Takasaki, Teruaki et al. (2013) International Worm Meeting "MRG-1 acts as an epigenome interpreter of Lys36 methylation on histone H3."

Sakamoto, Hiroshi, Strome, Susan, Takasaki, Teruaki, Rechtsteiner, Andreas, Egelhofer, Thea

[International Worm Meeting, 2013]

We previously identified the autosome-associated protein MRG-1 as an essential maternal factor for proper germline development, yet its molecular function remains unclear (Takasaki et al., 2007). MRG-1 contains a CHROMO domain, which is found in numerous proteins that associate with methylated histones. In our search for the element(s) that recruits MRG-1 to autosomes, we found that oocyte and sperm chromosomes arrive in the zygote marked with methylated H3K36, and that MRG-1 accumulates predominantly on those methylated chromosomes. Genome-wide chromatin immunoprecipitation analysis in early embryos revealed that methylation of H3K36 is propagated on the body of germline-expressed genes by the histone methyltransferase MES-4 in an epigenetic manner (Rechtsteiner et al., 2010; Furuhashi et al., 2010). The distribution of MRG-1 exhibits a high correlation with the distributions of both MES-4 and H3K36me, and the accumulation of MRG-1 on germline-specific genes depends on MES-4-generated H3K36me. These findings suggest that MRG-1 recognizes germline-expressed genes using the epigenetic mark H3K36me generated by MES-4. Strikingly, we found that germline-specific loci exhibit an atypical H4K16 acetylation pattern, which is dependent on MES-4. By analogy to the Drosophila homolog of MRG-1, which acts in the dosage compensation complex (the MSL complex) to generate H4K16ac on the X chromosome, it is likely that MRG-1 contributes to the germline-gene-specific H4K16ac pattern. Supporting this idea, we identified MYS-2, which is a homolog of another component of the MSL complex, as an H4K16 acetyltransferase. Furthermore, we found that loss of MYS-2, like loss of MES-4 and MRG-1, suppresses ectopic expression of germline genes in somatic cells of mep-1 mutant larvae. This suggests that H4K16 acetylation serves a pivotal role in germline development. Taken together, our results suggest that MRG-1 interprets the epigenetic H3K36me landmark and as part of an MSL-like complex generates H4K16ac specifically at germline-expressed genes.

Teruaki Takasaki et al. (2005) International Worm Meeting "MRG-1, a C.elegans chromodomain protein is essential for proper gene expression in the primordial germ cells"

Teruaki Takasaki, Kunio Inoue, Yasuaki Habara, Toshinobu Fujiwara, Kiyoji Nishiwaki, Hiroshi Sakamoto, Jun-ichi Nakayama

[International Worm Meeting, 2005]

During metazoan development, cell-type-specific gene expression is essential for cellular differentiation. In many animals, establishment of the primordial germ cell (PGC) depends on several maternal factors. However, how they regulate the germline gene expression is not fully understood. We previously reported that a novel maternal protein, MRG-1, is required for germline development in C.elegans (1). MRG-1 contains a chromodomain, which is often found in many chromatin-associated proteins, suggesting that MRG-1 is involved in chromatin remodeling in the germline. Here we show that MRG-1 is localized to nuclei and associated with chromosomes. In early embryos, MRG-1 is observed in all nuclei. In late embryos, MRG-1 staining is restricted to the nuclei of two PGCs. These observations suggest that MRG-1 has a function in chromatin remodeling in PGCs. To investigate whether MRG-1 is involved in germline gene expression in PGCs, we examined the expression of PGL-1, a component of P-glanules, which is known to be constitutively expressed in the germline. We found that mrg-1(RNAi) larvae have defects in zygotic expression of PGL-1 in PGCs. Our results suggest that MRG-1 plays an important role to ensure the proper gene expression in PGCs through chromatin remodeling. We also attempted to isolate the mrg-1 gene disruption mutants. Using a TMP/UV-based deletion mutant library, we screened 2,400,000 haploid genomes and found one candidate for the mrg-1 mutation containing 450bp deletion between exon 1 and exon 2, which may cause frameshift in the coding region. National BioResource Project (NBRP) also isolated another mrg-1 deletion mutant (2). The mutation (tm1227) deletes the 401bp region spanning the promoter region and a half of exon 1 of mrg-1. Characterization of these deletion mutants is currently in progress.(1) Fujita et al., Mech. Dev. 114, 61-69, 2002.(2) NBRP homepage, http://shigen.lab.nig.ac.jp/shigen/nbrp/nbrp.jsp

Teruaki Takasaki et al. (2010) East Asia Worm Meeting "MES-4 transmits the memory of germline gene expression to progeny via methylation of histone H3K36"

Teruaki Takasaki, William G. Kelly, Andreas Rechtsteiner, Susan Strome, Hirofumi Furuhashi, Thea A. Egelhofer, Jason D. Lieb, Sevinc Ercan

[East Asia Worm Meeting, 2010]

Germ cells transmit the genome from one generation to the next. The identity and immortality of germ cells are crucial for the perpetuation of species, yet the mechanisms that regulate these properties remain elusive. In C.elegans, the histone methyltransferase MES-4 is required for the survival of nascent germ cells in progeny. MES-4 methylates histone H3 at lysine 36 (H3K36), a modification previously linked to transcription elongation and involved in preventing aberrant transcription initiation within the body of genes. To understand how H3K36 methylation regulation contributes to germ cell immortality in progeny, we indentified the targets of MES-4 binding in embryos by genome-wide chromatin immunoprecipitation (ChIP) analysis. MES-4 overlies the coding regions of approximately 5000 genes. Although MES-4 is generally found over Pol II-bound genes, on-going transcription is neither necessary nor sufficient to recruit MES-4. Analysis of MES-4 association with genes in different temporal-spatial classes revealed that MES-4 associates with genes that were previously expressed in the maternal germ line and does not require Pol II to remain associated with those loci in embryos. In addition, genetic analysis suggests that MES-4 is predominantly responsible for maintenance, not de novo, methylation of H3K36. To our knowledge, this is the first example of transcription-uncoupled H3K36 methylation. We suggest that MES-4-generated H3K36 methylation serves an epigenetic role, by transmitting the memory of gene expression from one generation of germ cells to the next.

Teruaki Takasaki et al. (2003) International Worm Meeting "MRG-1, a member of the mortality factor-related gene family, is required for primordial germ cells to initiate mitotic proliferation"

Hiroshi Sakamoto, Teruaki Takasaki, Masaki Fujita, Noboru Nakajima, Yoshiro Shimura, Taizo Kawano

[International Worm Meeting, 2003]

MRG15, an evolutionally conserved chromodomain protein, is a member of human mortality factor 4 (MORF4)-related gene family. MORF4 is known to induce a senescence-like phenotype to a subset of immortal human cell lines such as HeLa cells. Although MRG15 is nearly identical to MORF4, it does not exhibit the senescence-inducing activity. Intriguingly, MRG15, but not MORF 4, has a chromatin organization modifier domain (chromodomain), a motif found in proteins associated with higher-order chromatin structure such as heterochromatin protein HP-1 and polycomb group (PcG) proteins. Members of MORF4-related gene family (MRGs) contain several predicted protein motifs, including a nuclear localization signal, a helix-loop-helix, and a leucine zipper. These motifs are often found in transcription factors, suggesting that MRGs may function as a transcriptional regulator. Recent studies of human MRG15 revealed that MRG15 regulates the transcription from some promoters in vitro. However the in vivo function(s) of MRG15 is still unknown. To investigate the in vivo function of MRG15, we utilized a Caenorhabditis elegans as a model organism. Here we show that MRG-1, a C. elegans homolog of MRG15, is essential for germ-line development. MRG-1 is expressed in the germ-line, and abundant in almost all cells in embryo. RNA-mediated interference (RNAi) of mrg-1 resulted in sterility caused by complete absence of germ cells. Examination of the expression of PGL-1, a component of germ-line specific P granules, revealed that two primordial germ cells (PGCs) are normally produced during embryogenesis in mrg-1(RNAi) animals, but these PGCs cannot undergo mitotic proliferation. To analyze the function of MRG-1 in post-embryonic germ-line development, we performed post-embryonic RNAi. The resultant larvae were able to produce fertile germ cells normally, but their F1 progeny lacked the germ cells. These results indicate that MRG-1 is required for PGCs to initiate mitotic proliferation, not to maintain germ cell proliferation.

Teruaki Takasaki et al. (2002) Japanese Worm Meeting "MRG-1, a mortality factor-related protein, is required maternally for primordial germ cells to initiate mitotic proliferation in C. elegans"

Masaki Fujita, Hiroshi Sakamoto, Taizo Kawano, Noboru Nakajima, Yoshiro Shimura, Teruaki Takasaki

[Japanese Worm Meeting, 2002]

In the course of study on cellular senescence, MRG15 has been identified as a human mortality factor 4 (MORF4)-related gene. Since it is highly conserved among a wide range of organisms, MRG15 is thought to have a fundamental function. Although MORF4 was known to induce a senescence-like phenotype in immortal cell lines, the function of MRG15 is still unknown. To investigate the physiological function of MRG15, we are using C. elegans as a model for multicellular organisms. RNA-mediated interference of mrg-1 , a nematode homologous gene of MRG15, resulted in a complete absence of the germline. Examination of the expression of PGL-1, a component of P granules, revealed that two primordial germ cells, Z2 and Z3, couldnt undergo mitotic proliferation in mrg-1 (RNAi) animals. It has been known that Z2 and Z3, which are generated from P4 cell at about 100-cell stage, do not divide further throughout embryogenesis. Our data suggest that MRG-1 is required for Z2 and Z3 to resume proliferation during post-embryonic development. Zygotic RNAi experiments using rde-1 , an RNAi-deficient mutant strain, demonstrated that MRG-1 functions maternally. Moreover, immunoblot analysis using mutant animals with germline deficiencies indicated that MRG-1 is expressed predominantly in oocyte. This expression pattern strongly supports that MRG-1 is a maternal protein. In immunoblot analysis, MRG-1 was clearly detected in embryo, L1 larvae and adult hermaphrodites, whereas it decreases drastically between the L2 and L4 larval stages. Recent studies in vitro, we found that MRG-1 protein is unstable in larval cell extracts. These results imply that some protease regulates the level of MRG-1 in different stages by a degradation mechanism.

Teruaki Takasaki et al. (2007) International Worm Meeting "MRG-1, an autosome-associated protein, silences X-linked genes and protects germline immortality in C. elegans."

Zheng Liu, Kiyoji Nishiwaki, Hiroshi Sakamoto, Yasuaki Habara, Susan Strome, Jun-ichi Nakayama, Teruaki Takasaki, Kunio Inoue

[International Worm Meeting, 2007]

In most sexually reproducing animals, germ cells are the only cells that can give rise to the next generation, and thus are responsible for perpetuation of species. To serve this role, germ cells must preserve the properties of totipotency and immortality. Understanding the molecular mechanisms that ensure the special properties of germ cells remains a central issue in biology. Mammalian mortality-factor-related MRG15 is required for cell proliferation and embryo survival. Our genetic analysis has revealed that the C. elegans ortholog MRG-1 serves similar roles. Maternal MRG-1 promotes embryo survival and is required for proliferation and immortality of the primordial germ cells (PGCs). As expected of a chromodomain protein, MRG-1 associates with chromatin. Unexpectedly, it is concentrated on the autosomes and not detectable on the X chromosomes. This association is not dependent on the autosome-enriched protein MES-4. Focusing on possible roles of MRG-1 in regulating gene expression, we determined that MRG-1 is required to maintain repression in the maternal germ line of transgenes on extrachromosomal arrays, and of several X-linked genes previously shown to depend on MES-4 for repression. MRG-1 is not required for PGCs to acquire transcriptional competence or for the turn-on of expression of several PGC-expressed genes (pgl-1, glh-1, glh-4 and nos-1). In contrast to this result in PGCs, MRG-1 is required for ectopic expression of those germline genes in somatic cells lacking the NuRD complex component MEP-1. We discuss how an autosome-enriched protein might repress genes on the X chromosome, promote PGC proliferation and survival, and influence the germ vs. soma distinction.

Kreher, Jeremy et al. (2015) International Worm Meeting "Transmitting an epigenetic 'memory of germline' across generations and through cell divisions in C. elegans."

Takasaki, Teruaki, Strome, Susan, Kreher, Jeremy

[International Worm Meeting, 2015]

In C. elegans, the germline is set apart from somatic lineages early and require­s the MES proteins for survival and proper development. The MES proteins are a group of chromatin regulators named for their Maternal Effect Sterile phenotype. It is well established that epigenetic mechanisms are involved in specifying and maintaining cell fates, yet it is still unclear if and how epigenetic marks are transmitted across generations and maintained through cell divisions. We are investigating the MES proteins and the histone modifications they generate to elucidate the mechanisms by which epigenetic information is transmitted from parents to progeny and to daughter chromosomes during DNA replication. We previously reported that MES-4 transmits an epigenetic 'memory of germline' from mother's germ cells to embryos, likely to guide gene expression in the primordial germ cells. MES-4 is a histone methyltransferase that catalyzes tri-methylation on Lys 36 of Histone H3 (H3K36me3), a mark associated with active transcription. Our data support a model in which MES-4 and H3K36me3 transmit a 'memory of germline gene expression' by maintaining marking in embryos of genes expressed in the maternal germline regardless of their expression status in embryos. We have used immunostaining to demonstrate that 1) H3K36me3 is delivered to embryos on the chromosomes from both the oocyte and the sperm, 2) MES-4 is delivered to embryos solely through the oocyte, 3) the recruitment of maternally supplied MES-4 to sperm chromosomes in the zygote requires that those chromosomes were marked with H3K36me3 during spermatogenesis, and 4) H3K36me3 inherited in the absence of MES-4 disappears after two rounds of DNA replication. Together, these results reveal that maternal MES-4 is required to maintain inherited H3K36me3 in early embryos. Remarkably, MES-4 maintains H3K36me3 in a gamete-of-origin manner: when H3K36me3 is inherited on the chromosomes from only one gamete, that pattern of half-marked/half-unmarked chromosomes persists through multiple rounds of cell division. Persistence of this gamete-of-origin pattern in early embryos suggests that MES-4 maintains the 'memory of germline' by propagating inherited H3K36me3 on germline-expressed genes. Our findings show that epigenetic information in the form of modified histones is transmitted to the embryo by both gametes, and is faithfully maintained through multiple rounds of DNA replication in the early embryo by maternally loaded enzyme. This epigenetic information is essential for primordial germ cell survival and development.

Strome, Susan et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Transmission and Antagonism of Germline Fate in C. elegans"

Takasaki, Teruaki, Rechtsteiner, Andreas, Gaydos, Laura, Egelhofer, Thea A, Lieb, Jason D, Petrella, Lisa, Ercan, Sevinc, Strome, Susan

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

Perpetuation of the C. elegans germ line from generation to generation depends on the MES proteins, named for their maternal-effect sterile phenotype. MES-2, MES-3, and MES-6 form the worm version of the Polycomb Repressive Complex 2 and methylate histone H3 on Lys 27 (H3K27), a modification associated with repression of gene expression. MES-4 is a homolog of the mammalian NSD proteins and methylates H3K36, a modification associated with active gene expression. Numerous studies have linked MES-2/3/6 and MES-4 to global repression of gene expression from the X chromosomes in the germ line, but have also suggested that MES-4 serves an additional critical role in early primordial germ cells (PGCs). Recent chromatin immunoprecipitation analysis of the distribution of MES-4, RNA Polymerase II, and H3K36 methyl marks across the genome in early embryos suggests that MES-4 serves a memory role. In contrast to previously studied H3K36 methyltransferases, which are targeted to genes by association with Pol II, MES-4 can associate with genes in a Pol II-independent manner. The genes that MES-4 binds and methylates in embryos are genes that were previously expressed in the maternal germline, many of which are no longer expressed in embryos. These and other findings suggest that MES-4 transmits the memory of gene expression in the parental germ line to offspring and ensures the ability of the PGCs to execute a proper germline program. Our findings raise the question of how somatic cells in the embryo, which also inherit MES-4 marking of germline genes, avoid following a germline fate. The synMuv B chromatin regulators play a key role, as their loss causes somatic cells to misexpress numerous germline-specific genes and causes larvae grown at elevated temperature to arrest. Concomitant loss of maternal MES-4 suppresses the germline potential of somatic cells and larval arrest. We will discuss the opposing roles of the MES and synMuv B proteins in the germ-soma decision.

Yamasaki, Akira et al. (2017) International Worm Meeting "Effects of Region-Specific Irradiation on Locomotion and Autophagy in C. elegans."

Zhang-Akiyama, Qiu-Mei, Suzuki, Michiyo, Kobayashi, Yasuhiko, Yamasaki, Akira, Funayama, Tomoo

[International Worm Meeting, 2017]

Ionizing radiation generates reactive oxygen species, and causes damage to cell components including DNA and protein. In C.elegans, radiation sensitivity is different from germline-cell and somatic cell. When adult C.elegans are irradiated with ten-fold higher dose than the dose that leads to germline-cell death, worms are still alive1. Locomotion using motor neurons and body-wall muscles was reduced immediately after irradiation with 0.5 kGy2. However the mechanism of reduction is not fully understood. In the present study, to investigate a tissue that is responsible for reduction of locomotion, we used region-specific microbeam irradiation. We used energetic carbon ions delivered from the AVF cyclotron at Takasaki Ion accelerators for Advanced Radiation Application facility of QST-Takasaki. After irradiation, a worm was replaced on NGM plate, and the locomotion was video-recorded and then the trajectory for 5-sec duration was derived by image processing of the movie. As a result, the same effects as whole-body irradiation were not observed after region-specific microbeam irradiation to pharynx or anterior half-body (the nerve ring, pharynx, intestine, and gonad) or posterior or posterior half-body (vulva, intestine, gonad, and tail). This suggests that the radiation effects on locomotion depend on the size of irradiation area. To detect the dose whether the reduction of locomotion is restored or leads to individual death, and we investigated alternation of locomotion after irradiation. We found the dose that locomotion of worms were completely stopped immediately after irradiation, and had been stopped at least twenty-four hours. Less than the dose, locomotion of the worms was reduced in a dose dependent manner, and was partially restored after twenty-four hours. Protein damage generated by irradiation may be involved in this reduction and restoration of locomotion, so we investigated whether autophagy is induced after irradiation. Using GFP reporter assay of lgg-1, one of the autophagosome genes, the increased level of GFP::LGG-1 was detected seven hours after irradiation in somatic cells. This suggests that autophagy may be one of the reasons of restoration of locomotion after irradiation. 1. Ishii,N., Suzuki, K., Int. J. Radiat. Biol., 58:827-833. (1990) 2. Suzuki, M., et al., J. Radiat. Res. 50, 119-125. (2009)

Suzuki, Michiyo et al. (2017) International Worm Meeting "Region specific irradiation of Caenorhabditis elegans with heavy ion microbeam."

Suzuki, Michiyo, Yokota, Yuichiro, Funayama, Tomoo

[International Worm Meeting, 2017]

Animals including humans are exposed to radiation from natural and artificial sources, cosmic rays, and nuclear accidents. Radiation may affect vital functions such as locomotion, feeding, learning. To develop effective methods to investigate the effects of radiation exposure at tissue level as well as whole body is needed. In the present study, we therefore aimed to establish a novel method for radiation-targeting of specific regions of Caenorhabditis elegans using a collimating microbeam system, thus allowing vital functions, especially locomotion, to be observed immediately after irradiation. We immobilized individual adult C. elegans in straight microfluidic channels (60 m in width) in a polydimethylsiloxane chip and subjected them to irradiation targeted to the nerve ring, mid, or tail region, respectively, using carbon ions. The ions were delivered from the AVF cyclotron at TIARA of QST-Takasaki and then were collimated using a f20 m beam exit (micro-aperture) of a collimating microbeam system. Furthermore, for wider area of the body, a f60 m micro-aperture was used. In the ventral half-body irradiation, we targeted to the f60 m semicircle area in order from head to tail and irradiated at fifteen or more times. For comparison, whole body was irradiated using a scan beam. Immediately after irradiation, we added a drop of buffer solution on a microfluidic chip and collected animals using a picker. Each animal was transferred to a NGM plate without food and the locomotion was evaluated. The effects of whole-body irradiation tended to be more effective than those of region-specific microbeam irradiation at the same dose (500 Gy). We believe that microbeam irradiation method is powerful tool for research into the cellular and molecular effects of radiation, and that the novel techniques developed will be of use in future studies.