CRISPR Cas9-guided chromatin immunoprecipitation identifies miR483 as an epigenetic modulator of<i>IGF2</i>imprinting in tumors

Zhang, Yi Qun; Hu, Ji-Fan; Wang, Hong; Cui, Jiuwei; Gao, Sujun; Hoffman, Andrew R.; Li, Wei

doi:10.18632/oncotarget.10918

Cited by 23 publications

(18 citation statements)

References 65 publications

(87 reference statements)

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“…Furthermore, microRNA may also play a role in IGF2 imprinting. The oncogenic miR‐483 upregulates IGF2 expression by binding the promoter P2, hereby reducing in histone H3K27 methylation and decreasing chromatin binding of CTCF and SUZ12 (Zhang et al ., ). In prostate carcinoma samples, we found that IGF2 expression can be best explained by differential IGF2 promoter usage.…”

Section: Discussionmentioning

confidence: 97%

Insulin‐like growth factor 2 expression in prostate cancer is regulated by promoter‐specific methylation

et al. 2018

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Deregulation of the insulin‐like growth factor (IGF) axis and dysbalance of components of the IGF system as potential therapeutic targets have been described in different tumor types. IGF2 is a major embryonic growth factor and an important activator of IGF signaling. It is regulated by imprinting in a development‐ and tissue‐dependent manner and has been implicated in a broad range of malignancies including prostate cancer (PCa). Loss of imprinting (LOI) usually results in bi‐allelic gene expression and increased levels of IGF2. However, the regulatory mechanisms and the pathophysiological impact of altered IGF2 expression in PCa remain elusive. Here, we show that in contrast to many other tumors, IGF2 mRNA and protein levels were decreased in 80% of PCa in comparison with non‐neoplastic adjacent prostate and were independent of LOI status. Instead, IGF2 expression in both tumors and adjacent prostate depended on preferential usage of the IGF2 promoters P3 and P4. Decreased IGF2 expression in tumors was strongly related to hypermethylation of these two promoters. Methylation of the A region in promoter P4 correlated specifically with IGF2 expression in the 20% of PCa where IGF2 was higher in tumors than in adjacent prostate. We conclude that IGF2 is downregulated in most PCa and may be particularly relevant during early stages of tumor development or during chemotherapy and androgen deprivation. PCa differs from other tumors in that IGF2 expression is mainly regulated through methylation of promoter‐specific and not by imprinting. Targeting of promoter‐specific regions may have relevance for the adjuvant treatment of PCa.

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Section: Discussionmentioning

confidence: 97%

Insulin‐like growth factor 2 expression in prostate cancer is regulated by promoter‐specific methylation

et al. 2018

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“…We constructed the Cas9-Sox2 gRNA vector by cloning two Sox2 promoter gRNAs into the lenti Cas9-IGF2 gRNA vector that contains the catalytically inactive Cas9 (dCAs9) (Zhang et al 2017). The pU6-gRNA1-pH1-gRNA2 cassette was synthesized by joining the H1 promoter with two oligonucleotides that contain the guiding RNA (gRNA) from the Sox2 promoter, including Sox2-gRNA1: 5 ′ -GGGGTTGAGGACACGTGCTG-3 ′ and Sox2-gRNA2: 5 ′ -GAGCCAATATTCCGTAGCAT-3 ′ , respectively (Supplemental Fig.…”

Section: Mapping Of the Promoter Lncrna Interacting Network By Crist mentioning

confidence: 99%

“…S2; Supplemental Table S3). The expression cassette was inserted downstream from the U6 promoter in the vector using PmeI and NotI (Zhang et al 2017). The Cas9 control vectors were constructed by replacing target gRNAs with two scrambled guiding RNAs: gCT1: 5 ′ -GTTCCCTGCAAGAGTGCCCA-3 ′ and gCT2: 5 ′ -GCACTACCAGAGCTAACTCA -3 ′ ).…”

Section: Mapping Of the Promoter Lncrna Interacting Network By Crist mentioning

confidence: 99%

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Profiling the long noncoding RNA interaction network in the regulatory elements of target genes by chromatin in situ reverse transcription sequencing

et al. 2019

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Long noncoding RNAs (lncRNAs) can regulate the activity of target genes by participating in the organization of chromatin architecture. We have devised a "chromatin-RNA in situ reverse transcription sequencing" (CRIST-seq) approach to profile the lncRNA interaction network in gene regulatory elements by combining the simplicity of RNA biotin labeling with the specificity of the CRISPR/Cas9 system. Using gene-specific gRNAs, we describe a pluripotency-specific lncRNA interacting network in the promoters of Sox2 and Pou5f1, two critical stem cell factors that are required for the maintenance of pluripotency. The promoter-interacting lncRNAs were specifically activated during reprogramming into pluripotency. Knockdown of these lncRNAs caused the stem cells to exit from pluripotency. In contrast, overexpression of the pluripotency-associated lncRNA activated the promoters of core stem cell factor genes and enhanced fibroblast reprogramming into pluripotency. These CRIST-seq data suggest that the Sox2 and Pou5f1 promoters are organized within a unique lncRNA interaction network that determines the fate of pluripotency during reprogramming. This CRIST approach may be broadly used to map lncRNA interaction networks at target loci across the genome.

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“…Even more scalable, as it does not require genetic editing, is the use of sgRNAs to target dCas9 to a locus of interest, and then performing ChIP experiments to dCas9 itself or a protein-tagged dCas9 (Fujii and Fujita, 2015). This approach enables profiling of all proteins interacting at single genomic locus, and has already been used to characterize site-specific proteomic binding patterns by combining dCas9-ChIP with by mass spectrometry (Zhang et al, 2016). The incorporation of selective isotopic labeling of amino acids in cell culture (SILAC) with dCas9-ChIP would further allow investigation of protein turnover rates, at any locus, and over defined periods of time (Fujita and Fujii, 2014).…”

Section: Future Directionsmentioning

confidence: 99%

Application of CRISPR/Cas9 to the study of brain development and neuropsychiatric disease

Powell

Gregory

Akbarian

et al. 2017

Molecular and Cellular Neuroscience

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CRISPR/Cas9 technology has transformed our abilities to manipulate the genome and epigenome, as applications have expanded from efficient genomic editing to include the targeted localization of effectors to specific genomic loci. By facilitating the manipulation of DNA- and histone-modifying enzyme activities, activation or repression of gene expression, and targeting of transcriptional regulators to defined loci, it is now possible to directly probe the role of gene-regulatory and epigenetic pathways in order to examine their roles in basic biology and disease processes. Here we discuss these emerging CRISPR-based methodologies, with specific consideration of neurobiological applications using human induced pluripotent stem cell (hiPSC)-based models.

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CRISPR Cas9-guided chromatin immunoprecipitation identifies miR483 as an epigenetic modulator ofIGF2imprinting in tumors

Cited by 23 publications

References 65 publications

Insulin‐like growth factor 2 expression in prostate cancer is regulated by promoter‐specific methylation

Insulin‐like growth factor 2 expression in prostate cancer is regulated by promoter‐specific methylation

Profiling the long noncoding RNA interaction network in the regulatory elements of target genes by chromatin in situ reverse transcription sequencing

Application of CRISPR/Cas9 to the study of brain development and neuropsychiatric disease

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