Ikaros is SUMOylated in lymphocytes.
To identify Ikaros family interactors, a thymocyte library was used in a yeast two-hybrid screen with the Aiolos and Ikaros proteins as baits as described previously (24
). Three different types of cDNA were isolated multiple times, which encoded Aiolos and Ikaros interactors belonging to the SUMO pathway (Fig. ). One was SUMO1, the second was the component of the E1-SUMO activating enzyme Uba2, and the third was the E2-conjugating enzyme Ubc9. These proteins exhibited stronger interactions with Ikaros in the yeast two-hybrid system (Fig. ). Delineation of the Ikaros-Ubc9 interaction interface revealed their association through the N-terminal (Y6, exons 2 to 7 [amino acids 1 to 282], and Y9, exons 2 to 4 [amino acids 1 to 141]) but not the C-terminal region (Y4, amino acids 364 to 518 of exon 8) of the Ikaros protein (Fig. ).
FIG. 1. Ikaros interacts with components of the SUMO pathway and is SUMOylated in primary lymphocytes. (A) SUMO1 and the SUMO enzymes E1 (Uba2 component) and E2 (Ubc9) were isolated in a yeast two-hybrid screen with Aiolos and Ikaros. These were found to interact (more ...)
Protein SUMOylation occurs on the lysine of a ψKxE protein motif, where ψ represents the hydrophobic residue isoleucine (I), leucine (L), or valine (V) and x is any amino acid. Analysis of the Ikaros protein sequence revealed four putative SUMOylation sites, at positions K58 and K240, located in the N-terminal half of the protein, and K425 and K459, located in the C-terminal region (Fig. ). Of these, the first three represent perfect consensus motifs for SUMOylation. The Ikaros SUMOylation motifs containing K58 and K240 are highly conserved across species from skates to humans (16
). These two motifs were also found to be conserved in Aiolos at positions K63 and K256. The other two Ikaros family members, Helios and Eos, had only one SUMOylation motif at their N termini, corresponding to Ikaros K58.
Given the potential of Ikaros for SUMOylation, we tested whether the protein was modified in vivo in primary T cells. Nuclear extracts, prepared from murine thymocytes in the presence or absence of 1% SDS and the SUMO-isopeptidase inhibitor NEM, were examined for Ikaros protein expression. Three slower-migrating Ikaros protein species were detected only in the presence of NEM, indicating that a fraction of the protein is SUMOylated (Fig. , +NEM/SDS, bands M1, M2, and B1; see next section). Other Ikaros-interacting proteins, i.e., mSin3A and HDAC2, were also tested and found not to be SUMOylated in thymocytes (data not shown).
Mapping of the Ikaros SUMOylation sites.
We next investigated which of the Ikaros SUMOylation motifs were used in vivo. For this study, the putative lysine-SUMO acceptors were mutated to arginine singly or in combination. Vectors expressing an untagged version of Ikaros (Ik-1) or the SUMOylation motif mutant forms and GFP-SUMO1 were cotransfected in the epithelial 293T cell line. Ikaros proteins were immunoprecipitated with anti-Ikaros antibody and immunoblotted with Ikaros and GFP antibodies to better reveal the modified forms (Fig. , α-Ikaros and α-GFP). In addition to the main Ikaros protein band, three slower-migrating species were observed with Ikaros antibodies (Fig. , lanes 3 and 5), similar to those observed in thymocytes (Fig. ). Antibodies to GFP confirmed that these protein species were the result of Ikaros-GFP-SUMO1 conjugation (Fig. , right half).
FIG. 2. Ikaros SUMOylation sites established in nonlymphoid and lymphoid cells. (A) GFP-tagged SUMO1 was coexpressed with wild-type Ik-1 (WT) or the Ikaros mutant forms Ik-1K58R, Ik-1K240R, Ik-1K425R, Ik-1K459R, Ik-1K240,425,459R, and Ik-1K58,240,459R into 293T (more ...)
The first of the modified bands contains Ikaros protein that is monoSUMOylated at K58 as this disappears upon the K58 mutation (Fig. , band M1, lane 6; also Fig. ). The second contains Ikaros that is monoSUMOylated at K240 as this is also affected by the corresponding mutation (Fig. , band M2, lane 7; also Fig. ). Ikaros protein that is biSUMOylated at both the K58 and K240 sites runs at the third position, and this is equally affected by either of the single mutations (Fig. , band B1, lane 12; also Fig. ). In sharp contrast to the Ikaros mutations at K58 and K240, mutations at K425 and K459 had no effect on the slower-migrating forms (Fig. , lanes 8 and 9). Similar findings on SUMOylation of Ikaros and its mutant forms were obtained with the human cell line U2 OS (data not shown). Other members of the SUMO family, SUMO2 and -3, were also tested for the ability to be conjugated to Ikaros. A series of expression and immunoprecipitation studies demonstrated that all three of the SUMO proteins could be conjugated to Ikaros at positions K58 and K240 (data not shown).
The Ikaros sites of SUMOylation were confirmed in lymphoid cells, where Ikaros is normally expressed. The T-cell line NA1, derived from Ikaros-null mice, was infected with pMX-GFP retroviruses harboring either Ik-1, the Ik-1 K58 and 240R SUMOylation mutant form, or the parental vector as a control (13
). pMX-GFP-Ik-1 and pMX-GFP-Ik-1 K58 and 240R, but not the parental control vector, reconstituted Ikaros protein expression, which was detected as a major band at ~65 kDa (Fig. ). Reconstitution with Ik-1, but not with the SUMOylation mutant form of Ikaros, also provided three Ikaros-related slower-migrating protein species (Fig. ). These were similar in size to those observed in primary thymocytes that express Ikaros (Fig. ). This study verifies that the higher-molecular-weight Ikaros species observed in thymocytes and in an Ikaros-reconstituted T-cell line represent SUMOylated forms of the Ikaros protein. Furthermore, it shows that Ikaros lysines 58 and 240 are major sites for SUMOylation in vivo in both lymphoid and nonlymphoid cells.
Ikaros SUMOylation is actively regulated by SUMO isopeptidases and E3 ligases.
SUMOylation is a dynamic process that is controlled by opposing enzymatic activities. In line with previous observations, Ikaros SUMOylation was examined in the presence of differing amounts of SUMOylase and deSUMOylase.
GFP-SUMO1-Ikaros proteins were detected upon coexpression of Ik-1 and GFP-SUMO1 in 293T cells (Fig. , Contr.). Ikaros SUMOylation was progressively reduced when the level of the isopeptidase Senp1 was increased (Fig. , MT-Senp1). A similar effect on Ikaros deSUMOylation was also observed when the expression of another isopeptidase, Axam, was increased (data not shown).
FIG. 3. SUMOylation of Ikaros is controlled by the relative activities of SUMOylases and deSUMOylases. (A) Ik-1 and GFP-SUMO1 were coexpressed in 293T cells with increasing amounts of a Myc-tagged version of the deSUMOylase Senp1 (MT-Senp1) or empty vector as (more ...)
In contrast to the effect of isopeptidases, an increase in SUMO E3 ligase (PIASxα) increased Ikaros SUMOylation (Fig. ; see also Fig. and ). Immunoprecipitation studies revealed a strong interaction between Ikaros and the E3 ligase PIASxα and a weaker interaction with PIAS3 (Fig. ). The PIASxβ and PIAS1 family members were not detected within the Ikaros immunoprecipitates (Fig. ). Thus, Ikaros SUMOylation is regulated by the PIAS family of ligases, probably through Ikaros' specific interactions with two members of its family.
FIG. 5. Functional interactions of Ikaros with the SUMO pathway. (A) One microgram of Gal4 (lanes 1 and 2) or Gal4-Ik-1 (lanes 3 and 4) was cotransfected, together with the 5XGal4-tk-CAT reporter, Flag-PIASxα (3 μg), and GFP-SUMO1 (2 μg) (more ...)
FIG. 7. SUMOylation interferes with Ikaros' ability to associate with corepressors of transcription. (A) CMV2 Flag-tagged Ikaros (Flag-Ik-1) was cotransfected together with (+) or without (−) GFP-SUMO1 into 293T cells. Ikaros proteins were immunoprecipitated (more ...) Ikaros SUMOylation mutant forms are more potent transcriptional repressors.
We next examined whether SUMOylation of Ikaros can influence its activity as a repressor or activator of transcription. Ikaros' function as a transcriptional repressor can be revealed upon its tethering to the Gal4 DNA-binding domain (29
). Gal4-Ik-1 or the Gal4-Ik-1-SUMO mutant form was coexpressed with the 5XGal4-tk-CAT reporter in U2 OS or NIH 3T3 cells (Fig. and data not shown). As previously shown (29
), Gal4-Ik-1 repressed transcription (Fig. , WT). Significantly, the Ikaros K58R (data not shown), K240R, and K58 and 240R SUMOylation mutant forms were stronger repressors of transcription than the wild-type Ikaros protein, a fraction of which is likely SUMOylated (Fig. ). Interestingly, a similar increase in repression was detected with either of the single-mutant forms or the double-SUMOylation mutant form of Ikaros, suggesting that co-occupancy of these SUMOylation sites is required to relieve repression. As shown by Western blot analysis, the effect of the mutations in Ikaros on its repression activity was not due to a change in protein expression or stability (Fig. , bottom, and data not shown).
FIG. 4. SUMOylation of Ikaros specifically interferes with its ability to repress transcription. (A) Gal4 (−) or Gal4-Ik-1 fusion proteins (wild type [WT], K240R, and K58,240R) were cotransfected with the 5XGal4-tk-CAT reporter and GH control into U2 (more ...)
The effect of SUMOylation on Ikaros' ability to potentiate gene expression was also examined in a transient transcription assay (28
). Ikaros and its SUMO mutant forms were cotransfected with a 4XIk-tk-CAT reporter (32
) in U2 OS cells (Fig. ). Expression of either the wild-type Ikaros protein or its SUMOylation mutant forms provided a similar strong potentiation of reporter activity (Fig. ).
Taken together, these data support the model in which SUMOylation of Ikaros at both the K58 and K240 sites specifically negates its ability to repress gene expression.
The SUMO pathway directly regulates Ikaros' activity as a repressor.
Importantly, Ikaros mutations that prevent its SUMOylation enhance its potential as a transcriptional repressor (Fig. ). The role of the SUMO pathway in regulating Ikaros' repressor function was examined by varying the level of enzymes that have disparate effects on SUMOylation.
Gal4-Ik-1 and the 5XGal4-tk-CAT reporter were coexpressed with or without GFP-SUMO1 and the E3 ligase PIASxα in U2 OS cells (Fig. ). Cell lysates were prepared for CAT assays and for Western blot analysis. As previously shown (29
), expression of Gal-4-Ikaros represses the Gal-4 reporter (Fig. , lane 3). However, when Gal-4-Ikaros was coexpressed with PIASxα and GFP-SUMO1, high levels of reporter activity were observed and very little repression was obtained (Fig. , lane 4). Importantly, coexpression of PIASxα resulted in a significant increase in both the level and complexity of Ikaros SUMOylation, whereas the overall Ikaros protein expressed in these two experimental points was not significantly different (Fig. , Western blot assays, compare lanes 3 and 4; also data not shown). As expression of the E3 SUMOylase did not alter Gal4-dependent transcription, the observed effect is Ikaros dependent (Fig. , lane 2).
Given that enzymes that promote SUMOylation attenuate Ikaros' repression activity, we set out to test whether the converse was also true; that is, whether deSUMOylases enhance Ikaros-mediated repression. To demonstrate an increase in an already strong repression potential, we adapted the experimental design to achieve lower levels of Ikaros protein. As shown in Fig. , the amount of protein generated by 0.5 μg of Gal4-Ik-1 gave moderate repression (Fig. , lane 3). Coexpression of the deSUMOylase Senp1 increased Ikaros-mediated repression and the level of unmodified Ikaros protein, possibly by antagonizing its SUMOylation (Fig. , compare lanes 3 and 4). It is important to note that the level of repression provided by wild-type Ikaros and deSUMOylases was similar to that generated by the Ikaros SUMO mutant forms when expressed at similar levels (Fig. , compare lanes 4 and 8).
As shown in Fig. , coexpression of PIASxα and GFP-SUMO1 increased Ikaros SUMOylation, decreased the level of unmodified protein, and reduced its repression activity (Fig. , lane 7). In contrast, coexpression of PIASxα with the Ikaros K58 and K240R SUMOylation mutant form had no such effect on its strong repression activity (Fig. , lane 8).
Taken together, these studies demonstrate that SUMOylation can directly and inversely regulate transcriptional repression by Ikaros. The more SUMOylated Ikaros protein there is, the less repression it can provide.
Ikaros SUMOylation does not interfere with its nuclear localization to pericentromeric heterochromatin.
To determine the mechanism by which SUMOylation regulates Ikaros-mediated repression, we examined the effects on the protein's nuclear localization. In proliferating cells, Ikaros undergoes a dynamic redistribution into pericentromeric heterochromatin, an event that has been correlated in the past with its function as a repressor and silencer of gene expression (1
Untagged Ikaros and its SUMOylation mutant form (K58 and K240R) were cotransfected together with GFP-SUMO1 and Senp1 or PIASxα into NIH 3T3 fibroblasts where, as in cycling lymphocytes, Ikaros localizes into pericentromeric heterochromatin (4
). Immunofluorescence analysis with Ikaros antibodies indicated that most of both wild-type Ikaros and its SUMOylation mutant form localized into pericentromeric heterochromatin (Fig. ). The localization of Ikaros (and its single- or double-SUMOylation mutant form) remained unaffected by the presence or absence of GFP-SUMO1 (Fig. and data not shown) or by the presence of the isopeptidase Senp1 or the E3 ligase PIASxα (Fig. and data not shown).
FIG. 6. SUMOylation of Ikaros does not interfere with its localization into pericentromeric heterochromatin. (A) Wild-type Ik-1 (WT) and the SUMO double-mutant form (Ik-1 K58,240R) were coexpressed together with GFP-SUMO1 and the isopeptidase Senp1 or GFP-SUMO1 (more ...)
The distribution of GFP-SUMO1 was also revealed by fluorescence. In the absence of Ikaros or in the presence of the K58 and 240R double-SUMOylation mutant form, GFP-SUMO1 was widely distributed throughout the nucleus with only a small selective presence in pericentromeric heterochromatin. However, in the presence of wild-type Ikaros or a single-SUMOylation mutant (K58 or K240) form of Ikaros, most of the GFP-SUMO1 protein was detected in pericentromeric heterochromatin together with Ikaros (Fig. ). This is likely the result of interaction and covalent association between Ikaros and GFP-SUMO1 that dictates the overall nuclear distribution of GFP-SUMO1.
Taken together, these results indicate that Ikaros SUMOylation does not influence its activity as a repressor by altering its nuclear localization. SUMOylated Ikaros, which has lost its function as a repressor, can still localize into pericentromeric heterochromatin.
SUMOylation of Ikaros inhibits its interactions with transcriptional corepressors.
Ikaros has been proposed to regulate transcription by associating with chromatin remodeling and other transcription regulators (24
). SUMOylation of Ikaros may specifically interfere with its ability to associate with some of these factors, which are engaged in repression.
To examine this possibility, Flag-tagged Ik-1 was expressed in 293T cells without and with GFP-SUMO1, and its association with its previously reported interactors Mi-2β (NuRD complex), Sin3A, Sin3B, CtBP, and Brg-1 (Swi/Snf complex) was tested by Ikaros immunoprecipitation, followed by Western blotting (Fig. ). In the absence of GFP-SUMO1, a small fraction of Ikaros was SUMOylated and strong interactions with endogenously expressed Sin3A, Sin3B, CtBP, Mi-2β, and Brg-1 were detected (Fig. , − GFP-SUMO1). In sharp contrast, when GFP-SUMO1 was expressed, a major fraction of Ikaros was SUMOylated and its interaction with Sin3A, Sin3B, CtBP, and Mi-2β was greatly reduced (Fig. , + GFP-SUMO1). The effect was most pronounced with CtBP, which was almost absent in the Ikaros immunoprecipitate. The interactions of Ikaros with its corepressors were also evaluated upon coexpression of GFP-SUMO1 and the SUMO E3 ligase PIASxα. As shown in Fig. dramatic increase in Ikaros SUMOylation disrupted Sin3A interactions.
In sharp contrast to Ikaros interactions with Sin3A, Sin3B, Mi-2β, and CtBP, which were greatly reduced upon its SUMOylation, Ikaros association with the Swi/Snf ATPase Brg-1 was mildly affected (Fig. ). A previous established interaction between Ikaros and the simian virus 40 (SV40) T antigen (TAg) in 293T cells was also not influenced by SUMOylation (Fig. ).
These data strongly support a scenario in which SUMOylation of Ikaros specifically inhibits its interactions with HDAC-dependent (Sin3 and Mi-2β), as well as HDAC-independent (CtBP), corepressors of transcription. It does not, however, significantly affect its interactions with components of the Swi/Snf complex or with the viral protein SV40 TAg.