Project description:Aberrant promoter DNA hypermethylation is a hallmark of cancer; however, whether this is sufficient to drive cellular transformation in the absence of genetic mutations is not clear. To investigate this question, we use a CRISPR/dCas9 based epigenetic editing tool, where an inactive form of Cas9 is fused to DNMT3A and its regulator DNMT3L. Using this system, we show simultaneous de novo DNA methylation of genes commonly methylated in cancer, CDKN2A, RASSF1, HIC1 and PTEN in primary myoepithelial 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 results in sustained repression of CDKN2A and RASSF1 transcripts which prevents cells from entering senescence. The phenotype is associated with retuned expression of a subset of genes to levels in early passage cells; however, the outgrowing myoepithelial cells are not immortal but proliferate for 25-30 population doublings before cell cycle arrest. Finally, we show that the key driver of this phenotype is repression of CDKN2A transcript p16, but prolonged proliferation is enhanced by combined hypermethylation and repression of both CDKN2A transcripts p16 and p14. This work demonstrates that hit-and-run epigenetic events can prevent senescence entry, a potential first step in the disease process.

2017-08-07 | GSE100184 | GEO

Hit-and-run epigenetic editing prevents senescence entry in primary breast cells from healthy donors [RNA-seq]

Project description:Aberrant promoter DNA hypermethylation is a hallmark of cancer; however, whether this is sufficient to drive cellular transformation in the absence of genetic mutations is not clear. To investigate this question, we use a CRISPR/dCas9 based epigenetic editing tool, where an inactive form of Cas9 is fused to DNMT3A and its regulator DNMT3L. Using this system, we show simultaneous de novo DNA methylation of genes commonly methylated in cancer, CDKN2A, RASSF1, HIC1 and PTEN in primary myoepithelial 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 results in sustained repression of CDKN2A and RASSF1 transcripts which prevents cells from entering senescence. The phenotype is associated with retuned expression of a subset of genes to levels in early passage cells; however, the outgrowing myoepithelial cells are not immortal but proliferate for 18-20 population doublings before cell cycle arrest. Finally, we show that the key driver of this phenotype is repression of CDKN2A transcript p16, but prolonged proliferation is enhanced by combined hypermethylation and repression of both CDKN2A transcripts p16 and p14. This work demonstrates that hit-and-run epigenetic events can prevent senescence entry, a potential first step in the disease process.

2017-08-07 | GSE100027 | GEO

Hit-and-run epigenetic editing prevents senescence entry in primary breast cells from healthy donors [EPIC methylation 2]

2017-08-07 | GSE100208 | GEO

Single-cell RNA sequencing of human nasal swab from healthy donors

Project description:Understanding on pathogenesis of COVID-19 is rapidly growing, but primary target cells of SARS-CoV-2 infection is still not known. Here, we performed single cell RNA sequencing on human nasal swab from healthy donors to investigate the expression patterns of host cell entry factors of SARS-CoV-2.

2021-04-06 | GSE171488 | GEO

Sorted normal bone marrow cells from healthy volunteers at Washington University

Project description:Normal healthy cells (monocytes, promyelocytes, polymorphonuclear cells, B cells, T cells, CD34+CD38- HSPCs) run as controls for TCGA AML marker publication. Bone marrow cells collected from healthy donors were sorted and DNA extracted at Washington University in St. Louis, microarrays were then run at USC

2013-08-07 | GSE49618 | GEO

Homo sapiens

Project description:Inheritable Silencing of Endogenous Genes by Hit-and-Run Targeted Epigenetic Editing

| PRJNA322643 | ENA

Durable and efficient gene silencing in vivo by hit-and-run epigenome editing

Project description:Durable and efficient gene silencing in vivo by hit-and-run epigenome editing

| PRJNA939164 | ENA