MMSET activates c-MYC by repressing miR-126*
Supplemental data
Supplemental Material and Methods:
Table S1. Sequence of the oligonucleotides used in this study.
Primers
Sequence (5'-3')
c-MYC F
TCGGATTCTCTGCTCTCCTCG
c-MYC R
CTGCGTAGTTGTGCTGATGTGTG
miR-126* mutation F
GCTGGCGACGGGACATGCTTACTTTTGGTACGCG
miR-126* mutation R
CGCGTACCAAAAGTAAGCATGTCCCGTCGCCAGC
c-Myc 3' UTR mutation F
GCCATTAAATGTAAATAACTTTGCTAAAACGTTTATAGCAGTTAC
c-Myc 3' UTR mutation R
GTAACTGCTATAAACGTTTTAGCAAAGTTATTTACATTTAATGGC
Tag F
TTTAGCTAGCATGAAGCGACGATGGAAAAAG
Tag R
ATTGCGGCCGCGTCGACGGTATCGATAAGCT
F5 F
ATTAGGATCCGAGCATGTCAGTGGAGGAG
F5 R
TACTCGAGTTGCCCTCTGTGACTCTCC
N-GFP F
GCGGATCCAGATCCAAAAAAGAAGAGAAAGGTAATGCCCGCCATGAAGAT
N-GFP R
TTCTCGAGTTATCATCGAGCTCGTGATCT
KAP1 shRNA F
GATCCGTGCAAGTGGATGTCAAGATTCAAGAGATCTTGACATCCACTTGCACTTTTTTACGCGT G
KAP1 shRNA R
AATTCACGCGTAAAAAAGTGCAAGTGGATGTCAAGATCTCTTGAATCTTGACATCCACTTGCACG
miR-126* TSS F
GGTTGTGAAGGGAGCTGTGT
miR-126* TSS R
CCTTGGGCACACAGAGTGTA
miR-126* 10 Kb upstream F
CAGCACGCTCTGGGAAGGTAAG
miR-126* 10 Kb upstream R
CATCTGGGCACTGGAAGGTCTG
Primary antibodies used for immunoblotting and immunoprecipitation:
MMSET (1), PARP1/2 (sc-7150, Santa Cruz), caspase-3 (9662, Cell Signaling),
KAP1 (ab22553, Abcam), HDAC1 (ab46985, Abcam), HDAC2 (ab7029, Abcam),
c-MYC (04-216, Millipore), GAPDH (ab347, Chemicon), H3-Ac (39139, Active
Motif), H4 (ab7311, Abcam), H3 (05-499, Millipore), H3K36me2 (07-369,
Millipore), H3K9me3 (ab8898, Abcam), and calmodulin binding protein (CBP)
(07-482, Millipore).
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MMSET activates c-MYC by repressing miR-126*
siRNAs:
MMSET-siRNA (cat. M-006571-01) and HDAC1-siRNA (cat. M-003493-02) were
purchased from Dharmacon.
miRNA microarray
Total RNA was isolated using the miRNeasy mini kit (Qiagen). miRNA screening
was
performed
at
the
MD
Anderson
Cancer
Center
(http://www.mdanderson.org/ncrnaprogram/). The data was normalized and
filtered based on the miRNA data analysis guidelines of Nanostring Technologies
(www.nanostring.com).
Tagged MMSET and Nuclear GFP Constructs
The plasmids SBP/CBP-pBIG2i-MMSET and SBP/CBP-pBIG2i-GFP were
constructed as follows: a DNA fragment containing the streptavidin binding
peptide (SBP) and a calmodulin binding peptide (CBP) tags were PCR amplified
from pNTAP vector (Stratagene), and inserted into pBIG2i(2) (“SBP/CBP-pBIG2i”
vector). A fragment of MMSET lacking the region encoding the N-terminal
PWWP domain of the protein (F5) was amplified from pCEFL-MMSET-II(3) and
inserted into the SBP/CBP-pBIG2i vector. A nuclear GFP DNA fragment was
amplified from pmaxGFP (Amaxa) and inserted into SBP/CBP-pBIG2i vector.
Generation of 293 Cells Producing Epitope Tagged MMSET (F5) and GFP
Doxycycline-inducible SBP/CBP-tagged MMSET and GFP constructs were
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MMSET activates c-MYC by repressing miR-126*
transfected into 293 cells, selected with 100 g/ml hygromycin B and screened
for inducible protein expression by immunoblot. To generate a tightly regulated
system, MMSET-expressing 293 cells were transfected with ptTS-Neo vector
(Clontech) encoding a tet-repressor. Stable colonies were selected wirh 500
g/ml G418 (Cellgro).
Protein identification by LC-tandem mass spectroscopy
Nuclear proteins were prepared from 2 x 108 293 cells expressing MMSET (F5)
or the nuclear GFP in a Dox-inducible manner (2 g/ml Dox), using the Nuclear
Complex Co-IP kit (Active Motif). The MMSET complex was batch purified by
Streptavidin beads (GE Healthcare) and D-biotin (United States Biochemical),
concentrated by trichloroacetic acid (Sigma), and dissolved in 2X loading buffer
(BioRad) with 2-mercaptoethanol. The proteins were resolved on a NuPAGE BisTris gel (Invitrogen) and were visualized by Colloidal Coomassie stain
(Invitrogen). Each lane was cut into 16 gel plugs and destained with 30%
methanol for 4h. Alkylation and trypsin digestion in-gel were performed as
described (4). The resulting peptides were resolved on a nano-capillary reverse
phase
column
(Picofrit
column,
New
Objective)
using
a
1%
acetic
acid/acetonitrile gradient at 300 nl/min and directly introduced into a linear iontrap mass spectrometer (LTQ XL, ThermoFisher). Data-dependent MS/MS
spectra on the 5 most intense ions from each full MS scan were collected
(relative CE ~35%). Proteins were identified by comparing the data to the Human
IPI database (v 3.65) appended with decoy (reverse) sequences using the
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MMSET activates c-MYC by repressing miR-126*
X!Tandem/Trans-Proteomic Pipeline (TPP) software suite. All proteins with a
ProteinProphet probability score of >0.8 (error rate <2%) were considered
positive identifications and manually verified.
References
1.
Martinez-Garcia E, Popovic R, Min DJ, Sweet SM, Thomas PM,
Zamdborg L, et al. The MMSET histone methyl transferase switches
global histone methylation and alters gene expression in t(4;14) multiple
myeloma cells. Blood 2011 Jan 6; 117(1): 211-220.
2.
Strathdee CA, McLeod MR, Hall JR. Efficient control of tetracyclineresponsive gene expression from an autoregulated bi-directional
expression vector. Gene 1999 Mar 18; 229(1-2): 21-29.
3.
Marango J, Shimoyama M, Nishio H, Meyer JA, Min DJ, Sirulnik A, et al.
The MMSET protein is a histone methyltransferase with characteristics of
a transcriptional corepressor. Blood 2008 Mar 15; 111(6): 3145-3154.
4.
Muntean AG, Tan J, Sitwala K, Huang Y, Bronstein J, Connelly JA, et al.
The PAF complex synergizes with MLL fusion proteins at HOX loci to
promote leukemogenesis. Cancer Cell 2010 Jun 15; 17(6): 609-621.
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MMSET activates c-MYC by repressing miR-126*
Supplemental figure legends
Figure S1. MMSET enhances growth of t(4;14) positive multiple myeloma
cells. (A) KMS11 cells harboring an inducible MMSET shRNA were grown in the
absence or presence of dox. The growth of parental KMS11 cells (wt) is
presented as a control. (B) KMS11 cells harboring an inducible MMSET shRNA
were previously grown in the presence of dox for 7 days. After that time, dox was
removed from the media (dox -) or kept as a control (dox +), and the proliferation
of the cells was evaluated. Day 0 represents the day dox removal. Live cells
were counted by trypan blue exclusion. Values represent mean SD from 3
experiments.
Figure S2. MMSET knockdown decreases c-MYC protein levels in t(4;14)
KMS11 cells. MMSET was depleted in KMS11 cells by transduction with a pool
of siRNA targeting different regions of the MMSET mRNA (cat. M-006571-01,
Dharmacon). After nuclear fractionation, MMSET and c-MYC protein levels were
assayed by immunoblot with histone H3 as a loading control.
Figure S3. MMSET knockdown in KMS11 cells affects the expression of
miRNAs. (A) Array-based miRNA profiling was performed in KMS11 cells
harboring a dox-inducible MMSET shRNA. Cells were grown in the absence or
presence of dox (dox- or dox+) for 7 days. Presented is a heat map indicating
relative levels of miRNAs that were significantly changed (≥ 2-fold) in response to
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MMSET activates c-MYC by repressing miR-126*
MMSET depletion. (B) Top: schematic structure of a c-MYC 3’UTR luciferase
reporter. Bottom: seed sequence of wild type miR-126* and the critical
complementary sequences of wild-type c-MYC 3’UTR reporter, and their
mutants.
Figure S4. Identification of MMSET protein partners. (A) Schematic
representation of streptavidin/calmodulin tagged MMSET (F5) and nuclear GFP
proteins. MMSET harbors PWWP, HMG, PHD, and SET domains, and has a
nuclear localization signal (NLS). (B) MMSET and GFP protein levels in the
absence or presence of 2 g/ml dox for 2 days were assayed by immunoblot
using MMSET or calmodulin binding protein (CBP) antibody, respectively, with
GAPDH as a loading control. (C) An epitope tagged form of MMSET was
expressed in 293 cells and subjected to affinity purification. Co-purifying proteins
were identified by mass spectroscopy. Presented is the list of MMSET partner
proteins identified from two independent experiments, and not detected in the
control purification of nuclear localized GFP.
Figure S5. Ectopic expression of MMSET in the t(4;14) negative RPMI-8226
cells binds to the miR-126* promoter and is associated with altered
chromatin modifications. Chromatin from RPMI-8226 cells harboring GFP
control vector, wild-type, or mutant MMSET was immunoprecipitated with antiMMSET (A), anti-H3K9me3 (B), or anti-acetyl histone H3 (C) antibodies.
Immunoglobulin G antibody was used as a negative control. Precipitated
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MMSET activates c-MYC by repressing miR-126*
chromatin was analyzed by real time PCR using primers amplifying regions near
the predicted miR-126* TSS or 10 kb upstream (10kb Up). Results are presented
as percentage of total input DNA precipitated.
Figure S6. HDACs are involved in the repression of miR-126*. (A) Chromatin
immunoprecipitation (ChIP) was performed using an anti-HDAC1 or HDAC2
antibody in KMS11 cells. Immunoglobulin G antibody was used as a negative
control. Precipitated chromatin was analyzed by real time PCR using primers
amplifying regions near the predicted miR-126* TSS or 10 kb upstream (10kb
Up). Results are presented as percentage of total input DNA precipitated. Values
of HDAC1 represent mean +/-SD from 2 experiments. (B) KMS11 cells were
transfected with a HDAC1 or scrambled siRNA. After nuclear fractionation,
HDAC1 and c-MYC protein levels were assayed by immunoblot, with histone H3
as a loading control.
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