The role of menin in the pathology of multiple endocrine neoplasia type 1, embryonic development, and the normal regulation of cell growth and/or survival has yet to be elucidated despite its demonstrated ability to modulate the activity of transcription factors such as JunD, NF-κB, and Smad3 (2
). The discovery of a menin-interacting protein such as RPA2, with its links to DNA replication, recombination, repair, and transcription, provides a new direction for the investigation of menin function.
The identification of multiple endocrine neoplasia type 1-associated missense mutations that disrupt binding of menin to RPA2 without affecting its binding to JunD (or vice versa), e.g., P12L, F144V, and W183S (Table ), raises the possibility that both of these proteins are important for menin's tumor suppressor activity. However, the finding that mutants such as W436R and F447S retain the ability to interact with both RPA2 and JunD indicates that multiple endocrine neoplasia type 1 can also develop through mechanisms that do not involve perturbations in menin or JunD binding. In the case of F447S, this mechanism may be loss of the ability to repress JunD activity (2
). However, the W436R mutant retains both RPA2/JunD binding and the ability to repress JunD activity; consequently, the mechanism for multiple endocrine neoplasia type 1 development in this case must lie downstream of menin-RPA2 or menin-JunD binding or involve an aspect of menin function that is unrelated to its interaction with RPA2 and JunD. Further study is needed to distinguish among these possibilities.
Similarities in the effects of N-terminal mutations such as H139D, A160P, A164D, A176P, and L286P on the interaction between menin and RPA2 or JunD are consistent with the finding that both of these interactors have binding sites within the N-terminal half of menin (Fig. ) (2
). Furthermore, C-terminal mutations such as W436R and F447S had no effect on RPA2 binding (Table ), consistent with gel filtration data indicating that RPA2 is capable of binding to menin C-terminally truncated at amino acid 316 (data not shown). The modest reduction in RPA2-menin binding observed as a result of the T344R mutation might be attributed to the influence of conformational changes on an upstream binding site for RPA2.
The menin-binding region of RPA2 was mapped to amino acids 43 to 171 (Fig. ), which also contains an ssDNA-binding domain and sequences required for interaction with RPA3 (10
). This domain is believed to be important in stabilizing the interaction between the RPA heterotrimer and ssDNA and is thought to play a role in establishing RPA-ssDNA binding polarity, with RPA1 and RPA2 oriented to the 5′ and 3′ ends, respectively, of the ssDNA (17
). This polarity is thought to be important in positioning 5′ and 3′ endonucleases in the incision step of nucleotide excision repair (17
) and for regulation of lagging-strand DNA synthesis during DNA replication (47
Mapping the menin-binding site to the ssDNA-binding region of RPA2 raised the possibility that menin could influence RPA ssDNA-binding activity and, consequently, its functions in DNA repair, replication, or recombination. However, menin did not have a significant effect on binding of the RPA heterotrimer or RPA2(43-171)-RPA3 complex to ssDNA or undamaged or damaged dsDNA (Fig. and data not shown) in vitro. This finding was consistent with the low efficiency of binding observed between menin and trimeric RPA or RPA2-RPA3 complexes relative to free RPA2 in vitro and could explain why simian virus 40 replication in vitro is not noticeably affected by the addition of purified menin (data not shown).
RPA3 was also shown to compete with menin for RPA2 binding in vitro (Fig. ), suggesting that they may have overlapping binding sites on RPA2. Although menin immunoprecipitates contained RPA1 as well as RPA2 (Fig. ), the presence of RPA3 could not be established due to the low reactivity of available RPA3-specific antibodies and/or the low expression level of RPA3 relative to the other two subunits. Thus, it is conceivable that menin forms a complex with RPA1 and RPA2 in vivo in the absence of RPA3. Although a complex containing only these two RPA subunits has yet to be demonstrated, a significant proportion of the RPA3 population seems to be localized separately from RPA1 and RPA2 in the nucleoli of G1
- or S-phase cells and in the cytoplasm during telophase (52
). However, the ability of menin-specific antibodies to coprecipitate RPA1 as well as RPA2 from mammalian cell extracts (Fig. ), together with the observed similarity in endogenous RPA1, RPA2, and menin intranuclear localization patterns (Fig. ), raises the possibility that menin may be able to bind to the RPA heterotrimer and influence its DNA-binding activity in vivo.
Interestingly, some studies have suggested that RPA also binds to undamaged dsDNA in vivo. For instance, the bulk of chromatin-bound RPA in HeLa extracts is released by DNase I and micrococcal nuclease but not by single-strand-specific endonucleases (66
), suggesting that most of this RPA is bound to dsDNA rather than ssDNA. RPA has also been identified as a repressor protein bound to the promoter of yeast metabolism and repair genes (20
), the human metallothionein type IIA promoter (63
), and a mutant endothelial nitrogen oxide synthase gene (50
), raising the possibility that RPA could be involved in menin-mediated repression of JunD and NF-κB activity on certain promoters.
As alluded to previously, RPA has also been shown to undergo conformational changes upon ssDNA binding that could cause changes in its ability to interact with other proteins. For example, ssDNA binding by RPA stimulates the phosphorylation of RPA2 by DNA-dependent protein kinase, independent of nucleic acid-mediated activation of kinase activity (8
). Practically nothing is known about the mechanism of potential RPA-dsDNA binding or conformational changes that might accompany this binding, but it seems likely that there are some proteins in the cell that interact preferentially with the form of RPA that binds dsDNA. Proteins that regulate transcription, such as menin, could fall into this category.
Links between RPA and transcription have also been postulated based on the observed interaction between RPA and Gal4 (25
), VP16 (25
), p53 (19
), Stat3 (35
), and RBT1 (14
), a putative transcriptional coactivator with homology to Drosophila
trithorax proteins involved in developmental gene regulation and/or chromatin remodeling (12
). Interestingly, Stat3 and Rbt1, like menin, have been shown to interact with the RPA2 subunit. RPA has also been identified as a component of a human RNA polymerase II complex (45
). However, further investigation is required to determine the significance of these interactions.
A number of studies have suggested that menin may have a role in DNA repair or maintenance of genome stability, based on findings of elevated frequencies of spontaneous or DNA damage-induced chromosome abnormalities in peripheral blood lymphocytes from multiple endocrine neoplasia type 1-affected individuals (7
), raising the tantalizing possibility that menin has a role in maintaining the genomic integrity of the cell. Many proteins involved directly in repair processes, including RPA (15
), form foci in response to agents that cause certain types of DNA damage. Therefore, the absence of noticeable changes in menin localization in response to a wide variety of DNA-damaging agents, such as UV and camptothecin (Fig. ) or ionizing radiation, diepoxybutane, cisplatin, ethyl methanesulfonate, and mitomycin C (data not shown), suggests that any role of menin in repair is likely to be regulatory rather than direct.
Since dsDNA break repair is thought to occur through homologous recombination and/or nonhomologous end joining, many proteins that form foci in response to dsDNA break-inducing agents and participate in DNA break repair (reviewed in reference 64
) are also involved in meiotic recombination. RPA, for example, forms foci in response to dsDNA break-inducing agents such as ionizing radiation (22
), camptothecin (58
), and etoposide (51
), has been localized to meiotic recombination nodules on mouse spermatocytes (56
), and is thought to participate in the process of DNA strand exchange (reviewed in reference 28
Although menin is ubiquitously expressed, its expression level is particularly high in the testis, consistent with a potential role in recombination (62
). However, the absence of menin focus formation in response to camptothecin, ionizing radiation, or bleomycin (Fig. and data not shown), suggests that any involvement of menin in recombination, as in the case of DNA repair, is likely to occur at the level of regulation rather than direct participation in the core reactions. A similar argument can be made with respect to DNA replication, based on the lack of menin mobilization into replication foci in cells synchronized at the G1
/S border or released into S phase (Fig. ). The detection of RPA2-containing foci in menin-null cells (data not shown) further indicates that menin is not essential for the assembly of DNA replication or repair complexes and that any regulation of these processes by menin is likely to occur via some other mechanism.
The requirement for RPA in DNA replication, recombination, and repair, as well as its potential role in the modulation of gene expression, makes it an attractive target for regulation by tumor suppressors or other proteins involved in the control of cell proliferation and/or apoptosis. Indeed, specific interaction between p53 and RPA is inversely correlated with the abilities of RPA to bind ssDNA and of p53 to bind transactivating sequences in the promoters of its target genes (19
). This interaction is disrupted by UV (1
), adozelesin (42
), and cisplatin (57
), and a correlation between RPA-p53 binding and RPA2 phosphorylation status has been observed. RPA2 has been shown to undergo cell cycle-dependent and damage-induced phosphorylation, and although the consequences of this phosphorylation are unclear, it may influence RPA conformation, DNA-binding characteristics, and/or its interactions with other proteins, as in the case of p53.
Although treatment of cultured cells with cell-synchronizing or DNA-damaging agents and the accompanying increase in RPA2 phosphorylation does not result in detectable changes in menin localization or binding to RPA2 (Fig. and data not shown), this interaction is likely to have some other important role in the control of cell growth and/or survival. The identification of RPA2 as a menin-interacting protein is thus an important step in the elucidation of menin's function as a tumor suppressor and may reveal new aspects of RPA's central role in the physiology of the cell.