Inactivation of the Mrg15 gene in mice has demonstrated that it has an essential role in embryogenesis, most likely through regulation of cell growth and differentiation. Mrg15−/− embryos display growth retardation and delayed development of many organs and tissues, which is reflected in the proliferative behavior of MEF cultures. MRG15 is involved in chromatin remodeling, and we have shown here that the protein is recruited to the α-globin promoter and correlates with increased histone acetylation around the α-globin promoter and increased expression of the α-globin gene. This could explain the paler appearance of Mrg15 null embryos.
MRG15 is evolutionarily conserved in organisms from yeast to human and is expressed ubiquitously in all tissues (7
). It encodes a chromodomain that is very similar to the msl3 chromodomain in Drosophila
, which is involved in dosage compensation and causes an increase in transcription along the entire X chromosome of male flies (23
). This suggests that MRG15 has the potential to be involved in global transcriptional control. Very similar MRG15-containing complexes exist in yeast and mammalian cells and are thought to be involved in transcriptional control through chromatin remodeling.
The MRG15 homologue in budding yeast, Eaf3p, has been found to be a component of the NuA4 HAT complex (19
), and a similar complex has been reported in human cells (13
). The Eaf3p null mutant is nonlethal, and the only phenotype is a decrease in the expression of a small number of target genes (19
). It is now proposed that Eaf3p is required for maintaining the normal pattern of global H3 and H4 acetylation (10
) because of the global histone acetylation pattern changes observed in the Eaf3p deletion mutant (52
), although the physiological impact of this in Saccharomyces cerevisiae
remains unknown. In contrast, deletion of Alp13, which is the MRG15 homologue in fission yeast (50
) and a component of the Clr6 histone deacetylase complex (24
), results in loss of viability and increased sensitivity to DNA damage-inducing agents. The Alpl3 mutant also exhibits impaired condensation and resolution of chromosomes during mitosis. The reasons for the differences in the phenotypes in budding versus fission yeast are not clear, but they may be due to differences in cofactors in the cells and thus in the outcome of inactivation of the MRG15 orthologs. The results suggest that Alp13 and other components of the Clr6 complex are involved in the maintenance of genomic integrity (24
). If this function is conserved in mammals, we might expect that cancer incidence will be higher in heterozygous MRG15 versus wild-type mice.
MRG-1, the MRG15 homologue in Caenorhabditis elegans
, is essential for mitotic proliferation of primordial germ cells during postembryonic development, and RNA interference knockdown of mrg-1
expression results in sterility in 100% of the injected worms (22
; A. Olgun, T. Aleksenko, O. M. Pereira-Smith, and D. K. Vassilatis, in press). A small percentage exhibit body wall defects, vulval protrusion, and posterior developmental defects that cause a blunt and shortened tail (Olgun et al., in press). Interestingly, as one analyzes more complex organisms, the Mrg15
null phenotype becomes more severe.
A loss-of-function mutation generated by a P element insertion into the Drosophila Mrg15
allele (dMrg15) results in recessive lethality (FlyBase Report [http://flybase.bio.indiana.edu
]). Although we do not know the precise mechanism and phenotype for lethality of this mutant, it is possible that dMrg15 is important for cell growth control during embryo development, similar to the Mrg15
null phenotype in mice. Recently, it has been shown that Drosophila
dMrg15 is one of the components of the dTip60 complex, similar to what has been found in yeast and human cells, and that it is essential for DNA repair of double-strand breaks by γ-irradiation (34
TRRAP (for transactivation-transformation domain-associated protein) is a component of HAT complexes, such as PCAF (59
), GCN5 (44
), and TIP60 (27
). A null mutation of Trrap
in mice results in peri-implantation lethality due to a blocked proliferation of blastocysts (26
-deficient cells exhibit chromosome missegregation, mitotic exit failure, and a compromised mitotic checkpoint. These defects are caused by transcriptional dysregulation of the mitotic checkpoint proteins Mad1 and Mad2 (38
), since TRRAP recruits HAT activities, including TIP60 and PCAF, to the promoters of these genes. Consistent with this is the fact that acetylation of histone H4 and H3 at these promoters is decreased in Trrap
-deficient cells. The phenotypes of Mrg15
, and Pcaf
) are milder than those of Trrap
deficiency. This may be due to the fact that TRRAP is shared by different HAT complexes and loss of Trrap
expression results in various overlapping phenotypes that are the consequence of decreased activity of multiple HATs.
MEFs exhibit impaired proliferation in culture, and the p21 protein is expressed at higher levels in early-passage Mrg15
null MEFs than in the wild type. Early-passage Mrg15
null MEFs also exhibit enlarged and flattened morphology compared with wild-type cells and enter the senescent state more rapidly. The molecular mechanism(s) that results in this growth defect and the increase in p21 expression in Mrg15
null MEFs, with no change in protein levels of p53, requires further investigation. However, it has been reported that the human HAT TIP60 and the budding yeast homologue, Esa1, are involved in DNA damage response (6
). If MRG15 is a canonical subunit in complexes involving TIP60 and essential for their function(s), it is possible that this p21 up-regulation and cell proliferation defect may be triggered by increasing genetic instability in Mrg15−/−
MEFs due to the loss of some TIP60 functions rather than induction by p53. Furthermore, the reduced growth may cause the small-size phenotype and tissue abnormalities observed during development in Mrg15
During development, many factors are involved in organogenesis, and their expression is tightly controlled in a spatiotemporal manner. Control of the α- and β-globin gene loci has been under analysis for many years, and intriguing models for the control of gene expression have been proposed (14
). At E11.5 of mouse gestation, the main site of erythropoiesis changes from the embryonic yolk sac (primitive erythropoiesis) to the fetal liver (definitive erythropoiesis) (57
). This change in site is coincident with a change from primitive to definitive gene expression in both the α- and β-globin gene clusters, leading to predominant expression of α1 and α2 and βmaj and βmin. At the α-globin locus, acetylation of histones H3 and H4 in ES and nonerythroid cells is maintained at low levels (4
), and these are dramatically increased during hemopoietic lineage commitment and differentiation. In the MEL cell differentiation model, although some fraction of H3 and H4 is already acetylated at the α-globin locus prior to induction of globin gene expression, possibly reflecting a primed condition, there is an increase just prior to α-globin gene induction (4
) (data not shown). We have found that recruitment of MRG15 protein to the α-globin locus precedes an increase in acetylation. Therefore, one possibility is that MRG15 recruits a HAT(s) to the α-globin locus and thereby controls α-globin expression levels.
We have demonstrated that MRG15 is present in multiple complexes in human cells (13
) and that it can act as an activator of the B-myb
). Both the chromodomain and the leucine zipper region of the protein are required for this activity (47
). Thus, it is possible that MRG15 acts through chromatin remodeling, as well as directly at promoters, to regulate the transcription of target genes. The results with the null mice support the hypothesis that MRG15 affects cell proliferation in many tissues and that it is required as a positive regulator for growth during mouse embryogenesis. Comparative studies using null versus wild-type embryos, MEFs, or other cell types should allow us to identify additional endogenous target genes. This will aid in the identification of other potential functions of MRG15 in the maintenance of genomic integrity, immortalization, and tumor formation.