Modulation of ATRA regulated transcription by EVI1

The EVI1 (ecotropic virus integration site 1) gene plays important roles in the normal development of many organs, e.g., of the nervous system, but also contributes as an oncogene to the emergence and particular aggressiveness of certain types of leukemias and solid tumors. EVI1 codes for a protein that has several properties typical of transcription factors, i.e., of proteins involved in the regulation of the expression of other genes. Nevertheless, only limited information is available about genes regulated by EVI1 in the context of either tumorigenesis or normal development. Also, only one physiological agent has so far has been shown to regulate the expression of the EVI1 gene itself: all-trans retinoic acid (ATRA), which promotes the differentiation of many cell types, including neuronal and hematopoietic cells, enhances the transcription of EVI1. During previous work, we have characterized the mechanism through which ATRA induces the expression of EVI1 in the human teratocarcinoma cell line NTERA-2, a cell line that undergoes neuronal differentiation upon stimulation with ATRA. In the course of these experiments, we discovered that EVI1 counteracts, and thereby limits, its own induction by ATRA, while it enhances the induction of another ATRA-regulated gene, the retinoic acid receptor ß (RARß) gene. The latter finding could be confirmed in a human leukemia cell line, U937T. In the work proposed here, we aim to investigate whether EVI1 is a more general regulator of the ATRA response, i.e., whether, in addition to its own and the RARß genes, it stimulates or represses the induction of others of the over 500 known ATRA regulated genes. Furthermore, we intend to characterize the mechanisms through which EVI1 exerts its effects on the ATRA response. Experiments will be performed to determine whether these effects involve binding of EVI1 to DNA and/or to RAR and RXR, the transcription factors responsible for retinoic acid dependent gene regulation. The impact of EVI1 on the association of transcriptional cofactors with its own and the RARß promoters (and/or the promoters of other genes with an EVI1 modified ATRA response), and the ensuing alterations of posttranslational modifications of histones will be investigated. Finally, we will ask whether and how EVI1 affects the association of the basal transcriptional machinery with ATRA regulated, EVI1 modified promoters. These studies will advance our understanding of the still poorly investigated ways in which EVI1 influences normal development, and also enhance our knowledge about the role of EVI1 in haematopoiesis and leukemogenesis. Furthermore, they will elucidate a novel aspect of the already intensely studied process of gene regulation by ATRA: even though other transcription factors have been reported to either stimulate or repress ATRA induced transcription, EVI1 is unique in that it can have both effects, in a manner that depends on the promoter context.

The "ecotropic viral integration site 1" (EVI1) gene codes for a transcription factor, i.e., a protein that regulates the activity ("expression") of other genes. Pathological overexpression of EVI1 contributes to the emergence of certain forms of leukemia with a particularly aggressive course of disease. However, like all cancer genes, EVI1 also has physiological roles in the healthy organism, e.g., in the hematopoietic (blood cell forming) system as well as in various developmental processes.all-trans retinoic acid (ATRA) is a derivative of vitamin A, and affects a multitude of biological processes. Among others, it promotes the maturation ("differentiation") of particular types of blood cells, which makes it a useful addition to the therapy of a genetically defined subgroup of leukemias. ATRA mainly acts by influencing the expression of certain target genes, which it achieves by binding to a specific receptor that in turn associates with the regulatory regions of these genes and acts as a transcription factor.Earlier work from the project leader's laboratory had shown that EVI1 can influence gene regulation by ATRA both in a positive and a negative manner, yet so far each of these modes had been observed only for a single gene. Based on these findings, two goals were defined for project P21401: 1) the identification of additional genes with an EVI1 modulated ATRA response through methods simultaneously interrogating all known human genes, and 2) elucidation of the molecular mechanism behind this type of gene regulation.A substantial number of genes with an EVI1 modulated ATRA response was indeed uncovered. Interestingly, EVI1 enhanced the effects of ATRA in the vast majority of cases, and the originally also observed negative influence of EVI1 on ATRA regulated gene expression was found only rarely. These results inspired a third question that transcended the original project plan, namely, whether EVI1 could also impact on cell biological responses to ATRA. This was in fact found to be the case: EVI1 enhanced the effects of ATRA on cellular proliferation, differentiation, and death. Furthermore, one of the genes that exhibited strongly synergistic regulation by EVI1 and ATRA was shown to contribute causally to some of these processes. Regarding the mechanism mediating cooperative gene regulation by EVI1 and ATRA, EVI1 was found to enhance the interaction between the retinoic acid receptor and one of its cofactors that are important in the process of gene regulation. Altogether, the results generated in the course of this project may be relevant to the therapy of certain forms of leukemia.