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PubMed1993

Activation of the human immunodeficiency virus type 1 long terminal repeat by BTEB, a GC box-binding transcription factor.

BTEB, a GC box-binding transcription factor, was tested for its ability to activate the human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR). An electrophoretic mobility shift assay demonstrated specific binding of BTEB to GC boxes of the HIV-1 LTR. When a BTEB expression vector was cotransfected into A3.01 cells with a fusion gene of HIV-1 LTR and chloramphenicol acetyltransferase (CAT) structural gene, the CAT activity was increased. This increase was accompanied by an increase in the content of CAT mRNA. Transcriptional activity of the HIV-1 LTR, stimulated by Tat, was further enhanced by the expression of BTEB. BTEB also activated the LTR activity in cooperation with phorbol 12-myristate 13-acetate. Northern blot analysis showed that various T cell and macrophage/monocyte cell lines expressed the BTEB mRNA to a level comparable with that of Sp1, another GC box-binding transcription factor. These results suggest that BTEB, like Sp1, is involved in transcriptional activation of the HIV-1 LTR.

Base Sequence↗

PubMed1998

Role of E and CArG boxes in developmental regulation of muscle glycogen phosphorylase promoter during myogenesis.

Muscle glycogen phosphorylase (MGP) transcript and protein levels increase during skeletal muscle development in tandem with the products of other muscle genes responsible for glucose and glycogen metabolism. Previous studies demonstrated that a 269 bp region 5' to exon 1 of MGP is sufficient for developmental regulation in the C2C12 myogenic cell line (Froman et al., 1994). This genomic region (-209 to +60) contains four consensus E box motifs, a CArG-like sequence, and a GC-rich domain. Native MGP transcripts were not detected in pluripotent CH310T1/2 fibroblasts, but low levels of MGP mRNA were measured in CH310T1/2 cells that were stably transfected with MyoD. Three of the E box motifs in the MGP proximal promoter interacted with C2C12 nuclear proteins. However, cotransfection of the MGP promoter with myogenic regulatory factors, including MyoD and myogenin, produced less than 2-fold activation compared with 20-fold activation of the desmin promoter. Mutational analyses of the MGP promoter demonstrated that increased expression in C2C12 myotubes did not require any of the E box motifs or the CArG-like element. A small region (-76 to -68) upstream of GC-rich domain (-64 to -51) significantly reduced promoter activities in both myoblasts and myotubes. The functional studies suggest that MGP is developmentally regulated during myogenesis by alternative pathways that utilize unidentified regulatory elements or ancillary factors.

Animals↗

PubMed1999

Isolation and characterization of rice MADS box gene homologues and their RFLP mapping.

Thirty-five MADS box gene homologues were identified through a large-scale cDNA analysis in rice. Based on the nucleotide sequences of the 3'-untranslated region, these clones were classified into 11 independent species. Seven species were found to be new among the rice MADS box gene family, and the other 4 corresponded to the previously reported OsMADS1, OsMADS2, OsMADS4, and OsMADS5. The full nucleotide sequences of the 7 new species were determined. Each clone encoded a deduced protein of 164-267 amino acids. The K-domain of the MADS protein was conserved in all clones though with lower degree in clone S10304. Reverse transcription PCR analysis showed that clones E31254 and E31864 were expressed mainly in panicles. Dendrogram analysis suggested that E31254 and E31864 are close to Arabidopsis AGL9 and AP1, respectively. Restriction fragment length polymorphism (RFLP) linkage mapping revealed that the rice MADS box gene homologues reported here are not clustered but are located throughout the genome. The locus of E31864 on the RFLP map was closely linked to the long sterile lemma gene, g-1.

Amino Acid Sequence↗

PubMed2001

Inhibition of p53-dependent transcription by BOX-I phospho-peptide mimetics that bind to p300.

The N-terminal BOX-I domain of p53 containing a docking site for the negative regulator MDM2 and the positive effector p300, harbours two recently identified phosphorylation sites at Thr18 or Ser20O whose affect on p300 is undefined. Biochemical assays demonstrate that although MDM2 binding is inhibited by these phosphorylations, p300 binding is strikingly stabilized by Thr18 or Ser20 phosphorylation. Introducing EGFP-BOX-I domain peptides with an aspartate substitution at Thr18 or Ser20 induced a significant inhibition of endogenous p53-dependent transcription in cycling cells, in irradiated cells, as well as in cells transiently co-transfected with p300 and p53. In contrast an EGFP-wild-type BOX-I domain peptide stimulated p53 activity via inhibition of MDM2 protein binding. These results suggest that phosphorylation of p53 at Thr18 or Ser20 can activate p53 by stabilizing the p300-p53 complex and also identify a class of small molecular weight ligands capable of selective discrimination between MDM2- and p300-dependent activities.

Amino Acid Substitution↗

PubMed1997

Cloning of an inr- and E-box-binding protein, TFII-I, that interacts physically and functionally with USF1.

The transcription factor TFII-I has been shown to bind independently to two distinct promoter elements, a pyrimidine-rich initiator (Inr) and a recognition site (E-box) for upstream stimulatory factor 1 (USF1), and to stimulate USF1 binding to both of these sites. Here we describe the isolation of a cDNA encoding TFII-I and demonstrate that the corresponding 120 kDa polypeptide, when expressed ectopically, is capable of binding to both Inr and E-box elements. The primary structure of TFII-I reveals novel features that include six directly repeated 90 residue motifs that each possess a potential helix-loop/span-helix homology. These unique structural features suggest that TFII-I may have the capacity for multiple protein-protein and, potentially, multiple protein-DNA interactions. Consistent with this hypothesis and with previous in vitro studies, we further demonstrate that ectopic TFII-I and USF1 can act synergistically, and in some cases independently, to activate transcription in vivo through both Inr and the E-box elements of the adenovirus major late promoter. We also describe domains of USF1 that are necessary for its independent and synergistic activation functions.

Adenoviridae↗

PubMed1999

PLENA and FARINELLI: redundancy and regulatory interactions between two Antirrhinum MADS-box factors controlling flower development.

We report the discovery of an Antirrhinum MADS-box gene, FARINELLI (FAR), and the isolation of far mutants by a reverse genetic screen. Despite striking similarities between FAR and the class C MADS-box gene PLENA (PLE), the phenotypes of their respective mutants are dramatically different. Unlike ple mutants, which show homeotic conversion of reproductive organs to perianth organs and a loss of floral determinacy, far mutants have normal flowers which are partially male-sterile. Expression studies of PLE and FAR, in wild-type and mutant backgrounds, show complex interactions between the two genes. Double mutant analysis reveals an unexpected, redundant negative control over the B-function MADS-box genes. This feature of the two Antirrhinum C-function-like genes is markedly different from the control of the inner boundary of the B-function expression domain in Arabidopsis, and we propose and discuss a model to account for these differences. The difference in phenotypes of mutants in two highly related genes illustrates the importance of the position within the regulatory network in determining gene function.

Amino Acid Sequence↗

PubMed2000

Solution structure of the MEF2A-DNA complex: structural basis for the modulation of DNA bending and specificity by MADS-box transcription factors.

The solution structure of the 33 kDa complex between the dimeric DNA-binding core domain of the transcription factor MEF2A (residues 1-85) and a 20mer DNA oligonucleotide comprising the consensus sequence CTA(A/T)(4)TAG has been solved by NMR. The protein comprises two domains: a MADS-box (residues 1-58) and a MEF2S domain (residues 59-73). Recognition and specificity are achieved by interactions between the MADS-box and both the major and minor grooves of the DNA. A number of critical differences in protein-DNA contacts observed in the MEF2A-DNA complex and the DNA complexes of the related MADS-box transcription factors SRF and MCM1 provide a molecular explanation for modulation of sequence specificity and extent of DNA bending ( approximately 15 versus approximately 70 degrees ). The structure of the MEF2S domain is entirely different from that of the equivalent SAM domain in SRF and MCM1, accounting for the absence of cross-reactivity with other proteins that interact with these transcription factors.

Amino Acid Sequence↗

PubMed2001

The B-box dominates SAP-1-SRF interactions in the structure of the ternary complex.

The serum response element (SRE) is found in several immediate-early gene promoters. This DNA sequence is necessary and sufficient for rapid transcriptional induction of the human c-fos proto-oncogene in response to stimuli external to the cell. Full activation of the SRE requires the cooperative binding of a ternary complex factor (TCF) and serum response factor (SRF) to their specific DNA sites. The X-ray structure of the human SAP-1-SRF-SRE DNA ternary complex was determined (Protein Data Bank code 1hbx). It shows SAP-1 TCF bound to SRF through interactions between the SAP-1 B-box and SRF MADS domain in addition to contacts between their respective DNA-binding motifs. The SAP-1 B-box is part of a flexible linker of which 21 amino acids become ordered upon ternary complex formation. Comparison with a similar region from the yeast MATalpha2-MCM1-DNA complex suggests a common binding motif through which MADS-box proteins may interact with additional factors such as Fli-1.

Amino Acid Sequence↗

PubMed2003

The crystal structure of the human polo-like kinase-1 polo box domain and its phospho-peptide complex.

Human polo-like kinase Plk1 localizes to the centrosomes, kinetochores and central spindle structures during mitosis. It plays an essential role in promoting mitosis and cytokinesis through phosphorylation of a number of different substrates. Kinase activity is regulated by a conserved C-terminal domain, termed the polo box domain (PBD), which acts both as an autoinhibitory domain and as a subcellular localization domain. We have determined the crystal structure of Plk1 PBD (residues 367-603) to 2.2 A resolution and the structure of a phospho-peptide-PBD (residues 345-603) complex to 2.3 A resolution. The two polo boxes of the PBD exhibit identical folds based on a six-stranded beta-sheet and an alpha-helix, despite only 12% sequence identity. The phospho-peptide binds at a site between the two polo boxes. It makes a short antiparallel beta-sheet connection and critical contacts to residues Trp414, Leu490, His538 and Lys540. Most of these residues had been shown to be important for biological activity through mutational studies. The results provide an explanation for phospho-peptide recognition and create the basis for new functional studies.

Amino Acid Sequence↗

PubMed1998

Mutational analysis of the yeast DEAH-box splicing factor Prp16.

Prp16 is an essential yeast splicing factor that catalyzes RNA-dependent hydrolysis of nucleoside triphosphates. Prp16 is a member of the DEAH-box protein family, which is defined by six collinear sequence motifs. The importance of residues within four of the conserved motifs was assessed by alanine-scanning mutagenesis. Mutant alleles of PRP16 were tested for in vivo function by complementation of a Deltaprp16 null strain. In motif I (GETGSGKT), alanine substitutions at Gly-378, Lys-379, and Thr-380 were lethal, whereas replacement of the amino acids in positions 373-377 were viable. In the signature DEAH-box (motif II), Asp-473 and Glu-474 were essential, whereas the H476A mutant was viable. The S505A and T507A mutants in motif III (SAT) were viable. In motif VI (QRSGRAGRTAPG), mutants Q685A, R686A, G688A, R689A, and R692A were lethal, whereas G691A, P695A, and G696A supported growth. Instructive structure-function relationships were established by conservative substitutions at essential residues identified by alanine scan. Overexpression of nonviable alleles impaired the growth of wild-type PRP16 cells. Deletion analysis of the 1071-amino-acid Prp16 protein revealed that the N-terminal 204 amino acids and the C-terminal 100 residues were dispensable for PRP16 function in vivo. These studies provide an instructive framework for functional analysis of other DEAH-box splicing factors.

Adenosine Triphosphatases↗

PubMed2002

In situ hybridization of the MADS-box gene POTM1 during potato floral development.

The potato MADS-box gene POTM1 is a member of the SQUA-like family of plant MADS-box genes. The amino acid sequence of POTM1 most closely matches the sequence of PFG (Petunia Flowering Gene), a petunia MADS-box gene involved in the transition from vegetative to reproductive development. To examine the likely role of POTM1 in potato reproductive tissue, in situ hybridizations were performed on sections of the potato shoot apex at various stages during the transition to flowering. Scanning electron micrographs provide a reference for each stage to illustrate the progression from vegetative meristem to inflorescence meristem and floral organs. POTM1 mRNA accumulates in vegetative, inflorescence and floral meristems. Transcripts are present in petal primordia during the later stages of petal development and they accumulate later during floral development inside the stamens and carpels, but not in carpel walls. This widespread pattern of mRNA accumulation is unique among the SQUA-like genes and indicates that POTM1 is active during a variety of stages of development, and may function in regulating cell determination in transitional phases of both vegetative and floral meristems.

In Situ Hybridization↗

PubMed2002

Cloning and characterization of four apple MADS box genes isolated from vegetative tissue.

With the aim of finding genes involved in the floral transition of woody species four MADS box genes containing cDNAs from apple (Malus domestica) have been isolated. Three genes were isolated from vegetative tissue of apple, but were homologues of known genes that specify floral organ identity. MdMADS13 is an AP3-like B class MADS box gene, and was mainly expressed in petals and stamens as demonstrated by Northern blot analysis. MdMADS14 and -15 are AGAMOUS-like genes. They differed slightly in expression patterns on Northern blots, with MdMADS15 mRNA levels equally high in stamens and carpels, but MdMADS14 preferably expressed in carpels. MdMADS14 is likely to be the apple orthologue of one of the Arabidopsis thaliana SHATTERPROOF genes, and MdMADS15 closely resembled the Arabidopsis AGAMOUS gene. It has been shown with RT-PCR that the three floral apple MADS box genes are expressed in vegetative tissues of adult as well as juvenile trees, albeit at low levels. MdMADS12 is an AP1-like gene that is expressed at similar levels in leaves, vegetative shoots, and floral tissues, and that may be involved in the transition from the juvenile to the adult stage.

Amino Acid Sequence↗

PubMed2003

MADS-box genes in Ginkgo biloba and the evolution of the AGAMOUS family.

MADS-box proteins are a large family of transcription factors. In plants, many genes belonging to this family are involved in the homeosis of the floral system. Up to now, they have mainly been studied in angiosperms, especially in the model species Arabidopsis thaliana and Antirrhinum majus. We undertook a study of MADS-box genes in Ginkgo biloba, the unique extant representative of a whole branch of the phylogenetic tree of the seed plants. A polymerase chain reaction (PCR) survey reveals the diversity of MADS-box genes present in the genome of the Ginkgo. Duplications probably occurred specifically in the ginkgophyte lineage. Phylogenetic analyses revealed that one of these genes, GBM5, is an orthologue of the AGAMOUS gene of A. thaliana. We cloned and sequenced the entire cDNA of the GBM5 gene and studied its intron/exon structure. We showed by reverse transcriptase-PCR that it is expressed in both floral and vegetative tissues. We discuss the molecular evolution of the AGAMOUS family of genes.

Amino Acid Sequence↗

PubMed2006

Promoter analysis of MADS-box genes in eudicots through phylogenetic footprinting.

The MIKC MADS-box gene family has been shaped by extensive gene duplications giving rise to subfamilies of genes with distinct functions and expression patterns. However, within these subfamilies the functional assignment is not that clear-cut, and considerable functional redundancy exists. One way to investigate the diversity in regulation present in these subfamilies is promoter sequence analysis. With the advent of genome sequencing projects, we are now able to exert a comparative analysis of Arabidopsis and poplar promoters of MADS-box genes belonging to the same subfamily. Based on the principle of phylogenetic footprinting, sequences conserved between the promoters of homologous genes are thought to be functional. Here, we have investigated the evolution of MADS-box genes at the promoter level and show that many genes have diverged in their regulatory sequences after duplication and/or speciation. Furthermore, using phylogenetic footprinting, a distinction can be made between redundancy, neo/nonfunctionalization, and subfunctionalization.

Arabidopsis↗

PubMed2007

Chimpanzee, orangutan, mouse, and human cell cycle promoters exempt CCAAT boxes and CHR elements from interspecies differences.

Mechanisms regulating the cell division cycle are well conserved among all eukaryotes. Consistently many proteins regulating the cell cycle are functionally interchangeable between many organisms. Cell division control is regulated on different levels of which the transcriptional level appears to be particularly important for controlling synthesis of many cell cycle proteins. We had earlier described transcription factor-binding sites essential for regulating genes important for the transition from the G(2) phase to mitosis. A tandem repressor site named cell cycle-dependent element (CDE) and cell cycle genes homology region (CHR) are responsible for the correct expression during the cell cycle. Another feature of these G(2)/M-specific promoters is the activation through 2 or 3 CCAAT boxes binding the transcription factor nuclear factor-Y (NF-Y). These major activating sites have to be spaced 32 or 33 bp apart to be fully functional. We were interested in looking at the evolutionary changes in regulatory elements and overall promoter structure of 3 well-characterized cell cycle genes. Here, we compare the DNA sequences and functional features of the cdc25C, cyclin B1, and cyclin B2 promoters from humans, mouse, chimpanzee, and orangutan. We find numerous differences in the nucleotide sequence between mouse and primate promoters. However, CHR and CCAAT boxes stand out in that they are perfectly conserved in all promoters tested. The CDE site contains nucleotide exchanges between mouse and primate promoters. Comparing sequences and functions of chimpanzee, orangutan, and human promoters, we observe a complete conservation in nucleotide sequence of the regulatory elements. Functional assays of the cyclin B1, cyclin B2, and cdc25C promoters yield moderate variations in activity and thereby a good conservation of function. Although we find nucleotide differences in cell cycle promoters between orangutan and humans of about 5%, there are never changes in any of the CCAAT boxes or CDE/CHR sites in the cyclin B1, cyclin B2, and cdc25C promoters. Furthermore, we describe the influence of the tumor suppressor p53 and the transcriptional activator NF-Y on regulation of the newly cloned primate promoters.

Animals↗

PubMed2001

Expression of a Y-box protein, YB2/RYB-a, precedes protamine 2 expression during spermatogenesis in rodents.

Y-box binding proteins, a large family of proteins, are involved in a variety of functions. The present study describes the expression of YB2, a rat Y-box binding protein, and/or RYB-a, an alternatively spliced product of the YB2 gene during spermatogenesis. YB2/RYB-a is thought to be the rat orthologue of mouse Y-box protein 3 (MSY3). An antibody which recognizes YB2/RYB-a was developed and applied in an immunochemical study of rat and mouse testes. We also carried out an in-situ hybridization study and Northern blot analysis of YB2/RYB-a and protamine 2 mRNA expression. Both YB2/RYB-a mRNA and the proteins appeared in prepubertal mouse testes, prior to the expression of the mouse protamine 2 mRNA. The mRNA and protein were present at high levels in spermatocytes, decreased in round to elongated spermatids, and were absent in spermatozoa. Since the protamine 2 mRNA was present at high levels in round and elongating spermatids, the proposed function of the YB2/RYB-a protein as a translational repressor of the mRNA was supported in mouse. The level and localization of YB2/RYB-a mRNA and protein expression in the rat testis was comparable to that in mouse testis, although rat testis is known to express a very low level of protamine 2, but is also likely to affect the expression of other proteins (including protamine 1) during spermatogenesis.

Animals↗

PubMed1988

Stage- and tissue-specific expression of two homeo box genes in sea urchin embryos and adults.

We report the isolation of two different homeo box genes, HB3 and HB4, from the Hawaiian sea urchin Tripneustes gratilla. DNA sequencing revealed a definitive Antennapedia (Antp) class homeo box in each gene. Southern transfer hybridizations showed the genes to be single-copy. A 5.7-kb transcript of the HB3 gene was found in ovary, testis, small intestine and gastrula poly(A)+ RNA. The HB4 gene produces three transcripts. A 3.7-kb and a 4.4-kb transcript are expressed during embryogenesis. A 3.5-kb transcript appears in each of the adult tissues studied. The HB4 gene appears to be the sea urchin cognate of the Drosophila infrabdominal-7 (iab-7) gene, the mouse Hox 1.7 and Hox 3.2 genes and the Xenopus X1Hbox 6 gene. An examination of Antp class homeo box genes in deuterostomes indicates that a chromosomal duplication has taken place in the evolutionary line leading to the vertebrates after the divergence of the echinoderms. Thus, the sea urchin represents the primitive condition.

Amino Acid Sequence↗

PubMed1990

A novel transcriptional activator (PO-B) binds between the TATA box and cap site of the pro-opiomelanocortin gene.

We demonstrate that a factor (PO-B), detected in a number of mammalian cell lines, binds specifically between the TATA box and the transcription initiation site of the pituitary-specific pro-opiomelanocortin (POMC) gene. Methylation interference and DNAse 1 footprint analysis revealed that the binding site of this protein, -3 to -15, does not overlap the POMC TATA box or cap site. Mutation of the PO-B binding site significantly decreased the transcriptional activity of the POMC promoter after transient transfection into the ATt-20 mouse pituitary tumor cell line and also in in vitro transcription assays. In contrast, mutation of the TATA box produced no overall decrease in transcription in vivo but induced multiple heterogeneous start sites in vitro. A vector harboring the PO-B site alone was unable to direct transcription initiation. PO-B represents a new transcription factor that may be able to facilitate POMC gene expression by interacting with components of the transcription initiation complex.

Animals↗