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Hit-and-run epigenetic editing prevents senescence entry in primary breast cells from healthy donors.

ABSTRACT: Aberrant promoter DNA hypermethylation is a hallmark of cancer; however, whether this is sufficient to drive cellular transformation is not clear. To investigate this question, we use a CRISPR-dCas9 epigenetic editing tool, where an inactive form of Cas9 is fused to DNA methyltransferase effectors. Using this system, here we show simultaneous de novo DNA methylation of genes commonly methylated in cancer, CDKN2A, RASSF1, HIC1 and PTEN in primary breast cells isolated from healthy human breast tissue. We find that promoter methylation is maintained in this system, even in the absence of the fusion construct, and this prevents cells from engaging senescence arrest. Our data show that the key driver of this phenotype is repression of CDKN2A transcript p16 where myoepithelial cells harbour cancer-like gene expression but do not exhibit anchorage-independent growth. This work demonstrates that hit-and-run epigenetic events can prevent senescence entry, which may facilitate tumour initiation.

SUBMITTER: Saunderson EA

PROVIDER: S-EPMC5684409 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Hit-and-run epigenetic editing prevents senescence entry in primary breast cells from healthy donors.

Saunderson Emily A EA Stepper Peter P Gomm Jennifer J JJ Hoa Lily L Morgan Adrienne A Allen Michael D MD Jones J Louise JL Gribben John G JG Jurkowski Tomasz P TP Ficz Gabriella G

Nature communications 20171113 1

Aberrant promoter DNA hypermethylation is a hallmark of cancer; however, whether this is sufficient to drive cellular transformation is not clear. To investigate this question, we use a CRISPR-dCas9 epigenetic editing tool, where an inactive form of Cas9 is fused to DNA methyltransferase effectors. Using this system, here we show simultaneous de novo DNA methylation of genes commonly methylated in cancer, CDKN2A, RASSF1, HIC1 and PTEN in primary breast cells isolated from healthy human breast ti ...[more]

PMID: 29133799

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Project description:Dynamic transcriptional regulation is critical for an organism's response to environmental signals and yet remains elusive to capture. Such transcriptional regulation is mediated by master transcription factors (TF) that control large gene regulatory networks. Recently, we described a dynamic mode of TF regulation named "hit-and-run". This model proposes that master TF can interact transiently with a set of targets, but the transcription of these transient targets continues after the TF dissociation from the target promoter. However, experimental evidence validating active transcription of the transient TF-targets is still lacking.Here, we show that active transcription continues after transient TF-target interactions by tracking de novo synthesis of RNAs made in response to TF nuclear import. To do this, we introduced an affinity-labeled 4-thiouracil (4tU) nucleobase to specifically isolate newly synthesized transcripts following conditional TF nuclear import. Thus, we extended the TARGET system (Transient Assay Reporting Genome-wide Effects of Transcription factors) to include 4tU-labeling and named this new technology TARGET-tU. Our proof-of-principle example is the master TF Basic Leucine Zipper 1 (bZIP1), a central integrator of metabolic signaling in plants. Using TARGET-tU, we captured newly synthesized mRNAs made in response to bZIP1 nuclear import at a time when bZIP1 is no longer detectably bound to its target. Thus, the analysis of de novo transcripomics demonstrates that bZIP1 may act as a catalyst TF to initiate a transcriptional complex ("hit"), after which active transcription by RNA polymerase continues without the TF being bound to the gene promoter ("run").Our findings provide experimental proof for active transcription of transient TF-targets supporting a "hit-and-run" mode of action. This dynamic regulatory model allows a master TF to catalytically propagate rapid and broad transcriptional responses to changes in environment. Thus, the functional read-out of de novo transcripts produced by transient TF-target interactions allowed us to capture new models for genome-wide transcriptional control.

Dataset Information

Hit-and-run epigenetic editing prevents senescence entry in primary breast cells from healthy donors.

Publications

Hit-and-run epigenetic editing prevents senescence entry in primary breast cells from healthy donors.

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