Targeted intragenic demethylation initiates chromatin rewiring for gene activation

Liu, Yanjing V.; Bassal, Mahmoud A.; Lin, Quy Xiao Xuan; Wu, Chan; Kwon, Junsu; Zhou, Qiling; Tan, Hong Kee; Ebralidze, Alexander K.; Chai, Li; Benoukraf, Touati; Ruscio, Annalisa Di; Tenen, Daniel G.

doi:10.1101/2020.07.16.205922

Cited by 8 publications

(9 citation statements)

References 46 publications

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“…In order tolocalize the critical CpG region that responsible for regulation of SALL4 expression, and to test the causal relationship between DNA methylation of SALL4 RNA expression, we applied the CRISPR-DNMT1-interacting RNA (CRISPR-DiR) technique 33 , a novel approach of inducing locus-specific demethylation by blocking DNMT1 activity 34 in cell lines with no/low SALL4 expression.…”

Section: Resultsmentioning

confidence: 99%

“…In the CRISPR-DiR method, two loops of the guide RNA which are not required for guide function have been replaced with sequences which specifically interact with and inhibit DNA methyltransferase 1 (DNMT1). 33,34 This modified guide RNA can achieve site-specific demethylation. 33 We tested several site-specific guide RNAs around the major CpG island of SALL4, and only sgSALL4_1 (named as DiR_SALL4 here), targeting around the CpG 16 region, demonstrated effective demethylation ability.…”

Section: Resultsmentioning

confidence: 99%

“…33,34 This modified guide RNA can achieve site-specific demethylation. 33 We tested several site-specific guide RNAs around the major CpG island of SALL4, and only sgSALL4_1 (named as DiR_SALL4 here), targeting around the CpG 16 region, demonstrated effective demethylation ability. 32 We further monitored the methylation of the 5’UTR-Exon 1-Intron 1 CpG region in HL-60 cells (no/low SALL4 expression) following treatment with CRISPR-DiR (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Demethylation and upregulation of an oncogene post hypomethylating treatment

Liu

Fabiani

Kwon

et al. 2020

Preprint

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While hypomethylating agents (HMA) are currently used to treat myelodysplastic syndrome (MDS) patients, their effects on reactivation and/or upregulation of oncogenes have not been previously described. SALL4 is a known oncogene that plays an important role in MDS. In this study, we examined the relationship between SALL4 methylation and expression, and evaluated changes of SALL4 expression and their prognostic value in MDS patients undergoing HMA treatment. In no/low-SALL4 expressing leukemic cell lines, we identified that demethylation of a critical CpG region was associated with increased SALL4 expression, and HMA treatment led to demethylation of this region and upregulation of SALL4. In MDS patients, we observed SALL4 upregulation after four cycles of azacytidine (AZA) treatment in 40% of the cases. Significantly, patients in the responder group with SALL4 upregulation had the worst outcome. This is the first study focusing on demethylation and upregulation of an oncogene after HMA treatment. Our data indicate that MDS patients receiving HMA treatment should be monitored for upregulation of oncogenes such as SALL4 for poor outcome.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Demethylation and upregulation of an oncogene post hypomethylating treatment

Liu

Fabiani

Kwon

et al. 2020

Preprint

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“…Interestingly, using a Crispr-dCas9-based gene locus-specific demethylation system, we have observed that demethylation of this 5 0 UTR-Exon 1 region could also lead to upregulation of SALL4 in several SALL4-negative cancer cell lines, suggesting a critical role of DNA methylation in regulating SALL4 expression (39)(40)(41).…”

Section: Upregulation Of Sall4 With Decreased Dna Methylation Post Hm...mentioning

confidence: 99%

Targeting an Inducible SALL4-Mediated Cancer Vulnerability with Sequential Therapy

et al. 2021

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Oncofetal protein SALL4 is critical for cancer cell survival. Targeting SALL4, however, is only applicable in a fraction of cancer patients who are positive for this gene. To overcome this limitation, we propose to induce a cancer vulnerability by engineering a partial dependency upon SALL4. Following exogenous expression of SALL4, SALL4-negative cancer cells became partially dependent on SALL4. Treatment of SALL4-negative cells with the FDA-approved hypomethylating agent 5-aza-2′-deoxycytidine (DAC) resulted in transient upregulation of SALL4. DAC pretreatment sensitized SALL4-negative cancer cells to entinostat, which negatively affected SALL4 expression through a microRNA, miRNA-205, both in culture and in vivo. Moreover, SALL4 was essential for the efficiency of sequential treatment of DAC and entinostat. Overall, this proof-of-concept study provides a framework whereby the targeting pathways such as SALL4-centered therapy can be expanded, sensitizing cancer cells to treatment by transient target induction and engineering a dependency. Significance: These findings provide a therapeutic approach for patients harboring no suitable target by induction of a SALL4-mediated vulnerability.

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“…In this system, an RNA aptamer that directly inhibits endogenous DNA methyltransferases is grafted onto the sgRNA stem-and tetraloops. The efficiency of this strategy was recently demonstrated in mammalian cells for DNMT1 inhibition using the CRISPR-DIR system [ 43 ].…”

Section: Advances In Crispr-dcas9 Epigenome Editing In Plantsmentioning

confidence: 99%

Deciphering Plant Chromatin Regulation via CRISPR/dCas9-Based Epigenome Engineering

Dubois

Roudier

2021

Epigenomes

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CRISPR-based epigenome editing uses dCas9 as a platform to recruit transcription or chromatin regulators at chosen loci. Despite recent and ongoing advances, the full potential of these approaches to studying chromatin functions in vivo remains challenging to exploit. In this review we discuss how recent progress in plants and animals provides new routes to investigate the function of chromatin regulators and address the complexity of associated regulations that are often interconnected. While efficient transcriptional engineering methodologies have been developed and can be used as tools to alter the chromatin state of a locus, examples of direct manipulation of chromatin regulators remain scarce in plants. These reports also reveal pitfalls and limitations of epigenome engineering approaches that are nevertheless informative as they are often associated with locus- and context-dependent features, which include DNA accessibility, initial chromatin and transcriptional state or cellular dynamics. Strategies implemented in different organisms to overcome and even take advantage of these limitations are highlighted, which will further improve our ability to establish the causality and hierarchy of chromatin dynamics on genome regulation.

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Targeted intragenic demethylation initiates chromatin rewiring for gene activation

Cited by 8 publications

References 46 publications

Demethylation and upregulation of an oncogene post hypomethylating treatment

Demethylation and upregulation of an oncogene post hypomethylating treatment

Targeting an Inducible SALL4-Mediated Cancer Vulnerability with Sequential Therapy

Deciphering Plant Chromatin Regulation via CRISPR/dCas9-Based Epigenome Engineering

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