PRDM16/MEL1: a Novel Smad Binding Protein Expressed in Murine Embryonic Orofacial Tissue ⁎ Dennis R

Biochimica et Biophysica Acta 1773 (2007) 814–820 www.elsevier.com/locate/bbamcr

PRDM16/MEL1: A novel Smad binding protein expressed in murine embryonic orofacial tissue ⁎ Dennis R. Warner , Kristin H. Horn, Lisa Mudd, Cynthia L. Webb, Robert M. Greene, M. Michele Pisano

Department of Molecular, Cellular, and Craniofacial Biology, University of Louisville Birth Defects Center, 501 South Preston Street, Suite 301, Louisville, KY 40292, USA

Received 24 October 2006; received in revised form 14 March 2007; accepted 15 March 2007 Available online 28 March 2007

Abstract

TGFβ signaling regulates central cellular processes such as proliferation and extracellular matrix production during development of the orofacial region. Extracellular TGFβ binds to cell surface receptors to activate the nucleocytoplasmic Smad proteins that, along with other transcription factors and cofactors, bind specific DNA sequences in the promoters of target genes to regulate their expression. To determine the identity of Smad binding proteins that regulate TGFβ signaling in developing murine orofacial tissue, a yeast two-hybrid screening approach was employed. The PR-domain containing protein, PRDM16/MEL1 was identified as a novel Smad binding protein. The interaction between PRDM16/MEL1 and Smad 3 was confirmed by GST pull-down assays. The expression of PRDM16/MEL1 was detected in developing orofacial tissue by both Northern blot and in situ hybridization. PRDM16/MEL1 was constitutively expressed in orofacial tissue on E12.5–E14.5 as well as other embryonic tissues such as heart, brain, liver, and limb buds. Taken together, these results demonstrate that PRDM16/MEL1 is a Smad binding protein that may be important for development of orofacial structures through modulation of the TGFβ signaling pathway. © 2007 Elsevier B.V. All rights reserved.

Keywords: TGFβ; Smad; PRDM16; MEL1; Orofacial

1. Introduction specific nucleotide sequences within the promoter of target genes [6,7]. The Smad/CBP/p300 complex binds to additional Members of the transforming growth factor β (TGFβ) family proteins such as histone acetyltransferases (HAT) and RNA of secreted cytokines modulate multiple developmental pro- polymerase II [8,9]. Although Smad 3 has intrinsic DNA cesses critical to orofacial ontogeny [1–4]. Extracellular TGFβ binding ability, the affinity is low, and Smad 2 has even weaker ligands bind to a type II TGFβ receptor (TβRII) to promote DNA affinity because of the presence of an amino acid insert heterodimerization with a TGFβ type I receptor (TβRI), that within the DNA binding domain [10]. Therefore, the interaction then phosphorylates Smads 2 and 3, thus exposing the Smad 4 between Smads and other transcription factors and cofactors is binding surface and the nuclear import signal [5]. In the nucleus, essential for high affinity, specific binding of Smads to DNA. Smads bind to additional transcription factors/cofactors such as The identification of these cofactors is required to define tissue FAST1 and CBP/p300, creating a complex which then binds to and cell-specific effects mediated by TGFβ. The secondary palate originates from the maxillary processes on days 11.5–12.5 in mice with the initial phase of growth Abbreviations: PRDM16, PR-domain containing protein-16; MEL1, MDS1/ characterized by cell proliferation and extracellular matrix β β Evi1-like protein; TGF , transforming growth factor ; GST, glutathione production. Growth proceeds through E13.5 and by E14.5 the S-transferase; CBP, CREB binding protein; DIG, digoxigenin; kDa, kiloDalton; kb, kilobase; E, embryonic day; GCNF, germ cell nuclear factor palatal processes reorient from their position alongside the ⁎ Corresponding author. Tel.: +1 502 852 3823; fax: +1 502 852 8309. tongue to meet and fuse above the tongue thereby separating the E-mail address: [email protected] (D.R. Warner). oral and nasal cavities. Perturbations of this sequence of events

0167-4889/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2007.03.016 D.R. Warner et al. / Biochimica et Biophysica Acta 1773 (2007) 814–820 815 will lead to a palatal cleft. TGFβ is involved in each process of 2.4. PRDM16/MEL1 riboprobe palate and orofacial development from control of cell proliferation [1], extracellular matrix production [4], and fusion For in situ hybridization and Northern blotting experiments, a 508-bp EcoRI–SacII restriction fragment of murine PRDM16/MEL1 corresponding to of the shelves [11]. In order to identify novel Smad binding bases 1963–2470 was subcloned from the yeast two hybrid vector, pGADT7, proteins which would better define the means by which TGFβ into the transcription vector, pSPT-18 (Roche Diagnostics, Indianapolis, IN). regulates this multiplicity of developmental processes in Sense and antisense riboprobes were prepared with the DIG RNA labeling kit orofacial tissue, a yeast two-hybrid screen was performed according to the manufacturer's instructions (Roche Diagnostics) and purified using the MH2 domain of Smad 3 as a “bait” to screen a cDNA by multiple LiCl precipitations. Yields were determined with a NanoDrop ND- – 1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE) and the expression library prepared from orofacial tissue from E11.5 integrity of the riboprobes was assessed by agarose gel electrophoresis. E13.5 murine embryos. Several novel Smad binding proteins Preparations that yielded a single transcript were used for these studies. have recently been reported [12–14]. Here we report the identification of PRDM16 (also called MEL1) as a Smad 2.5. Northern blotting binding protein and offer a preliminary analysis of its expression pattern in developing murine orofacial tissue and Total RNA was purified from dissected palatal tissue using the RNeasy μ discuss its possible function in TGFβ signaling. Protect RNA purification system (Qiagen, Inc., Valencia, CA). Ten g was separated on a 1.1% agarose-formaldehyde gel and transferred to a nylon membrane (Hybond-N+, Amersham Biosciences-GE Healthcare Bio-Sciences 2. Methods Corp. Piscataway, NJ) according to established procedures [16]. Following capillary transfer and UV crosslinking, the membrane was blocked with DIG 2.1. Animals Easy Hyb (Roche Diagnostics), hybridized to a DIG-labelled PRDM16/MEL1 antisense riboprobe (100 ng/ml, overnight, at 65 °C), washed and processed ICR mice were obtained from Harlan Laboratories (Indianapolis, IN) and with the DIG Wash and Block buffer set (Roche Diagnostics), and developed housed at an ambient temperature of 22 °C with a 12-h light/dark cycle with with CDP-Star (Roche Diagnostics). Membranes were exposed to film or access to food and water ad libitum. For timed matings, mature male and female images captured with a Kodak Image Station 440-CF and analyzed with the mice were housed overnight and the presence of a vaginal plug the following Kodak 1D 3.6 software package (Kodak, Rochester, NY). To assess the equality morning was taken as evidence of mating and designated embryonic day 0.5 of RNA loading and transfer, membranes were hybridized to a DIG-labelled (E0.5). Pregnant mice were euthanized on various days of gestation, embryos 338-nucleotide β-actin antisense riboprobe (Ambion, Inc., Austin, TX) which removed, and palatal tissue microdissected and prepared for either Western detects a 2.1-kb transcript. Densitometric analysis was performed with NIH blotting or Northern blotting as described below. Image 1.62f.

2.2. Yeast two-hybrid assay 2.6. In situ hybridization

Details of the yeast two-hybrid screen, including construction of the Murine embryos of the appropriate gestational age were removed and tissue expression library and bait vectors have been described in detail previously [12]. dissected (primary and secondary palates and maxilla), crosslinked overnight In brief, pGBKT7-Smad 3 MH2 was transformed into Saccharomyces with 4% paraformaldehyde-PBS, rinsed with PBT (PBS+0.1% Tween-20), and cerevisiae (strain AH109) along with the murine orofacial expression library bleached in 6% (v/v) H2O2 for 1 h. Tissue was then digested with 50 μg/ml in pGADT7. Transformants were plated on yeast medium in the absence of proteinase K for 10 min and post-fixed with 4% paraformaldehyde/0.2% histidine, leucine, tryptophan, and adenine and in the presence of 2.5 mM 3- glutaraldehyde for 20 min at room temperature. Tissue was then incubated amino-1,2,4-triazole and 20 μg/ml X-α-gal according to instructions provided overnight in a solution that contained 1 μg/ml DIG-labelled PRDM16/MEL1 by the manufacturer (Matchmaker System 3, Clontech Laboratories, Mountain riboprobe at 65 °C, washed thoroughly and incubated overnight again with View, CA). Approximately 5×105 yeast transformants were screened, and blue alkaline phosphatase-conjugated anti-digoxigenin antibody (1:2000 dilution; colonies maintaining the His+/Leu+/Trp+/Ade+ phenotype were sequenced and Roche Diagnostics). Color development was initiated by the addition of verified for interaction with Smad 3 MH2. 375 μg/ml nitro blue tetrazolium chloride, 475 μg/ml 5-Bromo-4-chloro-3- indolyl phosphate (added from a 50× stock solution, Roche Diagnostics) and 2.3. Glutathione S-transferase (GST) pull-down assay 2 mM tetramisole.

In vitro interaction between PRDM16/MEL1 and Smads was demonstrated 2.7. Luciferase reporter assay by a GST pull-down assay using GST-Smad fusion proteins and in vitro translated, [35S]methionine-labeled PRDM16/MEL1. The preparation and For luciferase reporter assays, primary cultures of murine embryonic purification of GST-fusion protein constructs for Smads 1, 2, 3, 4, and 7 has maxillary mesenchyme (MEMM) were established as previously reported [17] been described previously [13]. GST-GCNF was prepared in the same way and plated at a density of approximately 7,000 cells/cm2 in a 6-well tissue except that it was induced with 300 μM IPTG for 3 h at 37 °C. [35S]methionine- culture plate and incubated until they were ∼70% confluent. Each well was labeled PRDM16/MEL1 was prepared by in vitro translation using the TNT T7 transfected with DNA plasmids (1 μg p3TP-lux and 50 ng pRL-CMV) and Coupled Reticulocyte Lysate System (Promega Corp., Madison, WI). One μgof siRNAs (100 nM PRDM16/MEL1 or control) with the lipophilic transfection purified GST-Smad bound to glutathione–Sepharose (Amersham Biosciences, reagent, Effectene (Qiagen Inc.). p3TP-lux is a TGFß reporter construct that Piscataway, NJ) was mixed with 5 μl[35S]methionine-labeled PRDM16/MEL1 drives the Smad-dependent expression of firefly luciferase and pRL-CMV is a for 1 h at 4 °C in binding buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 0.1% NP- control plasmid for transfection efficiency that contains the cDNA for Renilla

40, 1 mM dithiothreitol, 10% glycerol, 1 mM EDTA, 2.5 mM MgCl2,1μg/ml luciferase (Promega Corp., Madison, WI). siRNAs targeting murine PRDM16/ aprotinin, and 1 μg/ml leupeptin). The complex was washed three times with MEL1 and, separately, a non-targeting control siRNA were obtained from binding buffer and immobilized proteins were liberated by heating in 2× Dharmacon, Inc. (Lafayette, CO). PRDM16/MEL1 ONTARGETplus™ Laemmli sample loading buffer [15] and separated on an 8–16% polyacrylamide siRNAs are composed of a pool of four individual siRNAs, specific to different gel under reducing/denaturing conditions. Gels were fixed for 30 min with 50% regions of their respective mRNA, and are designed to minimize off-target methanol:10% acetic acid and exposed to Kodak X-Omat AR film for 2 days at effects. Forty-eight hours post-transfection, cells were rinsed with fresh culture −80 °C. Following autoradiography, dried gels were stained with Coomassie medium and treated with 2 ng/ml porcine TGFβ1 (R&D Systems Inc., blue to compare the loading efficiency for each sample. Minneapolis, MN) for an additional 24 h. Each well was rinsed twice with PBS, 816 D.R. Warner et al. / Biochimica et Biophysica Acta 1773 (2007) 814–820

3. Results

3.1. Identification of PRDM16/MEL1 as a Smad binding protein

TGFβ signaling leads to disparate cellular outcomes in the developing embryo depending on the tissue and stage of gestation. Specificity is achieved through the interaction of Smads with cell- and stage-specific proteins. The goal of this study was to identify Smad binding proteins that are required for TGFβ regulation of orofacial development. A yeast two-hybrid screen with the MH2 domain of the TGFβ-regulated Smad 3 and an expression library constructed from E11.5 to E13.5 embryonic murine orofacial RNA was performed. Several unique Smad binding proteins were identified and their identities and potential role in TGFβ signaling have been recently published [12–14,19]. In the current study, evidence is provided to demonstrate that the PR domain containing protein Fig. 1. Results of a yeast two hybrid assay demonstrating interaction between Smad 3 and PRDM16/MEL1. Cultures of S. cerevisiae were co-transformed PRDM16 [PR domain containing protein-16; also known as with pGADT7-PRDM16/MEL1 and pGBKT7-Smad3 MH2, pGBKT7-Smad3 MEL1 [20]] is a Smad binding protein. PRDM16/MEL1 bound (full-length), pGBKT7-lamin C, or pGBKT7 and plated under low stringency to the MH2 domain and, to a lesser extent, full-length Smad 3 in conditions. Colonies that proliferated were streaked and grown under high a yeast two-hybrid assay (Fig. 1). No interaction was observed α stringency conditions and in the presence of X- -gal (shown). Yeast growth was with a control protein, Lamin C. When PRDM16/MEL1 was observed only when pGADT7-PRDM16/MEL1 was co-transformed with pGBKT7-Smad3 MH2 and, to a lesser extent, pGBKT7-Smad3 (full-length) co-expressed with the empty bait vector, pGBKT7, no yeast demonstrating a specific interaction between PRDM16/MEL1 and Smad 3. growth was observed, demonstrating that PRDM16/MEL1 alone was unable to activate transcription in this assay. The lysed, and luciferase activity measured using the Dual Luciferase Reporter PRDM16/MEL1 clone isolated from this screen contains Assay System (Promega). Firefly luciferase activity is reported after normal- nucleotide bases 1,957–3,825 (amino acids 655–1,275) of the ization to control, Renilla, luciferase activity for each sample. Each condition coding region of the published murine PRDM16/MEL1 se- was assayed in triplicate and the experiment was performed twice with quence (NCBI identifier NM_027504) and approximately comparable results. To assess the efficiency of PRDM16/MEL1 mRNA ′ 35 knockdown, duplicate wells were analyzed by RT-PCR. The sequences of the 600 bp of 3 untranslated sequence. [ S]-labeled PRDM16/ individual siRNAs in the non-targeting control were (sense strand): 5′- MEL1, in vitro translated and used for GST pull-down UGUUUACAUGUCGACUAA-3′;5′-UGGUUUACAUGUUUUCUGA-3′; contained the repressor domain, DNA binding domain-2, and 5′-UGGUUUACAUGUUUUCCUA-3′;and5′-UGGUUUACAUGUUGU- the activation domain, but was missing the PR domain and GUGA (sequences provided by Dharmacon, Inc.). DNA binding domain-1 as well as the proline-rich region (see Fig. 2). The interaction between PRDM16/MEL1 and Smad 3 2.8. RT-PCR was confirmed with a GST pull down assay (Fig. 3). Purified, Total RNA was purified from siRNA-transfected MEMM cells using the recombinant GST-Smad fusion proteins bound to glutathione 35 RNeasy Protect purification system (Qiagen Inc.). cDNAs were synthesized beads were incubated with in vitro translated, [ S]-methionine using the SuperScript first strand cDNA synthesis system (Gibco Invitrogen labeled PRDM16/MEL1 and isolated by centrifugation. Fig. 3A Corp., Gaithersburg, MD) and used as the template in real-time PCR assays demonstrates that PRDM16/MEL1 binds full-length Smads 2, with probe:primer pairs purchased from Applied Biosystems (Foster City, CA) and analyzed using the ABI Prism 7000 Sequence Detection System (Applied 3, and 7 (lanes 4, 6, and 10, respectively). Minimal binding was Biosystems). All data were normalized to the amplification signal from observed with full-length Smads 1 and 4 and a mutant Smad 3 GAPDH and fold-change values were determined by the ΔΔCt method [18]. that is missing the MH2 domain (Smad 3 ΔMH2) (lanes 2, 8,

Fig. 2. PRDM16/MEL1 domain structure. Murine PRDM16 (MEL1) contains at least 6 distinct amino acid domains, as indicated. It contains the characteristic PR domain (PRD) and two DNA binding domains (DBD1 and DBD2). In addition, it contains a proline-rich domain (PRR), a repressor domain (RD), and an acidic domain (AD). The PRDM16/MEL1 cDNA clone isolated from the yeast two hybrid screen with Smad 3 MH2 as the bait codes for a truncated protein that starts at valine-655 (indicated by the arrow) and continues through the carboxyl terminus (leucine-1275). D.R. Warner et al. / Biochimica et Biophysica Acta 1773 (2007) 814–820 817

Fig. 3. GST pull-down assay demonstrating interaction between Smad 3 and PRDM16/MEL1. In vitro translated, [35S]-labeled PRDM16/MEL1 was prepared and mixed with glutathione-Sepharose-immobilized GST-Smads. GST-Smad bound [35S]-PRDM16/MEL1 was detected by SDS-PAGE and autoradiography. Input (lane 1, panels A and B) represents the [35S]-PRDM16/MEL1 signal from 10% of the amount added to each sample. Panel A: PRDM16/MEL1 bound to full-length TGFß- regulated Smad 2 (lane 4) and Smad 3 (lane 6), and the inhibitory Smad 7 (lane 10). Weak or no interaction was observed with full-length Smad 1 (lane 2) and Smad 4 (lane 8) and a mutant Smad 3 missing the MH2 domain (Smad 3 ΔMH2, lane 12). A higher extent of binding was found with the MH2 domains of Smad 1 (lane 3), Smad 2 (lane 5), and Smad 3 (lane 7). Lane 13 is GST alone and represents background binding. Panel B: additional control demonstrating only background binding to an unrelated protein, GCNF (lane 3). and 12, respectively). Further, no binding to a control protein, robe (Fig. 4A). A major 10-kb transcript was detected in each GST-GCNF or GST alone was observed (Fig. 3B, lanes 3 and sample, with minor 7 kb and 5 kb transcripts also present with no 4). A higher extent of binding was found with the MH2 domains apparent differences in levels of expression. The slightly of Smads 1, 2, and 3 (lanes 3, 5 and 7, respectively). These data weaker-intensity band on E14.5 is likely due to loading and/or demonstrate that PRDM16/MEL1 interacts with the MH2 do- membrane transfer differences, since both densitometric analy- main of these Smads. sis and chemiluminescent detection with a CCD camera revealed To determine the temporal expression pattern of PRDM16/ no statistically significant differences in the ratios of the MEL1 mRNA, purified total RNA from microdissected PRDM16/MEL1 transcript to that of β-actin. RNA from tissue orofacial tissue from E12.5 to E14.5 murine embryos (three microdissected from E14.5 mouse embryo heart, liver, brain, independent litters from each day) was analyzed by Northern and limb bud was also analyzed by Northern blotting with the blotting with a PRDM16/MEL1 antisense, DIG-labelled, ribop- PRDM16/MEL1 riboprobe (Fig. 4B). PRDM16/MEL was expressed in each of these tissues, with signals for all three splice variants also in evidence.

3.2. Spatial expression of PRDM16/MEL1 mRNA in the primary and secondary palate

In situ hybridization experiments with a PRDM16/MEL1 antisense riboprobe detected expression in both the primary and secondary palates (Fig. 5). In addition, PRDM16/MEL1 transcripts were detected in the nasal septum and upper lip. Interestingly, the expression of PRDM16/MEL1 in the secondary palate was mainly in the anterior one-half with little expression detected in the posterior region of the palate. No signal was observed when a PRDM16/MEL1 sense riboprobe was used (not shown).

β Fig. 4. Northern blot analysis of RNA purified from isolated developing murine 3.3. Effect of PRDM16/MEL1 on TGF reporter activation embryonic orofacial tissue. To determine the temporal expression of PRDM16/ MEL1 mRNA transcripts, E12.5–E14.5 orofacial tissue (Panel A) or additional To determine the role of PRDM16/MEL1 in Smad-dependent tissues from E14.5 embryos (Panel B) was dissected and total RNA purified and TGFβ signaling, an siRNA-based strategy was used to reduce analyzed by Northern blotting with a PRDM16/MEL1 riboprobe. To assess the the level of PRDM16/MEL1 mRNA in primary cultures of loading/transfer efficiency, the RNA blots were also probed with a β-actin- specific riboprobe, which detects a 2.1-kb transcript. A transcript for the 10-kb murine embryonic mesenchyme (MEMM) cells derived from species was detected on the 3 days of gestation critical for development of the E13.5 embryonic orofacial tissue. The effect on TGFβ signaling secondary palate (Panel A). Based upon densitometric analysis and normal- was then determined in reporter assays with the plasmid, p3TP- ization to β-actin mRNA levels, there was no significant difference in the level lux, that encodes the plasminogen activator inhibitor promoter of expression across this developmental window. In addition, transcripts for upstream of firefly luciferase. p3TP-lux was co-transfected with PRDM16/MEL1 could be detected in embryonic heart, liver, brain, and limb β bud tissue, with possible higher expression of the 7-kb transcript in brain tissue PRDM16/MEL1 siRNAs and the effect on TGF -mediated (Panel B). The data presented are representative of three independent luciferase synthesis determined (Table 1). Based upon real-time preparations of RNA from each day of gestation. RT-PCR assays, 100 nM PRDM16/MEL1 siRNA reduced the 818 D.R. Warner et al. / Biochimica et Biophysica Acta 1773 (2007) 814–820

Smads. Binding of PRDM16/MEL1 to both regulatory Smads and to inhibitory Smads would at first appear contradictory. However, inhibitory Smads are regulated at the level of transcription by TGFβ itself [22]. Therefore, only after prolonged signaling would Smad 7 levels increase and potentially disrupt the interaction between PRDM16/MEL1 and regulatory Smads. PRDM16/MEL1 was first cloned by Mochizuki et al. and termed MEL1 because of its similarity to MDS1/Evi1 (MDS1/ Evi1-like) which share 56% amino acid identity [23]. Both proteins have a PR domain, a subclass of the SET domain, so called for the presence of the regulatory domain present in PRD1-BF-1 and RIZ1 that may be important in the regulation of oncogenic potential, particularly in certain types of leukemia in which PRDM16/MEL1 is aberrantly expressed [24]. The PRDM gene family contains at least 13 members, each containing a PR domain and a DNA binding domain which is necessary for direct DNA binding and function [25]. Based upon Northern blotting experiments, several PRDM16/ Fig. 5. In situ hybridization of embryonic murine orofacial tissue with a MEL1 transcripts have been observed [8 kb and 5 kb], leading biotinylated PRDM16/MEL1 riboprobe. Whole-mount in situ hybridization of palate and maxilla of E13.5 murine embryo with PRDM16/MEL1 antisense to the presumption that alternative splice variants exist [23,26]. riboprobe. Expression of PRDM16/MEL1 mRNA was detected in both the Indeed, two major isoforms of PRDM16/MEL1 have been primary (pp, green arrow) and secondary palates (ps, yellow arrows) in addition identified, a 170-kDa, long form (MEL1), and a 150-kDa short to the nasal septum (ns, yellow arrowhead) and upper lip (ul, green arrowhead). form (MEL1S) that is missing most of the PR domain [27].As The anterior one half of the secondary palate stained more intensely than the shown in Fig. 4, several PRDM16/MEL1 transcripts were posterior one half. expressed in murine embryos, including a major transcript of 10 kb with minor transcripts of 7 kb and 5 kb. Whether these are the same transcripts as previously observed in human tissue [23] level of mRNA an average of 2.2-fold compared to a control or whether these represent alternative forms not found in human siRNA. The relative luciferase activity following TGFβ stimu- tissue is unknown. There appeared to be no temporal regulation lation was ∼5-fold for control samples and 7.3-fold for of any of the splice variants. RT-PCR analysis of embryonic PRDM16/MEL1 siRNA-transfected MEMM cells. Although orofacial tissue with oligonucleotide primers targeting the exon suggestive that PRDM16/MEL1 knockdown increases TGFβ 1–2 boundary (the region coding for the PR domain) were signaling, the difference was not statistically significant (t-test, successful in amplifying cDNA from these same samples, p=0.238). Based upon similarities to both MDS1/Evi1 and suggesting that the long form is expressed (not shown). These Evi1, both known to affect TGFβ signaling, it is likely that the same primers, and primers spanning the exon 13–14 boundary weak knockdown of PRDM16/MEL1 by the siRNA was were also used to assess the knockdown efficiency of PRDM16/ insufficient to significantly alter TGFβ signaling. Western MEL1 with targeted siRNAs (Table 1). blotting with both Smad 2 and anti-phospho-Smad 2 antibodies PRDM16/MEL1 is similar in structure to Evi1 (minus the of parallel wells did not detect any change in the amount of PR domain) which has been previously demonstrated to phospho-Smad 2 as a result of PRDM16/MEL1 siRNA trans- bind the MH2 domain of Smad 3 through its DNA binding fection (not shown). domain-1 (Zinc finger domain-1) and repress TGFβ signaling

4. Discussion Table 1 The diverse outcomes resulting from activation of the TGFβ Effect of PRDM16/MEL1 knockdown on TGFβ-mediated transcription pathway in embryonic tissue is determined by the makeup of Relative luciferase activity mRNA knockdown proteins assembled into the transcriptional complex and bound (fold change) (fold change) β to target gene promoters. Therefore, to dissect TGF signaling Control siRNA 5.0±0.8 – specificity, identification of the individual proteins that are in PRDM16 siRNA 7.3±1.4 2.2±0.2 these complexes is critical. Many of the cellular processes Cultured murine embryonic maxillary mesenchymal (MEMM) cells were involved in orofacial development are under the influence of transfected with a pool of 4 control or PRDM16/MEL-specific siRNAs, the TGFβ [2,4,21]. The goal of the current work was to identify TGFβ reporter plasmid, p3TP-lux, and pRL-CMV. Forty-eight hours later, cells novel Smad binding proteins that confer TGFβ signaling were treated with 2 ng/ml TGFβ for 24 h, after which time cells were lysed and specificity during orofacial development. The results presented luciferase activity determined. Data are presented as the mean fold-change of relative luciferase activity (Firefly luciferase/Renilla luciferase)±standard error here demonstrate that PRDM16 (MEL1) is a Smad binding of the mean for three independent experiments, with triplicate samples in each protein that can bind a number of different Smads, including experiment. There were no statistically significant effects of PRDM16/MEL1 TGFβ- and BMP-regulated Smads, in addition to inhibitory knockdown on Smad-mediated TGFβ signaling (t-test, p=0.238). D.R. Warner et al. / Biochimica et Biophysica Acta 1773 (2007) 814–820 819

[28]. Conversely, MDS1/Evi1 has been shown to enhance TGFβ in the palate medial edge epithelium and is critical for palatal signaling [29]. Based upon these reports, we predicted that fusion [11]. A number of TGFβ and BMP receptors are also PRDM16/MEL1 would enhance TGFβ signaling based on its expressed in the palate in patterns that overlap that of PRDM16/ similarity to MDS1/Evi1. However, siRNA knockdown of MEL1: Alk-1, Alk-2, Alk-5, and Alk7 [38]. Dudas recently PRDM16/MEL1 led to a small, but statistically insignificant demonstrated that intact TGFβ signaling in palate epithelium increase in TGFβ-mediated reporter activity (Table 1). This and mesenchyme is required for palatal shelf fusion [39]. indicates the possibility that domains other than the PR domain, Therefore, PDM16/MEL1 may alter mesenchymal TGFβ or interaction with additional proteins, such as CtBP in the case signals in order to enhance the signaling potential of TGFß of Evi1 [30] will be important for the signaling outcome from the and thus alter the gene induction profile necessary for normal interaction between PRDM16/MEL1 and TGFβ Smads. The palate fusion. BMP also has important functions in palate weak effect of these siRNAs may be due to the relatively modest development [40] and the expression of Bmp2 and Bmp4 reduction in PRDM16/MEL1 (∼2-fold). Due to technical overlap significantly with that of PRDM16/MEL1. Therefore, limitations, higher concentrations could not be tested, and it is PRDM16/MEL1 may coordinate signaling through two impor- unclear at this point if greater efficacy of the siRNA approach tant pathways (TGFβ and BMP) necessary for proper palate would magnify the effect, if any, on TGFß reporter activity. We development. are currently investigating in more detail the nature of the impact Based upon similarities to Evi1, and the observation that of PRDM16/MEL1 on TGFβ signaling, with particular PRDM16/MEL1 is ectopically activated through translocations emphasis on mechanisms of orofacial development. Unlike in a number of cancer cells, we predict that at least one role for Evi1, the interaction between PRDM16/MEL1 and Smads is not PRDM16/MEL1 is in the control of the cell cycle progression. likely through the DNA binding domain-1 since the yeast two- This novel Smad binding protein is expressed in developing hybrid clone isolated from the screen was missing this domain, orofacial tissue and modulates TGFβ signaling. The specific the PR domain, and the proline-rich region. expression pattern within the developing orofacial region, and Whole-mount in situ hybridizations with a PRDM16/MEL1- in particular the primary and secondary palates, coupled with specific riboprobe clearly demonstrate that PRDM16/MEL1 is preliminary reports of its involvement in human cases of expressed in murine embryonic orofacial tissue, but appears to clefting provide strong evidence that this protein is important be restricted to the anterior portion of the secondary palate, along for normal orofacial development. Future studies will focus on with strong staining in the primary palate (Fig. 5). Recently, the role of PRDM16/MEL1 in mediating TGFβ dependent several studies have focused on the differential expression of cellular processes such as cell proliferation and extracellular genes along the anterior–posterior axis of the palate and their matrix synthesis, both of which are central to normal orofacial role in patterning of the secondary palate [31–33]. Strictly based development. upon its location within the developing palate, it is plausible to propose that PRDM16/MEL1 plays a role in anterior–posterior Acknowledgements patterning of the secondary palate and possibly in differentiation of the anterior (bony) and posterior (soft) palate. Recently, a The authors wish to thank the following investigators for murine mutant generated by whole-animal ENU mutagenesis plasmids: Dr. H. Brivanlou at the Rockefeller University New with a mutation in the gene for PRDM16/MEL1 [34] presented York, NY (pBluescript-Smad 7); Dr. M. Kato at the Japanese with a cleft palate [35]. In addition missense mutations of Foundation for Cancer Research, Tokyo, Japan (pGEX4T1- PRDM16/MEL1 were found in several cases of non-syndromic Smad 3 ΔMH2); Dr. H. Heldin at the Ludwig Institute for cleft lip/palate [35]. These findings suggest that PRDM16/ Cancer Research, Uppsala, Sweden (pcDNA-Smad 3); Dr. X. MEL1 is critical for palate development. Lin at Baylor University, Houston, TX (pGEX2TK- Smad 1); Few studies have examined the expression pattern of and Dr. Y. Chen at the Indiana University, Indianapolis, IN PRDM16/MEL1 in either adult or embryonic tissue. Recently, (pGEX4T2-Smad 2 and pGEX4T2-Smad 4), and Dr. JiJian Lan however, Van Campenhout, et al. demonstrated expression of at the University of Louisville (pGEX4T-GCNF). This work PRDM16/MEL1 mRNA in migrating hyoid crest cells and was supported through NIH Grants DE12363 (to M.M.P.) and in various stages of Xenopus development, in the otic vesicle, DE12858 and DE05550 (to R.M.G.), P20RR017702 from the the developing brain, retinal pigment epithelium of the eye, the COBRE Program of the National Center for Research developing heart, and in the developing kidney [36]. Prelimi- Resources (to R.M.G.), and by the Commonwealth of Kentucky nary analysis of the expression of PRDM16/MEL1 in develop- Research Challenge Trust Fund. ing murine embryos reveals a similar pattern of expression (Horn, manuscript in preparation). References PRDM16/MEL1 colocalizes with a number of signaling molecules that have been suggested, or demonstrated to [1] Y. Ito, J.Y. Yeo, A. Chytil, J. Han, P.J. Bringas, A. Nakajima, C.F. Shuler, function in, palatogenesis [e.g. Shox2 [31], Msx1 [33], Osr1, H.L. Moses, Y. Chai, Conditional inactivation of Tgfbr2 in cranial neural crest causes cleft palate and calvaria defects, Development 130 (2003) Osr2, and Pax9 [37]. Of particular note are those involved in the – β 5269 5280. TGF or BMP signaling pathways since both of these utilize [2] M.M. Pisano, P. Mukhopadhyay, R.M. Greene, Molecular fingerprinting Smads to transduce signals into the nucleus and have the of TGFbeta-treated embryonic maxillary mesenchymal cells, Orthod. potential to interact with PRDM16/MEL1. TGFβ3 is expressed Craniofac. Res. 6 (2003) 194–209. 820 D.R. Warner et al. / Biochimica et Biophysica Acta 1773 (2007) 814–820

[3] M. D'Angelo, J.M. Chen, K. Ugen, R.M. Greene, TGF beta 1 regulation of [23] N. Mochizuki, S. Shimizu, T. Nagasawa, H. Tanaka, M. Taniwaki, collagen metabolism by embryonic palate mesenchymal cells, J. Exp. J. Yokota, K. Morishita, A novel gene, MEL1, mapped to 1p36.3 is highly Zool. 270 (1994) 189–201. homologous to the MDS1/EVI1 gene and is transcriptionally activated in t [4] M. D'Angelo, R.M. Greene, Transforming growth factor-beta modulation (1;3)(p36;q21)-positive leukemia cells, Blood 96 (2000) 3209–3214. of glycosaminoglycan production by mesenchymal cells of the developing [24] M. Yoshida, K. Nosaka, J. Yasunaga, I. Nishikata, K. Morishita, M. murine secondary palate, Dev. Biol. 145 (1991) 374–378. Matsuoka, Aberrant expression of the MEL1S gene identified in [5] Z. Xiao, X. Liu, Y.I. Henis, H.F. Lodish, A distinct nuclear localization association with hypomethylation in adult T-cell leukemia cells, Blood signal in the N terminus of Smad 3 determines its ligand-induced nuclear 103 (2004) 2753–2760. translocation, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 7853–7858. [25] A.D. Keller, T. Maniatis, Identification and characterization of a novel [6] C. Pouponnot, L. Jayaraman, J. Massague, Physical and functional inter- repressor of beta-interferon gene expression, Genes Dev. 5 (1991) action of SMADs and p300/CBP, J. Biol. Chem. 273 (1998) 22865–22868. 868–879. [7] E. Weisberg, G.E. Winnier, X. Chen, C.L. Farnsworth, B.L. Hogan, M. [26] S. Barjesteh van Waalwijk van Doorn-Khosrovani, C. Erpelinck, B. Whitman, A mouse homologue of FAST-1 transduces TGF beta Lowenberg, R. Delwel, Low expression of MDS1-EVI1-like-1 (MEL1) superfamily signals and is expressed during early embryogenesis, Mech. and EVI1-like-1 (EL1) genes in favorable-risk acute myeloid leukemia, Dev. 79 (1998) 17–27. Exp. Hematol. 31 (2003) 1066–1072. [8] A. vonMikecz, S. Zhang, M. Montminy, E.M. Tan, P. Hemmerich, CREB- [27] I. Nishikata, H. Sasaki, M. Iga, Y. Tateno, S. Imayoshi, N. Asou, T. binding protein (CBP)/p300 and RNA polymerase II colocalize in Nakamura, K. Morishita, A novel EVI1 gene family, MEL1, lacking a PR transcriptionally active domains in the nucleus, J. Cell Biol. 150 (2000) domain (MEL1S) is expressed mainly in t(1;3)(p36;q21)-positive AML 265–273. and blocks G-CSF-induced myeloid differentiation, Blood 102 (2003) [9] S. Ait-Si-Ali, S. Ramirez, F.X. Barre, F. Dkhissi, L. Magnaghi-Jaulin, J.A. 3323–3332. Girault, P. Robin, M. Knibiehler, L.L. Pritchard, B. Ducommun, D. [28] T. Alliston, T.C. Ko, Y. Cao, Y.Y. Liang, X.H. Feng, C. Chang, R. Trouche, A. Harel-Bellan, Histone acetyltransferase activity of CBP is Derynck, Repression of bone morphogenetic protein and activin-inducible controlled by cycle-dependent kinases and oncoprotein E1A, Nature 396 transcription by Evi-1, J. Biol. Chem. 280 (2005) 24227–24237. (1998) 184–186. [29] R. Sood, A. Talwar-Trikha, S.R. Chakrabarti, G. Nucifora, MDS1/EVI1 [10] S. Dennler, S. Huet, J.M. Gauthier, A short amino-acid sequence in MH1 enhances TGF-beta1 signaling and strengthens its growth-inhibitory domain is responsible for functional differences between Smad2 and effect but the leukemia-associated fusion protein AML1/MDS1/EVI1, Smad3, Oncogene 18 (1999) 1643–1648. product of the t(3;21), abrogates growth-inhibition in response to TGF- [11] G. Proetzel, S.A. Pawlowski, M.V. Wiles, M. Yin, G.P. Boivin, P.N. beta1, Leukemia 13 (1999) 348–357. Howles, J. Ding, M.W. Ferguson, T. Doetschman, Transforming growth [30] K. Izutsu, M. Kurokawa, Y. Imai, K. Maki, K. Mitani, H. Hirai, The factor-β 3 is required for secondary palate fusion, Nat. Genet. 11 (1995) corepressor CtBP interacts with Evi-1 to repress transforming growth 409–414. factor beta signaling, Blood 97 (2001) 2815–2822. [12] D.R. Warner, M.M. Pisano, E.A. Roberts, R.M. Greene, Identification of [31] L. Yu, S. Gu, S. Alappat, Y. Song, M. Yan, X. Zhang, G. Zhang, Y. Jiang, three novel Smad binding proteins involved in cell polarity, FEBS Lett. Z. Zhang, Y. Zhang, Y. Chen, Shox2-deficient mice exhibit a rare type of 539 (2003) 167–173. incomplete clefting of the secondary palate, Development 132 (2005) [13] D.R. Warner, E.A. Roberts, R.M. Greene, M.M. Pisano, Identification of 4397–4406. novel Smad binding proteins, Biochem. Biophys. Res. Commun. 312 [32] J.Z. Jin, J. Ding, Analysis of Meox-2 mutant mice reveals a novel (2003) 1185–1190. postfusion-based cleft palate, Dev. Dyn. 235 (2006) 539–546. [14] D.R. Warner, V. Bhattacherjee, X. Yin, S. Singh, P. Mukhopadhyay, M.M. [33] Z. Zhang, Y. Song, X. Zhao, X. Zhang, C. Fermin, Y. Chen, Rescue of cleft Pisano, R.M. Greene, Functional interaction between Smad, CREB binding palate in Msx1-deficient mice by transgenic Bmp4 reveals a network of protein, and p68 RNA helicase, Biochem. Biophys. Res. Comm. 324 BMP and Shh signaling in the regulation of mammalian palatogenesis, (2004) 70–76. Development 129 (2002) 4135–4146. [15] U.K. Laemmli, Cleavage of structural proteins during the assembly of the [34] F.W. Buass, Mouse genomics gets the royal treatment, GenomeBiology 5 head of bacteriophage T4, Nature 227 (1970) 680–685. (2004) 310. [16] F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, S.J.A., [35] B.C. Bjork, A.R. Vieira, S. Faust, S.A. Camper, J.C. Murray, D.R. Beier, K. Struhl, Current Protocols in Molecular Biology, John Wiley and Sons Phenotypic, Genetic, and Developmental Characterization of CPO1, a Inc., New York, 1994. Recessive ENU-induced Mouse Model of Cleft Palate, Mouse Molecular [17] D.R. Warner, R.M. Greene, M.M. Pisano, Cross-talk between the TGFbeta Genetics Cold Spring Harbor Press, Woodbury, NY, 2006, p. 27. and Wnt signaling pathways in murine embryonic maxillary mesenchymal [36] C. Van Campenhout, M. Nichane, A. Antoniou, H. Pendeville, O.J. cells, FEBS Lett. 579 (2005) 3539–3546. Bronchain, J.C. Marine, A. Mazabraud, M.L. Voz, E.J. Bellefroid, Evi1 is [18] C.A. Heid, J. Stevens, K.J. Livak, P.M. Williams, Real time quantitative specifically expressed in the distal tubule and duct of the Xenopus PCR, Genome Res. 6 (1996) 986–994. pronephros and plays a role in its formation, Dev. Biol. 294 (2006) [19] L.R. Ellis, D.R. Warner, R.M. Greene, M.M. Pisano, Interaction of Smads 203–219. with collagen types I, III, and V, Biochem. Biophys. Res. Commun. 310 [37] Y.Lan, C.E. Ovitt, E.S. Cho, K.M. Maltby, Q. Wang, R. Jiang, Odd-skipped (2003) 1117–1123. related 2 (Osr2) encodes a key intrinsic regulator of secondary palate [20] N. Mochizuki, S. Shimizu, T. Nagasawa, H. Tanaka, M. Taniwaki, growth and morphogenesis, Development 131 (2004) 3207–3216. J. Yokota, K. Morishita, A novel gene, MEL1, mapped to 1p36.3 is highly [38] M. Dudas, A. Nagy, N.J. Laping, A. Moustakas, V. Kaartinen, TGFβ-3- homologous to the MDS1/EVI1 gene and is transcriptionally activated in t induced palatal fusion is mediated by Alk-5/Smad pathway, Dev. Biol. 266 (1;3)(p36;q21)-positive leukemia cells, Blood 96 (2000) 3209–3214. (2004) 96–108. [21] M. D'Angelo, J.-M. Chen, K. Ugen, R. Greene, TGFβ1 regulation of [39] M. Dudas, J. Kim, W.Y. Li, A. Nagy, J. Larsson, S. Karlsson, Y. Chai, V. collagen metabolism by embryonic palate mesenchymal cells, J. Exp. Kaartinen, Epithelial and ectomesenchymal role of the type I TGF-beta Zool. 270 (1994) 189–201. receptor ALK5 during facial morphogenesis and palatal fusion, Dev. Biol. [22] A. Nakao, M. Afrakhte, A. Moren, T. Nakayama, J.L. Christian, R. 296 (2006) 298–314. Heuchel, S. Itoh, M. Kawabata, C.H. Heldin, P. ten Dijke, Identification of [40] W. Liu, X. Sun, A. Braut, Y. Mishina, R.R. Behringer, M. Mina, J.F. Smad7, a TGF-beta-inducible antagonist of TGF-beta signalling. Nature Martin, Distinct functions for Bmp signaling in lip and palate fusion in 389 (1997) 631–635. mice, Development 132 (2005) 1453–1461.