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Originally published In Press as doi:10.1074/jbc.M512449200 on June 19, 2006

J. Biol. Chem., Vol. 281, Issue 34, 24544-24552, August 25, 2006
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Regulation of Murine TGFbeta2 by Pax3 during Early Embryonic Development*Formula

Chandra S. K. Mayanil{ddagger}1, Angela Pool{ddagger}2, Hiromichi Nakazaki{ddagger}, Anvesh C. Reddy{ddagger}, Barbara Mania-Farnell§, Beth Yun{ddagger}3, David George{ddagger}, David G. McLone{ddagger}, and Eric G. Bremer{ddagger}

From the {ddagger}Laboratory of Neural Tube Research, Department of Pediatric Neurosurgery, Children's Memorial Research Center and Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60614, the §Department of Biology, Purdue University at Calumet, Hammond, Indiana 46323, and the Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611

Received for publication, November 21, 2005 , and in revised form, April 27, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Previously our laboratory identified TGFbeta2 as a potential downstream target of Pax3 by utilizing microarray analysis and promoter data base mining (Mayanil, C. S. K., George, D., Freilich, L., Miljan, E. J., Mania-Farnell, B. J., McLone, D. G., and Bremer, E. G. (2001) J. Biol. Chem. 276, 49299-49309). Here we report that Pax3 directly regulates TGFbeta2 transcription by binding to cis-regulatory elements within its promoter. Chromatin immunoprecipitation revealed that Pax3 bound to the cis-regulatory elements on the TGFbeta2 promoter (GenBankTM accession number AF118263 [GenBank] ). Both TGFbeta2 promoter-luciferase activity measurements in transient cotransfection experiments and electromobility shift assays supported the idea that Pax3 regulates TGFbeta2 by directly binding to its cis-regulatory regions. Additionally, by using a combination of co-immunoprecipitation and chromatin immunoprecipitation, we show that the TGFbeta2 cis-regulatory elements between bp 741-940 and bp 1012-1212 bind acetylated Pax3 and are associated with p300/CBP and histone deacetylases. The cis-regulatory elements between bp 741 and 940 in addition to associating with acetylated Pax3 and HDAC1 also associated with SIRT1. Whole mount in situ hybridization and quantitative real time reverse transcription-PCR showed diminished levels of TGFbeta2 transcripts in Pax3-/- mouse embryos (whose phenotype is characterized by neural tube defects) as compared with Pax3+/+ littermates (embryonic day 10.0; 30 somite stage), suggesting that Pax3 regulation of TGFbeta2 may play a pivotal role during early embryonic development.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Pax3 encodes a paired homeobox containing transcription factor that is expressed in neuroepithelium, neural crest, and presomitic mesoderm (1). Homozygous mouse embryos carrying a loss-of-function Pax3 allele (Pax3-/-) develop open neural tube defects, such as exencephaly or spina bifida (2), and die around embryonic day 14 (E14)4 as a consequence of heart defects (3). More recently, Pax3 has been shown to function at the nodal point in melanocyte stem cell differentiation (4). Heterozygous embryos (Pax3+/-) are viable but exhibit white patches on their bellies caused by defective development of neural crest-derived melanocytes. This suggests that disruption of the Pax3-dependent developmental program may cause defects in the development of neural crest-derived structures.

Two main Pax3-binding sites have been identified and are found in most Pax3 target elements as follows: (i) a binding site derived from the Drosophila Paired (ATTA N5 GTTCC), and (ii) a Pax3 paired domain binding site (CGTCAC(G/A)(C/G)TT) identified by Epstein et al. (5) by CASTing (DNA-binding site selection assays) in the c-Met promoter region. In addition several paired domains, such as GTTCC, CAGTGT, GTTAT, GTGTGA, and CAAGG (6), as well as the homeodomain ATTA, have been suggested to be "putative Pax3-binding motifs" (7, 8). More recently, Corey and Underhill (9) demonstrated that Pax3 can regulate target genes through alternative modes of DNA recognition. They observed that although the microphthalmia-associated transcription factor element is characterized by suboptimal recognition motifs for the paired domain and homeodomain, it sustains a higher level of Pax3 binding than TRP-1, which contains a canonical paired domain site. The basis for this difference involved a context-dependent cooperative binding event requiring both the paired and homeodomain, whereas the paired domain alone was sufficient for TRP-1 recognition.

Since Pax3 is important in diverse cellular functions during development, we wanted to identify additional genes regulated by Pax3. To accomplish this we utilized oligonucleotide arrays and RNA isolated from Pax3-transfected cell lines and promoter-based data mining (6). Based on the putative Pax3-binding motif score (motif score is defined as the total number of times a paired or a homeodomain appears in the promoter sequence of a given gene) in the promoter regions of the genes whose expression was changed by Pax3 transfection, we postulated 17 new genes that may be direct Pax3 downstream targets and another 17 genes (whose promoter sequences are not known) as putative Pax3 downstream targets (6). The expression of one of these genes, human TGFbeta2, was increased in two of the three Pax3 transfectants studied, and this gene showed the highest putative Pax3-binding motif score, making it a likely gene regulated by Pax3.

The importance of TGFbeta2 in the process of neural tube closure was first demonstrated by Sanford et al. (10) who showed that TGFbeta2 knock-out mice had spina bifida occulta and unfused neural arches at the midline of neural tube along with other developmental abnormalities. In this paper, we demonstrate the following: (i) Pax3 regulates TGFbeta2 by binding to cis-regulatory elements of the promoter; (ii) acetylated Pax3 is associated with p300/CBP, HDAC1, and SIRT1; and (iii) TGFbeta2 transcript levels are diminished in the Pax3-/- embryos. Thus our microarray data (6), along with the present findings in this paper in combination with the results of Sanford et al. (10), indicate that TGFbeta2 is regulated by Pax3 and could be involved in early embryonic development.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Genotyping—The C57BL/6J-Pax3Sp/J (stock = 2469) male and C57BL/6J breeding pairs were obtained from The Jackson Laboratory. Genotyping of Pax3+/+, Pax3+/-, and Pax3-/- embryos was performed by isolating genomic DNA from embryonic membranes of embryos and performing PCR with a wild type Pax3+/+- or Pax3-/--specific reverse primer and a common forward primer in separate PCRs as described by Epstein et al. (5).

Chromatin Immunoprecipitation (ChIP)—ChIP assays were performed according to the procedure described by the manufacturer (Upstate%20Biotechnology">Upstate Biotechnology, Inc.). Briefly, E10.0 (30 somite stage) Pax3+/+ embryos were fixed in formalin (1% final concentration) for 4 h at 4°C, quenched in 0.125 M glycine, sonicated, and then processed according to manufacturer's protocol using a rabbit polyclonal Pax3 antibody (lot number D20123 [GenBank] -1, catalog number CA1010-50UL, Oncogene Sciences), rabbit polyclonal antibody to HDAC1 (catalog number ab7028, Abcam), rabbit polyclonal antibody to acetylated proteins (catalog number ab193, Abcam), rabbit polyclonal antibody against amino acids 448-747 of SIRT1 of human origin (catalog number sc-15404, Santa Cruz Biotechnology, Inc.), and IgG from rabbit serum (catalog number I-5006, Sigma). PCR was performed with primers to murine TGFbeta2 promoter region between bp 1 and 200 (primer set A-(forward), 5'-ggatccctgcttgtcacatg-3'; (reverse), 5'-ttatatacagcaatgaacat-3'); bp 121 and 321 (primer set B-(forward), 5'-tgtatgccagctatatcatt-3'; (reverse), 5'-tggccacgatgactacacag-3'); bp 481 and 681 (primer set C-(forward), 5'-agtttgagcaagttgaagta-3'; (reverse), 5'-ctctgtatcactgctttgaa-3'); bp 741 and 941 (primer set D-(forward), 5'-acagggctggattgtaaaca-3'; (reverse), 5'-aggcgtgtacacacacacac-3'); bp 1012 and 1212 (primer set E-(forward), 5'-tagaaacgggatgcttagct-3'; (reverse), 5'-gaacaaagtgacgcgctagg-3'); bp 1091 and 1291 (primer set F-(forward), 5'-atgccagtcgccctccctta-3'; (reverse), 5'-aaacctgctgccagcagata-3'), which amplified a 200-bp fragment in each case. All ChIP samples were tested for false-positive PCR amplification by sequencing the 200-bp amplified product to ascertain the specificity of the Pax3 binding to the cis-regulatory elements. Additionally, false positive PCR amplification was ruled out by amplifying the sequence from the murine beta-actin gene, which failed to give any PCR product in the immunoprecipitates.

Analysis of TGFbeta2 Promoter Activity—Plasmids containing TGFbeta2 promoter sequences (11) were kindly provided by Dr. Angie Rizzino (University of Nebraska Medical Center, Omaha). The plasmid pPGB11, which contains a luciferase reporter gene (12), was provided by Dr. S. Tsuji (Institute of Physical and Chemical Research, RIKEN, Saitama, Japan). TGFbeta2 promoter sequences (GenBankTM accession number AF118263 [GenBank] ) were subcloned into the pPGB11 plasmid and labeled as TG1 (bp 1226-1290), TG2 (bp 1191-1290), TG3 (bp 1143-1290), TG4 (bp 950-1290), TG6 (bp 734-1290), and TG7 (bp 1-1290), which is the full-length promoter sequence (11) (Fig. 2). Deletion and site-directed mutations of "putative Pax3-binding sites" were initially made in the TG4 and TG7 promoter constructs, with the QuickChangeXL site-directed mutagenesis kit (Stratagene). Mutated sequences are identified in Fig. 2. Plasmids (5 µg) with the mutated sequences were transiently co-transfected with Pax3-pcDNA3 or pcDNA3 vector control into wild type DAOY cell line (6, 13). Renilla luciferase plasmid, pRL-null (0.5 µg) (dual luciferase system, Promega), was simultaneously transfected as an insertional control for transfection efficiency. Cells were seeded at 5 x 104 cells/60-mm diameter dish in DMEM supplemented with 10% fetal calf serum for 24 h prior to transfection. Both plasmids were transfected into the cells using Lipofectamine 2000 reagent (Invitrogen). After 48 h, the cells were washed three times with phosphate-buffered saline (PBS) and then lysed with Passive Lysis Buffer (PLB). Luciferase activity was measured using a Vector 2 Luminometer (PerkinElmer Life Sciences).

Pax3-GST Fusion Protein Expression and Isolation—The Pax3-GST fusion cDNA was provided by Dr. Mary R. Loeken (Harvard Medical School, Boston). The Escherichia coli was transformed with Pax3-GST fusion protein construct in pcDNA3, and the cells were grown overnight in LB medium. Pax3-GST fusion protein expression and isolation were performed as described previously (6).

Electromobility Shift Assays (EMSA)—Probes were prepared for EMSA by annealing complementary oligonucleotides representing selected regions of murine TGFbeta2 promoter, followed by 5'-end labeling with [{gamma}-32P]ATP by T4 polynucleotide kinase. For EMSA experiments, 0.5 µg of purified Pax3-GST fusion protein was incubated in 20 µl of reaction mixture containing 15 mM Tris (pH 7.5), 6.5% glycerol, 90 mM KCl, 0.7 mM EDTA, 0.2 mM dithiothreitol, 1 mg/ml bovine serum albumin, 50 µM pyrophosphate, 300 ng of salmon sperm DNA as a competitor to reduce nonspecific DNA binding, and 30,000 dpm per nmol of 32P-labeled double-stranded oligonucleotide probe. Double-stranded 30-mer oligonucleotide probes covered the following regions: ON-a (bp 175-204) (5'-ataataatgttcattgctgtatataatgta-3'); mON-a (atgaaatgttcattgctgtatatgatgta-3'); ON-b (bp 211-240) (5'-ctatatcattacatatcattacagctccct-3'); mON-b (5'-ctatatcagtacatatcagtacagctccct-3'); ON-c (bp 504-533) (5'-aatgtcaagttatgatccttgtgtgaagga-3'); mON-c, (5'-aatgtcaagctatgatactgtttgacagga-3'); ON-d (bp 647-676) (5'-ataaagaaggcaacagttcctttattcaaa-3'); mON-d (5'-ataaagaaggcaacagtacctttattcaaa-3'); ON-e (bp 751-780) (5'-attgtaaacaggctaatgaaaaacaacctg-3'); mON-e (5'-attgtaaacaggctgatgaaaaacaacctg-3'); ON-f (bp 793-822) (5'-gaactcaagagttaggtcagcagtctcaga-3'); mON-f (5'-gaactcaagagtctaggtcagcagtctcaga-3'); ON-g (bp 1039-1070) (5'-ctccacacagacccttggtgatcacaga ca-3'); mON-g (5'-ctccacacagaccattggtgatcacaga ca-3'). The underlined sequences indicate the putative Pax3-binding sites. The boldface nucleotide in each probe was mutated to obtain the mutant oligonucleotides, for electromobility shift assays. The labeled oligonucleotides were incubated at room temperature with Pax3-GST fusion protein in the presence or absence of 100-fold molar excess cold oligonucleotide. After a 20-min incubation, free DNA and DNA-protein complexes were resolved in 8% polyacrylamide gels using 0.25x TBE as the running buffer. Electrophoresis was performed in the cold room at 300 mV and 30 mA for 3 h. The gels were dried and subjected to PhosphorImager (Amersham Biosciences) analysis to view the shifted bands.


Figure 1
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  		FIGURE 1.
ChIP assays using E10.0 Pax3+/+ embryos (30 somites) show that Pax3 binds to cis-regulatory elements of TGFbeta2 promoter. Rabbit polyclonal antibody against Pax3 was used to immunoprecipitate the Pax3-DNA complex. Lane 1 is input sample; lane 2 is immunoprecipitated DNA using IgG from normal rabbit serum; and lane 3 is immunoprecipitated DNA using rabbit polyclonal antibody against Pax3. The 200-bp amplified product using mTGFbeta2 primer sets A-F (see "Experimental Procedures") is shown. The bottom panel shows the schematic representation of the primer sets A-F. Murine beta-actin primers were used as negative controls, and the amplified product is present only in the input and not in the control IgG control or the immunoprecipitate. Each ChIP experiments (n = 4) was performed in triplicate.

 
Co-immunoprecipitation—Pax3 transfectants, B9 (6), were treated with or without 2 µM trichostatin A in serum-free DMEM for 4 h at 37 °Cand then washed three times with cold PBS. The washed cells were collected in cold PBS and then lysed on ice for 30 min in RIPA buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.1% SDS, and 0.5% sodium deoxycholate. Protease inhibitors (Calbiochem) were added just before lysis. Immunoprecipitations were carried our by using polyclonal HDAC1 (Abcam) and acetylated protein antibody (Abcam) as per the manufacturer's instructions. The immunoprecipitates were collected by 50 µl of 50% slurry of protein G-Sepharose (Sigma). The precipitates were washed three times in lysis buffer, and boiled in 2x SDS sample buffer for 5 min and centrifuged for 1 min. The proteins in the supernatant were resolved in 8% SDS-PAGE and immunoblotted with Pax3 monoclonal antibody (Developmental Studies Hybridoma Bank, University of Iowa) and with p300/CBP (Santa Cruz Biotechnology, Inc.).

Whole Mount in Situ Hybridization—Whole mount in situ hybridization was done according to the method described previously (14). Washes were performed at 65 °C. The plasmid pmTGFbeta2 containing mouse TGFbeta2 (442 bp) was kindly provided by Dr. M. Azhar (University of Cincinnati, Department of Molecular Genetics, Cincinnati, OH). The antisense digoxigenin labeled TGFbeta2 riboprobe was synthesized by linearizing the PCR products with EcoRI and using SP6 polymerase. The sense digoxigenin-labeled TGFbeta2 riboprobe was synthesized by linearizing the PCR products with XhoI and using T7 polymerase. Embryos were stained and photographed with a Spot Camera (Diagnostic Instruments Inc.).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Pax3 Binds to cis-Regulatory Regions of Murine TGFbeta2 Promoter—An unbiased survey of the entire murine TGFbeta2 promoter (GenBankTM accession number AF118263 [GenBank] ) to ascertain putative Pax3-binding elements was done by employing chromatin immunoprecipitation on E10.0 (30 somite stage) Pax3+/+ embryos using rabbit polyclonal Pax3 antibody. The forward and reverse primer sets A-F amplified 200-bp fragments from the input sample (Fig. 1, lane 1) and immunoprecipitates (Fig. 1, lane 3). No amplification was observed when beta-actin forward and reverse primers were used. Immunoprecipitation reactions using IgG from normal rabbit serum did not show any amplification of the TGFbeta2 promoter in the immunoprecipitated DNA (Fig. 1, lane 2). All ChIP samples were tested for false-positive PCR amplification by sequencing the 200-bp amplified product to ascertain the specificity of the Pax3 binding to the cis-regulatory elements. Additionally, false-positive PCR amplification was ruled out by amplifying the sequence from murine beta-actin gene, which failed to give any PCR product in the immunoprecipitates. With the exception of primer set A, all other primer sets (B-F) amplified a 200-bp fragment from the DNA extracted from the immunoprecipitates (Fig. 1, lane 3). It is important to note that even though the primers to probe specific regions are spaced at 200-bp intervals, the level of resolution is determined by the fragment DNA length following sonication. This is generally on the order of 200-600 bp, depending on the conditions of the sonication.


Figure 2
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  		FIGURE 2.
Murine TGFbeta2 promoter sequence (GenBankTM accession number AF118263) (11). The arrows indicate the following promoter-luciferase constructs, TG1, TG2, TG3, TG4, TG6, and TG7 as outlined in Fig. 3. The dinucleotide repeat (gt)32 is in boldface and underlined. To maintain the consistency of the nomenclature and to establish a connection between the different experiments, we have introduced a schematic figure as supplemental Fig. 1, where specific nomenclature regarding promoter regions used in luciferase studies, chromatin immunoprecipitation, and the oligonucleotides used in EMSA.

 


Figure 3
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  		FIGURE 3.
Pax3 regulation of murine TGFbeta2 promoter. Pax3-pcDNA3 and murine TGFbeta2 promoter-luciferase constructs TG1, TG2, TG3, TG4, TG6, and TG7 (refer to Fig. 2) or pcDNA3 and murine TGFbeta2 promoter constructs and pPGB11 (promoter-less luciferase gene) were co-transfected into wild type DAOY cells. The luciferase activity was assayed 48 h after transfection as described under "Experimental Procedures." Values of pPGB11 were subtracted from the TGFbeta2 promoter-luciferase values. Experiments were performed in quadruplicate with each data point in duplicate. Note the region of (gt)32 and the region between bp 1-734 and bp 950-1143 that may have contributed to increased TG4 and TG7 construct activity.

 
Functional Analysis of Murine TGFbeta2 Promoter Activity— To determine the functional interaction of Pax3 with murine TGFbeta2 promoter and the effect on the promoter activity, we employed transient co-transfection experiments using luciferase-reporter plasmids containing different murine TGFbeta2 promoter sequences as described by Wilder et al. (11) with Pax3 expression plasmids. Murine TGFbeta2 promoter plasmids were subcloned into the pPGB11 plasmid and labeled as TG1 (bp 1226-1290), TG2 (bp 1191-1290), TG3 (bp 1143-1290), TG4 (bp 950-1290), TG6 (bp 734-1290), and TG7 (bp 1-1290) (11). A full-length promoter sequence and each construct are shown in Fig. 2. The promoterless luciferase gene in pPGB11 (13) and the plasmid constructs containing murine TGFbeta2 promoter sequences were assayed for promoter activity by transient co-transfection of Pax3-pcDNA3 into wild type DAOY cells (Fig. 3). Luciferase activity was normalized to Renilla luciferase activity by cotransfecting an internal control plasmid, pRL-null, carrying cDNA encoding the Renilla luciferase gene. Our results showed that the promoter constructs TG4 and TG7 displayedmaximumluciferaseactivity with TG4 showing slightly higher activity than TG7 construct. These observations suggest that these constructs contain transcriptional activating elements that may be responsive to Pax3 binding. The promoter constructs TG1, TG2, TG3, and TG6, on the other hand, displayed no increase in luciferase activity. It is intriguing to know that TG6 promoter construct did not show any increase in promoter activity, although it encompassed the TG4 construct promoter region. This observation suggests that the TG6 promoter construct may contain silencing elements 5' to the boundary of the TG4 promoter construct that significantly decreases promoter activity. One plausible explanation for this latter observation could be due to the presence of a dinucleotide (GT)32 repeat region in this promoter sequence (Fig. 2). Previous studies by Yoshikawa and co-workers (15) have identified a functional and polymorphic (GT)n repeat in the 5'-regulatory region of N-methyl-D-aspartate receptor 2A subunit (GRIN2A) gene that resulted in the length-dependent inhibition of transcriptional activity. Similarly, Yamada et al. (16) suggested that the larger the size of a (GT)n repeat in the hemoxygenase-1 gene promoter, the greater the transcriptional inhibition. In sum, the observations from our promoter-luciferase study concluded that TG7 and TG4 promoter constructs may be having Pax3 responsive regions between bp 1-734 and bp 950-1143, respectively.


Figure 4
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  		FIGURE 4.
Identification of Pax3 response regions in murine TGFbeta2 promoter; point mutants of TGFbeta2 promoter. Point mutations in paired-(P) or homeodomain (H)-binding sites within the TG7 promoter construct included Tgm4 (A178)-H, Tgm6 (T514)-P, Tgm7 (T526)-P, Tgm8 (A569)-H, Tgm10 (T664)-P, Tgm11 (A766)-H, and Tgm14 (T1056)-P. These promoter constructs and pPGB11 (promoter-less luciferase gene) were co-transfected into wild type DAOY with Pax3-pcDNA3 or vector alone and assayed for luciferase activity 48 h after transfection as described under "Experimental Procedures." The luciferase activity values for pPGB1, pcDNA3 transfected cell, and DAOY wild type were subtracted from TGFbeta2 promoter-luciferase construct values. Experiments were performed in quadruplicate with each data point in duplicate. The {blacktriangledown} in the left-hand panel denotes the point mutation in either the paired domain (P) or homeodomain (H)-binding sites.

 


Figure 5
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  		FIGURE 5.
Identification of Pax3 response region in murine TGFbeta2 promoter; double mutants (deletion and point) of TGFbeta2 promoter. DM1, containing deletion TG7{Delta}2 (gttat, bp 512-516) and point mutation Tgm10 (T664) in the TG7 promoter construct; DM2, containing deletion TG{Delta}3 (gtgtga, bp 523-528) and point mutation Tgm10 (T664) in TG7 promoter construct; DM5, containing deletion TG7{Delta}2 (gttat, bp 512-516) and a point mutation Tgm14 (T1056) in the TG7 promoter construct; DM6, containing deletion TG7{Delta}3 (gtgtga, bp 523-528) and a point mutation Tgm14 (T1056) in the TG7 promoter construct. Mutant constructs and pPGB11 (promoter-less luciferase gene) were co-transfected into wild type DAOY with Pax3-pcDNA3 or pcDNA3 alone and assayed for luciferase activity 48 h after transfection as described under "Experimental Procedures." The luciferase activity values for pPGB1, pcDNA3 transfected cells, and DAOY wild type were subtracted from TGFbeta2 promoter-luciferase construct values. Experiments were performed in quadruplicate with each data point in duplicate. The {blacksquare} and {blacktriangledown} in the left-hand panel denotes deletion and point mutation, respectively.

 
Pax3 Regulates Murine TGFbeta2 Promoter—The above study prompted us to look for the paired or the homeodomain binding sequences within bp 1-734 (TG7) and bp 950-1143 (TG4) that contributed to the maximum activity. Therefore, we made several promoter-luciferase constructs on the TG7 backbone with point mutations, either in a paired domain binding site (PDBS) or homeodomain-binding site (HDBS) between bp 1-734 and bp 950-1143, representing the regions in TG7 and TG4 that showed highest luciferase activity in Fig. 3. The results (Fig. 4) show that point mutations T514 (Tgm6) in the PDBS "gttat" and T664 (Tgm10) in the PDBS "gttcc" showed a decrease in promoter activity with T1056 (Tgm14) in the PDBS "ccttg" showing a marginal decrease in promoter activity. The Tgm6 and Tgm10 mutants gave the maximum reduction in promoter activity followed by Tgm14. By contrast, a point mutation T526 (Tgm7) in the PDBS "gtgtga" did not show any decrease in promoter activity. Except for point mutation in the HDBS A178 (Tgm4), point mutations in HDBS represented by A569 (Tgm8) and A766 (Tgm11) showed no appreciable decrease in promoter activity. These results suggest that not all paired domains or homeodomain-binding sites contribute equally to TGFbeta2 promoter activity by Pax3. Thus, our data show that the PDBS "gttat" (bp 512-516) and ccttg (bp 1053-1057) and HDBS "atta" (bp 176-179) contribute maximally to promoter activity.

The above results as well as our chromatin immunoprecipitation data suggested that there may be more than one Pax3-response elements in the TG7 promoter construct. To evaluate how different regions may combine to influence promoter activity, we introduced several deletion as well as point mutations into the TG7 promoter construct (as outlined in Fig. 5). Deletion and point mutation locations were based on the mutation studies mentioned above. These studies defined maximal promoter activation located in the PDBS gttat (bp 512-516) and ccttg (bp 1053-1057). We also chose another PDBS gtgtga (bp 523-528) adjacent to gttat. This PDBS was described by Epstein and co-workers (16, 17) as binding to Pax3 and mediating activation of the c-RET enhancer. We introduced a deletion mutation to verify whether this region was important for transcriptional activity of the TGFbeta2 promoter. Our results (Fig. 5) showed that deletion of the paired domain binding motif, gttat (TG7{Delta}2, bp 512-516), in the TG7 construct decreased promoter activity marginally by 42%, whereas deletion of another paired domain binding motif, gtgtga (TG7{Delta}3, bp 523-528), did not reduce promoter activity. These results suggested that the PDBS gttat (bp 512-516) may be part of a Pax3-response element in the murine TGFbeta2 promoter.


Figure 6
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  		FIGURE 6.
Identification of TGFbeta2 as a biological target of Pax3. Electromobility shift assays of binding reactions performed with Pax3-GST fusion protein and 32P-labeled double-stranded oligonucleotides in the absence or presence of 100-fold molar excess of unlabeled oligonucleotide, ON-a through ON-g and mutated oligonucleotides mON-a through mON-g, as described under "Experimental Procedures."

 
We then wanted to examine how gttat (TG7{Delta}2, bp 512-516) deletion or a gtgtga (TG7{Delta}3, bp 523-528) deletion together with a point mutation T664 (Tgm10) in a PDBS gttcc (depicted by DM1 and DM2 promoter constructs, respectively) or a point mutation T1056 (Tgm14) in a PDBS ccttg (depicted by DM5 and DM6, respectively) would affect the promoter activity. The results (Fig. 5) showed that both the double mutants DM1 and DM2 decreased the promoter activity significantly. It is interesting to note that the mutant DM5 decreased promoter activity significantly, whereas DM6, which has a mutation in an adjacent PDBS gtgtga (bp 523-528), did not change promoter activity significantly. These studies suggested that murine TGFbeta2 promoter has at least three Pax3-responsive PDBS gttat (bp 512-516), gttcc (bp 662-666), and ccttg (bp 1053-1057) that work in conjunction with each other.

Identification of Pax3-binding Sequences in Murine TGFbeta2 Promoter—To identify Pax3-binding sequences in the murine TGFbeta2 promoter, we used EMSA with 32P-labeled 30-mer oligonucleotides and purified Pax3-GST fusion protein. Following preliminary screening, we identified seven double-stranded oligonucleotides with putative Pax3-binding sites which were named as follows: ON-a (bp 175-204); ON-b (bp 211-240); ON-c (bp 504-533); ON-d (bp 647-676); ON-e (bp 751-780); ON-f (bp 793-822); and ON-g (bp 1039-1070).

In our initial screen for the choice of oligonucleotides, we found that oligonucleotide containing gtgtga weakly bound Pax3-GST in EMSA (data not shown). These data corroborated those of Lang et al. (17). Because the ON-c (bp 504-533) oligonucleotide used in our EMSA studies also contains a gtgtga region, we wanted to confirm the specificity of the Pax3 binding by introducing a point mutation (T526) within the gtgtga region of ON-c. However, even after introducing a point mutation (T526), Pax3-GST still bound the oligonucleotide (data not shown). This clearly suggested an additional region within the oligonucleotide ON-c-bound Pax3-GST. Our promoter luciferase data suggested that the Pax3-GST binding region in ON-c might be gttat (bp 512-516) (Fig. 6). Deleting gtgtga (bp 523-528) as in TG7{Delta}3 (Fig. 6) and a point mutation (T526) as in the mutant promoter construct Tgm7 (Fig. 5) did not change the activity substantially when compared with the native TG7 promoter construct. These data suggested that gtgtga (bp 523-528), while binding to Pax3-GST, was not as transcriptionally significant as gttat (bp 512-516) in the context of our experiments using the TGFbeta2 promoter. On the contrary, the sequence gtgtga was reported to be significant for the activation of the c-RET enhancer, where Pax3 binding is modified by the Sox10-binding site adjacent to the Pax3-binding site (17, 18). We therefore chose to introduce two point mutations, one being T514 within the gttat region and the other being T526 within the gtgtga region in the ON-c oligonucleotide for EMSA and not in the luciferase assays. Our results showed that introducing two point mutations in ON-c abolished binding to purified Pax3-GST.


Figure 7
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  		FIGURE 7.
Acetylated Pax3 is associated with HDAC1 and p300/CBP in Pax3 transfectants. Pax3 transfectants, B9 was treated with (lane 1) or without (lane 2) 2 µM trichostatin A in serum-free DMEM for 4 h at 37 °C,and cells were washed twice with cold PBS. The washed cells were lysed in RIPA buffer as described under "Experimental Procedures." The immunoprecipitating (IP) antibodies (Ab) were against HDAC1 and acetylated protein, and the immunoblotting (WB) antibodies were against Pax3 and p300/CBP. Each experiment (n = 4) was done in duplicate.

 
The TGFbeta2 cis-Regulatory Elements Bind Acetylated Pax3 and Are Associated with p300/CBP and Histone Deacetylases— The above studies demonstrated that Pax3 not only bound the TGFbeta2 promoter but also participated in its transcriptional regulation. To understand the molecular mechanisms that underlie the regulation of the TGFbeta2 promoter by Pax3, we used a combination of co-immunoprecipitation assays coupled with chromatin immunoprecipitations to map the trans-acting factors that might modulate Pax3 function. To demonstrate that Pax3 gets acetylated in cells, we used stable Pax3 transfectants B9 and incubated with or without trichostatin A. The cells were harvested and subjected to immunoprecipitation with acetylated protein antibody and then immunoblotted with Pax3 antibody. The results (Fig. 7) showed that Pax3 gets acetylated in stable Pax3 transfectant B9. To ascertain whether HDAC1 is associated with acetylated Pax3, we performed immunoprecipitation assays using HDAC1 antibody and immunoblotted with Pax3 antibody. The presence of Pax3 in the immunoprecipitates showed that HDAC1 is associated with acetylated Pax3. To ascertain whether p300/CBP also forms the part of the transcriptional regulatory complex, we performed immunoblotting of HDAC1 immunoprecipitates with p300/CBP. The presence of p300/CBP in the immunoprecipitates showed that p300/CBP forms the part of transcriptional regulatory complex. Thus our co-immunoprecipitation data showed the following: (i) Pax3 gets acetylated, and (ii) Pax3 associates with p300/CBP and HDAC1.


Figure 8
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  		FIGURE 8.
Chromatin immunoprecipitation of E10.0 (30 pair of somites) Pax3+/+ embryos show that HDAC1 and SIRT1 associate with certain cis-regulatory elements of the TGFbeta2 promoter that bound Pax3. ChIP assays were performed as described under "Experimental Procedures" using rabbit polyclonal HDAC1, SIRT1, and acetylated protein antibodies. The primers sets A-F used were the same as in Fig. 1. Lane 1, input DNA; lane 2, immunoprecipitated DNA using normal rabbit IgG; lane 3, immunoprecipitated DNA using HDAC1 antibody; lane 4, immunoprecipitated DNA using SIRT1 antibody; lane 5, immunoprecipitated DNA using acetylated protein antibody. Primer sets amplified a 200-bp PCR fragment from the immunoprecipitated DNA. Each ChIP experiment (n = 4) was performed in triplicate.

 


Figure 9
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  		FIGURE 9.
Whole mount in situ hybridization of Pax3+/+ and Pax3-/- E10.0 (30 somite) embryos using digoxigenin-labeled murine TGFbeta2 riboprobe. A shows the expression of TGFbeta2 mRNA in migratory neural crest cells in Pax3+/+ embryo; B shows the magnified field denoted by a dashed rectangle in A. C shows the expression of TGFbeta2 mRNA in migratory neural crest cells in Pax3-/- embryos. D shows the magnified field denoted by a dashed rectangle in C. The arrows show the migrating neural crest cells. Note that there is a significant reduction in the TGFbeta2 expression in Pax3-/- embryos. The sense riboprobe did not show any staining (data not shown). Each experiment was done at least three times for each genotype from 3 to 5 different pregnant heterozygous females.

 
Next we wanted to know whether chromatin immunoprecipitation can identify HDAC1, SIRT1, or acetylated protein interaction domains in the murine TGFbeta2 promoter, which was earlier defined by Pax3. By using the same set of TGFbeta2 promoter forward and reverse primers as used in Fig. 1, which defined the Pax3 recognition domains, we were able to map the HDAC1 interaction domains to the 200-bp PCR-amplified product, with the exception of primer set A (Fig. 8). However, when SIRT1 antibody was used for chromatin immunoprecipitation, only primer sets C, D, and F amplified the 200-bp fragment from the immunoprecipitates. When acetylated protein antibody was used for immunoprecipitation, only primer sets D and F amplified the 200-bp fragment from the immunoprecipitates. These observations suggest that not all regions of the TGFbeta2 promoter behave equally even though Pax3 might bind to at least seven oligonucleotides (Fig. 6) spanning five cis-regulatory regions of the TGFbeta2 promoter (Fig. 1). The presence of deacetylases such as HDAC1 and SIRT1 might also mean association of other acetylated transcription factors such as p53 (19-21) within the 200-bp cis-regulatory region of TGFbeta2 promoter. The 200-bp fragment amplified by primer set D (between bp 741 and 940) is note-worthy because this region of the promoter as defined by promoter construct TG6 (Fig. 3) has the (gt)32 repeats that showed diminished promoter activity in our promoter-luciferase reporter assays. p53 is a known substrate for SIRT1 (21, 22) involved in transcriptional repression of promoters. On the other hand, the 200-bp fragment amplified by primer set E (between bp 1012 and 1212) does not seem to have SIRT1 associated to it. Interestingly, the TGFbeta2 promoter construct TG4 (Fig. 3), which showed an increase in the promoter-luciferase activity, houses this 200-bp fragment. Further evaluation of which other trans-acting factors modulate the TGFbeta2 promoter activity along with Pax3 during early embryonic development is currently underway in our laboratory.

TGFbeta2 mRNA Expression Is Reduced in Pax3-/- Mice—Based on the above findings it was tempting to ask whether Pax3-/- embryos, whose phenotype is characterized by neural tube defects, show diminished levels of TGFbeta2 expression. To test the hypothesis that Pax3 regulates the expression of TGFbeta2 in vivo during early embryonic development, we used Pax3+/+ and Pax3-/- mouse embryos (E10.0; 30 somites). The neural tube in these genotypes is closed in the thoracicolumbar region, whereas the caudal region is open in Pax3-/- mouse embryos. We performed whole mount in situ hybridization of E10.0 (30 somites) of Pax3+/+ and stage-matched Pax3-/- littermates with digoxigenin-labeled murine TGFbeta2 riboprobe. We observed a significant reduction of TGFbeta2-positive staining in the migratory neural crest cells in Pax3-/- embryos (Fig. 9, C and D) as compared with the age-matched Pax3+/+ embryos (Fig. 9, A and B). In sum, our data suggest that Pax3 is involved in the regulation of TGFbeta2 expression in the migrating neural crest cells. Taken together, our data suggest that Pax3 is involved in the regulation of TGFbeta2 expression during early embryonic development.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Implications for TGFbeta2 Regulation by Pax3—Early embryonic development involves neural crest induction, epithelial-to-mesenchymal transition neural crest cell migration, and subsequent neural tube formation (23). Failure of any of these processes results in a neural tube defect, characterized by exencephaly and anencephaly in the rostral part and as spina bifida in the caudal part of the neural tube (24). Pax3 is expressed in the developing neural crest (25). Neural crest cells emerge from the dorsal neural tube and migrate throughout the developing embryo, where they give rise to a range of cell types, including neurons and glial cells of the peripheral nervous system and melanocytes. In Pax3-/- embryos, neural crest cells fail to migrate correctly, resulting in either a severe reduction or a complete absence of neural crest derivatives (26). Studies by Serbedzija and McMahon (27) indicate that the defect in the Splotch (Pax3+/-) mutation is not intrinsic to the neural crest cells themselves but appears to reflect inappropriate cell interactions either within the neural tube or between the neural tube and the somite. When caudal neural tubes from Splotch (Pax3+/-) embryos were grafted in a nonsplotch host, normal neural crest migration was observed, suggesting that these interactions are defective in Splotch (Pax3+/-) mice and that neural crest generation requires interaction with neighboring tissue (27). These experiments imply that a soluble mediator or the extracellular matrix is involved in neural crest cell migration. TGFbeta2 may be a candidate for this soluble mediator. Several studies discussed below support this possibility.

TGFbetas have been shown to modify neural crest cell differentiation and migration (28). TGFbetas regulate key aspects of embryonic development and major human diseases (29). TGFbeta2 mediate effects on cell migration by regulating genes responsible for remodeling the cell-extracellular matrix as well as genes related to adhesion molecules/receptors or the cytoskeleton. In support of this view, Col2A(I) has been shown to be a downstream target of TGFbeta2 signaling (30). Similarly, an increase in versican, a chondroitin sulfate proteoglycan, and associated proteins in response to growth factors, such as platelet-derived growth factor and TGFbeta1, have been shown to cause an increase in the pericellular matrix of the cells and expansion of the extracellular matrix (31). Previously, we showed that Pax3 overexpression causes an up-regulation of the V2 splice variant of versican and down-regulation of the V3 splice variant (6). It is quite plausible that Pax3 may affect versican regulation via TGFbeta2 signaling. Zavadil et al. (32) have shown increased gene expression of a host of extracellular matrix molecules, such as VEGF, tenascin, lamininB1, lamininA3, fibronectin1, thrombospondin, integrin{alpha}2, following TGFbeta treatment. Similarly, genes important for cytoskeletal remodeling have been shown to be up-regulated following TGFbeta treatment (32).

In addition to acting on genes involved in cell migration, TGFbeta2 signaling has also been shown to act on genes involved in neural crest generation and epithelial-mesenchymal transformation. For instance, Slug encoding a zinc finger transcription factor, is a downstream target of TGFbeta signaling (33). Slug is expressed in both pre-migratory and migratory chick neural crest cells (17) and promotes desmosome dissociation (33), a key step in epithelial-to-mesenchymal transition. Recently, Msx1 and Pax3 (34) and Pax3 and Zic1 (35) have been shown to be responsible for Slug induction, a direct TGFbeta2 target. TGFbeta also up-regulates Sox4 and Sox9, key regulators in the differentiation of early mesenchymal progenitor cells into cardiac (36) and chondrogenic lineage (37). Thus, Pax3 regulation of TGFbeta2 at the time of neural tube closure seems quite plausible in the light of the above findings.

Pax3 is not the only functional regulator of TGFbeta2, but one important regulator of neural crest cell migration. Developmental events such as neural tube closure are tightly modulated by gene regulatory networks (23, 38, 39, 41). The relatively small change in the expression of a key regulator gene such as TGFbeta2 can either be amplified by a regulatory network of genes to affect the expression of downstream genes or can signal a counter-regulatory loop to compensate for the change. Earlier we had shown that a 2-4-fold difference in the expression of ST8SiaII/STX in Pax3 transfectants had a profound effect on NCAM polysialylation. Furthermore, an increase in PSA-NCAM resulted in a complete phenotypic change by inhibiting NCAM-dependent aggregation and increasing PSA-NCAM heterophilic adhesion to heparin sulfate proteoglycan (13). In summary, our results show the following: (i) Pax3 regulates TGFbeta2 gene by binding to the cis-regulatory elements on the promoter, and (ii) the expression of TGFbeta2 is reduced in migratory neural crest cells in Pax3-/- mice. Overall, these results support the hypothesis that Pax3 regulates TGFbeta2 expression during early embryonic development.

Trans-regulatory Factors Affecting Pax3 Regulation of TGFbeta2 Promoter—The question is, "Does TGFbeta2 belong to a Pax3-dependent developmental program?" Our data support that Pax3 regulates TGFbeta2 but does not suggest that TGFbeta2 belongs to a Pax3-dependent developmental program. TGFbeta2 regulation by Pax3 may represent an aspect of the Pax3-dependent developmental program, such as migration of neural crest cells, but is not necessarily responsible for all the downstream events. The studies by Pani et al. (42) is particularly noteworthy because they suggested that neural tube closure is a Pax3-independent process, and the sole required function of Pax3 in neural tube closure is to inhibit p53-dependent apoptosis. Similarly, SIRT1 has been shown to inhibit p53-dependent apoptosis (43). Cordenonsi et al. (44) have shown that p53 partners with Smad2 in the activation of multiple TGFbeta target genes. Wilkinson et al. (45) have shown that there is a direct cooperation between p53 and TGFbeta effectors in chromatin modification and transcription repression. Secreted TGFbeta2 can activate NF-{kappa}B, block apoptosis, and is essential for the survival of some tumor cells (40). These observations and our finding that Pax3 regulates TGFbeta2 may imply that Pax3 may be performing the same role of blocking apoptosis the way it does to block p53-mediated apoptosis.

In summary, we show the following: (i) Pax3 regulates TGFbeta2 directly by binding to cis-regulatory elements on the promoter; (ii) certain regions of the cis-regulatory elements in TGFbeta2 promoter bind acetylated Pax3, which is associated with deacetylases and p300/CBP; and (iii) TGFbeta2 expression is diminished in migrating neural crest cells in Pax3-/- embryos.


   FOOTNOTES
 
* This work was supported in part by a State of Illinois Excellence in Academic Medicine award (to C. S. K. M.) and a grant from Spastic Paralysis Research Foundation of Illinois-Eastern Iowa District of Kiwanis (to C. S. K. M. and D. G. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1. Back

2 Present address: Graduate Program in Neurobiology, UCLA, Los Angeles, CA 90095. Back

3 Present address: Dept. of Neurology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611. Back

1 To whom correspondence should be addressed: Neurobiology Program, Children's Memorial Research Center, M/c 226, 2430 N. Halsted, Chicago, IL 60614. Tel.: 773-755-6530; Fax: 773-755-6363; E-mail: smayanil{at}northwestern.edu.

4 The abbreviations used are: E, embryonic day; ChIP, chromatin immunoprecipitation; EMSA, electromobility shift assay; DMEM, Dulbecco's modified Eagle's medium; HDAC1, histone deacetylases 1; SIRT1, silent information regulator 1 (Sir2{alpha}); TRP-1, tyrosinase-related protein-1; PDBS, paired domain binding site; HDBS, homeodomain-binding site; PBS, phosphate-buffered saline; TGFbeta, transforming growth factor-beta. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Peter Gruss (Max Planck Institute, Gottingen, Germany) for the pBH3.2 plasmid containing Pax3; Dr. Angie Rizzino (University of Nebraska Medical Center) for the murine TGFbeta2 promoter; Dr. Mary R. Loeken (Harvard Medical School, Boston) for the Pax3-GST fusion cDNA; Dr. M. Azhar for plasmid containing mouse TGFbeta2 clone; and Dr. John A. Kessler (Feinberg School of Medicine, Northwestern University) and Dr. Sara Ahlgren (Children's Memorial Medical Center) for valuable discussions.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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