SUMO (small ubiquitin-related modifier) modification is emerging as an important post-translational control in transcription. In general, SUMO modification is associated with transcriptional repression. Although many SUMO-modified transcription factors and co-activators have been identified, little is known about the mechanism underlying SUMOylation-elicited transcriptional repression. Here, we summarize that SUMO modification of transcription factors such as androgen receptor, glucocorticoid receptor, Smad4 and CBP [CREB (cAMP-response-element-binding protein)-binding protein] co-activator results in the recruitment of a transcriptional co-repressor Daxx, thereby causing transcriptional repression. Such a SUMO-dependent recruitment of Daxx is mediated by the interaction between the SUMO moiety of SUMOylated factors and Daxx SUMO-interacting motif. Interestingly, the transrepression effect of Daxx on these SUMOylated transcription factors can be relieved by SUMOylated PML (promyelocytic leukaemia) via altering Daxx partition from the targeted gene promoter to PML nuclear bodies. Because Daxx SUMO-interacting motif is a common binding site for SUMOylated factors, a model of competition for Daxx recruitment between SUMOylated PML and SUMOylated transcription factors was proposed. Together, our findings strongly suggest that Daxx functions as a SUMO reader in the SUMO-dependent regulation of transcription and subnuclear compartmentalization.
- compartmental regulation
- promyelocytic leukaemia (PML)
- small ubiquitin-related modifier (SUMO) reader
- transcriptional repression
SUMO (small ubiquitin-related modifier) modification regulates diverse cellular processes, including signal transduction, nuclear transport, genome instability and transcription [1–4]. Remarkably, over half of the identified SUMO substrates are transcriptional factors or co-regulators. In most cases, SUMOylation leads to transcriptional repression. Although the molecular mechanism by which conjugated SUMO elicits transcriptional repression largely remains unclear, recent studies suggest that SUMOylation is to promote the recruitment of transcriptional co-repressor(s), causing inhibition of transcription. For instance, SUMOylation of the transcriptional co-activator p300 and Elk-1 mediates the recruitment of HDAC6 (histone deacetylase 6) and HDAC2 respectively, leading to transcriptional repression [5,6]. Similarly, we recently demonstrated that a transcriptional co-repressor Daxx can be recruited by several SUMOylated factors for SUMO-dependent repression. Here, we summarize the current understanding of Daxx in SUMOylation-mediated transcriptional regulation.
The role of Daxx in transcriptional control
Daxx was initially identified by a yeast two-hybrid screen by using the death domain of the Fas receptor as bait and shown to act as a cytoplasmic molecule linking Fas signalling to the JNK (c-Jun N-terminal kinase) pathway via ASK1 (apoptosis signal-regulating kinase 1) . Besides its role in the cytoplasm, Daxx mainly functions as a transcriptional co-repressor in the nuclear compartment through its interaction with a growing number of transcription factors and other nuclear factors. Daxx was shown to associate with multiple proteins involved in transcriptional repression, such as HDAC1 , HDAC2 , DNMT1 (DNA methyltransferase 1) and its associated protein DMAP1 (DNMT1-associated protein) , a chromatin-associated factor DEK  and ATRX (α-thalassaemia/mental retardation syndrome X-linked), a protein associated with HP1 (heterochromatin protein 1) and part of a chromatin-remodelling complex [11–13]. Daxx was also reported to interact and suppress the activity of several transcription factors, including ETS1 (E twenty-six 1) , Pax3 [15,16], GR (glucocorticoid receptor) [17,18], p53 family proteins [19,20], MR (mineralocorticoid receptor) , AR (androgen receptor) , Smad4  and NF-κB (nuclear factor κB) [24,25].
Among Daxx-interacting transcription factors, we demonstrated that Daxx can associate with and repress AR transcriptional activity via inhibition of AR DNA binding activity and SUMOylation-dependent transrepression . We also showed that Daxx suppresses TGF-β (transforming growth factor-β)-induced Smad4-mediated transactivation via its interaction with Smad4 SUMOylation at Lys159 . Likewise, we recently reported that Daxx binds to SUMO-modified CBP [CREB (cAMP-response-element-binding protein)-binding protein] and suppresses CBP transcriptional potential via HDAC2 recruitment . More recently, we also demonstrated that Daxx inhibits GR transcriptional activity through binding to SUMOylated GR . These findings led us to propose that Daxx acts as a common SUMO reader in repressing the transcriptional potential of SUMOylated transcription factors (Figure 1).
The subnuclear compartmentalization of Daxx
In general, Daxx is found in the nucleoplasm and some distinct subnuclear structures via its interacting proteins as illustrated in Figure 1. Daxx was reported to associate with SUMO-modified PML (promyelocytic leukaemia), which serves as a key component of PML-NBs (PML nuclear bodies), also known as PODs (PML oncogenic domains) [8,17,28,29]. Daxx can also be targeted to the nucleolus through its interaction with a nucleolar protein MSP58 (microspherule protein of 58 kDa) . In addition, Daxx was shown to interact with CENP-C (centromere protein-C) for centromere localization during interface, and to co-localize with ATRX at the heterochromatin in the late S-phase of the cell cycle. These observations suggest that Daxx may have a different function when associated with distinct subnuclear compartments.
It is conceivable that alteration of Daxx subnuclear partition would influence its function. Indeed, Daxx-mediated transcriptional repression can be modulated by subnuclear compartmentalization. We showed that expression of MSP58 can sequester Daxx to the nucleolus, reversing Daxx-mediated transcriptional repression on GR . Similar to this scenario, Daxx-mediated transcriptional repression can be attenuated by SUMOylated PML proteins, whereas such a de-repression correlates well with the sequestration of Daxx from the nucleoplasm to the PML-NBs . Others  and we  have reported that wild-type but not SUMOylation-defective PML can relieve the repressive effect of Daxx on the transcriptional activity of GR and Pax3 via targeting Daxx to PML-NBs. Furthermore, arsenic trioxide treatment induces the SUMOylation extent of PML, causing a change of endogenous Daxx partitioning from GR-regulated gene promoter to PML-NBs and a relief of Daxx repression on GR-regulated gene expression [17,27]. These findings indicate that Daxx may exert its repressive effect outside of the PML-NB compartment. In line with this notion, Daxx was found to concentrate at condensed heterochromatin in PML−/− cells . More importantly, these observations suggest a notion that alteration of PML SUMOylation in response to different cellular milieu provides a novel cross-regulation of the activities of a variety of SUMOylated transcription factors by changing Daxx subnuclear partition.
The role of Daxx SUMO-interacting motif
Apparently, the capacity of Daxx binding to SUMOylated factors is important for its transcriptional control and PML-NB association. To dissect such regulatory events, we recently defined a SIM (SUMO-interacting motif), I733IVLSDSD740, within Daxx and demonstrated that Daxx SIM is essential for the recognition of the SUMO moiety of SUMOylated factors . Our findings indicate that Daxx SIM binds to a groove surface of SUMO-1 formed by four basic residues, Lys37, Lys39, Lys46 and Arg54, at four corners lined with hydrophobic and aromatic residues, which also serves as an interaction surface for other SUMO-binding sequences of RanBP2 (Ran-binding protein 2), thymine DNA glycosylase and PIASx [(protein inhibitor of activated STAT (signal transducer and activator of transcription) x] [30–32]. It should be noted that several basic and hydrophobic residues of SUMO-2, equivalent to these SUMO-1 residues in the groove area, have also been shown to be critical for SUMO-mediated transcriptional repression , suggesting that this specific groove area on SUMO molecules serves as a docking site for recruiting transcriptional co-repressor(s). In line with this notion, several factors were identified to contact these basic residues in the groove area of SUMO-2 and may contribute to the SUMO-mediated repression . As expected, the Daxx SIM was shown to be essential for targeting Daxx to PML-NBs via SUMOylated PML interaction and for its interaction with and repression of SUMOylated transcription factors such as Smad4, AR, GR and CBP co-activator . Our results reveal a mechanistic insight into Daxx–SUMO interaction and provide a common mode of Daxx activity in SUMO-dependent interaction and regulation.
Daxx can accumulate at condensed heterochromatin areas in PML−/− cells . Interestingly, such localization at condensed heterochromatin is completely lost with a Daxx SIM mutant , indicating the involvement of a SUMO-dependent recruitment of Daxx to the condensed heterochromatin. A recent study in fission yeast revealed that SUMO modification plays a direct role in modulating heterochromatin proteins such as HP1 homologue Swi6 and Suv39h homologue Clr4 . Whether these conserved heterochromatin proteins facilitate Daxx targeting to the condensed heterochromatin requires further investigation. Intriguingly, Daxx was also reported to co-localize with ATRX via its N-terminal domain at the heterochromatin only during cell cycle S-phase, which may involve the phosphorylation of ATRX by cellular kinase(s) . Thus, temporal phosphorylation and/or SUMOylation of distinct heterochromatin factors may facilitate Daxx localization at the heterochromatin via Daxx SIM-dependent and -independent manners.
The Daxx SIM is also essential for Daxx SUMO modification . Such a non-covalent SUMO interaction affecting substrate SUMOylation was also observed in TDG (thymine DNA glycosylase) . Currently, the underlying mechanism for such a regulation remains unknown. It is possible that Daxx SIM can enhance the recruitment of Ubc9 (ubiquitin-conjugating enzyme 9)–SUMO thioester via SUMO interaction, leading to a productive SUMOylation of Daxx. Notably, Daxx contains 15 lysine residues that are accessible for SUMO modification . Studies of a Daxx SUMOylation-defective mutant revealed that SUMOylation of Daxx is irrelevant to its PML-NB targeting and transcriptional repression on GR . Thus the capability of SUMO binding rather than conjugation is critical for Daxx in the SUMO-dependent transcriptional and compartmental regulation.
Is Daxx a SUMO reader for more SUMO-modified factors?
Given that Daxx can bind to a few SUMO-modified factors, is Daxx a SUMO reader for more SUMOylated factors? The answer is obvious from the list of Daxx-interacting proteins. Most of the Daxx-interacting proteins were used as bait in yeast two-hybrid screens and pulled out the Daxx C-terminal domain containing SIM. These include cellular factor Fas , GLUT4 , Pax3 , ETS1 , p53-family proteins [19,38], MR , CENP-C , DJ-1  and viral protein HIV type 1 p6 , adenovirus E1B 55-kDa , and Puumala hantavirus nucleocapsid protein PUUV-N . All these proteins have been shown either to be SUMO-modified or to interact with SUMO-1 or Ubc9 in yeast two-hybrid experiments, suggesting that Daxx may interact with these factors in a SUMOylation-dependent manner. For instance, Daxx can suppress the transcriptional activity of ETS-1  and recent reports showed that the region of ETS1 involved in Daxx interaction can be SUMO-modified [44,45], strongly implying that Daxx can repress the transcriptional activity of the ETS1 in a SUMOylation-dependent manner. Similar to this scenario, Daxx may target to the centromere via SUMOylation of CENP-C because CENP-C has the capacity to be modified by SUMO molecules . Furthermore, we also found that several nuclear factors other than PML can form nuclear speckles to recruit Daxx in a SUMOylation-dependent fashion (T.-T. Chao and H.-M. Shih, unpublished work). These observations suggest that Daxx functions as a SUMO reader for many SUMOylated factors involved in diverse cellular and viral processes.
Although Daxx has the capability to recognize the SUMO moiety of SUMOylated factors via its SIM, it is very unlikely that Daxx can bind all SUMOylated factors. Indeed, Daxx can interact with Smad4 through SUMO modification at Smad4 Lys159 but not Lys113 . Similarly, Daxx was reported to preferentially recognize SUMOylated PML at Lys160 and associate with the cell transformation phenotype of acute PML . These findings imply that the context of SUMO modification and the residues outside of Daxx SIM control the specificity of interaction.
The present studies indicate that Daxx is dynamically recruited by SUMOylated factors and involved in transcriptional regulation and compartmental distribution. The Daxx SIM is a common binding motif for diverse SUMOylated factors; it is thus conceivable that various SUMOylated factors can compete with each other for Daxx interaction, such as SUMOylated PML and SUMOylated GR, causing a cross-regulation of cellular events. We anticipate that more examples will be revealed. In summary, we suggest that Daxx functions as a SUMO reader in the SUMO-dependent regulation of transcription and subnuclear compartmentalization.
This work was supported by National Science Council grants 95-3112-B-001-020 and 95-2311-B-001-027, a thematic research programme of Academia Sinica (AS-95-TP-B02) and National Health Research Institute extramural grant EX95-9529NI (to H.-M.S.).
Regulation of Protein Function by SUMO Modification: A Biochemical Society Focussed Meeting held at Manchester Conference Centre, Manchester, U.K., 25–27 June 2007. Organized and Edited by R. Hay (Dundee, U.K.) and A. Sharrocks (Manchester, U.K.).
Abbreviations: AR, androgen receptor; ATRX, α-thalassaemia/mental retardation syndrome X-linked; CBP, CREB (cAMP-response-element-binding protein)-binding protein; CENP-C, centromere protein-C; DNMT1, DNA methyltransferase 1; ETS1, E twenty-six 1; GR, glucocorticoid receptor; HDAC, histone deacetylase; MR, mineralocorticoid receptor; MSP58, microspherule protein of 58 kDa; PML, promyelocytic leukaemia; PML-NB, PML nuclear body; SUMO, small ubiquitin-related modifier; SIM, SUMO-interacting motif; Ubc9, ubiquitin-conjugating enzyme 9
- © The Authors Journal compilation © 2007 Biochemical Society