Determination of local chromatin interactions using a combined CRISPR and peroxidase APEX2 system

Wan-hua, Qiu; Xu, Zhijiao; Zhang, Min; Zhang, Dandan; Li, Taotao; Wang, Qianfeng; Liu, Peiru; Zhu, Zaihua; Du, Duo; Tan, Minjia; Wen, Bo; Liu, Yun

doi:10.1093/nar/gkz134

Cited by 38 publications

(31 citation statements)

References 74 publications

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“…Nevertheless, ingenious BioID variations (such as ChromID), continue to be designed [readers are referred to other recent reviews on proximity-based labeling (Kim and Roux, 2016;Samavarchi-Tehrani et al, 2020)]. Of particular interest are those that fuse APEX (CASPEX, C-BERST, and CAPLOCUS) or biotin ligases (CasID) to dCas9, to label proteins at a specific genomic locus, via sgRNA targeting (Schmidtmann et al, 2016;Gao et al, 2018;Myers et al, 2018;Qiu et al, 2019). In initial experiments, CASPEX was localized to the hTERT promoter and identified known interactors, such as TP53 and MAZ (Myers et al, 2018).…”

Section: Locus-specific Chromatin Analysismentioning

confidence: 99%

Interactions With Histone H3 & Tools to Study Them

Scott

Campos

2020

Front. Cell Dev. Biol.

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Histones are an integral part of chromatin and thereby influence its structure, dynamics, and functions. The effects of histone variants, posttranslational modifications, and binding proteins is therefore of great interest. From the moment that they are deposited on chromatin, nucleosomal histones undergo dynamic changes in function of the cell cycle, and as DNA is transcribed and replicated. In the process, histones are not only modified and bound by various proteins, but also shuffled, evicted, or replaced. Technologies and tools to study such dynamic events continue to evolve and better our understanding of chromatin and of histone proteins proper. Here, we provide an overview of H3.1 and H3.3 histone dynamics throughout the cell cycle, while highlighting some of the tools used to study their protein-protein interactions. We specifically discuss how histones are chaperoned, modified, and bound by various proteins at different stages of the cell cycle. Established and select emerging technologies that furthered (or have a high potential of furthering) our understanding of the dynamic histone-protein interactions are emphasized. This includes experimental tools to investigate spatiotemporal changes on chromatin, the role of histone chaperones, histone posttranslational modifications, and histone-binding effector proteins.

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Section: Locus-specific Chromatin Analysismentioning

confidence: 99%

Interactions With Histone H3 & Tools to Study Them

Scott

Campos

2020

Front. Cell Dev. Biol.

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“…These include nucleic acid hybridization-based approaches (Ide and Dejardin 2015;Déjardin and Kingston 2009;Antão et al 2012;Kennedy-Darling et al 2014) and approaches that utilize DNA-binding proteins including LexA (Byrum et al 2012;Fujita and Fujii 2011), TetR (Pourfarzad et al 2013), Cas9 (Waldrip et al 2014;Fujita and Fujii 2013;X. Liu et al 2017;Gao et al 2018;Schmidtmann et al 2016;Tsui et al 2018;Qiu et al 2019) or TALE proteins (Fujita et al 2013;Byrum, Taverna, and Tackett 2013;Fang et al 2018). Although nucleic acid hybridization-based approaches pioneered the field, they require intensive probe-specific optimizations (Ide and Dejardin 2015;Déjardin and Kingston 2009;Antão et al 2012), which may account for why this approach has not yet been widely adopted nor translated to single copy elements in mammalian cells.…”

Section: Introductionmentioning

confidence: 99%

“…To target such challenging genomic regions in the mammalian genome, TALE (Fang et al 2018) and CRISPR/Cas9 (Waldrip et al 2014;Fujita and Fujii 2013;X. Liu et al 2017;Gao et al 2018;Schmidtmann et al 2016;Tsui et al 2018;Qiu et al 2019) approaches are starting to emerge as the systems of choice as, unlike TetR and LexA proteins, they do not rely on genetic engineering of binding sites into the target sequence.…”

Section: Introductionmentioning

confidence: 99%

TINC - a method to dissect transcriptional complexes at single locus resolution - reveals novelNanogregulators in mouse embryonic stem cells

Knaupp

Mohenska

Larcombe

et al. 2020

Preprint

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Cellular identity is ultimately controlled by transcription factors (TFs), which bind to specific regulatory elements (REs) within the genome to regulate gene expression and cell fate changes. While recent advances in genome-wide epigenetic profiling techniques have significantly increased our understanding of which REs are utilized in which cell type, it remains largely unknown which TFs and cofactors interact with these REs to modulate gene expression. A major hurdle in dissecting the whole composition of a multi-protein complex formed at a specific RE is the shortage of appropriate techniques. We have developed a novel method termed TALE-mediated Isolation of Nuclear Chromatin (TINC). TINC utilizes epitope-tagged TALEs to isolate a specific genomic region from the mammalian genome and includes a nuclei isolation and chromatin enrichment step for increased specificity. Upon crosslinking of the cells and isolation of the chromatin, the target region is purified based on affinity purification of the TALE and associated nucleic acid and protein molecules can be subjected to further analyses. A key TF in the pluripotency network and therefore in embryonic stem cells (ESCs) is NANOG. It is currently not fully understood how Nanog expression is regulated and consequently it remains unclear how the ESC state is maintained. Using TINC we dissected the protein complex formed at the Nanog promoter in mouse ESCs and identified many known and numerous novel factors. Enrichment and other statistical analysesProtein classification analyses were performed with Panther ( Thomas et al. 2003), and all other ontology analyses were performed with Metascape (Zhou et al. 2019). Transcription factors were defined by collected annotation from Riken and fantom five mouse TF datasets (Carninci et al. 2005;Kanamori et al. 2004). All other epigenetic modifiers and respective metadata were obtained from the epifactors database (Medvedeva et al. 2015). The pluripotency network was obtained from ESCAPE (Embryonic Stem Cells Atlas of Pluripotency Evidence) (Xu et al. 2013(Xu et al. , 2014. Enrichment of TINC proteins within the pluripotency network was calculated with Fisher's exact test. RNA-seq data of reprogramming fibroblasts into iPSCs was obtained from Knaupp et al. 2017. Raw sequencing files were processed to raw counts as described in the original publication. The raw counts were then TMM normalized and converted to log2 CPM for all analyses. The standardized log2CPM values were centred to the mean expression for each gene. Pearson's correlation coefficients were calculated on standardized log2CPM values for all genes corresponding to proteins identified in all three TINC replicates. Protein-protein enrichment networks were visualized with Cytoscape v3.7.1 (Shannon et al. 2003). ComplexHeatmap was used for any visualization of heatmaps with default clustering parameters and all other analyses outputs were graphed using GGplot2 unless otherwise stated (Gu, Eils, and Schlesner 2016;Wickham 2016). A set seed of 123 was used in all analys...

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“…The ease-of-use and flexibility to target any genomic loci by simply changing the sequence of the single guide RNA (sgRNA) sets this technology apart from other traditionally used technologies like zinc finger nucleases (ZFNs) and transcription activatorlike effector nucleases (TALENs), which requires extensive protein engineering to target specific DNA bases [3][4][5] . The catalytically inactive mutant of Cas9, also called dead Cas9 (dCas9), has further harnessed the utility of this genome targeting tool for localized delivery of functional tags to enable transcriptional activation/repression 6,7 , base-editing 8,9 , chromatin pull-down 10,11 , and gene visualization 12 . These applications greatly rely on dCas9 derivatized with genetically encoded fusion proteins (e.g., eGFP, APOBEC, VP64) 13 and epitope tags (e.g., Flag and SunTag) [14][15][16] .…”

Section: Introductionmentioning

confidence: 99%

TUTase mediated site-directed access to clickable chromatin employing CRISPR-dCas9

George

Azhar

Aich

et al. 2019

Preprint

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Locus-specific interrogation of the genome using programmable CRISPR-based technologies is tremendously useful in dissecting the molecular basis of target gene function and modulating its downstream output. Although these tools are widely utilized in recruiting genetically encoded functional proteins, display of small molecules using this technique is not well developed due to inadequate labeling technologies. Here, we report the development of a modular technology, sgRNA-Click (sgR-CLK), which harnesses the power of bioorthogonal click chemistry for remodeling CRISPR to display synthetic molecules on target genes. A terminal uridylyl transferase (TUTase) was repurposed to construct an sgRNA containing multiple minimally invasive bioorthogonal clickable handles, which served as a Trojan horse on CRISPR-dCas9 system to guide synthetic tags site-specifically on chromatin employing copper-catalyzed or strainpromoted click reactions. Our results demonstrate that sgR-CLK could provide a simplified solution for site-directed display of small molecules to study as well as modulate the function of gene targets.

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Determination of local chromatin interactions using a combined CRISPR and peroxidase APEX2 system

Cited by 38 publications

References 74 publications

Interactions With Histone H3 & Tools to Study Them

Interactions With Histone H3 & Tools to Study Them

TINC - a method to dissect transcriptional complexes at single locus resolution - reveals novelNanogregulators in mouse embryonic stem cells

TUTase mediated site-directed access to clickable chromatin employing CRISPR-dCas9

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