Transcriptional Control in Cardiac Progenitors: Tbx1 Interacts with the BAF Chromatin Remodeling Complex and Regulates Wnt5a

Mutations of the Wnt5a gene, encoding a ligand of the non-canonical Wnt pathway, and the Ror2 gene, encoding its receptor, have been found in patients with cardiac outflow tract defects. We found that Wnt5a is expressed in the second heart field (SHF), a population of cardiac progenitor cells destined to populate the cardiac outflow tract and the right ventricle. Because of cardiac phenotype similarities between Wnt5a and Tbx1 mutant mice, we tested potential interactions between the two genes. We found a strong genetic interaction in vivo and determined that the loss of both genes caused severe hypoplasia of SHF–dependent segments of the heart. We demonstrated that Wnt5a is a transcriptional target of Tbx1 and explored the mechanisms of gene regulation. Tbx1 occupies T-box binding elements within the Wnt5a gene and interacts with the Baf60a/Smarcd1 subunit of a chromatin remodeling complex. It also interacts with the Setd7 histone H3K4 monomethyltransferase. Tbx1 enhances Baf60a occupation at the Wnt5a gene and enhances its H3K4 monomethylation status. Finally, we show that Baf60a is required for Tbx1–driven regulation of target genes. These data suggest a model in which Tbx1 interacts with, and probably recruits a specific subunit of, the BAF complex as well as histone methylases to activate or enhance transcription. We speculate that this may be a general mechanism of T-box function and that Baf60a is a key component of the transcriptional control in cardiac progenitors.


Introduction
The second heart field (SHF) provides progenitor cells for the development of several segments of the mature heart, such as the outflow tract, right ventricles and atria [1,2]. Mouse models of congenital heart disease suggest that perturbation of SHF development may be the basis of relatively common heart defects in humans, such as conotruncal anomalies, but the transcriptional mechanisms driving SHF development are not well defined. An important example of a congenital heart disease gene that functions within the SHF is TBX1 encoding a T-box transcription factor. This is haploinsufficient in the DiGeorge/Velocardiofacial/22q11.2 deletion syndrome, which is associated with various types of cardiac outflow tract (OFT) and vascular defects [3]. Tbx1 mouse mutants recapitulate well the human phenotype, which has facilitated a detailed analysis of the role of the gene in heart development. In particular, Tbx1 is expressed in the SHF where it sustains cell proliferation and inhibits differentiation [4]. However, there is considerable less information about the effectors of these developmental roles, and about the mechanisms for target gene regulation.
SHF cardiac progenitors, which reside outside the heart, are thought to migrate into the heart and differentiate as they are incorporated into the OFT. Thus, it is reasonable to expect that transcriptional regulation of SHF progenitors should involve cell polarity and cell migration, functions that in many cases are regulated by the non-canonical Wnt pathway [5]. Here we show that Wnt5a, which encodes a ligand of the non-canonical pathway, is expressed in the SHF. It has been shown that Wnt5a 2/2 animals have OFT abnormalities, though generally not as severe as in Tbx1 2/2 animals [6]. In addition, OFT defects have also been shown in Ror2 2/2 mice, Ror2 being a receptor of Wnt5a. Furthermore, mutations of WNT5a or ROR2 in humans are associated with Robinow syndrome [7], which includes OFT defects, though at a low penetrance. Therefore, we postulated that there may be interaction between Tbx1 and the non-canonical Wnt pathway. We have crossed Tbx1 and Wnt5a mouse mutants and found that there is indeed a genetic interaction and, most interestingly, the loss of both genes caused developmental failure of the SHF-dependent heart segments, indicating that the two genes are required for SHF function. Next, we investigated the transcriptional mechanisms underlying this interaction. We found that Wnt5a is a transcriptional target of Tbx1. Previous data showing a genetic interaction between Tbx1 and the gene encoding the chromodomain protein Chd7 [8] and physical interaction with the histone methyltransferase Ash2l [9] prompted us to investigate the role of chromatin remodeling and histone modifiers. We found that Tbx1 co-immunoprecipitates with Baf60a, a component of the SWI-SNF-like BAF chromatin remodeling complex, and with the Setd7 histone 3, Lysin 4 monomethyltransferase. Tbx1 expression increases the occupation of the Wnt5a gene by Baf60a, and enhances the H3K4 monomethylation status of the chromatin in the T-box binding element (TBE)-harboring regions of the Wnt5a gene. Finally, we found that Baf60a is required for Tbx1-induced regulation of Wnt5a. Overall, our data support a mechanism by which Tbx1 enhances transcription of Wnt5a (and possibly other target genes) by interacting with a chromatin remodeling complex and enhancing H3K4 de novo methylation. This is the first evidence of an impact of Tbx1 on chromatin status and of its interaction with the BAF complex. Our data also raises the intriguing possibility that Baf60a is a critical BAF subunit for the transcriptional program of cardiac progenitors.

Wnt5a is regulated by Tbx1 in the SHF
We carried out in situ hybridization of Wnt5a in embryos from E8.0 to E11.5 and found strong expression in the pharyngeal and splanchnic mesoderm including the SHF region ( Figure 1). In addition, we found expression in the OFT, as previously noted [6,10]. We next tested whether Wnt5a expression may be altered by Tbx1 mutation. At E8.0-8.5, Wnt5a expression was reduced in the SHF of Tbx1 2/2 embryos (n = 3; Figure 1), while it was not affected in the OFT or pharyngeal arch core mesoderm ( Figure 1). 3D reconstruction analysis of section images provides an overall view of the expression domains affected by loss of Tbx1 ( Figure 1K-1P). Similar results were obtained at later stages (E9.0-11.5, not shown).
To confirm that Wnt5a is expressed in cardiac progenitors, we carried out qRT-PCR on the P19CL6 mouse embryonal carcinoma cell line that is able to differentiate into cardiomyocyte progenitors upon 5-azacytidine and/or DMSO treatment [11]. We found that Wnt5a is robustly expressed after 3 days of differentiation ( Figure S1). The activation of Wnt5a expression roughly coincides with the up-regulation of Tbx1 expression, suggesting that Tbx1 may have a role in the activation of Wnt5a rather than in its expression maintenance. We also found that loss of Wnt5a in these cells reduces cell migration, as tested by a wound-healing assay ( Figure S2). Next we tested if Tbx1 can regulate Wnt5a expression in these cells. For this, we transfected graded amounts of a Tbx1-expressing vector into P19CL6 undifferentiated cells and evaluated expression of the endogenous Wnt5a gene by qRT-PCR. Results revealed that Wnt5a expression responds positively to Tbx1 in a dosage-dependent manner ( Figure  S3).
Analysis of Wnt5a 2/2 embryos (n = 5) revealed ventricular septal defects (VSD) and abnormal positioning of the great arteries (aorta and pulmonary trunk), which were side-by-side ( Figure 2B-2D). However, in none of these embryos did we observe truncus arteriosus communis (TAC), although this defect was previously reported for most Wnt5a 2/2 embryos [6]. This difference may be due to different genetic backgrounds. However, the extracardiac phenotype that we found was consistent with previously reported data (cleft palate, cleft lip, small thymus, truncated tail and limbs) ( Figure 2A and data not shown). Analysis of Tbx1 +/2 ;Wnt5a 2/2 embryos (n = 22) revealed a more severe phenotype than in Wnt5a 2/2 embryos from the same crosses. In particular, out of 22 embryos examined, 13 (59%) showed additional abnormalities compared to Wnt5a 2/2 embryos, namely TAC, small ear and edema ( Figure 2A9-2D9). The other embryos (9 out of 22 or 41%) were phenotypically indistinguishable from Wnt5a 2/2 embryos. Together, these results indicate a genetic interaction between Tbx1 and Wnt5a.
We did not retrieve any double homozygous embryos at E18.5 (n = 162), suggesting that this genotype is lethal during early embryogenesis. Therefore, we harvested embryos at earlier embryonic stages. The latest stage at which we found live Tbx1 2/2 ; Wnt5a 2/2 embryos was E9.5. At this stage, double homozygous embryos (n = 10) showed a severe cardiac phenotype characterized by severe hypoplasia of the OFT and right ventricle (RV), structures that sometimes appeared to be absent by visual inspection (Figure 3). Histological sections confirmed that the development of these structures is severely affected ( Figure 3A0-3D0). We carried out in situ hybridization with a probe for CyclinD2, a marker of the proximal OFT and the right ventricle (RV) [12]. Results showed that CyclinD2 was normally expressed in control, Tbx1 2/2 and Wnt5a 2/2 E9.5 embryos, but it was greatly reduced or undetectable in Tbx1 2/2 ;Wnt5a 2/2 embryos (Figure 4), confirming the severe hypoplasia of the OFT and of the RV. We asked whether the hypoplasia of these structures could be due to increased apoptosis. We carried out immunohistochemistry on E9.5 embryos using an anti-cleaved Caspase 3 antibody. Results showed very few positive cells in the OFT and RV of WT and

Author Summary
We have demonstrated a novel interaction between the Tbx1 gene, the mutation of which causes DiGeorge syndrome, and Wnt5a, another human disease gene, which is important for oriented cell migration and cell polarity. We found that, in mice, reduced dosage of each of the two genes enhances the phenotype caused by the mutation of the other. Loss of the two genes in mice has very severe consequences for heart development. Our genetic and biochemical data determined that Tbx1, a transcription factor of the T-box family, regulates Wnt5a expression. We found that Tbx1 targets the BAF chromatin remodeling complex to the Wnt5a gene and interacts with a histone monomethyltransferase. Tbx1 expression increases Baf60a occupation of the Wnt5a gene and enhances its H3K4 monomethylation status, while Baf60a knockdown abolishes the ability of Tbx1 to regulate Wnt5a and other target genes. Overall, our data identify Wnt5a as an important effector of Tbx1 function in heart development and demonstrate that Tbx1 regulates the gene by interacting with the chromatin remodeling and histone methylation machinery.

Wnt5a is a target of Tbx1
To understand whether Wnt5a may be a direct target of Tbx1, we searched for T-Box binding elements (TBEs) in a 20 kbp genomic sequence encompassing the mouse Wnt5a gene. We found three putative TBEs, two of which closely located in intron 3-4 (TBE1: AAGGGGTGAA, TBE2: GTAGGTGCCAGG) and one in the 39-UTR (TBE3: AGAGGTGTTGCA) ( Figure 6A). We next cloned two evolutionarily conserved (in human and mouse) DNA segments containing TBE1-2 and TBE3, into a luciferase reporter plasmid with a basic promoter ( Figure 6B-6C). We also generated mutagenized constructs in which the three TBEs were mutated (the core sequence GTG was changed into AAA). We then carried out luciferase assays. Results showed that the two DNA segments responded well to transfected Tbx1 ( Figure 6B-6C). However, mutation of the TBEs, individually or combined, all had a significant impact on the ability of Tbx1 to activate the constructs ( Figure 6B-6C), although the mutation of TBE1 had a relatively lower effect. Thus, these TBEs are required for Tbx1induced activation in this assay.
Next, we tested whether the endogenous Tbx1 protein occupies these TBE sites in P19Cl6 cells by standard and quantitative chromatin immunoprecipitation (ChIP) assays using a Tbx1 antibody. TBE1 and TBE2 were assayed together because they are too close to be assayed independently. Results demonstrated that indeed these sites are occupied by endogenous Tbx1 in P19CL6 cells ( Figure 7A-7B). Next, we repeated the same assay using chromatin from E9.5 wild type embryos and again we could demonstrate enrichment at the Wnt5a TBE loci ( Figure 7C).

Tbx1 interacts with the BAF chromatin remodeling complex and with the Setd7 histone methyltransferase
To begin to dissect the transcriptional machinery within which Tbx1 exerts its transcriptional functions, we tested by coimmunoprecipitation (co-IP) or affinity purification chromatin remodeling and histone modification proteins, Baf60a, Baf60c, Baf155, Setd7 and p300. Under the experimental conditions tested, we found that Baf60a and Setd7, but not Baf60c, Baf155 and p300, co-immunoprecipitated with Tbx1 ( Figure 8 and Figure  S4A)). The Baf60a-Tbx1 interaction was also confirmed using a GST pull down assay ( Figure S4B). Furthermore, we confirmed Baf60a-Tbx1 co-IP using E9.5 embryo tissues ( Figure 9A). At this stage, the Baf60a gene is expressed broadly, including the SHF, but is very weakly expressed or absent in the heart ( Figure 9B). In  Table 1. Summary of phenotyping results of E18.5 embryos from Tbx1 +/2 ;Wnt5a +/2 X Tbx1 +/2 ;Wnt5a +/2 intercrosses.

Total 162
AoA Abn: Abnormalities of the aortic arch and of the origin of the right subclavian artery. Ao-PT: abnormal relative position of the aorta and pulmonary trunk, arranged side-by-side. contrast, Baf60c, an alternative component of the BAF complex, is mainly expressed in the heart and somites, and it has been previously shown to interact with another T-box transcription factor, Tbx5 [15]. While Tbx1 is mainly required in cardiac progenitors, where it is important to keep cells proliferating and to inhibit their differentiation, Tbx5 is important for cardiomyocyte differentiation. We compared expression of Tbx1, Baf60a and Baf60c mRNA during P19Cl6 cell differentiation. Results showed that Tbx1 is expressed in the early phases of differentiation. Baf60a is particularly strong in these early phases and then its expression is reduced (although still robustly expressed). In contrast, Baf60c expression is very low in the early phases of differentiation and becomes quite strongly expressed as differentiation proceeds ( Figure 1). If the Baf60a-Tbx1 interaction were a feature of the transcriptional machinery at target genes, we would expect Baf60a to occupy the regions of the Wnt5a gene harboring the TBEs. To test this, we used ChIP with a Baf60a antibody on P19CL6 cells. Results demonstrated that indeed this protein occupies the TBE1/ 2 region as well as the TBE3 region ( Figure 10A). The same results were obtained with a ChIP assay using chromatin from E9.5 embryos ( Figure 10B).

Tbx1 affects the chromatin status of TBE regions of Wnt5a and requires Baf60a for its function
Given the interactions with the BAF complex and with Setd7, we postulated that Tbx1 would recruit Baf60a to the target gene and would affect histone methylation. Indeed, quantitative ChIP using a Baf60a antibody showed that Tbx1 increases significantly the enrichment of Wnt5a TBE sequences ( Figure 10C). Thus, increased dosage of Tbx1 is sufficient to enrich the Wnt5a gene with a chromatin remodeling complex. Next, we asked whether Tbx1 dosage can modulate H3K4me1 at TBE regions of the Wnt5a gene. To this end, we carried out quantitative ChIP with an anti H3K4me1 antibody (Setd7 is a monomethyltransferase) on P19Cl6 cells transfected with Tbx1. Results indicated that there is indeed enrichment of H3K4me1 after increased dosage of Tbx1 ( Figure 11A-11B). Next, we tested whether H3K4me1 is also affected by loss of Tbx1 in vivo. To this end, we carried out qChIP assays using chromatin from WT and Tbx1 2/2 E9.5 embryos. Results indicated that there is a significantly higher enrichment of H3K4me1 in WT embryos compared to mutant embryos, but limited to the TBE1/2 locus ( Figure 11C).
Next, we tested additional histone H3 modifications associated with gene activation. In particular, we carried out qChIP using chromatin from P19Cl6 cells transfected with an empty vector or with a vector over-expressing Tbx1 using antibodies against H3K4me2, H3K4me3, or AcH3 (recognizing acetylation of histone 3). Results indicated that none of these modifications are enriched in correspondence of the TBE loci of the Wnt5a gene, regardless of Tbx1 transfection ( Figure 12).
Finally, we asked whether Baf60a is important for Tbx1induced upregulation of the Wnt5a gene. To this end, we knockeddown Baf60a expression by RNA interference and determined the ability of Tbx1 to regulate Wnt5a in the presence of reduced Baf60a dosage in P19CL6 cells. Results showed that after a knock down of approx. 80% of Baf60a, Tbx1 was unable to regulate Wnt5a ( Figure 13). Interestingly, without Baf60a, Tbx1 was not able to activate other candidate targets (Fgf8, Fgf10 and Cyp26a1) indicating that Baf60a is required for Tbx1 transcriptional activity in this context. Baf60a knock-down per se did not affect significantly the basal expression of endogenous Tbx1, Wnt5a, Fgf8, Fgf10 and Cyp26a1 in control experiments ( Figure S5).

Discussion
This work identifies Wnt5a as a novel and important transcriptional target of Tbx1. Combined loss of the two genes caused severe hypoplasia of SHF-derived heart segments, and early embryonic lethality. This is a much more severe phenotype than that caused by loss of the individual genes. The exact cause of the dramatic heart phenotype will require further investigation. However, our data suggest at least two possible mechanisms. Loss of Tbx1 impairs the ability to expand the heart progenitors pool of the SHF [16], the loss of Wnt5a impairs their ability to migrate (and/or to be correctly orientated) into the heart. Thus, a double loss would essentially amount to a double hit upon cardiac progenitors, which would be fewer in number and less capable of contributing to the heart. Another possible cause of the severe phenotype might be an interference with the Wnt canonical signaling pathway in the double mutants, as suggested by the observed upregulation of b-catenin expression. Constitutive activation of Wnt canonical signaling in the SHF leads to severe heart abnormalities that are similar to those observed in the double homozygous mutants [17].
The importance of the non-canonical Wnt signaling for OFT development has already been illustrated by the study of Wnt11 mouse mutants [18]. Wnt5a and Wnt11 are both expressed in the OFT but, in contrast to Wnt5a, Wnt11 is not expressed in the SHF. It would be interesting to cross the two mutants to see whether there is functional redundancy in the OFT proper.
Because of the clear in vivo importance of the Tbx1-.Wnt5a transcriptional control for SHF function, we decided to focus our attention on the mechanisms regulating this control, as a possible paradigm for transcriptional control in the SHF.
We found an interaction between Tbx1 and Baf60a. The latter is a component of the BAF (Brg1-Brm Associated Factor) complex, which is related to the yeast and fly SWI-SNF chromatin remodeling complex. The canonical function of the BAF complex is to utilize its ATPase activity to rearrange the nucleosome distribution of chromatin, thus playing a crucial role in regulating accessibility to components of the transcriptional machinery, and thereby gene expression, either positively or negatively [19]. One of the critical issues concerning the biology of the BAF complex, is how it is targeted to genes that need to be regulated, i.e. how is specificity achieved despite the apparent homogeneity of the core   components of the complex. One possibility is that specificity is provided by non-core subunits and by the recruitment activity operated by transcription factors that target specific genes. Our ChIP data suggest that this may be the case, as Tbx1 appears to recruit a BAF subunit onto the Wnt5a gene. Interaction with a histone methyltransferase would further help transcription, for example by stabilizing the remodeling machinery on the locus. We show that indeed Tbx1 expression correlates with increased H3K4 monomethylation of the Wnt5a TBE loci in cultured cells and in vivo. Interestingly, Tbx1 co-immunoprecipitates with Baf60a but not Baf60c. These are two alternative subunits of the BAF complex, possibly associated with different target genes and different cellular differentiation states. Baf60a has been associated with undifferentiated/multipotent status [20,21], while Baf60c has been associated with differentiating muscle cells (cardiac or skeletal) [22,23]. We also show here that Baf60a tends to be downregulated during P19Cl6 cell differentiation, while Baf60c is upregulated. This finding is supported by in vivo expression data indicating that Baf60a is poorly represented in differentiated heart tissue in contrast to Baf60c, which is mostly expressed in heart and somite tissue [15]. Thus, it is possible that an exchange of BAF subunits plays a role in the passage from the progenitor state to the differentiated cardiomyocyte state. Exchange of BAF subunits has already been described during differentiation from neural progenitors to neurons (Baf53a to Baf53b, and Baf45a to Baf45b) [24].
Exactly what promoters or enhancers exchange BAF subunits during cardiac differentiation would be an interesting question to address using genome-wide ChIP-seq studies.
Setd7 is a H3K4 monomethyltransferase [25][26][27] already shown to interact with another T-box transcription factor named Tbx21 (also known as Tbet) [28]. It is thus tempting to speculate that interaction with the BAF complex and histone methyltransferases is a common feature of T-box proteins and at the core of the transcriptional function of these important transcription factors.
Interestingly, our tissue culture experiments evidentiated that Tbx1 has a positive effect on H3K4me1 enrichment, but not H3K4me2, H3K4me3, or H3 acetylation. This suggests that Tbx1 promotes de novo methylation H3K4 at TBE enhancers perhaps making them a target for additional regulators.
Overall, our data support a model by which Tbx1 regulates Wnt5a by interacting with and perhaps recruiting a specific subunit of the BAF complex, along with the histone modifier enzyme Setd7, resulting in activation or enhancement of transcription of the target gene. While a number of important molecular details remain to be clarified, our data using a tissue culture model indicate the importance of Baf60a for Tbx1-induced regulation of Wnt5a and perhaps other target genes. Hence it is reasonable to speculate that this BAF subunit is a key cofactor for Tbx1 function in cardiac progenitors.

Ethics statement
All animal experimentations were carried out according to animal welfare regulations and guidelines of the USA and of the European Union.

Mouse lines
We used the mouse lines Tbx1 +/lacZ (here cited as Tbx1 +/2 ) [29], available through the EMMA repository, and Wnt5a +/2 [10] available through the Jackson Laboratories. Both are null alleles. Genotyping was carried out according to instructions provided by the original reports.
All crosses were carried out in conventional, clean facilities in a C57Bl6/129SvEv mixed genetic background.

Mouse embryo phenotyping
Embryos were examined after manual dissection under a stereo microscope. In most cases we also carried histological sectioning of paraffin-embedded specimens. Whole mount in situ hybridization according to standard methods. Embryos were photographed and then sectioned. In some cases we used images of sections for 3D reconstruction using the Amira software.

Constructs and cell lines
P19CL6 cells were grown in Dulbecco-Modified Minimal Essential Medium supplemented with 10% fetal bovine serum. For cardiomyocytes differentiation the cells were plated at a density of 5.0610 5 cells/well on a 35-mm tissue culture dish. The next day, when cells reached ,90% confluence, the medium was replaced with a growth medium containing 10 mM 5-Azacytidine for 24 h [11]. After treatment with 5-Azacytidine, cells were incubated in the growth medium containing 1.0% DMSO that was changed daily in order to remove the cell debris resulting from cell death. The experimental days were numbered consecutively beginning from the day of treatment with 5-Azacytidine (day 0).
The generation of the stably transfected cell lines P19-Tbx1-TEV-PA and P19-TEV-PA has been described [30]. These cell lines have been used for affinity purification experiments (see below).
For transient transfection, cells were cultured in 10 cm dishes until 60-70% confluent and transfected with Fugene6 (Roche) following the manufacturer protocol. Dharmacon ON-TARGETplus SMARTpool Baf60a/Smarcd1 siRNA was used to knockdown Baf60a expression using Fugene6 transfection reagent. ON-TARGETplus siCONTROL Non-targeting pool was used for control transfections.

Wound healing assay
P19Cl6 cells were cultured to confluent monolayer in 12-well. Two hours before the experiment, we treated cells with mitomycin c (10 mg/ml) and then we made a scratch wound using a standard 200-ml pipette tip. Wounded monolayers were washed with PBS and digitally photographed for the 0 hour timepoint using an inverted microscope equipped with a digital camera (Leica AF6000LX time-lapse). Images of the wound were acquired every 30 min for 24 h. Subsequently, pictures were analyzed using the ''Image J'' software. The wound healing effect was calculated as area wound closure compared with the area of the initial wound. Briefly, the distance between the wound margins was measured at 0 hours and again every 4 hours post-wounding for 24 h. 12 hours post-wounding the following formula was used to evaluate the area wound closure: distance t = 12 h -distance t = 0 h . Data represent the average of at least 3 independent experiments (2 wells/experiment).

Affinity purification
Native affinity purification was performed with strains P19-Tbx1-PA and P19-PA as previously described [30,31]. Briefly, nuclear extracts were transferred to Poly-Propylene Chromatography Columns with IgG sepharose beads (Amersham) to capture Tbx1-TEV-Protein A-containing complexes. Then, the columns were subjected to protease TEV cleavage to release the Tbx1- containing protein complexes, which were recovered and transferred into PVDF membrane (Amersham) for Western blotting analyses. The antibodies used for immunoblotting were the monoclonal anti-Baf60a antibody (BD Biosciences, #611728) and the anti-p300 (BD Biosciences, #554215).

Chromatin immunoprecipitation
P19Cl6 cells were cross-linked using 1% formaldehyde at room temperature for 15 min, and the reaction was stopped using glycine at a final concentration of 0.125 M for 5 min. Cells were then lysed in 1 ml of lysis buffer (10 mM HEPES, 60 mM KCl, 1 mM EDTA, 0.075% v/v NP40, 1 mM DTT and 1X protease inhibitors, pH 7.6) on ice for 10 min, dounced using a 2 ml B dounce to release nuclei. Isolated nuclei were suspended in Nuclei lysis buffer (20 mM TrisHCl, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% Glycerol and 1X protease inhibitors, adjusted to pH 8.0), incubated on ice for 30 min, washed in nuclei lysis buffer and sonicated to obtain 200-500 bp. Sonicated chromatin was immunoprecipitated 10 mg of a Tbx1 antibody Figure 11. Tbx1 enhances H3K4 monomethylation of TBE regions of Wnt5a in cultured cells and in vivo. (A) ChIP assays of P19Cl6 cells using an anti-H3K4me1 antibody or mouse IgG. DNA isolated from immunoprecipitated material was amplified by standard PCR with primers to amplify the TBE1-2 and TBE3 regions of Wnt5a. The amplified PCR fragments were analyzed on 2% agarose gel. (B) q-ChIP assay from P19Cl6 cells transfected with an empty vector (EV) or with a vector over-expressing Tbx1 (Tbx1), followed by quantitative real-time PCR. The immunoprecipitation was carried out with an anti-H3K4me1 antibody. The enrichment of TBE1-2 and TBE3 regions is evaluated compared to an internal control. TBE regions are significantly more enriched in the Tbx1 overexpressing sample, indicating that increased dosage of Tbx1 correlates with increased methylation of H3K4 cross-linked to these DNA sequences. (C) q-ChIP assay from E9.5 Tbx1 2/2 and WT mouse embryos followed by quantitative realtime PCR. The immunoprecipitation was carried out with an anti-H3K4me1 antibody. The enrichment of TBE1-2 and TBE3 regions is evaluated compared to an internal control and expressed as percentage of input. Note the reduced enrichment of H3K4me1 at the Wnt5a TBE1-2 in Tbx1 2/2 embryos compared to WT. No change could be detected at the TBE3 locus. Values are from 3 experiments (mean6S.D.). Asterisks indicate statistically significant difference (P-value less than 0.05). doi:10.1371/journal.pgen.1002571.g011 (Abcam, #ab18530), 2 mg of Baf60a antibody (BD Biosciences, #611728), 2 mg of H3K4me1 antibody (Abcam, #ab8895), 2 mg of Dimethyl-Histone H3 (Lys4) (Millipore, #07-030), 5 mg of Antitrimethyl-Histone H3 (Lys4) (Millipore, #07-473), 5 mg of Antiacetyl-Histone H3 (Millipore, #06-599), or normal rabbit/mouse IgG (Santa Cruz Biotechnology, #2027). Samples were then incubated with 20 ml of protein A/G PLUS agarose (Santa Cruz Biotechnology) at 4uC ON. The samples were extensively washed and incubated in an elution buffer (1% SDS and 0.1 M NaHCO3) at 30uC for 20 min. Cross-linking of protein-DNA complexes was reversed at 65uC ON, followed by treatment with DNase-fre RNase A for 30 min at 37uC and 100 mg/ml proteinase K for 2 h at 55uC. Phenol/chloroform-extracted, and ethanol-precipitated DNA was resuspended in 50 ml of H2O and subjected to PCR amplification (see below). ChIP experiments using embryo tissue was carried out as follows. WT and Tbx1 2/2 E9.5 mouse embryos were dissected, their heads were cut off and the trunks were stored at 280uC. Pools of 5 embryos were used for each ChIP experiment. Each ChIP experiment was performed using 5 mg of H3K4me1 (Abcam, #ab8895), 10 mg of TBX1 antibody (Abcam, #ab18530) and 5 mg or 10 mg of normal rabbit IgG (Santa Cruz Biotechnology, #2027) as negative control with the LowCell ChIP Kit (Diagenode). In brief, the embryos were fixed in 1% formaldehyde at room temperature for 15 min and then glycine was added to stop the reaction to a final concentration of 0.125 M for 5 min. Subsequently, embryos were washed with PBS, disaggregated by dounce homogenization in cold PBS and then resuspended in cell lysis buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% Igepal supplemented with protease inhibitors). Next, nuclei were resuspended in 130 ml of Buffer B (LowCell ChIP Kit reagent) and chromatin was sheared into 200-500 bp long fragments using the Covaris S2 Sample Preparation System (Duty Cycle: 5%, Cycles: 6, Intensity: 2, Figure 12. Effects of Tbx1 on H3K4 di-and tri-methylation and H3 acethylation status of the TBE regions of Wnt5a. q-ChIP assay from P19Cl6 cells transfected with an empty vector (EV) or with a vector over-expressing Tbx1 (Tbx1) followed by quantitative real-time PCR. The immunoprecipitation was carried out with anti-H3K4me2, anti-H3K4me3 or anti-AcH3 antibodies. (A) TBE1/2 region, (B) TBE3 region, and (C) p16 promoter region (positive control). There is no enrichment for any of these histone modifications at the TBE regions. Values are from 3 experiments (mean6S.D.). doi:10.1371/journal.pgen.1002571.g012 Temperature: 4uC, Cycles per Burst: 200, Power mode: Frequency Sweeping, Cycle Time: 60 seconds, Degassing mode: Continuous). Following steps included incubation of the sheared chromation with antibody coated beads over night, several washing steps and reverse crosslinking. Next, equal DNA amounts of input and immunoprecipitated DNA were initially used as a template for conventional PCR amplification of the TBE1-2 region of Wnt5a (Wnt5a TBE1-2_1_F 59-CTTCCCCTGGTGTGGATATG-39, Wnt5a TBE1-2_1_R 59-AGAGGCTCCTTCCAGTCCTC-39) and TBE3 region (wnt5a TBE-3_1_F 59-ACTGCTGGTAGGGCAGAAAA-39, wnt5a TBE-3_1_R 59-TCAGGCACCATTAAACCACA-39).
For quantitative ChIP, we next carried out real-time PCR of the immunoprecipitated DNA and inputs, using the FastStart Universal SYBR Green Master kit (Roche) and the 7900HT Fast Real-Time PCR System (Applied Biosystems). ChIP signals were normalized to that of an internal control amplimer that we have selected from an ORF-free region of mouse chromosome 14 (INT-XIV-1F 59-TTCTTGTCCACAGCCCTCTT-39, INT-XIV-1R 59-TGGTG-GAAGAGGAGACATCC-39). PCR efficiencies were determined for each primer pair using standard curves. PCR was carried out on 2 ml of 1/100 dilution of the input or 2 ml of immunoprecipitated samples.

Expression and purification of GST-Tbx1 fusion proteins
A GST-Tbx1 expression vector was kindly provided by Dr. Amendt (IBT, Texas A&M University, Houston, TX USA) and transformed into E. coli BL21(DE3) cells. Protein synthesis was induced by the addition of IPTG to a final concentration of 0.1 mM. GST pull-down assay was performed using ProFound Pull-Down GST Protein: Protein Interaction Kit (PIERCE).
RNA extraction, cDNA synthesis, and Q-RT-PCR RNA was extracted from P19Cl6 cells using TRI-Reagent (Ambion/Applied Biosystems) according to the manufacturer's protocol. Extracted RNA was treated with DNA-free Kit (Ambion/Applied Biosystems). cDNA was synthesized from 2 mg total RNA (normalized via UV spectroscopy) using the High Capacity cDNA Reverse Transcription Kit, according to the manufacturer's instructions (Applied Biosystems). Target cDNA levels were compared by Q-RT-PCR in 20-ml reactions containing 16 SYBR green (Applied Biosystems), 100 mm of each primer, and we used the 7900HT Fast Real-Time PCR System (Applied Biosystems). Results were normalized against glyceraldehyde-3phosphate dehydrogenase (GAPDH) expression, unless otherwise indicated. Relative expression was evaluated using the delta-deltacycle threshold method.