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Cardiovascular Research Advance Access first published online on February 19, 2008
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Cardiovascular Research, doi:10.1093/cvr/cvn049
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Msx1 and Msx2 are functional interacting partners of T-box factors in the regulation of Connexin43

Kees-Jan Boogerd, L.Y. Elaine Wong, Vincent M. Christoffels, Meinke Klarenbeek, Jan M. Ruijter, Antoon F.M. Moorman and Phil Barnett*

Department of Anatomy and Embryology, Heart Failure Research Centre, Academic Medical Centre, University of Amsterdam, Meibergdreef 9, 1105AZ Amsterdam, The Netherlands

* Corresponding author. Tel: +31 20 566 7821; fax: +31 20 697 6177. E-mail address: p.barnett{at}amc.uva.nl

Received 13 November 2007; revised 12 February 2008; accepted 17 February 2008

Time for primary review: 21 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Aims: T-box factors Tbx2 and Tbx3 play key roles in the development of the cardiac conduction system, atrioventricular canal, and outflow tract of the heart. They regulate the gap-junction-encoding gene Connexin43 (Cx43) and other genes critical for heart development and function. Discovering protein partners of Tbx2 and Tbx3 will shed light on the mechanisms by which these factors regulate these gene programs.

Methods and results: Employing an yeast 2-hybrid screen and subsequent in vitro pull-down experiments we demonstrate that muscle segment homeobox genes Msx1 and Msx2 are able to bind the cardiac T-box proteins Tbx2, Tbx3, and Tbx5. This interaction, as that of the related Nkx2.5 protein, is supported by the T-box and homeodomain alone. Overlapping spatiotemporal expression patterns of Msx1 and Msx2 together with the T-box genes during cardiac development in mouse and chicken underscore the biological significance of this interaction. We demonstrate that Msx proteins together with Tbx2 and Tbx3 suppress Cx43 promoter activity and down regulate Cx43 gene activity in a rat heart-derived cell line. Using chromatin immunoprecipitation analysis we demonstrate that Msx1 can bind the Cx43 promoter at a conserved binding site located in close proximity to a previously defined T-box binding site, and that the activity of Msx proteins on this promoter appears dependent in the presence of Tbx3.

Conclusion: Msx1 and Msx2 can function in concert with the T-box proteins to suppress Cx43 and other working myocardial genes.

KEYWORDS T-box transcription factor; Heart development; Tbx2; Tbx3; Msx1; Msx2; Connexin43; Atrioventricular canal; Working myocardium; Cardiac conduction system; Mouse; Chicken


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
During looping and septation of the heart, a coordinated chamber specific gene program is initiated which sees the development of the atria and ventricles (chambers). This event is earmarked by the induced expression of working myocardial genes including the gap-junction gene Connexin43 (Cx43), which is required for the rapid conduction of the electrical impulse over the working myocardium. The remaining parts of the heart tube, the inflow tract, atrioventricular canal and outflow tract (OFT) initially do not differentiate into working myocardium, and will not activate the expression of the associated gene program. These structures play important roles in septation and derive the major components of the central cardiac conduction system.1

Arrhythmias are a major cause of sudden cardiac death. Conditional ablation of Cx43 in mouse heart has been shown to result in ventricular arrhythmias.2 Further, Cx43 homozygous knockout mice die at birth and display a number of cardiac defects such as conotruncal malformations, outflow obstruction and coronary anomalies3,4 some of which may have their origins in the cardiac neural crest (CNC) cell population.5 Although the precise mechanisms of Cx43 regulation during development and in the adult heart remain largely unexplored, its correct expression in the working myocardium of the heart, alongside its strict exclusion from components of the conduction system and OFT, is key to the formation and function of the heart. The T-box transcription factors Tbx2 and Tbx3 have been shown to play critical roles in the transcriptional repression of Cx43 in the heart.6–10

The T-box gene family, so-named because of a 180 amino acid residue conserved DNA binding T domain, represents a large group of transcription factors present in all metazoans and known to play key roles in embryonic heart development.11 Mutations in several T-box family members are associated with different human congenital heart diseases.12 The T-box members Tbx2 and Tbx3 repress the expression of the working myocardial specific genes Cx43, Connexin40 (Cx40), and Nppa in the developing conduction system and suppress cardiac chamber differentiation.8,9,13–15 Tbx5, a T-box protein which is highly similar to Tbx2 and Tbx3, serves a chiefly opposite role during heart development in that it largely induces myocardial differentiation, transactivating many of the genes which Tbx2 and Tbx3 are known to down-regulate.16–18

Combinatorial interactions between cardiac-specific and ubiquitous transcriptional regulators define the tissue-specific activity of gene programs and are most certainly required to provide the driving force for correct development. Perhaps one of the major discoveries to this end has been the interaction of Tbx5 with the ‘cardiac’ homeobox factor Nkx2.5.18 Tbx5 has now also been shown to interact with other developmentally important transcription factors such as GATA4,19 chicken LMP4,20 the homeo- and paired domain containing Pax621 and most recently the WW-domain containing protein TAZ.22 Paradoxically, Nkx2.5 whilst being essential for myocardial differentiation, forming an interaction complex with Tbx5 and GATA4, its interaction with Tbx3 appears just as important for repression of the working myocardial phenotype. It can be hypothesized, therefore, that correct functioning of T-box proteins is dictated, in part, by the complexes it forms with other protein partners. Therefore, identifying partners involved in protein interaction networks will prove crucial for assigning specific gene regulatory and developmental functional roles to proteins and provide insight into the genetic-linked diseases they cause.

In order to obtain a clearer picture of the regulatory functions of Tbx2 and Tbx3 in the regulation of differentiation and gene expression, focussing our studies on the Cx43 because of its important role in conduction and cardiac development, we used various forms of Tbx2 as bait in an yeast 2-hybrid screen in order to isolate functional protein partners. Using this approach we have identified the homeobox proteins Msx1 and Msx2 as new T-box interacting partners cooperating in the regulation of Cx43.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No.85-23, revised 1996).

2.1 Expression constructs
All fusion constructs used in this study were PCR generated using primer combinations shown in Supplementary material online, Table S1, and sequenced to verify sequence integrity, unless stated otherwise.

Mouse Tbx2 bait constructs were generated to derive pGBkt7 (Clontech) bait vectors encoding full-length Tbx2 (pGBkt7-Tbx2), residues 1–286 (pGBkt7-Tbx2-N), residues 91–701 (pGBkt7-Tbx2-C) and residues 91–286 (pGBkt7-Tbx2-T-box) (Figure 1A). pGBkt7-LAM (Clontech) was used as a negative control. pGADt7-Msx1 and pGADt7-Msx2 encode full-length Msx1 and Msx2, respectively.


Figure 1
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  		Figure 1 Yeast 2-hybrid constructs and retransformation. (A) Yeast 2-hybrid and pull-down fusion constructs. Position of the T-box or homeodomain is labelled in italics. Numerical indicators are amino acids of the protein that are present in each construct. (B) Yeast 2-hybrid retransformation of Tbx2 together with Msx1 and Msx2. (i) All transformants (auxotrophic strain AH109) grow on media lacking leucine and tryptophan. (ii) Only 2-hybrid combinations of pGBkt7-Tbx2 and pGADt7-Msx1 or Msx2 give growth on media also lacking histidine and (iii) beta galactosidase activity of all transformants in the strain Y187. GBD, Gal4-DNA-binding-domain; MBP, maltose-binding protein; GAD, Gal4-activation domain; GST, glutathione-S-transferase; hd, homeodomain.

 
Constructs encoding maltose-binding protein (MBP)-fusions with Tbx2 and Tbx5 (pMAL2C-Tbx2-N, pMAL2C-Tbx2-T-box, pMAL2C-Tbx5-T-box), based on pMAL2C (Clontech) are shown in Figure 1A. pRP265nb-based23 glutathione-S-transferase (GST) fusion constructs were generated to express GST-MSX1 (pRP265nb-Msx1), GST-MSX2 (pRP265nb-Msx2), GST-NKX2.5 (pRP265nb-Nkx2.5), GST-MSX1hd (pRP265nb-Msx1-hd), GST-MSX2hd (pRP265nb-Msx2-hd, Figure 1A). Fusion proteins were expressed in Escherichia coli (E. coli) strain BL21 RIL(DE3).

The mutation (Msx2M) Arg146Cys in the homeodomain of MSX2 was generated by PCR using the mismatch primer; 5'-AGAGGATCCACCGGAAGCCATGCACACCC-3'.

Full-length Msx1, Msx2, and Msx2M were cloned into pcDNA3.1 (Invitrogen) using extended primers to generate N-terminal FLAG-tagged fusions. The pcDNA-HA-Tbx2, pcDNA-HA-Tbx3, and pGL3-Cx43-luciferase constructs have been described previously.9,13,24

2.2 Yeast 2-hybrid screen
Tbx2 bait constructs were tested for self-activation by co-transfection to yeast strain AH109 (Clontech, Matchmaker systems) with empty activation domain (AD) plasmid pGADT7. Individual bait constructs were transformed to AH109 and screening carried out according to the manufacturers instructions. The total mated library (mouse total cDNA e.d. 11.5, Clontech) was plated to a triple-drop-out selection media, Leu– Trp– His–, in the presence of the galactoside X-{alpha}-Gal. Potential surviving colonies were replated to triple drop out medium and subsequently picked for AD-plasmid rescue and sequencing.

2.3 Maltose-binding protein pull-down and co-immunoprecipitation assays
E. coli BL21 cells were transformed with bacterial expression constructs. Cells were induced with 1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) (Gibco-BRL) and after 2 h growth at 30°C, harvested by centrifugation and resuspended in 5 mL of ice-cold phosphate-buffered saline containing 0.05% v/v Triton X-100 (Sigma) (PBSTr). Cell suspensions were lysed by sonication and centrifuged to pellet cell debris. GST containing fusion constructs were purified on glutathione 4B-Sepharose following the manufacturer’s instructions (Pharmacia). Binding assays were set-up as described previously23 except that a total 2 µg of target GST-fusion was passed over the MBP-fusion bound amylose column in 1 mL PBSTr. Western-blots were probed with alkaline phosphatase (AP) conjugated {alpha}-GST (Cat#A5838, Sigma), horseradish peroxidase (HRP) conjugated {alpha}-HA (Cat# 1667475, Roche) or mouse-{alpha}-FLAG and goat-{alpha}-mouse-AP (Cat#200472, Stratagene and Cat#A3562, Sigma) antibodies, and visualized using enhanced chemiluminescence (Amersham) or AP-staining.

Co-immunoprecipitations were performed using a neonatal rat heart-derived cell line, H10.25 Cells were seeded in 10 cm plates and transfected with 10 µg of pcDNA3.1-HA-Tbx3, pcDNA3.1-FLAG-Msx2 or both. After 48 h cellular lysates were immunoprecipitated as described26 using 10 µL {alpha}-hTBX3 (Cat#SC17871, SantaCruz).

2.4 In situ hybridizations
In situ hybridizations on mouse embryonic day (ED) 11.5 or 12.5 and chicken Hamburger–Hamilton (HH) stage 24 sections were performed as described previously,27 using probes described in Supplementary methods.

2.5 Luciferase assay
H10 cells, grown in standard 6-wells plates in Dulbeccos Modified Eagles Medium supplemented with 10% foetal calf serum (Gibco-BRL) and glutamine, were transfected in duplo using the Sigma Escort-V transfection kit. Standard 700 ng Cx43-luciferase construct was co-transfected with 3 ng of phRG-TK vector, as normalization control (Promega), together with appropriate combinations of Tbx2, Msx1, Msx2, and Msx2M pcDNA3.1 expression constructs. Measurements were performed on a Turner TD20/20 luminometer. Duplo transfection experiments were repeated at least twice.

2.6 Quantitative real-time polymerase chain reaction
Triplo transfections in H10 cells (and H9C2) were set up essentially as for luciferase assays, using 100 ng plasmid DNA for each transcription factor. For Tbx3 RNA interference (RNAi)-directed studies, an assayed optimum transfection concentration of 10pM of RNAi-Tbx3 or Control was (co)-transfected using Lipofectamine2000 (Invitrogen) according to manufacturers protocol. RNAi duplexes used were: RNAi-Tbx3, 5'-GCAUGGCCUAUCAUCCGUUUU and RNAi-Control, 5'-CGUAGGUACCCUCUAGCUUUU. Total RNA was isolated and cDNA synthesized using the Reverse Transcriptase kit Superscript (Invitrogen) using polyT-priming. Samples were analysed by quantitative real-time polymerase chain reaction (qRTPCR) on a Roche systems Light-cycler480. Experiments were repeated at least twice. Primers used for amplification were: GAPDH, 5'-GTCGGTGTGAACGGATTTGG and 5'-TTCCCGTTGATGACCAGCTT; Tbx3, 5'-CTGCGTTACAGCCCGTATTC and 5'-AGCGGCTATTCAGTTCCGAC; Cx43, 5'-ATGGGTGACTGGAGTGCCCTTG and 5'-GAAGCGCACGTGAGAGATGG. Data were analysed using the LinRegPCR software. Statistical analysis was carried out as described in ‘Statistical analysis’ section.

2.7 Chromatin immunoprecipitation studies
H10 cells, grown in a standard 10 cm plate, were transfected in triplo with 20 µg pcDNA-Flag-Msx1, non-transfected cells were used as a control. Chromatin was fragmented by sonication (20 20-s pulses) and immunoprecipitated with 1 µg anti-Msx1 antibody (Cat#M0944, Sigma) using chromatin immunoprecipitation (ChIP)-IT kit (Active Motif). DNA was analysed by qRTPCR (Roche LightCycler480) using primers 5'-CCGACGAGTAGACATACCCCT and 5'-GGGTGTGCGTGATCTTTCTTATG, at position -587 and -454 bp in rat Cx43 promoter, respectively. Primers for a non-specific gene (HPRT 5'-GGTCCATTCCTATGACTGTAGATTTT F 5'-CAATCAAGACGTTCTTTCCAGTT) were used as negative control. DNA concentration (N0) after ChIP was calculated using LinReqPCR software and statistical analysis carried out as in ‘Statistical analysis’ section. Data are presented as [N0ChIP : N0input]transfected cells divided by [N0ChIP : N0input]untransfected cells.

2.8 Statistical analysis
Luciferase measurements and qRTPCR data were corrected for inter-session variation as described before.28 Subsequently, data were analysed using one-way ANOVA, followed by Student–Newman–Keuls comparison of groups. All results shown are significant (P < 0.05), unless stated otherwise. Two-way ANOVA was used to compare Msx2 and Msx2M for all concentrations in the luciferase assay.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Supplementary material
 Funding
 References
 
3.1 Mouse embryonic day 11.5 yeast 2-hybrid screen
Using various length bait fusions of the T-box transcription factor Tbx2, a pretransformed mouse ED 11.5 yeast 2-hybrid library was screened for potential interacting partners. From an initial screen of 1.5 x 106 colonies, a final 20 surviving clones revealed a GAL4 fusion to a peptide in a reading frame coding for a BLASTP genome identifiable sequence. The majority of clones picked up in this screen were done so with pGBkt7-Tbx2-N and pGBkt7-Tbx2-T-box (Figure 1A). One of these clones, picked up twice, encoded the muscle segment homeobox protein Msx1. This interaction could be verified in a direct yeast 2-hybrid assay (Figure 1B). Msx1 has previously been shown to be present during development of the heart and in particular has been associated by localization with septation, valvulation, and development of the conduction system29–31 thus warranting closer examination of this interaction.

3.2 In situ hybridization
Localization studies (Figure 2, Supplementary material online, Figures S1 and S2) show that the expression patterns of the T-box genes Tbx2 and Tbx3, and those of Msx1 and a second muscle segment homeobox protein, Msx2, closely overlap in distinct regions of the developing mouse and chicken heart13,14,32 providing evidence for a biologically significant protein–protein interaction. Notably, expression overlap is not merely confined to the heart, but appears in other areas such as the developing limbs (Figure 2A) and body wall mesenchyme. Msx1 and Msx2 together with Tbx2 and Tbx3 can be seen to be expressed in the cushion mesenchyme, the endocardium, and the epicardium of the atrioventricular canal (Figure 2B and C), though in chicken, Msx2 appears absent from the cushion mesenchyme. Notably, in this region, Msx2, but not Msx1, is also expressed in a limited region of cTnI positive myocardium overlapping the expression pattern that can be noted for Tbx2 and Tbx3. The working myocardial markers Nppa, Cx40, and Cx43 are strictly excluded from areas of myocardium expressing Msx2, Tbx2, and Tbx3 (Figure 2B and C, Supplementary material online, Figure S1), a feature that is conserved between mouse and chicken (Figure 2D, Supplementary material online, Figure S2). This part of the myocardium will derive central parts of the cardiac conduction system, including the atrioventricular node, and the left and right atrioventricular ring bundles.29 This expression overlap and the high level of amino acid identity shared between Msx1 and Msx2, warranted testing the ability of Msx2 to associate with Tbx2. Using a direct assay we were indeed able to demonstrate this interaction (Figure 1B).


Figure 2
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  		Figure 2 In situ hybridization showing overlapping expression patterns of Tbx2 and Tbx3 with Msx1 and Msx2 in mouse and chicken hearts. (A) mouse ED(embryonic day) 11.5, Msx1, Msx2, Tbx2 and Tbx3 expression patterns correspond closely in the heart and other areas of the body. Arrows mark the limb bud and the body wall mesenchyme. (B) mouse ED11.5, Msx1 is expressed in atrioventricular cushions, whereas Msx2 and Tbx3 are expressed in the myocardium (marked by cardiac Troponin I [cTnI] expression) of the atrioventricular canal (AVC). (C) mouse ED12.5, Cx43 is not expressed in the AVC myocardium, whereas Msx2, Tbx2 and Tbx3 are. (D) Expression patterns of Tbx2 and Tbx3 with Msx1 and Msx2 in chicken embryo HH24 sections. Tbx2, Tbx3 and Msx2 are expressed in the AVC myocardium, whereas Msx1 and the chamber marker Nppb are not. Dashed lines in B and C are used to indicate the border between myocardium and other tissue layers. Scalebar is 1mm (A) or 0.1 mm (B, C, D). Abbreviations are: nt, neural tube; ht, heart; lb, limb bud; ra, right atrium; la, left atrium; rv, right ventricle; lv, left ventricle; ac, atrioventricular cushion; lac, lateral atrioventricular cushion, myo, myocardium; en, endocardium; cus, cushion mesenchyme; epi, epicardium.

 
3.3 Verification of 2-hybrid results
An MBP pull-down approach was chosen to verify 2-hybrid interaction results in vitro. Purified GST-fusions of Msx1, Msx2, and Nkx2.5, were able to interact with MBP-Tbx2-N (Figure 3A). GST alone and GST fused to a non-interacting protein were unable to bind. Further to this, we tested the hypothesis that Msx2 could form a complex with Tbx3 in a cellular context. Therefore Msx2, Tbx3 or both were transfected to a neonatal rat heart-derived cell line, H10.25 Subsequent co-immunoprecipitation using a polyclonal antibody raised against human Tbx3 reveals that Msx2 is indeed coprecipitated with Tbx3 (Figure 3B).


Figure 3
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  		Figure 3 Msx proteins bind to T-box proteins in vitro and in H10 cells. (A), Maltose-binding protein (MBP)-pull-down experiments using purified, equivalent amounts (Inputs) of either; glutathione-S-transferase (GST), GST fused 25 kDa protein identified in the 2-hybrid screen (UIP), GST-Msx1, GST-Msx2, GST-Nkx2.5. The eluted fraction is subjected to denaturing polyacrylamide gelelectrophoresis (SDS–PAGE) followed by total protein staining with Coomassie (eluate). Only in the presence of Tbx2 are Msx1, Msx2, and Nkx2.5 retained, as shown by the blot probed with anti-GST. Controls, GST alone and UIP, are not retained. (B), Co-immunoprecipitation of Msx2 with Tbx3 from H10 cell-lysate (Input). Flag-Msx2 co-transfected with Tbx3, could be co-immunoprecipitated with {alpha}-Tbx3. (C) MBP-pull-down assays using Tbx2 T-box and Tbx5 T-box. T-boxes of both Tbx2 and Tbx5 are able to bind the homeodomains of Msx1 and Msx2. GST and Msx2 with a point-mutation at Arg146 (Msx2M) are not retained. Sizes of molecular weight marker proteins (M, kDa) are shown on the right.

 
3.4 The T-box and the Msx homeodomain support the protein–protein interaction in vitro
MBP pull-down experiments using constructs possessing the T-box encoding regions from both Tbx2 and Tbx5 fused to MBP and GST fusions of the homeodomain regions of both Msx1 and Msx2 are shown in Figure 3C. This clearly demonstrates that both the T-box and the homeodomain are on their own sufficient to support this interaction and that Tbx5 is also able to bind both Msx1 and Msx2.

A recent biochemical analysis of eight Nkx2.5 mutations33 from patients with cardiac anomalies revealed a conserved Arg residue (Arg142)34 which when mutated to a Cys negatively affected both DNA binding capacity and the ability of the homeodomain to associate with Tbx5. We subsequently mutated the equivalently positioned Arg to Cys in Msx2 (Arg146) and examined its effect on interaction with Tbx2 and Tbx5. This experiment demonstrates that whereas wild-type Msx2 homeodomain is able to specifically associate in vitro with the T-box (Figure 3C), the mutated Msx2M has lost this capacity.

3.5 Suppression of Connexin43 promoter activity
Previously, it has been shown that expression of Cx43 can be down-regulated by Tbx2 and Tbx3.6–8,10,35 Further, cooperative interaction between Tbx2 and Tbx3 with the homeobox Nkx2.5 is able to down-regulate the expression of another chamber marker, Nppa.10,13 The colocalization of Msx2 and Tbx2 and Tbx3 expression in the atrioventricular canal myocardium, a region of the developing heart in which Cx43 and Nppa are not expressed, lead to our questioning whether Msx1 and Msx2 could act together with Tbx2 and Tbx3 in the regulation of such genes. Therefore we transfected a 1.6 kb Cx43 promoter fragment reporter construct36 to H10-cells, which results in expression of luciferase (Figure 4A). Notably, this cell line expresses Cx43 endogenously (Figure 4B) though it does not express detectable levels of Nppa and only low levels of Cx40 (data not shown). Co-transfection of either Msx1 or Msx2 results in the down-regulation of promoter activity, in a dose dependent manner. The mutated Msx2 displays a significant loss of repressive activity (P < 0.001) relative to wild-type Msx2. Further, transfection of small amounts of Tbx2 show a decrease in measurable luciferase activity, but increasing levels of Tbx2 fail to repress the Cx43 promoter fragment any further, indicating system saturation.


Figure 4
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  		Figure 4 Downregulation of Connexin43 (Cx43) expression by Msx1 and Msx2 depends on Tbx3. (A) Co-transfections with a 1.6 kb Cx43 promoter-driven luciferase construct were carried out using pCDNA3.1 (Cont) expressing Tbx2, Msx1, Msx2 and Msx2R146C mutant (Msx2M). (B), Tbx2, Msx1 and Msx2 can down-regulate endogenous Cx43. H10 cells were transfected with either empty vector (Cont), Msx1, Msx2, Msx2M, Tbx2 or Tbx5 (100 ng each). Total mRNA was isolated and levels of endogenous Cx43 measured by qRTPCR. Cells transfected with Tbx2, Msx1 or Msx2 show lower levels of Cx43. (C) 10 pmol RNAi, directed against Tbx3, was used to downregulate the endogenous production of Tbx3 as measured by qRTPCR. Scrambled RNAi (RNAi C) was used as a control. (D) Following knock-down of Tbx3, Cx43 levels can be seen to increase. Co-transfection of Msx1 or Msx2 (100 ng each) in this case no longer shows a decrease in endogenous Cx43 levels. Symbols: #, not significant; §, no significant difference between samples within this group.

 
3.6 RNAi knockdown of endogenous Tbx3 in H10 cells
The expression of endogenous Cx43 and Tbx3 by the H10 cell line provided an opportunity to study the effect of Msx on the expression of Cx43, in a cardiac like context. Figure 4B shows, in line with our luciferase assay, that transfection of Msx1, Msx2, and Tbx2 can reduce the expression of Cx43, as measured by qRTPCR. As expected, mutant Msx2M is unable to reduce endogenous levels of Cx43. Near identical results were obtained using a second cardiomyocyte cell line, H9C2 (data not shown). We next examined the effect of removing the endogenous Tbx3 using RNAi directed against the coding sequence of Tbx3 (Figure 4C). Knocking-down Tbx3 expression in this cell line resulted in an up-regulation of Cx43 (Figure 4D). Interestingly, when Tbx3 is suppressed, Msx1 and Msx2 are no longer capable of repressing Cx43 expression. These experiments show that a functional complex between Tbx and Msx can form within the cell and functions synergistically to down-regulate Cx43.

3.7 Chromatin immunoprecipitation of the Connexin43 promoter
Examination of the upstream promoter region of Cx43 reveals the presence of mammalian species conserved (and many non-conserved) potential T-box and Msx binding motifs (Figure 5).36,37 ChIPs have previously demonstrated that indeed Tbx3 can bind to this region of the Cx43 promoter.10 In an electrophoretic mobility shift assay, Msx1 binds to this region of the mouse Cx43 promoter (Supplementary material online, Figure S3). To determine if Msx1 can also bind the Cx43 promoter in a cellular context, we performed ChIP experiments on H10 cells using an anti-Msx1 antibody. Cx43 was found enriched in the ChIP experiments only when Msx1 is over-expressed in the cells (Figure 5A), whereas this is not the case for the control locus (HPRT). This establishes that Msx1 can also bind the endogenous Cx43 promoter in H10 cells.


Figure 5
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  		Figure 5 Chromatin immunoprecipitation of the endogenous Connexin43 (Cx43) promoter. (A) Msx1 transfected H10 cells were chromatin immunoprecipitated with {alpha}-Msx1 antibody and co-precipitated DNA was analysed for the presence of Cx43 promoter or HPRT (Control) using qRTPCR (quantitative real-time polymerase chain reaction). Cx43 shows at least a 2.5x enrichment compared with non-transfected cells, whereas HPRT yield is comparable between Msx1-transfected and control cells. (B) Sequence alignment of the Cx43 promoter region possessing both conserved putative Msx1 (underlined) and T-box (bold italic) binding consensus sites. Position is relative to transcription start site.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Supplementary material
 Funding
 References
 
Searching for functional and biologically relevant protein–protein interactions is an initial step towards elucidating a transcription factor’s function during organogenesis. Spurred by our interest in the functional role of the T-box transcription factor Tbx2 during cardiac conduction system development,1,8 we carried out an ED 11.5 mouse 2-hybrid screen to search for Tbx2 interacting proteins. From this screen we identified the muscle segment homeobox protein Msx1 and subsequently Msx2 as T-box interacting partners. This group of homeobox proteins, generally considered to function as transcriptional repressors, are essential for normal craniofacial, limb, and ectodermal organ morphogenesis.38–40

The spatiotemporal overlap of Tbx2/3/5 and Msx1/2 during cardiac development, in particular, in the atrioventricular canal, suggests a role for this interaction in development of this region of the heart. The presence of Msx1 and Msx2 in the heart has been known for some time,29–31 though the functional significance of each has not been gained from homozygous knockout mouse studies. The similar patterns of expression and high level of functional redundancy between Msx1 and Msx2 probably explains the lack of cardiac phenotype in homozygous single Msx mutants.41,42 In support of this hypothesis, Msx1/Msx2 double knockout mice reveal the presence of severe cardiac anomalies.30 These mice display OFT defects in cushion development with eventual malalignment of the truncus, septal defects of both the OFT and atrioventricular canal regions, and hypoplasia of the ventricles. Moreover, this spectrum of cardiac defects resembles those displayed in the homozygous Tbx214 and Tbx3 knockout mice, the latter displaying double outlet right ventricle (DORV), a phenotype possibly due to loss of Tbx3 expression in the CNC (unpublished results). Therefore, although Msx proteins display redundancy, their functions during cardiac development are essential and appear linked to those of Tbx2 and Tbx3. On the basis of our results, which show that Tbx2, Tbx3, and Tbx5 are all able to interact with Msx1 and Msx2, this redundancy also appears to hold for the Tbx–Msx interaction itself. This, combined with the spatiotemporal overlap of all these potential partners, may reflect stringent fail-safe mechanisms regulating differentiation of the myocardium of the atrioventricular canal and OFT.

Using a similar yeast 2-hybrid approach, Hiroi and co-workers identified the T-box protein Tbx5 as interacting partner of the cardiac-specific homeobox Nkx2.5. Like the Nkx2.5–Tbx5 interaction, the homeodomains alone of Msx1 and Msx2 are able to support the interaction directly with the conserved T-box of Tbx2, Tbx3, and Tbx5. Mutation of a conserved Arg residue, shared between a large number of homeodomain proteins34 including Nkx2.533 and Msx, results in a significant loss of interaction with the T-box domain (Figure 3), demonstrating that T-box-homeodomain interaction as well as being specific, maybe a more widespread conserved feature of these domain types.

Revealing the functional consequence of this interaction for the development of the conduction system is challenging, mainly due to the redundancy displayed by the protein partners and their interactions. However, based on previous studies and reports from other systems as to the nature of Msx1 and Msx2 function, namely as transcriptional repressor, we examined the effect of Msx on the promoter of a well-studied cardiac chamber differentiation marker, Cx43.8,43,44 Cx43, like several other cardiac markers, e.g. Nppa, and Cx40, is known to be regulated in vivo by the T-box proteins. Our in situ hybridization results demonstrate the exclusion of chamber markers such as Nppa, Cx40, and Cx43 from areas expressing Msx1 and Msx2 together with T-box factors (Figure 2). Using the rat neonatal heart cell line H10, we were able to demonstrate that a 1.6 kb Cx43 promoter construct could be down-regulated using either Tbx2, Msx1, or Msx2. No up- or down-regulation was observed using Tbx5, as expected,16 and a point-mutation in Msx2 significantly reduces its repressive capacity. Similar results were obtained in an in vivo situation using the endogenous Cx43 expression in the H10 cell line via qRTPCR. As with the plasmid promoter assays, levels of endogenous Cx43 mRNA could be decreased by transfecting small amounts of Tbx2, Msx1, or Msx2.

The H10 cell line also expresses Tbx3, though not Tbx2 or Tbx5. Knocking down Tbx3 using RNAi transfection results in an up-regulation of endogenous Cx43 levels. Transfection of Msx proteins along with RNAi demonstrated that by knocking-down endogenous Tbx3, both Msx1 and Msx2 are no longer able to down-regulate Cx43, thus showing that the previous down-regulation of Cx43 by Msx proteins depends on the presence of Tbx3. Further to this, using ChIP we demonstrated that Tbx310 and Msx1 can bind the endogenous Cx43 promoter in a heart cell line, probably by binding a conserved putative Msx binding site37 located close to the T-box binding elements in all mammalian Cx43 promoters examined. This fact combined with the overlap in spatiotemporal expression of T-box proteins and Msx proteins we observe in the heart, is supportive of a potential synergism in the regulation of gene expression during heart development.

During cardiac development, the OFT of the heart receives a considerable contribution of cells from the neural crest. The critical regulation of Cx43 levels in these cells appears crucial to their correct migration and distribution in the OFT. Both over-expression and ablation of Cx43 in these cells results in malformations of the OFT.3–5 The Msx1/Msx2 combinatorial knockout mice also display OFT defects including DORV,30 postulated to be as a result of increase in CNC proliferation in this region. Interestingly, previous studies have shown that loss of Msx1 in neural crest can be compensated for by over-expression of Msx2,45 demonstrating again the functional redundancy of these proteins. Although the underlying mechanisms for the observed defects in the CNC may differ somewhat, the critical regulation of Cx43 levels and the necessary presence of Msx and Tbx proteins in this population of cells is self-evident. Extrapolating this and functions reported in literature for both Tbx2/Tbx3 and Msx1/Msx2 leads us to the postulation that these proteins work in a complex, perhaps ensuring that strict stoichiometric levels of downstream targets are maintained by robust, multiple redundant, fail-safe mechanisms.


   Supplementary material
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
Funding
 References
 
Supplementary material is available at Cardiovascular Research online.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
References
 
This project was funded by the Netherlands Heart Foundation grant 1996M002 and European Community’s Sixth Framework Programme contract HeartRepair LSHM-CT-2005-018630.


   Acknowledgements
 
We thank Irma Thesleff (University of Helsinki, Finland) and Juan-Carlos Izpisúa-Belmonte for providing reagents.

Conflict of interest: the authors declare no conflict of interest.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 

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