Oct-3/4 is a POU domain homeobox gene that is expressed during gametogenesis and in early embryonic cells, where it has been shown to be important for maintaining pluripotency. Following implantation, this gene undergoes a novel multi-step programme of inactivation. Transcriptional repression is followed by a pronounced increase in histone H3 methylation on Lys 9 that is mediated by the SET-containing protein, G9a. This step sets the stage for local heterochromatinization via the binding of HP1 and is required for subsequent de novo methylation at the promoter by the enzymes Dnmt3a/3b. Genetic studies show that these epigenetic changes actually have an important role in the inhibition of Oct-3/4 re-expression, thereby preventing reprogramming.

The pluripotency determining gene, Oct-3/4 (also called Pou5f1) undergoes post implantation silencing in a process mediated by the histone methyltransferase (HMT) G9a. Microarray analysis now shows that this enzyme may operate as a master regulator that inactivates multiple early embryonic genes by bringing about methylated-histone H3K9 heterochromatinization and de novo DNA methylation. Genetic studies in differentiating ES cells demonstrate that a point mutation in the G9a SET domain prevents heterochromatinization, but still allows de novo methylation, while biochemical and functional studies indicate that G9a itself is capable of bringing about de novo methylation through its ankyrin (ANK) domain, by recruiting Dnmt3a/3b independently of its HMT activity. These modifications appear to be programmed for carrying out two separate biological functions, with histone methylation blocking target-gene reactivation in the absence of transcriptional repressors, while DNA methylation prevents reprogramming to the undifferentiated state.

Cyclins D1, D2 and D3 play important roles in cell proliferation and differentiation. Although their abnormal expression has been linked to cancer development and progression in a number of tissues, the expression of cyclin D2 and D3 proteins in colon cancer has not yet been characterised. In this study, we examined cyclin D1, D2 and D3 protein expression by Western blot analysis in tumour and adjacent normal colon tissues of 57 patients. In addition, we examined D-type cyclins protein expression in HT29 and LoVo39 cell lines from colon carcinomas, as a function of induced proliferation and differentiation. In both cell lines, the expression of the three D-type cyclins increased as a result of induced proliferation, whereas the expression of cyclin D3 increased as a result of induced differentiation. In colon tumours, cyclin D1 was overexpressed in 44%, cyclin D2 was overexpressed in 53% and cyclin D3 was overexpressed in 35% of the cases. We also found that in 16% of the cases, cyclin D3 protein expression was reduced in the tumour, as compared to the adjacent normal tissue. Examination of D-type cyclin protein overexpression in relation to the TNM stage of the tumours revealed that overexpression of cyclins D1 and/or D2, but not cyclin D3, is linked to colon carcinogenesis and that overexpression of cyclin D2 may be related to a higher TNM stage of the tumour.

Although the transcriptional regulatory events triggered by Oct-3/4 are well documented, understanding the proteomic networks that mediate the diverse functions of this POU domain homeobox protein remains a major challenge. Here, we present genetic and biochemical studies that suggest an unexpected novel strategy for Oct-3/4-dependent regulation of embryogenesis and cell lineage determination. Our data suggest that Oct-3/4 specifically interacts with nuclear b-catenin and facilitates its proteasomal degradation, resulting in the maintenance of an undifferentiated, early embryonic phenotype both in Xenopus embryos and embryonic stem (ES) cells. Our data also show that Oct-3/4-mediated control of b-catenin stability has an important function in regulating ES cell motility. Down-regulation of Oct-3/4 increases b-catenin protein levels, enhancing Wnt signalling and initiating invasive cellular activity characteristic of epithelialmesenchymal transition. Our data suggest a novel mode of regulation by which a delicate balance between b-catenin, Tcf3 and Oct-3/4 regulates maintenance of stem cell identity. Altering the balance between these proteins can direct cell fate decisions and differentiation.