MMSET activates c-MYC by repressing miR-126*

Supplemental data

Supplemental Material and Methods:

Table S1. Sequence of the oligonucleotides used in this study.

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).

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 ( The data was normalized and filtered based on the miRNA data analysis guidelines of Nanostring Technologies (

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 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 Bis-Tris 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 ion-trap 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 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 tetracycline-responsive 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.

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 apool 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 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 partnerproteins 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 anti-MMSET (A), anti-H3K9me3 (B), or anti-acetyl histone H3 (C) antibodies. 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.

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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