Published: September 20th, 2018
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.
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.
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 expression1,2,3. 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....
1) Cell Line Culture for Producing Lentivirus
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.
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 .......
The authors would like to thank the members of the Hathaway and Jin laboratories for their helpful discussions. The authors also thank Dan Crona and Ian MacDonald for their critical reading of the manuscript. This work was supported in part by Grant R01GM118653 from the U.S. National Institutes of Health (to N.A.H.); and by Grants R01GM122749, R01CA218600, and R01HD088626 from the U.S. National Institutes of Health (to J.J.). This work was also supported by a tier 3 and a student grant from the UNC Eshelman Institute for Innovation (to N.A.H and A.M.C, respectively). Additional funding from a T-32 GM007092 (to A.M.C) supported this work. Flow cytometry data was obtain....
|HEPES (for tissue culture)
|Individual lots tested for quality
|Transfection media (Opti-mem)
|Virus centrifuge tubes
|Virus filter membrane
|Santa Cruz Biotechnology
|0.25 % Trypsin
|0.05 % Trypsin
|SYBR Green Master Mix (asymmetrical cyanine dye)
|(ChIP kit) ChIP-IT High Sensitivity Kit
|SW-32 centrifuge rotor
|SW-32 Ti centrifuge buckets
|LentiX 293 Human Embryonic Kidney
|HEPES (for ChIP)
|Flow cytometer, Attune NxT
|Protease Inhibitors (Leupeptin)
|Protease Inhibitors (Chymostatin)
|Protease Inhibitors (Pepstatin)
|The kind gift of G. Crabtree
|Lif-1C-alpha - producing Cos cells
|The kind gift of J. Wysocka
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