Inheritable Silencing of Endogenous Genes by Hit-and-Run Targeted Epigenetic Editing.

Authors:
Amabile A, Migliara A, Capasso P, Biffi M, Cittaro D, Naldini L, Lombardo A.
In:
Source: Cell
Publication Date: (2016)
Issue: 167(1): 219-232
Research Area:
Immunotherapy / Hematology
Gene Expression
Cells used in publication:
K-562
Species: human
Tissue Origin: blood
NIH/3T3
Species: mouse
Tissue Origin: embryo
T cell, human stim.
Species: human
Tissue Origin: blood
293T
Species: human
Tissue Origin: kidney
B-cell lymphoma cell line
Species: human
Tissue Origin:
Platform:
4D-Nucleofectorâ„¢ X-Unit
Experiment

Transfections were performed using the 4D-Nucleofector System(Lonza) and following manufacturer’s instructions for K-562, HEK-293T, NIH/3T3 and T-lymphocytes, or using the pulse programEW-113 and SF solution for B-lymphoblastoid cells.

Abstract

Gene silencing is instrumental to interrogate gene function and holds promise for therapeutic applications. Here, we repurpose the endogenous retroviruses' silencing machinery of embryonic stem cells to stably silence three highly expressed genes in somatic cells by epigenetics. This was achieved by transiently expressing combinations of engineered transcriptional repressors that bind to and synergize at the target locus to instruct repressive histone marks and de novo DNA methylation, thus ensuring long-term memory of the repressive epigenetic state. Silencing was highly specific, as shown by genome-wide analyses, sharply confined to the targeted locus without spreading to nearby genes, resistant to activation induced by cytokine stimulation, and relieved only by targeted DNA demethylation. We demonstrate the portability of this technology by multiplex gene silencing, adopting different DNA binding platforms and interrogating thousands of genomic loci in different cell types, including primary T lymphocytes. Targeted epigenome editing might have broad application in research and medicine.