Claudia Gebert

Extracellular annexins and dynamin are important for sequential steps in myoblast fusion

Annexins A1 and A5 are important for initial lipid mixing, whereas subsequent stages of myoblast fusion depend on dynamin, phosphatidylinositol(4,5)bisphosphate, and cellular metabolism.

DNA methylation in the IGF2 intragenic DMR is re-established in a sex-specific manner in bovine blastocysts after somatic cloning

The recent identification of an intragenic differentially methylated region (DMR) within the last exon of the bovine Insulin-like growth factor 2 (IGF2) gene provides a diagnostic tool for in-depth investigation of bovine imprinting and regulatory mechanisms which are active during embryo development. Here, we used bisulfite sequencing to compare sex-specific DNA methylation patterns within this DMR in bovine blastocysts produced in vivo, by in vitro fertilization and culture, SCNT, androgenesis or parthenogenesis. In in vivo derived embryos, DNA methylation was removed from this intragenic DMR after fertilization, but partially replaced by the time the embryo reached the blastocyst stage. Among embryos developing in vivo, the level of DNA methylation was significantly lower in female than in male blastocysts. This sexual dimorphism was also found between parthenogenetic and androgenetic embryos, and followed the donor cell sex in SCNT derived blastocysts and is evidence for correct methylation reprogramming in SCNT embryos.

H19 Imprinting Control Region Methylation Requires an Imprinted Environment Only in the Male Germ Line

ABSTRACT The 2.4-kb H19 imprinting control region (H19ICR) is required to establish parent-of-origin-specific epigenetic marks and expression patterns at the Igf2/H19 locus. H19ICR activity is regulated by DNA methylation. The ICR is methylated in sperm but not in oocytes, and this paternal chromosome-specific methylation is maintained throughout development. We recently showed that the H19ICR can work as an ICR even when inserted into the normally nonimprinted alpha fetoprotein locus. Paternal but not maternal copies of the ICR become methylated in somatic tissue. However, the ectopic ICR remains unmethylated in sperm. To extend these findings and investigate the mechanisms that lead to methylation of the H19ICR in the male germ line, we characterized novel mouse knock-in lines. Our data confirm that the 2.4-kb element is an autonomously acting ICR whose function is not dependent on germ line methylation. Ectopic ICRs become methylated in the male germ line, but the timing of methylation is influenced by the insertion site and by additional genetic information. Our results support the idea that DNA methylation is not the primary genomic imprint and that the H19ICR insertion is sufficient to transmit parent-of-origin-dependent DNA methylation patterns independent of its methylation status in sperm.

Promoter cross-talk via a shared enhancer explains paternally biased expression of Nctc1 at the Igf2/H19/Nctc1 imprinted locus

Developmentally regulated transcription often depends on physical interactions between distal enhancers and their cognate promoters. Recent genomic analyses suggest that promoter–promoter interactions might play a similarly critical role in organizing the genome and establishing cell-type-specific gene expression. The Igf2/H19 locus has been a valuable model for clarifying the role of long-range interactions between cis-regulatory elements. Imprinted expression of the linked, reciprocally imprinted genes is explained by parent-of-origin-specific chromosomal loop structures between the paternal Igf2 or maternal H19 promoters and their shared tissue-specific enhancer elements. Here, we further analyze these loop structures for their composition and their impact on expression of the linked long non-coding RNA, Nctc1. We show that Nctc1 is co-regulated with Igf2 and H19 and physically interacts with the shared muscle enhancer. In fact, all three co-regulated genes have the potential to interact not only with the shared enhancer but also with each other via their enhancer interactions. Furthermore, developmental and genetic analyses indicate functional significance for these promoter–promoter interactions. Altogether, we present a novel mechanism to explain developmental specific imprinting of Nctc1 and provide new information about enhancer mechanisms and about the role of chromatin domains in establishing gene expression patterns.

Annexin A1 Deficiency does not Affect Myofiber Repair but Delays Regeneration of Injured Muscles.

Repair and regeneration of the injured skeletal myofiber involves fusion of intracellular vesicles with sarcolemma and fusion of the muscle progenitor cells respectively. In vitro experiments have identified involvement of Annexin A1 (Anx A1) in both these fusion processes. To determine if Anx A1 contributes to these processes during muscle repair in vivo, we have assessed muscle growth and repair in Anx A1-deficient mouse (AnxA1−/−). We found that the lack of Anx A1 does not affect the muscle size and repair of myofibers following focal sarcolemmal injury and lengthening contraction injury. However, the lack of Anx A1 delayed muscle regeneration after notexin-induced injury. This delay in muscle regeneration was not caused by a slowdown in proliferation and differentiation of satellite cells. Instead, lack of Anx A1 lowered the proportion of differentiating myoblasts that managed to fuse with the injured myofibers by days 5 and 7 after notexin injury as compared to the wild type (w.t.) mice. Despite this early slowdown in fusion of Anx A1−/− myoblasts, regeneration caught up at later times post injury. These results establish in vivo role of Anx A1 in cell fusion required for myofiber regeneration and not in intracellular vesicle fusion needed for repair of myofiber sarcolemma.

Cell-surface phosphatidylserine regulates osteoclast precursor fusion

Bone-resorbing multinucleated osteoclasts that play a central role in the maintenance and repair of our bones are formed from bone marrow myeloid progenitor cells by a complex differentiation process that culminates in fusion of mononuclear osteoclast precursors. In this study, we uncoupled the cell fusion step from both pre-fusion stages of osteoclastogenic differentiation and the post-fusion expansion of the nascent fusion connections. We accumulated ready-to-fuse cells in the presence of the fusion inhibitor lysophosphatidylcholine and then removed the inhibitor to study synchronized cell fusion. We found that osteoclast fusion required the dendrocyte-expressed seven transmembrane protein (DC-STAMP)-dependent non-apoptotic exposure of phosphatidylserine at the surface of fusion-committed cells. Fusion also depended on extracellular annexins, phosphatidylserine-binding proteins, which, along with annexin-binding protein S100A4, regulated fusogenic activity of syncytin 1. Thus, in contrast to fusion processes mediated by a single protein, such as epithelial cell fusion in Caenorhabditis elegans, the cell fusion step in osteoclastogenesis is controlled by phosphatidylserine-regulated activity of several proteins.

H19ICR mediated transcriptional silencing does not require target promoter methylation

Transcription of the reciprocally imprinted genes Insulin-like growth factor 2 (Igf2) and H19 is orchestrated by the 2.4-kb H19 Imprinting Control Region (H19ICR) located upstream of H19. Three known functions are associated with the H19ICR: (1) it is a germline differentially methylated region, (2) it is a transcriptional insulator, and (3) it is a transcriptional silencer. The molecular mechanisms of the DMR and insulator functions have been well characterized but the basis for the ICRs silencer function is less well understood. In order to study the role the H19ICR intrinsically plays in gene silencing, we transferred the 2.4-kb H19ICR to a heterologous non-imprinted location on chromosome 5, upstream of the alpha fetoprotein (Afp) promoter. Independent of its orientation, the 2.4-kb H19ICR silences transcription from the paternal Afp promoter. Thus silencing is a function intrinsic to this DNA element. Further, ICR mediated silencing is a developmental process that, unexpectedly, does not occur through DNA methylation at the target promoter.

Chromosome choice for initiation of V–(D)–J recombination is not governed by genomic imprinting

V–(D)–J recombination generates the antigen receptor diversity necessary for immune cell function, while allelic exclusion ensures that each cell expresses a single antigen receptor. V–(D)–J recombination of the Ig, Tcrb, Tcrg and Tcrd antigen receptor genes is ordered and sequential so that only one allele generates a productive rearrangement. The mechanism controlling sequential rearrangement of antigen receptor genes, in particular how only one allele is selected to initiate recombination while at least temporarily leaving the other intact, remains unresolved. Genomic imprinting, a widespread phenomenon wherein maternal or paternal allele inheritance determines allele activity, could represent a regulatory mechanism for controlling sequential V–(D)–J rearrangement. We used strain‐specific single‐nucleotide polymorphisms within antigen receptor genes to determine if maternal vs paternal inheritance could underlie chromosomal choice for the initiation of recombination. We found no parental chromosomal bias in the initiation of V–(D)–J recombination in T or B cells, eliminating genomic imprinting as a potential regulator for this tightly regulated process.

234 IMPRINTING STATUS OF DEVELOPMENTALLY IMPORTANT GENES INBOVINE PREIMPLANTATION EMBRYOS

Several imprinted genes have been identified in different species such as mouse and man but in cattle imprinting has only been confirmed for the Insulin-like growth factor 2 receptor gene (Igf2r). DNA methylation is one of the most common mechanisms of imprinting. Imprinting is correlated with the methylation of normally unmethylated CpG islands, and aberrations in methylation are thought to be involved in the Large Offspring Syndrome (LOS) which is frequently observed in offspring derived from in vitro-produced and/or cloned embryos. The imprinting of the bovine Insulin-like growth factor 2 gene (Igf2), the Igf2r gene and the Mammalian achaete scute homologue 2 gene (Mash2) was analyzed in bovine preimplantation blastocysts cultured in SOF + BSA. mRNA levels of the Igf2r gene and the Mash2 gene in in vitro-produced (IVP) and parthenogenetic expanded single blastocysts were analyzed by semi-quantitative RT-PCR (Wrenzycki C et al., 2001 Biol. Reprod. 65, 309–317). Genes expressed exclusively from the maternal allele (i.e. paternally methylated) should be represented by a higher relative abundance of transcriptional products in parthenogenetic embryos whereas paternally expressed genes (i.e. maternally methylated) should be correlated with a higher gene expression in IVP embryos carrying one paternal and one maternal allele. For determination of methylation patterns by bisulfite sequencing, sequences of several DNA fragments from the bovine Igf2 gene were identified in samples from bovine uterus and kidney. The primer pairs were generated from the ovine and bovine Igf2 sequences available in the Genebank database (accession number U00664/X53553). DNA fragments identified were from the 5′ untranslated region and the 3′ translated region of the bovine Igf2 gene. All fragments consist of a high number of CG dinucleotides, and computer analysis using the CpGwin program (Anbazhagan R et al., 2001 BioTechniques 30, 110–114) revealed CpG islands within these fragments. Relative abundances of transcriptional products were statistically analyzed using the SigmaStat 2.0 (Jandel Scientific, San Rafael, CA, USA) software package. After testing for normality, an ANOVA followed by multiple pairwise comparisons using the Tukey test was employed. Results from the semi-quantitative RT-PCR analyses revealed no difference (P > 0.05) in gene expression pattern of the Igf2r gene, suggesting that the Igf2r gene is biallelically expressed during bovine preimplantation development. In contrast, there was a significant difference (P < 0.05) in the relative abundance of mRNA of the Mash2 gene, with a lower relative abundance of transcriptional products in parthenogenetic expanded blastocysts. Thus, in cattle as reported in mice (Guillemot F et al., 1995 Nat. Genet. 9, 235–242), the paternal allele of the Mash2 gene seems to participate in the expression of the gene prior to implantation.