Researchers map how transcription factors interact to create a heart.

It has long been known that transcriptional regulation during embryonic development relies on the regulation of thousands of genes. Within a particular cell type defined sets of DNA-binding transcription factors (TFs), proteins that direct gene expression, functioning on defined genomic elements are involved in determining cellular identity.  Combinatorial interactions of small numbers of TFs have been proposed to underlie tissue-specific gene expression in mammalian organogenesis.
However, the functional output of interactions between developmentally important TFs and their genomic

basis have not been explored on a broad scale or in sufficient detail.  Now, a study led by researchers at the Gladstone Institutes has shown that three transcription factors (TFs) interact with each other and the genome to influence how a heart forms in an embryo. The team state that without these protein interactions, severe congenital heart defects can occur and by understanding how the transcription factors work together during heart development, the global medical community may discover new ways to treat heart disease. The opensource study is published in the journal Cell.

Previous studies show that transcription factors dictate which genes are turned on or off in a cell during embryo formation, thereby controlling the type of organ the cell will form.  It is known that the T-box TF TBX5 and the homeodomain TF NKX2-5 regulate several aspects of heart development.  TBX5 and NKX2-5 can interact physically, with this interaction proposed as the basis for congenital heart defects caused by mutations in either TBX5 or NKX2-5 in humans.  Similarly, mutations in the TF gene GATA4 result in congenital heart defects, with GATA4 suggested as a functional partner of TBX5 and NKX2-5.  Therefore, a mechanistic understanding of the collaborative function of TBX5, NKX2-5, and GATA4 will have important implications for understanding congenital heart disease and for developing strategies for cardiac regeneration.  The current study tracked NKX2-5, TBX5, and GATA4 to reveal for the first time how they interact on a genomic and physical level.

The current study developed mouse embryos that were missing one or two of the proteins. Results show that a loss of one transcription factor resulted in known heart defects, while embryos missing two transcription factors had almost no heart, demonstrating the importance of both factors.

Next, the group created cardiac cells missing the same transcription factors to investigate how their interactions affect gene expression. Data findings show that the transcription factors often sat next to each other on the genome and required the presence of the other to bind to the DNA.  Results show that if one of the proteins was absent, at times the other transcription factors would go rogue, binding to places on the genome where they were not supposed to be; this migration turned on genes that were supposed to remain off and silenced other genes when they should have been activated.

The lab explain that where and when the transcription factors bound to the genome and how dependent they were on each other is written in the cell’s DNA, which acts like an instruction manual by dictating the proteins’ behaviour. They go on to add that by learning the rules for these three proteins, they can infer the rules of other transcription factors that are also important for heart development.

In a final step, the researchers created a crystal structure, or 3D representation, of the transcription factors co-binding on the genome. Data shows that the proteins sat near each other on the genome and they were also physically touching.  The lab state that the crystal structure critically shows the interaction between two of the transcription factors and how they influence one another’s binding to a specific stretch of DNA.  They go on to conclude that their detailed structural analysis revealed a direct physical connection between TBX5 and NKX2-5 and demonstrated that DNA plays an active role in mediating the interaction between the two proteins.

The team surmise that their findings have important implications for both congenital and adult heart disease.  They go on to add that the better the understanding of these transcription factors, the closer they’ll come to a treatment for heart disease. For the future, the researchers state they’re using this knowledge to search for small molecules that can affect gene regulation and reverse some of the problems caused by the loss of these transcription factors.

Source: Gladstone Institutes


Image of a mouse embryo, different cell types shown by different colors [Image: B0008305 Mouse embryo under a CC BY-NC-ND 2.0 license].
Image of a mouse embryo, different cell types shown by different colors [Image: B0008305 Mouse embryo under a CC BY-NC-ND 2.0 license].

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