Biochemical Analysis of Genome Functions Using Locus-Specific Chromatin Immunoprecipitation Technologies

Fujita, Toshitsugu; Fujii, Hodaka

doi:10.4137/grsb.s32520

Cited by 12 publications

(10 citation statements)

References 45 publications

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“…A comprehensive understanding of epigenetic signaling cascades is hindered by the lack of methods that enable a dynamic and targeted readout of epigenetic modifications in living cells at the level of endogenous loci. Affinity-based enrichment methods are frequently employed to map the genome-wide distributions of 5mC and histone modifications 8 , 9 but these procedures require cell lysis, thereby providing only a snapshot of the dynamic epigenetic landscape and obstructing information on cellular physiology. In histological sections, locus specific readout of histone marks has been addressed in a proximity ligation assay by combining antibody detection of the epigenetic mark with fluorescence in situ hybridization (FISH) for locus resolution 10 .…”

Section: Introductionmentioning

confidence: 99%

Modular fluorescence complementation sensors for live cell detection of epigenetic signals at endogenous genomic sites

et al. 2017

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Investigation of the fundamental role of epigenetic processes requires methods for the locus-specific detection of epigenetic modifications in living cells. Here, we address this urgent demand by developing four modular fluorescence complementation-based epigenetic biosensors for live-cell microscopy applications. These tools combine engineered DNA-binding proteins with domains recognizing defined epigenetic marks, both fused to non-fluorescent fragments of a fluorescent protein. The presence of the epigenetic mark at the target DNA sequence leads to the reconstitution of a functional fluorophore. With this approach, we could for the first time directly detect DNA methylation and histone 3 lysine 9 trimethylation at endogenous genomic sites in live cells and follow dynamic changes in these marks upon drug treatment, induction of epigenetic enzymes and during the cell cycle. We anticipate that this versatile technology will improve our understanding of how specific epigenetic signatures are set, erased and maintained during embryonic development or disease onset.

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Section: Introductionmentioning

confidence: 99%

Modular fluorescence complementation sensors for live cell detection of epigenetic signals at endogenous genomic sites

et al. 2017

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“…An alternative approach to detecting physical interactions between genomic regions is to purify specific genomic regions engaged in molecular interactions and then analyze the genomic DNA in the purified complexes. To purify specific genomic regions, we recently developed two locus-specific chromatin immunoprecipitation (locus-specific ChIP) technologies, insertional ChIP (iChIP) (Hoshino & Fujii 2009;Fujita & Fujii 2011, 2012a, 2014aFujita et al 2015a) (see review (Fujita & Fujii 2016)) and engineered DNA-binding molecule-mediated ChIP (enChIP) , 2014bFujita et al , 2015bFujita et al , 2016a) (see reviews ). enChIP consists of the following steps ( Fig.…”

Section: Introductionmentioning

confidence: 99%

“…To purify specific genomic regions, we recently developed two locus‐specific chromatin immunoprecipitation (locus‐specific ChIP) technologies, insertional ChIP (iChIP) (Hoshino & Fujii ; Fujita & Fujii , 2012a, ; Fujita et al . ) (see review (Fujita & Fujii )) and engineered DNA‐binding molecule‐mediated ChIP (enChIP) (Fujita & Fujii , ; Fujita et al . , , ,b) (see reviews (Fujii & Fujita ; Fujita & Fujii )).…”

Section: Introductionmentioning

confidence: 99%

Identification of physical interactions between genomic regions by enChIP‐Seq

et al. 2017

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Physical interactions between genomic regions play critical roles in the regulation of genome functions, including gene expression. Here, we show the feasibility of using engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) in combination with next-generation sequencing (NGS) (enChIP-Seq) to detect such interactions. In enChIP-Seq, the target genomic region is captured by an engineered DNA-binding complex, such as a clustered regularly interspaced short palindromic repeats (CRISPR) system consisting of a catalytically inactive form of Cas9 and a single guide RNA. Subsequently, the genomic regions that physically interact with the target genomic region in the captured complex are sequenced by NGS. Using enChIP-Seq, we found that the 5'HS5 locus, which is involved in the regulation of globin genes expression at the β-globin locus, interacts with multiple genomic regions upon erythroid differentiation in the human erythroleukemia cell line K562. Genes near the genomic regions inducibly associated with the 5'HS5 locus were transcriptionally up-regulated in the differentiated state, suggesting the existence of a coordinated transcription mechanism mediated by physical interactions between these loci. Thus, enChIP-Seq might be a potentially useful tool for detecting physical interactions between genomic regions in a nonbiased manner, which would facilitate elucidation of the molecular mechanisms underlying regulation of genome functions.

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“…Such enzymatically dead Cas9 variants continue to be delivered via gRNA to prespecified DNA addresses even when attached to additional proteins with novel functions; for example, a readily traceable fluorescent marker ligated to dCas9 can be addressed to any gRNA‐specified address such that the absolute and relative locations of genes within the nucleus can be revealed (Chen et al , ; Cheng et al , ): a mapping capacity that alone should lead to a vastly improved understanding of the intranuclear spatial relationships contributing to gene regulation. Other proteins similarly delivered to pre‐assigned DNA addresses have been used to support chromatin purification strategies (Fujita and Fujii, ) making it possible to capture and characterize the specific regulatory components controlling gene expression programs. Even more remarkably, ligating activators, repressors, or chromatin modifiers to dCas9 allows experimental control over the normal expression programming of targeted genes (Thakore et al , ; Tost, ).…”

Section: The Crispr Revolutionmentioning

confidence: 99%

CRISPR: express delivery to any DNA address

Peterson

2016

Oral Diseases

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The sudden emergence and worldwide adoption of CRISPR gene‐editing technology confronts humanity with unprecedented opportunities and choices. CRISPR's transformative impact on our future understanding of biology, along with its potential to unleash control over the most fundamental of biological processes, is predictable by already achieved applications. Although its origin, composition, and function were revealed only recently, close to 3000 CRISPR‐based publications have appeared including insightful and diversely focused reviews referenced here. Adding further to scientific and public awareness, a recent symposium addressed the ethical implications of interfacing CRISPR technology and human biology. However, the magnitude of CRISPR's rapidly emerging power mandates its broadest assessment. Only with the participation of a diverse and informed community can the most effective and humanity‐positive CRISPR applications be defined. This brief review is aimed at those with little previous exposure to the CRISPR revolution. The molecules that constitute CRISPR's core components and their functional organization are described along with how the mechanism has been harnessed to edit genome structure and modulate gene function. Additionally, a glimpse into CRISPR's potential to unleash genetic changes with far‐reaching consequences is presented.

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Biochemical Analysis of Genome Functions Using Locus-Specific Chromatin Immunoprecipitation Technologies

Cited by 12 publications

References 45 publications

Modular fluorescence complementation sensors for live cell detection of epigenetic signals at endogenous genomic sites

Modular fluorescence complementation sensors for live cell detection of epigenetic signals at endogenous genomic sites

Identification of physical interactions between genomic regions by enChIP‐Seq

CRISPR: express delivery to any DNA address

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