Abstract
Transcription factors are crucial in the regulation of heart development and function.
FOG-2 is one such protein, which in conjunction with a conserved network of inter-regulating
transcription factors, is important in areas of cardiac development and cardiac hypertrophy. This
study aimed to identify novel post-translational modification mechanisms for FOG-2. Two main
areas were investigated. First, novel transcription factor protein partners were pursued and the
prefoldin-like protein Androgen Receptor Trapped clone 27 (Art27) was isolated through an
interaction with FOG-2. This protein was then tested in cardiac gene regulation and cardiomyocyte
differentiation. Second, the post-translational modifier Small Ubiquitin-Related MOdifier (SUMO)
was identified to target FOG-2. Subsequently this modification was functionally characterised in
cardiac hypertrophy.
In addition to interacting FOG-2, Art27 was identified to physically interact other crucial
cardiac transcription factors, GATA-4, GATA-6, Nkx2.5 and haemopoietic transcription factor
GATA-1. The transcriptional activity of Art27 was also assessed, demonstrating an intrinsic
transcriptional repression capacity. To achieve this function Art27 required a means to associate
with gene promoters. This was exhibited with mutual cofactor interaction with GATA-4, Nkx2.5,
GATA-6 and GATA-1, whereby the respective transcriptional activation by these proteins was
markedly diminished on targets genes. Similarly, Art27 down-regulates an artificial GAL-4
responsive gene promoter when fused with the DNA binding domain of GAL-4, indicative of its
intrinsic transcriptional repression capacity. However, Art27 alone or with other transcriptional
repressors like FOG-2 shows little to no apparent function, suggesting that Art27 is a specific
repressor of transcriptional activators by a means of physical interaction in cardiac gene
regulation.
To establish if Art27 regulation of cardiac genes corresponds to a function in regulation of
cardiomyocyte differentiation, the P19CL6-Mlc2vGFP cell system was utilised. Optimisation of this
system revealed a suitable efficiency of cardiomyocyte differentiation with or without stimulation
by pro-differentiation factor GATA-4, as measured by multiple techniques at the morphological
and genetic level. However, introduction of Art27 alone resulted in no discernable difference
compared to control, probably indicating that Art27 does not function in this setting.
Analysis of FOG-2 revealed that it is sumoylated at four amino acid residues (K324, K471,
K915 and K955) and mutation of these residues prevents sumoylation by exogenously introduced
SUMO-1 and endogenously expressed SUMO. When SUMO deficient FOG-2 was functionally
characterised, it showed normal function in heterologous cells, with nuclear localisation and
normal transcriptional activity. However, analysis of the same mutant in neonatal rat
cardiomyocytes revealed aberrant cellular localisation and a deficiency in protection against cell
enlargement and pro-hypertrophic gene expression in endothelin-1 induced cardiac hypertrophy.
The studies of this thesis extend the knowledge of transcriptional regulation in cardiac
development and cardiac hypertrophy, which may contribute to therapies based around cardiac
regeneration, congenital heart disease and cardiac hypertrophy.