PubMed Health⌕ Search

Biomedical subjects

M Crossley

Publications and source records attributed to M Crossley.

At least 19 recordsLinked to original sources
PubMed2005

Monitoring emergence from coma following severe brain injury in an octogenarian using behavioural indicators, electrophysiological measures and metabolic studies: a demonstration of the potential for good recovery in older adults.

This case study describes a multi-disciplinary investigation of the emergence from coma of an 80-year old female (KE) following severe traumatic brain injury. The relationship between cognitive/behavioural ability and the integrity of cerebral function was assessed using neuropsychological measures, positron emission tomography, electroencephalography, somatosensory evoked potentials and trans-cranial magnetic stimulation. These investigations were performed as KE was beginning to emerge from coma (4 weeks) and, again, approximately 1 year following brain injury, when she was judged to have achieved her maximum level of recovery. Neuropsychological measures revealed improvement during the first year post-injury in KE's speed of information processing, memory and executive abilities. Electrophysiological and metabolic studies indicated a restoration of functional integrity that was consistent with the gradual recovery in higher brain function documented using behavioural procedures. This case study demonstrates the rehabilitation potential of pre-morbidly healthy older adults following severe traumatic brain injury.

Accidents, Traffic↗
PubMed2004

Public service values.

Explore the source record for details and available documents.

Attitude of Health Personnel↗
PubMed2004

Chromatin modifiers in transcription and DNA repair.

In eukaryotes, the packaging of DNA into nucleosomes and the organization of chromatin fibres generate constraints for all nuclear processes involving DNA, including replication, repair, recombination and transcription. The three major processes that regulate chromatin structure and counterbalance its repressive effects are ATP-dependent chromatin remodeling, post-translational modification of histones and histone replacement. While many of these processes have been intensively studied with respect to their effects on transcription, there is also evidence that they affect other nuclear processes involving DNA. This review explores the functions of chromatin-remodeling factors and histone-modifying enzymes in gene regulation and summarizes recent findings which suggest a role for these chromatin modifiers in DNA repair.

Animals↗
PubMed2001

X-linked thrombocytopenia caused by a novel mutation of GATA-1.

A family with recessive X-linked thrombocytopenia affecting 4 males in 2 generations, characterized by macrothrombocytopenia, profound bleeding, and mild dyserythropoiesis, is described. Microsatellite linkage analysis identified a region of the X chromosome including the GATA-1 gene, which encodes a critical transcription factor involved in erythrocyte and megakaryocyte development. By sequencing the entire coding region of GATA-1, a 2-base mutation was detected that results in a single amino acid substitution (glycine 208 to serine) within a highly conserved portion of the N-terminal zinc finger domain. Restriction fragment length polymorphism confirmed that this novel mutation segregated with the affected males and female carrier. Although not required for DNA binding, Gly208 of GATA-1 is involved in direct interaction with Friend of GATA-1 (FOG), a cofactor required for normal megakaryocytic and erythroid development. These results demonstrate that the GATA-1-FOG interaction is partially disrupted by the mutation and that the greatest effect involves contact with the FOG zinc finger 9. These findings help describe a novel mutation of GATA-1 in humans as a cause of X-linked thrombocytopenia, and they confirm the vital role played by this transcription factor during in vivo megakaryocyte development.

Bone Marrow↗
PubMed2001

The role of the coiled-coil motif in interactions mediated by TPD52.

TPD52 (D52)-like proteins are small coiled-coil motif-bearing proteins first identified through their expression in human breast carcinoma that mutually interact in hetero- and homomeric fashions. However, it has been unclear whether the coiled-coil motif is sufficient, or even necessary, for these interactions to occur. We have therefore examined the binding activities of a panel of C-terminally deleted D52 proteins in both the yeast two-hybrid system and pull-down assays. In the yeast two-hybrid system, interactions were only detected when regions C-terminal to the coiled-coil motif were also present. However, using pull-down assays, interactions were detected for all deletion mutants which included the coiled-coil motif. This suggests that the coiled-coil motif is indeed necessary for interactions mediated by D52 proteins, but that C-terminal protein regions facilitate and/or stabilize these interactions.

Amino Acid Motifs↗
PubMed2001

The N-terminal zinc finger of the erythroid transcription factor GATA-1 binds GATC motifs in DNA.

The mammalian transcription factor GATA-1 is required for normal erythroid and megakaryocytic development. GATA-1 contains two zinc fingers, the C-terminal finger, which is known to bind (A/T)GATA(A/G) motifs in DNA and the N-finger, which is important for interacting with co-regulatory proteins such as Friend of GATA (FOG). We now show that, like the C-finger, the N-finger of GATA-1 is also capable of binding DNA but recognizes distinct sequences with the core GATC. We demonstrate that the GATA-1 N-finger can bind these sequences in vitro and that in cellular assays, GATA-1 can activate promoters containing GATC motifs. Experiments with mutant GATA-1 proteins confirm the importance of the N-finger, as the C-finger is not required for transactivation from GATC sites. Recently four naturally occurring mutations in GATA-1 have been shown to be associated with familial blood disorders. These mutations all map to the N-finger domain. We have investigated the effect of these mutations on the recognition of GATC sites by the N-finger and show that one mutation R216Q abolishes DNA binding, whereas the others have only minor effects.

Amino Acid Sequence↗
PubMed2001

Induction of AP-2alpha expression by adenoviral infection involves inactivation of the AP-2rep transcriptional corepressor CtBP1.

AP-2 transcription factors execute important functions during embryonic development and malignant transformation. Recently, we have isolated a transcriptional repressor of AP-2alpha expression, the novel Krüppel-related zinc finger protein AP-2rep (Klf12). Here, we show that repression of AP-2alpha transcription by AP-2rep is dependent on an N-terminal PVDLS motif that interacts specifically with the corepressor CtBP1 both in vivo and in vitro. This interaction motif was previously identified in the C-terminal region of the adenoviral oncoprotein E1A. Infection of both HeLa and PA-1 cells with adenovirus type 5 strongly induced AP-2alpha mRNA. Consistently, E1A was necessary and sufficient to mediate up-regulation of AP-2alpha. Transiently transfected wild-type E1A protein activated an AP-2rep sensitive cis-regulatory element of the AP-2alpha promoter, but E1A protein harboring a mutation in the PVDLS motif failed to activate. In summary, we conclude that the adenoviral oncoprotein E1A activates transcription from the endogenous AP-2alpha gene, an effect that involves transcriptional derepression of the AP-2alpha promoter by interaction of E1A with the AP-2rep corepressor CtBP1.

Adenoviridae↗
PubMed2001

Evi-1 transforming and repressor activities are mediated by CtBP co-repressor proteins.

Ectopic production of the EVI1 transcriptional repressor zinc finger protein is seen in 4--6% of human acute myeloid leukemias. Overexpression also transforms Rat1 fibroblasts by an unknown mechanism, which is likely to be related to its role in leukemia and which depends upon its repressor activity. We show here that mutant murine Evi-1 proteins, lacking either the N-terminal zinc finger DNA binding domain or both DNA binding zinc finger clusters, function as dominant negative mutants by reverting the transformed phenotype of Evi-1 transformed Rat1 fibroblasts. The dominant negative activity of the non-DNA binding mutants suggests sequestration of transformation-specific cofactors and that recruitment of these cellular factors might mediate Evi-1 transforming activity. C-terminal binding protein (CtBP) co-repressor family proteins bind PLDLS-like motifs. We show that the murine Evi-1 repressor domain has two such sites, PFDLT (site a, amino acids 553--559) and PLDLS (site b, amino acids 584--590), which independently can bind CtBP family co-repressor proteins, with site b binding with higher affinity than site a. Functional analysis of specific CtBP binding mutants show site b is absolutely required to mediate both transformation of Rat1 fibroblasts and transcriptional repressor activity. This is the first demonstration that the biological activity of a mammalian cellular transcriptional repressor protein is mediated by CtBPs. Furthermore, it suggests that CtBP proteins are involved in the development of some acute leukemias and that blocking their ability to specifically interact with EVI1 might provide a target for the development of pharmacological therapeutic agents.

Animals↗
PubMed2001

The CtBP family: enigmatic and enzymatic transcriptional co-repressors.

Transcription factors that associate with DNA sequences in promoters and enhancers often recruit co-regulators that modulate their activity. Many of these co-regulators have intrinsic enzymatic activity and influence gene expression by modifying chromatin and altering its structure. Recently, a new family of co-repressors, the C-terminal binding proteins, has been described. These proteins recognize Pro-X-Asp-Leu-Ser (PXDLS) motifs in DNA-binding proteins and function as transcriptional co-repressors in Drosophila, Xenopus and mammals. The precise mechanisms by which they influence transcription are still under investigation. CtBP proteins dimerize and can contact histone deacetylases; hence they may operate by linking deacetylases to DNA-bound factors. But it appears that CtBP proteins also have intrinsic enzymatic activity. They have significant homology to D-isomer-specific 2-hydroxy acid dehydrogenases, and remarkably one family member, rat CtBP, has been shown to have a second role, functioning as an acyl transferase in Golgi maintenance. These observations raise the possibility that CtBP proteins might regulate gene expression directly by means of their enzymatic activities, in addition to serving as simple bridging proteins. Supplementary material for this article can be found on the BioEssays homepage at http://www.interscience.wiley.com/jpages/0265-9247/suppmat/v23_8.684.

Alcohol Oxidoreductases↗
PubMed2000

Eos and pegasus, two members of the Ikaros family of proteins with distinct DNA binding activities.

Members of the Ikaros family of transcription factors, Ikaros, Aiolos, and Helios, are expressed in lymphocytes and have been implicated in controlling lymphoid development. These proteins contain two characteristic clusters of zinc fingers, an N-terminal domain important for DNA recognition, and a C-terminal domain that mediates homo- and heterotypic associations between family members. The conservation of these domains is such that all three proteins recognize related DNA sequences, and all are capable of dimerizing with other family members. Here we describe two additional Ikaros family proteins, Eos and Pegasus. Eos is most highly related to Helios and shares its DNA binding and protein association properties. Pegasus is related to other Ikaros proteins in its C-terminal dimerization domain but contains a divergent N-terminal zinc finger domain. Pegasus self-associates and binds to other family members but recognizes distinct DNA-binding sites. Eos and Pegasus repress the expression of reporter genes containing their recognition elements. Our results suggest that these proteins may associate with previously described Ikaros family proteins in lymphoid cells and play additional roles in other tissues.

3T3 Cells↗
PubMed2000

Solution structures of two CCHC zinc fingers from the FOG family protein U-shaped that mediate protein-protein interactions.

BACKGROUND: Zinc finger domains have traditionally been regarded as sequence-specific DNA binding motifs. However, recent evidence indicates that many zinc fingers mediate specific protein-protein interactions. For instance, several zinc fingers from FOG family proteins have been shown to interact with the N-terminal zinc finger of GATA-1. RESULTS: We have used NMR spectroscopy to determine the first structures of two FOG family zinc fingers that are involved in protein-protein interactions: fingers 1 and 9 from U-shaped. These fingers resemble classical TFIIIA-like zinc fingers, with the exception of an unusual extended portion of the polypeptide backbone prior to the fourth zinc ligand. [15N,(1)H]-HSQC titrations have been used to define the GATA binding surface of USH-F1, and comparison with other FOG family proteins indicates that the recognition mechanism is conserved across species. The surface of FOG-type fingers that interacts with GATA-1 overlaps substantially with the surface through which classical fingers typically recognize DNA. This suggests that these fingers could not contact both GATA and DNA simultaneously. In addition, results from NMR, gel filtration, and sedimentation equilibrium experiments suggest that the interactions are of moderate affinity. CONCLUSIONS: Our results demonstrate unequivocally that zinc fingers comprising the classical betabetaalpha fold are capable of mediating specific contacts between proteins. The existence of this alternative function has implications for the prediction of protein function from sequence data and for the evolution of protein function.

Amino Acid Sequence↗
PubMed2000

The transactivation domain within cysteine/histidine-rich region 1 of CBP comprises two novel zinc-binding modules.

cAMP-response element-binding protein-binding protein (CBP) is a transcriptional coactivator that interacts with a number of DNA-binding proteins and cofactor proteins involved in the regulation of transcription. Relatively little is known about the structure of CBP, but it has been noted that it contains three domains that are rich in cysteine and histidine (CH1, CH2, and CH3). The sequence of CH2 conforms to that of a leukemia-associated protein domain (PHD finger), and it has been postulated that this and both CH1 and CH3 may be zinc finger domains. This has not, however, been demonstrated experimentally. We have studied CH1 and show that it is composed of two novel zinc-binding modules, which we term "zinc bundles." Each bundle contains the sequence Cys-X(4)-Cys-X(8)-His-X(3)-Cys, and we show that a synthetic peptide comprising one zinc bundle from CH1 can fold in a zinc-dependent manner. CH3 also appears to contain two zinc bundles, one with the variant sequence Cys-X(2)-Cys-X(9)-His-X(3)-Cys, and we demonstrate that this variant motif also undergoes Zn(II)-induced folding. CH1 acts as a transcriptional activation domain in cellular assays. We show that mutations in any of the four zinc-chelating residues in either zinc bundle of CH1 significantly impair this activity and that these mutations also interfere with certain protein-protein interactions mediated by CH1. Our results indicate that CBP is a genuine zinc-binding protein and introduce zinc bundles as novel protein interaction domains.

Amino Acid Sequence↗
PubMed2000

Human Krüppel-like factor 8: a CACCC-box binding protein that associates with CtBP and represses transcription.

CACCC-boxes are recognised by transcription factors of the Sp/Krüppel-like Factor (Sp1/KLF) family. Here we describe one member of this family, KLF8/ZNF741/BKLF3 (KLF8). KLF8 contains a characteristic C-terminal DNA-binding domain comprised of three Krüppel-like zinc fingers, but also has limited homology to another family member, KLF3/Basic Krüppel-like Factor (KLF3/BKLF), in its N-terminus. Most significantly, it shares with KLF3/BKLF a Pro-Val-Asp-Leu-Ser/Thr motif. In KLF3/BKLF this motif mediates contact with the co-repressor protein C-terminal Binding Protein (CtBP). We demonstrate that the KLF8 Pro-Val-Asp-Leu-Ser motif also contacts CtBP. We show that the N-terminus of KLF8 functions as a repression domain and that its activity relies on the integrity of the CtBP recognition motif. We demonstrate that the zinc fingers of KLF8 recognize CACCC elements in DNA and that full-length KLF8 can repress a CACCC-dependent promoter. Finally we determine that KLF8 is broadly expressed in human tissues. These results establish KLF8 as a CACCC-box binding protein that associates with CtBP and represses transcription.

3T3 Cells↗
PubMed2000

A class of zinc fingers involved in protein-protein interactions biophysical characterization of CCHC fingers from fog and U-shaped.

Zinc fingers (ZnFs) are extremely common protein domains. Several classes of ZnFs are distinguished by the nature and spacing of their zinc-coordinating residues. While the structure and function of some ZnFs are well characterized, many others have been identified only through their amino acid sequence. A number of proteins contain a conserved C-X2-C-X12-H-X1-5-C sequence, which is similar to the spacing observed for the 'classic' CCHH ZnFs. Although these domains have been implicated in protein-protein (and not protein-nucleic acid) interactions, nothing is known about their structure or function at a molecular level. Here, we address this problem through the expression and biophysical characterization of several CCHC-type zinc fingers from the erythroid transcription factor FOG and the related Drosophila protein U-shaped. Each of these domains does indeed fold in a zinc-dependent fashion, coordinating the metal in a tetrahedral manner through the sidechains of one histidine and three cysteine residues, and forming extremely thermostable structures. Analysis of CD spectra suggests an overall fold similar to that of the CCHH fingers, and indeed a point mutant of FOG-F1 in which the final cysteine residue is replaced by histidine remains capable of folding. However, the CCHC (as opposed to CCHH) motif is a prerequisite for GATA-1 binding activity, demonstrating that CCHC and CCHH topologies are not interchangeable. This demonstration that members of a structurally distinct subclass of genuine zinc finger domains are involved in the mediation of protein-protein interactions has implications for the prediction of protein function from nucleotide sequences.

Amino Acid Sequence↗
PubMed1999

hFOG-2, a novel zinc finger protein, binds the co-repressor mCtBP2 and modulates GATA-mediated activation.

We have identified a novel human zinc finger protein, hFOG-2, which is related to but distinct from the murine transcription factor Friend-of-GATA-1 (mFOG-1). The hFOG-2 gene was initially detected in K562 cells using a polymerase chain reaction approach with degenerate primers corresponding to zinc finger regions of mFOG-1. A murine homologue of hFOG-2 was also identified in the mouse expressed sequence tag data banks, indicating that a family of FOG genes exists in mammals. hFOG-2 appears to be widely expressed, while mFOG-1 is expressed primarily in erythroid and megakaryocytic cells and plays a fundamental role in the development of these lineages. Sequencing of the full-length hFOG-2 cDNA indicates that the interaction domains for transcription factors GATA-1 and mCtBP2 are both conserved and we have shown that this new FOG protein also physically interacts with these factors. We have demonstrated that hFOG-2, like mFOG-1, can act in concert with GATA-1 to activate gene expression from the p45 NF-E2 promoter region, but that it can also act to repress GATA-mediated activation of additional reporter constructs. Finally, we have identified a repression domain in hFOG-2 and show that repression is dependent upon the integrity of the mCtBP2 interaction motif Pro-Ile-Asp-Leu-Ser.

Alcohol Oxidoreductases↗
PubMed1999

Transcriptional cofactors of the FOG family interact with GATA proteins by means of multiple zinc fingers.

Friend of GATA-1 (FOG-1) is a zinc finger protein that has been shown to interact physically with the erythroid DNA-binding protein GATA-1 and modulate its transcriptional activity. Recently, two new members of the FOG family have been identified: a mammalian protein, FOG-2, that also associates with GATA-1 and other mammalian GATA factors; and U-shaped, a Drosophila protein that interacts with the Drosophila GATA protein Pannier. FOG proteins contain multiple zinc fingers and it has been shown previously that the sixth finger of FOG-1 interacts specifically with the N-finger but not the C-finger of GATA-1. Here we show that fingers 1, 5 and 9 of FOG-1 also interact with the N-finger of GATA-1 and that FOG-2 and U-shaped also contain multiple GATA-interacting fingers. We define the key contact residues and show that these residues are highly conserved in GATA-interacting fingers. We examine the effect of selectively mutating the four interacting fingers of FOG-1 and show that each contributes to FOG-1's ability to modulate GATA-1 activity. Finally, we show that FOG-1 can repress GATA-1-mediated activation and present evidence that this ability involves the recently described CtBP co-repressor proteins that recognize all known FOG proteins.

Alcohol Oxidoreductases↗