Structural and functional characterization of the phosphorylation-dependent interaction between PML and SUMO1.

PML and several other proteins localizing in PML-nuclear bodies (PML-NB) contain phosphoSIMs (SUMO-interacting motifs), and phosphorylation of this motif plays a key role in their interaction with SUMO family proteins. We examined the role that phosphorylation plays in the binding of the phosphoSIMs of PML and Daxx to SUMO1 at the atomic level. The crystal structures of SUMO1 bound to unphosphorylated and tetraphosphorylated PML-SIM peptides indicate that three phosphoserines directly contact specific positively charged residues of SUMO1. Surprisingly, the crystal structure of SUMO1 bound to a diphosphorylated Daxx-SIM peptide indicate that the hydrophobic residues of the phosphoSIM bind in a manner similar to that seen with PML, but important differences are observed when comparing the phosphorylated residues. Together, the results provide an atomic level description of how specific acetylation patterns within different SUMO family proteins can work together with phosphorylation of phosphoSIM's regions of target proteins to regulate binding specificity.

Identification of a non-covalent ternary complex formed by PIAS1, SUMO1, and UBC9 proteins involved in transcriptional regulation.

Post-translational modifications with ubiquitin-like proteins require three sequentially acting enzymes (E1, E2, and E3) that must unambiguously recognize each other in a coordinated fashion to achieve their functions. Although a single E2 (UBC9) and few RING-type E3s (PIAS) operate in the SUMOylation system, the molecular determinants regulating the interactions between UBC9 and the RING-type E3 enzymes are still not well defined. In this study we use biochemical and functional experiments to characterize the interactions between PIAS1 and UBC9. Our results reveal that UBC9 and PIAS1 are engaged both in a canonical E2·E3 interaction as well as assembled into a previously unidentified non-covalent ternary complex with SUMO as evidenced by bioluminescence resonance energy transfer, nuclear magnetic resonance spectroscopy, and isothermal titration calorimetry studies. In this ternary complex, SUMO functions as a bridge by forming non-overlapping interfaces with UBC9 and PIAS1. Moreover, our data suggest that phosphorylation of serine residues adjacent to the PIAS1 SUMO-interacting motif favors formation of the non covalent PIAS1·SUMO·UBC9 ternary complex. Finally, our results also indicate that the non-covalent ternary complex is required for the known transcriptional repression activities mediated by UBC9 and SUMO1. Taken together, the data enhance our knowledge concerning the mode of interaction of enzymes of the SUMOylation machinery as well as their role in transcriptional regulation and establishes a framework for investigations of other ubiquitin-like protein systems.

Differential Roles of PML Isoforms.

The tumor suppressor promyelocytic leukemia (PML) protein is fused to the retinoic acid receptor alpha in patients suffering from acute promyelocytic leukemia (APL). Treatment of APL patients with arsenic trioxide (As2O3) reverses the disease phenotype by a process involving the degradation of the fusion protein via its PML moiety. Several PML isoforms are generated from a single PML gene by alternative splicing. They share the same N-terminal region containing the RBCC/tripartite motif but differ in their C-terminal sequences. Recent studies of all the PML isoforms reveal the specific functions of each. Here, we review the nomenclature and structural organization of the PML isoforms in order to clarify the various designations and classifications found in different databases. The functions of the PML isoforms and their differential roles in antiviral defense also are reviewed. Finally, the key players involved in the degradation of the PML isoforms in response to As2O3 or other inducers are discussed.

Requirement of PML SUMO interacting motif for RNF4- or arsenic trioxide-induced degradation of nuclear PML isoforms.

PML, the organizer of nuclear bodies (NBs), is expressed in several isoforms designated PMLI to VII which differ in their C-terminal region due to alternative splicing of a single gene. This variability is important for the function of the different PML isoforms. PML NB formation requires the covalent linkage of SUMO to PML. Arsenic trioxide (As2O3) enhances PML SUMOylation leading to an increase in PML NB size and promotes its interaction with RNF4, a poly-SUMO-dependent ubiquitin E3 ligase responsible for proteasome-mediated PML degradation. Furthermore, the presence of a bona fide SUMO Interacting Motif (SIM) within the C-terminal region of PML seems to be required for recruitment of other SUMOylated proteins within PML NBs. This motif is present in all PML isoforms, except in the nuclear PMLVI and in the cytoplasmic PMLVII. Using a bioluminescence resonance energy transfer (BRET) assay in living cells, we found that As2O3 enhanced the SUMOylation and interaction with RNF4 of nuclear PML isoforms (I to VI). In addition, among the nuclear PML isoforms, only the one lacking the SIM sequence, PMLVI, was resistant to As2O3-induced PML degradation. Similarly, mutation of the SIM in PMLIII abrogated its sensitivity to As2O3-induced degradation. PMLVI and PMLIII-SIM mutant still interacted with RNF4. However, their resistance to the degradation process was due to their inability to be polyubiquitinated and to recruit efficiently the 20S core and the ß regulatory subunit of the 11S complex of the proteasome in PML NBs. Such resistance of PMLVI to As2O3-induced degradation was alleviated by overexpression of RNF4. Our results demonstrate that the SIM of PML is dispensable for PML SUMOylation and interaction with RNF4 but is required for efficient PML ubiquitination, recruitment of proteasome components within NBs and proteasome-dependent degradation of PML in response to As2O3.

Identification of estrogen receptor ß as a SUMO-1 target reveals a novel phosphorylated sumoylation motif and regulation by glycogen synthase kinase 3ß.

SUMO conjugation has emerged as a dynamic process in regulating protein function. Here we identify estrogen receptor ß (ERß) to be a new target of SUMO-1. ERß SUMO-1 modification occurs on a unique nonconsensus sumoylation motif which becomes fully competent upon phosphorylation of its contained serine residue, which provides the essential negative charge for sumoylation. This process is further regulated by phosphorylation of additional adjacent serine residues by glycogen synthase kinase 3ß (GSK3ß), which maximizes ERß sumoylation in response to hormone. SUMO-1 attachment prevents ERß degradation by competing with ubiquitin at the same acceptor site and dictates ERß transcriptional inhibition by altering estrogen-responsive target promoter occupancy and gene expression in breast cancer cells. These findings uncovered a novel phosphorylated sumoylation motif (pSuM), which consists of the sequence ψKXS (where ψ represents a large hydrophobic residue) and which is connected to a GSK3-activated extension that functions as a SUMO enhancer. This extended pSuM offers a valuable signature to predict SUMO substrates under protein kinase regulation.

Role of SUMO in RNF4-mediated promyelocytic leukemia protein (PML) degradation: sumoylation of PML and phospho-switch control of its SUMO binding domain dissected in living cells.

Promyelocytic leukemia protein (PML) is a tumor suppressor acting as the organizer of subnuclear structures called PML nuclear bodies (NBs). Both covalent modification of PML by the small ubiquitin-like modifier (SUMO) and non-covalent binding of SUMO to the PML SUMO binding domain (SBD) are necessary for PML NB formation and maturation. PML sumoylation and proteasome-dependent degradation induced by the E3 ubiquitin ligase, RNF4, are enhanced by the acute promyelocytic leukemia therapeutic agent, arsenic trioxide (As2O3). Here, we established a novel bioluminescence resonance energy transfer (BRET) assay to dissect and monitor PML/SUMO interactions dynamically in living cells upon addition of therapeutic agents. Using this sensitive and quantitative SUMO BRET assay that distinguishes PML sumoylation from SBD-mediated PML/SUMO non-covalent interactions, we probed the respective roles of covalent and non-covalent PML/SUMO interactions in PML degradation and interaction with RNF4. We found that, although dispensable for As2O3-enhanced PML sumoylation and RNF4 interaction, PML SBD core sequence was required for As2O3- and RNF4-induced PML degradation. As confirmed with a phosphomimetic mutant, phosphorylation of a stretch of serine residues, contained within PML SBD was needed for PML interaction with SUMO-modified protein partners and thus for NB maturation. However, mutation of these serine residues did not impair As2O3- and RNF4-induced PML degradation, contrasting with the known role of these phosphoserine residues for casein kinase 2-promoted PML degradation. Altogether, these data suggest a model whereby sumoylation- and SBD-dependent PML oligomerization within NBs is sufficient for RNF4-mediated PML degradation and does not require the phosphorylation-dependent association of PML with other sumoylated partners.

Evolution of C2H2-zinc finger genes and subfamilies in mammals: species-specific duplication and loss of clusters, genes and effector domains.

BACKGROUND: C2H2 zinc finger genes (C2H2-ZNF) constitute the largest class of transcription factors in humans and one of the largest gene families in mammals. Often arranged in clusters in the genome, these genes are thought to have undergone a massive expansion in vertebrates, primarily by tandem duplication. However, this view is based on limited datasets restricted to a single chromosome or a specific subset of genes belonging to the large KRAB domain-containing C2H2-ZNF subfamily. RESULTS: Here, we present the first comprehensive study of the evolution of the C2H2-ZNF family in mammals. We assembled the complete repertoire of human C2H2-ZNF genes (718 in total), about 70% of which are organized into 81 clusters across all chromosomes. Based on an analysis of their N-terminal effector domains, we identified two new C2H2-ZNF subfamilies encoding genes with a SET or a HOMEO domain. We searched for the syntenic counterparts of the human clusters in other mammals for which complete gene data are available: chimpanzee, mouse, rat and dog. Cross-species comparisons show a large variation in the numbers of C2H2-ZNF genes within homologous mammalian clusters, suggesting differential patterns of evolution. Phylogenetic analysis of selected clusters reveals that the disparity in C2H2-ZNF gene repertoires across mammals not only originates from differential gene duplication but also from gene loss. Further, we discovered variations among orthologs in the number of zinc finger motifs and association of the effector domains, the latter often undergoing sequence degeneration. Combined with phylogenetic studies, physical maps and an analysis of the exon-intron organization of genes from the SCAN and KRAB domains-containing subfamilies, this result suggests that the SCAN subfamily emerged first, followed by the SCAN-KRAB and finally by the KRAB subfamily. CONCLUSION: Our results are in agreement with the "birth and death hypothesis" for the evolution of C2H2-ZNF genes, but also show that this hypothesis alone cannot explain the considerable evolutionary variation within the subfamilies of these genes in mammals. We, therefore, propose a new model involving the interdependent evolution of C2H2-ZNF gene subfamilies.

Sumoylation of the transcriptional intermediary factor 1beta (TIF1beta), the Co-repressor of the KRAB Multifinger proteins, is required for its transcriptional activity and is modulated by the KRAB domain.

Small ubiquitin-related modifier (SUMO) has emerged as a key post-translational modulator of protein functions. Here we show that TIF1beta, a developmental regulator proposed to act as a universal co-repressor for the large family of KRAB domain-containing zinc finger proteins, is a heavily SUMO-modified substrate. A combined analysis of deletion and punctual mutants identified TIF1beta as a multilysine acceptor for SUMO which specifically targets six lysine residues (Lys(554), Lys(575), Lys(676), Lys(750), Lys(779), and Lys(804)) within the TIF1beta C-terminal repressive region. Reporter gene assays indicate that TIF1beta requires SUMO-modification for its repressive activity. Indeed, sumoylation-less mutants failed to recapitulate TIF1beta-dependent repression. TIF1beta homodimerization properties and interaction with the KRAB domain are preserved in the mutants with lysine to arginine substitutions as confirmed by in vivo bioluminescence resonance energy transfer (BRET). Using histone deacetylase (HDAC) inhibitors, we also demonstrate that TIF1beta sumoylation is a prerequisite for the recruitment of HDAC and that TIF1beta SUMO-dependent repressive activity involves both HDAC-dependent and HDAC-independent components. Finally, we report that, in addition to relying on the integrity of its PHD finger and on its self-oligomerization, TIF1beta sumoylation is positively regulated by its interaction with KRAB domain-containing proteins. Altogether, our results provide new mechanistic insights into TIF1beta transcriptional repression and suggest that KRAB multifinger proteins not only recruit TIF1beta co-repressor to target genes but also increase its repressive activity through enhancement of its sumoylation.

Real-time monitoring of ubiquitination in living cells by BRET.

Ubiquitin has emerged as an important regulator of protein stability and function in organisms ranging from yeast to mammals. The ability to detect in situ changes in protein ubiquitination without perturbing the physiological environment of cells would be a major step forward in understanding the ubiquitination process and its consequences. Here, we describe a new method to study this dynamic post-translational modification in intact human embryonic kidney cells. Using bioluminescence resonance energy transfer (BRET), we measured the ubiquitination of beta-arrestin 2, a regulatory protein implicated in the modulation of G protein-coupled receptors. In addition to allowing the detection of basal and GPCR-regulated ubiquitination of beta-arrestin 2 in living cells, real-time BRET measurements permitted the recording of distinct ubiquitination kinetics that are dictated by the identity of the activated receptor. The ubiquitination BRET assay should prove to be a useful tool for studying the dynamic ubiquitination of proteins and for understanding which cellular functions are regulated by this post-translational event.

Oligomerization of transcriptional intermediary factor 1 regulators and interaction with ZNF74 nuclear matrix protein revealed by bioluminescence resonance energy transfer in living cells.

Transcriptional intermediary factor 1 (TIF1) alpha and KAP-1/TIF1beta, two members of the TIF1 family of nuclear cofactors, are ubiquitous co-regulators of nuclear receptors and KRAB motif-containing zinc finger transcription factors, respectively. Despite the functional evidence suggesting a role for TIF1 proteins as modulators of transcription, the study of their interactions with transcriptional machineries in physiologically relevant systems has been difficult. Here, we have developed a bioluminescence resonance energy transfer (BRET) biophysical approach to study protein-protein interactions in the nuclear compartment of living mammalian cells. We report that TIF1alpha and KAP-1 form homo- and hetero-oligomers in intact mammalian cells. BRET titration experiments indicate that both homo- and hetero-oligomers occur with relatively high affinity suggesting that they could co-exist in cells. Furthermore, we demonstrate that KAP-1 but not TIF1alpha interacts with the KRAB multifinger ZNF74 in the nuclear matrix. Splice variants and point mutants of ZNF74 that lack transcriptional activity were found not to interact with KAP-1 confirming the physiological importance of this interaction in living cells. The interaction of ZNF74 with KAP-1 did not prevent KAP-1 homomerization indicating that the oligomers most likely represent the transcriptionally active species. Furthermore, the detection of ternary ZNF74.KAP-1.TIF1alpha complexes suggests the existence of cross-talk between KAP-1-interacting KRAB proteins and TIF1alpha-interacting nuclear receptors. In addition to providing new insights into the molecular interactions involved in the transcriptional activities of these proteins, this study shows that BRET can be advantageously used as a non-transcription-based oligomerization detection system to study the interaction of transcriptionally active proteins, including nuclear matrix proteins, in living cells.