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1.  MRG15 activates the cdc2 promoter via histone acetylation in human cells 
Experimental cell research  2011;317(11):1534-1540.
Chromatin remodeling is required for transcriptional activation and repression. MRG15 (MORF4L1), a chromatin modulator, is a highly conserved protein and is present in complexes containing histone acetyltransferases (HATs) as well as histone deacetylases (HDACs). Loss of expression of MRG15 in mice and Drosophila results in embryonic lethality and fibroblast and neural stem/progenitor cells cultured from Mrg15 null mouse embryos exhibit marked proliferative defects when compared with wild type cells. To determine the role of MRG15 in cell cycle progression we performed chromatin immunoprecipitation with an antibody to MRG15 on normal human fibroblasts as they entered the cell cycle from a quiescent state, and analyzed various cell cycle gene promoters. The results demonstrated a 3-fold increase in MRG15 occupancy at the cdc2 promoter during S phase of the cell cycle and a concomitant increase in acetylated histone H4. H4 lysine 12 was acetylated at 24 hours post serum stimulation while there was no change in acetylation of lysine 16. HDAC1 and 2 were decreased at this promoter during cell cycle progression. Over-expression of MRG15 in HeLa cells activated a cdc2 promoter-reporter construct in a dose dependent manner, whereas knockdown of MRG15 resulted in decreased promoter activity. In order to implicate HAT activity, we treated cells with the HAT inhibitor anacardic acid and determined that HAT inhibition results in loss of expression of cdc2 mRNA. Further, chromatin immunoprecipitation with Tip60 localizes the protein to the same 110 bp stretch of the cdc2 promoter pulled down by MRG15. Additionally, we determined that co-transfection of MRG15 with the known associated HAT Tip60 had a cooperative effect in activating the cdc2 promoter. These results suggest that MRG15 is acting in a HAT complex involving Tip60 to modify chromatin via acetylation of histone H4 at the cdc2 promoter to activate transcription.
PMCID: PMC3410549  PMID: 21324423
MRG15; Tip60; cdc2; normal human fibroblasts
2.  MrgX Is Not Essential for Cell Growth and Development in the Mouse 
Molecular and Cellular Biology  2005;25(12):4873-4880.
MRGX is one of the members of MORF4/MRG family of transcriptional regulators, which are involved in cell growth regulation and cellular senescence. We have shown that MRGX and MRG15 associate with Rb in nucleoprotein complexes and regulate B-myb promoter activity. To elucidate the functions of MRGX and to explore its potential role in modulating cell growth in vivo, we have generated MrgX-deficient mice. Characterization of the expression pattern of mouse MrgX demonstrated it was ubiquitously expressed in all tissues of adult mice and also during embryogenesis and overlapped with its homolog Mrg15. MRGX and MRG15 proteins localize predominantly to the chromatin fraction in the nucleus, although a small amount of both proteins localized to the nuclear matrix. Whereas disruption of Mrg15 results in embryonic lethality, absence of MrgX did not impair mouse development and MrgX null mice are healthy and fertile. MrgX-deficient and wild-type mouse embryonic fibroblasts (MEFs) also had similar growth rates and showed no differences in cell cycle-related gene expression in response to serum stimulation. Mrg15 expression in MrgX-deficient tissues and MEFs was not upregulated compared with wild-type tissues and MEFs. MRG15 is highly conserved with orthologs present from humans to yeast and is essential for survival of mice. In contrast, MRGX, which evolved later, is expressed only in vertebrates, suggesting that the lack of phenotype of MrgX-deficient mice is secondary to a compensatory effect by the evolutionarily conserved MRG15 protein but not vice versa.
PMCID: PMC1140578  PMID: 15923606
3.  The Emerging Role of the MORF/MRG Gene Family in Various Biological Processes Including Aging 
Cellular senescence is the dominant phenotype over immortality. In our studies to identify senescence related genes we cloned Morf4 that induced senescence in a subset of tumor cells. Morf4 is a member of a family of 7 genes, and the Morf related genes (Mrg) on chromosomes 15 (Mrg15) and X (MrgX) are also expressed. In contrast to MORF4, MRG15 and MRGX are positive regulators of cell division. All three proteins interact with histone acetylases (HATs) and acetyltransferase (HDACs), suggesting they function in regulation of chromatin dynamics. Mrg15 knockout mice are embryonic lethal, and MEFs derived from Mrg15 null embryos proliferate poorly, enter senescence rapidly and have impaired DNA repair compared to wild type. Mrg15 null embryonic neural stem/progenitor cells also have a decreased capacity for proliferation and differentiation. Further studies are needed to determine the function of this gene family in various biological processes including neural stem/progenitor cell aging.
PMCID: PMC2918256  PMID: 20536842
cellular senescence; Mrg (Morf related genes); MRG15 (Morf related gene on chromosome 15); chromatin remodelling; neural stem/progenitor cells; proliferation; differentiation; DNA damage; aging
4.  Conditional inactivation of MRG15 gene function limits survival during larval and adult stages of Drosophila melanogaster 
Experimental gerontology  2010;45(11):825-833.
The mammalian MRG15 gene encodes a chromodomain protein predicted to bind to chromatin via methylated histone tails. Human MORF4 encodes a related but truncated protein that is capable of promoting cellular senescence in a subset of human tumor cell lines. Drosophila contains a single homolog of human MRG15, called DmMRG15. Null mutation of MRG15 is embryonic lethal in mice and Drosophila, making study of MRG15 requirements in adults difficult. In these studies the DmMRG15 gene was over-expressed in Drosophila, during developmental stages and in adults, using a doxycycline-regulated system (Tet-on). In addition an inverted-repeated construct was designed to inactivate DmMRG15 via the RNAi pathway, and RNAi constructs were expressed using both the Tet-on system and Geneswitch system. The DmMRG15 protein was readily expressed in adult flies in a doxycycline-dependent manner. A truncated form of DmMRG15 (called DmMT1) was designed to mimic the structure of human MORF4, and expression of this mutant protein or the inverted repeat constructs inhibited fertility in females. Conditional expression of the DmMRG15 inverted-repeat constructs during larval development or in adults caused reductions in survival. These experiments indicate that Drosophila DmMRG15 gene function is required for female fertility, larval survival and adult life span, and provide reagents that should be useful for further dissecting the role of DmMRG15 in cell proliferation and aging.
PMCID: PMC2952352  PMID: 20600782
senescence; chromatin; epigenetics; stem cells; aging
5.  MRG15, a component of HAT and HDAC complexes, is essential for proliferation and differentiation of neural precursor cells 
Journal of neuroscience research  2009;87(7):1522-1531.
Neurogenesis during development depends on the coordinated regulation of self-renewal and differentiation of neural precursor cells. Chromatin regulation is a key step in self-renewal activity and fate decision of neural precursor cells. However, the molecular mechanism(s) of this regulation is not fully understood. Here, we demonstrate for the first time that MRG15, a chromatin regulator, is important for proliferation and neural fate decision of neural precursor cells. Neuroepithelia from Mrg15 deficient embryonic brain are much thinner than those from control, and apoptotic cells increase in this region. We isolated neural precursor cells from Mrg15 deficient and wild-type embryonic whole brains and produced neurospheres to measure the self-renewal and differentiation abilities of these cells in vitro. Neurospheres culture from Mrg15 deficient embryo grew less-efficiently than those from wild-type. Measurement of proliferation, using BrdU incorporation, revealed that Mrg15 deficient neural precursor cells have reduced proliferation ability and apoptotic cells do not increase during in vitro culture. The reduced proliferation of Mrg15 deficient neural precursor cells most likely accounts for the thinner neuroepithelia in Mrg15 deficient embryonic brain. Moreover, we also demonstrate Mrg15 deficient neural precursor cells are defective in differentiation into neurons in vitro. Our results demonstrate that MRG15 has more than one function in neurogenesis and defines a novel role for this chromatin regulator that integrates proliferation and cell-fate determination in neurogenesis during development.
PMCID: PMC2913448  PMID: 19115414
Neural precursor cell; development; chromatin; epigenetics; gene expression
6.  Mrg15 null and heterozygous mouse embryonic fibroblasts exhibit DNA repair defects post exposure to gamma ionizing radiation 
FEBS letters  2007;581(27):5275-5281.
MRG15 is a core component of the NuA4/Tip60 histone acetyltransferase complex that modifies chromatin structure. We here demonstrate that Mrg15 null and heterozygous mouse embryonic fibroblasts exhibit an impaired DNA damage response post gamma irradiation, when compared to wild-type cells. Defects in DNA repair and cell growth, and delayed recruitment of repair proteins to sites of damage were observed. Formation of phosphorylated H2AX and 53BP1 foci was delayed in Mrg15 mutant versus wild-type cells following irradiation. These data implicate a novel role for MRG15 in DNA damage repair in mammalian cells.
PMCID: PMC2132445  PMID: 17961556
MORF4; NuA4; Sin3-HDAC; ATM; 53BP1
7.  Regulation of Arabidopsis Flowering by the Histone Mark Readers MRG1/2 via Interaction with CONSTANS to Modulate FT Expression 
PLoS Genetics  2014;10(9):e1004617.
Day-length is important for regulating the transition to reproductive development (flowering) in plants. In the model plant Arabidopsis thaliana, the transcription factor CONSTANS (CO) promotes expression of the florigen FLOWERING LOCUS T (FT), constituting a key flowering pathway under long-day photoperiods. Recent studies have revealed that FT expression is regulated by changes of histone modification marks of the FT chromatin, but the epigenetic regulators that directly interact with the CO protein have not been identified. Here, we show that the Arabidopsis Morf Related Gene (MRG) group proteins MRG1 and MRG2 act as H3K4me3/H3K36me3 readers and physically interact with CO to activate FT expression. In vitro binding analyses indicated that the chromodomains of MRG1 and MRG2 preferentially bind H3K4me3/H3K36me3 peptides. The mrg1 mrg2 double mutant exhibits reduced mRNA levels of FT, but not of CO, and shows a late-flowering phenotype under the long-day but not short-day photoperiod growth conditions. MRG2 associates with the chromatin of FT promoter in a way dependent of both CO and H3K4me3/H3K36me3. Vice versa, loss of MRG1 and MRG2 also impairs CO binding at the FT promoter. Crystal structure analyses of MRG2 bound with H3K4me3/H3K36me3 peptides together with mutagenesis analysis in planta further demonstrated that MRG2 function relies on its H3K4me3/H3K36me3-binding activity. Collectively, our results unravel a novel chromatin regulatory mechanism, linking functions of MRG1 and MRG2 proteins, H3K4/H3K36 methylations, and CO in FT activation in the photoperiodic regulation of flowering time in plants.
Author Summary
The photoperiodic flowering in Arabidopsis requires the key regulator CO and its target gene FT. However, how CO regulates FT expression in the context of chromatin remains largely obscure. In this work, we present Arabidopsis MRG1/2 as novel chromatin effectors directly involved in the CO-FT photoperiodic flowering. Firstly, MRG1/2 proteins are identified as recognition factors of H3K4 and H3K36 methylation via their chromodomains. The mrg1 mrg2 double mutant shows a late-flowering phenotype only under long-day conditions through down-regulation of FT but not of CO. MRG2 can directly target in vivo the FT promoter chromatin in a H3K4me3/H3K36me3-level dependent manner. More importantly, MRG2 and CO physically interact and enhance each other's binding to the FT promoter in planta. Determination of co-crystal structures of MRG2 with H3K4me3/H3K36me3 peptides and mutagenesis of a key amino acid residue involved in structural interaction demonstrate that MRG2 reader activity is essential for in planta function. Taken together, our findings uncover a novel mechanism of FT activation in flowering promotion and provide a striking example of mutual interplay between a transcription factor and a histone methylation reader in transcription regulation.
PMCID: PMC4161306  PMID: 25211338
8.  Structure of human MRG15 chromo domain and its binding to Lys36-methylated histone H3 
Nucleic Acids Research  2006;34(22):6621-6628.
Human MRG15 is a transcription factor that plays a vital role in embryonic development, cell proliferation and cellular senescence. It comprises a putative chromo domain in the N-terminal part that has been shown to participate in chromatin remodeling and transcription regulation. We report here the crystal structure of human MRG15 chromo domain at 2.2 Å resolution. The MRG15 chromo domain consists of a β-barrel and a long α-helix and assumes a structure more similar to the Drosophila MOF chromo barrel domain than the typical HP1/Pc chromo domains. The β-barrel core contains a hydrophobic pocket formed by three conserved aromatic residues Tyr26, Tyr46 and Trp49 as a potential binding site for a modified residue of histone tail. However, the binding groove for the histone tail seen in the HP1/Pc chromo domains is pre-occupied by an extra β-strand. In vitro binding assay results indicate that the MRG15 chromo domain can bind to methylated Lys36, but not methylated Lys4, Lys9 and Lys27 of histone H3. These data together suggest that the MRG15 chromo domain may function as an adaptor module which can bind to a modified histone H3 in a mode different from that of the HP1/Pc chromo domains.
PMCID: PMC1747190  PMID: 17135209
9.  Loss of the chromatin regulator MRG15 limits neural stem/progenitor cell proliferation via increased expression of the p21 Cdk inhibitor 
Stem cell research  2011;7(1):75-88.
Chromatin regulation is crucial for many biological processes such as transcriptional regulation, DNA replication, and DNA damage repair. We have found it is also important for neural stem/progenitor cell (NSC) function and neurogenesis. Here, we demonstrate that expression of the cyclin-dependent kinase inhibitor p21 is specifically up-regulated in Mrg15 deficient NSCs. Knockdown of p21 expression by p21 shRNA results in restoration of cell proliferation. This indicates that p21 is directly involved in the growth defects observed in Mrg15 deficient NSCs. Activated p53 accumulates in Mrg15 deficient NSCs and this most likely accounts for the up-regulation of p21 expression in the cells. We observed decreased p53 and p21 levels and a concomitant increase in the percentage of BrdU positive cells in Mrg15 null cultures following expression of p53 shRNA. DNA damage foci, as indicated by immunostaining for γH2AX and 53BP1, are detectable in a sub-population of Mrg15 deficient NSC cultures under normal growing conditions and the majority of p21-positive cells are also positive for 53BP1 foci. Furthermore, Mrg15 deficient NSCs exhibit severe defects in DNA damage response following ionizing radiation. Our observations highlight the importance of chromatin regulation and DNA damage response in NSC function and maintenance.
PMCID: PMC3130620  PMID: 21621175
. Neural precursor cell; cell proliferation; chromatin; epigenetics; gene expression; DNA damage response
10.  MRG-1, an autosome-associated protein, silences X-linked genes and protects germline immortality in Caenorhabditis elegans 
Development (Cambridge, England)  2007;134(4):757-767.
MRG15, a mammalian protein related to the mortality factor MORF4, is required for cell proliferation and embryo survival. Our genetic analysis has revealed that the Caenorhabditis 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). By 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 versus soma distinction.
PMCID: PMC2435364  PMID: 17215300
C. elegans; MRG-1; Germ line; X chromosome silencing
11.  Sequence Requirements for Combinatorial Recognition of Histone H3 by the MRG15 and Pf1 Subunits of the Rpd3S/Sin3S Corepressor Complex 
Journal of molecular biology  2012;422(4):519-531.
The transcriptional output at a genomic locus in eukaryotes is determined, in part, by the pattern of histone modifications that are read and interpreted by key effector proteins. The histone deacetylase activity of the evolutionarily-conserved Rpd3S/Sin3S complex is crucial for suppressing aberrant transcription from cryptic start sites within intragenic regions of actively transcribed genes. Precise targeting of the complex relies on the chromatin binding activities of the MRG15 and Pf1 subunits. Whereas the molecular target of the MRG15 chromodomain (CD) has been suggested to be H3K36me2/3, the precise molecular target of the Pf1 plant homeodomain 1 (PHD1) has remained elusive. Here we show that Pf1 PHD1 binds preferentially to the unmodified extreme N-terminus of histone H3 (H3K4me0) but not to H3K4me2/3, which are enriched in the promoter and 5′ regions of genes. Unlike previously characterized CD and PHD domains that bind to their targets with micromolar affinity, both MRG15 CD and Pf1 PHD1 bind to their targets with >100 μM affinity, offering an explanation for why both MRG15 CD and Pf1 PHD1 domains are required to target the Rpd3S/Sin3S complex to chromatin. Our results also suggest that bivalency, rather than cooperativity, is the operative mechanism by which Pf1 and MRG15 combine to engage H3 in a biologically significant manner. Finally, the studies reveal an unanticipated role of Pf1 PHD1 in engaging the MRG15 MRG domain, albeit in a Pf1 MRG-binding domain (MBD)-dependent manner, implying a key role for the MRG15 MRG-Pf1 MBD interaction in chromatin targeting of the Rpd3S/Sin3S complex.
PMCID: PMC3428507  PMID: 22728643
Transcription repression; combinatorial readout; histone code; histone interactions; NMR
12.  Structural Basis for Molecular Interactions Involving MRG Domains: Implications in Chromatin Biology 
Structure(London, England:1993)  2012;20(1):151-160.
MRG15 is a member of the mortality family of transcription factors that targets a wide variety of multi-protein complexes involved in transcription regulation, DNA repair, and alternative splicing to chromatin. The structure of the apo-MRG15 MRG domain implicated in interactions with diverse proteins has been described, but not in complex with any of its targets. Here we structurally and functionally characterize the interaction between MRG15 and Pf1, two constitutively-associated subunits of the histone deacetylase-associated Rpd3S/Sin3S corepressor complex. The MRG domain adopts a structure reminiscent of the apo-state whereas the Pf1 MRG-binding domain engages two discrete hydrophobic surfaces on the MRG domain via a bipartite motif comprising an α-helix and a segment in an extended conformation, both of which are critical for high-affinity interactions. Multiple MRG15 interactors share an FxLP motif in the extended segment but equivalent sequence/helical motifs are not readily evident, implying potential diversity in MRG-recognition mechanisms.
PMCID: PMC3259534  PMID: 22244764
13.  PAM14, a Novel MRG- and Rb-Associated Protein, Is Not Required for Development and T-Cell Function in Mice 
Molecular and Cellular Biology  2004;24(19):8366-8373.
PAM14 has been found to associate in complexes with the MORF4/MRG family of proteins as well as Rb, the tumor suppressor protein. This suggested that it might be involved in cell growth, immortalization, and/or senescence. To elucidate the in vivo function of PAM14, we characterized the expression pattern of mouse Pam14 and generated PAM14-deficient (Pam14−/−) mice. Pam14 was widely expressed in all mouse tissues and as early as 7 days during embryonic development. Despite this ubiquitous expression in wild-type mice, Pam14−/− mice were healthy and fertile. Response to mitogenic stimulation and production of interleukin-2 were the same in stimulated splenic T cells from Pam14−/− mice as in control littermates. Cell growth rates of mouse embryonic fibroblasts (MEFs) from all three genotypes were the same, and immortalized cells were obtained from all cell cultures during continuous culture. There was also no difference in expression of growth-related genes in response to serum stimulation in the null versus control MEFs. These data demonstrate that PAM14 is not essential for normal mouse development and cell cycle control. PAM14 likely acts as an adaptor protein in nucleoprotein complexes and is probably compensated for by another functionally redundant protein(s).
PMCID: PMC516751  PMID: 15367658
14.  Exploring the link between MORF4L1 and risk of breast cancer 
Martrat, Griselda | Maxwell, Christopher A | Tominaga, Emiko | Porta-de-la-Riva, Montserrat | Bonifaci, Núria | Gómez-Baldó, Laia | Bogliolo, Massimo | Lázaro, Conxi | Blanco, Ignacio | Brunet, Joan | Aguilar, Helena | Fernández-Rodríguez, Juana | Seal, Sheila | Renwick, Anthony | Rahman, Nazneen | Kühl, Julia | Neveling, Kornelia | Schindler, Detlev | Ramírez, María J | Castellà, María | Hernández, Gonzalo | Easton, Douglas F | Peock, Susan | Cook, Margaret | Oliver, Clare T | Frost, Debra | Platte, Radka | Evans, D Gareth | Lalloo, Fiona | Eeles, Rosalind | Izatt, Louise | Chu, Carol | Davidson, Rosemarie | Ong, Kai-Ren | Cook, Jackie | Douglas, Fiona | Hodgson, Shirley | Brewer, Carole | Morrison, Patrick J | Porteous, Mary | Peterlongo, Paolo | Manoukian, Siranoush | Peissel, Bernard | Zaffaroni, Daniela | Roversi, Gaia | Barile, Monica | Viel, Alessandra | Pasini, Barbara | Ottini, Laura | Putignano, Anna Laura | Savarese, Antonella | Bernard, Loris | Radice, Paolo | Healey, Sue | Spurdle, Amanda | Chen, Xiaoqing | Beesley, Jonathan | Rookus, Matti A | Verhoef, Senno | Tilanus-Linthorst, Madeleine A | Vreeswijk, Maaike P | Asperen, Christi J | Bodmer, Danielle | Ausems, Margreet GEM | van Os, Theo A | Blok, Marinus J | Meijers-Heijboer, Hanne EJ | Hogervorst, Frans BL | Goldgar, David E | Buys, Saundra | John, Esther M | Miron, Alexander | Southey, Melissa | Daly, Mary B | Harbst, Katja | Borg, Åke | Rantala, Johanna | Barbany-Bustinza, Gisela | Ehrencrona, Hans | Stenmark-Askmalm, Marie | Kaufman, Bella | Laitman, Yael | Milgrom, Roni | Friedman, Eitan | Domchek, Susan M | Nathanson, Katherine L | Rebbeck, Timothy R | Johannsson, Oskar Thor | Couch, Fergus J | Wang, Xianshu | Fredericksen, Zachary | Cuadras, Daniel | Moreno, Víctor | Pientka, Friederike K | Depping, Reinhard | Caldés, Trinidad | Osorio, Ana | Benítez, Javier | Bueren, Juan | Heikkinen, Tuomas | Nevanlinna, Heli | Hamann, Ute | Torres, Diana | Caligo, Maria Adelaide | Godwin, Andrew K | Imyanitov, Evgeny N | Janavicius, Ramunas | Sinilnikova, Olga M | Stoppa-Lyonnet, Dominique | Mazoyer, Sylvie | Verny-Pierre, Carole | Castera, Laurent | de Pauw, Antoine | Bignon, Yves-Jean | Uhrhammer, Nancy | Peyrat, Jean-Philippe | Vennin, Philippe | Ferrer, Sandra Fert | Collonge-Rame, Marie-Agnès | Mortemousque, Isabelle | McGuffog, Lesley | Chenevix-Trench, Georgia | Pereira-Smith, Olivia M | Antoniou, Antonis C | Cerón, Julián | Tominaga, Kaoru | Surrallés, Jordi | Pujana, Miguel Angel
Proteins encoded by Fanconi anemia (FA) and/or breast cancer (BrCa) susceptibility genes cooperate in a common DNA damage repair signaling pathway. To gain deeper insight into this pathway and its influence on cancer risk, we searched for novel components through protein physical interaction screens.
Protein physical interactions were screened using the yeast two-hybrid system. Co-affinity purifications and endogenous co-immunoprecipitation assays were performed to corroborate interactions. Biochemical and functional assays in human, mouse and Caenorhabditis elegans models were carried out to characterize pathway components. Thirteen FANCD2-monoubiquitinylation-positive FA cell lines excluded for genetic defects in the downstream pathway components and 300 familial BrCa patients negative for BRCA1/2 mutations were analyzed for genetic mutations. Common genetic variants were genotyped in 9,573 BRCA1/2 mutation carriers for associations with BrCa risk.
A previously identified co-purifying protein with PALB2 was identified, MRG15 (MORF4L1 gene). Results in human, mouse and C. elegans models delineate molecular and functional relationships with BRCA2, PALB2, RAD51 and RPA1 that suggest a role for MRG15 in the repair of DNA double-strand breaks. Mrg15-deficient murine embryonic fibroblasts showed moderate sensitivity to γ-irradiation relative to controls and reduced formation of Rad51 nuclear foci. Examination of mutants of MRG15 and BRCA2 C. elegans orthologs revealed phenocopy by accumulation of RPA-1 (human RPA1) nuclear foci and aberrant chromosomal compactions in meiotic cells. However, no alterations or mutations were identified for MRG15/MORF4L1 in unclassified FA patients and BrCa familial cases. Finally, no significant associations between common MORF4L1 variants and BrCa risk for BRCA1 or BRCA2 mutation carriers were identified: rs7164529, Ptrend = 0.45 and 0.05, P2df = 0.51 and 0.14, respectively; and rs10519219, Ptrend = 0.92 and 0.72, P2df = 0.76 and 0.07, respectively.
While the present study expands on the role of MRG15 in the control of genomic stability, weak associations cannot be ruled out for potential low-penetrance variants at MORF4L1 and BrCa risk among BRCA2 mutation carriers.
PMCID: PMC3219203  PMID: 21466675
15.  MRG-1 is required for genomic integrity in Caenorhabditis elegans germ cells 
Cell Research  2012;22(5):886-902.
During meiotic cell division, proper chromosome synapsis and accurate repair of DNA double strand breaks (DSBs) are required to maintain genomic integrity, loss of which leads to apoptosis or meiotic defects. The mechanisms underlying meiotic chromosome synapsis, DSB repair and apoptosis are not fully understood. Here, we report that the chromodomain-containing protein MRG-1 is an important factor for genomic integrity in meiosis in Caenorhabditis elegans. Loss of mrg-1 function resulted in a significant increase in germ cell apoptosis that was partially inhibited by mutations affecting DNA damage checkpoint genes. Consistently, mrg-1 mutant germ lines exhibited SPO-11-generated DSBs and elevated exogenous DNA damage-induced chromosome fragmentation at diakinesis. In addition, the excessive apoptosis in mrg-1 mutants was partially suppressed by loss of the synapsis checkpoint gene pch-2, and a significant number of meiotic nuclei accumulated at the leptotene/zygotene stages with an elevated level of H3K9me2 on the chromatin, which was similarly observed in mutants deficient in the synaptonemal complex, suggesting that the proper progression of chromosome synapsis is likely impaired in the absence of mrg-1. Altogether, these findings suggest that MRG-1 is critical for genomic integrity by promoting meiotic DSB repair and synapsis progression in meiosis.
PMCID: PMC3343660  PMID: 22212480
apoptosis; DSB repair; synapsis; meiosis; genomic integrity; C. elegans
16.  Repression of Germline RNAi Pathways in Somatic Cells by Retinoblastoma Pathway Chromatin Complexes 
PLoS Genetics  2012;8(3):e1002542.
The retinoblastoma (Rb) tumor suppressor acts with a number of chromatin cofactors in a wide range of species to suppress cell proliferation. The Caenorhabditis elegans retinoblastoma gene and many of these cofactors, called synMuv B genes, were identified in genetic screens for cell lineage defects caused by growth factor misexpression. Mutations in many synMuv B genes, including lin-35/Rb, also cause somatic misexpression of the germline RNA processing P granules and enhanced RNAi. We show here that multiple small RNA components, including a set of germline-specific Argonaute genes, are misexpressed in the soma of many synMuv B mutant animals, revealing one node for enhanced RNAi. Distinct classes of synMuv B mutants differ in the subcellular architecture of their misexpressed P granules, their profile of misexpressed small RNA and P granule genes, as well as their enhancement of RNAi and the related silencing of transgenes. These differences define three classes of synMuv B genes, representing three chromatin complexes: a LIN-35/Rb-containing DRM core complex, a SUMO-recruited Mec complex, and a synMuv B heterochromatin complex, suggesting that intersecting chromatin pathways regulate the repression of small RNA and P granule genes in the soma and the potency of RNAi. Consistent with this, the DRM complex and the synMuv B heterochromatin complex were genetically additive and displayed distinct antagonistic interactions with the MES-4 histone methyltransferase and the MRG-1 chromodomain protein, two germline chromatin regulators required for the synMuv phenotype and the somatic misexpression of P granule components. Thus intersecting synMuv B chromatin pathways conspire with synMuv B suppressor chromatin factors to regulate the expression of small RNA pathway genes, which enables heightened RNAi response. Regulation of small RNA pathway genes by human retinoblastoma may also underlie its role as a tumor suppressor gene.
Author Summary
In metazoans, soma and germline have specialized functions that require differential tissue-specific gene expression. In C. elegans, explicit chromatin marks deposited by the MES-4 histone methyltransferase and the MRG-1 chromodomain protein allow germline expression of particular suites of target genes. Conversely, the expression of germline-specific genes is repressed in somatic cells by other chromatin regulatory factors, including the retinoblastoma pathway genes. We characterized the distinct profiles of somatic misexpression of normally germline-specific genes in these mutants and mapped out three chromatin complexes that prevent misexpression. We demonstrate that one of the complexes closely counteracts the activity of MES-4 and MRG-1, whereas another complex interacts with additional regulators that are yet to be identified. We show that these intersecting chromatin complexes prevent the upregulation of a suite of germline-specific as well as ubiquitous small RNA pathway genes, which contributes to the enhanced RNAi response in retinoblastoma pathway mutant worms. We suggest that this function of the retinoblastoma pathway chromatin factors to prevent germline-associated gene expression programs in the soma and the upregulation of small RNA pathways may also underlie their role as tumor suppressors.
PMCID: PMC3297578  PMID: 22412383
17.  Role for the Mortality Factors MORF4, MRGX, and MRG15 in Transcriptional Repression via Associations with Pf1, mSin3A, and Transducin-Like Enhancer of Split 
Molecular and Cellular Biology  2002;22(22):7868-7876.
mSin3A and Transducin-Like Enhancer of Split (TLE) are two histone deacetylase (HDAC)-containing corepressors that function to repress transcription at targeted genes. Pf1 is a plant homeodomain zinc finger protein that interacts with both mSin3A and TLE, suggesting that it coordinates their function. Here we show that mSin3A and TLE interact with members of the mortality factor (MORF) family of putative transcriptional regulators. This family comprises MORF on chromosome 4 (MORF4) and MORF-related genes on chromosomes X and 15 (MRGX and MRG15, respectively) and is proposed to contribute to cellular senescence. Consistent with a role in transcription, we demonstrate that Gal4 fusions to each MORF family member repress transcription from a Gal4-dependent luciferase reporter. By using both mapping experiments and a dominant negative form of TLE, we show that repression by MORFs requires associations with mSin3A and TLE. Therefore, common functions of the MORFs are likely elicited through the action of a MORF/mSin3A/TLE complex. While the MORFs may have common functions, MRG15, but not MRGX or MORF4, interacted with Pf1. Therefore, MRG15 may have functions that are distinct from those of MRGX and MORF4. Consistent with this hypothesis, Pf1 reduced transcriptional repression by Gal4-MRG15 but it had no effect on repression by MRGX and MORF4. Pf1 has independent binding sites for MRG15 and mSin3A. In addition, Pf1 and MRG15 bind different domains on mSin3A. Together, these data suggest that the unique functions of MRG15 are elicited through the action of an MRG15/Pf1/mSin3A complex.
PMCID: PMC134742  PMID: 12391155
18.  Maintenance of Interphase Chromosome Compaction and Homolog Pairing in Drosophila Is Regulated by the Condensin Cap-H2 and Its Partner Mrg15 
Genetics  2013;195(1):127-146.
Dynamic regulation of chromosome structure and organization is critical for fundamental cellular processes such as gene expression and chromosome segregation. Condensins are conserved chromosome-associated proteins that regulate a variety of chromosome dynamics, including axial shortening, lateral compaction, and homolog pairing. However, how the in vivo activities of condensins are regulated and how functional interactors target condensins to chromatin are not well understood. To better understand how Drosophila melanogaster condensin is regulated, we performed a yeast two-hybrid screen and identified the chromo-barrel domain protein Mrg15 to interact with the Cap-H2 condensin subunit. Genetic interactions demonstrate that Mrg15 function is required for Cap-H2-mediated unpairing of polytene chromosomes in ovarian nurse cells and salivary gland cells. In diploid tissues, transvection assays demonstrate that Mrg15 inhibits transvection at Ubx and cooperates with Cap-H2 to antagonize transvection at yellow. In cultured cells, we show that levels of chromatin-bound Cap-H2 protein are partially dependent on Mrg15 and that Cap-H2-mediated homolog unpairing is suppressed by RNA interference depletion of Mrg15. Thus, maintenance of interphase chromosome compaction and homolog pairing status requires both Mrg15 and Cap-H2. We propose a model where the Mrg15 and Cap-H2 protein–protein interaction may serve to recruit Cap-H2 to chromatin and facilitates compaction of interphase chromatin.
PMCID: PMC3761296  PMID: 23821596
condensin; homolog pairing; Mrg15; chromosome structure; transvection
19.  Arabidopsis MRG domain proteins bridge two histone modifications to elevate expression of flowering genes 
Nucleic Acids Research  2014;42(17):10960-10974.
Trimethylation of lysine 36 of histone H3 (H3K36me3) is found to be associated with various transcription events. In Arabidopsis, the H3K36me3 level peaks in the first half of coding regions, which is in contrast to the 3′-end enrichment in animals. The MRG15 family proteins function as ‘reader’ proteins by binding to H3K36me3 to control alternative splicing or prevent spurious intragenic transcription in animals. Here, we demonstrate that two closely related Arabidopsis homologues (MRG1 and MRG2) are localised to the euchromatin and redundantly ensure the increased transcriptional levels of two flowering time genes with opposing functions, FLOWERING LOCUS C and FLOWERING LOCUS T (FT). MRG2 directly binds to the FT locus and elevates the expression in an H3K36me3-dependent manner. MRG1/2 binds to H3K36me3 with their chromodomain and interact with the histone H4-specific acetyltransferases (HAM1 and HAM2) to achieve a high expression level through active histone acetylation at the promoter and 5′ regions of target loci. Together, this study presents a mechanistic link between H3K36me3 and histone H4 acetylation. Our data also indicate that the biological functions of MRG1/2 have diversified from their animal homologues during evolution, yet they still maintain their conserved H3K36me3-binding molecular function.
PMCID: PMC4176166  PMID: 25183522
20.  Dissection of Hippocampal Dentate Gyrus from Adult Mouse 
The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.
PMCID: PMC3142893  PMID: 19920804
21.  Chfr and RNF8 synergistically regulate ATM activation 
Protein ubiquitination is a critical component of the DNA damage response. To study the mechanism of the DNA damage-induced ubiquitination pathway, we analyzed the impact of the loss of two E3 ubiquitin ligases, RNF8 and Chfr. Interestingly, DNA damage-induced ATM activation is suppressed in RNF8 and Chfr double-deficient (DKO) cells, and DKO mice develop thymic lymphomas that are nearly diploid but harbor clonal chromosome translocations. Moreover, DKO mice and cells are hypersensitive to ionizing radiation. We show evidence that RNF8 and Chfr synergistically regulate histone ubiquitination to control histone H4K16 acetylation through MRG15-dependent acetyltransferase complexes. Through these complexes, RNF8 and CHFR affect chromatin relaxation and modulate ATM activation and DNA damage response pathways. Collectively, our findings demonstrate that two chromatin remodeling factors, RNF8 and Chfr, function together to activate ATM and maintain genomic stability in vivo.
PMCID: PMC3130800  PMID: 21706008
22.  Structural and Functional Conservation of the NuA4 Histone Acetyltransferase Complex from Yeast to Humans 
Molecular and Cellular Biology  2004;24(5):1884-1896.
The NuA4 histone acetyltransferase (HAT) multisubunit complex is responsible for acetylation of histone H4 and H2A N-terminal tails in yeast. Its catalytic component, Esa1, is essential for cell cycle progression, gene-specific regulation and has been implicated in DNA repair. Almost all NuA4 subunits have clear homologues in higher eukaryotes, suggesting that the complex is conserved throughout evolution to metazoans. We demonstrate here that NuA4 complexes are indeed present in human cells. Tip60 and its splice variant Tip60b/PLIP were purified as stable HAT complexes associated with identical polypeptides, with 11 of the 12 proteins being homologs of yeast NuA4 subunits. This indicates a highly conserved subunit composition and the identified human proteins underline the role of NuA4 in the control of mammalian cell proliferation. ING3, a member of the ING family of growth regulators, links NuA4 to p53 function which we confirmed in vivo. Proteins specific to the human NuA4 complexes include ruvB-like helicases and a bromodomain-containing subunit linked to ligand-dependent transcription activation by the thyroid hormone receptor. We also demonstrate that subunits MRG15 and DMAP1 are present in distinct protein complexes harboring histone deacetylase and SWI2-related ATPase activities, respectively. Finally, analogous to yeast, a recombinant trimeric complex formed by Tip60, EPC1, and ING3 is sufficient to reconstitute robust nucleosomal HAT activity in vitro. In conclusion, the NuA4 HAT complex is highly conserved in eukaryotes, in which it plays primary roles in transcription, cellular response to DNA damage, and cell cycle control.
PMCID: PMC350560  PMID: 14966270
23.  Methanol May Function as a Cross-Kingdom Signal 
PLoS ONE  2012;7(4):e36122.
Recently, we demonstrated that leaf wounding results in the synthesis of pectin methylesterase (PME), which causes the plant to release methanol into the air. Methanol emitted by a wounded plant increases the accumulation of methanol-inducible gene mRNA and enhances antibacterial resistance as well as cell-to-cell communication, which facilitates virus spreading in neighboring plants. We concluded that methanol is a signaling molecule involved in within-plant and plant-to-plant communication. Methanol is considered to be a poison in humans because of the alcohol dehydrogenase (ADH)-mediated conversion of methanol into toxic formaldehyde. However, recent data showed that methanol is a natural compound in normal, healthy humans. These data call into question whether human methanol is a metabolic waste product or whether methanol has specific function in humans.
Here, to reveal human methanol-responsive genes (MRGs), we used suppression subtractive hybridization cDNA libraries of HeLa cells lacking ADH and exposed to methanol. This design allowed us to exclude genes involved in formaldehyde and formic acid detoxification from our analysis. We identified MRGs and revealed a correlation between increases in methanol content in the plasma and changes in human leukocyte MRG mRNA levels after fresh salad consumption by volunteers. Subsequently, we showed that the methanol generated by the pectin/PME complex in the gastrointestinal tract of mice induces the up- and downregulation of brain MRG mRNA. We used an adapted Y-maze to measure the locomotor behavior of the mice while breathing wounded plant vapors in two-choice assays. We showed that mice prefer the odor of methanol to other plant volatiles and that methanol changed MRG mRNA accumulation in the mouse brain.
We hypothesize that the methanol emitted by wounded plants may have a role in plant-animal signaling. The known positive effect of plant food intake on human health suggests a role for physiological methanol in human gene regulation.
PMCID: PMC3338578  PMID: 22563443
24.  G Protein coupled Receptor Specificity for C3a and Compound 48/80-induced degranulation in human mast cells: Roles of Mas-related genes MrgX1 and MrgX2 
European journal of pharmacology  2011;668(1-2):299-304.
Although human mast cells express G protein coupled receptors for the anaphylatoxin C3a, previous studies indicated that C3a causes mast cell degranulation, at least in part, via a C3a receptor-independent mechanism similar to that proposed for polycationic molecules such as compound 48/80. The purpose of the present study was to delineate the receptor specificity of C3a-induced degranulation in human mast cells. We found that C3a, a C3a receptor “superagonist” (E7) and compound 48/80 induced Ca2+ mobilization and degranulation in a differentiated human mast cell line, LAD2. However, C3a and E7 caused Ca2+ mobilization in an immature mast cell line, HMC-1 but compound 48/80 did not. We have previously shown that LAD2 cells express MrgX1 and MrgX2 but HMC-1 cells do not. To delineate the receptor specificity for C3a and compound 48/80 further, we generated stable transfectants expressing MrgX1 and MrgX2 in a rodent mast cell line, RBL-2H3 cells. We found that compound 48/80 caused degranulation in RBL-2H3 cells expressing MrgX1 and MrgX2 but C3a did not. By contrast, E7 activated RBL-2H3 cells expressing MrgX2 but not MrgX1. These findings demonstrate that in contrast to previous reports, C3a and compound 48/80 do not use a shared mechanism for mast cell degranulation. It shows that while compound 48/80 utilizes MrgX1 and MrgX2 for mast cell degranulation C3a does not. It further reveals the novel finding that the previously characterized synthetic peptide, C3a receptor “superagonist” E7 activates human mast cells via two mechanisms; one involving the C3a receptor and the other MrgX2.
PMCID: PMC3169012  PMID: 21741965
Mast cell; degranulation; C3a; Compound 40/80; MrgX1; MrgX2
25.  A Novel Mammalian Complex Containing Sin3B Mitigates Histone Acetylation and RNA Polymerase II Progression within Transcribed Loci▿  
Molecular and Cellular Biology  2010;31(1):54-62.
Transcription requires the progression of RNA polymerase II (RNAP II) through a permissive chromatin structure. Recent studies of Saccharomyces cerevisiae have demonstrated that the yeast Sin3 protein contributes to the restoration of the repressed chromatin structure at actively transcribed loci. Yet, the mechanisms underlying the restoration of the repressive chromatin structure at transcribed loci and its significance in gene expression have not been investigated in mammals. We report here the identification of a mammalian complex containing the corepressor Sin3B, the histone deacetylase HDAC1, Mrg15, and the PHD finger-containing Pf1 and show that this complex plays important roles in regulation of transcription. We demonstrate that this complex localizes at discrete loci approximately 1 kb downstream of the transcription start site of transcribed genes, and this localization requires both Pf1's and Mrg15's interaction with chromatin. Inactivation of this mammalian complex promotes increased RNAP II progression within transcribed regions and subsequent increased transcription. Our results define a novel mammalian complex that contributes to the regulation of transcription and point to divergent uses of the Sin3 protein homologues throughout evolution in the modulation of transcription.
PMCID: PMC3019848  PMID: 21041482

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