A novel isoform of human lymphoid enhancer-binding factor-1 (LEF-1) gene transcript encodes a protein devoid of HMG domain and nuclear localization signal.
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Lymphoid enhancer-binding factor-1 (LEF-1), a member of the high mobility group (HMG) family of proteins, regulates expression of T-cell receptor-alpha gene and is one of the key regulatory molecules in the epithelial-mesenchymal interactions during embryonic development. Among others, LEF-1 regulates expression of cytokeratin genes involved in formation of hair follicles and the gene encoding the cell-adhesion molecule E-cadherin. Transcription factor LEF-1, which acts as a dimer, binds beta-catenin and is involved in signal transduction by the wnt pathway. We have cloned and sequenced a novel isoform of human LEF-1 gene transcript. This isoform encodes a truncated protein devoid of HMG domain and nuclear localization signal but retaining beta-catenin binding domain. This isoform might either act in a dominant-negative manner by interfering with native LEF-1, or might bind beta-catenin in the cytosol, which would result in attenuation of the signals transmitted by the LEF-beta-catenin pathway. (+info)
Establishment of dorsal-ventral polarity of the Drosophila egg requires capicua action in ovarian follicle cells.
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The dorsal-ventral pattern of the Drosophila egg is established during oogenesis. Epidermal growth factor receptor (Egfr) signaling within the follicular epithelium is spatially regulated by the dorsally restricted distribution of its presumptive ligand, Gurken. As a consequence, pipe is transcribed in a broad ventral domain to initiate the Toll signaling pathway in the embryo, resulting in a gradient of Dorsal nuclear translocation. We show that expression of pipe RNA requires the action of fettucine (fet) in ovarian follicle cells. Loss of maternal fet activity produces a dorsalized eggshell and embryo. Although similar mutant phenotypes are observed with regulators of Egfr signaling, genetic analysis suggests that fet acts downstream of this event. The fet mutant phenotype is rescued by a transgene of capicua (cic), which encodes an HMG-box transcription factor. We show that Cic protein is initially expressed uniformly in ovarian follicle cell nuclei, and is subsequently downregulated on the dorsal side. Earlier studies described a requirement for cic in repressing zygotic target genes of both the torso and Toll pathways in the embryo. Our experiments reveal that cic controls dorsal-ventral patterning by regulating pipe expression in ovarian follicle cells, before its previously described role in interpreting the Dorsal gradient. (+info)
Distorted DNA structures induced by HMGB2 possess a high affinity for HMGB2.
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HMGB2 (HMG2) protein binds with DNA duplex in a sequence-nonspecific manner, then bends and unwinds the DNA. In DNA cyclization analyses for the bending activity of HMGB2, two unidentified bands, denoted alpha and beta, were observed in addition to monomer circular DNA (1C) on the gel. Re-electrophoresis and proteinase K digestion revealed that alpha and beta are complexes of circularized probe DNA (seeming 1C) with HMGB2 (K(d) approximately 10(-10) M). The DNA components of alpha and beta (alpha- and beta-DNA) showed higher affinities to HMGB2 than did the linear probe DNA (K(d) approximately 10(-7) M). The DNAs have distorted structures containing partial single-stranded regions. Nicked circular molecules presumably due to severe DNA distortion by HMGB2 were observed in alpha- and beta-DNA, in addition to closed circular double-stranded molecules. The alpha and beta bands were not formed in the presence of sole DNA binding regions which are necessary for DNA bending, indicating that the acidic C-tail in the HMGB2 molecule is necessary for inducing the peculiar distorted structures of higher affinity to HMGB2. HMGB2 binds with linker DNA and/or the entry and exit of nucleosomes fixed at both ends likewise mini-circles similar to alpha-DNA and beta-DNA. Thus, the distorted structures present in alpha-DNA and beta-DNA should be important in considering the functional mechanisms in which HMGB2 participates. (+info)
The modulation of the biological activities of mitochondrial histone Abf2p by yeast PKA and its possible role in the regulation of mitochondrial DNA content during glucose repression.
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The mitochondrial histone, Abf2p, of Saccharomyces cerevisiae is essential for the maintenance of mitochondrial DNA (mtDNA) and appears to play an important role in the recombination and copy number determination of mtDNA. Abf2p, encoded by a nuclear gene, is a member of HMG1 DNA-binding protein family and has two HMG1-Box domains, HMG1-Box A and B. To investigate the role of Abf2p in the control of mtDNA copy number, we asked if the in vivo functions of Abf2p are regulated by the possible modification such as phosphorylation. We found that the N-terminal extended segment (KRPT(21)S(22)) of HMG1-Box A is rapidly and specifically phosphorylated by cAMP-dependent protein kinase (PKA) in vitro. The phosphorylation in this region inhibits the binding of Abf2p to all kinds of DNA including four-way junction DNA and the supercoiling activity of Abf2p itself. The abf2 mutant cells with an abf2(T21A/S22A) allele defective in the phosphorylation site have a severe defect in the regulation of mtDNA content during glucose repression in vivo. These observations suggest that the phosphorylation via PKA, that is activated during glucose repression, may regulate the in vivo functions of Abf2p for the control of mtDNA content during shift from gluconeogenic to fermentative growth. (+info)
Interactions between HMG boxes.
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Many proteins consist of subdomains that can fold and function independently. We investigate here the interaction between the two high mobility group (HMG) box subdomains of the nuclear protein rHMG1. An HMG box is a conserved amino acid sequence of approximately 80 amino acids rich in basic, aromatic and proline side chains that is active in binding DNA in a sequence or structure-specific manner. In the case of HMG1, each box can bind structural DNA substrates including four-way junctions (4WJs) and branched or kinked DNA duplexes. Since proteins containing up to six HMG boxes are known, the question arises whether linking subdomains together influences the folding or function of individual boxes. In an effort to understand interactions between individual DNA-binding domains in HMG1, we created new fusion proteins: one is an inversion of the order of the AB di-domain in HMG1 (BA); in the second, we added a third A domain C-terminal to the AB di-domain (ABA). Pairs of boxes, AB or BA, behave similarly and are functionally active. By contrast, the ABA triple subdomain construct is partially unfolded and is less active than individual boxes or di-domains. Thus, long-range inter-domain effects can influence the activity of HMG boxes. (+info)
Recognition and repair of DNA-cisplatin adducts.
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Anticancer activity of cisplatin (cis-diamminedichloroplatinum) is believed to result from its interaction with DNA. The drug reacts with nucleophilic sites in DNA forming monoadducts as well as intra- and interstrand crosslinks. DNA-cisplatin adducts are specifically recognized by several proteins. They can be divided into two classes. One constitutes proteins which recognize DNA damage as an initial step of the nucleotide excision and mismatch repair pathways. The other class contains proteins stabilizing cellular DNA-protein and protein-protein complexes, including non-histone proteins from the HMG (high-mobility-group) family. They specifically recognize 1,2-interstrand d(GpG) and d(ApG) crosslinks of DNA-cisplatin adducts and inhibit their repair. Many HMG-domain proteins can function as transcription factors, e.g. UBF, an RNA polymerase I transcription factor, the mammalian testis-determining factor SRY and the human mitochondrial transcription factor mtTFA. Moreover, it seems that some proteins, which probably recognize DNA-cisplatin adducts non-specifically, e.g. actin and other nuclear matrix proteins, can disturb the structural and functional organization of the nucleus and whole cell. The formation of complexes between DNA and proteins in the presence of cisplatin and the changes in the cell architecture may account for the drug cytotoxicity. (+info)
TOX defines a conserved subfamily of HMG-box proteins.
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BACKGROUND: HMG-box proteins are a large and diverse superfamily of architectural factors that share one or more copies of a sequence- and structurally-related DNA binding domain. These proteins can modify chromatin structure by bending and unwinding DNA. HMG-box proteins can be divided into two subfamilies based on whether they recognize DNA in a sequence-dependent or sequence-independent manner. We recently identified an HMG-box protein involved in T cell development, designated TOX, which is highly conserved in humans and mice. RESULTS: We show here that based on sequence alignment, TOX best fits into the sequence-independent HMG-box family. Three other human and murine predicted proteins are identified that share a common HMG-box domain with TOX, as well as other features. The gene encoding one of these additional family members has a distinct but overlapping pattern of tissue expression when compared to TOX. In addition, we identify genes encoding predicted TOX HMG-box subfamily members in pufferfish and mosquito. CONCLUSIONS: We have identified a novel subfamily of HMG-box proteins that is related to the recently described TOX protein. The highly conserved nature of the TOX family of proteins in humans and mice and differences in the pattern of expression between family members suggest non-overlapping functions of individual proteins. In addition, our data suggest that the TOX subtype of HMG-box domain first appeared in invertebrates, was duplicated in early vertebrates and likely took on new functions in mammalian species. (+info)
Ste11p, a high-mobility-group box DNA-binding protein, undergoes pheromone- and nutrient-regulated nuclear-cytoplasmic shuttling.
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The high-mobility-group (HMG) box is a conserved DNA-binding domain found in a family of transcription factors that regulate growth and development. One family member, Ste11p, directs sexual differentiation of Schizosaccharomyces pombe by binding specific DNA sequences upstream of genes required for mating and meiosis. Here, we show that Ste11p is a shuttling protein. In growing cells, Ste11p is present in low levels and is pancellular. Mating pheromones and nutrient limitation trigger nuclear accumulation and increased expression of the transcription factor. Several mechanisms likely control Ste11p localization. First, the 14-3-3 protein, Rad24p, binds phosphorylated Ste11p and inhibits its nuclear accumulation. Second, the HMG domain of Ste11p contains a basic cluster nuclear localization signal. Finally, treatment of cells with leptomycin B, an exportin inhibitor, results in the nuclear accumulation of Ste11p. A Ste11p deletion mutation, DeltaC54, mimics the effects of leptomycin B. The C54 region contains no identifiable nuclear export signal but instead is required for biological activity and to stimulate Ste11p target gene expression. These results provide evidence that both nuclear import and export mechanisms operate to regulate cellular localization of an HMG box protein. In addition, they establish a paradigm for the potential role of pheromone/hormone-like polypeptides in cellular localization of this important class of developmental regulators. (+info)