Activation of multiple pathways is associated with cardiac hypertrophy and heart failure. Repression of antihypertrophic pathways has rarely been demonstrated to cause cardiac hypertrophy in vivo. Hop is an unusual homeodomain protein that is expressed by embryonic and postnatal cardiac myocytes. Unlike other homeodomain proteins, Hop does not bind DNA. Rather, it modulates cardiac growth and proliferation by inhibiting the transcriptional activity of serum response factor (SRF) in cardiomyocytes. Here we show that Hop can inhibit SRF-dependent transcriptional activation by recruiting histone deacetylase (HDAC) activity and can form a complex that includes HDAC2. Transgenic mice that overexpress Hop develop severe cardiac hypertrophy, cardiac fibrosis, and premature death. A mutant form of Hop, which does not recruit HDAC activity, does not induce hypertrophy. Treatment of Hop transgenic mice with trichostatin A, an HDAC inhibitor, prevents hypertrophy. In addition, trichostatin A also attenuates hypertrophy induced by infusion of isoproterenol. Thus, chromatin remodeling and repression of otherwise active transcriptional processes can result in hypertrophy and heart failure, and this process can be blocked with chemical HDAC inhibitors.

Activation of multiple pathways is associated with cardiac hypertrophy and heart failure. Repression of antihypertrophic pathways has rarely been demonstrated to cause cardiac hypertrophy in vivo. Hop is an unusual homeodomain protein that is expressed by embryonic and postnatal cardiac myocytes. Unlike other homeodomain proteins, Hop does not bind DNA. Rather, it modulates cardiac growth and proliferation by inhibiting the transcriptional activity of serum response factor (SRF) in cardiomyocytes. Here we show that Hop can inhibit SRF-dependent transcriptional activation by recruiting histone deacetylase (HDAC) activity and can form a complex that includes HDAC2. Transgenic mice that overexpress Hop develop severe cardiac hypertrophy, cardiac fibrosis, and premature death. A mutant form of Hop, which does not recruit HDAC activity, does not induce hypertrophy. Treatment of Hop transgenic mice with trichostatin A, an HDAC inhibitor, prevents hypertrophy. In addition, trichostatin A also attenuates hypertrophy induced by infusion of isoproterenol. Thus, chromatin remodeling and repression of otherwise active transcriptional processes can result in hypertrophy and heart failure, and this process can be blocked with chemical HDAC inhibitors.

GATA-1 and PU.1 are transcription factors that control erythroid and myeloid development, respectively. The two proteins have been shown to function in an antagonistic fashion, with GATA-1 repressing PU.1 activity during erythropoiesis and PU.1 repressing GATA-1 function during myelopoiesis. It has also become clear that this functional antagonism involves direct interactions between the two proteins. However, the molecular basis for these interactions is not known, and a number of inconsistencies exist in the literature. We have used a range of biophysical methods to define the molecular details of the GATA-1-PU.1 interaction. A combination of NMR titration data and extensive mutagenesis revealed that the PU.1-Ets domain and the GATA-1 C-terminal zinc finger (CF) form a low affinity interaction in which specific regions of each protein are implicated. Surprisingly, the interaction cannot be disrupted by single alanine substitution mutations, suggesting that binding is distributed over an extended interface. The C-terminal basic tail region of CF appears to be sufficient to mediate an interaction with PU.1-Ets, and neither acetylation nor phosphorylation of a peptide corresponding to this region disrupts binding, indicating that the interaction is not dominated by electrostatic interactions. The CF basic tail shares significant sequence homology with the PU.1 interacting motif from c-Jun, suggesting that GATA-1 and c-Jun might compete to bind PU.1. Taken together, our data provide a molecular perspective on the GATA-1-PU.1 interaction, resolving several issues in the existing data and providing insight into the mechanisms through which these two proteins combine to regulate blood development.

Homeodomain-only protein (HOP) is an 8-kDa transcriptional corepressor that is essential for the normal development of the mammalian heart. Previous studies have shown that HOP, which consists entirely of a putative homeodomain, acts downstream of Nkx2.5 and associates with the serum response factor (SRF), repressing transcription from SRF-responsive genes. HOP is also able to recruit histone deacetylase (HDAC) activity, consistent with its ability to repress transcription. Unlike other classic homeodomain proteins, HOP does not appear to interact with DNA, although it has been unclear if this is because of an overall divergent structure or because of specific amino acid differences between HOP and other homeodomains. To work toward an understanding of HOP function, we have determined the 3D structure of full-length HOP and used a range of biochemical assays to define the parts of the protein that are functionally important for its repression activity. We show that HOP forms a classical homeodomain fold but that it cannot recognize double stranded DNA, a result that emphasizes the importance of caution in predicting protein function from sequence homology alone. We also demonstrate that two distinct regions on the surface of HOP are required for its ability to repress an SRF-driven reporter gene, and it is likely that these motifs direct interactions between HOP and partner proteins such as SRF- and HDAC-containing complexes. Our results demonstrate that the homeodomain fold has been co-opted during evolution for functions other than sequence-specific DNA binding and suggest that HOP functions as an adaptor protein to mediate transcriptional repression.