HomeLarge Type Edition
HOME ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Services
Right arrow Download to citation manager
PubMed
Right arrow PubMed Citation
The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 60:543-548 (2005)
© 2005 The Gerontological Society of America

Functional Analysis of MRG-1: The Ortholog of Human MRG15 in Caenorhabditis elegans

Abdullah Olgun1, Tetyana Aleksenko2, Olivia M. Pereira-Smith3 and Demetrios K. Vassilatis4

1 Roy M. and Phyllis Gough Huffington Center on Aging, Baylor College of Medicine, Houston, Texas.
2 Tetyana Aleksenko is now with the Department of Neuroscience, Baylor College of Medicine, Houston, Texas 77030-3498.
3 Olivia M. Pereira-Smith is now with the Department of Cellular and Structural Biology, Sam and Ann Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, STCBM, 15355 Lambda Drive, San Antonio, Texas 78245-3207.
4 Demetrios K. Vassilatis is now with the Foundation for Biomedical Research of the Academy of Athens, Soranou Efessiou 4, 115 27 Athens, Greece.

Address correspondence to Abdullah Olgun, MD, Department of Biochemistry and Clinical Biochemistry, Gülhane School of Medicine, Etlik-06018, Ankara, Turkey. E-mail: aolgun{at}yahoo.com


   Abstract
Top
 Abstract
Method
 Results and Discussion
 References
 
Mortality Factor on Chromosome 4 (MORF4) induces senescence in several immortal human cell lines. MORF-related gene on chromosome 15 (MRG15), another expressed family member, is highly conserved and expressed in yeast to humans. To determine the biological functions of human MRG15 (hMRG15) we used RNA-mediated interference (RNAi) to silence mrg-1, the Caenorhabditis elegans ortholog, and its closest homolog Y37D8A.11. Expression of mrg-1 RNAi resulted in sterility, body wall defects, vulval protrusion, and posterior developmental defects in worms. We expressed mrg-1 under its own and the cytomegalovirus promoter in human cells. Both constructs were expressed, indicating that C. elegans promoter elements are functional in mammalian cells. Overexpression from the cytomegalovirus promoter eventually resulted in cell death, possibly due to competition with hMRG15 in endogenous nucleoprotein complexes. Recent data indicate a role for yeast and human MRG15 in transcriptional regulation via chromatin remodeling. Here we demonstrate the importance of mrg-1 in development and reproduction in C. elegans and discuss its potential to impact the aging process.

ONE of the powerful experimental approaches used to study cellular senescence has been cell fusion, which demonstrated that the senescent phenotype is dominant over immortality (1). Based on this dominance, different immortal cell lines were fused with each other and assigned to four complementation groups (A, B, C, and D) for indefinite division, indicating that there are at least four independent genetic mechanisms for senescence. The introduction of a normal human chromosome 4 by microcell transfer was shown to induce senescence in group B cells while having no effect on cell lines assigned to the other groups (2). Mortality Factor on Chromosome 4 (MORF4) was then cloned as the gene responsible for this effect. Two other expressed MORF-related gene (MRG) family members were identified on chromosomes 15 (MRG15) and X (MRGX). The structure of these genes revealed that MRGX and MORF4 are possible gene duplications of MRG15. These three genes are highly similar in the two-thirds of the C terminus containing a nuclear localization signal (NLS), helix-loop-helix motif (H-L-H), and leucine zipper (LZ). MRG15 has a chromodomain in the N-terminal region of the protein, and in MRGX this is a novel sequence. MORF4 lacks the N termini of the other proteins (3,4). MRG15 is highly conserved throughout evolution with orthologs present in yeast to humans, whereas MRGX is expressed only in vertebrates (5).

The Caenorhabditis elegans MRG family protein-1 is the ortholog of human MRG15 (hMRG15) and has moderate similarity to the human protein (27% identity, 45% similarity over 350 residues) (Figure 1). The mrg-1 gene is located on chromosome III and the complementary DNA (cDNA) predicts a protein of 337 amino acids that contains the five motifs conserved in other hMRG15 related proteins, including the drosophila male specific lethal-3 (MSL-3) proteins (6–8).



View larger version (74K):
[in this window]
[in a new window]
  		Figure 1. Comparison of human and invertebrate MORF-related genes (MRGs). The Caenorhabditis elegans Y37D8A.11 gene is also included in the comparison. A, ClustalW alignment. The identical amino acids are shaded in black; the conserved amino acids are shaded in gray. B, Phylogenetic tree of protein sequences. The invertebrate genes form a separate branch and the C. elegans Y37D8A.11 protein is the most divergent

 
Double-stranded RNA-mediated interference (RNAi) is a powerful reverse genetics technique that specifically silences gene expression (9). In this study we silenced mrg-1 using RNAi to gain insights into its biological function. We also silenced the Y37D8A.11 gene because of its similarity to mrg-1 (Figure 1).

Expression of mrg-1 RNAi caused sterility in all the worms, and some developmental defects in a small percentage of the animals. We observed no phenotype when Y37D8A.11 RNAi was used. Additionally, mrg-1 was efficiently expressed from its own promoter following transfection into mammalian cells and, when overexpressed, caused cell death possibly due to competition with hMRG15 in endogenous nucleoprotein complexes.


   METHOD
Top
 Abstract
 Method
Results and Discussion
 References
 
Wild type N2 and green fluorescent protein (GFP) transgenic worms were maintained under standard conditions (10). Young adult hermaphrodites were used for the injections. HeLa, an immortal human cell line derived from a cervical carcinoma, was used in this study. Details of cell culture conditions have been described previously (11).

The yk415f12 and yk635d7 lambda clones were provided by Yuji Kohara (National Institute of Genetics, Mishima, Japan). Amplification and in vivo excision were performed according to Lambda ZAP II Library and ZAP Express cDNA Synthesis Kit manuals obtained from Stratagene's (La Jolla, CA) web page. After confirmation by sequencing, the phagemids were linearized with Eco0109I for T3 reactions of both yk415f12 and yk635d7; and for T7 reactions with EcoRI for yk415f12 and with SpeI for yk635d7. In vitro transcription was performed using Ribomax Large Scale RNA Production Systems (Promega, Madison, WI) according to the manufacturer's instructions. Double-stranded RNA (ds-RNA) representing full-length cDNA, was obtained after mixing equal amounts of single-stranded sense and antisense RNA from T3 and T7 reactions and incubation at 68°C for 10 minutes followed by 37°C for 30 minutes. The double-strand formation was confirmed by agarose gel electrophoresis. The final concentration of ds-RNA was 1 µg/µL.

Worms expressing GFP in their body wall muscles were used for the injections. Injections were performed using a final concentration of 0.1 µg/µL ds-RNA in 10X injection solution (20% polyethylene glycol (molecular weight: 6000–8000 Da), 200 mM potassium phosphate (pH 7.5), and 30 mM potassium citrate (pH 7.5) by standard techniques. GFP ds-RNA was used as an injection control. The injected worms were transferred to a new plate the next morning, and the F1 progeny were analyzed for various phenotypes.

The pEGFP-N1 Y37D8A.9 (mrg-1 is registered as Y37D8A.9 in the genome database) construct was made to determine the localization of mrg-1 in mammalian cells and compared with the localization of human MRG15. The yk415f12 cDNA was amplified from the phagemid with high fidelity polymerase chain reaction (PCR) using the primers CEMrg5' (5'-CCGGAATTCGCCACCATGTCTTCAAAGAAGAACTTCGAAGTCGGCG-3') and CEMrg3' ('-CGCGGATCCCGCTATTGATTCGCTCCAACTCCGTC-3') and gel purified. The PCR product and the pEGFP-N1 vector were cut with EcoRI and BamHI, gel purified, and ligated.

The pPD95.79 Y37D8A.9 was constructed to check the expression pattern of mrg-1 in C. elegans. The pPD95.79 vector is a promoterless GFP fusion vector from the Andrew Fire Laboratory (Carnegie Institution, Washington, DC). The fragment covering 54604 -> 59061 (reverse complement) region in the y37d8a cosmid was PCR cloned with a PCR-XL-TOPO kit (Stratagene) using primers CEMrgene5' (5'-TTCCCCCCGGGGGATGGAACAGCCGTTACGATGAG-3') and CEMrgene3' (5'-AAACGTACGCGCTATTGATTCGCTCCAACCCGTCG-3') to obtain the PCR-XL-TOPO-Y37D8A.9 construct. This cloned region contains both the promoter/regulatory and entire coding sequence of the Y37D8A.10 gene (between 58117 and 58713) which is read in the reverse direction of mrg-1. The start codon is at position 57740, and the coding region ends at position 54604. All four exons and three introns are present, and the region from 59061 to 57740 includes the promoter/regulatory region. This region also contains approximately one third of the last exon of the Y37D8A.11 gene, which is read in the same direction as mrg-1. We specifically included all of the coding region of mrg-1 because it might have included some regulatory sequences in the intron. PCR-XL-TOPO-Y37D8A.9 was cut with BsiWI and XmaI; pPD95.79 was cut with Acc65I and XmaI. The cut vector and the fragment were gel purified and ligated to obtain pPD95.79 Y37D8A.9, and the sequence was confirmed. pPD95.79 y37d8a.9 (final concentration, 60 ng/µL) was injected into wild type N2 worms, and pRF4 (rol-6) DNA (final concentration, 80 ng/µL) was injected as a control. Human cells were transfected using LipofectAMINE Plus transfection reagent (Stratagene) according to the manufacturer's instructions.


   RESULTS AND DISCUSSION
Top
 Abstract
 Method
 Results and Discussion
References
 
We silenced both the mrg-1 and Y37D8A.11 genes independently by injecting ds-RNA into the distal gonads of hermaphrodite worms expressing GFP. The rationale for silencing Y37D8A.11 in addition to mrg-1 was because of their high similarity (44% identity and 60% similarity over 134 residues) (8); also, we wanted to ensure that the phenotype observed with the mrg-1 RNAi was specific. We used GFP RNAi as a positive control for the injections and observed that GFP expression was silenced in the body wall muscle cells (Figure 2a).



View larger version (104K):
[in this window]
[in a new window]
  		Figure 2. RNA-mediated interference (RNAi) with whole length mrg-1 complementary DNA. a, Green fluorescence protein RNAi as the injection control; b, sterile worms with empty gonads; c, leaky worms characterized with body wall defects causing the leakage of body contents; d, vulval protrusion; e, posterior defects characterized with short and blunt tail; and f, normal worm. Arrows point to the phenotypes

 
We analyzed all the F1 progeny from five independent experiments in each of which approximately 15 worms were injected with RNAi and the same number served as controls. The silencing of mrg-1 resulted in sterility in 100% of the F1 progeny, and the adult hermaphrodites completely lacked eggs (Figure 2b). This finding is in accordance with the published RNAi phenotype of mrg-1 described by Fujita and colleagues (6), who reported that mrg-1 is required for mitotic division of primordial germ cells (PGCs) Z2 and Z3 and that silencing causes complete loss of the germline in both hermaphrodites and males. No significant differences in growth rate, size, and life span (15–20 days) were observed in the RNAi injected worms compared to controls (data not shown). However, unlike Fujita and colleagues (6), in a small percentage of animals we observed additional deformities, such as loss of body wall integrity, that resulted in the leakage of the body contents (10%–20%) (Figure 2c), vulval protrusion (10%–20%) (Figure 2d), and a posterior developmental defect that caused a blunt and shortened tail (<5%) (Figure 2e). The fact that we did not observe these phenotypes in all the animals was most likely due to differences in permeability to ds-RNA of the various cell types involved. The silencing of the Y37D8A.11 gene caused no detectable defects, and when both genes were silenced together, the resulting phenotype was the same as that observed with mrg-1 RNAi alone.

Injection of pPD95.79 Y37D8A.9 (a GFP tagged-mrg-1 construct under the control of its own promoter) was performed to allow us to determine the expression pattern of the injected DNA, but we were unable to detect expression of this construct and it is possible this was a result of germ line silencing. Determination of the expression pattern might therefore require other approaches such as in situ hybridization or immunostaining. Fujita and colleagues (6) reported the localization of mrg-1 to oocytes by immunostaining. However, the antibody used cross-reacts with another protein of 55 kD in Western blot analysis of whole worm lysate, making this interpretation difficult. This antibody most likely recognizes the protein produced by the gene Y37D8A.11, which is predicted to have a molecular weight of 54.8 kD. It will be necessary to develop a new antibody which reacts only with mrg-1 to definitively answer questions of expression in specific cells.

We cloned mrg-1 into the pEGFP-N1 mammalian expression vector to analyze its localization and function in human HeLa cells and to compare this with the pattern observed with hMRG15 (3). After transfection with pEGFP-N1 MRG15 and pEGFP-N1 Y37D8A.9, HeLa cells were analyzed by fluorescent and confocal microscopy and hMRG15-GFP was found to be localized in the nucleus, whereas mrg-1-GFP was present in both the nucleus and cytoplasm (Figure 3). Almost all the cells that were expressing high levels of mrg-1 detached from the surface of the plate and died starting at 24 hours post transfection. This could be due to competition of mrg-1 with hMRG15 in endogenous nucleoprotein complexes. In the case of pPD95.79 Y37D8A.9, in which MRG1 was expressed from its own promoter, transfection into HeLa cells resulted in lower levels of expression, but the localization was similar to that observed with pEGFP-N1 Y37D8A.9 (Figure 2). No significant cell death was observed most likely because of the lower levels of expression obtained with this construct. This result indicates that the promoter elements of the nematode mrg-1 can function in human cells. However, the possible presence of cryptic promoters in the vector that may become active in HeLa cells and permit expression cannot be excluded.



View larger version (43K):
[in this window]
[in a new window]
  		Figure 3. HeLa cells transfected with pEGFP-N1-MRG15, pEGFP-N1-Y37D8A.9, pEGFP-N1 (empty vector), and pPD95.79-Y37D8A.9 were analyzed with fluorescent and confocal microscope. Human MORF-related gene on chromosome 15 (MRG15) is localized in the nucleus, mrg-1 is localized in both nucleus and cytoplasm. The mrg-1 gene expressed by its own promoter in HeLa cells has a similar localization to pEGFP-N1-Y37D8A.9. *The nucleus is not visible with 4'6-diamidino-2-phenylindole (DAPI). {dagger}This cell is different from the cell in the left three rows. {ddagger}This picture was obtained with DAPI + green fluorescent protein (GFP) filter

 
It has been reported that a cluster of 189 genes, including Y37D8A.9, are coregulated during the development of C. elegans. These genes are expressed primarily in oocytes very early in development (0–6 hours) and are involved in oogenesis, mitosis and meiosis, chromosome chromatin structure modulation (e.g., isomerases, helicases), RNA association, DNA binding, and cell cycle regulation. The probability of expression of this group of genes was found to be higher than that expected from random selection of this cluster from the genome as a whole (12).

Recently, it was shown that MRG15 is a component of the TIP60 histone acetyl transferase complex, which is implicated in DNA repair and apoptosis as well as transcriptional control in human cells (13–15). It is interesting that a homologous complex (NuA4) exists in yeast and that this complex contains Esa1, a HAT with high homology to TIP60, as well as a MRG15 ortholog (15,16). Esa1 is the only essential HAT in yeast and is involved in many processes including cell cycle progression. The high conservation of this MRG15-containing complex in yeast and humans, together with the fact that it regulates some gene promoters by interaction with other proteins such as Rb and PAM14, suggests that MRG15 plays a role in fundamental cellular processes.

Our results demonstrate that mrg-1 is required for oogenesis and aspects of development of C. elegans that include body wall, vulva, and tail development. Some of these phenotypes were not previously observed, probably because of incomplete RNAi penetrance. Nevertheless, they are not the result of partial silencing of Y37D8A.11, the closest homolog of mrg-1, because RNAi injections directed to this gene revealed no detectable defects. Generation of strains that completely lack mrg-1 due to the deletion of the gene will provide definitive answers regarding the role of mrg-1 in development, because the conserved function of mrg-1 and MRG15 extends to nuclear localization, as demonstrated in HeLa cell expression experiments, which also suggest the presence of conserved cis requlatory elements across species.

The question that is of greatest interest to gerontologists is the role of MRG15 in the process of aging. We performed a knockout of the gene in Saccharomyces cerevisiae and determined life span by mother–daughter analyses (with M. Jazwinski, Louisiana State University) and continuous replating experiments (with V. Lundblad, Baylor College of Medicine), and observed no effects (unpublished data). Subsequent experiments analyzing phenotypes in general also revealed no striking effect of deletion of MRG15 in S. cerevisiae (17). Similarly, a knockout in Schizosaccharomyces pombe (done by us with S. Sazer, Baylor College of Medicine) revealed no phenotype (unpublished data), although another group using a different strain reported progressive loss of viability and increased sensitivity to DNA-damaging agents following inactivation of Alp13 (MRG15) (18). No life-span studies were performed in the latter study. However, P element mutants in dmrg15 in the drosophila data base are described as lethal and require balancer chromosomes for survival. Similarly, we have found that inactivation of the gene is lethal in fetal mice and that MRG15-null mouse embryo fibroblasts proliferate poorly when compared with those of wild type mice (20). One could argue that the effects in all cases impact only developmental processes. However, the defects observed are not caused by proliferation alone but involve abberant expression of genes related to differentiation and DNA repair. The life span of heterozygous mice and flies should therefore be of interest. It could be argued that the heterozygous mice genetically modified in the manganese superoxide dismutase gene had life spans similar to those of wild type mice (19). However, this is a gene involved in the very specific pathway involving oxidative damage. MRG15, in contrast, is a highly multifunctional gene that affects transcription of other genes involved in multiple pathways, via multiple nucleoprotein complexes, some of which act by chromatin remodeling. We have preliminary analyses of blood cells from null embryos, which appear anemic compared with wild type and heterozygotic embryos; this finding indicates that there are many abnormal cells in nulls and intermediate numbers in heterozygotes when compared with wild type embryos (Robetorye et al., unpublished data). If hemizygous expression of MRG15 impacts the function of multiple organ systems and tissues, as one would predict from these analyses of null embryos, partial loss of MRG15 expression has the potential to affect life span.


   Acknowledgments
 
This work was supported by National Institutes of Health grant PO1AG20752 and by the Ellison Medical Foundation (to O.M.P.-S.), and by a Turkish Government grant (to A. Olgun).

We thank the members of the Vassilatis Laboratory for helping with the maintenance and injections of worms, Dr. Kaoru Tominaga for helping with the production of the constructs, Dr. Anna P. Newman for allowing use of her laboratory for the injections, Dr. Pam Larsen for critical reading of the manuscript, and Dr. Yuji Kohara for providing yk415f12 and yk635d7 cDNA clones.


   Footnotes
 
Decision Editor: James R. Smith, PhD

Received September 7, 2004

Accepted December 15, 2004


   References
Top
 Abstract
 Method
 Results and Discussion
 References
 

  1. Pereira-Smith OM, Smith JR. Evidence for the recessive nature of cellular immortality. Science. 1983;221:964-966.[Abstract/Free Full Text]
  2. Tominaga K, Olgun A, Smith JR, Pereira-Smith OM. Genetics of cellular senescence. Mech Ageing Dev. 2002;123:927-936.[Medline]
  3. Bertram MJ, Berube NG, Hang-Swanson X, et al. Identification of a gene that reverses the immortal phenotype of a subset of cells and is a member of a novel family of transcription factor-like genes. Mol Cell Biol. 1999;19:1479-1485.[Abstract/Free Full Text]
  4. Ran Q, Pereira-Smith OM. Genetic approaches to the study of replicative senescence. Exp Gerontol. 2000;35:7-13.[Medline]
  5. Koonin EV, Zhou S, Lucchesi JC. The chromo superfamily: new members, duplication of the chromo domain and possible role in delivering transcription regulators to chromatin. Nucleic Acids Res. 1995;23:4229-4233.[Abstract/Free Full Text]
  6. Fujita M, Takasaki T, Nakajima N, Kawano T, Shimura Y, Sakamoto H. mrg-1, a mortality factor-related chromodomain protein, is required maternally for primordial germ cells to initiate mitotic proliferation in C. elegans. Mech Dev. 2002;114:61-69.[Medline]
  7. Bertram MJ, Pereira-Smith OM. Conservation of the MORF4 related gene family: identification of a new chromo domain subfamily and novel protein motif. Gene. 2001;266:111-121.[Medline]
  8. Costanzo MC, Hogan JD, Cusick ME, et al. The yeast proteome database (YPD) and Caenorhabditis elegans proteome database (WormPD): comprehensive resources for the organization and comparison of model organism protein information. Nucleic Acids Res. 2000;28:73-76.[Abstract/Free Full Text]
  9. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998;391:806-811.[Medline]
  10. Brenner S. The genetics of Caenorhabditis elegans. Genetics. 1974;77:71-94.[Abstract/Free Full Text]
  11. Pereira-Smith OM, Smith JR. Phenotype of low proliferative potential is dominant in hybrids of normal human fibroblasts. Somatic Cell Genet. 1982;8:731-742.[Medline]
  12. Hill AA, Hunter CP, Tsung BT, Tucker-Kellogg G, Brown EL. Genomic analysis of gene expression in C. elegans. Science. 2000;290:809-812.[Abstract/Free Full Text]
  13. Doyon Y, Selleck W, Lane WS, Tan S, Cote J. Structural and functional conservation of the NuA4 histone acetyltransferase complex from yeast to humans. Mol Cell Biol. 2004;24:1884-1896.[Abstract/Free Full Text]
  14. Cai Y, Jin J, Tomomori-Sato C, et al. Identification of new subunits of the multiprotein mammalian TRRAP/TIP60-containing histone acetyltransferase complex. J Biol Chem. 2003;278:42733-42736.[Abstract/Free Full Text]
  15. Eisen A, Utley RT, Nourani A, et al. The yeast NuA4 and Drosophila MSL complexes contain homologous subunits important for transcription regulation. J Biol Chem. 2001;276:3484-3491.[Abstract/Free Full Text]
  16. Doyon Y, Cote J. The highly conserved and multifunctional NuA4 HAT complex. Curr Opin Genet Dev. 2004;14:147-154.[Medline]
  17. Eisen A, Utley RT, Nourani A, et al. The yeast NuA4 and Drosophila MSL complexes contain homologous subunits important for transcription regulation. J Biol Chem. 2001;276:3484-3491.
  18. Nakayama J, Xiao G, Noma K, et al. Alp13, an MRG family protein, is a component of fission yeast Clr6 histone deacetylase required for genomic integrity. EMBO J. 2003;22:2776-2787.[Medline]
  19. Van Remmen H, Ikeno Y, Hamilton M, et al. Life-long reduction in MnSOD activity results in increased DNA damage and higher incidence of cancer but does not accelerate aging. Physiol Genomics. 2003;16:29-37.[Abstract/Free Full Text]
  20. Tominaga K, Kirtane B, Jackson JG, et al.. Mol Cell Biol. 2005;25:2924-2937.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Services
Right arrow Download to citation manager
PubMed
Right arrow PubMed Citation

HOME ARCHIVE SEARCH TABLE OF CONTENTS