The mechanism and functional role of DNA methylation in tumors of early childhood
Zusammenfassung der Projektergebnisse
Epigenetic programming in its main three manifestations - DNA methylation, genomic imprinting, and histone modification - has been known to influence many cellular and organismal processes, especially during growth and development. Data accumulate that loss of imprinting plays a pivotal role in the genesis of cancer, especially in embryonal tumors arising in eariy childhood. However, the molecular machinery, including the DNA methyltransferase (Dnmt) complexes, the mechanistic basis of DNA methylation in chromatin, and targeting of these complexes to the sites of imprinting are not known. We suggested that alterations of these meta-stable DNA modifications are caused by defects in the catalytic activities or by mis-targeting of Dnmt complexes. Our joint research project led to numerous novel results, unveiling novel tumor suppressor genes in hepatoblastoma, analyzing the mechanism of DNA methylation in chromatin and identifying a novel DNA methyltransferase complex required for efficient DNA methylation in tumor cells. A screen for DNA methylation dependent changes in hepatoblastoma primary tumors and tumor cell lines led to the identification of significant downregulation of the SFRP1, HHIP and IGFBPS genes in hepatoblastoma cells. Downregulation was DNA methylation dependent and re-expression of these genes resulted in reduced malignant potential of these cells. An additional screen for mis-expression of chromatin modifying, DNA methylation and chromatin remodeling components in hepatoblastoma compared to normal liver cells revealed the overexpression of EZH2 and LSH chromatin remodeling machines that are directly involved in the DNA methylation process in vivo. These screens revealed novel markers for the progression from normal cellular states to the epigenetically modified, embryonal tumor cell. Molecular mechanistic studies addressed the mechanism of DNA methylation in chromatin. We could show that unpackaging of the DNA is absolutely required to allow DNA methylation. Chromatin remodeling machines, ATP-dependent helicases, do functionally interact with the DNA methyltransferases in order to allow DNA methylation. These results are confirmed by our observation that the chromatin remodeling complexes LSH and EZH2 were identified as tumor markers that allow efficient DNA methylation. Our results suggest that accessory factors are required for targeting and efficient DNA methylation, as we did initially suggested in our working hypothesis. In search for the accessory factors, we identified a novel methyltransferase complex consisting of Dnmtl, UHRFI and USP7. A combined functional analysis and epigenetic analysis revealed that UHRF1 is strongly overexpressed in tumor cells and required for efficient DNA methylation. We identified the complex on the hepatoblastoma marker genes. Biochemical studies showed that USP7 is an activator of Dnmtl methylation efficiency required for the activity of the complex. Another important result is the observation that Dnmtl exists as a dimer in the cell, giving an explanation for the previous misinterpretation in literature that numerous Dnmtl complexes exist. Our results, using native human tissue, suggest that Dnmt1/USP7 is the major soluble complex in the cell. All other Dnmtl interactions are only established when this complex is targeted to the DNA. Taken together, our combined biochemical, epigenetical and tumor biological approach revealed a novel Dnmtl complex, novel target genes and revealed insights into the molecular mechanism of DNA methylation. We strongly believe that our interdisciplinary approach of unraveling the molecular mechanisms of DNA methylation and the screening for relevant tumor markers will lead to novel therapeutic strategies in the treatment of embryonal tumors.
Projektbezogene Publikationen (Auswahl)
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(2007) DNMT1 but not its interaction with the replication machinery is required for maintenance of DNA methylation in human cells. J . Cell, Biol., 176, 565-71
Spada F, Haemmer A. Kuch D, Rothbauer U, Schermelleh L, Kremmer E, Carell T, Längst G and Leonhardt H
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(2007) Epigenetic disruption of ribosomal RNA genes and nucleolar architecture in DNA methyltransferase 1 (Dnmtl) deficient cells. NAR 35, 2191-8
Espada J, Ballestar E, Santoro R, Fraga MF, Villar-Garea A, Nemeth A, Lopez-Serra L, Ropero S, Aranda A, Orozco H, Moreno V, Juarranz A, Stockert JC, Längst G. Grummt I, Bickmore W and Esteller M
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DNA sequence and conformation directed positioning of nucleosomes by chromatin remodeling complexes. (2007) PNAS 104(40), 15635-15640
Rippe, K., Schrader, A., Riede, P., Strohner, R., Lehmann, E. and Längst, G.
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(2009) DNA methyltransferase 1 forms a stable head-to-head dimer. Journal of Cellular Biochemistry, 106, 521-8
Fellinger K., Rothbauer U., Felle M., Längst G.. Leonhardt H.
