Although the Sin3A-HDAC corepressor complex has been studied extensively, the roles of the various members of this complex are poorly understood. In this study, we have explored the functions of two members of this complex, the Sin3A-associated proteins SAP30L and SAP30, which share 70% sequence identity. We have discovered three types of interactions that illuminate the functional roles of these proteins. First, both SAP30L and SAP30 interact directly with the core histones 2A/2B. Second, we demonstrate that both proteins have intrinsic DNA-binding activity which is partly mediated through a novel N-terminal zinc-containing structure consisting of a C2CH module and a coordinated zinc ion. Binding to DNA is sequence independent and induces strong bending of the DNA. Third, we have identified a PI-binding site, a basic domain which binds monophosphorylated PIs specifically, adjacent to the zinc-binding element. Intriguingly, we have found that PI binding has a strong influence on the proteins' affinity for DNA in vitro, which leads us to suggest that the DNA binding is actually regulated by PIs. An increase in the concentration of PIs in the nucleus caused by hydrogen peroxide leads to reduced repression activity and cytoplasmic relocalization of SAP30L.
Previously, SAP30 has been assigned the role of a linker protein that mediates interactions of the Sin3-HDAC complex with various transcriptional repressors (e.g., YY1) or corepressors (e.g., N-CoR, CIR, and RBP1). Specifically, the interaction of SAP30 with N-CoR was demonstrated to occur through the N terminus, whereas the C terminus bound mSin3a (28
). Our results suggest a second function for the N terminus, which we found to bind DNA. Our results do not necessarily contradict the previous reports, because one can imagine that SAP30 can either bridge different multiprotein complexes or anchor a specific complex to nucleosomes, depending on the circumstances. Functional diversity of this kind is not unprecedented in Sin3A-associated proteins, since Fleischer et al. (13
) have observed at least three separate Sin3A-containing complexes. Furthermore, the C terminus also seems to carry multiple functions. Huang et al. (21
) identified SAP30 as a binding partner for the transcription factor YY1 and showed that it is able to enhance YY1-mediated repression in a dose-dependent manner. This interaction was mapped to the C-terminal region, i.e., the same region which also binds Sin3A, prompting the authors to suggest that the interactions of SAP30 with YY1 and Sin3A are mutually exclusive. Huang et al. (21
) suggested that HDAC activity could be brought to the YY1-SAP30 complex through a direct interaction of SAP30 with HDAC1 (61
). If SAP30L and SAP30 were to bind DNA and histones independently of Sin3A, it easy to envision that their N-terminal domains could participate in anchoring the YY1-SAP30-HDAC complex to chromatin to induce repression of transcription.
Sin3A by itself does not bind DNA or repress transcription but instead mediates gene silencing through the enzymes that it associates with (52
). Targeting of the Sin3A complex is carried out by DNA sequence-specific repressor proteins. Here, we show that SAP30L and SAP30 are able to bind DNA without any sequence specificity. This binding is dependent on an intact N terminus that contains a C2CH-type zinc module, whose disruption abolishes the DNA binding. Zinc fingers were originally identified as DNA-binding motifs, but they are now known to bind RNA, protein, and lipid substrates as well (6
). A zinc finger consists of two antiparallel β strands and an α helix, and the zinc ion is crucial for its stability. Usually, a single zinc finger does not bind DNA with very high affinity and can recognize only two or three base pairs, but when several, up to 60, zinc fingers are strung together, the group binds more tightly and can recognize longer DNA sequences. In the cases of both SAP30L and SAP30, only a single zinc-coordinating element was identified. Moreover, the stability of SAP30L was dependent on the zinc module since mutations in its zinc-coordinating residues led to rapid degradation of the protein. We have previously observed that N-terminally truncated SAP30L is poorly expressed in transient transfections, but this could be overcome by using MG132, a proteasome inhibitor, and now this can be explained by the loss of the stabilizing zinc-dependent module in the N terminus (57
). Zinc-binding domains are usually relatively short, i.e., 20 to 30 residues, and the spacing of 35 residues between the C2 and CH coordinating residues in SAP30L is unusually long. There is, however, a precedent for a large zinc-binding module, since THAP domains, which are conserved zinc-dependent modules capable of sequence-specific DNA binding, have a loop of 35 to 53 residues in the middle of the zinc-binding motif (8
). The THAP domain, however, contains other conserved elements in addition to the C2CH module, making it distinct from the zinc-binding motif in SAP30L.
The sequence-independent nature of the DNA binding rules out a sequence-specific targeting role for SAP30 and SAP30L and suggests a more general role in anchoring to nucleosomal/linker DNA. In addition, we demonstrated that this DNA binding results in strong bending of the DNA. Classical examples of proteins that bind and bend DNA in a sequence-independent manner are the HMG proteins (45
), which interact transiently with DNA. They are thought to antagonize histone H1 binding by competing for the same chromatin sites. Generally, they are thought to open up chromatin, although some HMG proteins may also compact chromatin (43
). We find interesting parallels between the HMG proteins and SAP30/SAP30L. Both are small and localized in the nucleus. Their domain structures are also similar, as both contain an N-terminal DNA-binding domain followed by an acidic region which contributes to histone interactions. This could imply functional similarity as well, and it seems likely that SAP30/SAP30L have roles in stabilizing the multiprotein complex on its target, increasing the availability of enzymatic targets to the complex, or promoting the recruitment of interacting proteins, the cumulative effect being increased repression activity.
Perhaps one of the most intriguing features of SAP30L and SAP30 is the presence of a PI-binding site. PIs are known to function in nuclear signaling, and local changes in PI concentrations are sensed by proteins with specific PI-binding domains, such as PH, ENTH, FYVE, and PHOX domains and lysine/arginine-rich patches (34
). A number of PI kinases and phosphatases translocate to the nucleus upon activation, and many PI species have been shown to be intranuclear (10
). Intervention of chromatin biology by signaling lipids is not unprecedented, since ATP-dependent chromatin-remodeling complexes, such as NURF, ISW2, INO80, and SWI/SNF, are also modulated by specific inositol polyphosphates, the cleavage products generated by PI-specific phospholipase C (50
). Additionally, another SWI/SNF-like chromatin remodeling complex, BAF, is targeted to chromatin and the nuclear matrix specifically by a PIP2-dependent mechanism upon lymphocyte activation (62
). Pf1, a recently identified nuclear binding partner for the corepressors mSin3A and TLE, has a PBR which binds specific monophosphoinositides (24
). The Sin3A-binding tumor suppressor ING2 binds PI(3), PI(4)P, and PI(5)P and shows PI(5)P-dependent association with chromatin and induction of p53-dependent apoptosis (16
). In response to cellular stress by UV irradiation or hydrogen peroxide, ING2 associates with chromatin through a PI(5)P-mediated mechanism (23
). Initially, the PHD domain of ING2 was reported to be sufficient for PI binding, but later, the PBR motif was demonstrated to be both necessary and sufficient on its own (16
). Even though the PBRs of Pf1 and ING2 were deemed critical for the binding activity and specificity, the preceding zinc-binding PHD domain contributed some specificity to the interaction with PIs (24
). SAP30 and SAP30L have a number of similarities with the PI-binding proteins Pf1 and ING. First, the domain architecture of SAP30/SAP30L resembles that of Pf1, with a zinc-binding element followed by a basic PI-binding module in both cases. Second, like SAP30/SAP30L, Pf1 and ING2 are part of the Sin3A complex. Third, all three proteins are nuclear and bind monophosphorylated PIs, albeit with different preferences. Fourth, in the cases of SAP30/SAP30L and ING2, the subcellular localization and chromatin association are modified by PI binding. However, in the cases of SAP30/SAP30L (but not in ING2), PI binding competes with DNA binding in vitro so that an increase in the concentration of monophosphorylated PIs causes SAP30L to detach from DNA. Furthermore, an increase in the concentration of nuclear PIs elicited with hydrogen peroxide leads to reduced repression activity and cytoplasmic relocalization of SAP30L. Although we note that these results are preliminary and mostly based on in vitro experiments, they suggest the intriguing possibility that changes in the concentration of nuclear monophosphorylated PIs may regulate transcriptional repression through SAP30/SAP30L in vivo. The site of PI binding was mapped to a region containing a motif previously shown to act as an NLS, and this motif is necessary for the PI-binding activity. However, the adjacent zinc-coordinating module also contributes to this interaction, a result that is in agreement with studies of other proteins (47
). The binding interface may reside on one side of the loop region of the zinc module, in a region which contains a stretch of basic residues. The specificity of PI binding is partly determined by the amino acid residue composition of the binding motif, since replacing the NLS motif with another basic motif (NoLS motif) in SAP30L led to changes in PI binding. Differences in binding specificity between different proteins are also evident. SAP30/SAP30L prefer PI(5)P over PI(3)P/PI(4)P, whereas Pf1 prefers PI(3)P, with some binding activity toward PI(3,5)P species (24
We propose a model in which SAP30L/SAP30 are actively involved in multiple protein-protein and protein-DNA interactions that modulate transcriptional repression. The domain structures of SAP30L/30 and the proposed model are depicted in Fig. , respectively. Briefly, we suggest that the DNA-binding activity plays a role in anchoring the Sin3A complex to nucleosomal and/or linker DNA in chromatin and that this binding is further strengthened by the interaction with core histone 2A/2B dimers. One consequence of DNA binding is bending of the DNA, and we envision that this leads to enhanced accessibility of nucleosomes and histone tails to deacetylating enzymes. Moreover, our results provide new evidence for the regulatory role played by nuclear PIs in transcriptional repression and relocalization of nuclear proteins.