Members of the MORF family are implicated as regulators of cellular senescence (7
). MORFs have motifs common to transcription factors and localize to the nucleus, suggesting that they likely function in transcriptional regulation (7
). In support of this hypothesis, we demonstrate that MRG15, MRGX, and MORF4 associate with multiple corepressors. MORF4, MRGX, and MRG15 all associate with mSin3A and TLE, whereas only MRG15 interacts with Pf1. Therefore, the MORFs are likely to have both shared and unique functions. We have uncovered a restricted interaction between MRG15 and Pf1, but we cannot rule out the possibility of the existence of specific partners for MORF4 and MRGX.
MORF4, MRGX, and MRG15 all bind mSin3A and TLE, suggesting that one shared characteristic of the MORFs is to contribute to the functions of these abundant corepressor complexes. TLE has been shown to interact with mSin3A (17
), and we report that MRG15 and MORF4 interact with the HID of mSin3A. As such, we propose that the interaction between the MORFs and TLE is bridged by mSin3A as opposed to being direct and independent. Repression by mSin3A is thought to occur close to the site of targeting (28
), whereas TLE can likely repress transcription at a distance (18
). Whereas both mSin3A and TLE depend, in part, on HDAC activity to drive transcriptional repression (11
), TLE also binds to the N-terminal tails of the histones to repress transcription (39
). Therefore, a ternary complex between mSin3A, TLE, and the different MORFs may be capable of repressing transcription by different mechanisms at short and long ranges. As TLE and the MORFs do not appear to be stoichiometric components of an mSin3A complex (Fleischer and Ayer, submitted) we propose that this ternary complex will be involved only in regulating a subset of mSin3A-dependent genes. Pf1 can also tether TLE to mSin3A, but surprisingly, Pf1 overexpression did not alter the interaction between MRG15, TLE, and mSin3A (Fig. ), suggesting that a quaternary complex does not form. The binding sites for both MRG15 and TLE are located between amino acids 102 and 273 of Pf1, raising the possibility that their interaction with Pf1 is mutually exclusive.
Pf1 binding is restricted to MRG15, expanding the number of regulatory complexes formed by MRG15 relative to those formed by MRGX and MORF4. MRG15 and Pf1 have independent binding sites on mSin3A, suggesting the formation of a ternary complex. In the presence of Pf1, Gal4-MRG15 repression is reduced whereas repression by the other MORFs is unaffected (Fig. ). These data are consistent with differential roles for MRG15/Pf1/mSin3A complexes and MRG15 (or MORF4 or MRGX)/mSin3A/TLE complexes in transcriptional regulation. Because MORFs are implicated in senescence, we examined pf1 mRNA levels in young and old fibroblasts and found that pf1 expression increased in senescent cells compared to that in presenescent cells (data not shown). Therefore, one function of Pf1 may be to specialize MRG15 transcriptional regulatory complexes during cellular senescence.
Transcriptional repression by Gal4-MRG15 correlated with its binding to mSin3A (Fig. ); however, this activity was insensitive to the HDAC inhibitor trichostatin A (TSA) (data not shown). Therefore, it is likely that MRG15 can interact with HDAC-independent corepressors in addition to the HDAC-dependent mSin3A. Transcriptional repression by mSin3A is not solely dependent on HDAC activity (24
), suggesting the possibility that mSin3A itself may provide MRG15 with an HDAC-independent repression capability. Whether the HDAC-independent functions of MRG15 can be attributed to interactions with mSin3A, TLE, or another at-present-unidentified corepressor remains to be determined.
The MORFs are found within multiple transcriptional complexes; however, their functions within these complexes are currently unknown. All MORFs have putative protein-protein interaction motifs, including HLH and leucine zipper domains (7
). MORFs may contribute to the stability or assembly of the corepressor complex or mediate interactions between their different corepressor complexes and other transcriptional regulators. Furthermore, only MRG15 interacts with Pf1, suggesting that it has unique functions compared to MORF4 and MRGX. In addition to binding Pf1, MRG15 is the only MORF with a chromodomain (7
). The chromodomain of MRG15 is most closely related to the chromodomain of the male specific lethal (MSL) proteins (8
). Within the MSL-3 protein, the chromodomain has been shown to bind roX2
RNA and contribute to the regulation of dosage compensation (2
). These findings raise the intriguing possibility that MRG15-containing complexes also regulate transcription via interactions with RNA.
In addition to binding the mSin3A and TLE complexes, MRG15 binds the Rb transcriptional corepressor (36
). MRG15 relieved E2F-mediated repression of the myb
promoter, presumably by affecting Rb function (36
). Whether this effect was unique to MRG15 or shared with the other MORFs was not tested. Transcriptional repression by Rb depends in part on association with HDACs (12
), and recent reports demonstrate that mSin3A is tethered to Rb through interactions with SAP30 and RBP1 (34
). As such, it is possible that MRG15 is recruited to Rb indirectly by interactions with the mSin3A complex.
While most of the existing data suggest a role for the MORFs in transcriptional repression, recent data from studies of S. cerevisiae
suggest a potential role in activation as well. For example, the yeast homolog of the MORF proteins, Eaf3p, is a component of the NuA4 HAT complex (8
). The catalytic acetyltransferase subunit of the NuA4 complex is Esa3p, and Esa3p is the yeast homolog of mammalian TIP60 acetyltransferase. Therefore, the TIP60 HAT complex and the NuA4 complex might function analogously. Not all of the components of the TIP60 complex have been characterized, but these findings suggest that the MORFs are likely to be components of the human TIP60 complex and contribute to its transcriptional activation function (8
mSin3A, TLE, and Rb are abundant transcriptional corepressors that control diverse cellular programs. Interaction between the MORFs and these corepressors implies that they have widespread functions as well. Unraveling the contribution of each MORF will require a careful analysis of the distinct MORF-containing corepressor complexes and the transcriptional targets of these complexes. Recently, SIN3 complexes that contain the yeast orthologs of TLE, HDAC1, MRG15, and Pf1 were purified from S. cerevisiae
). As such, S. cerevisiae
provides an attractive model system to address the functional roles of MRG15-containing complexes in transcriptional regulation.