Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Summary

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

Abstract

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Introduction

Chromatin consists of DNA wrapped around histone octamer proteins that form the core nucleosome particle. Regulation of chromatin compaction is an essential mechanism for proper DNA repair, replication, and expression^1,2,3. One way in which cells control the level of compaction is through the addition or removal of various post-translational histone tail modifications. Two such modifications include (1) lysine acetylation, which is most commonly associated with gene activation, and (2) lysine methylation, which can be associated with either gene activation or repression, depe....

Protocol

1) Cell Line Culture for Producing Lentivirus

1. Grow fresh, low-passage (less than passage 30) 293T human embryonic kidney (HEK) cells in high-glucose Dulbecco's modified Eagle's medium (DMEM) base media supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, 1x non-essential amino acids (NEAA), Pen/Strep, 2-mercaptoenthanol in a 37 °C incubator with 5% CO₂. Split the cells every 3 - 5 d and before they become > 95% confluent.
2. Passage 293T HEK cells with 18 x 10⁶ per 15-cm plate (one 15-cm plate for each virus produced).
   1. For a 15-cm format, aspirate the old media, add 10 mL of 1x phosphate-buf....

Results

We recently developed CEMs and demonstrated that this technology can be applied to regulate gene expression and the chromatin environment at a reporter locus in a dose-dependent and reversible manner. In Figure 1, a model of the lead CEM, CEM23, is shown. HDAC machinery is recruited to the reporter locus by the HDAC inhibitor which, in this case, is the GFP reporter inserted at the Oct4 locus.

Discussion

Here, we described the recently developed CEM system being applied to regulate gene expression and chromatin environment at a specific gene in a dose-dependent manner. We provide an accurate method to study the dynamics involved in regulating gene expression through the selective recruitment of specific endogenous chromatin regulatory proteins. This is a highly modular technology that can be applied to investigate how different protein- and chromatin-modifying complexes work in concert to properly regulate the chromatin .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Corning	10-013CV
NEAA	Gibco	11140-050
HEPES (for tissue culture)	Corning	25-060-Cl
BME (2-Mercaptoethanol)	Gibco	21985-023
FBS	Atlantic Biologicals	S11550	Individual lots tested for quality
Covaris tubes	ThermoScientific	C4008-632R
Covaris caps	ThermoScientific	C4008-2A
PEI (polyethylenimine)	Polysciences	23966-1
Transfection media (Opti-mem)	Gibco	31985070
Virus centrifuge tubes	Beckman Coulter	344058
Virus filter membrane	Corning	431220
Polybrene	Santa Cruz Biotechnology	SC134220
0.25 % Trypsin	Gibco	25200-056	with EDTA
0.05 % Trypsin	Gibco	25400-054	with EDTA
Puromycin	InvivoGen	ant-pr
Blasticidin	InvivoGen	ant-bl
SYBR Green Master Mix (asymmetrical cyanine dye)	Roche	4913914001
H3K27ac antibody	Abcam	ab4729
(ChIP kit) ChIP-IT High Sensitivity Kit	Active Motif	53040
Sonicator	Covaris	E110
Ultracentrifuge	Optima	XPN-80
SW-32 centrifuge rotor	Beckman Coulter	14U4354
SW-32 Ti centrifuge buckets	Beckman Coulter	130.2
LentiX 293 Human Embryonic Kidney	Clontech	632180
PBS	Corning	46-013-CM
BSA	Fisher	BP9700
EGTA	Sigma	E3889
EDTA	Fisher	S311-100
NaCl	Fisher	S271-1
Glycerol	Fisher	G33-1
Tris	Fisher	BP152
NP40	Roche	11754599001
Triton	MP Biomedicals	807426
Glycine	Fisher	BP381-500
TE buffer	Fisher	BP2474-1
HEPES (for ChIP)	Fisher	BP310-100
Gelatin	Sigma	G1890
Flow cytometer, Attune NxT	Thermo Fisher	A24858
FKBP-Gal4 plasmid	Addgene	44245
psPAX2 plasmid	Addgene	12260
pMD2.G plasmid	Addgene	12259
Nanodroplets	MegaShear	N/A
DNA Nanodrop	Thermo Fisher	ND1000
Protease Inhibitors (Leupeptin)	Calbiochem/EMD	108975
Protease Inhibitors (Chymostatin)	Calbiochem/EMD	230790
Protease Inhibitors (Pepstatin)	Calbiochem/EMD	516481
CiA:Oct4 mESC	The kind gift of G. Crabtree
Lif-1C-alpha - producing Cos cells	The kind gift of J. Wysocka