Formation of a TBX20-CASZ1 protein complex is protective against dilated cardiomyopathy and critical for cardiac homeostasis

By the age of 40, one in five adults without symptoms of cardiovascular disease are at risk for developing congestive heart failure. Within this population, dilated cardiomyopathy (DCM) remains one of the leading causes of disease and death, with nearly half of cases genetically determined. Though genetic and high throughput sequencing-based approaches have identified sporadic and inherited mutations in a multitude of genes implicated in cardiomyopathy, how combinations of asymptomatic mutations lead to cardiac failure remains a mystery. Since a number of studies have implicated mutations of the transcription factor TBX20 in congenital heart diseases, we investigated the underlying mechanisms, using an unbiased systems-based screen to identify novel, cardiac-specific binding partners. We demonstrated that TBX20 physically and genetically interacts with the essential transcription factor CASZ1. This interaction is required for survival, as mice heterozygous for both Tbx20 and Casz1 die post-natally as a result of DCM. A Tbx20 mutation associated with human familial DCM sterically interferes with the TBX20-CASZ1 interaction and provides a physical basis for how this human mutation disrupts normal cardiac function. Finally, we employed quantitative proteomic analyses to define the molecular pathways mis-regulated upon disruption of this novel complex. Collectively, our proteomic, biochemical, genetic, and structural studies suggest that the physical interaction between TBX20 and CASZ1 is required for cardiac homeostasis, and further, that reduction or loss of this critical interaction leads to DCM. This work provides strong evidence that DCM can be inherited through a digenic mechanism

A Gro/TLE-NuRD Corepressor Complex Facilitates Tbx20-Dependent Transcriptional Repression

The cardiac transcription factor Tbx20 has a critical role in the proper morphogenetic development of the vertebrate heart, and its misregulation has been implicated in human congenital heart disease. Although it is established that Tbx20 exerts its function in the embryonic heart through positive and negative regulation of distinct gene programs, it is unclear how Tbx20 mediates proper transcriptional regulation of its target genes. Here, using a combinatorial proteomic and bioinformatic approach, we present the first characterization of Tbx20 transcriptional protein complexes. We have systematically investigated Tbx20 protein-protein interactions by immunoaffinity purification of tagged Tbx20 followed by proteomic analysis using GeLC-MS/MS, gene ontology classification, and functional network analysis. We demonstrate that Tbx20 is associated with a chromatin remodeling network composed of TLE/Groucho co-repressors, members of the Nucleosome Remodeling and Deacetylase (NuRD) complex, the chromatin remodeling ATPases RUVBL1/RUVBL2, and the T-box repressor Tbx18. We determined that the interaction with TLE co-repressors is mediated via an eh1 binding motif in Tbx20. Moreover, we demonstrated that ablation of this motif results in a failure to properly assemble the repression network and disrupts Tbx20 function in vivo. Importantly, we validated Tbx20-TLE interactions in the mouse embryonic heart, and identified developmental genes regulated by Tbx20:TLE binding, thereby confirming a primary role for a Tbx20-TLE repressor complex in embryonic heart development. Together, these studies suggest a model in which Tbx20 associates with a Gro/TLE-NuRD repressor complex to prevent inappropriate gene activation within the forming heart

The TBX20^Avi-BirA system for isolation of the TBX20 interactome.

<p>(A) Schematic diagram of ESC transduction with Lenti-BirA and subsequent differentiation. (B) RT-PCR panel showing changes in gene expression that ESCs undergo during 8-day differentiation into iCMs. (C) Immunohistochemistry of iCMs stained with cardiomyocyte marker Myosin heavy chain (MHC) and counterstained with DAPI. (D) Schematic of BirA-dependent biotinylation and streptavidin affinity isolation of TBX20 from iCMs. (E) Steptavidin affinity isolation of TBX20^Avi complexes confirmed in BirA-expressing iCMs by western blot analysis.</p

The TBX20-CASZ1 interaction is required for maintaining cardiac homeostasis in young adult mice (4–7 weeks old).

<p>The TBX20-CASZ1 interaction is required for maintaining cardiac homeostasis in young adult mice (4–7 weeks old).</p

<i>Tbx20</i>^{<i>flox/+</i>}<i>; Casz1</i>^{<i>flox/+</i>} compound heterozygotes display decreased cardiac function.

<p>(A-D) Echocardiography in mice aged 8–11 weeks, (A) left ventricular ejection fraction, (B) fractional shortening, (C) left ventricular inner diameter at diastole (LIVD-D), and (D) left ventricular inner diameter at systole (LIVD-S) was measured for mice in indicated cohorts. Each red circle represents one mouse analyzed within that cohort. *p<0.05, **p<0.005, ***p<0.0005. Statistical significance between pairs was calculated using Student’s t-test.</p

A Gro/TLE-NuRD Corepressor Complex Facilitates Tbx20-Dependent Transcriptional Repression

The cardiac transcription factor Tbx20 has a critical role in the proper morphogenetic development of the vertebrate heart, and its misregulation has been implicated in human congenital heart disease. Although it is established that Tbx20 exerts its function in the embryonic heart through positive and negative regulation of distinct gene programs, it is unclear how Tbx20 mediates proper transcriptional regulation of its target genes. Here, using a combinatorial proteomic and bioinformatic approach, we present the first characterization of Tbx20 transcriptional protein complexes. We have systematically investigated Tbx20 protein–protein interactions by immunoaffinity purification of tagged Tbx20 followed by proteomic analysis using GeLC-MS/MS, gene ontology classification, and functional network analysis. We demonstrate that Tbx20 is associated with a chromatin remodeling network composed of TLE/Groucho corepressors, members of the Nucleosome Remodeling and Deacetylase (NuRD) complex, the chromatin remodeling ATPases RUVBL1/RUVBL2, and the T-box repressor Tbx18. We determined that the interaction with TLE corepressors is mediated via an eh1 binding motif in Tbx20. Moreover, we demonstrated that ablation of this motif results in a failure to properly assemble the repression network and disrupts Tbx20 function <i>in vivo</i>. Importantly, we validated Tbx20–TLE interactions in the mouse embryonic heart, and identified developmental genes regulated by Tbx20–TLE binding, thereby confirming a primary role for a Tbx20-TLE repressor complex in embryonic heart development. Together, these studies suggest a model in which Tbx20 associates with a Gro/TLE-NuRD repressor complex to prevent inappropriate gene activation within the forming heart

Double heterozygous hearts undergo pathological remodeling.

<p>(A-D) Hearts from mice aged 8–11 weeks. (A) Transverse sections of hearts stained with hematoxylin and counter-stained with eosin. (B) Transverse sections of hearts stained with Picrosirius red and fast green, and visualized using bright field microscopy. (C) Polarizing light microscopy of Picrosirius red-stained sections. Thin collagen fibers stain green to yellow, while thicker collagen fibers stain orange to red. (D) Transverse heart sections were immunostained with tropomyosin (red, cardiomyocytes) and WGA (green, cell membranes). Region of left ventricular free wall shown. (E) Quantification of cardiomyocyte cross-sectional areas, shown as mean ± SEM of 450+ cardiomyocytes per heart, n = 3 hearts per genotype. *p<0.05, **p<0.0005. Statistical significance between pairs was calculated using Student’s t-test. A-C, scale bar = 300 μm. D, scale bar = 20 μm.</p

The TBX20-CASZ1 interaction is required for maintaining cardiac homeostasis in 8-11wk old mice.

<p>The TBX20-CASZ1 interaction is required for maintaining cardiac homeostasis in 8-11wk old mice.</p

TBX20 and CASZ1 interact through their DNA binding domains.

<p>(A) (Top) Schematic of full-length TBX20 and truncations. NLS, Nuclear Localization Signal. Putative activation domain shown in green and putative repression domain in orange. (Bottom) Co-immunoisolations from <i>X</i>. <i>laevis</i> embryos expressing full-length CASZ1-V5 and either full-length HA-TBX20 or deletions shown in top panel. (B) (Top) Schematic of full-length CASZ1 and the truncations used in the co-immunoisolations. (Bottom) Co-immunoisolations from X. laevis embryos expressing full-length HA-TBX20 alone, or in combination with either full-length CASZ1-V5 or truncations shown in top panel. (C) (Top) Sequence alignment of TBX20 position 248–288 across 90 TBX20 orthologs. Height of letters is relative to conservation at that residue. (Bottom) Sequence alignment of CASZ1 at positions 601–650 across 90 CASZ1 orthologs.</p

TBX20^F245I mutant displays impaired interaction with CASZ1.

<p>(A) (Top) Schematic of full-length TBX20 with location of <i>F256I</i> mutation shown. (Bottom) Co-immunoisolations of full-length wild-type CASZ1 with wild-type TBX20 or TBX20^F256I. (B) Ribbon models of the average structures of TBX20 calculated from the 100 ns molecular dynamics simulations of the T-box domain. Left panel: starting structure of wild-type TBX20 (cyan) including DNA for reference. Right panel: an overlay of wild-type TBX20 (green) and TBX20^F256I (magenta). F256 and I256 side chains are displayed in stick form. Regions designated as 1, 2, and 3 undergo mutation-induced conformational changes in the unbound form. (C) Enlargement of the F256I residue shown in (B) in a different side chain rotamer that also induces steric clashes. The I256 mutation is rendered in purple stick form, with small red discs indicating steric clashes between the side chain of I256 and T-box residues E258 and T259.</p