An Engineered Split-TET2 Enzyme for Chemical-inducible DNA Hydroxymethylation and Epigenetic Remodeling

Summary

Detailed protocols for introducing an engineered split-TET2 enzyme (CiDER) into mammalian cells for chemical inducible DNA hydroxymethylation and epigenetic remodeling are presented.

Abstract

DNA methylation is a stable and heritable epigenetic modification in the mammalian genome and is involved in regulating gene expression to control cellular functions. The reversal of DNA methylation, or DNA demethylation, is mediated by the ten-eleven translocation (TET) protein family of dioxygenases. Although it has been widely reported that aberrant DNA methylation and demethylation are associated with developmental defects and cancer, how these epigenetic changes directly contribute to the subsequent alteration in gene expression or disease progression remains unclear, largely owing to the lack of reliable tools to accurately add or remove DNA modifications in the genome at defined temporal and spatial resolution. To overcome this hurdle, we designed a split-TET2 enzyme to enable temporal control of 5-methylcytosine (5mC) oxidation and subsequent remodeling of epigenetic states in mammalian cells by simply adding chemicals. Here, we describe methods for introducing a chemical-inducible epigenome remodeling tool (CiDER), based on an engineered split-TET2 enzyme, into mammalian cells and quantifying the chemical inducible production of 5-hydroxymethylcytosine (5hmC) with immunostaining, flow cytometry or a dot-blot assay. This chemical-inducible epigenome remodeling tool will find broad use in interrogating cellular systems without altering the genetic code, as well as in probing the epigenotype−phenotype relations in various biological systems.

Introduction

DNA methylation, mostly refers to the addition of a methyl group to the carbon 5 position of cytosine to form 5-methylcytosine (5mC), is catalyzed by DNA methyl-transferases (DNMTs). 5mC acts as a major epigenetic mark in the mammalian genome that often signals for transcriptional repression, X-chromosome inactivation and transposon silencing¹. The reversal of DNA methylation is mediated by the Ten-elven translocation (TET) protein family. TET enzymes belong to the iron (II) and 2-oxoglutarate dependent dioxygenases which catalyze the successive oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5c....

Protocol

1. Cell Culture, Plasmid Transfection and Chemical Induction

1. Culture an adherent cell line (e.g., the human embryonic kidney HEK293T cell) in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin and streptomycin at 37 °C with 5% CO₂.
2. Transfect cells with the CiDER plasmid.
   1. At least 18 h before transfection, seed 0.3 x 10⁶ cells in 2.5 mL of appropriate DMEM per well in a 6-well plate and incubate cells at 37 °C overnight to reach 60-80% confluency.
   2. Pre-warm the transfection reagent to room temperatur....

Results

Chemical inducible 5mC-to-5hmC conversion can be validated by immunostaining at single-cell level, flow cytometry analysis on transfected (mCherry-positive) cell populations, or a more quantitative dot-blot assay as illustrated in Figure 1. The catalytic domain of TET2, or TET2CD, were used throughout the study as a positive control. See references 18-20 for further details regarding the genome-wide mapping of rapamycin-induced changes in 5hmC and chromatin a.......

Discussion

Here we have illustrated the use of an engineered split-TET2 enzyme to achieve temporal control of DNA hydroxymethylation. Following the discovery of TET family of 5-methycytosine dioxygenase, many studies have been performed to decipher the biological functions of TET proteins and their major catalytic product 5hmC^1,2,3,4,5,6

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Lipofectamine 2000 Transfection Reagent	Thermo Fisher Scientific	11668027
Rapamycin	Sigma-Aldrich	37094
Paraformaldehyde, 16% Solution	VWR	100503-916
Triton X-100	Amresco	0694-1L
Hydrochloric Acid	Amresco	0369-500ML
Albumin, Bovine	Amresco	0332-100G
Tween 20	Amresco	0777-1L
5-Hydroxymethylcytosine (5-hmC) antibody (pAb)	Active Motif	39769
Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488	Thermo Fisher Scientific	A-11008
4,6-Diamidino-2-phenylindolde (DAPI)	Biotium	40043
SVAC1E SHEL LAB Economy Vacuum Oven, 0.6 Cu.Ft. (17 L)	SHEL LAB	SVAC1E
UltraPure SSC, 20X	Thermo Fisher Scientific	15557036
Bio-Dot Apparatus	BIO-RAD	1706545
Anti-5-methylcytosine (5mC) Antibody, clone 33D3	Millipore	MABE146
Anti-mouse IgG, HRP-linked Antibody	Cell Signaling Technology	7076S
Anti-rabbit IgG, HRP-linked Antibody	Cell Signaling Technology	7074S
West-Q Pico Dura ECL Solution	Gendepot	W3653-100