Tim Mohun: Projects

Nkx2-5 transcriptional targets

Characterising the genetic programme regulating cardiogenesis is important not only for understanding of heart development, but also because several of the same transcriptional regulators are now implicated in cardiac pathologies and disease.

The homeodomain protein NKX2-5 is an important early cardiac transcription factor implicated in many aspects of cardiogenesis including regulating the size and proliferation of cardiac precursor populations, terminal differentiation of the myocardium, establishment of the ventricular conduction system and postnatal conduction function.

In humans, mutations in the NKX2-5 gene results in a spectrum of congenital heart disease of varying phenotypic penetrance, that is mirrored in mouse models. Despite its importance, remarkably little is known about the place of NKX2-5 in the cardiac genetic regulatory network and few direct target genes have been unambiguously identified.

Figure

Overview of ChIP-chip method for mapping transcription factor binding sites in vivo.

 

The goal of this project is to identify direct target genes of NKX2-5 during cardiac development taking advantage of genomic resources and techniques for genome-wide analysis of transcription that have become available.

We have combined microarray studies with genome-wide chromatin immunoprecipitation (ChIP) using heart tissue from mouse and frog embryos to identify putative target genes of NKX2-5 in the developing embryonic heart. Using E11.5 mouse hearts, this analysis has identified several hundred novel Nkx2-5 binding sites within the mouse genome within close proximity to gene transcription units. Over 70% of the identified genomic regions contain an NKX2-5 binding motif and the genes identified include most of the previously identified NKX2-5 gene targets.

 

ChIP analysis with Xenopus tadpole hearts

ChIP analysis with Xenopus tadpole hearts using AcH4, SRF and NKX2-5 specific antisera. Cardiac actin and thyroid receptor promoters used as positive and negative controls, respectively.

 

In order to identify functionally important binding sites (and hence Nkx2-5 target genes) we are taking several approaches. ChIP hits are being classified into syn-expression groups and bioinformatic approaches are being used to test for evolutionary conservation of the identified Nkx2-5 recognition motifs. The functional role of such motifs within the target gene proximal promoter is being tested using transgenesis in fish and frog embryos.

ChIP results are also being compared with gene expression data obtained from DNA microarray studies of wild-type, Nkx2-5 null and NKX2-5 hypomorphic cardiac cells in order to identify those target genes showing functional analysis of identified Nkx2-5-target genes in embryonic heart development is being undertaken in zebrafish and frog embryos, using gene overexpression and morpholino-oligonucleotide knockdown approaches.

3D models of NKX2-5 hypomorph hearts

3D models of NKX2-5 hypomorph hearts from HREM data, showing enlarged right atrium, ventricular walls and ventricular septal defects (arrowed).

Collaborators - Richard Harvey, Victor Chang Cardiac Research Institute