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First published online 10 January 2007
doi: 10.1242/dev.02771

Development 134, 757-767 (2007)
Published by The Company of Biologists 2007
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MRG-1, an autosome-associated protein, silences X-linked genes and protects germline immortality in Caenorhabditis elegans

Teruaki Takasaki1,*, Zheng Liu2,*, Yasuaki Habara1,{dagger}, Kiyoji Nishiwaki3, Jun-ichi Nakayama3, Kunio Inoue1, Hiroshi Sakamoto1 and Susan Strome2,{ddagger}

1 Department of Biology, Graduate School of Science and Technology, Kobe University, 1-1 Rokkodaicho, Nadaku, Kobe 657-8501, Japan.
2 Department of Biology, Indiana University, Bloomington, IN 47405, USA.
3 RIKEN Center for Developmental Biology, Chuo-ku, Kobe 650-0047, Japan.


Figure 1
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  		Fig. 1. Deletion alleles of mrg-1. (A) The mrg-1 gene structure with exons (boxes), introns (joining lines), and the 3' UTR (gray). Black bars below the gene indicate regions deleted by each allele. (B) Wild-type and mrg-1(qa6200) M-Z-embryos co-stained with rabbit anti-MRG-1 antibody (red) and DAPI (blue). Scale bar: 10 µm. (C) Western blot analysis of 100 N2, mrg-1(tm1227) and mrg-1(ok1262) M+Z-adult hermaphrodites reacted with rabbit anti-MRG-1 (upper panel) and mouse anti-tubulin as a loading control (lower panel).

 

Figure 2
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  		Fig. 2. Germ cell proliferation and degeneration in mrg-1 mutants. (A) Number of germ cells in mrg-1(M-Z-) mutants. Each data point represents the average of 3-18 worms; see Table S1 in the supplementary material for values. Error bars show standard deviations. (B) Nomarski photomicrographs of live L3 larvae. (C) Germ nuclei containing GFP-tagged histone H2B. Enlarged nuclei were observed in the gonads of mrg-1 mutant and RNAi larvae (compare brackets). Scale bars: 10 µm.

 

Figure 3
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  		Fig. 3. Distribution of MRG-1 protein. (A-L) Wild-type embryos and L1 larva triply stained with DAPI (A,D,G,J), rabbit anti-MRG-1 antibody (B,E,H,K) and mouse anti-P-granule antibody K76 (C,F,I,L). (M-O) Extruded gonad co-stained with mouse PA3 to stain DNA (M and red in merge) and rabbit anti-MRG-1 (N and green in merge). Scale bars: 10 µm.

 

Figure 4
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  		Fig. 4. MRG-1 associates with autosomes independently of the MES proteins. (A-D) One-cell wild-type embryo at pronuclear meeting doubly stained with mouse PA3 to stain DNA (A) and rabbit anti-MES-4 (C) and imaged for GFP::MRG-1 (B). (D) Merged image of DNA (red) and GFP::MRG-1 (green). Arrows point to chromatin lacking both MRG-1 and MES-4. (E-M) One-cell embryos at pronuclear meeting stained with mouse PA3 (E,H,K and red in merge) and rabbit anti-MRG-1 (F,I,L and green in merge). (E-G) Wild-type embryo. (H-J) mes-2(bn76)rol-1 M-Z-embryo. (K-M) mes-4(bn73) M-Z-embryo. Arrows point to presumptive X chromosomes. (N-S) One-cell embryos at pronuclear meeting stained with mouse PA3 (N,Q and red in merge) and rabbit anti-MES-4 (O,R and green in merge). (N-P) Wild-type embryo. (Q-S) mrg-1(qa6200) M-Z-embryo. Arrows point to X chromosomes. Scale bars: 10 µm.

 

Figure 5
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  		Fig. 5. MRG-1 is not required for the appearance of elongating RNA polymerase II in early PGCs. (A-H) Embryos (~28-cell and ~100-cell) triply stained with antibodies to RNA Pol II CTD phosphorylated on Ser2 (green) and PGL-1 (red) and with DAPI (blue). In both wild-type and mrg-1(qa6200) M-Z-embryos, phosphorylated Pol II was not detected in the germline blastomere P4 (A,C), but appeared in Z2 and Z3 (E,G). Scale bar: 10 µm.

 

Figure 6
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  		Fig. 6. MRG-1 is not required for activation of transcription of at least some germline genes in PGCs. (A) RT-PCR comparison of mRNA levels in wild-type and mrg-1(qa6200) M-Z-L1 larvae. All transcript levels were normalized to ama-1 mRNA. Histograms show mean±s.e. of three independent experiments. (B-D) Expression of a paternally contributed pgl-1(+) allele in pgl-1/+ heterozygous L1s derived from mating pgl-1/pgl-1 mutant hermaphrodites, untreated (B) or treated with mrg-1 RNAi (C,D), with wild-type males. L1s were stained with anti-PGL-1. (E,F) Appearance of newly synthesized GLH-1 in wild-type and mrg-1 M-Z-L1s, seen by staining with anti-GLH-1 (green). PA3 stain of DNA is in red. Values in the upper right corners are numbers of L1s that displayed that staining pattern. Scale bar: 10 µm.

 

Figure 7
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  		Fig. 7. MRG-1 is required for several mep-1 mutant phenotypes. (A-F) L1 larvae stained with DAPI (blue) and anti-PGL-1 (red) (A-C) or anti-GLH-1 (red) (D-F). mep-1(RNAi) larvae displayed ectopic expression of PGL-1 and GLH-1 in somatic cells (B,E). Ectopic expression was reduced in mep-1(RNAi); mrg-1(RNAi) larvae (C,F). Arrowheads point to the position of PGCs. Scale bar: 50 µm. (G) RT-PCR comparison of pgl-1, glh-1, nos-1 and myo-2 mRNA levels in L1 and L2 larvae with the genotype indicated. ama-1 was used as the internal reference. Histograms show mean±s.e. of four independent experiments. (H) Percent of larvae that developed into adults after treatment of their mothers with the RNAi reagents shown. RNAi of mep-1 induced larval arrest. RNAi of mrg-1 but not dpy-5 strongly suppressed the mep-1(RNAi) larval arrest phenotype.

 

Figure 8
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  		Fig. 8. MRG-1 is required for silencing of a transgene and several X-linked genes in the germ line of mrg-1 M+Z-mutants. (A,B) Control adult hermaphrodite stained with Hoechst 33342 [as done in Garvin et al. (Garvin et al., 1998Go); B] and displaying expression of LET-858::GFP fluorescence in somatic nuclei but not in the germ line. None of 27 worms analyzed displayed GFP in the germ line. (C,D) mrg-1 (M+Z-) adult hermaphrodite stained with Hoechst 33342 (D) and displaying LET-858::GFP fluorescence in germ nuclei as well as somatic nuclei (C). Of 26 worms analyzed, 25 displayed GFP in the germ line. Brackets indicate the germ line. (E) RT-PCR comparison of mRNA levels from five X-linked and four autosomal genes in dissected wild-type and mrg-1(qa6200) M+Z-gonads. #1 and #2 show the results from two independent sets of dissected gonads. For each gene, RT-PCR was performed in triplicate. Transcript levels are relative to the reference gene ZK381.1/him-3 (Bender et al., 2006Go). The five X-linked genes are upregulated in mes-4 mutant gonads compared with wild type (Bender et al., 2006Go). Upregulation of those X-linked genes was also observed in mrg-1 gonads. The four autosomal genes are expressed at approximately equivalent levels in mes-4 and wild-type gonads (Bender et al., 2006Go). mrg-1 and wild-type gonads also accumulate similar levels of those genes.

 




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