Rationale: Mutations in the MYBPC3 gene encoding cardiac myosin-binding protein (cMyBP)-C are frequent causes of hypertrophic cardiomyopathy, but the mechanisms leading from mutations to disease remain elusive. Objective: The goal of the present study was therefore to gain insights into the mechanisms controlling the expression of MYBPC3 mutations. Methods and Results: We developed a cMyBP-C knock-in mouse carrying a point mutation. The level of total cMyBP-C mRNAs was 50% and 80% lower in heterozygotes and homozygotes, respectively. Surprisingly, the single G>A transition on the last nucleotide of exon 6 resulted in 3 different mutant mRNAs: missense (exchange of G for A), nonsense (exon skipping, frameshift, and premature stop codon) and deletion/insertion (as nonsense but with additional partial retention of downstream intron, restoring of the reading frame, and almost full-length protein). Inhibition of nonsense-mediated mRNA decay in cultured cardiac myocytes or in vivo with emetine or cycloheximide increased the level of nonsense mRNAs severalfold but not of the other mRNAs. By using sequential protein fractionation and a new antibody directed against novel amino acids produced by the frameshift, we showed that inhibition of the proteasome with epoxomicin via osmotic minipumps increased the level of (near) full-length mutants but not of truncated proteins. Homozygotes exhibited myocyte and left ventricular hypertrophy, reduced fractional shortening, and interstitial fibrosis; heterozygotes had no major phenotype. Conclusions: These data reveal (1) an unanticipated complexity of the expression of a single point mutation in the whole animal and (2) the involvement of both nonsense-mediated mRNA decay and the ubiquitin-proteasome system in lowering the level of mutant proteins. (Circ Res. 2009;105:239-248.)Key Words: cardiomyopathy Ⅲ hypertrophic cardiomyopathy Ⅲ mRNA stability Ⅲ transgenic mice Ⅲ ubiquitin C ardiac myosin-binding protein (cMyBP)-C is a major component of the A-band of the sarcomere, where it interacts with myosin, actin and titin (see elsewhere 1,2 and reviewed previously 3 ). It is exclusively expressed in the heart in humans and mice. 4,5 Its role has been enigmatic for long, but accumulating recent evidence suggests that cMyBP-C is essential for normal diastolic relaxation by inhibiting actin-myosin interactions at low intracellular Ca 2ϩ concentrations. 6 -10 Mutations in MYBPC3 encoding cMyBP-C cause hypertrophic cardiomyopathy (HCM) (reviewed previously 3,11 ).HCM is an autosomal-dominant disease characterized by left ventricular (LV) hypertrophy, which predominantly involves the interventricular septum and is associated with myocardial disarray and interstitial fibrosis. 12 HCM involves more than 450 mutations in at least 13 genes encoding sarcomeric proteins. 11,13 Out of them, mutations in MYBPC3 are frequent. 14 In contrast to other disease genes, in which the majority of the mutations are missense, Ϸ70% of MYBPC3 mutations result in a frameshift creating a premature termination...

Laboratoire de Recherche sur la réparation et la transcription dans les cellules souches, 6 INSERM, UMR 967, Fontenay-aux-roses, F-92265, 7 Univ Paris Diderot, Sorbonne Paris cité, UMR S967, Fontenay-aux-roses, F-92265 and 8 Univ Paris-Sud, UMR S967, Fontenay-aux-roses, F-92265 Correspondence should be addressed to M Vernet; Email: muriel.vernet@cea.fr Abstract Anti-silencing function 1 (ASF1) is an evolutionarily conserved histone H3-H4 chaperone involved in the assembly/disassembly of nucleosome and histone modification. Two paralogous genes, Asf1a and Asf1b, exist in the mouse genome. Asf1a is ubiquitously expressed and its loss causes embryonic lethality. Conversely, Asf1b expression is more restricted and has been less studied. To determine the in vivo function of Asf1b, we generated a Asf1b-deficient mouse line (Asf1b GT(ROSA-bgeo)437) in which expression of the lacZ reporter gene is driven by the Asf1b promoter. Analysis of b-galactosidase activity at early embryonic stages indicated a correlation between Asf1b expression and cell differentiation potential. In the gonads of both male and female, Asf1b expression was specifically detected in the germ cell lineage with a peak expression correlated with meiosis. The viability of Asf1b-null mice suggests that Asf1b is dispensable for mouse development. However, these mice showed reduced reproductive capacity compared with wild-type controls. We present evidence that the timing of meiotic entry and the subsequent gonad development are affected more severely in Asf1b-null female mice than in male mice. In female mice, in addition to subfertility related to altered gamete formation, variable defects compromising the development and/or survival of their offspring were also observed. Altogether, our data indicate the importance of Asf1b expression at the time of meiotic entry, suggesting that chromatin modifications may play a central role in this process. [477][478][479][480][481][482][483][484][485][486][487][488][489] 478 S Messiaen and others Reproduction (2016) 151 [477][478][479][480][481][482][483][484][485][486][487][488][489] www.reproduction-online.org Reproduction (2016) 151In eukaryotes, Asf1 exists either as a single gene (e.g. in yeast) or as two paralogs (Abascal et al. 2013). In mammals, these paralogs are called Asf1a and Asf1b. Due to the high sequence identity of the two ASF1 proteins, their functional redundancy or specialization has been investigated in vitro in human cell lines. Based on these studies, human ASF1A is preferentially involved in DNA repair and cell senescence, while ASF1B primarily contributes to cell proliferation (Groth et al. 2005(Groth et al. , 2007. Moreover, Asf1a-knockout mouse embryos die at mid-gestation (E9.5) (Hartford et al. 2011), indicating that Asf1b cannot compensate for the loss of Asf1a in vivo. The lack of redundancy between the Asf1 paralogs might be partially explained by their different expression patterns. In both humans and mice, Asf1a is ubiquitously expressed, whereas Asf1b expression is ...

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