This study has shown that in addition to the effects elicited by altering the quantitative balance between the small Mafs and their heterodimeric partners, sumoylation is a critical determinant in the acquisition of the bidirectional (positive and negative) regulatory capacity of MafG. We found that MafG is specifically modified by SUMO-2/3 in mouse bone marrow. Transgenic analysis demonstrated that sumoylation-defective MafG loses its repressor activity. In contrast, transgenic complementation rescue analyses demonstrated that sumoylation is dispensable for the participation of MafG in transcriptional activation. These results suggest that SUMO modification promotes the incorporation of MafG into a transcriptional repressor complex, likely through the recruitment of an HDAC-containing moiety.
Reports on the sumoylation of transcription factors have shown that such modification can promote either activation or repression depending on the acceptor proteins involved (7
). However, most of the previous work was performed using transient overexpression in cultured cells, and thus, the biological significance of those phenomena was not entirely clear. Two genetic studies with Caenorhabditis elegans
have demonstrated that the sumoylation of polycomb group proteins is essential for their physiological repression of Hox genes (39
) and that the sumoylation of LIN-11, a LIM homeobox protein, is important for its function in uterine/vulval morphogenesis (2
To address the basic mechanistic concern regarding the in vivo consequences of sumoylation, the status of sumoylation was examined in mice. As far as we are aware, MafG is the first example of a transcription factor modified specifically by SUMO-2/3 in vivo. The effects of MafG sumoylation on transcriptional activity were examined with mice by comparing MafG to the nonsumoylated MafG mutant, MafG K14R. This represents one of the rare trials for testing the in vivo significance of SUMO modification on the function of mammalian transcription factors. However, these data also indicate that the link between sumoylation and its functional consequences should be further explored by manipulating the sumoylation efficiency of MafG in megakaryocytes (see below).
A consequence of small Maf sumoylation seemed to reside in the differential recruitment of larger transcriptional complexes. Based on the result that MafG-mediated repression was sensitive to TSA (see Fig. ) and a report that MafK, another small Maf protein, associated with HDAC1 and HDAC2 in undifferentiated MEL cells (1
), we envisage that sumoylated MafG achieves transcriptional repression by recruiting a corepressor complex containing HDAC(s) (Fig. , bottom panel). A surprising finding was that MafG K14R satisfied the prerequisites for competitive passive repression (i.e., as an inactive homodimer), namely, being sufficient in protein abundance and having proper nuclear localization (H. Motohashi, unpublished observation) and comparable DNA binding (Fig. , lanes 1 to 10), and yet the mutant molecule did not antagonize the activity of MafG-p45. An intriguing notion that emerges from this observation is that the inhibition of MafG-p45 activity by excessive MafG is not attained by a simple competition with the MafG homodimer but likely involves changes at the structural chromatin level. The permissive chromatin environment, once generated by the MafG-p45 heterodimer with a coactivator complex (Fig. , top panel), might be maintained even when a homodimer of nonsumoylated MafG competes with the activating heterodimer for the target site (Fig. , middle panel). While we originally thought that MafG behaved as a typical passive repressor, the data presented here strongly suggest that MafG becomes qualified as an active repressor via sumoylation.
FIG. 9. Model of the functional conversion of MafG by sumoylation. MafG activates transcription by forming a heterodimer with p45, thereby recruiting a coactivator complex containing CBP (top panel). MafG homodimer competes with the heterodimer for DNA binding (more ...)
A series of in vitro analyses revealed that MafG sumoylation did not dramatically affect the DNA binding or formation of a homodimer or a heterodimer with p45. The DNA binding of homodimers with or without SUMO modification was easily resolved in EMSA. On the contrary, EMSA did not provide conclusive data on the heterodimer binding, since the shifted band of sumoylated heterodimer displayed a mobility similar to that of the nonsumoylated heterodimer, which is often the case with larger proteins (14
). Alternatively, a DNA pull-down assay successfully demonstrated the comparable DNA binding of two species of heterodimer. Our next critical question was whether sumoylated MafG binds to MARE as a homodimer or as a heterodimer when it exerts transcriptional repression in vivo. Our preliminary result indicated that the efficiency of MafG sumoylation by PIASy seemed to be decreased in the presence of p45 (Motohashi, unpublished observation), which suggested that MafG is preferentially sumoylated in the form of a homodimer, thereby acquiring repressor activity. Therefore, we contemplate that sumoylated MafG has little chance of existing in the form of a heterodimer with p45 but rather preferentially exists in the form of a homodimer in vivo.
Identification of the SUMO E3 ligase for MafG operating in vivo is an important future objective, since it will help to elucidate the mechanism of MafG sumoylation. This will also provide a strategy for inhibiting the modification of MafG, which would constitute another convincing method for investigating its significance. A recent report demonstrated a rhythmic sumoylation of BMAL1 synchronizing with the circadian cycle (3
), which implies an interesting regulatory mechanism for sumoylation. It would be significant if the ratio of sumoylated to nonsumoylated MafG was regulated in a tissue-specific manner and/or in response to various stimuli through differential activation of E3 ligase. PIASy was originally identified as an interacting partner of LEF1 (27
) and found to be effective in sumoylating MafG in cultured cells. Therefore, we expect that examination of the status of MafG sumoylation in the megakaryocytes of PIASy-deficient mice (26
) may reveal an important clue that could lend credence to this proposed mechanism.
An essential concept requiring further clarification is the physiological role that sumoylation serves in the function of MafG. There are few proteins whose functional alteration by sumoylation has been assigned a specific biological role. Sp3 and Elk1 are good examples of proteins that activate or repress transcription depending on their biological context, and for each protein it was clearly shown that SUMO modification is important for their repressive effects (25
). In the case of MafG, our present data seem to suggest that the frequency of PPF is finely tuned by positive and negative MARE-dependent regulation exerted by nonsumoylated and sumoylated MafG, respectively, since the PPF ratios observed in mafG−/−
::G1HRD-His-MafG K14R mice (lines 11, 45, and 71) were slightly higher than those of mafG−/−
::G1HRD-His-MafG mice (Fig. , panel E). We surmise that the higher PPF ratios observed in megakaryocytes containing exclusively nonsumoylatable MafG could result from the loss of negative regulation normally exerted by the sumoylated population of MafG. Our preliminary data also imply that MafG represses the expression of megakaryocyte-specific MARE-dependent genes in immature hematopoietic cells through sumoylation (Motohashi, unpublished observation). A comparison between immature cells in mafG
-null mutant bone marrow supplemented with MafG or MafG K14R would be a good starting point for demonstrating the physiological contribution of MafG sumoylation.
In summary, we previously found that small Maf proteins are regulated quite specifically at the transcriptional level (12
) and that their abundance is a critical determinant of positive and negative gene regulation through MARE (20
). This study revealed that the sumoylation of MafG is an indispensable posttranslational modification that controls the negative regulatory capacity of MafG. We envisage that small Maf sumoylation may be mechanistically utilized in a similar context where CNC partner molecules other than p45 are involved. Thus, another layer of complexity and specificity in transcriptional control by the network of heterodimeric CNC-small Maf and homodimeric small Maf interactions is achieved through the effect of this posttranslational modification of small Maf proteins.