Biomedical subjects

Merlin Crossley

Publications and source records attributed to Merlin Crossley.

At least 19 recordsLinked to original sources

PubMed2007

Sticky fingers: zinc-fingers as protein-recognition motifs.

Zinc-fingers (ZnFs) are extremely abundant in higher eukaryotes. Once considered to function exclusively as sequence-specific DNA-binding motifs, ZnFs are now known to have additional activities such as the recognition of RNA and other proteins. Here we discuss recent advances in our understanding of ZnFs as specific modules for protein recognition. Structural studies of ZnF complexes reveal considerable diversity in terms of protein partners, binding modes and affinities, and highlight the often underestimated versatility of ZnF structure and function. An appreciation of the structural features of ZnF-protein interactions will contribute to our ability to engineer and to use ZnFs with tailored protein-binding properties.

Amino Acid Motifs↗

PubMed2006

Solution structure of the THAP domain from Caenorhabditis elegans C-terminal binding protein (CtBP).

The THAP (Thanatos-associated protein) domain is a recently discovered zinc-binding domain found in proteins involved in transcriptional regulation, cell-cycle control, apoptosis and chromatin modification. It contains a single zinc atom ligated by cysteine and histidine residues within a Cys-X(2-4)-Cys-X(35-53)-Cys-X(2)-His consensus. We have determined the NMR solution structure of the THAP domain from Caenorhabditis elegans C-terminal binding protein (CtBP) and show that it adopts a fold containing a treble clef motif, bearing similarity to the zinc finger-associated domain (ZAD) from Drosophila Grauzone. The CtBP THAP domain contains a large, positively charged surface patch and we demonstrate that this domain can bind to double-stranded DNA in an electrophoretic mobility-shift assay. These data, together with existing reports, indicate that THAP domains might exhibit a functional diversity similar to that observed for classical and GATA-type zinc fingers.

Alcohol Oxidoreductases↗

PubMed2006

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

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.

Alcohol Oxidoreductases↗

PubMed2006

Specific recognition of ZNF217 and other zinc finger proteins at a surface groove of C-terminal binding proteins.

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.

Alcohol Oxidoreductases↗

PubMed2006

Molecular analysis of the interaction between the hematopoietic master transcription factors GATA-1 and PU.1.

GATA-1 and PU.1 are transcription factors that control erythroid and myeloid development, respectively. The two proteins have been shown to function in an antagonistic fashion, with GATA-1 repressing PU.1 activity during erythropoiesis and PU.1 repressing GATA-1 function during myelopoiesis. It has also become clear that this functional antagonism involves direct interactions between the two proteins. However, the molecular basis for these interactions is not known, and a number of inconsistencies exist in the literature. We have used a range of biophysical methods to define the molecular details of the GATA-1-PU.1 interaction. A combination of NMR titration data and extensive mutagenesis revealed that the PU.1-Ets domain and the GATA-1 C-terminal zinc finger (CF) form a low affinity interaction in which specific regions of each protein are implicated. Surprisingly, the interaction cannot be disrupted by single alanine substitution mutations, suggesting that binding is distributed over an extended interface. The C-terminal basic tail region of CF appears to be sufficient to mediate an interaction with PU.1-Ets, and neither acetylation nor phosphorylation of a peptide corresponding to this region disrupts binding, indicating that the interaction is not dominated by electrostatic interactions. The CF basic tail shares significant sequence homology with the PU.1 interacting motif from c-Jun, suggesting that GATA-1 and c-Jun might compete to bind PU.1. Taken together, our data provide a molecular perspective on the GATA-1-PU.1 interaction, resolving several issues in the existing data and providing insight into the mechanisms through which these two proteins combine to regulate blood development.

Acetylation↗

PubMed2006

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.

Alcohol Oxidoreductases↗

PubMed2006

Human KLF17 is a new member of the Sp/KLF family of transcription factors.

The Sp/KLF transcription factors perform a variety of biological functions, but are related in that they bind GC-box and CACCC-box sequences in DNA via a highly conserved DNA-binding domain. A database homology search, using the zinc finger DNA-binding domain characteristic of the family, has identified human KLF17 as a new family member that is most closely related to KLFs 1-8 and 12. KLF17 appears to be the human orthologue of the previously reported mouse gene, zinc finger protein 393 (Zfp393), although it has diverged significantly. The DNA-binding domain is the most conserved region, suggesting that both the murine and the human forms recognize the same binding sites in DNA and may retain similar functions. We show that human KLF17 can bind G/C-rich sites via its zinc fingers and is able to activate transcription from CACCC-box elements. This is the first report of the DNA-binding characteristics and transactivation activity of human KLF17, which, together with the homology it displays to other KLF proteins, put it in the Sp/KLF family.

Amino Acid Sequence↗

PubMed2006

Mechanisms directing the nuclear localization of the CtBP family proteins.

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.

Alcohol Oxidoreductases↗

PubMed2005

KAI1 promoter activity is dependent on p53, junB and AP2: evidence for a possible mechanism underlying loss of KAI1 expression in cancer cells.

A molecular mechanism to explain reduced KAI1 expression in invasive and metastatic tumour cells remains elusive. In this report, we extend an earlier study in bladder cells to confirm that a 76 bp region of the KAI1 promoter (residues -922 to -847), with binding motifs for p53, AP1 and AP2, is required for high level activity of a KAI1 reporter in prostate cancer cell lines. Gel shift and supershift experiments supported binding of p53, junB and heterodimers of AP2alpha/AP2gamma or AP2beta/AP2gamma to this sequence. Introduction of mutations into specific motifs demonstrated an essential requirement for p53 and junB to reporter activity, and that functional synergy between these two factors enhanced activity. A further elevation of reporter activity required AP2. Roles of individual p53, junB and AP2 proteins, as well as functional synergy between p53 and junB, were confirmed in transfection experiments. Western blotting analysis showed that an absence of wild-type p53, and/or a loss of junB and AP2 protein expression, correlated with downregulation of KAI1 mRNA levels in a series of prostate cancer cell lines. A loss of p53 function and/or expression of junB, combined with reduced expression of specific AP2 proteins may underly downregulated KAI1 expression in tumour cells.

Antigens, CD↗

PubMed2005

Zinc fingers as protein recognition motifs: structural basis for the GATA-1/friend of GATA interaction.

GATA-1 and friend of GATA (FOG) are zinc-finger transcription factors that physically interact to play essential roles in erythroid and megakaryocytic development. Several naturally occurring mutations in the GATA-1 gene that alter the FOG-binding domain have been reported. The mutations are associated with familial anemias and thrombocytopenias of differing severity. To elucidate the molecular basis for the GATA-1/FOG interaction, we have determined the three-dimensional structure of a complex comprising the interaction domains of these proteins. The structure reveals how zinc fingers can act as protein recognition motifs. Details of the architecture of the contact domains and their physical properties provide a molecular explanation for how the GATA-1 mutations contribute to distinct but related genetic diseases.

Binding Sites↗

PubMed2005

Assessment of the robustness of a serendipitous zinc binding fold: mutagenesis and protein grafting.

Zinc binding motifs have received much attention in the area of protein design. Here, we have tested the suitability of a recently discovered nonnative zinc binding structure as a protein design scaffold. A series of multiple alanine mutants was created to investigate the minimal requirements for folding, and solution structures of these mutants showed that the original fold was maintained, despite changes in approximately 50% of the sequence. We next attempted to transplant binding faces from chosen bimolecular interactions onto one of these mutants, and many of the resulting "chimeras" were shown to adopt a native-like fold. These results both highlight the robust nature of small zinc binding domains and underscore the complexity of designing functional proteins, even using such small, highly ordered scaffolds as templates.

Alanine↗

PubMed2005

Role for SUMO modification in facilitating transcriptional repression by BKLF.

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 Kruppel-like factor/Kruppel-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, PIASgamma, PIASxalpha, and PIASxbeta 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.

Alcohol Oxidoreductases↗

PubMed2004

Structural studies on a protein-binding zinc-finger domain of Eos reveal both similarities and differences to classical zinc fingers.

The oligomerization domain that is present at the C terminus of Ikaros-family proteins and the protein Trps-1 is important for the proper regulation of developmental processes such as hematopoiesis. Remarkably, this domain is predicted to contain two classical zinc fingers (ZnFs), domains normally associated with the recognition of nucleic acids. The preference for protein binding by these predicted ZnFs is not well-understood. We have used a range of methods to gain insight into the structure of this domain. Circular dichroism, UV-vis, and NMR experiments carried out on the C-terminal domain of Eos (EosC) revealed that the two putative ZnFs (C1 and C2) are separable, i.e., capable of folding independently in the presence of Zn(II). We next determined the structure of EosC2 using NMR spectroscopy, revealing that, although the overall fold of EosC2 is similar to other classical ZnFs, a number of differences exist. For example, the conformation of the C terminus of EosC2 appears to be flexible and may result in a major rearrangement of the zinc ligands. Finally, alanine-scanning mutagenesis was used to identify the residues that are involved in the homo- and hetero-oligomerization of Eos, and these results are discussed in the context of the structure of EosC. These studies provide the first structural insights into how EosC mediates protein-protein interactions and contributes to our understanding of why it does not exhibit high-affinity DNA binding.

Amino Acid Sequence↗

PubMed2004

A classic zinc finger from friend of GATA mediates an interaction with the coiled-coil of transforming acidic coiled-coil 3.

Classic zinc finger domains (cZFs) consist of a beta-hairpin followed by an alpha-helix. They are among the most abundant of all protein domains and are often found in tandem arrays in DNA-binding proteins, with each finger contributing an alpha-helix to effect sequence-specific DNA recognition. Lone cZFs, not found in tandem arrays, have been postulated to function in protein interactions. We have studied the transcriptional co-regulator Friend of GATA (FOG), which contains nine zinc fingers. We have discovered that the third cZF of FOG contacts a coiled-coil domain in the centrosomal protein transforming acidic coiled-coil 3 (TACC3). Although FOG-ZF3 exhibited low solubility, we have used a combination of mutational mapping and protein engineering to generate a derivative that was suitable for in vitro and structural analysis. We report that the alpha-helix of FOG-ZF3 recognizes a C-terminal portion of the TACC3 coiled-coil. Remarkably, the alpha-helical surface utilized by FOG-ZF3 is the same surface responsible for the well established sequence-specific DNA-binding properties of many other cZFs. Our data demonstrate the versatility of cZFs and have implications for the analysis of many as yet uncharacterized cZF proteins.

Amino Acid Sequence↗

PubMed2004

Activation and repression of interleukin-12 p40 transcription by erythroid Kruppel-like factor in macrophages.

Transcription of interleukin (IL)-12 p40 in myeloid cells is attributed to the recruitment of multiple activated transcription factors such as nuclear factor kappaB (NFkappaB), CCAAT enhancer-binding protein beta, ets-2, PU.1, and so forth. We now provide the first description of the human erythroid Kruppel-like factor (EKLF) in human primary macrophages and identify the role of EKLF in IL-12 p40 expression. EKLF-specific binding to the CACCC element (-224 to -220) on the human IL-12 p40 promoter was observed in resting human primary macrophages. Functional analysis of the CACCC element revealed a dependent role for EKLF binding in activating IL-12 p40 transcription in resting RAW264.7 cells, whereas EKLF overexpression in the presence or absence of this element repressed IL-12 p40 transcription in interferon gamma/lipopolysaccharide-stimulated RAW264.7 cells. Murine endogenous IL-12 p40 mRNA was consistently induced by overexpressed EKLF in resting RAW264.7 cells, whereas EKLF suppressed IL-12 p40 expression in activated RAW264.7 cells. Modulation of nuclear binding activities at the IL-12 p40 NFkappaB half-site was induced by EKLF for down-regulation of IL-12 p40 transcription in activated RAW264.7 cells, but no effect of EKLF on NFkappaB activity was observed in resting RAW264.7 cells. Taken together, we identify EKLF as a transcription factor in macrophages able to regulate IL-12 p40 transcription depending on the cellular activation status. The bifunctional control of IL-12 p40 by EKLF and its modulation of NFkappaB support a potential function for this factor in orchestrating IL-12 p40 production in macrophages.

Animals↗

PubMed2003

Pentaprobe: a comprehensive sequence for the one-step detection of DNA-binding activities.

The rapid increase in the number of novel proteins identified in genome projects necessitates simple and rapid methods for assigning function. We describe a strategy for determining whether novel proteins possess typical sequence-specific DNA-binding activity. Many proteins bind recognition sequences of 5 bp or less. Given that there are 4(5) possible 5 bp sites, one might expect the length of sequence required to cover all possibilities would be 4(5) x 5 or 5120 nt. But by allowing overlaps, utilising both strands and using a computer algorithm to generate the minimum sequence, we find the length required is only 516 base pairs. We generated this sequence as six overlapping double-stranded oligonucleotides, termed pentaprobe, and used it in gel retardation experiments to assess DNA binding by both known and putative DNA-binding proteins from several protein families. We have confirmed binding by the zinc finger proteins BKLF, Eos and Pegasus, the Ets domain protein PU.1 and the treble clef N- and C-terminal fingers of GATA-1. We also showed that the N-terminal zinc finger domain of FOG-1 does not behave as a typical DNA-binding domain. Our results suggest that pentaprobe, and related sequences such as hexaprobe, represent useful tools for probing protein function.

Algorithms↗

PubMed2003

The C-terminal domain of Eos forms a high order complex in solution.

Ikaros family transcription factors play important roles in the control of hematopoiesis. Family members are predicted to contain up to six classic zinc fingers that are arranged into N- and C-terminal domains. The N-terminal domain is responsible for site-specific DNA binding, whereas the C-terminal domain primarily mediates the homo- and hetero-oligomerization between family members. Although the mechanisms of action of these proteins are not completely understood, the zinc finger domains are known to play a central role. In the current study, we have sought to understand the physical and functional properties of these domains, in particular the C-terminal domain. We show that the N-terminal domain from Eos, and not its C-terminal region, is required to recognize GGGA consensus sequences. Surprisingly, in contrast to the behavior exhibited by Ikaros, the C-terminal domain of Eos inhibits the DNA-binding activity of the full-length protein. In addition, we have used a range of biophysical techniques to demonstrate that the C-terminal domain of Eos mediates the formation of complexes that consist of nine or ten molecules. These results constitute the first direct demonstration that Ikaros family proteins can form higher order complexes in solution, and we discuss this unexpected result in the context of what is currently known about the family members and their possible mechanism of action.

Amino Acid Sequence↗

PubMed2003

CCHX zinc finger derivatives retain the ability to bind Zn(II) and mediate protein-DNA interactions.

Classical (CCHH) zinc fingers are among the most common protein domains found in eukaryotes. They function as molecular recognition elements that mediate specific contact with DNA, RNA, or other proteins and are composed of a betabetaalpha fold surrounding a single zinc ion that is ligated by two cysteine and two histidine residues. In a number of variant zinc fingers, the final histidine is not conserved, and in other unrelated zinc binding domains, residues such as aspartate can function as zinc ligands. To test whether the final histidine is required for normal folding and the DNA-binding function of classical zinc fingers, we focused on finger 3 of basic Krüppel-like factor. The structure of this domain was determined using NMR spectroscopy and found to constitute a typical classical zinc finger. We generated a panel of substitution mutants at the final histidine in this finger and found that several of the mutants retained some ability to fold in the presence of zinc. Consistent with this result, we showed that mutation of the final histidine had only a modest effect on DNA binding in the context of the full three-finger DNA-binding domain of basic Krüppel-like factor. Further, the zinc binding ability of one of the point mutants was tested and found to be indistinguishable from the wild-type domain. These results suggest that the final zinc chelating histidine is not an essential feature of classical zinc fingers and have implications for zinc finger evolution, regulation, and the design of experiments testing the functional roles of these domains.

Amino Acid Sequence↗