Name: repressing gene transcription by redirecting cellular machinery with chemical epigenetic modifiers
Uploaded: 2018-09-20T14:00:00
Description: Watch this Scientific Journal Video about Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers at JoVE.com

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 obtained at the UNC Flow Cytometry Core Facility funded by a P30 CA016086 Cancer Center Core Support Grant to the UNC Lineberger Comprehensive Cancer Center.

1) Cell Line Culture for Producing Lentivirus
<ol>
	<li>Grow fresh, low-passage (less than passage 30) 293T human embryonic kidney (HEK) cells in high-glucose Dulbecco&#39;s modified Eagle&#39;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 &#176;C incubator with 5% CO2. Split the cells every 3 - 5 d and before they become &#62; 95% confluent.</li>
	<li>Passage 293T HEK cells with 18 x 106<...

<ol>
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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 ...

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, depending on the amino acid context. The addition of the acetylation and methylation marks is catalyzed by histone acetyltransferases (HATs) and histone methyltransferases (HMTs), respectively, whereas the removal of the mark is done by histone deacetylases (HDACs) and histone demethylases (HDMs), respectively4,5. Although the existence of these proteins has been known for decades, many mechanisms of how chromatin-modifying machinery works to properly regulate gene expression remain to be defined. Since chromatin regulatory processes are dysregulated in many human diseases, new mechanistic insights could lead to future therapeutic applications.
The chromatin in vivo assay (CiA) is a recently described technique that uses a chemical inducer of proximity (CIP) to control chromatin-modifying machinery recruitment to a specific locus6. This technology has been used to study an expanding list of chromatin dynamics, including histone-modifying proteins, chromatin remodelers, and transcription factors6,7,8,9. CIP-based chromatin tethering of exogenously expressed proteins has also been extended past CiA to modulate non-modified genetic loci by use with a deactivated Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein (dCas9)10,11. In the CiA system, mESCs have been modified to express a GFP reporter gene at the Oct4 locus, a highly expressed and strictly regulated area of the mESC genome. Previously, this system has been applied to describe the dose-dependent and reversible recruitment of exogenously expressed heterochromatin protein 1 (HP1), an enzyme that binds H3K9me3 and propagates repressive marks (e.g., H3K9me3) and DNA methylation6. To accomplish this type of recruitment strategy, the mESCs are infected with a plasmid that expresses an FK506-binding protein (FKBP) linked to a Gal4, which is a DNA-protein anchor that binds to a Gal4-binding array upstream of the GFP reporter. The cells are also infected with an FKBP-rapamycin-binding domain (FRB) connected to HP1. When the mESCs are exposed to low nanomolar concentrations of the CIP, rapamycin, both FRB and FKBP, are brought together at the target locus. Within days of rapamycin treatment, expression of the target gene is repressed, as evidenced by fluorescence microscopy and flow cytometry, and histone acetylation is decreased, shown by ChIP and bisulfite sequencing. While the development and validation of this approach have been significant advances in the field of chromatin research and regulation, one drawback is the required exogenous expression of chromatin-modifying machinery.
To make the technology based on recruitment of only endogenous physiologically-relevant enzymes and make a more modular system, we designed and characterized novel bifunctional molecules, termed CEMs (Figure 1)12. One component of the CEMs includes FK506 which, similar to rapamycin, binds tightly and specifically to FKBP. Thus, the CEMs will still be recruited to the Oct4 locus in the CiA mESCs. The other component of the CEMs is a moiety that binds endogenous chromatin-modifying machinery. In a pilot study, we tested CEMs that contain HDAC inhibitors. While the concept of using an inhibitor to recruit HDACs seems counter-intuitive, the inhibitor is nonetheless able to recruit HDAC activity to the gene of interest. This is accomplished by (1) redirecting the HDAC to the locus, releasing the enzyme, and increasing the density of un-inhibited HDACs in the area and (2) maintaining HDAC inhibition at the locus, but recruiting repressive complexes that bind to the inhibited HDACs, or (3) a combination of both. In a previous study, we showed that the CEMs were able to successfully repress the GFP reporter in a dose- and time-dependent manner, as well as in a manner that was rapidly reversible (i.e., within 24 hours)12. We characterized the ability of the CEM technology presented here to control gene expression using fluorescence microscopy, flow cytometry, and the ability to control the chromatin environment using ChIP-qPCR6. Here, we describe a method for using and characterizing the CEMs, which will facilitate the adaptation of this system to answer additional questions related to chromatin biology.

<table border="0" cellpadding="0" cellspacing="0" style="width:697px;" width="698">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">DMEM</td>
			<td style="width:164px;">Corning</td>
			<td style="width:119px;">10-013CV</td>
			<td style="width:183px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">NEAA</td>
			<td style="width:164px;">Gibco</td>
			<td style="width:119px;">11140-050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">HEPES (for tissue culture)</td>
			<td style="width:164px;">Corning</td>
			<td style="width:119px;">25-060-Cl</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">BME (2-Mercaptoethanol)</td>
			<td style="width:164px;">Gibco</td>
			<td style="width:119px;">21985-023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">FBS</td>
			<td>Atlantic Biologicals</td>
			<td style="width:119px;">S11550</td>
			<td>Individual lots tested for quality</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Covaris tubes</td>
			<td style="width:164px;">ThermoScientific</td>
			<td style="width:119px;">C4008-632R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Covaris caps</td>
			<td style="width:164px;">ThermoScientific</td>
			<td style="width:119px;">C4008-2A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">PEI (polyethylenimine)</td>
			<td>Polysciences</td>
			<td>23966-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Transfection media (Opti-mem)</td>
			<td>Gibco</td>
			<td style="width:119px;">31985070</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Virus centrifuge tubes</td>
			<td style="width:164px;">Beckman Coulter</td>
			<td style="width:119px;">344058</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Virus filter membrane</td>
			<td>Corning</td>
			<td>431220</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Polybrene</td>
			<td>Santa Cruz Biotechnology</td>
			<td style="width:119px;">SC134220</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">0.25 % Trypsin</td>
			<td style="width:164px;">Gibco</td>
			<td style="width:119px;">25200-056</td>
			<td>with EDTA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">0.05 % Trypsin</td>
			<td style="width:164px;">Gibco</td>
			<td style="width:119px;">25400-054</td>
			<td>with EDTA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Puromycin</td>
			<td style="width:164px;">InvivoGen</td>
			<td style="width:119px;">ant-pr</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Blasticidin</td>
			<td style="width:164px;">InvivoGen</td>
			<td style="width:119px;">ant-bl</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:232px;">SYBR Green Master Mix (asymmetrical cyanine dye)</td>
			<td style="width:164px;">Roche</td>
			<td style="width:119px;">4913914001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">H3K27ac antibody</td>
			<td style="width:164px;">Abcam</td>
			<td style="width:119px;">ab4729</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:232px;">(ChIP kit) ChIP-IT High Sensitivity Kit</td>
			<td style="width:164px;">Active Motif</td>
			<td style="width:119px;">53040</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Sonicator</td>
			<td style="width:164px;">Covaris</td>
			<td style="width:119px;">E110</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Ultracentrifuge</td>
			<td style="width:164px;">Optima</td>
			<td style="width:119px;">XPN-80</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">SW-32 centrifuge rotor</td>
			<td>Beckman Coulter</td>
			<td style="width:119px;">14U4354</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">SW-32 Ti centrifuge buckets</td>
			<td>Beckman Coulter</td>
			<td style="width:119px;">130.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LentiX 293 Human Embryonic Kidney</td>
			<td style="width:164px;">Clontech</td>
			<td align="right">632180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">PBS</td>
			<td style="width:164px;">Corning</td>
			<td style="width:119px;">46-013-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">BSA</td>
			<td style="width:164px;">Fisher</td>
			<td style="width:119px;">BP9700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">EGTA</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:119px;">E3889</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">EDTA</td>
			<td style="width:164px;">Fisher</td>
			<td style="width:119px;">S311-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">NaCl</td>
			<td style="width:164px;">Fisher</td>
			<td style="width:119px;">S271-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Glycerol</td>
			<td style="width:164px;">Fisher</td>
			<td style="width:119px;">G33-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Tris</td>
			<td style="width:164px;">Fisher</td>
			<td style="width:119px;">BP152</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">NP40</td>
			<td style="width:164px;">Roche</td>
			<td style="width:119px;">11754599001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Triton</td>
			<td style="width:164px;">MP Biomedicals</td>
			<td style="width:119px;">807426</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Glycine</td>
			<td style="width:164px;">Fisher</td>
			<td style="width:119px;">BP381-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">TE buffer</td>
			<td style="width:164px;">Fisher</td>
			<td style="width:119px;">BP2474-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">HEPES (for ChIP)</td>
			<td style="width:164px;">Fisher</td>
			<td style="width:119px;">BP310-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Gelatin</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:119px;">G1890</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Flow cytometer, Attune NxT</td>
			<td style="width:164px;">Thermo Fisher</td>
			<td style="width:119px;">A24858</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">FKBP-Gal4 plasmid</td>
			<td style="width:164px;">Addgene</td>
			<td style="width:119px;">44245</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">psPAX2 plasmid</td>
			<td style="width:164px;">Addgene</td>
			<td style="width:119px;">12260</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">pMD2.G plasmid</td>
			<td style="width:164px;">Addgene</td>
			<td style="width:119px;">12259</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Nanodroplets</td>
			<td style="width:164px;">MegaShear</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">DNA Nanodrop</td>
			<td style="width:164px;">Thermo Fisher</td>
			<td style="width:119px;">ND1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Protease Inhibitors (Leupeptin)</td>
			<td style="width:164px;">Calbiochem/EMD</td>
			<td style="width:119px;">108975</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Protease Inhibitors (Chymostatin)</td>
			<td style="width:164px;">Calbiochem/EMD</td>
			<td style="width:119px;">230790</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Protease Inhibitors (Pepstatin)</td>
			<td style="width:164px;">Calbiochem/EMD</td>
			<td style="width:119px;">516481</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:232px;">CiA:Oct4 mESC</td>
			<td style="width:164px;">The kind gift of G. Crabtree</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:232px;">Lif-1C-alpha - producing Cos cells</td>
			<td>The kind gift of J. Wysocka</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
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repressing gene transcription by redirecting cellular machinery with chemical epigenetic modifiers

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.

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.
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Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

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 ...

This method can help answer key questions in the epigenetics field such as the mechanisms of gene regulation and cause and effect relationships between different chromatin regulatory elements. The main advantage of this technique is that for the first time it is possible to module a chromatin loci with physiologically relevant endogenous enzymes. Generally individuals new to this method will struggle because mouse ESL culture is inherently difficult for those who have not been trained in the technique.Further demonstration of this method is critical as cell viral infection steps are difficult to learn. The researcher must time two separate lines of cell culture and it is necessary to vigilantly monitor safety during these steps. To begin the protocol, passage 18 million of 293T HEK cells per 50 centimeter plate.Aspirate the old media and add 10 milliliters of one X phosphate buffered saline, or PBS. Aspirate the PBS and add three milliliters of 0.05%trypsin. Incubate the cells in trypsin for eight minutes at 37 degrees Celsius.Rock and tap the cells halfway through the incubation. The following day, when the cells have reached 60 to 80%confluency, perform a polyethylenimine, or PEI, transfection. Next, gently mix the DNA, PEI, and transfection media in a 15 milliliter conical tube.Incubate the sample at room temperature for 15 minutes. After incubation, add the solution dropwise to the 15 centimeter plate. Then, incubate the sample for 16 hours in a 37 degree Celsius incubator with 5%carbon dioxide.The next day, aspirate the media and gently replace it with prewarmed, fresh 293 HEK media to the 15 centimeter plates. After the media exchange, incubate the cells in a 37 degree Celsius incubator with 5%carbon dioxide for 48 hours. Count the cells with a hemocytometer.Passage previously prepared mESC into a 12 well plate format with 100, 000 cells per well one day prior to infection. For cells grown in a 10 centimeter format, aspirate the old media, add five milliliters of one X PBS, aspirate the PBS, and add one milliliter of 0.25%trypsin. Incubate the cells in trypsin for eight minutes at 37 degrees Celsius and rock and tap the cells halfway through the incubation.48 hours after the media exchange, remove and transfer the supernatant of the 293T HEK cells to a 50 milliliter conical tube. Centrifuge the supernatant at 300 times G for five minutes to pellet the cell debris. Next, filter the supernatant through a 0.45 micron membrane.Concentrate the virus via ultracentrifuge with an SW32 rotor and spin the sample at 72, 000 times G for two and a half hours at four degrees Celsius. While the virus concentrates under ultracentrifugation, treat the CIA mESC's in the 12 well plate with fresh embryonic stem or ES media containing five micrograms per milliliter of Polybrene. When the virus concentration has finished, carefully aspirate the supernatant and carefully suspend the virus pellet in 100 microliters of one X PBS to avoid excess bubbles.Add the virus and one X PBS to a 1.5 milliliter Microfuge tube. Vortex the tube at the lowest setting to fully suspend the virus. Spin down the tube in a mini tabletop centrifuge for five to 10 seconds to remove bubbles.Add 30 microliters of the virus to each well of a 12 well plate. Swirl the plate and then centrifuge the samples at 1000 times G for 20 minutes. Place the 12 well plate back in the 37 degree Celsius incubator overnight.The following morning, exchange the media with fresh ES media and allow infection to take place for 48 hours. After 48 hours of infection, select for cells that integrated the viral plasmid by adding the appropriate antibiotic. Change the media daily.Keep the selection media on the cells for 72 to 96 hours in a 37 degree Celsius incubator with 5%carbon dioxide. Continue with chemical epigenetic modifier, or CEM, preparation and treatment by suspending the powdered CEM and dimethyl sulphoxide, or DMSO, to a stock concentration of one millimolar and a working concentration of 100 micromolar. After the cells have undergone selection for 72 to 96 hours, split the mESC's into a 12 well format with 100, 000 cells per well.Incubate the cells in a 37 degree Celsius incubator with 5%carbon dioxide for 24 hours. After incubation, prepare a media solution of 100 nanomolar CEM with ES media. Use the media to feed the CIA mESC with one milliliter well.Keep a well of cells without any added CEM's to serve as a control. Next, change the media by aspirating the old media and adding one milliliter per well off freshly prepared CEM containing or CEM free ES media every 24 hours for the duration of the experiment. After 48 hours of CEM treatment, image the cells without CEM treatment in the cells treated with 100 nanomolar CEM using a fluorescence microscope.Take representative images using phase or bright-field to record the mESC morphology at 10 to 40 times magnification. The cells should have formed around colonies with a few differentiated cells. Under the FITC fluorescence channel, image both cell conditions.Next, isolate the control and CEM treated cells for flow cytometry by aspirating the media, washing the cells with one milliliter of one X PBS, aspirating the PBS and adding 0.25 milliliters of 0.25%trypsin with EDTA for eight to 10 minutes. Confirm that the cells are no longer bunched in large clumps through the microscope using 20 times magnification. If the cells do not appear to be in a single cell suspension, gently pipette them up and down with a P1000 pipette to fully trypsinize the cells.Quench the trypsin with one milliliter of fresh media and resuspend the cells at high speed with a serological pipette until the cells are in a single cell suspension. Then, spin down the cells at 300 times G for five minutes at room temperature. Aspirate the supernatant, wash the cells with one X PBS, and centrifuge the cells.Aspirate the PBS supernatant and resuspend the cell pellet in 200 microliters of FACS buffer. Perform flow cytometry on approximately more than 50, 000 cells with the suspended cells and run the samples at a sheath speed of around 5, 000 cells per second. Finally, export the data for further analysis.Phase images of the CEM system at the Oct4 locus and CIA mESC's are imaged after 48 hours of treatment with 100 nanomolar of CEM23 or with untreated cells indicate specific GFP repression. Fluorescent images show a bright GFP expression in the control cells and a reduced GFP expression in mESC's treated with CEM23. Flow cytometry consistently revealed a greater than 30%decrease in GFP expression in the CIA mESC's treated with 100 nanomolar of CEM23 for 48 hours.Chromatin immunoprecipitation, or CHIP, reveled that 48 hours of treatment with 100 nanomolar of CEM23 decreased the level of H3K27ac at the target locus when compared to control cells. While attempting this procedure, it's important to remember to be careful with the mouse embryonic stem cells. If they differentiate during the protocol, the results will be nullified.After its development, this technique paved the way for researchers in the field of synthetic biology to explore the in vivo mechanisms in the mammalian genome.

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