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

Tbx1 regulates Smad signaling.

To have insight into the molecular mechanisms governing the function of Tbx1, a transcription factor involved in DiGeorge syndrome (DGS) and cardiovascular development, we searched for proteins interacting with it. Using an affinity purification protocol followed by a candidate protein approach, we found that Smad1/5/8 interacted with Tbx1. To confirm this interaction, protein extracts from mouse embryos were co-immunoprecipitated with an antibody anti Tbx1 and analyzed by western-blot. Results confirmed the interaction and revealed that Smad1 is the protein directly interacting with Tbx1. Next, we tested TBX1 missense mutations found associated with a DGS phenotype but. Results indicate that one of the mutant isoforms is unable to bind Smad1. To examine the role of Tbx1 in the regulation of transcriptional responses induced by Smad1 we performed luciferase assays and the results show that Tbx1 is capable of suppressing the activity of a Smad signaling reporter in a dosage-dependent manner. We also found, using a Co-IP approach, that Tbx1 inhibits Smad signaling activity by competing with Smad4 for binding to Smad1. Using a Tbx1 mutant isoform that prevents DNA binding, we found that this suppression is not dependent upon Tbx1-DNA binding. We in addition provided evidence that Tbx1 overexpression in mice causes a phenotype similar to that caused by loss of Smad1-dependent signaling in the same tissues (including cleft lip and outflow heart defects) and downregulates Smad1 target genes such as Msx1 and 15 Msx2. In conclusion, our data demonstrate a mechanism by which Tbx1 interferes with the Bmp/Smad1 signal transduction pathway in tissue culture and during mammalian development. In addition, we provide evidence that a T-box transcription factor can have functions not directly related to or mediated by its transactivation activity

Rebalancing gene haploinsufficiency in vivo by targeting chromatin

Congenital heart disease (CHD) affects eight out of 1,000 live births and is a major social and health-care burden. A common genetic cause of CHD is the 22q11.2 deletion, which is the basis of the homonymous deletion syndrome (22q11.2DS), also known as DiGeorge syndrome. Most of its clinical spectrum is caused by haploinsufficiency of Tbx1, a gene encoding a T-box transcription factor. Here we show that Tbx1 positively regulates monomethylation of histone 3 lysine 4 (H3K4me1) through interaction with and recruitment of histone methyltransferases. Treatment of cells with tranylcypromine (TCP), an inhibitor of histone demethylases, rebalances the loss of H3K4me1 and rescues the expression of approximately one-third of the genes dysregulated by Tbx1 suppression. In Tbx1 mouse mutants, TCP treatment ameliorates substantially the cardiovascular phenotype. These data suggest that epigenetic drugs may represent a potential therapeutic strategy for rescue of gene haploinsufficiency phenotypes, including structural defects

Tbx1 represses Mef2c gene expression and is correlated with histone 3 deacetylation of the anterior heart field enhancer

The TBX1 gene is haploinsufficient in 22q11.2 deletion syndrome (22q11.2DS), and genetic evidence from human patients and mouse models points to a major role of this gene in the pathogenesis of this syndrome. Tbx1 can activate and repress transcription, and previous work has shown that one of its functions is to negatively modulate cardiomyocyte differentiation. Tbx1 occupies the anterior heart field (AHF) enhancer of the Mef2c gene, which encodes a key cardiac differentiation transcription factor. Here, we show that increased dosage of Tbx1 correlates with downregulation of Mef2c expression and reduced acetylation of its AHF enhancer in cultured mouse myoblasts. Consistently, 22q11.2DS-derived and in vitro-differentiated human induced pluripotent stem cells (hiPSCs) expressed higher levels of MEF2C and showed increased AHF acetylation, compared with hiPSCs from a healthy donor. Most importantly, we show that in mouse embryos, loss of Tbx1 enhances the expression of the Mef2c-AHF-Cre transgene in a specific region of the splanchnic mesoderm, and in a dosage-dependent manner, providing an in vivo correlate of our cell culture data. These results indicate that Tbx1 regulates the Mef2c AHF enhancer by inducing histone deacetylation

<i>Wnt5a</i> is expressed in the second heart field (SHF) and is downregulated in <i>Tbx1^−/−</i> embryos.

<p>(A–D′) The expression of <i>Wnt5a</i> was downregulated in the SHF, but not in the outflow tract (OFT) or the pharyngeal arch (PA) core mesoderm of <i>Tbx1^−/−</i> embryos at E8.0–11.5 (A′–D′), compared with the littermate controls (A–D), shown by whole-mount <i>in situ</i> hybridization (n = 3). Right view; arrowheads indicate the SHF. (E–J′) Transverse or sagittal sections cut from whole-mount <i>in situ</i> hybridized E8.0–9.0 <i>Tbx1^−/−</i> embryos (F, H, J) showed the downregulation of <i>Wnt5a</i> expression, compared with the littermate control (E, G, I, n = 3); squared regions are magnified on the adjacent panels; arrows indicate the SHF/splanchnic mesoderm domain. Arrowheads on panel E′ indicate a mesodermal expression domain strongly affected by the loss of <i>Tbx1</i>. (K–P) 3D reconstructions of E8.0 WT and <i>Tbx1^−/−</i> embryos provide a schematic, overall view of the expression domains regulated by <i>Tbx1</i>. Reconstructions were built using sections from embryos shown in E and F. Arrows indicate the SHF. PA: pharyngeal arch; OFT: outflow tract; SHF: second heart field; RV: right ventricle; ST: somite; NT, notochord; RA, right atrium; V: common ventricle; LV, left ventricle. Scale bars: 100 µm.</p

β-Catenin is upregulated in the SHF of <i>Tbx1^−/−;Wnt5a^−/−</i> embryos at E9.5.

<p>(A–D) Immunohistochemistry using an anti-cleaved Caspase 3 antibody showed no obvious difference in apoptosis between <i>Wnt5a</i>^−/− and <i>Tbx1</i>^−/−; <i>Wnt5a</i>^−/− embryos at E9.5. (E–K) Immunofluorescence using a β-catenin antibody showed no change of expression in the SHF (arrows) of <i>Tbx1</i>^−/− or <i>Wnt5a</i>^−/− embryos (E–G), while it was strongly upregulated in the SHF of <i>Tbx1</i>^−/−; <i>Wnt5a</i>^−/− embryos at E9.5 (H–K panels from independent embryos). Squared regions are magnified in the bottom row of panels (E′–K′). Red: β-catenin; Blue: DAPI. PE: pharyngeal endoderm; OFT: outflow tract; RA: right atrium; Scale bars: 100 µm.</p

Effects of Tbx1 on H3K4 di- and tri-methylation and H3 acethylation status of the TBE regions of <i>Wnt5a</i>.

<p>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) <i>p16</i> promoter region (positive control). There is no enrichment for any of these histone modifications at the TBE regions. Values are from 3 experiments (mean±S.D.).</p

<i>Tbx1</i> occupies the TBEs of <i>Wnt5a</i> in P19Cl6 cells.

<p>Chromatin immunoprecipitation (ChIP) assays of P19Cl6 using an anti-Tbx1 antibody. (A) Standard PCR results using primers for the TBE1–2 and the TBE3 regions of <i>Wnt5a</i>. Amplification is clearly visible from the Tbx1-immunoprecipitated material and from the input sample but not from the IgG-immunoprecipitated material (negative control). (B) Quantitative ChIP assay (q-ChIP) on a similar experiment as the one shown in (A) but followed by quantitative real-time PCR. (C) Q-ChIP assay of E9.5 mouse embryos using an anti-Tbx1 antibody. The enrichment of TBE1–2 and TBE3 regions is evaluated compared to an internal control, which is a primer pair amplifying an ORF-free segment of mouse chromosome 14, and expressed as percentage of input. Values are from 3 experiments (mean±S.D.).</p

Tbx1 and Wnt5a interact genetically.

<p>(A–A′) Extracardiac phenotypes in <i>Wnt5a^−/−</i> embryos included cleft palate, cleft lip, small thymus, truncated tail and truncated limbs at E18.5 (A, n = 5). In addition to the extracardiac phenotypes seen in <i>Wnt5a^−/−</i> embryos, <i>Tbx1^+/−</i>; <i>Wnt5a</i>^−/− embryos had small ears and edema at E18.5 (A′, n = 22). (B–D′) Coronal sections of the heart revealed ventricular septal defects (VSD) and abnormal (side-by-side) positioning of the ascending aorta (AAo) and pulmonary trunk (PT) in <i>Wnt5a</i>^−/− embryos (B–D, n = 5). 13 out of 22 (59%) <i>Tbx1^+/−; Wnt5a^−/−</i> embryos showed truncus arteriosus communis (TAC), which was not observed in the <i>Wnt5a</i>^−/− littermate (B′–D′). T: truncus arteriosus communis. Scale bars: 1 mm in A–B′; 100 µm in C–D′.</p