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1.  Structural insights into ChpT, an essential dimeric histidine phosphotransferase regulating the cell cycle in Caulobacter crescentus  
The cell-cycle regulator ChpT of C. crescentus is a dimeric histidine phosphotransferase that resembles the typical structure of histidine kinases.
Two-component and phosphorelay signal-transduction proteins are crucial for bacterial cell-cycle regulation in Caulobacter crescentus. ChpT is an essential histidine phosphotransferase that controls the activity of the master cell-cycle regulator CtrA by phosphorylation. Here, the 2.2 Å resolution crystal structure of ChpT is reported. ChpT is a homodimer and adopts the domain architecture of the intracellular part of class I histidine kinases. Each subunit consists of two distinct domains: an N-terminal helical hairpin domain and a C-terminal α/β domain. The two N-terminal domains are adjacent within the dimer, forming a four-helix bundle. The ChpT C-terminal domain adopts an atypical Bergerat ATP-binding fold.
PMCID: PMC3433190  PMID: 22949187
bacterial cell cycle; Caulobacter crescentus; histidine kinases; histidine phosphotransferases
2.  Specific Recognition of ZNF217 and Other Zinc Finger Proteins at a Surface Groove of C-Terminal Binding Proteins▿  
Molecular and Cellular Biology  2006;26(21):8159-8172.
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 CtBP's 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 ZNF217's 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.
PMCID: PMC1636751  PMID: 16940172
3.  Role of the C-Terminal Binding Protein PXDLS Motif Binding Cleft in Protein Interactions and Transcriptional Repression▿  
Molecular and Cellular Biology  2006;26(21):8202-8213.
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 CtBP's 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.
PMCID: PMC1636740  PMID: 16940173
4.  Mechanisms Directing the Nuclear Localization of the CtBP Family Proteins 
Molecular and Cellular Biology  2006;26(13):4882-4894.
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.
PMCID: PMC1489157  PMID: 16782877
5.  Role for SUMO Modification in Facilitating Transcriptional Repression by BKLF†  
Molecular and Cellular Biology  2005;25(4):1549-1559.
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.
PMCID: PMC548027  PMID: 15684403
6.  Functional Cross-Antagonism between Transcription Factors FLI-1 and EKLF 
Molecular and Cellular Biology  2003;23(4):1390-1402.
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.
PMCID: PMC141137  PMID: 12556498

Results 1-6 (6)