Previously, we identified the novel Max-like protein Mlx. Here we describe the novel partner protein for Mlx, termed Mondo. The predicted primary amino acid sequence of the MondoA protein reveals a large BHLHZip protein with two regions of sequence similarity at the amino- and carboxy-terminal regions that are conserved among Homo sapiens, D. melanogaster, and C. elegans mondo homologues. The carboxy-terminal conserved domain is striking because it is conserved among all of the mondo and mlx family members. This suggests that mlx and mondo genes may have a common evolutionary ancestor and that this region may be crucial for aspects of Mlx and Mondo function. The amino terminus of the MondoA protein has at least two functional domains, one that regulates cytoplasmic localization of MondoA-Mlx heterocomplexes and another involved in transactivation (see below). Thus, the mlx and mondo genes form a new family of cytoplasmically localized BHLHZip transcription factors.
Several lines of evidence support the physiological relevance of the interaction between MondoA and Mlx. First, 39 of 40 positive clones in our two-hybrid screen to detect Mlx-interacting proteins included the BHLHZip domain of murine MondoA. Second, murine MondoA did not interact with other members of the Max network in a directed two-hybrid screen. Third, MondoA-Mlx heterodimers bound specifically to CACGTC sites and activated transcription from CACGTG-dependent reporters in a manner that required dimerization and DNA binding. Fourth, MondoA and Mlx localize to the cytoplasm and redistribute to the nucleus together. Finally, we have detected association between MondoA and Mlx in cytoplasmic extracts of P19 cells by coimmunoprecipitation and DNA pulldown assays. Together, these experiments provide compelling evidence that MondoA-Mlx heterocomplexes exist in vivo.
Regulated nuclear import of many transcription factors is a well-established biological control mechanism (51
). MondoA-Mlx heterodimers localize predominantly to the cytoplasm; however, we propose that these proteins function in the nucleus. Our strongest experimental support for this proposal comes from targeting the heterocomplex to the nucleus with a strong NLS grafted onto the amino terminus of Mlx. The targeted heterocomplex activated transcription from CACGTG-driven reporter genes. We have not yet identified the conditions or cell types in which wild-type MondoA-Mlx heterocomplexes accumulate in the nucleus, and therefore it is possible that the transcriptional activation attributed to MondoA resulted from artificial nuclear targeting. However, as we observed both nuclear localization and transcriptional activation with wild-type Mlx and a MondoA mutant that lacks the CLD (Δ322MondoA), it is unlikely that the 9-amino-acid NLS supplies an artificial activation domain to Mlx. Furthermore, additional evidence supports a nuclear function for MondoA-Mlx heterodimers: (i) MondoA-Mlx heterodimers shuttle through the nucleus and therefore are not constitutively cytoplasmic proteins, (ii) the DBDs of both MondoA and Mlx are highly conserved across species and are capable of specific CACGTG binding, and (iii) the amino terminus of MondoA has a potent autonomous TAD. Therefore, we propose that MondoA-Mlx heterocomplexes function as transcriptional activators whose nuclear activity is under tight control via their cytoplasmic localization. A signal(s), as yet unidentified, may be required to redistribute the heterocomplex from the cytoplasm to the nucleus. Alternatively, MondoA-Mlx heterodimers may shuttle rapidly through the nucleus and not reach high levels at steady state. We are attempting to distinguish between these two mechanisms.
While we propose a nuclear function for Mondo-Mlx heterodimers, we cannot rule out the possibility that the heterocomplex has additional activities in the cytoplasm. A cytoplasmic function for MondoA might include the sequestration of Mlx, which would limit the access of Mlx to other nuclear dimerization partners, such as Mad1 and Mad4. When expressed on its own, however, Mlx localized to the cytoplasm (A. L. Eilers, unpublished data), suggesting that cytoplasmic localization of Mlx is an intrinsic property and is independent of Mondo. However, at this time we cannot rule out a dominant-negative role for MondoA in regulating the access of Mlx to as-yet-unidentified transcription factors. Mondo-Mlx heterodimers may have cytoplasmic functions, but as our evidence points strongly to a nuclear function for the heterocomplex, it seems unlikely that it has exclusively cytoplasmic functions.
Other members of the Max network BHLHZip superfamily are primarily nuclear but can also be regulated by subcellular localization, and thus this type of regulation is not unique to Mlx and Mondo. For example, though c-Myc is usually nuclear, it is sequestered in the cytoplasm of Purkinje cells via an interaction with the CDR-2 protein. This interaction is thought to block c-Myc-induced cell death in Purkinje cells (43
To decipher the regulation of cytoplasmic localization by Mondo-Mlx heterodimers, we identified a CLD in MondoA. The CLD (amino acids 125 to 321) provides a strong cytoplasmic localization signal that is capable of overriding the activity of both Gal4 and the SV40 large-T-antigen NLS. Treatment of cells with leptomycin B resulted in the nuclear accumulation of Mlx and MondoA. Leptomycin B is an inhibitor of CRM-dependent nuclear export (54
), suggesting that the CLD of MondoA functions via a known export pathway to maintain MondoA-Mlx heterocomplexes in the cytoplasm. The CLD does not show obvious sequence similarity to known export signals (39
) and likely constitutes a novel regulatory domain.
Wild-type MondoA, N-terminal deletions of MondoA lacking the CLD, and Mlx all localize to the cytoplasm when expressed individually (data not shown), suggesting that additional sequences in both MondoA and Mlx regulate their subcellular localization. When coexpressed, wild-type Mlx and MondoA also localize to the cytoplasm. However, when Mlx is coexpressed with any of the MondoA deletions lacking the CLD, they localize predominantly to the nucleus. Therefore, in the absence of the CLD, heterodimerization of MondoA and Mlx results in their nuclear localization. It is possible that heterodimerization blocks the cytoplasmic localization functions of monomeric MondoA and Mlx, or heterodimerization may unmask a dominant NLS(s) in either MondoA, Mlx, or both proteins. It is also possible that monomers of MondoA and Mlx are rapidly degraded in the nucleus. However, as we can detect NLSMlx in the nucleus when it is expressed alone (data not shown), we consider this possibility less likely. Therefore, our data suggest that in order for MondoA and Mlx to accumulate in the nucleus multiple negative regulatory steps must be overcome; the function of the CLD of MondoA must be inactivated, perhaps by extracellular signaling, and the two proteins must heterodimerize. Interestingly, a similar dependence on heterodimerization for nuclear accumulation has been observed for the Drosophila
clock proteins PER and TIM (46
The amino terminus of MondoA also contains a TAD between amino acids 322 and 445 which is extremely active when fused to the Gal4DBD. This activity is much greater than that seen with the wild-type MondoA protein, possibly due to the high affinity of the Gal4 homodimer for DNA. The MondoA TAD is rich in serine, threonine, and proline, a characteristic shared with TADs of the PAX family (49
). In spite of the other functional similarities between Myc and Mondo, their TADs have little specific sequence similarity. However, a segment of c-Myc's activation domain, amino acids 41 to 103, is proline rich (33
), suggesting that there may be functional similarities shared by Myc and Mondo transactivation.
We have described a new transcription factor network whose center is Mlx. Like Max, Mlx is a common partner for both transcriptional activators, the Mondo proteins, and repressors, the Mad proteins. Given the similarities between the Max network and the Mlx network, it is likely that the Mlx network will also function in the regulation of cell proliferation and differentiation. Our preliminary data suggest that transcriptional cross talk between the Max network and the Mlx network regulates their activity. For example, in Mad1 null mice, Mlx is upregulated in the granulosa cells of the ovary, and Drosophila Myc appears to regulate Drosophila Mondo mRNA levels (data not shown). Thus, it is likely that a web of regulatory interactions between the Max and Mlx networks regulates the functions of these two transcription factor families. Future experiments will be directed at understanding the complicated interplay of these two networks.