LB10
PAX3 isoforms differentially regulate Id gene expression in mouse ESCs
Qiuyu Wang1, Shant Kumar2, Patricia Kumar1
1Manchester Metropolitan University, UK; 2Manchester University, UK
Background
The transcription factor PAX3 has key roles in lineage commitment and
differentiation during embryonal development. PAX3 is involved in myogenesis,
melanogenesis and neurogenesis. Abnormal PAX3 expression is found in human
tumours including melanoma, neuroblastoma and rhabdomyosarcoma. PAX3 has seven
different spliced isoforms, PAX3a-h. Studies by us have shown PAX3 isoforms
differ in functions in mouse melanocytes [1-3]; however the mechanisms by which
they contribute to stem cell differentiation, development and tumourigenesis
remain unclear.
Method
In this study, the mouse embryonic stem cell line E14 was transfected with PAX3c,
PAX3e or PAX3g. Transfectants with pcDNA4 empty vector were used
as controls. Cell proliferation assay and akaline phosphatase assay were
performed on the transfectants. Affymetrix microarray analyses were used to
identify the alteration of gene expression profiles in E14-PAX3 isoform
transfectants. Quantitative RT-PCR and western blotting were used to confirm
microarray data.
Results
PAX3 isoforms had differential effects on the activity of stem cell marker,
akaline phosphatase (AP). AP activities decreased in PAX3c and PAX3g but
increased in PAX3e transfectants. Affymetrix microarray analysis identified a
number of genes involved in cell differentiation were differentially regulated
by PAX3 isoforms. This included changing the expression of the inhibitor of DNA
binding genes (Id1-4). These results were confirmed by quantitative
RT-PCR and western blotting.
Conclusion
Id proteins work primarily as dominant negative regulators of bHLH (basic
helix-loop-helix) transcription factors, inhibit lineage commitment, cell
differentiation and control cell growth [4]. Our results suggest PAX3 isoforms
may exert biological effects that contribute to stem cell differentiation by
differentially regulating the expression of Id genes, although this requires
further investigation.
References
[1] Parker CJ et al. Int J Cancer 2004; 108: 314-20.
[2] Wang Q et al. Cancer Res 2006; 66: 8574-80.
[3] Wang Q et al. Int J Cancer 2007; 120: 1223-31.
[4] Ling MT et al. Differentiation 2006; 481-7.
