SUPPLEMENTARY MATERIALS (Molecular Therapy, Gonçalves et al., 2010)
Rationale for generating the hybrid TF MyoDCD
MyoCD is one of the most powerful eukaryotic transcriptional activators identified so far.1 Of
note, the TAD at the COOH terminus of MyoCD has a general, non-specific, transcriptionactivating role and its deletion confers a dominant-negative phenotype to this TF. MyoCD’s
activity is modulated by posttranslational modifications and by its association with other
proteins. The central and COOH-terminal parts of MyoCD, for instance, possess binding sites
for transcription-repressing class II histone deacetylases (e.g. HDAC5) and for transcriptionpromoting histone acetyltransferases (e.g. p300 and CBP), respectively.2,3 Equally well
established are the fundamental roles played by the centrally located bHLH domains and
contiguous amino acid sequences4 in conferring DNA-binding specificity and myogenic
activity to the MRFs. Indeed, Mef2C and the bHLH motif-containing E proteins (e.g. E12 and
E47) stimulate skeletal muscle gene expression by cooperating with MyoD through
interactions with its bHLH domain. Heterodimerization of MyoD with the ubiquitous E
proteins via their bHLH domains greatly increases its affinity for the consensus sequence
CANNTG, referred to as an E-box, present in the control regions of many skeletal musclespecific genes. Finally, interactions between the bHLH domain of MyoD and the MADS and
MEF2 motifs at the NH2 terminus of Mef2C are key in potentiating each others transcriptional
activity and, possibly, also in recruiting MyoD to skeletal muscle genes with Mef2-binding
sites in their regulatory sequences.5
Thus, in search of a dominant myogenic TF, we investigated whether by covalently
tethering the TAD of human MyoCD to the bHLH motif of human MyoD and its flanking
sequences including the NH2-terminal TAD, would result in a chimeric protein (MyoDCD)
1
superior to MyoD in triggering cell-autonomous myogenic differentiation and fusion of nonmuscle cells.
References
1. Wang, D, Chang, PS, Wang, Z, Sutherland, L, Richardson, JA, Small, E et al. (2001).
Activation of cardiac gene expression by myocardin, a transcriptional cofactor for
serum response factor. Cell 105: 851-862.
2. Cao, D, Wang, Z, Zhang, CL, Oh, J, Xing, W, Li, S et al. (2005). Modulation of
smooth muscle gene expression by association of histone acetyltransferases and
deacetylases with myocardin. Mol Cell Biol 25: 364-376.
3. Taurin, S, Sandbo, N, Yau, DM, Sethakorn, N, Kach, J and Dulin, NO (2009).
Phosphorylation of myocardin by extracellular signal-regulated kinase. J Biol Chem
284: 33789-33794.
4. Chakraborty, T and Olson, EN (1991). Domains outside of the DNA-binding domain
impart target gene specificity to myogenin and MRF4. Mol Cell Biol 11: 6103-6108.
5. Molkentin, JD, Black, BL, Martin, JF and Olson, EN (1995) Cooperative activation of
muscle gene expression by MEF2 and myogenic bHLH proteins. Cell 83: 1125-1136.
2
Supplementary Results
Investigation of differential myogenic TF effects on GAPDH promoter activity
Control experiments were carried out to establish that the PpLuc signals retrieved from the
cell fusion experiments (Fig. 5a) do reflect the differential activities of MyoD:HA and
MyoDCD::HA in stimulating cell-to-cell fusion instead of being caused by a different effect
of the two TFs on the GAPDH promoter driving PpLuc expression. For this purpose,
hfMSCsGS.Luc were transduced with the FLPe-encoding gutless adenovirus vector
HD.FLPe.F501 to assemble functional GAPDH promoter-driven PpLuc expression units
(Supplementary Fig. S3, three rightmost solid bars). Next, the FLPe-positive hfMSCsGS.Luc
as well as their FLPe-negative counterparts (Supplementary Fig. S3, three rightmost open
bars) were either mock-treated or transduced with LV.MyoD::HA.eGFP or with
LV.MyoDCD::HA.eGFP.
Negative
controls
consisted
of
hfMSCsFLPe
that
had
(Supplementary Fig. S3, second bar from left) or had not (Supplementary Fig. S3, leftmost
bar) been exposed to HD.FLPe.F50. After 3 days in differentiation medium,
chemiluminescence measurements showed that the activity of the human GAPDH promoter in
LV.MyoD::HA.eGFP-transduced cells is similar to that in their LV.MyoDCD::HA.eGFPtransduced counterparts (Supplementary Fig. S3, two rightmost solid bars).
References
1. Gonçalves, MAFV, Holkers, M, van Nierop, GP, Wieringa, R, Pau, MG and de
Vries, AA (2008) Targeted chromosomal insertion of large DNA into the human
genome by a fiber-modified high-capacity adenovirus-based vector system. PLoS
One 3: e3084.
3
Supplementary Materials and Methods
DNA transfections. HeLa cells were seeded in wells of 24-well plates (Greiner Bio-One,
Alphen aan den Rijn, The Netherlands) at a density of 0.8-1.0×105 cells per well. After an
overnight incubation period, each sample received 350 ng of DNA mixtures in a total volume
of 50 l of 150 mM NaCl. The DNA mixtures contained 280 ng of pGL3.MCK.Luc, 4.6 nmol
of LV shuttle plasmid and 4.6 nmol of either pcDNA3.1/Hygro(+) (Invitrogen, Breda, The
Netherlands) or a human Mef2C-encoding derivative thereof. A total amount of 350 ng of
DNA was obtained by adding a plasmid lacking eukaryotic promoter elements. After the
addition of 2.4 l of ExGen 500 (Fermentas, St. Leon-Rot, Germany), the DNA mixtures
were immediately shaken vigorously for 10 s and incubated for 15 min at room temperature.
The DNA-polycation complexes were then diluted 10-fold in culture medium and added onto
the HeLa cell monolayers. After 20 h at 37°C in a humidified air-10% CO2 atmosphere, the
transfection medium was replaced by regular culture medium. Three days post-transfection,
cell lysates were prepared for measuring luciferase activity (see main text).
LV production. The VSV-G-pseudotyped self-inactivating HIV-1-based vectors CMVPRES1
(herein referred to as LV.eGFP), LV.MyoD.eGFP, LV.MyoG.eGFP, LV.Myf5.eGFP,
LV.Myf6.eGFP,
LV.LacZ.eGFP,
LV.MyoD::HA.eGFP,
LV.MyoDCD::HA.eGFP
and
LV.MyoCD::HA.eGFP were produced in 293T cells by using the packaging constructs
psPAX2 (Addgene, Cambridge, MA) and pLP/VSVG (Invitrogen) together with the proper
LV vector shuttle plasmid as previously described.2 The gene transfer activities of eGFPencoding LV stocks was determined by limiting dilution assays in HeLa cells using flow
cytometric analysis as read-out and were expressed in terms of HTU/ml. The physical particle
titers
of
LV.pA+.CK8/coh.Dys,
LV.PRE.pA+.CK8.Luc,
LV.PRE.pA+.Luc,
LV.PRE.pA+.CMV.Luc,
4
LV.PRE.pA+.MHCK7.Luc,
LV.FLPe.PurR
and
LV.GS.Luc
preparations were determined by using the RETRO-TEK HIV-1 p24 ELISA kit (Gentaur
Molecular Products, Brussels, Belgium) according to the instructions given by the
manufacturer. Titers of these LV stocks expressed in terms of TU/ml were derived by using a
conversion factor of 2500 TU per ng of HIV-1 capsid/p24 protein.
Intracellular HA tag-specific staining and flow cytometry. To ascertain that the
transduction levels of cells expressing the various TF-encoding genes were similar, we
employed
the
LVs
LV.MyoCD::HA.eGFP,
LV.MyoD::HA.eGFP
and
LV.MyoDCD::HA.eGFP encoding HA epitope-tagged versions of MyoCD, MyoD and
MyoDCD, respectively. Expression of each of these proteins in target cells was measured by
flow cytometry as detailed below. Suspensions of hMSCs transduced with each of the vectors
were fixed for 10 min at 37°C in 400 l of phosphate-buffered 4% formaldehyde solution (pH
7.0-7.4; KLINIPATH, Duiven, The Netherlands) and, subsequently, chilled on ice for 1 min.
After being spun down for 5 min at 400 g and 4°C, the cells were resuspended in 400 l of
ice-cold 90% methanol and incubated on ice for 30 min while being gently swirled every 5-10
min. Next, aliquots containing 5-10×104 cells were diluted in 4 ml of PBS-0.1% bovine serum
albumin (BSA), collected by centrifugation for 5 min at 400 g and 4°C and then incubated for
1 h at room temperature with a 1:20 dilution of AlexaFluor 647-conjugated anti-HA mouse
monoclonal antibody HA-Tag (clone 6E2; BIOKÉ, Leiden, The Netherlands) in PBS-0.1%
BSA. Parallel samples to which no antibody was added served as negative controls. The cell
suspensions were gently swirled every 10-15 min. After two consecutive washing steps
consisting of adding 4 ml of PBS-0.1% BSA followed by 5-min centrifugations at 400 g and
4°C, the cells were resuspended in 250 l of PBS-0.1% BSA and kept on ice until flow
cytometric analysis for eGFP- and HA-specific fluorescence.
5
Quantification of MHCK7 promoter activity in haMSCs. Thirty thousand haMSCs were
incubated
overnight
in
wells
of
24-well
plates
with
LV.PRE.pA+.Luc
or
LV.PRE.pA+.MHCK7.Luc at an MOI of 30 TU/cell. Mock-transduced haMSCs served as
controls. The resulting haMSCLuc and haMSCMHCK7.Luc populations were amplified by subculturing. Subsequently, the cells were incubated with LV.LacZ.eGFP, LV.MyoD::HA.eGFP
or LV.MyoDCD::HA.eGFP at an MOI of 25 HTU/cell and cultured in growth medium for 6
days. After another 6-day culture period in differentiation medium, cell lysates were prepared
for measuring PpLuc activity. Mock-transduced haMSCs subjected to the same culture
regimen served as negative control.
Quantification of CK8 promoter activity in human myocytes before and after
differentiation. DMD myoblasts were seeded in wells of 24-well plates at a density of 2 ×
105 cells per well. The following day these cells were transduced in duplicate with LVs
carrying a promoterless, a CMV promoter-driven or a CK8 promoter-based PpLuc expression
unit at MOIs of 0.5, 2.0 and 8.0 TU/cell. At 48 h post-infection, the cells were rinsed once
with 2 ml of PBS after which the cells in one sample of each pair of cell cultures were
trypsinized and stored at -20°C whilst the cells in the other sample were exposed to
differentiation medium for 4 days prior to trypsinization and storage at -20°C. After thawing
and lysis of the cells, chemiluminescence measurements were performed.
Establishment and deployment of hfMSCCK8.Dys cultures. Thirty thousand hfMSCs in
wells of 24-well plates were incubated overnight with LV.pA+.CK8.coh/Dys at an MOI of
25 TU/cell. After expansion, the resulting hfMSCsCK8.Dys were seeded in wells of 24-well
plates at a density of 3×104 cells/well and were incubated overnight with either
LV.MyoD::HA.eGFP or with LV.MyoDCD::HA.eGFP at 25 HTU/cell. Unmodified hfMSCs
6
seeded in parallel were transduced with the same dose of either of the two TF-encoding LVs.
Mock-infected hfMSCs and hfMSCsCK8.Dys served as negative controls. Four days later, 35×104 cells from each of the various cell populations were seeded on glass cover slips in wells
of 6-well plates either alone or together with 2×105 DMD myoblasts. After 4 days in their
respective growth media, both monocultures and co-cultures were rinsed once with PBS and
incubated in differentiation medium. After 4 additional days of culture, the cells were fixed
and processed for dystrophin immunofluorescence microscopy as specified in the main text.
Testing the effect of MyoD and MyoDCD on GAPDH promoter activity
The effects of MyoD and MyoDCD on the activity of the GAPDH promoter driving FLPedependent PpLuc expression were evaluated as follows. Thirty thousand hfMSCsGS.Luc and
hfMSCsFLPe were transduced with the FLPe-encoding gutless
adenovirus vector
HD.FLPe.F50 at an MOI of 40 gene switch-activating units/cell. Parallel cultures of these
cells not exposed to HD.FLPe.F50 served as negative controls. Both the FLPe-positive
hfMSCsGS.Luc as well as their FLPe-negative counterparts were subsequently either mockinfected or transduced with LV.MyoD::HA.eGFP or with LV.MyoDCD::HA.eGFP at an MOI
of 25 HTU/cell. The cells were exposed to the different LVs for approximately 24 h. After 7
days in growth medium and an additional 3-day period in differentiation medium, PpLuc
activity in the lysates of the cell cultures was determined by luminometry.
References
1 Seppen, J, Rijnberg, M, Cooreman, MP and Oude Elferink, RP (2002). Lentiviral
vectors for efficient transduction of isolated primary quiescent hepatocytes. J Hepatol
36: 459-465.
7
2 Gonçalves, MAFV, Swildens, J, Holkers, M, Narain, A, van Nierop, GP, van de
Watering, MJM et al. (2008). Genetic complementation of human muscle cells via
directed stem cell fusion. Mol Ther 16: 741-748.
8