We demonstrated a critical, conserved and dosage-sensitive role for the BAF chromatin remodelling complex ATPase Brg1 in vertebrate heart development. Our results further show an important interdependency between BAF complexes and disease-related cardiac transcription factors, which suggests a mechanism for CHDs caused by mutations in these transcription factor genes and implications for multigenic inheritance of CHDs.
In mouse and zebrafish, Brg1
is required for important and specific aspects of heart development. In particular, chamber morphogenesis is disrupted in both mice and fish lacking Brg1, and proliferation of cardiac progenitors is reduced. A key subset of cardiac genes was affected by the loss of Brg1
, consistent with specific roles for BAF complexes in differentiation of other cell types9
. In many of these cases, Brg1
is required for differentiation after specification. Our results support a clear role for Brg1
in cardiac differentiation. As the function of Gata4, Nkx2–5 and Tbx5 relies on BAF complexes, in part via Baf60c7
, loss of Brg1
likely affects target genes primarily by reducing the activation potential of cardiac transcription factors. Loss of brg1
in zebrafish demonstrates a strikingly conserved role for BAF complexes in vertebrate cardiac development. The morphogenetic and gene expression defects in brg1
mutant fish closely resemble that seen in mice lacking Brg1
, suggesting that the gene networks regulated by BAF complexes are well conserved in vertebrate evolution.
Notably, mice heterozygous for a deletion of Brg1
have heart defects. This important finding indicates that Brg1
is haploinsufficient in the heart. Haploinsufficiency of Brg1
has been reported in the brain13
and immune system43
, indicating that certain cell types are sensitive to the dosage of Brg1
. The important dose dependency of Brg1
function in cardiogenesis shows that, as for DNA-binding transcription factors, normal formation of the heart relies on precise levels of functional BAF complexes. Furthermore, the genetic interaction between Brg1
and several cardiac transcription factor genes shows that transcription factor function is intimately linked to levels of Brg1. The resulting CHDs are likely to reflect these interactions in multiple cell types. Our promoter occupancy data indicate that, for at least a portion of co-regulated genes, a direct cell-autonomous dosage-related interaction exists. Specific genetic programmes are sensitive to the dosage of each transcription factor, and there is considerable complexity in the genomic regulation of gene expression by Brg1 and cardiac transcription factors. In some cases, a clear interrelationship exists between the dosage effects of either factor, resulting in a more profound effect in embryos haploinsufficient for Brg1
. In other words, diminution of levels of one factor will reduce that factor's chance of interaction with BAF complexes, and this can be accentuated when Brg1 levels are also lower. Our molecular data support a model (, model a) for direct interactions between Brg1 and cardiac transcription factors, and imply that reduced recruitment of BAF complexes may be a significant molecular mechanism underlying transcription factor haploinsufficiency in congenital heart disease.
Models for the interaction between DNA-binding transcription factors and Brg1.
The observation that expression levels of several transcripts that are altered in heart heterozygous for a null allele of Nkx2–5, Tbx5 or Brg1 are restored to normal or near-normal levels in Nkx2–5lacz/+;Brg1+/− or Tbx5lacz/+;Brg1+/− compound heterozygous hearts indicates that for some genes, the relative allelic balance of Nkx2–5 (or Tbx5) and Brg1 may be more important than their absolute levels. The mechanism underlying this dosage interdependence is not known, but may include increased potential for interaction with other proteins that would alter the functional output on target genes (, model b). Thus, haploinsufficiency of cardiac transcription factors in CHDs is predicted to result in an imbalance between transcription factors and BAF complexes, likely resulting in impaired transcriptional activation at loci sensitive to this balance. The reason for distinct dosage-sensitive responses from one group of genes to the next is not known, but perhaps the chromatin status of some genes requires different dosage-related interactions between BAF complexes and DNA-binding transcription factors.
We cannot discern which specific group of genes, between those that respond more in the compound heterozygotes, those that return to WT levels in compound versus single heterozygotes, or combinations thereof, is responsible for the phenotypic output of transcription factor or Brg1 haploinsufficiency. It is likely that deregulation of many different genes in various functional classes would be responsible for the complex altered morphology and function that we observe.
In conclusion, we demonstrated a critical requirement for Brg1 in the development of the vertebrate heart, and importantly showed that there exists a fine balance between Brg1 levels and those of cardiac transcription factors that have been implicated in human CHD. The genetic interactions between Brg1 and cardiac transcription factor genes predict that, in the developing heart, maintaining relative levels of BAF complexes and transcription factors is critical for the timely and precise activation of groups of genes during development. In human congenital heart disease, disruption of these dosage-sensitive interactions would predict the impaired activation of specific gene networks that are essential for specific aspects of cardiac morphogenesis and function. We propose that an imbalance in this relationship is a molecular basis underlying CHDs caused by mutations in cardiac transcription factors. Although such a finely regulated interrelationship is perhaps more prone to disruption, it confers significant advantages in the fine quantitative regulation of transcript levels, which is an essential component of complex morphogenesis. These results further underscore the potential for multigenic effects on dominant mutations, and suggest that polymorphisms in BAF complex subunit genes, including but not restricted to Brg1, may modulate the penetration and phenotypic consequence of disease-causing mutations in transcription factor genes in human populations.