A chemical tool for in vitro and in vivo precipitation of lysine methyltransferase G9a

Article abstract of DOI:10.1002/cmdc.201300450

Here we report the design, synthesis, and biochemical characterization of a new chemical tool, UNC0965. UNC0965 is a biotinylated version of our previously reported G9a chemical probe, UNC0638. Importantly, UNC0965 maintains high in vitro potency and is c

Full text of DOI:10.1002/cmdc.201300450

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DOI: 10.1002/cmdc.201300450
A Chemical Tool for In Vitro and In Vivo Precipitation of
Lysine Methyltransferase G9a
Kyle D. Konze,[a] Samantha G. Pattenden,[a] Feng Liu,[a] Dalia Barsyte-Lovejoy,[b] Fengling Li,[b]
Jeremy M. Simon,[c] Ian J. Davis,[c] Masoud Vedadi,[b] and Jian Jin*[a]
Here we report the design, synthesis, and biochemical charac-
terization of a new chemical tool, UNC0965. UNC0965 is a bio-
tinylated version of our previously reported G9a chemical
probe, UNC0638. Importantly, UNC0965 maintains high in vitro
potency and is cell penetrant. The biotinylated tag of UNC0965
enables “chemiprecipitation” of G9a from whole cell lysates.
Further, the cell penetrance of UNC0965 allowed us to explore
the localization of G9a on chromatin both in vitro and in vivo
through chemical inhibitor-based chromatin immunoprecipita-
tion (chem-ChIP).
pitation”). In addition, UNC0965 is a useful tool for exploring
the localization of G9a on chromatin both in vitro and in vivo.
We previously reported the X-ray co-crystal structure of the
G9a–UNC0638–S-adenosyl-l-homocysteine (SAH) complex.[13] In
this structure, UNC0638 adopted a conformation that buried
the 7-(3-pyrrolidin-1-yl-)propoxy side chain in the lysine bind-
ing channel, and the isopropyl group of the 4-(N-isopropylpi-
peridin-4-ylamino) moiety was solvent exposed. We reiterated
our previous efforts with the histone-lysine N-methyltransfer-
ase enhancer of zeste homologue 2 (EZH2)[14] by appending
a biotin tag to the solvent exposed region of UNC0638, which
yielded UNC0965, a functionalized chemical tool for biological
applications (Figure 1a). We hypothesized that a relatively long
linker (ꢀ20 atoms from the piperidinyl nitrogen to the biotin
carbonyl) would ensure that the biotin was at a sufficient dis-
tance from the surface of G9a to permit access to streptavidin-
conjugated magnetic beads. This hypothesis was supported by
docking studies using the G9a X-ray structure (Figure 1b). As
expected, UNC0965 docked in a nearly identical orientation to
UNC0638, with the 7-(3-pyrrolidin-1-yl)propoxy side chain em-
bedded in the lysine binding channel, and the biotin solvent
exposed, well away from the surface of the protein.
G9a is a lysine methyltransferase (KMT) responsible for catalyz-
ing the mono- and dimethylation of histone H3 lysine 9
(H3K9).[1–6] Its activity is linked to multiple biological pathways
and human diseases including cancer (reviewed in [7]). G9a is
overexpressed in a number of tumors;[8] specifically, it is associ-
ated with increased metastasis and invasion of tumor cells in
lung cancer.[9] Surprisingly, loss of G9a-dependent dimethylated
H3K9 (H3K9me2) has also been observed in various cancer cell
lines.[10] This discrepancy underscores the importance of further
research into the biological role of G9a.
Chemical probes that demonstrate sufficient cellular potency
and target selectivity can greatly accelerate the understanding
of protein function.[11,12] Here, we report the design, synthesis,
and biochemical characterization of the chemical tool,
UNC0965, which is a biotinylated version of our previously re-
ported G9a chemical probe UNC0638.[13] UNC0965 displays low
nanomolar in vitro potency, is active in cellular assays, and se-
lectively precipitates G9a from whole cell lysates (“chemipreci-
The synthesis of UNC0965 is outlined in Scheme 1. Com-
pound 6 was prepared from commercially available starting
material 1 according to the synthetic route developed previ-
ously.[13,15–17] Compound 8 was synthesized from commercially
available tert-butyl piperidin-4-ylcarbamate (7) in two straight-
forward steps. Nucleophilic aromatic substitution of 6 with 8
gave UNC0638 derivative 9, which contains a terminal alkyne.
We used this terminal alkyne to perform a copper-catalyzed
click reaction with commercially available biotin-PEG3-azide to
give UNC0965 (10).
[a] K. D. Konze,+ Dr. S. G. Pattenden,+ Dr. F. Liu, Prof. Dr. J. Jin
Center for Integrative Chemical Biology and Drug Discovery
Division of Chemical Biology and Medicinal Chemistry
UNC Eshelman School of Pharmacy
We next confirmed that UNC0965 binds G9a and inhibits its
catalytic activity. We used a scintillation proximity assay as pre-
viously reported[18] to determine the IC50 value of UNC0965
(Figure 2a). This assay is a direct measurement of the G9a-cata-
University of North Carolina at Chapel Hill
120 Mason Farm Road, Chapel Hill, NC 27599 (USA)
E-mail: jianjin@email.unc.edu
3
lyzed transfer of a tritiated methyl group from H-S-adenosyl-l-
[b] Dr. D. Barsyte-Lovejoy, Dr. F. Li, Prof. Dr. M. Vedadi
Structural Genomics Consortium, University of Toronto
Toronto, Ontario (Canada)
methionine (3H-SAM) to the histone peptide substrate.
UNC0965 showed potent inhibition, with an IC50 value of
<2.5 nm. This strong inhibition of the G9a catalytic activity im-
plied that UNC0965 would bind the protein tightly enough to
efficiently chemiprecipitate G9a from cell lysates.
[c] Dr. J. M. Simon, Prof. Dr. I. J. Davis
Departments of Genetics and Pediatrics
Carolina Center for Genome Sciences
Lineberger Comprehensive Cancer Center
University of North Carolina at Chapel Hill
Chapel Hill, NC (USA)
UNC0965 was coupled to streptavidin-conjugated magnetic
beads to chemiprecipitate G9a from HEK293T whole cell ex-
tracts (Figure 2b). Beads pre-incubated in the presence of 1%
dimethyl sulfoxide (DMSO) did not pull down G9a (Figure 2b,
lane 2). UNC0965-coated streptavidin-conjugated beads effec-
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300450.
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coated beads to chemiprecipi-
tate G9a (Figure 2b, lanes 3 and
5). Therefore, UNC0965 chemi-
precipitates G9a from cell ly-
sates, which further confirms
that the biotin moiety of
UNC0965 does not interfere with
its binding to G9a. Future appli-
cations for this chemiprecipita-
tion technique could include
proteomic analysis of precipitat-
ed proteins to identify G9a-bind-
ing partners.
To further characterize this
chemical tool, we investigated
whether UNC0965 could capture
G9a from formaldehyde cross-
linked chromatin. Chromatin is
a highly dynamic structure, and
an effective barrier to gene tran-
scription. As such, it is frequently
remodeled to create or prevent
access of transcription factors to
underlying DNA sequences. Tra-
ditional antibody-based chroma-
tin immunoprecipitation (ChIP)
was developed as a tool to pro-
vide a snapshot of protein–DNA
interactions in the dynamic chro-
matin environment (reviewed in
Figure 1. Design of UNC0965. a) G9a-UNC0638-SAH co-crystal structure (PDBID: 3RJW) was previously solved and
demonstrated that the N-isopropyl group of UNC0638 is solvent exposed. We took advantage of this chemical
handle and designed the biotinylated derivative UNC0965. b) Docking of UNC0965 into the G9a crystal structure
predicts a similar binding mode as UNC0638. Importantly, the biotin moiety of UNC0965 is solvent exposed and
suitable for streptavidin capture.
tively pulled down G9a, while pre-incubation of the lysate with
UNC0638 blocked this interaction (Figure 2b, lanes 3 and 4).
Additionally, UNC1152, a negative control of UNC0638 that
does not significantly inhibit G9a catalytic activity (compound
26a in ref. [18]), was unable to block the ability of UNC0965-
ref. [19–21]). In a standard ChIP assay, a cross-linking reagent
such as formaldehyde is used to covalently attach proteins to
DNA, the cells are lysed, and the chromatin is fragmented by
sonication or enzymatic digestion. An antibody is then used to
immunoprecipitate a protein of interest from the chromatin
Scheme 1. Synthesis of UNC0965. Reagents and conditions: a) 1-chloro-3-iodopropane, K2CO3, CH3CN, reflux; b) HNO3, Ac2O, 08C to RT, 75% over two steps;
c) pyrrolidine, K2CO3, NaI, cat. tetrabutylammonium iodide, CH3CN, reflux, 81%; d) Fe dust, NH4OAc, EtOAc/H2O, reflux, 63%; e) 4n HCl, dioxane, cyclohexane-
carbonitrile, RT; f) N,N-diethylaniline, POCl3, reflux, 47% over two steps; g) hex-5-yn-1-yl 4-methylbenzenesulfonate, K2CO3, DMF, 608C, 88%; h) TFA, CH2Cl2, RT,
99%; i) 1-(hex-5-yn-1-yl)piperidin-4-amine·2TFA (8), K2CO3, DMF, 608C, 81%; j) biotin-PEG3-azide, cat. Cu(CH3CN)4PF6, CH2Cl2, RT, 92%.
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Figure 2. In vitro characterization of UNC0965. a) UNC0965 shows potent in-
hibition of G9a catalytic activity in an in vitro scintillation proximity assay
(IC50 <2.5 nm). Experiments were performed in duplicate. b) Streptavidin-
conjugated magnetic beads pre-coated with UNC0965 selectively chemipre-
cipitate G9a from HEK293T cell lysates. G9a western blot indicates that
UNC0965-treated beads (lane 3) chemiprecipitate G9a compared to DMSO-
treated beads (lane 2). This interaction can be blocked or retained by pre-
treating lysate with UNC0638 (lane 4) or its negative control UNC1152
(lane 5), respectively. The large (G9aL) and small (G9aS) isoforms of G9a are
indicated. The asterisk (*) denotes a background band. These data are repre-
sentative of three biological replicates.
Figure 3. G9a occupancy on chromatin can be determined using antibody
ChIP or in vitro chem-ChIP with UNC0965. a) ChIP using G9a antibody or IgG
control followed by qPCR. b) In vitro chem-ChIP with UNC0965-coated strep-
tavidin-conjugated magnetic beads followed by qPCR. PCR primer sets cor-
respond to chromosomal regions (CR) occupied by G9a as well as a GAPDH
control region that shows no G9a occupancy. Chromosomal regions assayed
include: CR1 (white), CR2 (checkered), CR3 (grey), and CR4 (black), and
GAPDH (light grey). Error bars represent the standard deviation of three bio-
logical replicates. Statistical significance between DMSO and UNC0965
chem-ChIP for each region was determined by an unpaired t test and indi-
cated by asterisks (*=p<0.05, **=p<0.01, ***=p<0.001).
extract, and the associated DNA sequences are analyzed by
methods such as quantitative PCR (qPCR) or next-generation
sequencing (ChIP-seq). We hypothesized that UNC0965 could
substitute for an antibody to precipitate G9a protein in
a method referred to as chemical inhibitor-based chromatin
precipitation (chem-ChIP).[22]
region near the GAPDH gene (Figure 3a). We performed in
vitro chem-ChIP using the protocol for antibody ChIP, replacing
the G9a antibody and IgG control with streptavidin-conjugated
magnetic beads pre-incubated with UNC0965 and DMSO, re-
spectively. We then examined the identified G9a-occupied
chromosomal regions by qPCR (Figure 3b). The UNC0965 in
vitro chem-ChIP showed a greater than 5-fold increase over
DMSO at each chromosomal region. The difference between
the G9a antibody ChIP signal and the IgG signal was less sig-
nificant (p<0.5 to p<0.01; Figure 3a), than the signal for
UNC0965 in vitro chem-ChIP compared to DMSO (p<0.01 to
p<0.001). Thus, UNC0965 effectively captures G9a in an in
vitro ChIP assay, and in this case with a better G9a signal over
background than the antibody ChIP.
Use of a functionalized chemical tool for chromatin precipi-
tation has distinct advantages over an antibody. First, such
chemical tools can be easily synthesized at low cost, and elimi-
nate the batch-to-batch variability common with antibody re-
agents. Second, the biotin tag can be designed to be solvent
exposed for chemical tools that have a well-defined mode of
binding. This design has a potential added advantage over tra-
ditional ChIP, where cross-linking can prevent access of an anti-
body to the protein of interest. Third, chemical tools can be
cell permeable, which allows cell treatment prior to cross-link-
ing. This in vivo chem-ChIP method would be particularly well
suited for proteins that are difficult to precipitate after cross-
linking due to antibody epitope masking. Therefore, we tested
the ability of UNC0965 to precipitate G9a from chromatin ex-
tracted from cross-linked cells (in vitro chem-ChIP), and chro-
matin generated from cells treated with the compound prior
to cross-linking (in vivo chem-ChIP) and compared these re-
sults to the normal antibody method.
As noted earlier, unlike antibodies, cell-permeable small-mol-
ecule tools can pre-bind the target in cells prior to cross-link-
ing. We assessed cellular activity of UNC0965 with a cell-based
western assay reported previously.[13] Treatment of cells for
48 h with increasing concentrations of UNC0965 resulted in
a decrease in H3K9me2 (IC50 =(3.3Æ1.2) mm), with low cell tox-
icity (EC50 >50 mm; Figure 4a). While UNC0965 has lower cell
permeability compared to UNC0638,[13] it shows sufficient in-
hibition of H3K9me2 to be useful for cell-based assays.
Using traditional antibody ChIP, we identified four chromo-
somal regions in HEK293T cells (table S1 in the Supporting In-
formation) that showed a significant fold change in G9a occu-
pancy over background when compared to a negative control
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Our success with UNC0965 and G9a highlights methods to
convert highly selective chemical probes into extremely valua-
ble tools that may aid in understanding biological questions.
The in vivo chem-ChIP technique could be an alternative
method for targets that have no suitable ChIP-grade antibody
due to masking of the appropriate epitope after crosslinking.
In fact, use of a labeled chemical tool could prevent the need
for overexpression of a tagged protein. We noted that the
signal to noise ratio was lower for the antibody ChIP compared
to both the in vitro and in vivo chem-ChIP assays. Thus, it
would be interesting to compare genome-wide occupancy of
G9a between all three methods to determine how the overall
signal for UNC0965 chem-ChIP compares to that of traditional
ChIP.
Experimental Section
Methods for chemical synthesis, in vitro scintillation proximity
assay, chemiprecipitation of G9a, western blotting, antibody chro-
matin immunoprecipitation (ChIP), quantitative PCR (qPCR), cell-
based western, and cell toxicity assays are described in the Sup-
porting Information.
Figure 4. UNC0965 is cell penetrant and can be used for in vivo chem-ChIP
assays. a) Cell-based western assay of MDA-MB-231 cells treated with
UNC0965 and assayed for total cellular H3K9me2 levels. UNC0965 reduces
cellular levels of H3K9me2 (light grey squares; IC50 =(3.3Æ1.2) mm) and is
not toxic in the compound concentration range tested (grey circles;
EC50 >50 mm). Error bars represent the standard deviation of three biological
replicates. These data confirm that UNC0965 is cell penetrant. b) In vivo
chem-ChIP. HEK293T cells were treated with UNC0965 (5 mm) or DMSO
(0.05%) for 15 min, cross-linked with formaldehyde, and then chromatin was
isolated. ChIP was performed using streptavidin-conjugated magnetic beads
followed by qPCR. PCR primer sets correspond to chromosomal regions (CR)
occupied by G9a as well as a GAPDH control region that shows no G9a oc-
cupancy. Chromosomal regions assayed include: CR1 (white), CR2 (check-
ered), CR3 (grey), and CR4 (black), and GAPDH (light grey). Error bars repre-
sent the standard deviation of three biological replicates. Statistical signifi-
cance between DMSO and UNC0965 chem-ChIP for each region was deter-
mined by an unpaired t-test and indicated by asterisks (**=p<0.01,
***=p<0.001).
In vitro chem-ChIP: Chromatin was prepared from HEK293T cells as
described for the antibody ChIP (see Supporting Information). Each
ChIP contained chromatin from approximately 8ꢁ106 cells. Strepta-
vidin-conjugated magnetic beads (Dynabeads M-280 streptavidin,
Life Technologies #11205D, 250 mg per chemiprecipitation) were
washed 3ꢁ with 500 mL TBST (20 mm Tris-HCl, pH 8.0, 150 mm
NaCl, 0.1% Tween-20) and diluted in 100 mL TBST. UNC0965 (4 mL,
10 mm in DMSO) was added, and the suspension was rotated on
an end-over-end rotator for 1 h at RT to saturate the beads. After
binding, the beads were washed with TBST (3ꢁ500 mL) to remove
excess unbound compound. Chromatin (200 mL representing ~8 ꢁ
106 cells) was added to the beads, and the mixture was placed on
a rotator at 48C overnight. The supernatant was aspirated from the
beads, which were then washed with ChIP wash buffer (3ꢁ500 mL;
20 mm Tris, pH 8.0, 150 mm NaCl, 0.1% SDS, 1% TritonX-100, 2 mm
EDTA) followed by one wash with ChIP final wash buffer (500 mL;
20 mm Tris-HCl, pH 8.0, 500 mm NaCl, 1% SDS, 1% TritonX-100,
2 mm EDTA). During each wash, the sample was rotated at RT for
3 min. The washed beads were resuspended in 200 mL TE (10 mm
Tris-HCl, pH 8.0, 1 mm EDTA) with 20 mg proteinase K, and the con-
tents were transferred into a PCR tube. The mixture was incubated
for 30 min at 558C followed by 2 h at 808C to reverse the cross-
links. The DNA in the collected supernatant was then purified as
described in the Active Motif ChIP-IT High Sensitivity kit (Active
Motif #53040).
For the in vivo chem-ChIP assay, we wanted to pulse the
cells with the compound, rather than treat for several days, in
order to minimize any effects of G9a inhibition on its chroma-
tin localization. Therefore, we grew HEK293T cells to 80% con-
fluence, and then exposed the cells to 5 mm UNC0965 or
0.05% (volume equivalent) DMSO for 15 min prior to formalde-
hyde cross-linking. We then performed the standard antibody
ChIP protocol (see Supporting Information), with the exception
of the precipitation step, which was performed using streptavi-
din-conjugated magnetic beads. Cells treated with UNC0965
showed a significant average change in G9a occupancy over
DMSO-treated cells (p<0.01; Figure 4b), which was very com-
parable to the in vitro chem-ChIP results (Figure 3b). These
data demonstrate that even with compound pre-treatment of
the cells, the biotin moiety on UNC0965 remains solvent ex-
posed after formaldehyde cross-linking, confirming that
UNC0965 is a useful chemical tool for in vivo chem-ChIP stud-
ies and could be applied to additional cellular studies.
In vivo chem-ChIP: HEK293T cells were grown to 80% confluence
on 15 cm plates as described (see Supporting Information). Cells
were treated with 5 mm UNC0965 for 15 min prior to formaldehyde
cross-linking. Chromatin was prepared as described for the anti-
body ChIP. Each chem-ChIP represented approximately 8ꢁ106 cells.
Streptavidin-conjugated magnetic beads (Dynabeads M-280 strep-
tavidin, Life Technologies #11205D, 500 mg per chemiprecipitation)
were washed 3ꢁ with 500 mL TBST. Chromatin was then added to
the beads, and the mixture was placed on a rotator at 48C over-
night. Wash and DNA purification steps were performed as de-
scribed above for the method for in vitro chem-ChIP.
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methyltransferase · medicinal chemistry
·
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·
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·
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Received: November 6, 2013
Published online on January 17, 2014
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