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First published online January 11, 2007
Stem Cells Vol. 25 No. 5 May 2007, pp. 1178 -1185
doi:10.1634/stemcells.2006-0106; www.StemCells.com
© 2007 AlphaMed Press

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TECHNOLOGY DEVELOPMENT

A Cassette System to Study Embryonic Stem Cell Differentiation by Inducible RNA Interference

Daniel Wegmüllera, Ines Raineria, Brigitte Grossa, Edward J. Oakeleyb, Christoph Moronia

aInstitute for Medical Microbiology, Department of Biological and Clinical Sciences, University of Basel, Basel, Switzerland;
bFriedrich Miescher Institute for Biomedical Research, Basel, Switzerland

Key Words. Embryonic stem cells • Cardiac development • RNA interference • Tetracycline • Zfp36L1 • mRNA turnover

Correspondence: Christoph Moroni, M.D., University of Basel, Institute for Medical Microbiology, Petersplatz 10, 4051 Basel, Switzerland. Telephone: +41 61 2673262; Fax: +41 61 267 32 83; e-mail: Christoph.Moroni{at}unibas.ch

Received February 24, 2006; accepted for publication January 2, 2007.
First published online in STEM CELLS EXPRESS   January 11, 2007.


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Although differentiation of pluripotent embryonic stem cells is restricted by a hierarchy of transcription factors, little is known about whether post-transcriptional mechanisms similarly regulate early embryoid differentiation. We developed a system where small hairpin (sh)RNAs can be induced in embryonic stem (ES) cells from a defined locus following integration by Flp recombinase-mediated DNA recombination. To verify the system, the key transcription factor Stat3, which maintains pluripotency, was downregulated by shRNA, and the expected morphological and biochemical markers of differentiation were observed. Induction of shRNA specific for the post-transcriptional regulator Brf1 (Zfp36L1) amplified the cardiac markers with strong stimulation of cardiomyocyte formation within embryoid bodies. These findings identify Brf1 as a novel potential regulator of cardiomyocyte formation and suggest that post-transcriptional mechanisms are of importance to early development and, possibly, to regenerative medicine. The inducible RNA interference system presented here should also allow assignment of function for candidate genes with suspected roles in ES cell development.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Embryonic stem (ES) cell lines provide an attractive system to study the basically unresolved question of how stem cells decide between self-renewal and differentiation [1]. From a clinical perspective, they provide a promising tool for the emerging field of regenerative medicine. The pluripotency of cultured murine ES cells is maintained by the cytokine leukemia inhibitory factor (LIF), which restrains ES cells from differentiation and acts via LIF-receptor-dependent activation of the transcription factor Stat3 [2–5]. How the Stat3 targets maintain pluripotency and why loss of Stat3 activation leads to differentiation is not known, although recent work assigns a key role to c-myc [6]. In addition to Stat3, other transcription factors, including Oct4, nanog, Sox2, and the BMP4 regulatory protein, also play important roles in maintaining the pluripotent state of ES cells [7–13]. Recent results from human ES cells indicate that promoter regions of 353 genes, including Stat3, are co-occupied by Oct4, nanog, and Sox2 [14]. This is consistent with a model where a hierarchical system of transcription factors controls the balance between self-renewal and differentiation and where subtle changes may be sufficient for triggering differentiation [1, 15]. Although it is plausible that post-transcriptional forms of regulation may also play a role in ES differentiation, this aspect has, to the best of our knowledge, not been addressed. The control of mRNA turnover for transcripts containing an AU-rich element (ARE) in their 3'-untranslated region (UTR) is of particular interest, as this element is present in many transcription factors, cytokines, chemokines, and other regulators [16, 17]. ARE-binding proteins such as AUF1, TTP, or Brf1 have been identified, which promote ARE-dependent mRNA decay [18–21], whereas HuR acts as a stabilizer [22]. These proteins regulate access of decapping enzymes and RNases including deadenylases and exosomal enzymes to the transcripts [23–25]. The presence of common signals such as the ARE on many transcripts has suggested the concept of a "post-transcriptional operon" [26], and it may well be that a similar form of regulation also operates in ES cells and embryogenesis. We report here a system based on inducible RNA interference to assess effects of suspected regulators, post-transcriptional or otherwise. Small hairpin (sh)RNA, first introduced into a defined green fluorescent protein (GFP)-marked locus by Flp recombinase ("flipase") [27], is induced by doxycycline and triggers decay of the mRNA target. In addition to describing a generally applicable system, we report that doxycycline-induced RNA interference directed at Brf1 led to expression of cardiomyocyte-specific markers and massive amplification of beating areas within embryoid bodies (EBs).


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Cell Culture
CCE ES cells [28, 29] were cultured on gelatin-coated dishes in 250 U/ml LIF (Chemicon, Temecula, CA, http://www.chemicon.com) containing medium consisting of high glucose Dulbecco's modified Eagle's medium (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) supplemented with 15% fetal calf serum (FCS; Gibco, Grand Island, NY, http://www.invitrogen.com), 2 mM L-glutamine (Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com), 0.1 mM nonessential amino acids (Stem Cell Technologies), 1 mM sodium pyruvate (Stem Cell Technologies), and 100 µM monothioglycerol (Sigma-Aldrich). Cells were frozen in medium containing 50% FCS, 40% culture medium, and 10% dimethyl sulfoxide (Sigma-Aldrich). Experiments with CCE-TR-FRT cells (see below) were performed in ES medium containing 100 U/ml LIF in the presence or absence of 2 µg/ml doxycycline (Dox).

To generate cardiomyocytes from EBs, ES cells were cultured for 2 days in hanging drops followed by a 2-day suspension culture. Then, 30 or 2 EBs were plated into either gelatin-coated 24- or 48-well plates, respectively, and cultured for 24 hours in maintenance medium containing Iscove's modified Dulbecco's medium (Sigma-Aldrich), 20% FCS, 2 mM L-glutamine, 0.1 mM nonessential amino acids, and 100 µM monothioglycerol to allow attachment of EBs to the culture dish. Thereafter, cells were kept for 48 hours in starvation medium consisting of maintenance medium supplemented with only 0.2% FCS, followed by culture in supplemented medium corresponding to maintenance medium containing serum replacement (Sigma-Aldrich) instead of FCS.

Plasmids
Tet repressor plasmid (pCAG-TR-IRESpuro3): pCAG and pIRESpuro3 (Clontech, Palo Alto, CA, http://www.clontech.com) plasmids were digested (SpeI and EcoRI) and the IRESpuro3 fragment ligated into pCAG. Digestion of pcDNA6/TR (Invitrogen) vector (AflII, blunt ending, NotI) releases the Tet repressor (tetR)-intervening sequence (IVS) insert. This fragment was cloned into pCAG-IRESpuro3 digested by NotI.

To generate the FRTd2EGFP plasmid (pCAG-FRTd2EGFP-IRESneo3), the d2EGFP was amplified without the start codon from pd2EGFP-N1 (Clontech) with BglII and NotI linkers. The CAG promoter was cut from pCAG with EcoRI and SpeI and ligated with an oligo containing an ATG, a FRT site [30], EcoRI, and BglII linkers to the d2EGFP fragment. This insert was finally inserted into pIRESneo3 (Clontech) digested with SpeI and NotI.

To generate short hairpin (sh)RNA plasmids (pTER-shRNA-FRT), the shRNAs (Stat3, Brf1) were cloned into pTER-Ni [31] using BglII and NotI. Plasmids were opened with NsiI and SapI and blunted, followed by insertion of an FRT-Hygro-SV40pA fragment from pcDNA5/FRT (Invitrogen) digested with PvuII. To generate the flipase plasmid (pCAG-Flipase), flipase was polymerase chain reaction (PCR) amplified from the pOG44 vector (Invitrogen) and the product digested with BsaI and blunt ended (IVS-Flipase-pA) and was then inserted into pCAG vector opened with HindIII and blunt ended.

Transient and Stable Transfection of CCE Cells
All transfections were done using Lipofectamine 2000 according to the standard protocol from Invitrogen; however, cells were incubated with liposome/plasmid complexes for only 3 hours at 37°C/5% CO2. For generation of cells exhibiting Dox inducible expression of shRNAs, CCE ES cells were first transfected with the pCAG-TR-IRESpuro3 vector and selected with 1 µg/ml puromycin (Calbiochem, San Diego, http://www.emdbiosciences.com). Clones were identified by Western blot with mouse anti-tetR monoclonal antibody mix (MoBiTec, Göttingen, Germany, http://www.mobitec.de). A high expressing clone (TR8) was chosen for further transfection with pCAG-FRT-d2EGFP-IRESneo3. Selection was done with Geneticin (Gibco) at a concentration of 600 µg/ml.

Recombination of shRNA was done by cotransfection of the flipase containing vector (pCAG-Flipase) and the vector containing the shRNA (pTER-shRNA-FRT). Cells were then selected with hygromycin (Calbiochem) at a concentration of 165 U/ml. Cells were then further screened for loss of both GFP and G418 resistance.

shRNA, Small Interfering RNA, and Primers
Murine Stat3-specific oligonucleotides: 5' GAT CTG AGT CAC ATG CCA CGT TGG TTC AAG AGA CCA ACG TGG CAT GTG ACT CTT TTT A 3' and 5' AGC TTA AAA AGA GTC ACA TGC CAC GTT GGT CTC TTG AAC CAA CGT GGC ATG TGA CTC A 3'.

Murine Stat3 small interfering (si)RNA: 5' GAG UCA CAU GCC ACG UUG G 3' (XM_109608).

Control siRNA (human β-globin): 5' CAA GAA AGU GCU CGG UGC C 3' (V00497.1 [GenBank] ).

Murine Brf1-specific oligonucleotides: 5' GAT CTG TCC GAA TCC CCT CAC ATG TTC AAG AGA CAT GTG AGG GGA TTC GGA CTT TTT A 3' and 5' AGC TTA AAA AGT CCG AAT CCC CTC ACA TGT CTC TTG AAC ATG TGA GGG GAT TCG GAC A 3'.

Murine Stat3 primer: 5' AGT CAC ATG CCA CGT TGG T 3'.

Murine {alpha}-cardiac actin PCR primers: forward 5' GCT TTG GTG TGT GAC AAT 3' GG, reverse 5' GTG ATA ATG CCA TGT TCA ATG G 3'.

Murine Nkx2.5 PCR primers: forward 5' CGG AAC GAC TCC CAC CTT TAG G 3', reverse 5' GGA ATC CGT CGA AAG TGC CC 3'.

Murine Gata4 PCR primers: forward 5' CGA GAT GGG ACG GGA CAC T 3', reverse 5' CTC ACC CTC GGC CAT TAC GA 3'.

Murine myogenin PCR primers: forward 5' ACA AGC CAG ACT CCC CAC TC 3', reverse 5' GCA CTC ATG TCT CTC AAA CGG T 3'.

Murine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) PCR primers: forward 5' CAC CAC CAA CTG TTA GCC 3', reverse 5' CCT GCT TCAC CAC CTT CTT G 3'.

Murine 18S ribosomal RNA primers: forward 5' CGG CTA CCA CAT CCA AGG AA 3', reverse 5' GCT GGA ATT ACC GCG GCT 3'.

Northern Blot
Total RNA was harvested using TRIzol (Invitrogen). To detect Brf1, Oct4, Rex-1, Fgf-4, and β-actin, Northern blots were hybridized overnight with [{alpha}32P]-dCTP-labeled PCR fragments generated from cDNA of the aforementioned genes (Brf1: nucleotide [nt] 945-1328, number M58566 [GenBank] ; GAPDH: nt 589-1246, number M33197 [GenBank] ; β-actin: nt 516-1144, number NM_008085 [GenBank] ; Oct4: nt 731-1101, number NM_013633 [GenBank] ; Rex-1: nt 687-1059, number NM_009556 [GenBank] ; Fgf-4: nt 250-583, number NM_010202 [GenBank] ) [32]. To analyze expression of shRNA in F3-1-Stat3 and F3-1-Brf1 clones, 30 µg of total RNA were separated on 15% polyacrylamide gels containing 8 M urea (Anamed Elektrophorese GmbH, Darmstadt-Arheiligen, Germany, http://www.anamed-gele.com). Gels were stained with ethidium bromide to check for equal loading before RNA was transferred by electroblotting onto Hybond-N+ (Amersham Biosciences, Piscataway, NJ, http://www.amersham.com) membranes. After UV-cross-linking, filters were hybridized at 45°C in 0.5 M sodium phosphate buffer pH 7.2 containing 1% bovine serum albumin (Fraction V, Sigma-Aldrich), 7% sodium dodecyl sulfate, and 5 mM EDTA using a [{gamma}32P]-ATP labeled Stat3 or Brf1 specific oligonucleotide of 19 nucleotides. Blots were analyzed using the Personal Molecular Imager FX (Bio-Rad, Hercules, CA, http://www.bio-rad.com) and the Quantity One software (Bio-Rad).

Western Blot
The Western blot protocol employed and generation of Brf1 antibodies has recently been described [32]. A monoclonal anti-tetR antibody mix was used to detect expression of the Tet repressor. A monoclonal antibody against {alpha}-tubulin (clone 236-10501; Molecular Probes, Carlsbad, CA, http://probes.invitrogen.com) was used. Stat3 (Cell Signaling Technology, Beverly, MA, http://www.cellsignal.com), Nkx2.5 (clone N-19; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), Gata4 (Clone H-112; Santa Cruz Biotechnology Inc.), and horseradish peroxidase-coupled GAPDH (Abcam, Cambridge, U.K., http://www.abcam.com) polyclonal antibodies were utilized. Alkaline phosphatase-coupled goat-anti-rabbit IgG (SouthernBiotech, Birmingham, AL, http://www.southernbiotech.com) and horseradish peroxidase-coupled goat-anti-mouse IgG (DAKO, Glostrup, Denmark, http://www.dako.com) and rabbit anti-goat IgG (SouthernBiotech) were used as secondary antibodies. Development was performed using CDP-Star (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com) or ECL Advance (Amersham Biosciences).

Microscopy
A Nikon Eclipse TE200 (Nikon Corporation, Tokyo, http://www.nikon.com) microscope equipped with a Hamamatsu digital camera (C4742–95; Hamamatsu Photonics, Hamamatsu City, Japan, http://www.hamamatsu.com) was used, and all pictures were made at a x40 magnification. Beating areas were marked under live-microscopy using Openlab 2.2 Software (Improvision Inc., Lexington, MA, http://www.improvision.com) allowing quantification.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
We were interested to see whether the expression of the post-transcriptional ARE-dependent regulators Brf1, AUF1, and HuR would change under conditions at which ES cells are triggered to differentiate. When LIF was removed, CCE cells lost the compact dome-like colony morphology characteristic of undifferentiated cells and assumed the morphology of differentiated cells (supplemental online Fig. 1A; see also below). In parallel, Brf1 protein levels dropped progressively, whereas expression of HuR and AUF1 was not affected (Fig. 1A). Northern blot analysis suggested that this regulatory effect on Brf1 expression occurred at the mRNA level (Fig. 1B). The same general conclusion was obtained when a second ES line, CGR8, was similarly examined (supplemental online Fig. 1A, 1B). As LIF supports the pluripotent phenotype by activating the transcription factor Stat3, we tested whether this effect would be mimicked by siRNA downregulation of Stat3. Indeed, treatment with Stat3 specific siRNA led to a concomitant downregulation of both Stat3 and Brf1 protein levels, assessed at day 2 (Fig. 1C). As it appears that Brf1 is a target gene of the LIF-gp130-Stat3 pathway, we hypothesized that a reduction of its expression may be linked to a role in ES cell differentiation. To address this possibility, we decided to set up a system in CCE cells, in which a shRNA of choice can be inducibly expressed from a defined locus by the addition of a drug. To achieve this, we used a recently described vector system [31], illustrated in Figure 2A, where the Tet repressor protein binds to the Tet operator within the H1 promoter and acts as a "roadblock" for the RNA polymerase III. Following abrogation of repressor binding by addition of doxycycline, the downstream shRNA sequence is transcribed by RNA polymerase III and terminated by a run of five T residues [33]. CCE cells were first transfected with a plasmid encoding the Tet repressor [34, 35], and after puromycin selection a stable clone was selected, which maintained Tet repressor expression well over the time required for embryoid body formation (data not shown). This clone was further transfected with a construct where GFP, under control of a CAG promoter, is flanked by a Flp-recombinase-target (frt) recombination site [27] and the neomycin resistance gene. This strategy was chosen as it allows the use of a defined locus, marked by frt-GFP-neo and allowing continuous stable gene expression, as a target for a Flp-recombinase to integrate an inducible shRNA. The GFP expressing clone F3-1, which is resistant to G418 and expresses GFP well past day 10 after induction of differentiation (data not shown), serves as the host system for a flipase-encoding plasmid together with the shRNA plasmid containing a frt site and the selectable marker hygromycin B phosphotransferase (hph). After successful recombination, the GFP gene of F3-1 is displaced by hph and inducible shRNA can now be expressed from the H1 promoter together with hygromycin resistance from the CAG promoter (Fig. 2B).


Figure 1
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  		Figure 1. Brf1 expression is controlled by LIF and Stat3. (A): LIF removal experiment. CCE cells were kept for 4 days either with or without LIF as indicated. Protein levels of Brf1, AUF1, HuR, and {alpha}-tubulin are shown. (B): Brf1 mRNA levels monitored by Northern blot; GAPDH served as loading control. (C): Effect of Stat3 siRNA. Cells were plated with or without LIF and, as indicated, treated for 48 hours with Stat3 or β-globin (control) siRNA. Expression of Stat3 and Brf1 was monitored by Western blotting. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LIF, leukemia inhibitory factor; siRNA, small interfering RNA.

 


Figure 2
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  		Figure 2. Schematic view of doxycycline-inducible shRNA expression. (A): The tetR binds to the TO, blocking RNA polymerase III. Addition of Dox removes tetR, and shRNA is produced and processed intracellularly to 21-nucleotide siRNA as described by van de Wetering [31]. (B): A modified Flip-In system is used to recombine a vector containing the shRNA of interest into a defined locus marked by green fluorescent protein (GFP) and flanked by the frt and the neomycin resistance gene. After successful recombination following cotransfection with plasmids encoding flipase and frt-containing Tet-shRNA, cells lose both GFP expression and G418 resistance after dislocation from the promoter but acquire hygromycin resistance and inducible shRNA expression. Frt is marked by an asterisk. Abbreviations: d2EGFP, destabilized enhanced green fluorescent protein; Dox, doxycycline; frt, Flp-recombinase-target; Hygro, hygromycin B phosphotransferase; IRES, internal ribosome entry site; Neo, neomycin phosphotransferase; pA, polyA signal; shRNA, small hairpin RNA; siRNA, small interfering RNA; tetR, Tet repressor; TO, Tet operator.

 
We tested this strategy first by introducing a shRNA targeting Stat3, as successful downregulation of Stat3 would be expected to trigger differentiation [5] and, hence, can be easily monitored. We selected two F3-1-derived Stat3-shRNA clones (F3-1-S2; F3-1-S3), which displayed the expected profile of GFP negative, G418 insensitive, hygromycin resistant, and correct recombination as verified by PCR (data not shown). When cells were treated for 7 days with doxycycline, both clones displayed strong siRNA induction with negligible background expression (Fig. 3A). Notably, Stat3 protein levels were downregulated in both clones tested, whereas doxycycline had no effect on F3-1 control cells (Fig. 3B). The effect was detectable after 1–2 days and was more pronounced in F3-1-S3 cells, apparently reflecting clonal variation. In contrast, LIF removal downregulated Stat3 in all three clones, including F3-1. Consistent with the result from Figure 1, arguing that Brf1 is controlled at least in part by LIF-Stat3, we observed that addition of doxycycline to clones F3-1-S2 and F3-1-S3 and in additional Stat3 shRNA expressing clones also led to downregulation of Brf1 (supplemental online Figs. 2, 3A). As with Stat3 expression, we observed some clonal variation (not shown).


Figure 3
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  		Figure 3. Inducible downregulation of Stat3. (A): After 7 days in culture (+LIF; ±Dox), RNA from indicated cells was isolated and processed for Northern blotting using a 5'-labeled Stat3 oligonucleotide probe. Markers shown on the right include synthetic 21-nucleotide (nt) Stat3 siRNA and 58-nt Stat3 shRNA. (B): Time-course experiment over 7 days in presence or absence of LIF and doxycycline as indicated. Lysates from F3-1 control cells and the two Stat3 shRNA clones F3-1-S2 and F3-1-S3 were analyzed by Western blot against Stat3. GAPDH served as loading control. Abbreviations: as, antisense strand; Dox, doxycycline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LIF, leukemia inhibitory factor; shRNA, small hairpin RNA; siRNA, small interfering RNA.

 
Next, we checked whether addition of doxycycline would trigger the morphological changes characteristic for differentiation. As shown in Figure 4A, both shRNA clones displayed vigorous morphological differentiation upon addition of doxycycline despite the presence of LIF (right panels). To corroborate this finding, we assessed three known markers of undifferentiated ES cells (Oct4, Fgf-4, Rex-1). As expected, their levels dropped following LIF removal in all three clones tested, whereas addition of doxycycline reproduced the same effect as LIF removal in the two shRNA containing clones but not in F3-1 control cells (Fig. 4B). The stem cell marker nanog, measured at day 3, was reduced by LIF removal but not by doxycycline in both F3-1 and F3-1-S2 cells (supplemental online Fig. 3B), consistent with the fact that nanog is not controlled by Stat3 [10]. Together, these data indicated that the morphological changes induced by shRNA were accompanied by reprogramming gene expression of established regulators and show that the frt-GFP locus functions reliably as an acceptor of the shRNA cassette with the expected doxycycline inducible response. We concluded that the system can be used as a tool to investigate other genes with suspected roles in differentiation and concentrated our further studies on Brf1.


Figure 4
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  		Figure 4. Induction of differentiation by Stat3 small hairpin RNA. (A): After 7 days in culture (+LIF/–Dox; –LIF/–Dox; +LIF/+Dox), F3-1-S2 and F3-1-S3 cells were inspected for morphological changes after doxycycline addition. F3-1 cells served as control. Colony morphology of undifferentiated cells is compact (left panels), that of differentiated cells spread-out and extended. Magnification is 40-fold. (B): At the indicated time of culture (+LIF/–Dox; –LIF/–Dox; +LIF/+Dox), RNA from parallel cultures shown in (A) was extracted and processed for Northern blotting. Northern blots probed for three stem cell markers (Fgf-4, Oct4, Rex-1) are shown on the left and quantification on the right. RNA levels were normalized against β-actin; the RNA levels of cells at day 3 +LIF/–Dox were set as 100%. Abbreviations: d, day; Dox, doxycycline; LIF, leukemia inhibitory factor.

 
We used the same strategy as with Stat3 and isolated clones containing doxycycline-inducible Brf1 shRNA. Shown in Figure 5A are Northern blots from two representative clones (F3-1-B9 and F3-1-B14). Again, siRNA is strongly induced in both clones with negligible background expression and no signal in control cells. A parallel Western blot at day 3 showed that doxycycline led to downregulation of Brf1 protein (Fig. 5B). However, treatment with doxycycline for up to 7 days produced no morphological changes (data not shown). We wondered whether Brf1 shRNA might affect differentiation at later stages. Thus, we produced EBs and cultured these further by allowing them to attach. Unexpectedly, EBs from F3-1-B14, but not from control cells, displayed a markedly altered morphology when cultured with doxycycline. Shown in Figure 6 are representative examples photographed at day 10. Although EBs from control cells appeared as compact cellular masses surrounded by a halo of outgrowing cells, doxycycline treatment of F3-1-B14 cells led to an apparent loosening of the central mass, increased outgrowth, and formation of satellite "microbodies." Interestingly, these changes were observed when doxycycline was present until day 4 but not when added after day 4. These data argue that early but not later changes in Brf1 levels affect the architecture of an embryoid body.


Figure 5
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  		Figure 5. Doxycycline-inducible downregulation of Brf1. (A): After 7 days in culture (+leukemia inhibitory factor [LIF]; ±Dox), RNA from indicated cells was isolated and processed for Northern blotting using a 5'-labeled Brf1 oligonucleotide probe. Markers shown on the right include synthetic 21-nucleotide (nt) Brf1 siRNA and 58-nt Brf1 shRNA. (B): After 3 days in culture in the presence of LIF (±Dox) as indicated, Brf1 was examined by Western blot analysis. {alpha}-Tubulin served as loading control. Abbreviations: as, antisense strand; Dox, doxycycline; shRNA, small hairpin RNA; siRNA, small interfering RNA.

 


Figure 6
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  		Figure 6. Embryoid body morphology. Morphology of embryoid bodies (EBs) at d10 from F3-1 and F3-1-B14 cells either kept with or without doxycycline from day 0 to day 18. Additionally, F3-1-B14 EBs were either kept with doxycycline during the first 4 days or from day 4 until the end of the experiment. Abbreviations: d, day; Dox, doxycycline.

 
It is well established that cardiomyocyte formation occurs spontaneously in cultured EBs, which is easily recognizable under microscopy as "beating areas" or detectable biochemically by measuring cardiac specific markers such as the transcription factor Nkx2.5 [36, 37]. We were surprised to observe that induction of Brf1 shRNA by doxycycline led in F3-1-B14 cells to a substantial increase in number and size of beating areas (Fig. 7). We also noticed the elevated background in these cells (140 areas) compared with the F3-1 control cells, which may reflect some leakiness. Addition of doxycycline had also a low but reproducible effect on F3-1 control cells, an effect we cannot explain but which is negligible compared with the very strong doxycycline effect in Brf1 shRNA expressing cells. To substantiate this effect of cardiomyocyte formation, we examined the expression of various markers. Eighteen days following addition of doxycycline, Nkx2.5 and Gata4 expression was weak in controls but strong in F3-1-B14 cells as shown by Western blot and PCR analysis (Fig. 8A and supplemental online Fig. 4A). The skeletal muscle marker myogenin was not expressed in ES cells (supplemental online Fig. 4A). Additionally, we monitored the cardiac marker {alpha}-cardiac actin by semiquantitative reverse transcription-PCR at days 4 and 18 and observed weak induction in F3-1 at day 18 and higher levels in response to doxycycline in F3-1-B14 (Fig. 8B, supplemental online Fig. 4A). These findings are in agreement with the microscopic data and indicate that the appropriate change in gene expression took place.


Figure 7
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  		Figure 7. Increase of beating areas within embryoid bodies (EBs). Embryoid bodies from F3-1 and F3-1-B14 cells were plated with or without doxycycline into 24-well plates. For each cell and condition, approximately 720 EBs were plated, and beating areas were counted at day 18. Upper panel: Shown in red are beating areas. Note that a single EB may contain more than one beating area, and beating areas may become confluent with time. Numbers indicate beating areas from 720 bodies plated. Lower panel: The size of beating areas from three experiments was quantified, averaged, and is shown with standard error of the mean. Abbreviation: Dox, doxycycline.

 


Figure 8
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  		Figure 8. Brf1 downregulation is correlated with stimulation of cardiomyocyte formation. (A): Control cells (F3-1) and Brf1 small hairpin RNA cells (F3-1-B14) were cultured in the absence of LIF with or without doxycycline for 18 days. Western blot analysis was performed for Nkx2.5, Gata4, and GAPDH. Also shown are undifferentiated F3-1 and F3-1-B14 cells grown in LIF and mouse heart as controls. (B): Semiquantitative RT-PCR of the cardiac marker {alpha}-cardiac actin at indicated days after LIF removal. GAPDH served as control. (C): F3-1 and F3-1-B14 embryoid bodies (EBs) were plated into 48-well plates at two bodies per well and cultured with or without doxycycline. EBs were checked daily for onset of beating and scored. The data are shown as percentage of beating bodies to total plated. Upper panel: F3-1 and F3-1-B14 EBs cultured with doxycycline for the total length of the experiment. For each time point, at least three experiments are averaged, and standard errors of the mean are shown. Lower panel: As above, but doxycycline was present where indicated only from day 0 to day 4. For each time point the average and standard errors of the mean of at least three experiments are shown. Abbreviations: d, day; Dox, doxycycline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LIF, leukemia inhibitory factor; RT-PCR, reverse transcription-polymerase chain reaction.

 
It was of interest now whether downregulation of Brf1 would affect both the kinetics and/or the magnitude of cardiomyocyte formation and if doxycycline has to be present throughout the experiment. Thus, we plated two EBs into each well of a 48-well plate, which allowed daily inspection of individual beating bodies and scoring of the percentage of beating. As shown in Figure 8C (upper panel), beating body formation in doxycycline-treated F3-1-B14 cells was first observed on day 9 and reached a plateau at approximately day 14. In the absence of doxycycline, beating body formation was observed in the range of control cells. Note that, in these experiments, the number of beating bodies was scored rather than beating areas as in Figure 7. The difference between the two stimulation indices is explained by the fact that a given beating body generally contains more than one beating area that is also of larger size. As with the morphological changes (Fig. 6), this effect on beating bodies required the presence of doxycycline early (day 0–day 4) in the experiment (Fig. 8C, lower panel), and no effect was seen when the drug was added from day 4 onwards (data not shown).


   DISCUSSION
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 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
This work describes a Tet-based inducible RNA interference system, which allows the downregulation of any transcript of choice following flp recombinase mediated DNA recombination in ES cells. The power of the system is documented in experiments in which Stat3, the known regulator of ES cell differentiation, was targeted. In addition, it allowed assignment of a novel function to the post-transcriptional regulator Brf1 in ES-cell-derived cardiomyocyte formation. Clone F3-1, carrying both the Tet repressor and an frt-GFP-marked locus that integrated into genomic regions favorable for long-term gene expression (even long after onset of differentiation), is central to our system. Establishment of the doxycycline-inducible shRNA system in ES cells is of significance in view of the reported difficulties in establishing the Tet system in ES cells, thought to be due to a tendency in ES cells toward gene silencing. The efficiency of successful recombination assessed by the three selection markers GFP, neomycin, and hygromycin resistance and confirmatory PCR was remarkably high and approached approximately 50% with Stat3 and 70% with Brf1 plasmids (data not shown). With Stat3, we observed strong doxycycline-dependent induction of 21 nt siRNA formation, negligible background, and a strong morphological and biochemical response including signs of differentiation and appropriate changes of the stem-cell markers Oct4, Fgf-4, and Rex-1. Niwa et al. [5] have previously described an ES system where tetracycline induction triggered differentiation via the formation of a dominant-negative form of Stat3 protein expressed from a chromosomal site. As our system compared well with this dominant-negative system, we concluded that the RNA interference approach is suitable to probe the possible function of other suspected regulators and concentrated on Brf1, a regulator of mRNA turnover.

Brf1, originally discovered as an immediate-early gene [38], is a member of a small family of RNA-binding proteins with a conserved and characteristic CCCH zinc-finger domain recognizing AREs in the 3'UTR and promoting mRNA decay. We reported recently that the mRNA decay promoting activity of Brf1 is negatively regulated by phosphorylation via protein kinase B, which promotes complex formation to 14-3-3 [39]. Target mRNAs of Brf1 are not known but play a role in development, as mice lacking both alleles die at day 11 [40]. That Brf1 is controlled at least in part by Stat3 was suggested by the LIF removal experiment where Brf1 levels dropped (Fig. 1) and supported by transient transfection of Stat3 siRNA, which led to concomitant downregulation of Stat3 and Brf1 (Fig. 1C). In several Stat3 shRNA clones tested, doxycycline led to downregulation of Brf1 protein and transcripts (supplemental online Figs. 2, 3A). These results are consistent with DNA microarray data of others who reported Brf1 (Zfp36L1) downregulation after LIF removal [41, 42]. That the effect in some clones was less intense than that following LIF removal suggests that not all effects on gene expression by LIF removal are mediated by Stat3. Although a Stat3 site in the promoter region of Brf1 has been reported, support for additional control elements was provided in a recent paper on the human ES cell transcriptome where chromatin immunoprecipitation was combined with DNA microarray analysis to identify promoters of transcripts expressed in stem cells that are co-occupied by Oct4, nanog, and Sox2. Interestingly, both Stat3 and Brf1 were among the 353 transcripts that fulfilled these criteria [14]. Oct4 and Sox2 bind the Brf1 promoter 2497 nt upstream of the transcription initiation site and nanog at position 1733.

When we tested the effects of Brf1 shRNA induction in ES long-term cultures, we made the unexpected observation that the morphology of EBs, the number of beating areas within an embryoid body, and also the total number of beating areas was dramatically altered. We do not know whether this reflects increased survival or enhanced promotion into myogenic tissue. A time-course revealed that downregulation of Brf1 did not change the kinetics of cardiomyocyte formation, but rather amplified the spontaneous formation known to occur in cultures of murine and human EBs [37]. It is significant that both effects on general EB morphology and on cardiomyocyte formation do not occur when Brf1 is downregulated only at day 4 or later in EB cultures. This supports the hypothesis that a transient early change in Brf1 levels may set the stage for downstream processes executed by Brf1 target genes. Our data suggests the following model: Brf1, a target of Stat3, is a suppressor of differentiation expressed in undifferentiated pluripotent stem cells. Reducing its levels physiologically via reduction of LIF-Stat3 signaling or by inducible RNA interference leads to changes in embryoid body architecture and enhancement of cardiomyocyte formation. Whether generation of other cell types is similarly enhanced remains to be examined. As Brf1 regulates ARE-dependent mRNA turnover, the model predicts the existence of ARE-containing transcripts favoring cardiomyogenesis, a possibility that can now be addressed by DNA microarray analysis and suitable follow-up experiments with candidate genes. Overexpression of Brf1 would be expected to suppress cardiomyogenesis, which, however, could not be tested, as transfected Brf1 is toxic to ES cells (supplemental online Fig. 5). If, as may well be the case, Brf1 also regulates cardiomyocyte formation in human ES cells, the protocol described here may prove useful in regenerative cardiology for replacement therapy. In addition, transgenic mice eventually generated from the lines described here might circumvent the lethality of Brf1 knockouts [40] and allow the downregulation of Brf1 in adult tissue to reveal further functional aspects of this post-transcriptional regulator.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
The authors indicate no potential conflicts of interest.


   REFERENCES
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 Abstract
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 Materials and Methods
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 Disclosure of Potential...
 References
 

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J. Wang, T. W. Theunissen, and S. H. Orkin
Site-directed, virus-free, and inducible RNAi in embryonic stem cells
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