managers of the cellular factory, and key components to many 1 diseases. Many non-coding single nucleotide polymorphisms affect transcription factors, either 2 by directly altering the protein or its functional activity at individual binding sites. Here we first 3 briefly summarize high throughput approaches to studying transcription factor activity. We then 4 demonstrate, using published chromatin accessibility data (specifically ATAC-seq), that the genome 5 wide profile of TF recognition motifs relative to regions of open chromatin can determine the key 6 transcription factor altered by a perturbation. Our method of determining which TF are altered by a 7 perturbation is simple, quick to implement and can be used when biological samples are limited. In 8 the future, we envision this method could be applied to determining which TFs show altered activity 9 in response to a wide variety of drugs and diseases. 10

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Chromatin Immunoprecipitation Assay as a Tool for Analyzing Transcription Factor Activity

Cited by 69 publications

References 40 publications

Hepatic IRF2BP2 Mitigates Nonalcoholic Fatty Liver Disease by Directly Repressing the Transcription of ATF3

Hepatic IRF2BP2 Mitigates Nonalcoholic Fatty Liver Disease by Directly Repressing the Transcription of ATF3

TGF-β1 induces Fstl1 via the Smad3-c-Jun pathway in lung fibroblasts

Detecting differential transcription factor activity from ATAC-seq data

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