Concurrent Genome and Epigenome Editing by CRISPR-Mediated Sequence Replacement

Concurrent genome and epigenome editing by CRISPR-mediated sequence replacement

BMC Biology ◽

10.1186/s12915-019-0711-z ◽

2019 ◽

Vol 17 (1) ◽

Cited By ~ 3

Author(s):

Jes Alexander ◽

Gregory M. Findlay ◽

Martin Kircher ◽

Jay Shendure

Keyword(s):

Genome Editing ◽

Cpg Island ◽

Strand Break ◽

End Joining ◽

Exogenous Dna ◽

Cpg Dinucleotides ◽

Epigenome Editing ◽

Homology Directed Repair ◽

Non Homologous End Joining

Abstract Background Recent advances in genome editing have facilitated the direct manipulation of not only the genome, but also the epigenome. Genome editing is typically performed by introducing a single CRISPR/Cas9-mediated double-strand break (DSB), followed by non-homologous end joining (NHEJ)- or homology-directed repair-mediated repair. Epigenome editing, and in particular methylation of CpG dinucleotides, can be performed using catalytically inactive Cas9 (dCas9) fused to a methyltransferase domain. However, for investigations of the role of methylation in gene silencing, studies based on dCas9-methyltransferase have limited resolution and are potentially confounded by the effects of binding of the fusion protein. As an alternative strategy for epigenome editing, we tested CRISPR/Cas9 dual cutting of the genome in the presence of in vitro methylated exogenous DNA, with the aim of driving replacement of the DNA sequence intervening the dual cuts via NHEJ. Results In a proof of concept at the HPRT1 promoter, successful replacement events with heavily methylated alleles of a CpG island resulted in functional silencing of the HPRT1 gene. Although still limited in efficiency, our study demonstrates concurrent epigenome and genome editing in a single event. Conclusions This study opens the door to investigations of the functional consequences of methylation patterns at single CpG dinucleotide resolution. Our results furthermore support the conclusion that promoter methylation is sufficient to functionally silence gene expression.

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Demethylation of somatic and testis-specific histone H2A and H2B genes in F9 embryonal carcinoma cells

Molecular and Cellular Biology ◽

10.1128/mcb.13.9.5538-5548.1993 ◽

1993 ◽

Vol 13 (9) ◽

pp. 5538-5548

Author(s):

Y C Choi ◽

C B Chae

Keyword(s):

Embryonal Carcinoma ◽

Cpg Island ◽

Cpg Islands ◽

High Density ◽

Carcinoma Cells ◽

Embryonal Carcinoma Cells ◽

Cpg Dinucleotides ◽

F9 Embryonal Carcinoma Cells ◽

Methylation Patterns

In contrast to many other genes containing a CpG island, the testis-specific H2B (TH2B) histone gene exhibits tissue-specific methylation patterns in correlation with gene activity. Characterization of the methylation patterns within a 20-kb segment containing the TH2A and TH2B genes in comparison with that in a somatic histone cluster revealed that: (i) the germ cell-specific unmethylated domain of the TH2A and TH2B genes is defined as a small region surrounding the CpG islands of the TH2A and TH2B genes and (ii) somatic histone genes are unmethylated in both liver and germ cells, like other genes containing CpG islands, whereas flanking sequences are methylated. Transfection of in vitro-methylated TH2B, somatic H2B, and mouse metallothionein I constructs into F9 embryonal carcinoma cells revealed that the CpG islands of the TH2A and TH2B genes were demethylated like those of the somatic H2A and H2B genes and the metallothionein I gene. The demethylation of those CpG islands became significantly inefficient at a high number of integrated copies and a high density of methylated CpG dinucleotides. In contrast, three sites in the somatic histone cluster, of which two sites are located in the long terminal repeat of an endogenous retrovirus-like sequence, were efficiently demethylated even at a high copy number and a high density of methylated CpG dinucleotides. These results suggest two possible mechanisms for demethylation in F9 cells and methylation of CpG islands of the TH2A and TH2B genes at the postblastula stage during embryogenesis.

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Demethylation of somatic and testis-specific histone H2A and H2B genes in F9 embryonal carcinoma cells.

Molecular and Cellular Biology ◽

10.1128/mcb.13.9.5538 ◽

1993 ◽

Vol 13 (9) ◽

pp. 5538-5548 ◽

Cited By ~ 24

Author(s):

Y C Choi ◽

C B Chae

Keyword(s):

Embryonal Carcinoma ◽

Cpg Island ◽

Cpg Islands ◽

High Density ◽

Carcinoma Cells ◽

Embryonal Carcinoma Cells ◽

Cpg Dinucleotides ◽

F9 Embryonal Carcinoma Cells ◽

Methylation Patterns

In contrast to many other genes containing a CpG island, the testis-specific H2B (TH2B) histone gene exhibits tissue-specific methylation patterns in correlation with gene activity. Characterization of the methylation patterns within a 20-kb segment containing the TH2A and TH2B genes in comparison with that in a somatic histone cluster revealed that: (i) the germ cell-specific unmethylated domain of the TH2A and TH2B genes is defined as a small region surrounding the CpG islands of the TH2A and TH2B genes and (ii) somatic histone genes are unmethylated in both liver and germ cells, like other genes containing CpG islands, whereas flanking sequences are methylated. Transfection of in vitro-methylated TH2B, somatic H2B, and mouse metallothionein I constructs into F9 embryonal carcinoma cells revealed that the CpG islands of the TH2A and TH2B genes were demethylated like those of the somatic H2A and H2B genes and the metallothionein I gene. The demethylation of those CpG islands became significantly inefficient at a high number of integrated copies and a high density of methylated CpG dinucleotides. In contrast, three sites in the somatic histone cluster, of which two sites are located in the long terminal repeat of an endogenous retrovirus-like sequence, were efficiently demethylated even at a high copy number and a high density of methylated CpG dinucleotides. These results suggest two possible mechanisms for demethylation in F9 cells and methylation of CpG islands of the TH2A and TH2B genes at the postblastula stage during embryogenesis.

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Clonal expansion and epigenetic reprogramming following deletion or amplification of mutant IDH1

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1708914114 ◽

2017 ◽

Vol 114 (40) ◽

pp. 10743-10748 ◽

Cited By ~ 54

Author(s):

Tali Mazor ◽

Charles Chesnelong ◽

Aleksandr Pankov ◽

Llewellyn E. Jalbert ◽

Chibo Hong ◽

...

Keyword(s):

Copy Number ◽

Cpg Island ◽

Clonal Expansion ◽

Low Grade ◽

Copy Number Alterations ◽

Wild Type ◽

Mutant Idh1

IDH1 mutation is the earliest genetic alteration in low-grade gliomas (LGGs), but its role in tumor recurrence is unclear. Mutant IDH1 drives overproduction of the oncometabolite d-2-hydroxyglutarate (2HG) and a CpG island (CGI) hypermethylation phenotype (G-CIMP). To investigate the role of mutant IDH1 at recurrence, we performed a longitudinal analysis of 50 IDH1 mutant LGGs. We discovered six cases with copy number alterations (CNAs) at the IDH1 locus at recurrence. Deletion or amplification of IDH1 was followed by clonal expansion and recurrence at a higher grade. Successful cultures derived from IDH1 mutant, but not IDH1 wild type, gliomas systematically deleted IDH1 in vitro and in vivo, further suggestive of selection against the heterozygous mutant state as tumors progress. Tumors and cultures with IDH1 CNA had decreased 2HG, maintenance of G-CIMP, and DNA methylation reprogramming outside CGI. Thus, while IDH1 mutation initiates gliomagenesis, in some patients mutant IDH1 and 2HG are not required for later clonal expansions.

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145 THE METHYLATION PATTERNS OF POTENTIAL DIFFERENTIALLY METHYLATED REGIONS IN BOVINE Xist, Impact, NDN, AND H19 GENES

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab145 ◽

2007 ◽

Vol 19 (1) ◽

pp. 190

Author(s):

N. T. D'Cruz ◽

K. J. Wilson ◽

M. K. Holland

Keyword(s):

Genomic Dna ◽

Bisulfite Sequencing ◽

Cpg Island ◽

Methylation Pattern ◽

Imprinted Genes ◽

Aberrant Methylation ◽

Differentially Methylated Regions ◽

Methylation Patterns ◽

The Impact

Clinical and laboratory-assisted reproductive techniques such as ICSI have recently been associated with an increased incidence of several syndromes associated with defects in genomic imprinting. Genomically imprinted genes are expressed from only one parental allele and act to regulate growth of the fetus and placenta and brain development/ function. Imprinted genes are controlled by differentially methylated regions (DMRs), whereby one parental allelle (i.e. either maternal or paternal) is epigenetically silenced via methylation. Studies conducted in vitro suggest that culture of embryos and embryo manipulations may perturb the imprinting process. In the current study, the genomic DNA methylation patterns of CpG islands within bovine H19 (27 CpGs analyzed), Impact (36 CpGs), NDN (22 CpGs), and Xist (21 CpGs) were analyzed by bisulfite sequencing. Genomic DNA from a female fibroblast cell line and sperm were chosen for analysis. Potential DMRs for the 4 genes were identified, and semi-nested PCR primers were designed surrounding those regions. Second-round PCR products (2 separate reactions) were mixed, subcloned, and sequenced (n ≥ 10). The fibroblast methylation pattern of the Xist DMR showed consistent methylation in 50% of sequenced clones, with no methylation observed in sperm. The H19 DMR in fibroblast DNA also showed consistent methylation in 25% of sequenced clones, with sperm DNA fully methylated. These results confirm previous studies showing that Xist and H19 are imprinted in cattle. Sequencing of the putative Impact DMR clones indicated no methylation in either cell type, suggesting no imprinting in cattle, tissue-specific imprinting, or that this CpG island (15 bp post ATG) is not the DMR that controls imprinted expression of the Impact gene. The NDN DMR (500 bp post ATG) in sperm was not methylated, whereas the fibroblast cells had a variable methylation pattern. This may be for the same reasons suggested for Impact, but the variability within the CpG island may also be due to in vitro culture conditions resulting in aberrant methylation. This possible culture effect is currently being confirmed through bisulfite sequencing of the gene in an adult tissue. The investigation of methylation patterns in oocytes is also underway. Together, the information gathered will be used to determine the imprinting status of several bovine genes and, in the future, whether any of these imprinted genes are responsible for the increased pregnancy loss and calf abnormalities associated with advanced reproductive technologies.

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Phospho-dependent phase separation of FMRP and CAPRIN1 recapitulates regulation of translation and deadenylation

Science ◽

10.1126/science.aax4240 ◽

2019 ◽

Vol 365 (6455) ◽

pp. 825-829 ◽

Cited By ~ 50

Author(s):

Tae Hun Kim ◽

Brian Tsang ◽

Robert M. Vernon ◽

Nahum Sonenberg ◽

Lewis E. Kay ◽

...

Keyword(s):

Phase Separation ◽

Rna Processing ◽

Intrinsically Disordered Protein ◽

Resonance Spectroscopy ◽

Specific Interactions ◽

Intrinsically Disordered ◽

Functional Consequences ◽

Translational Regulators

Membraneless organelles involved in RNA processing are biomolecular condensates assembled by phase separation. Despite the important role of intrinsically disordered protein regions (IDRs), the specific interactions underlying IDR phase separation and its functional consequences remain elusive. To address these questions, we used minimal condensates formed from the C-terminal disordered regions of two interacting translational regulators, FMRP and CAPRIN1. Nuclear magnetic resonance spectroscopy of FMRP-CAPRIN1 condensates revealed interactions involving arginine-rich and aromatic-rich regions. We found that different FMRP serine/threonine and CAPRIN1 tyrosine phosphorylation patterns control phase separation propensity with RNA, including subcompartmentalization, and tune deadenylation and translation rates in vitro. The resulting evidence for residue-specific interactions underlying co–phase separation, phosphorylation-modulated condensate architecture, and enzymatic activity within condensates has implications for how the integration of signaling pathways controls RNA processing and translation.

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p38β - MAPK11 and its role in female cancers

10.21203/rs.3.rs-107231/v1 ◽

2020 ◽

Author(s):

Periklis Katopodis ◽

Rachel Kerslake ◽

Athanasios Zikopoulos ◽

Nefeli Eirini Beri ◽

Vladimir Anikin

Keyword(s):

Cpg Island ◽

Methylation Status ◽

Cpg Islands ◽

Her2 Status ◽

Tissue Specific ◽

Cancer Data ◽

Signalling Molecules ◽

Significant Difference

Abstract Background The p38MAPK family of Mitogen Activated Protein Kinases are a group of signalling molecules involved in cell growth, survival, proliferation and differentiation. The widely studied p38α isoform is ubiquitously expressed and is implicated in a number of cancer pathologies, as are p38γ and p38δ. However, the mechanistic role of the isoform, p38β, remains fairly elusive. Recent studies suggest a possible role of p38β in both breast and endometrial cancer with research suggesting involvement in bone metastasis and cancer cell survival. Female tissue specific cancers such as breast, endometrial, uterine and ovary account for over 3,000,000 cancer related incidents annually; advancements in therapeutics and treatment however require a deeper understanding of the molecular aetiology associated with these diseases. This study provides an overview of the MAPK signalling molecule p38β (MAPK11) in female cancers using an in-silico approach. Methods A detailed gene expression and methylation analysis was performed using datasets from cBioportal, CanSar and MEXPRESS. Breast, Uterine Endometrial, Cervical, Ovarian and Uterine Carcinosarcoma TCGA cancer datasets were used and analysed.Results Data using cBioportal and CanSAR suggest that expression of p38β is lower in cancers: BRCA, UCEC, UCS, CESC and OV compared to normal tissue. Methylation data from SMART and MEXPRESS indicate significant probe level variation of CpG island methylation status of the gene MAPK11. Analysis of the genes’ two CpG islands shows that the gene was hypermethylated in the CpG1 with increased methylation seen in BRCA, CESC and UCEC cancer data sets with a slight increase of expression recorded in cancer samples. CpG2 exhibited hypomethylation with no significant difference between samples and high levels of expression. Further analysis from MEXPRESS revealed no significance between probe methylation and altered levels of expression. In addition, no difference in the expression of BRCA oestrogen/progesterone/HER2 status was seen. Conclusion This data provides an overview of the expression of p38β in female tissue specific cancers, showing a decrease in expression of the gene in BRCA, UCEC, CESC, UCS and OV, increasing the understanding of p38β MAPK expression and offering insight for future in-vitro investigation and therapeutic application.

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Downregulation of Dual-specificity phosphatase 9 promotes tumor progression and contributes to poor prognosis in colorectal cancer

10.21203/rs.2.23210/v1 ◽

2020 ◽

Author(s):

Zhaoyan Qiu ◽

Ning Liang ◽

Tao Sun ◽

Hongyuan Xue ◽

Tianyu Xie ◽

...

Keyword(s):

Colorectal Cancer ◽

Functional Role ◽

Cpg Island ◽

Functional Study ◽

Dual Specificity Phosphatase ◽

Dual Specificity ◽

Low Expression

Abstract Background Dual-specificity phosphatase 9 (DUSP9) belongs to the dual-specificity protein phosphatase subfamily. Recently, increasing attention has been paid on the role of DUSP9 in a variety of cancers. However, its functional role in tumor development is still unclear, especially in colorectal cancer (CRC). Methods The functional role of DUSP9 in inhibiting the progression of CRC was verified both in vivo and in vitro using colony formation assay, EdU incorporation assay, wound healing assay, nude mice xenograft model, and et al. RNA-seq was performed to assess the gene expression profiling in SW480 cells with DUSP9 stable knockdown and shControl cells. Bisulfite sequencing (BSE) was performed to reveal methylation status of CpG island in promoter of DUSP9. Results DUSP9 was significantly down regulated in tumor tissues compared with peritumor tissues. Moreover, low DUSP9 expression in CRC was closely associated with tumor size, depth of invasion and advanced TNM stage, indicating that DUSP9 may be involved in the progression of CRC. Kaplan–Meier survival analysis showed that the overall survival (OS) and recurrence-free survival (RFS) of patients with low expression of DUSP9 were significantly shorter than that of patients with high expression of DUSP9. Functional study revealed that DUSP9 inhibited tumor migration, invasion and metastasis both in vitro and in vivo . Mechanistically, low expression of DUSP9 in CRC was caused by the upregulation of miR-1246 and hypermethylation status of CpG island in promoter of DUSP9. Conclusion Our findings demonstrate that DUSP9 plays a critical role in the progression of CRC and therapeutic intervention to increase the expression or activity of DUSP9 may be a potential target for CRC treatment in the future.

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The Role of H3K4 Trimethylation in CpG Islands Hypermethylation in Cancer

Biomolecules ◽

10.3390/biom11020143 ◽

2021 ◽

Vol 11 (2) ◽

pp. 143

Author(s):

Giuseppe Zardo

Keyword(s):

Cpg Islands ◽

Alternative Promoters ◽

Cpg Dinucleotides ◽

H3k4 Trimethylation ◽

Mammalian Embryogenesis ◽

Intergenic Regions ◽

Methylation Patterns ◽

Unmethylated State

CpG methylation in transposons, exons, introns and intergenic regions is important for long-term silencing, silencing of parasitic sequences and alternative promoters, regulating imprinted gene expression and determining X chromosome inactivation. Promoter CpG islands, although rich in CpG dinucleotides, are unmethylated and remain so during all phases of mammalian embryogenesis and development, except in specific cases. The biological mechanisms that contribute to the maintenance of the unmethylated state of CpG islands remain elusive, but the modification of established DNA methylation patterns is a common feature in all types of tumors and is considered as an event that intrinsically, or in association with genetic lesions, feeds carcinogenesis. In this review, we focus on the latest results describing the role that the levels of H3K4 trimethylation may have in determining the aberrant hypermethylation of CpG islands in tumors.

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Role of DNA Methylation and CpG Sites in the Viral Telomerase RNA Promoter during Gallid Herpesvirus 2 Pathogenesis

Journal of Virology ◽

10.1128/jvi.01488-20 ◽

2020 ◽

Vol 94 (23) ◽

Author(s):

Srđan Pejaković ◽

André Claude Mbouombouo Mfossa ◽

Laëtitia Wiggers ◽

Ahmed Kheimar ◽

Damien Coupeau ◽

...

Keyword(s):

Dna Methylation ◽

Telomerase Activity ◽

Tumor Formation ◽

Telomerase Rna ◽

Viral Promoter ◽

Cpg Dinucleotides ◽

Cpg Sites ◽

Infected Cells ◽

Methylation Patterns

ABSTRACT Gallid herpesvirus type 2 (GaHV-2) is an oncogenic alphaherpesvirus that induces malignant T-cell lymphoma in chicken. GaHV-2 encodes a viral telomerase RNA subunit (vTR) that plays a crucial role in virus-induced tumorigenesis, enhances telomerase activity, and possesses functions independent of the telomerase complex. vTR is driven by a robust viral promoter, highly expressed in virus-infected cells, and regulated by two c-Myc response elements (c-Myc REs). The regulatory mechanisms involved in controlling vTR and other genes during viral replication and latency remain poorly understood but are crucial to understanding this oncogenic herpesvirus. Therefore, we investigated DNA methylation patterns of CpG dinucleotides found in the vTR promoter and measured the impact of methylation on telomerase activity. We demonstrated that telomerase activity was considerably increased following viral reactivation. Furthermore, CpG sites within c-Myc REs showed specific changes in methylation after in vitro reactivation and in infected animals over time. Promoter reporter assays indicated that one of the c-Myc REs is involved in regulating vTR transcription, and that methylation strongly influenced vTR promoter activity. To study the importance of the CpG sites found in c-Myc REs in virus-induced tumorigenesis, we generated recombinant virus containing mutations in CpG sites of c-Myc REs together with the revertant virus by two-step Red-mediated mutagenesis. Introduced mutations in the vTR promoter did not affect the replication properties of the recombinant viruses in vitro. In contrast, replication of the mutant virus in infected chickens was severely impaired, and tumor formation completely abrogated. Our data provides an in-depth characterization of c-Myc oncoprotein REs and the involvement of DNA methylation in transcriptional regulation of vTR. IMPORTANCE Previous studies demonstrated that telomerase RNAs possess functions that promote tumor development independent of the telomerase complex. vTR is a herpesvirus-encoded telomerase RNA subunit that plays a crucial role in virus-induced tumorigenesis and is expressed by a robust viral promoter that is highly regulated by the c-Myc oncoprotein binding to the E-boxes. Here, we demonstrated that the DNA methylation patterns in the functional c-Myc response elements of the vTR promoter change upon reactivation from latency, and that demethylation strongly increases telomerase activity in virus-infected cells. Moreover, the introduction of mutation in the CpG dinucleotides of the c-Myc binding sites resulted in decreased vTR expression and complete abrogation of tumor formation. Our study provides further confirmation of the involvement of specific DNA methylation patterns in the regulation of vTR expression and vTR importance for virus-induced tumorigenesis.

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