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At least 361 records · Page 20Linked to original sources

PubMed2005

Transcriptional modulation of the pre-implantation embryo-specific Rnf35 gene by the Y-box protein NF-Y/CBF.

Maternal-to-zygotic transition of a fertilized egg and the subsequent pre-implantation development of the embryo involve zygotic genome activation and reprogramming of gene expression. The goal of the present study is to establish a model suitable for the characterization of transcriptional modulation of mammalian pre-implantation development. Rnf35 is a mouse RING-finger protein gene that is temporally transcribed in the early embryo, but is permanently silenced before the blastocyst stage of development. We first show that the Chinese-hamster ovary-K1 cells are unique in supporting Rnf35 promoter activities in transient transfection assays. Using the permissive Chinese-hamster ovary-K1 cell line, we show that Rnf35 transcription is driven by an Inr (initiator) core promoter element in the absence of a TATA box; the Inr promoter function is confirmed by direct microinjection of mouse one-cell embryos. This is the first demonstration of the involvement of an Inr core promoter element in transcription in pre-implantation development. We show that the Rnf35 promoter is regulated by three obligatory Y-box (CCAAT-box) elements: two Y boxes (Y(I) and Y(II)) located at -81 are coupled in a palindrome and act synergistically in contributing to Rnf35 transcription; the third Y box (Y(III)) is situated at -13, just upstream of the Inr element, and may be an integral part of the Inr function. Electrophoretic mobility-shift assays and competition experiments further reveal that the Y(I) box is bound by the ubiquitous NF-Y (nuclear factor-Y)/CBF (CCAAT-binding factor) and that Y(II) is targeted by an unidentified protein(s) that acts synergistically with the NF-Y. We suggest that the NF-Y, targeting at a Y-box sequence, may function as an important activator in transcriptional regulation of the Rnf35 gene in the pre-implantation embryo.

Animals↗

PubMed1996

Identification of a transcription factor that binds to the S box of the I-A beta gene of the major histocompatibility complex.

Class II genes of the MHC show a striking homology upstream of the transcription start site that is composed of three conserved sequences (S, X and Y boxes, each separated by 15-20 bp). The presence of the S-box sequence in the mouse MHC class II gene I-A Beta was examined for its influence on the expression of this gene. Deletion or mutation of the S box decreased the induction of chloramphenicol acetyltransferase (CAT) activity in B lymphocytes by 32%. In macrophages, deletion or mutation of the S box abolished interferon-gamma (IFN-gamma) inducibility of CAT activity. Using a gel-retardation assay, we have identified a nuclear factor whose binding site overlaps the 7-mer conserved sequence of the S box. This factor is present in lymphocytes, macrophages, mastocytes and fibroblasts. Surprisingly, binding of this nuclear factor to DNA was induced by IFN-gamma in bone-marrow-derived macrophages, but not in macrophage-like cell lines. The binding site for this factor was defined by DNase I footprinting and partially purified by using an affinity column containing double-stranded oligonucleotides containing a sequence of the S box. A prominent protein of 43 kDa was found that bound specifically to the S-box sequence.

Animals↗

PubMed1997

Pollen-specific expression of DEFH125, a MADS-box transcription factor in Antirrhinum with unusual features.

MADS-box genes encode transcription factors that regulate different processes of early and late floral development. A novel type of MADS-box gene, DEFH125, was isolated from a stamen specific cDNA library from Antirrhinum majus. The DEFH125 protein shows extensive similarity over the entire length to AGL17, a root-specific MADS-box protein of Arabidopsis. By sharing amino acid deviations from the consensus MADS-box sequence not found in other MADS-box families, these two proteins constitute a novel MADS-box subfamily. However, in contrast to members of other subfamilies the overall structural similarity between the DEFH125 and AGL17 proteins does not coincide with a similarity of expression patterns and functions. The DEFH125 gene is expressed at detectable levels only in the third whorl when the meiotic division of the pollen mother cell is already accomplished. The DEFH125 protein has been located in the cytoplasm of the vegetative cell within the maturing pollen. Surprisingly, after pollination, the DEFH125 protein is also found in nuclei of cells within the transmitting tract of the carpel. The intriguing role of DEFH125, the first MADS-box transcription factor of this type, in aspects of fertilization, such as pollen maturation, pollen tube formation or pollen tube guidance in the carpel, is discussed.

Amino Acid Sequence↗

PubMed1998

An endosperm-specific DOF protein from barley, highly conserved in wheat, binds to and activates transcription from the prolamin-box of a native B-hordein promoter in barley endosperm.

A cDNA encoding a DNA-binding protein of the DOF class of transcription factors was isolated from a barley endosperm library. The deduced amino acid sequence for the corresponding protein is 94% identical through the DOF domain to the prolamin-box (P-box) binding factor PBF from maize. The gene encoding the barley PBF (BPBF) maps to chromosome 7H, and its expression is restricted to the endosperm where it precedes that of the hordein genes. The BPBF expressed in bacteria as a GST-fusion binds a P-box 5'-TGTAAAG-3' containing oligonucleotide derived from the promoter region of an Hor2 gene. Binding was prevented when the P-box motif was mutated to 5'-TGTAgAc-3'. A P-box binding activity, present in barley and wheat endosperm nuclei, interacted similarly to BPBF with this synthetic oligonucleotide, and the binding was abolished by 1,10-phenanthroline. Transient expression experiments in developing barley endosperms demonstrate that BPBF transactivates transcription from the P-box element of a native Hor2 promoter and that direct binding of BPBF to its target site is essential for transactivation since mutations in the DOF DNA-binding domain or in the P-box motif of this promoter abolished both binding and transactivation. Evidence was also obtained for the presence in wheat of a Pbf homologue having similar DNA-binding properties to that of BPBF. These results strongly implicate this endosperm-specific DOF protein from barley as an important activator of hordein gene expression and suggest the evolutionary conservation of the Pbf gene function among small grain cereals.

Amino Acid Sequence↗

PubMed1999

The tomato I-box binding factor LeMYBI is a member of a novel class of myb-like proteins.

The RBCS3A gene of tomato belongs to a small gene family consisting of five members. Although the RBCS1, RBCS2 and RBCS3A promoters contain closely related cis regulatory sequences, the expression patterns of the genes are different. Whereas the RBCS1 and RBCS2 genes are expressed in both leaves and young fruit, the RBCS3A promoter is highly active in leaves, but not in young fruit. This lack of transcription could be due to a mutation in the RBCS3A promoter creating the so-called F-box, a protein binding site located between the activating cis elements, the I-box and G-box. In order to identify proteins that bind to the RBCS3A I-box/F-box region, the yeast one-hybrid system was used. One clone, LeMYBI was isolated which contains strong similarity to plant myb transcription factors. The encoded LeMYBI protein is at least 188 amino acids in length and contains two myb-like domains located at the amino terminus and close to the carboxy terminus, separated by a negatively charged domain. The protein contains a SHAQKYF amino acid signature motif in the second myb-like repeat, which is highly conserved in a number of recently identified plant myb-related genes, thus defining a new class of plant DNA-binding proteins. LeMYBI binds specifically to the I-box sequence of the RBCS1, RBCS2 and RBCS3A promoters, therefore representing the first cloned I-box binding factor. LeMYBI acts as a transcriptional activator in yeast and plants, and binds to the I-box with a DNA-binding domain located in the carboxyterminal domain.

Amino Acid Sequence↗

PubMed2000

MADS-box gene evolution beyond flowers: expression in pollen, endosperm, guard cells, roots and trichomes.

MADS-box genes encode transcriptional regulators involved in diverse aspects of plant development. Here we describe the cloning and mRNA spatio-temporal expression patterns of five new MADS-box genes from Arabidopsis: AGL16, AGL18, AGL19, AGL27 and AGL31. These genes will probably become important molecular tools for both evolutionary and functional analyses of vegetative structures. We mapped our data and previous expression patterns onto a new MADS-box phylogeny. These analyses suggest that the evolution of the MADS-box family has involved a rapid and simultaneous functional diversification in vegetative as well as reproductive structures. The hypothetical ancestral genes had broader expression patterns than more derived ones, which have been co-opted for putative specialized functions as suggested by their expression patterns. AGL27 and AGL31, which are closely related to the recently described flowering-time gene FLC (previously AGL25), are expressed in most plant tissues. AGL19 is specifically expressed in the outer layers of the root meristem (lateral root cap and epidermis) and in the central cylinder cells of mature roots. AGL18, which is most similar in sequence to the embryo-expressed AGL15 gene, is expressed in the endosperm and in developing male and female gametophytes, suggesting a role for AGL18 that is distinct from previously characterized MADS-box genes. Finally, AGL16 RNA accumulates in leaf guard cells and trichomes. Our new phylogeny reveals seven new monophyletic clades of MADS-box sequences not specific to flowers, suggesting that complex regulatory networks involving several MADS-box genes, similar to those that control flower development, underlie development of vegetative structures.

Arabidopsis Proteins↗

PubMed1998

DNA-binding properties of the tandem HMG boxes of high-mobility-group protein 1 (HMG1).

High-mobility-group protein 1 (HMG1) is a conserved chromosomal protein with two homologous DNA-binding HMG-box domains, A and B, linked by a short basic region to an acidic carboxy-terminal tail. NMR spectroscopy on the free didomain (AB) shows that the two HMG boxes do not interact. The didomain has a higher affinity for all DNA substrates tested than single HMG-box domains and has a significantly higher ability to distort DNA by bending and supercoiling. The interaction of the didomain with DNA is stabilized by the presence of the basic region (approximately 20 residues, 9 of which are Lys) that links the second HMG box to the acidic tail in intact HMG1; this may be, at least in part, why this region also enhances supercoiling of relaxed circular DNA by the didomain and circularization of short DNA fragments (in the presence of ligase). Competition assays suggest significantly different structure-specific preferences of single and tandem HMG boxes for four-way junction and supercoiled plasmid DNA. Binding to supercoiled DNA appears to be promoted by protein oligomerization, which is pronounced for the didomains. Electron microscopy suggests that the oligomers are globular aggregates, associated with DNA looping. One box versus two (or several) is likely to be an important determinant of the properties of (non-sequence specific) HMG-box proteins.

Binding Sites↗

PubMed2003

Testing for the D zygosity with three different methods revealed altered Rhesus boxes and a new weak D type.

BACKGROUND: The discrimination of D+/D+ from D+/D- partners of D- mothers with anti-D is important to estimate the risk for HDN. This may be achieved if the presence or absence of the hybrid Rhesus box in the father can be demonstrated. STUDY DESIGN AND METHODS: A new PCR-SSP method specific for the hybrid Rhesus box comprising an internal amplification control was compared with two published PCR-based methods (PCR-SSP and PCR-RFLP) in 83 D+, 13 D-, and 37 weak D samples. RESULTS: The deletion of RHD was detectable in all D- and weak D samples. By all three methods, concordant results were obtained in 82 of 83 D+ samples, with one sample showing discrepant results. The control band in the PCR-RFLP method, specific for the downstream Rhesus box, was missing in two weak D samples, namely a weak D type 4.0 and a novel weak D type dubbed weak D type 29. Further investigations revealed an altered downstream Rhesus box in the weak D type 29 sample. In the weak D type 4.0 sample, no amplicon was achieved with any primer specific for the upstream and downstream Rhesus box. CONCLUSION: A PCR-SSP method with internal control was established for the detection of the hybrid Rhesus box. Polymorphisms in the downstream Rhesus box may interfere with the detection of RHD.

Alleles↗

PubMed2002

Analysis of MADS box protein-protein interactions in living plant cells.

Over the last decade, the yeast two-hybrid system has become the tool to use for the identification of protein-protein interactions and recently, even complete interactomes were elucidated by this method. Nevertheless, it is an artificial system that is sensitive to errors resulting in the identification of false-positive and false-negative interactions. In this study, plant MADS box transcription factor interactions identified by yeast two-hybrid systems where studied in living plant cells by a technique based on fluorescence resonance energy transfer (FRET). Petunia MADS box proteins were fused to either cyan fluorescent protein or yellow fluorescent protein and transiently expressed in protoplasts followed by FRET-spectral imaging microscopy and FRET-fluorescence lifetime imaging microscopy to detect FRET and hence protein-protein interactions. All petunia MADS box heterodimers identified in yeast were confirmed in protoplasts. However, in contrast to the yeast two-hybrid results, homodimerization was demonstrated in plant cells for three petunia MADS box proteins. Heterodimers were identified between the ovule-specific MADS box protein FLORAL BINDING PROTEIN 11 and members of the petunia FLORAL BINDING PROTEIN 2 subfamily, which are also expressed in ovules, suggesting that these dimers play a role in ovule development. Furthermore, the role of dimerization in translocation of MADS box protein dimers to the nucleus is demonstrated, and the nuclear localization signal of MADS box proteins has been mapped to the N-terminal region of the MADS domain by means of mutant analyses.

Active Transport, Cell Nucleus↗

PubMed1986

Cognate homeo-box loci mapped on homologous human and mouse chromosomes.

The homeotic genes of Drosophila, which regulate pattern formation during larval development, contain a 180-base-pair DNA sequence termed the "homeo-box." Nucleotide sequence comparisons indicate that the homeo-box motif is highly conserved in a variety of motazoan species. As in Drosophila, homeo-box sequences of mammalian species are expressed in a temporal and tissue-specific pattern during embryogenesis. These observations suggest functional homologies between dipteran and mammalian homeo-box gene products. To identify possible relationships between homeo-box genes of mice and humans, we have compared the chromosomal location of homeo-box genes in these species. Using in situ hybridization and somatic cell genetic techniques, we have mapped the chromosome 6-specific murine Hox-1 homolog to the region p14-p21 on human chromosome 7. We have also regionally mapped the murine Hox-3 locus to 15F1-3 and its human cognate to 12q11-q21. These comparative mapping data indicate that a syntenic relationship in mice and humans is maintained for all homeo-box loci examined to date. We suggest these regions represent evolutionarily conserved genomic domains encoding homologous protein products that function in regulating patterns of mammalian development.

Animals↗

PubMed1992

Conserved motifs in a divergent nod box of Azorhizobium caulinodans ORS571 reveal a common structure in promoters regulated by LysR-type proteins.

Nodulation of leguminous plants by Rhizobium, Bradyrhizobium, and Azorhizobium spp. is dependent on the induction by the plant host of different bacterial nodulation (nod) loci. The transcription of these nod loci is activated in the presence of plant-produced flavonoids upon binding of the NodD protein--a LysR-type activator--to specific sequences present in the nod promoters. Originally, a 47-base-pair (bp) region called the nod box was shown to be the target sequence for binding of NodD. From the comparison of the nod box sequences of (brady)rhizobia with a more divergent nod box from Azorhizobium caulinodans, we now propose a modular build-up of the nod box with the sequence A-T-C-N9-G-A-T as the binding target of the NodD protein (the NodD box). More generally, we show that LysR-type-regulated promoters contain the characteristic sequence T-N11-A as the core of an inverted repeat and propose this to be the "LysR motif" involved in specific binding to LysR-type proteins. Data obtained upon site-specific mutagenesis of this motif in the NodD box sustains this proposal. Further, we provide strong arguments that the inducer flavonoid, involved in transcriptional activation of Azorhizobium nod genes, interacts directly with the NodD protein, thereby increasing its binding affinities for the NodD box.

Base Sequence↗

PubMed2000

An ancestral MADS-box gene duplication occurred before the divergence of plants and animals.

Changes in genes encoding transcriptional regulators can alter development and are important components of the molecular mechanisms of morphological evolution. MADS-box genes encode transcriptional regulators of diverse and important biological functions. In plants, MADS-box genes regulate flower, fruit, leaf, and root development. Recent sequencing efforts in Arabidopsis have allowed a nearly complete sampling of the MADS-box gene family from a single plant, something that was lacking in previous phylogenetic studies. To test the long-suspected parallel between the evolution of the MADS-box gene family and the evolution of plant form, a polarized gene phylogeny is necessary. Here we suggest that a gene duplication ancestral to the divergence of plants and animals gave rise to two main lineages of MADS-box genes: TypeI and TypeII. We locate the root of the eukaryotic MADS-box gene family between these two lineages. A novel monophyletic group of plant MADS domains (AGL34 like) seems to be more closely related to previously identified animal SRF-like MADS domains to form TypeI lineage. Most other plant sequences form a clear monophyletic group with animal MEF2-like domains to form TypeII lineage. Only plant TypeII members have a K domain that is downstream of the MADS domain in most plant members previously identified. This suggests that the K domain evolved after the duplication that gave rise to the two lineages. Finally, a group of intermediate plant sequences could be the result of recombination events. These analyses may guide the search for MADS-box sequences in basal eukaryotes and the phylogenetic placement of new genes from other plant species.

Amino Acid Sequence↗

PubMed1995

Differential binding of the NFE3 and CP1/NFY transcription factors to the human gamma- and epsilon-globin CCAAT boxes.

Naturally occurring nondeletional mutations affecting the distal CCAAT box of the human gamma-globin gene promoter result in hereditary persistence of fetal hemoglobin in adult life. Although the distal CCAAT box is the target of several factors, including CP1/NFY, CDP, GATA-1 and NFE3, only NFE3 binding activity is consistently sensitive to well characterized mutations in this region such as G-117-->A, C-114-->T, and delta 13 hereditary persistence of fetal hemoglobin. We extensively characterized the binding specificities of NFE3 and demonstrated that NFE3 has unique properties with respect to other CCAAT box-binding proteins. Affinity-purified NFE3 from erythroid K562 cells binds the distal but not the proximal human gamma-globin CCAAT box, the single CCAAT box of the human epsilon-globin promoter, and the proximal CCAAT box of the evolutionarily related Galago crassicaudatus gamma-globin gene. Within the epsilon-globin CCAAT box, NFE3 represents the major and almost exclusive binding activity. Disruption of such a binding site essentially inactivates the epsilon-globin promoter, suggesting that NFE3 plays an important role in the embryonic expression of this gene.

Adult↗

PubMed1995

Solution structure of the sequence-specific HMG box of the lymphocyte transcriptional activator Sox-4.

Two groups of HMG box proteins are distinguished. Proteins in the first group contain multiple HMG boxes, are non-sequence-specific, and recognize structural features as found in cruciform DNA and cross-over DNA. The abundant chromosomal protein HMG-1 belongs to this subgroup. Proteins in the second group carry a single HMG box with affinity for the minor groove of the heptamer motif AACAAAG or variations thereof. A solution structure for the non-sequence-specific C-terminal HMG box of HMG-1 has recently been proposed. Now, we report the solution structure of the sequence-specific HMG-box of the SRY-related protein Sox-4. NMR analysis demonstrated the presence of three alpha-helices (Val10-Gln22, Glu30-Leu41 and Phe50-Tyr65) connected by loop regions (Ser23-Ala49 and Leu42-Pro49). Helices I and II are positioned in an antiparallel mode and form one arm of the HMG box. Helix III is less rigid, makes an average angle of about 90 degrees with helices I and II, and constitutes the other arm of the molecule. As in HMG1B, the overall structure of the Sox-4 HMG box is L-shaped and is maintained by a cluster of conserved, mainly aromatic residues.

Amino Acid Sequence↗

PubMed1997

Conserved E boxes function as part of the enhancer in hypersensitive site 2 of the beta-globin locus control region. Role of basic helix-loop-helix proteins.

The human beta-globin gene cluster is regulated in part by a distal locus control region that is required for opening a chromatin domain in erythroid cells and enhancing expression of the beta-like globin genes at the correct developmental stages. One part of the locus control region, called hypersensitive site 2 (HS2), functions as a strong enhancer. Matches to the consensus binding sites for basic helix-loop-helix (bHLH) proteins (E boxes) are well conserved within the HS2 core. We show that mutations of the HS2 core that alter an invariant E box cause a 3.5-fold reduction in enhancement of expression of an epsilon-globin reporter gene in transiently transfected K562 cells, both before and after induction. Mutations of the HS2 core that alter a less-highly conserved E box cause a more modest reduction in enhancement. Footprint analysis shows binding of erythroid nuclear proteins in vitro to the invariant E box as well as an adjacent CAC/GTG box. Probes containing the E box regions form sequence-specific complexes with proteins from both K562 and MEL nuclear extracts; these are disrupted by the same mutations that decrease enhancement. Some of these latter complexes contain known bHLH proteins, as revealed by specific loss of individual complexes when treated with antibodies against TAL1 and USF. Interaction between the E boxes and the bHLH proteins, as well as other binding proteins, could account for the role of these sites in enhancement by HS2.

Base Sequence↗

PubMed1998

Unique mode of GCC box recognition by the DNA-binding domain of ethylene-responsive element-binding factor (ERF domain) in plant.

Ethylene-responsive element-binding proteins (EREBPs)have novel DNA-binding domains (ERF domains), which are widely conserved in plants, and interact specifically with sequences containing AGCCGCC motifs (GCC box). Deletion experiments show that some flanking region at the N terminus of the conserved 59-amino acid ERF domain is required for stable binding to the GCC box. Three ERF domain-containing fragments of EREBP2, EREBP4, and AtERF1 from tobacco and Arabidopsis, bind to the sequence containing the GCC box with a high binding affinity in the pM range. The high affinity binding is conferred by a monomeric ERF domain fragment, and DNA truncation experiments show that only 11-base pair DNA containing the GCC box is sufficient for stable ERF domain interaction. Systematic DNA mutation analyses demonstrate that the specific amino acid contacts are confined within the 6-base pair GCCGCC region of the GCC box, and the first G, the fourth G, and the sixth C exhibit highest binding specificity common in all three ERF domain-containing fragments studied. Other bases within the GCC box exhibit modulated binding specificity varying from protein to protein, implying that these positions are important for differential binding by different EREBPs. The conserved N-terminal half is likely responsible for formation of a stable complex with the GCC box and the divergent C-terminal half for modulating the specificity.

Amino Acid Sequence↗

PubMed1999

Interaction between the skeletal muscle type 1 Na+ channel promoter E-box and an upstream repressor element. Release of repression by myogenin.

We have defined how four elements that regulate expression of the rat skeletal muscle type 1 sodium channel (SkM1) gene cooperate to yield specific expression in differentiated muscle. A basal promoter region containing within it a promoter E-box (-31/-26) is broadly expressed in many cells, including myoblasts and myotubes; mutations within the promoter E-box that disrupt binding of the myogenic basic helix-loop-helix (bHLH) factors reduce expression in all cell types only slightly. Sequential addition of upstream elements to the wild-type promoter confer increasing specificity of expression in differentiated cells, even though all three upstream elements, including a positive element (-85/-57), a repressor E-box (-90/-85), and upstream repressor sequences (-135/-95), bind ubiquitously expressed transcription factors. Mutations in the promoter E-box that disrupt the binding of the bHLH factors counteract the specificity conferred by addition of the upstream elements, with the greatest interaction observed between the upstream repressor sequences and the promoter E-box. Forced expression of myogenin in myoblasts releases repression exerted by the upstream repressor sequences in conjunction with the wild-type, but not mutant, promoter E-box, and also initiates expression of the endogenous SkM1 protein. Our data suggest that particular myogenic bHLH proteins bound at the promoter E-box control expression of SkM1 by releasing repression exerted by upstream repressor sequences in differentiated muscle cells.

Animals↗

PubMed2001

Interaction of the ring finger-related U-box motif of a nuclear dot protein with ubiquitin-conjugating enzymes.

The U-box domain has been suggested to be a modified RING finger motif where the metal-coordinating cysteines and histidines have been replaced with other amino acids. Known U-box-containing proteins have been implicated in the ubiquitin/proteasome system. In a search for proteins interacting with the ubiquitin-conjugating enzyme UbcM4/UbcH7, we have identified a novel U-box containing protein, termed UIP5, that is exclusively found in the nucleus as part of a nuclear dot-like structure. Interaction between UbcM4 and UIP5 was observed in vivo and in vitro with bacterially expressed proteins. In addition to UbcM4, several other ubiquitin-conjugating enzymes (E2s) that share the same sequence within the L1 loop bind to UIP5. Mutational analysis showed that the U-box, like the RING finger in other proteins, forms the physical basis for the interaction with E2 enzymes. Further support for the structural similarity between U-box and RING finger comes from the observation that, in both cases, the same regions within the UbcM4 molecule are required for interaction. Our results establish at the molecular level a link between the U-box and the ubiquitin conjugating system and strongly suggest that proteins containing U-box domains are functionally closely related to RING finger proteins.

Adaptor Proteins, Signal Transducing↗