Epigenetic regulation is crucial for normal embryonic development and plays a role in a number of diverse biologic mechanisms such as genomic imprinting, X inactivation, DNA recombination and transcription. Epigenetic control is established by changes in chromatin structure and involves histone modifications and DNA methylation of cytosine in the context of CpG dinucleotides. A major focus of our group is to study the role of Lsh in epigentics using a mouse model with a targeted deletion of Lsh. Lsh, (for lymphoid specific helicase) is a member of the SNF2 family of chromatin remodeling proteins. Although Lsh was originally identified based on its high expression levels in murine lymphocytes, Lsh is ubiquitously expressed and plays a crucial role during embryonic development. Lsh is required for normal DNA methylation pattern and we have previosuly shown that Lsh is directy involved in the de novo methylation pathway. The establishment of DNA methylation is critical for normal heterochromatin strcuture and transcriptional repression. Biologic consequences of Lsh deletion are defects in mitosis, bi-allelic gene expression at specific imprinted loci, and a loss of retroviral silencing and control of parasitic elements in the genome. Role of Lsh in tumorigensis Human tumors show frequently a global reduction of DNA methylation. It remains unclear whether genomic hypomethylation contributes to tumorigenesis or whether DNA methylation is a consequence of human cancer. To explore the functional link between genomic hypomethylation and cancer, we studied the development of hematopoietic neoplasm using the Lsh-/- mouse model. Since Lsh -/- mice die shortly after birth, tumor development cannot be studied using adult Lsh-/- mice. Therefore we created radiation chimeras by injecting Lsh-/- hematopoietic precursor cells into irradiated recipient wild type mice. These transplantation experiments revealed that Lsh is required for normal hematopoiesis, since most Lsh-/- recipients showed reduced cellular levels of all blood lineages and suffered from a severely suppressed immune response. About 10% of mice succumbed to erythroleukemia. The development of erythroleukemia was accompanied by global DNA hypomethylation, reactivation of endogenous retroviral elements and elevations of the transcription factor PU.1, an oncogene that is frequently overexpressed in murine erythroid tumors. Moreover, a slight increase in PU.1 transcripts was already detectable in Lsh deleted hematopoietic precursor cells. Analysis of the PU.1 genomic locus revealed DNA hypomethylation of endogenous retroviral sequences and showed their specific activation upon deletion of Lsh. Examination of the de novo DNA methyltransferase Dnmt3b demonstrated that Lsh controls the recruitment of Dnmt3b to the PU.1 locus. Thus, Lsh is a critical component of the molecular machinery for DNA methylation. In addition, Lsh is involved in the establishment of retroviral silencing and important to prevent dangerous oncogene activation. In summary, Lsh depletion resulted in genome wide DNA hypomethylation as well as specific hypomethylation at retroviral elements located in the PU.1 locus. This was associated with transcriptional activation of those endogenous retroviral elements and a slight increase in mRNA and protein levels of the tumor promoting factor PU.1. Taken together our study suggests that genomic hypomethylation after Lsh deletion can cause a dual threat; on the one hand it may lead to severe hematopoietic defects with the danger of reduced immune responses and on the other hand, it may create an enhanced risk of developing hematopoietic neoplasm. Since demethylating agents are currently used in the clinic our study makes a contribution in therapeutic risks assessments and identifies some of the molecular pathways associated with DNA hypomethylation. In collaboration with De la Fuente (University of Pennsylvania) we furthermore contributed to studies on epigenetic changes during female meiosis and in collaboration with Richard Meehan (MRC Edinburgh) we contributed to work on Lsh and interaction with the DNA methyltransferase Dnmt1. In summary, studies on the molecular mechanisms of Lsh and the biologic consequences of DNA hyomethylation caused by Lsh deletion enhance our understanding about basic mechanisms of epigenetic regulation and their role in development and may help to identify novel molecuar targets involved in silencing of tumor suppressor genes and cancer development.
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