Alexis Verger

Modification with SUMO: A role in transcriptional regulation

Small ubiquitin‐related modifier (SUMO) is a protein moiety that is ligated to lysine residues in a variety of target proteins. The addition of SUMO can modulate the ability of proteins to interact with their partners, alter their patterns of subcellular localization and control their stability. It is clear that SUMO influences many different biological processes, but recent data suggest that it is particularly important in the regulation of transcription. Indeed, several transcription factors, such as Sp3, c‐Jun, c‐Myb and various nuclear receptors, have recently been shown to be subject to sumoylation and, although this modification can have a positive influence, a growing body of evidence highlights its role in the negative regulation of transcription. This review summarizes recent experiments focusing on sumoylation and transcriptional repression.

Functional Cross-Antagonism between Transcription Factors FLI-1 and EKLF

ABSTRACT FLI-1 is an ETS family transcription factor which is overexpressed in Friend erythroleukemia and contributes to the blockage of differentiation of erythroleukemic cells. We show here that FLI-1 represses the transcriptional activity of the β-globin gene promoter in MEL cells and interacts with two of its critical transactivators, GATA-1 and EKLF. Unexpectedly, FLI-1 enhances the stimulating activity of GATA-1 on a GATA-1-responsive promoter but represses that of EKLF on β-globin and an EKLF-responsive artificial promoters. This repressive effect of FLI-1 requires the ETS DNA binding domain and its association with either the N- or C-terminal domain, which themselves interact with EKLF but not with GATA-1. Furthermore, the FLI-1 ETS domain alone behaves as an autonomous repression domain when linked to the Gal4 DNA binding domain. Taken together, these data indicate that FLI-1 represses EKLF-dependent transcription due to the repression activity of its ETS domain and its indirect recruitment to erythroid promoters by protein-protein interaction with EKLF. Reciprocally, we also show that EKLF itself represses the FLI-1-dependent megakaryocytic GPIX gene promoter, thus further suggesting that functional cross-antagonism between FLI-1 and EKLF might be involved in the control of the erythrocytic versus megakaryocytic differentiation of bipotential progenitors.

Role for SUMO Modification in Facilitating Transcriptional Repression by BKLF

ABSTRACT Small ubiquitin-like modifier (SUMO) is a protein moiety that is ligated to lysine residues on a variety of target proteins. Many known SUMO substrates are transcription factors or coregulators of transcription, and in most cases, modification with SUMO leads to the attenuation of transcriptional activation. We have examined basic Krüppel-like factor/Krüppel-like factor 3 (BKLF), a zinc finger transcription factor that is known to function as a potent transcriptional repressor. We show that BKLF recruits the E2 SUMO-conjugating enzyme Ubc9 and can be modified by the addition of SUMO-1 in vitro and in vivo. The SUMO E3 ligases PIAS1, PIASγ, PIASxα, and PIASxβ but not Pc2 enhance the sumoylation of BKLF. Site-directed mutagenesis identified two lysines (K10 and K197) of BKLF as the sumoylation sites. Sumoylation does not detectably affect DNA binding by BKLF, but mutation of the sumoylation sites reduces transcriptional repression activity. Most interestingly, when mutations preventing sumoylation are combined with an additional mutation that eliminates contact with the C-terminal binding protein (CtBP) corepressor, BKLF becomes an activator of transcription. These results link SUMO modification to transcriptional repression and demonstrate that both recruitment of CtBP and sumoylation are required for full repression by BKLF.

Specific Recognition of ZNF217 and Other Zinc Finger Proteins at a Surface Groove of C-Terminal Binding Proteins

ABSTRACT Numerous transcription factors recruit C-terminal binding protein (CtBP) corepressors. We show that the large zinc finger protein ZNF217 contacts CtBP. ZNF217 is encoded by an oncogene frequently amplified in tumors. ZNF217 contains a typical Pro-X-Asp-Leu-Ser (PXDLS) motif that binds in CtBPs PXDLS-binding cleft. However, ZNF217 also contains a second motif, Arg-Arg-Thr (RRT), that binds a separate surface on CtBP. The crystal structure of CtBP bound to an RRTGAPPAL peptide shows that it contacts a surface crevice distinct from the PXDLS binding cleft. Interestingly, both PXDLS and RRT motifs are also found in other zinc finger proteins, such as RIZ. Finally, we show that ZNF217 represses several promoters, including one from a known CtBP target gene, and mutations preventing ZNF217s contact with CtBP reduce repression. These results identify a new CtBP interaction motif and establish ZNF217 as a transcriptional repressor protein that functions, at least in part, by associating with CtBP.

Mechanisms directing the nuclear localization of the CtBP family proteins

ABSTRACT The C-terminal binding protein (CtBP) family includes four proteins (CtBP1 [CtBP1-L], CtBP3/BARS [CtBP1-S], CtBP2, and RIBEYE) which are implicated both in transcriptional repression and in intracellular trafficking. However, the precise mechanisms by which different CtBP proteins are targeted to different subcellular regions remains unknown. Here, we report that the nuclear import of the various CtBP proteins and splice isoforms is differentially regulated. We show that CtBP2 contains a unique nuclear localization signal (NLS) located within its N-terminal region, which contributes to its nuclear accumulation. Using heterokaryon assays, we show that CtBP2 is capable of shuttling between the nucleus and cytoplasm of the cell. Moreover, CtBP2 can heterodimerize with CtBP1-L and CtBP1-S and direct them to the nucleus. This effect strongly depends on the CtBP2 NLS. PXDLS motif-containing transcription factors, such as BKLF, that bind CtBP proteins can also direct them to the nucleus. We also report the identification of a splice isoform of CtBP2, CtBP2-S, that lacks the N-terminal NLS and localizes to the cytoplasm. Finally, we show that mutation of the CtBP NADH binding site impairs the ability of the proteins to dimerize and to associate with BKLF. This reduces the nuclear accumulation of CtBP1. Our results suggest a model in which the nuclear localization of CtBP proteins is influenced by the CtBP2 NLS, by binding to PXDLS motif partner proteins, and through the effect of NADH on CtBP dimerization.

Engineering a Protein Scaffold from a PHD Finger

The design of proteins with tailored functions remains a relatively elusive goal. Small size, a well-defined structure, and the ability to maintain structural integrity despite multiple mutations are all desirable properties for such designer proteins. Many zinc binding domains fit this description. We determined the structure of a PHD finger from the transcriptional cofactor Mi2beta and investigated the suitability of this domain as a scaffold for presenting selected binding functions. The two flexible loops in the structure were mutated extensively by either substitution or expansion, without affecting the overall fold of the domain. A binding site for the corepressor CtBP2 was also grafted onto the domain, creating a new PHD domain that can specifically bind CtBP2 both in vitro and in the context of a eukaryotic cell nucleus. These results represent a step toward designing new regulatory proteins for modulating aberrant gene expression in vivo.

Role of the C-terminal binding protein PXDLS motif binding cleft in protein interactions and transcriptional repression

ABSTRACT C-terminal binding proteins (CtBPs) are multifunctional proteins that can mediate gene repression. CtBPs contain a cleft that binds Pro-X-Asp-Leu-Ser (PXDLS) motifs. PXDLS motifs occur in numerous transcription factors and in effectors of gene repression, such as certain histone deacetylases. CtBPs have been depicted as bridging proteins that self-associate and link PXDLS-containing transcription factors to PXDLS-containing chromatin-modifying enzymes. CtBPs also recruit effectors that do not contain recognizable PXDLS motifs. We have investigated the importance of the PXDLS binding cleft to CtBPs interactions with various partner proteins and to its ability to repress transcription. We used CtBP cleft mutant and cleft-filled fusion derivatives to distinguish between partner proteins that bind in the cleft and elsewhere on the CtBP surface. Functional assays demonstrate that CtBP mutants that carry defective clefts retain repression activity when fused to heterologous DNA-binding domains. This result suggests that the cleft is not essential for recruiting effectors. In contrast, when tested in the absence of a fused DNA-binding domain, disruption of the cleft abrogates repression activity. These results demonstrate that the PXDLS binding cleft is functionally important but suggest that it is primarily required for localization of the CtBP complex to promoter-bound transcription factors.

A L225A substitution in the human tumour suppressor HIC1 abolishes its interaction with the corepressor CtBP

HIC1 (hypermethylated in cancer) is a tumour suppressor gene located in 17p13.3, a region frequently hypermethylated or deleted in many types of prevalent human tumour. HIC1 is also a candidate for a contiguous‐gene syndrome, the Miller–Dieker syndrome, a severe form of lissencephaly accompanied by developmental anomalies. HIC1 encodes a BTB/POZ‐zinc finger transcriptional repressor. HIC1 represses transcription via two autonomous repression domains, an N‐terminal BTB/POZ and a central region, by trichostatin A‐insensitive and trichostatin A‐sensitive mechanisms, respectively. The HIC1 central region recruits the corepressor CtBP (C‐terminal binding protein) through a conserved GLDLSKK motif, a variant of the consensus C‐terminal binding protein interaction domain PxDLSxK/R. Here, we show that HIC1 interacts with both CtBP1 and CtBP2 and that this interaction is stimulated by agents increasing NADH levels. Furthermore, point mutation of two CtBP2 residues forming part of the structure of the recognition cleft for a PxDLS motif also ablates the interaction with a GxDLS motif. Conversely, in perfect agreement with the structural data and the universal conservation of this residue in all C‐terminal binding protein‐interacting motifs, mutation of the central leucine residue (leucine 225 in HIC1) abolishes the interaction between HIC1 and CtBP1 or CtBP2. As expected from the corepressor activity of CtBP, this mutation also impairs the HIC1‐mediated transcriptional repression. These results thus demonstrate a strong conservation in the binding of C‐terminal binding protein‐interacting domains despite great variability in their amino acid sequences. Finally, this L225A point mutation could also provide useful knock‐in animal models to study the role of the HIC1–CtBP interaction in tumorigenesis and in development.

Mutual repression of transcriptional activation between the ETS-related factor ERG and estrogen receptor.

Transcription factors are known to interact with each other to modulate their transcriptional activity. In this study, we found that the transcriptional activity of human Erg (one of the Ets family-transcription factors) was repressed by several nuclear receptors, including human estrogen receptor ERα, nonsteroid receptors and orphan receptors. Conversely, Erg inhibited ERα-dependent transcription. These reciprocal functional interactions extended to other nuclear receptors such as thyroid hormone and retinoic acid receptors, as well as to Fli1, an ERG-related ETS factor. Although similarly inhibited by overexpression of the orphan nuclear receptors ERR1 and RORα, ERG activity was unaffected by either REV-ERBα1 or COUP-TFII. The antagonism between ERG and ERα did not depend on DNA binding inhibition or direct protein–protein interactions. Repression of ERα-dependent transcription required the carboxyterminal and aminoterminal transactivation domains of Erg whereas the carboxyterminal AF-2 domain of ERα was necessary for repression of Erg activity. Reciprocal inhibition between Erg and ERα was not alleviated by overexpressing CBP, SRC-1 or RIP 140, three nuclear coactivator proteins. A negative cross-talk observed between Erg and ERα expands their potential range of regulation and may be relevant in vivo, particularly in endothelial, urogenital and cartilaginous tissues where both factors are expressed.

CtBP and Hematopoietic Transcriptional Regulators

The C-terminal binding proteins (CtBPs) are ubiquitous corepressors that recruit histone-modifying enzymes to a variety of sequence specific DNA-binding proteins and other transcriptional regulators. CtBPs appear to play an important role in mediating repression and transforming activities of a variety of hematopoietic transcription factors such as Basic Kruppel-like Factor/Kruppel-like Factor 3 (BKLF/KLF3), Friend of GATA (FOG), Evi-1 and members of the Ikaros family. Mice lacking CtBPs die during embryonic development and exhibit defects in a wide range of developmental processes, including aberrant heart formation and absence of blood vessels in the yolk sac. The ongoing identification of repressed target genes and interacting transcriptional partners will help to unravel the contributions of CtBP proteins to hematopoiesis.