Jaenisch Lab Research Summary

Our long range goals are to understand epigenetic regulation of gene expression in mammalian development and disease. Faulty epigenetic reprogramming is the main problem in the development of cloned mammals produced by nuclear transfer and understanding its molecular basis is a major focus of our work. DNA methylation is a crucial component of the epigenetic control of gene activity through the regulation of chromatin state. A number of factors, such as methyl-binding proteins and histone-deacetylases, have been identified that are involved in this process. We are deriving mice carrying targeted mutations in the different components of the epigenetic machinery to understand how stable expression states of the genome are established and maintained. In particular we are focusing on the role of DNA methylation in cancer, genomic imprinting and in the function of the postnatal brain and are employing gene-targeting methods to generate embryonic stem cells and mice with lineage-specific and inducible gene deletions.

Nuclear Cloning and the Reprogramming of the Genome. A major issue raised some 50 years ago in seminal frog cloning experiments was the question of whether nuclei of terminally differentiated cells can be reprogrammed to generate animals after nuclear transfer. We have shown recently that monoclonal mice can be derived from mature B and T cells by nuclear transfer, demonstrating that the genome of terminally differentiated cells can be reprogrammed to direct development of a new animal. The efficiency is, however, very low, suggesting that most cloned animals may be derived from somatic stem cells rather than terminally differentiated cells as assumed. Nuclear transfer represents an unbiased experimental approach that allows distinguishing between epigenetic and genetic alterations of the genome. We have used this approach to derive animals from postmitotic neurons to investigate whether neural functions such as olfactory receptor choice or memory involve genetic in addition to epigenetic changes. In our most recent experiments we have derived cloned mice from nuclei of cancer cells in an effort to distinguish between epigenetic (=reversible) and genetic (=irreversible) changes that are involved in malignant transformation. Finally, we have performed a "proof of principle" experiment in a mouse disease model to provide evidence that therapeutic cloning combined with gene therapy represents a valid strategy for transplantation therapy.

Embryonic Stem Cells and the Control of Self-renewal. The transcription factors Oct4, Sox2, and Nanog have essential roles in early development and are required for the propagation of undifferentiated embryonic stem (ES) cells in culture. To gain insights into transcriptional regulation of human ES cells, we have, in collaboration with the Young lab, identified Oct4, Sox2, and Nanog target genes using genome-scale location analysis. We found, surprisingly, that Oct4, Sox2, and Nanog co-occupy a substantial portion of their target genes. These target genes frequently encode transcription factors, many of which are developmentally important homeodomain proteins. Our data also show that Oct4, Sox2, and Nanog collaborate to form regulatory circuitry in ES cells consisting of autoregulatory and feedforward loops. These results provide new insights into the transcriptional regulation of stem cells and reveal how Oct4, Sox2, and Nanog contribute to pluripotency and self-renewal.

Oct4 and Its Role in Proliferation, Pluripotency and Cancer.Oct-4 is normally expressed in pluripotent mammalian cells including epiblast cells, embryonic stem cells and primordial germ cells. In addition, many germ cell tumors and a few somatic tumors show detectable expression of Oct-4. While Oct-4’s role during preimplantation development is to maintain embryonic cells in a pluripotent state, little is known about its oncogenic properties. Here, we investigate the effect of ectopic Oct-4 expression on somatic tissues of adult mice using a doxycycline-dependent expression system. We find that activation of Oct-4 results in dysplastic cell proliferation in the intestine, skin and stomach, and this phenotype is dependent on continuous Oct-4 expression. Dysplastic lesions show an expansion of progenitor cells which is accompanied by increased b-catenin transcriptional activity. Loss of Oct-4 expression in dysplastic cells of the intestine results in their differentiation,suggesting that Oct-4’s tumorigenic potential is mediated by the inhibition of differentiation similar to its function in embryonic cells. These data show that Oct-4 can function as a potent oncogene in vivo and support the notion that progenitor cells are a driving force in tumorigenesis.

DNA Methylation.The importance of DNA methylation in vertebrate gene control has remained a controversial issue because of the indirect and correlative nature of many studies in the field. The targeted inactivation of the Dnmt1 and Dnmt3 genes results in embryonic lethality, establishing the essential role of DNA methylation in mammals. We are creating systems that allow the tissue specific and inducible inactivation of the methyl-transferases and methyl-binding proteins to assess the role of methylation in normal development and cancer.

Genomic Imprinting. Imprinted genes are expressed either from the maternal or the paternal allele but not from both and we have shown that monoallelic expression of imprinted genes depends on DNA methylation. It has been suggested that faulty imprinting in cloned animals derived by nuclear transfer contributes to the abnormal phenotype (the “Larger Offspring syndrome”). Indeed, the analysis of cloned pups showed widespread dysregulation of imprinted genes. To directly test whether imprinting has an intrinsic role in development we are generating “non-imprinted” mice from “non-imprinted” ES cells by nuclear transplantation.

Cancer. The involvement of DNA methylation in cancer has been controversial: both hypomethylation as well as hypermethylation have been associated with malignant transformation. When the MTase mutation was introduced into mice with a genetic predisposition to colon cancer, a surprising result was seen: the MTase enzyme level directly correlated with the development of cancer. This argued that the MTase enzyme itself may act as an oncogenic determinant and may be a potentially attractive drug target for cancer prevention and treatment.

Genomic hypomethylation is a widely observed and early step in human tumorigenesis. Using different mutant alleles of the Dnmt1 gene we have shown that hypomethylation results in a substantial increase in the genomic mutation rates, the mutations being caused by enhanced mitotic recombination. These results are significant as they may explain the selective advantage of hypomethylation in early stages of transformation: hypomethylation leading to genomic instability may provide the incipient tumor cell with a mechanism to efficiently delete tumor suppressor genes by LOH. Indeed, the great majority of mice carrying a hypomorphic Dnmt1 allele develop aggressive thymic tumors. Our results indicate that therapeutic inhibition of Dnmt1 may prevent some forms of cancer such as intestinal tumors, but may promote cancer in other tissues. Therefore, to assess the consequences of Dnmt inhibitors as potential cancer drugs, it will be crucial to define the role of hypomethylation in diverse tumor models as Dnmt inhibitors may have opposite effects on cancer incidence when arising in different tissues. The MTases Dnmt3a and b are the best candidates to cause the silencing of tumor suppressor genes by de novo methylation. We have generated mice carrying conditional alleles of Dnmt3a and Dnmt3b that are being introduced into mice prone to develop intestinal and prostate cancer to directly test their role in carcinogenesis.

Epigenetic control of brain function: Both Dnmt1 or Dnmt3 are highly expressed in the post-mitotic neurons, raising the possibility that methylation may have an important role in the physiology and disease of the postnatal brain. This possibility has been dramatically supported by the recent discovery that the mutation of the methyl binding protein, MECP2, leads to RETT syndrome, one of the most frequent causes of severe mental retardation in girls. We have generated mice carrying a conditional mutation of the Mecp2 gene and found that the phenotype of mutant animals resembles that of patients. We are using this mouse strain to investigate the pathology of neuronal dysfunction and to devise potential strategies for therapeutic intervention. Finally, we are deriving mice carrying a Dnmt1 or Dnmt3 deletion in specific regions of the brain to directly assess the potential role of methylation in the brain physiology of processes such as memory or aging.

Last updated August 2005.

SELECTED PUBLICATIONS

Hochedlinger, K., Yamada, Y., Beard, C. & Jaenisch, R. Ectopic expression of Oct-4 blocks progenitor cell differentiation and causes dysplastic growth in epithelial tissues. Cell 121, 465-472 (2005).

Eggan, K., Baldwin, K., Tackett, M., Osborne, J., Gogos, J., Chess, A., Axel, R., & Jaenisch, R. Mice cloned from olfactory sensory neurons. Nature 428, 44-49 (2004).

Hochedlinger, K., Blelloch, R., Brennan, C., Yamada, Y., Kim, M., Chin, L. & Jaenisch, R. Reprogramming of a melanoma genome by nuclear transplantation. Genes Dev. 18, 1875-1885 (2004).

Jaenisch, R. Human cloning – The science and ethics of nuclear transplantation. N.Eng.J. Med. 351, 2787-2791 (2004).

Blelloch, R., Hochedlinger, K., Yamado, Y., Brennan, C. Kim, M., Mintz, B., Chin, L. & Jaenisch, R. Nuclear cloning of embryonal carcinoma cells. PNAS 101, 13985-13990 (2004).

Eggan, K., Baldwin, K., Tackett, M., Osborne, J., Gogos, J., Chess, A., Axel, R. and Jaenisch, R. Mice cloned from olfactory sensory neurons. Nature 428, 44-49 (2004).

Gaudet, F., Hodgson, J.G., Eden, A., Jackson-Grusby, L., Dausman, J., Gray, J.W., Leonhardt, H. and Jaenisch, R. Induction of tumors in mice by genomic hypomethylation. Science 300: 489-492 (2003).

Hochedlinger, K. & Jaenisch, R. Nuclear transplantation, embryonic stem cells, and the potential for cell therapy. N. Engl. J. Med. 349, 275-86 (2003).

Bortvin, A., Eggan, K., Skaletsky, H., Akutsu, H., Berry, D.L., Yanagimachi, R., Jaenisch, R. and Page, D.C. Nuclear cloning, reprogramming of gene expression and the differentiation state of the donor cell. Development 130: 1673-80 (2003).

Rideout, W.M., Hochedlinger, K., Kyba, M., Daley, G., & Jaenisch, R. Correction of a genetic defect by nuclear transfer and combined cell and gene therapy. Cell 109: 17-27 (2002).

Hochedlinger, K. & Jaenisch, R. Generation of monoclonal mice by nuclear transfer from mature B and T donor cells. Nature 415, 1035-1038 (2002).



For additional publications, visit the PubMed database.