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M Crossley

Publications and source records attributed to M Crossley.

At least 37 records · Page 2Linked to original sources
PubMed1999

Mammalian Krüppel-like transcription factors: more than just a pretty finger.

The transcription factor SP1 contains three Krüppel-like zinc fingers. Recently, several related proteins, including erythroid, lung and gut-enriched Krüppel-like factors, have been identified. Together with SP1, these proteins form a sizeable family of transcription factors that share homology in their zinc-finger domains but differ elsewhere. Analysis of these differences is illuminating specific mechanisms by which transcription is regulated.

Amino Acid Sequence↗
PubMed1999

Basic Krüppel-like factor functions within a network of interacting haematopoietic transcription factors.

Basic Krüppel-like Factor (BKLF) is a recently recognized member of a small group of transcription factors that bind CACCC motifs in DNA, by means of three highly conserved C-terminal Krüppel-like (typically Cys-X2-4-Cys-X12-His-X3-4-His) zinc fingers. Together with Erythroid Krüppel-like Factor (EKLF), it is one of the most abundant CACCC-binding proteins in erythroid cells. In contrast to EKLF, BKLF can act to repress transcription and thus may serve to moderate EKLF activity in vivo. Interestingly, it has also been shown that BKLF expression in erythroid cells is dependent on EKLF. Analysis of proteins interacting with BKLF indicates that it represses transcription by recruiting the general co-repressor protein CtBP, a cofactor that also associates with other haematopoietic transcriptional repressors such as Evi-1 and ZEB/AREB6. The observation that mice deficient in BKLF exhibit a myeloproliferative disorder suggests that BKLF regulates important processes involved in haematopoietic differentiation.

Amino Acid Sequence↗
PubMed1999

The solution structure of the N-terminal zinc finger of GATA-1 reveals a specific binding face for the transcriptional co-factor FOG.

Zinc fingers (ZnFs) are generally regarded as DNA-binding motifs. However, a number of recent reports have implicated particular ZnFs in the mediation of protein-protein interactions. The N-terminal ZnF of GATA-1 (NF) is one such finger, having been shown to interact with a number of other proteins, including the recently discovered transcriptional co-factor FOG. Here we solve the three-dimensional structure of the NF in solution using multidimensional 1H/15N NMR spectroscopy, and we use 1H/15N spin relaxation measurements to investigate its backbone dynamics. The structure consists of two distorted beta-hairpins and a single alpha-helix, and is similar to that of the C-terminal ZnF of chicken GATA-1. Comparisons of the NF structure with those of other C4-type zinc binding motifs, including hormone receptor and LIM domains, also reveal substantial structural homology. Finally, we use the structure to map the spatial locations of NF residues shown by mutagenesis to be essential for FOG binding, and demonstrate that these residues all lie on a single face of the NF. Notably, this face is well removed from the putative DNA-binding face of the NF, an observation which is suggestive of simultaneous roles for the NF; that is, stabilisation of GATA-1 DNA complexes and recruitment of FOG to GATA-1-controlled promoter regions.

Amino Acid Sequence↗
PubMed1999

Positional treatment vs continuous positive airway pressure in patients with positional obstructive sleep apnea syndrome.

OBJECTIVES: The aim of this study was to compare the relative efficacy of continuous positive airway pressure (CPAP) and positional treatment in the management of positional obstructive sleep apnea (OSA), using objective outcome measures. DESIGN: A prospective, randomized, single blind crossover comparison of CPAP and positional treatment for 2 weeks each. SETTING: A university teaching hospital. PATIENTS: Thirteen patients with positional OSA, aged (mean+/-SD) 51+/-9 years, with an apnea-hypopnea index (AHI) of 17+/-8. MEASUREMENTS: (1) Daily Epworth Sleepiness Scale scores; (2) overnight polysomnography, an objective assessment of sleep quality and AHI; (3) maintenance of wakefulness testing; (4) psychometric test battery; (5) mood scales; (6) quality-of-life questionnaires; and (7) individual patient's treatment preference. RESULTS: Positional treatment was highly effective in reducing time spent supine (median, 0; range, 0 to 32 min). The AHI was lower (mean difference, 6.1; 95% confidence interval [CI], 2 to 10.2; p = 0.007), and the minimum oxygen saturation was higher (4%; 95% CI, 1% to 8%; p = 0.02) on CPAP as compared with positional treatment. There was no significant difference, however, in sleep architecture, Epworth Sleepiness Scale scores, maintenance of wakefulness testing sleep latency, psychometric test performance, mood scales, or quality-of-life measures. CONCLUSION: Positional treatment and CPAP have similar efficacy in the treatment of patients with positional OSA.

Cross-Over Studies↗
PubMed1998

Key residues characteristic of GATA N-fingers are recognized by FOG.

Protein-protein interactions play significant roles in the control of gene expression. These interactions often occur between small, discrete domains within different transcription factors. In particular, zinc fingers, usually regarded as DNA-binding domains, are now also known to be involved in mediating contacts between proteins. We have investigated the interaction between the erythroid transcription factor GATA-1 and its partner, the 9 zinc finger protein, FOG (Friend Of GATA). We demonstrate that this interaction represents a genuine finger-finger contact, which is dependent on zinc-coordinating residues within each protein. We map the contact domains to the core of the N-terminal zinc finger of GATA-1 and the 6th zinc finger of FOG. Using a scanning substitution strategy we identify key residues within the GATA-1 N-finger which are required for FOG binding. These residues are conserved in the N-fingers of all GATA proteins known to bind FOG, but are not found in the respective C-fingers. This observation may, therefore, account for the particular specificity of FOG for N-fingers. Interestingly, the key N-finger residues are seen to form a contiguous surface, when mapped onto the structure of the N-finger of GATA-1.

3T3 Cells↗
PubMed1998

Involvement of the N-finger in the self-association of GATA-1.

Zinc fingers are recognized as small protein domains that bind to specific DNA sequences. Recently however, zinc fingers from a number of proteins, in particular the GATA family of transcription factors, have also been implicated in specific protein-protein interactions. The erythroid protein GATA-1 contains two zinc fingers: the C-finger, which is sufficient for sequence-specific DNA-binding, and the N-finger, which appears both to modulate DNA-binding and to interact with other transcription factors. We have expressed and purified the N-finger domain and investigated its involvement in the self-association of GATA-1. We demonstrate that this domain does not homodimerize but instead makes intermolecular contacts with the C-finger, suggesting that GATA dimers are maintained by reciprocal N-finger-C-finger contacts. Deletion analysis identifies a 25-residue region, C-terminal to the core N-finger domain, that is sufficient for interaction with intact GATA-1. A similar subdomain exists C-terminal to the C-finger, and we show that self-association is substantially reduced when both subdomains are disrupted by mutation. Moreover, mutations that impair GATA-1 self-association also interfere with its ability to activate transcription in transfection studies.

Amino Acid Sequence↗
PubMed1998

Transcription factor erythroid Krüppel-like factor (EKLF) is essential for the erythropoietin-induced hemoglobin production but not for proliferation, viability, or morphological maturation.

The erythroid Krüppel-like factor (EKLF) is essential for the transcription of betamaj globin in erythroid cells. We show here that RNA for this transcription factor did not alter during erythropoietin-induced differentiation of J2E cells; however, EKLF protein content decreased and was inversely related to globin production. This unexpected result was also observed during chemically induced maturation of two murine erythroleukemia cell lines. To explore the role of EKLF in erythroid terminal differentiation, an antisense EKLF construct was introduced into J2E cells. As a consequence EKLF RNA and protein levels fell by approximately 80%, and the cells were unable to manufacture hemoglobin in response to erythropoietin. The failure to produce hemoglobin was due to reduced transcription of not only globin genes but also key heme enzyme genes. However, numerous other genes, including several erythroid transcription factors, were unaffected by the decrease in EKLF. Although hemoglobin synthesis was severely impaired with depleted EKLF levels, morphological maturation in response to erythropoietin continued normally. Moreover, erythropoietin-induced proliferation and viability were unaffected by the decrease in EKLF levels. We conclude that EKLF affects a specific set of genes, which regulates hemoglobin production and has no obvious effect on morphological changes, cell division, or viability in response to erythropoietin.

Animals↗
PubMed1998

Cloning and characterization of mCtBP2, a co-repressor that associates with basic Krüppel-like factor and other mammalian transcriptional regulators.

Basic Krüppel-like factor (BKLF) is a zinc finger protein that recognizes CACCC elements in DNA. It is expressed highly in erythroid tissues, the brain and other selected cell types. We have studied the activity of BKLF and found that it is capable of repressing transcription, and have mapped its repression domain to the N-terminus. We carried out a two-hybrid screen against BKLF and isolated a novel clone encoding murine C-terminal-binding protein 2 (mCtBP2). mCtBP2 is related to human CtBP, a cellular protein which binds to a Pro-X-Asp-Leu-Ser motif in the C-terminus of the adenoviral oncoprotein, E1a. We show that mCtBP2 recognizes a related motif in the minimal repression domain of BKLF, and the integrity of this motif is required for repression activity. Moreover, when tethered to a promoter by a heterologous DNA-binding domain, mCtBP2 functions as a potent repressor. Finally, we demonstrate that mCtBP2 also interacts with the mammalian transcripition factors Evi-1, AREB6, ZEB and FOG. These results establish a new member of the CtBP family, mCtBP2, as a mammalian co-repressor targeting diverse transcriptional regulators.

Alcohol Oxidoreductases↗
PubMed1998

Expression of the parathyroid Ca(2+)-sensing receptor in cytotrophoblasts from human term placenta.

Fura-2-loaded human cytotrophoblasts responded to elevated extracellular Ca2+ concentration ([Ca2+]o) with monophasic or, in the case of large (> 20 microns) extravillous cells, biphasic elevations in intracellular free Ca2+ ion concentration ([Ca2+]i) that returned to baseline levels after restoration of control [Ca2+]o. Large extravillous cytotrophoblasts also responded to elevated [Mg2+]o with transient elevations in [Ca2+]i, consistent with the behaviour of the parathyroid Ca2(+)-sensing receptor. Expression of the parathyroid Ca2(+)-sensing receptor in placental cells was confirmed using Northern blot and reverse transcription (RT)-PCR analysis. However, the major transcript in human placental cells (6.2 kb) differed from that expressed by human parathyroid cells (5.6 kb). RT-PCR analysis and DNA sequencing of key PCR products also revealed the presence of a splice variant in placental and parathyroid cells that lacks exon 3.

Blotting, Northern↗
PubMed1997

FOG, a multitype zinc finger protein, acts as a cofactor for transcription factor GATA-1 in erythroid and megakaryocytic differentiation.

The hematopoietic transcription factor GATA-1 is essential for development of the erythroid and megakaryocytic lineages. Using the conserved zinc finger DNA-binding domain of GATA-1 in the yeast two-hybrid system, we have identified a novel, multitype zinc finger protein, Friend of GATA-1 (FOG), which binds GATA-1 but not a functionally inactive mutant lacking the amino (N) finger. FOG is coexpressed with GATA-1 during embryonic development and in erythroid and megakaryocytic cells. Furthermore, FOG and GATA-1 synergistically activate transcription from a hematopoietic-specific regulatory region and cooperate during both erythroid and megakaryocytic cell differentiation. These findings indicate that FOG acts as a cofactor for GATA-1 and provide a paradigm for the regulation of cell type-specific gene expression by GATA transcription factors.

3T3 Cells↗
PubMed1997

Letter and category fluency in community-dwelling Canadian seniors: a comparison of normal participants to those with dementia of the Alzheimer or vascular type.

Measures of letter (FAS) and category (Animal Naming) fluency were completed by community-dwelling, cognitively normal seniors (n = 635), and by individuals diagnosed with dementia of the Alzheimer type (DAT) (n = 155), or with vascular dementia (n = 39). Among normal seniors, category, but not letter fluency, declined with age, females performed better than males on letter but not on category fluency, and performance increased with education on both tasks. Among participants with DAT, letter fluency was influenced by level of education, whereas category fluency was education-, age-, and gender-invariant. Both measures differentiated normal seniors from those with dementia. Category fluency was more impaired than letter fluency at both mild and moderate stages of dementia, but neither task differentiated DAT from vascular dementia.

Aged↗
PubMed1997

Use of the Brief Psychiatric Rating Scale to measure success in a psychosocial day program.

This study contrasted six subscales of the Brief Psychiatric Rating Scale (BPRS) to determine their sensitivity to psychosocial treatment outcome. An expanded version of the BPRS was administered to 216 clients on admission to a day program. The subscale measuring hostility and suspiciousness discriminated at intake clients who were therapeutically discharged from clients who did not complete the program and predicted discharge status after the investigators controlled for the effects of demographic variables. Significant reductions in scores were obtained on five subscales for a subset of clients to whom the BPRS was readministered before discharge. The results support the use of the expanded BPRS as an evaluative tool in psychosocial rehabilitation programs.

Adult↗
PubMed1996

Isolation and characterization of the cDNA encoding BKLF/TEF-2, a major CACCC-box-binding protein in erythroid cells and selected other cells.

CACCC boxes are among the critical sequences present in regulatory elements of genes expressed in erythroid cells, as well as in selected other cell types. While an erythroid cell-specific CACCC-box-binding protein, EKLF, has been shown to be required in vivo for proper expression of the adult beta-globin gene, it is dispensable for the regulation of several other globin and nonglobin erythroid cell-expressed genes. In the work described here, we searched for additional CACCC-box transcription factors that might be active in murine erythroid cells. We identified a major gel shift activity (termed BKLF), present in yolk sac and fetal liver erythroid cells, that could be distinguished from EKLF by specific antisera. Through relaxed-stringency hybridization, we obtained the cDNA encoding BKLF, a highly basic, novel zinc finger protein that is related to EKLF and other Krüppel-like members in its DNA-binding domain but unrelated elsewhere. BKLF, which is widely but not ubiquitously expressed in cell lines, is highly expressed in the midbrain region of embryonic mice and appears to correspond to the gel shift activity TEF-2, a transcriptional activator implicated in regulation of the simian virus 40 enhancer and other CACCC-box-containing regulatory elements. Because BKLF binds with high affinity and preferentially over Sp1 to many CACCC sequences of erythroid cell expressed genes, it is likely to participate in the control of many genes whose expression appears independent of the action of EKLF.

Amino Acid Sequence↗
PubMed1995

The C-terminal zinc finger of GATA-1 or GATA-2 is sufficient to induce megakaryocytic differentiation of an early myeloid cell line.

The GATA-1 and GATA-2 transcription factors, which each contain two homologous zinc fingers, are important hematopoietic regulators expressed within the erythroid, mast cell, and megakaryocytic lineages. Enforced expression of either factor in the primitive myeloid line 416B induces megakaryocytic differentiation. The features of their structure required for this activity have been explored. The ability of 12 GATA-1 mutants to promote 416B maturation was compared with their DNA-binding activity and transactivation potential. Differentiation did not require any of the seven serine residues that are phosphorylated in vivo, an N-terminal region bearing the major transactivation domain, or a C-terminal segment beyond the fingers. Removal of a consensus nuclear localization signal following the second finger did not block differentiation or nuclear translocation. The N-terminal finger was also dispensable, although its removal attenuated differentiation. In contrast, the C-terminal finger was essential, underscoring its distinct function. Remarkably, only 69 residues spanning the C-terminal finger were required to induce limited megakaryocytic differentiation. Analysis of three GATA-2 mutants led to the same conclusion. Endogenous GATA-1 mRNA was induced by most mutants and may contribute to differentiation. Because the GATA-1 C-terminal finger could bind its target site but not transactivate a minimal reporter, it may direct megakaryocytic maturation by derepressing specific genes and/or by interacting with another protein which provides the transactivation function.

3T3 Cells↗
PubMed1995

Self-association of the erythroid transcription factor GATA-1 mediated by its zinc finger domains.

GATA-1, the founding member of a distinctive family of transcription factors, is expressed predominantly in erythroid cells and participates in the expression of numerous erythroid cell-expressed genes. GATA-binding sites are found in the promoters and enhancers of globin and nonglobin erythroid genes as well as in the alpha- and beta-globin locus control regions. To elucidate how GATA-1 may function in a variety of regulatory contexts, we have examined its protein-protein interactions. Here we show that GATA-1 self-associates in solution and in whole-cell extracts and that the zinc finger region of the molecule is sufficient to mediate this interaction. This physical interaction can influence transcription, as GATA-1 self-association is able to recruit a transcriptionally active but DNA-binding-defective derivative of GATA-1 to promoter-bound GATA-1 and result in superactivation. Through in vitro studies with bacterially expressed glutathione S-transferase fusion proteins, we have localized the minimal domain required for GATA-1 self-association to 40 amino acid residues within the C-terminal zinc finger region. Finally, we have detected physical interaction of GATA-1 with other GATA family members (GATA-2 and GATA-3) also mediated through the zinc finger domain. These findings have broad implications for the involvement of GATA factors in transcriptional control. In particular, the interaction of GATA-1 with itself and with other transcription factors may facilitate its function at diverse promoters in erythroid cells and also serve to bring together, or stabilize, loops between distant regulatory elements, such as the globin locus control regions and downstream globin promoters. We suggest that the zinc finger region of GATA-1, and related proteins, is multifunctional and mediates not only DNA binding but also important protein-protein interactions.

Amino Acid Sequence↗
PubMed1994

Phosphorylation of the erythroid transcription factor GATA-1.

GATA-1 is a zinc finger DNA-binding protein thought to be involved in the expression of the vast majority of erythroid specific genes. We have examined the phosphorylation of GATA-1 in murine erythroleukemia (MEL) cells and have mapped the sites of phosphorylation by overexpression of GATA-1 in monkey kidney COS cells. We show that GATA-1 is phosphorylated on 6 serines within its amino terminus in uninduced MEL cells and that a 7th site, serine 310, becomes phosphorylated after MEL cells are induced to differentiate by exposure to dimethyl sulfoxide. This site lies near the carboxyl boundary of the DNA-binding domain in a conserved region of the protein believed to be involved in DNA bending. Detailed analyses indicate, however, that phosphorylation at this site, or the other sites identified, does not significantly influence DNA-binding affinity or specificity, DNA bending, or transcriptional transactivation by GATA-1.

3T3 Cells↗