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FOG-2 is a transcriptional co-regulator that is required for cardiac morphogenesis as mice deficient in this factor die during mid-gestation of cardiac malformations. FOG-2 interacts with GATA4 to attenuate GATA4-dependent gene expression. The first 12 amino acids of FOG-2 (the FOG Repression Motif) are necessary to mediate this repression. To determine the mechanism by which the FOG Repression Motif functions, we identified 7 polypeptides from rat cardiac nuclear extracts that co-purified with a GST-FOG-2 fusion protein. All proteins identified are members of the NuRD nucleosome-remodeling complex. Using in vitro binding and co-immunoprecipitation assays, we demonstrate that Metastasis-Associated proteins (MTA)-1, 2 and 3 and Retinoblastoma binding proteins RbAp46 and RbAp48 interact with FOG-2, but not with a mutant form of FOG-2 that is unable to repress transcription. Further, we define a novel domain located in the C-terminal portion of MTA-1 that mediates the FOG-2/MTA-1 interaction. We also demonstrate that knockdown of MTA protein expression dramatically impairs the ability of FOG-2 to repress GATA4 activity. Finally, we show that the zinc finger domain of MTA-1 is required for FOG-2 mediated transcriptional repression and that this domain interacts with RbAp46 and RbAp48 subunits of the NuRD complex. Together, these results demonstrate the importance of FOG-2/MTA/RbAp interactions for FOG-2 mediated transcriptional repression and further define the molecular interactions between the FOG Repression Motif and the NuRD complex.

No abstract

Mutations in several transcription factors that regulate cardiac development have recently been described to cause congenital heart disease in humans. A greater understanding of the molecular mechanisms that regulate heart formation may help identify other genes that when mutated will result in human congenital heart disease. FOG-2 is one such gene that encodes a transcriptional corepressor expressed in the developing heart. It is critical for proper cardiac morphogenesis as mice deficient in this factor die during midgestation of cardiac malformations. We have previously shown that FOG-2 physically interacts with GATA4 and attenuates GATA4's ability to activate cardiac specific gene expression. This repression is mediated by a domain of FOG-2 localized to its N-terminus, termed the FOG repression motif. To gain further insights into the molecular mechanism responsible for this repression, we took a biochemical approach to identify factors that interact with FOG repression motif. Using MALDI-TOF mass spectrometry, we identified seven proteins from rat neonatal cardiac nuclear extracts that copurified with a FOG-2-GST fusion protein. All of these proteins have been previously described to be subunits of a nucleosome-remodeling complex called the NuRD complex. To determine which of the NuRD subunits directly interact with the FOG repression motif, we used a series of in vitro binding assays. We found that MTA-1 and MTA-2 specifically bound to the N-terminus of FOG-2 but not to a mutant form of the N-terminus that is unable to mediate repression. In situ hybridization revealed that both MTA-1 and MTA-2 are expressed in the developing heart during mouse embryogenesis. Taken together, these results suggest that FOG-2 mediates transcriptional repression during cardiac development by the recruitment of the NuRD complex to GATA-dependent promoters leading to the remodeling of the local chromatin structure and the attenuation of gene expression.This work was supported by NIH HL071063 and a grant from the Schweppe Foundation.