Raregulated Genes

There has been great effort over the last 25 years by many investigators to elucidate the cascade of gene expression events that ultimately results in the differentiated phenotype displayed by EC and ES cells following RA treatment. The first step to achieve this goal has been to identify genes whose expression is altered by RA during differentiation along a number of different pathways. The approaches used to address this question have changed over the years taking advantage of the major technical developments in molecular and cellular biology. Among the technical approaches used include differential screening of cDNA libraries, subtractive cDNA libraries, differential display, microarray analysis, and proteomic analysis. An extremely long list of genes and expressed sequence tags (ESTs) that display altered expression upon treatment of EC and/or ES cells following RA exposure has resulted from these numerous studies. A listing of genes and ESTs differentially regulated in EC and ES cells induced by RA to differentiate along a number of different pathways can be obtained from a variety of sources including Gudas (1991), Gudas et al. (1994), Nishiguchi et al. (1994), Bouillet et al. (1995), Faria et al. (1998), Bain et al. (2000), Harris and Childs (2002), Wei et al. (2002), Sangster-Guity et al. (2004), and An et al. (2005). Several general trends are readily apparent on examination of these lists of genes and ESTs.

First, RA treatment of EC and ES cells results in both an increase and a decrease in the expression of particular genes. In some cases, the fold changes in expression are rather modest while in others the fold changes are very large. Furthermore, RA treatment causes changes in the expression of genes with a variety of different functions including transcription factors, RA metabolism and transport proteins, extracellular matrix proteins, protooncogenes, growth factors and their receptors, cytoskeletal proteins, proteins involved in cell metabolism, cell surface antigens, apoptosis-related proteins, cell-cycle control proteins, and proteins that mediate intracellular and extracellular signaling.

Second, the temporal pattern of gene expression during RA-induced differentiation has several phases. Regardless of the pathway of differentiation, there is a subset of genes whose expression is increased rapidly (within the first 12-16 h) upon RA treatment in the presence of cycloheximide indicating that they are primary response genes to RA. Promoter analysis of many of these genes demonstrates that they contain a RARE and that the RAR/ RXR heterodimer mediates the increase in expression upon RA treatment. Finally, many of the same primary RA response genes are regulated in both EC and ES cells irrespective of the pathway of differentiation and are most often either transcription factors or proteins involved in RA metabolism and transport.

A much larger number of other genes display altered expression at later time points (1 or more days) following RA treatment and these changes in expression require new protein synthesis. Hence, these genes are secondary responders and are indirectly regulated by RA treatment. Furthermore, many of these genes are associated with a specific differentiation pathway. Differentiation of F9 cells to primitive endoderm is biphasic (early and late responding genes). A third phase occurs when these cells are induced to differentiate in the presence of RA and dibutyryl cAMP to parietal endo-derm or to differentiate upon treatment of embryoid bodies with RA to visceral endoderm. Similarly P19 cells induced to differentiate to neuronal cells display three phases of gene expression changes, the initial primary response phase (0-16 h following RA treatment), the neural differentiation phase (16 h to 2 days following RA treatment), and the terminal differentiation phase (5-6 days following RA treatment).

Third, a final point to keep in mind is that the differentiation process often involves RA treatment along with additional treatments/factors such as dibutyryl cAMP or aggregation of cells (embryoid bodies). Therefore, some of the gene expression changes are mediated solely by RA, some by one of the additional treatments/factors, and a third group by a combination of RA and one or more of the additional treatments/factors. Some studies have attempted to address this issue, while others have not (Teramoto et al., 2005).

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