Role of Lsh in meiosis One focus of our group is to study the molecular role of Lsh, a member of the SNF2 family of chromatin remodeling proteins. Lsh (for lymphoid specific helicase) is important for normal heterochromatin structure and we have previosuly shown that Lsh is directy involved in the de novo methylation pathway. Though Lsh is critical for heterochromatin formation and transcriptional silencing in somatic cells, it wasu nknown, whether Lsh is involved in the regulation of other chromatin-mediated processes such as meiosis in germ cells. During meiosis dynamic changes of chromatin structure are critical, leading to the generation of double strand DNA breaks (DSBs), and allowing the reciprocal recombination between homologous chromosomes of paternal and maternal origin. Defects in centromeric heterochromatin can interfere with normal meiosis and result in aneuploidy. Recent reports suggested that a lack of epigenetic silencing of retrotransposon in DNA methyltransferase 3-L (Dnmt3L) deficient male germ cells severely affected meiosis and resulted in male sterility. This prompted the question whether DNA methylation controlled by Lsh would play a similar role in the meiotic processes of the female germ cells and if so, what would be the underlying mechanism for the meiotic defect. This study was performed in collaboration with Dr Rabindranath from the University of Pennsylvania. Since Lsh deletion is perinatal lethal, fetal female germ cells obtained from Lsh-/-embryos on day 18 of gestation were analyzed by in vitro culture. Although Lsh-/- gonads showed normal presence of germ cells, suggesting that Lsh is not required for primordial germ cell formation, ovary cultures in the absence of Lsh failed to generate primary oocytes, indicating an important role for Lsh in post-natal oocyte growth and differentiation. Immunostaining of the axial/lateral elements of the synaptonemal complex revealed signs of incomplete and abnormal pairing of homologous chromosomes in the absence of Lsh.To address the mechanism of the incomplete synapsis in the absence of Lsh, the ability to form double strand DNA breaks (DSBs) and to repair these breaks were analyzed. Lsh-/- oocytes showed increases in phosphorylated H2AX staining and persistent RAD51 foci indicating that DSB are initiated in the absence of Lsh but cannot be properly resolved. In addition, MLH1 (a protein important for reciprocal recombination during crossover) was not properly loaded onto fully synapsed bivalents suggesting impaired crossovers in the absence of Lsh. In situ hybridization revealed defects in synapsis of the X chromosomes suggesting an inability for normal homologous pairing. To determine a role for DNA methylation in meiosis, we analyzed the methylation status of repetitive DNA sequences. Major and minor satellite sequences and transposobale elements showed signs of demethylation in the absence of Lsh and transposable elements were transcriptionally de-repressed in the absence of Lsh suggesting a greatly perturbed heterochromatin structure in the absence of Lsh. Taken together, our results indicate that, Lsh is crucial for retroviral silencing in female germ cells and is essential for homologous chromosome synapsis during meiosis. Possibly, replicative retrotransposition leads to DSB and impaired synapsis, or perturbed heterochromatin interferes with the proper recognition and pairing of homologous chromosomes. Our results suggest that Lsh and DNA methylation are critical factors in germ-line specific epigenetic regulation and the maintenance of genomic stability in germ cells. Role of Lsh in polycomb mediated silencing In a second approach we were interested in the question whether Lsh mediated DNA methylation would interact with another well studied gene silencing pathway known as polycomb silencing. The polycomb proteins that act in a large protein complex (PRC, polycomb repressive complex) are crucial for developmental control of Hox gene expression. Recently, several connections were reported between DNA methylation and PRC mediated silencing in cancer cells. It was suggested that PRC mediated histone modification (such as H3-K27 methylation) may pre-mark sites for de novo DNA methylation at genes methylated in cancer, but this may be a rare and aberrant event in cells pre-disposed to cancer. We tested the idea whether PRC mediated silencing and DNA methylation is normally linked during development using our Lsh knockout model. Using various tissues derived from Lsh-/- embryos we found several Hox genes de-repressed. Since Hox genes are usually silenced in wild type tissue, our results suggested that Lsh plays an important role in transcriptional regulation of some selected polycomb targets during normal development. To address the molecular mechanism of Lsh action, we examined the DNA methylation pattern at Hox genes. We found that Hox genes that were de-repressed in the absence of Lsh showed also a decrease in DNA methylation whereas unaffected genes were also unchanged in their DNA methylation pattern. This suggested a close correlation of DNA methylation pattern and gene expression in dependence of Lsh. Using ChIPs, we found Lsh specifically associated to selected Hox sites and furthermore, the presence of Lsh determined the recruitment of Dnmt3b to Hox sites. This suggested that Lsh may play a direct role in the methylation at selected Hox genes. In addition, we found evidence for interaction of Lsh with several polycomb proteins. Furthermore, Chips analysis revealed that several PRC mediated histone modifications were affected in the absence of Lsh, for example selected Hox genes that showed reduced binding of the PRC component EZH2 and a decrease in H3-K27 methylation in Lsh deleted cells in comparison with wild type. This suggested that Lsh/DNA methylation is important for complete assembly and activity of the PRC complex and that there exist a feedback loop between DNA methylation and PRC mediated histone modifications. In summary our data suggests, that DNA methylation and polycomb mediated histone modifications are not only aberrantly connected in cancer cells but are part of an ordinary physiologic pathway involving Lsh. Though it is currently still unknown, why selecetd sites are methylated in cancer (while they stay unmethylated in normal tissue), Lsh is certainly a possible candidate that could play a role in the aberrant recruitment of DNA methylatransferases to tumor suppressor regions in cancer cells. On the other hand, cancer is also associated with global DNA hypomethylation which in turn may de-repress some Hox genes. Deregulation of Hox genes has been implied in several hematopoietic malignancies and in ovarian cancer. The suggested connection between the two epigenetic pathways sheds new light on the molecular mechanisms involved in tumorigenesis and may help to improve strategies for cancer treatment or prevention in future
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