Deletion of Lsh perturbs DNA methylation patterns in mice yet it is unknown whether Lsh plays a direct role in the methylation process. Two types of methylation pathways have been distinguished: maintenance methylation by Dnmt1 occurring at the replication fork, and de novo methylation established by the methyltransferases Dnmt3a and Dnmt3b. Using an episomal vector in LshÀ/À embryonic fibroblasts, we demonstrate that the acquisition of DNA methylation depends on the presence of Lsh. In contrast, maintenance of previously methylated episomes does not require Lsh, implying a functional role for Lsh in the establishment of novel methylation patterns. Lsh affects Dnmt3a as well as Dnmt3b directed methylation suggesting that Lsh can cooperate with both enzymatic activities. Furthermore, we demonstrate that embryonic stem cells with reduced Lsh protein levels show a decreased ability to silence retroviral vector or to methylate endogenous genes. Finally, we demonstrate that Lsh associates with Dnmt3a or Dnmt3b but not with Dnmt1 in embryonic cells. These results suggest that the epigenetic regulator, Lsh, is directly involved in the control of de novo methylation of DNA.

We investigated structural and functional aspects of the first mutation in TNNC1, coding for the calcium‐binding subunit (cTnC) of cardiac troponin, which was detected in a patient with hypertrophic cardiomyopathy [ Hoffmann B, Schmidt‐Traub H, Perrot A, Osterziel KJ & Gessner R (2001) Hum Mut17, 524]. This mutation leads to a leucine–glutamine exchange at position 29 in the nonfunctional calcium‐binding site of cTnC. Interestingly, the mutation is located in a putative interaction site for the nonphosphorylated N‐terminal arm of cardiac troponin I (cTnI) [ Finley NL, Abbott MB, Abusamhadneh E, Gaponenko V, Dong W, Seabrook G, Howarth JW, Rana M, Solaro RJ, Cheung HC et al. (1999) EJB Lett453, 107–112]. According to peptide array experiments, the nonphosphorylated cTnI arm interacts with cTnC around L29. This interaction is almost abolished by L29Q, as observed upon protein kinase A‐dependent phosphorylation of cTnI at serine 22 and serine 23 in wild‐type troponin. With CD spectroscopy, minor changes are observed in the backbone of Ca2+‐free and Ca2+‐saturated cTnC upon the L29Q replacement. A small, but significant, reduction in calcium sensitivity was detected upon measuring the Ca2+‐dependent actomyosin subfragment 1 (actoS1)‐ATPase activity and the sliding velocity of thin filaments. The maximum actoS1‐ATPase activity, but not the maximum sliding velocity, was significantly enhanced. In addition, we performed our investigations at different levels of protein kinase A‐dependent phosphorylation of cTnI. The in vitro assays mainly showed that the Ca2+ sensitivity of the actoS1‐ATPase activity, and the mean sliding velocity of thin filaments, were no longer affected by protein kinase A‐dependent phosphorylation of cTnI owing to the L29Q exchange in cTnC. The findings imply a hindered transduction of the phosphorylation signal from cTnI to cTnC.

Lymphoid specific helicase (Lsh) is a major epigenetic regulator that is essential for DNA methylation and transcriptional silencing of parasitic elements in the mammalian genome. However, whether Lsh is involved in the regulation of chromatin-mediated processes during meiosis is not known. Here, we show that Lsh is essential for the completion of meiosis and transcriptional repression of repetitive elements in the female gonad. Oocytes from Lsh knockout mice exhibit demethylation of transposable elements and tandem repeats at pericentric heterochromatin, as well as incomplete chromosome synapsis associated with persistent RAD51 foci and gammaH2AX phosphorylation. Failure to load crossover-associated foci results in the generation of non-exchange chromosomes. The severe oocyte loss observed and lack of ovarian follicle formation, together with the patterns of Lsh nuclear compartmentalization in the germ line, demonstrate that Lsh has a critical and previously unidentified role in epigenetic gene silencing and maintenance of genomic stability during female meiosis.