Our current focus is on Lsh, a novel chromatin remodeling protein. Lsh is a component of pericentromeric heterochromatin and controls several characteristics of heterochromatin such as histone acetylation, histone methylation and DNA methylation levels. Upon Lsh depletion heterochromatin structure is greatly disturbed and retroviral elements that are usually repressed get reactivated. As a consequence of abnormal heterochromatin we observed defects in mitosis and in embryogenesis in general.To address the question whether Lsh plays a role in other chromatin mediated processes we studied germ cell development in collaboration with delaFuente (University of Pennsylvania). Lsh depleted ovaries showed severe oocyte loss and lacked normal ovarian follicle formation. Ovaries from Lsh knockout mice exhibited DNA hypomethylation and transcriptional reactivation of retroviral elements. Furthermore, Lsh-/- oocytes showed incomplete chromosome synapsis associated with persistent Rad51 foci and gammaH2AX phosphorylation. Failure to load crossover-associated (Mlh1) foci results in the generation of non-exchange chromosomes and incomplete meiosis. Thus Lsh plays also a critical role in epigenetic silencing and maintenance of genomic stabiliy during female meiosis.Most recently we have addressed the molecular function of Lsh with respect to DNA methylation. Two types of methylation pathways have been distinguished: maintenance methylation by Dnmt1 and de novo methylation established by Dnmt3a and Dnmt3b. Using an episomal vector system, we demonstrated that the acquisition of novel DNA methylation pattern requires the presence of Lsh. In contrast, maintenance of previously methylated episomes does not depend on Lsh, implying a functional role for Lsh in the establishment of de novo methylation patterns. Lsh associates with Dnmt3a or Dnmt3b and affects Dnmt3a as well as Dnmt3b directed methylation suggesting that Lsh can cooperate with both enzymatic activities. Furthermore, embryonic stem cells with reduced Lsh protein levels show a decreased ability to silence retroviral elements. This suggests that the epigenetic regulator, Lsh, is directly involved in the control of de novo methylation of DNA and that other epigenetic modifications observed in the absence of Lsh are subsequent changes of DNA methylation.Since de novo methylation is thought to be an important mechanism for silencing of tumor suppressor genes, we are currently testing the role of Lsh in tumor formation. These studies should provide insights in a number of basic biologic processes that involve epigenetic modifications such as transcription, imprinting, mitosis and cellular transformation.
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