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PubMed2025

Genome-wide identification and expression profiling of the MADS-box gene family in Lavandula angustifolia.

BACKGROUND: MADS-box genes encode transcription factors critical for plant development, particularly floral organogenesis, flowering time regulation, and adaptation to environmental stresses. Among these, the MIKCC-type genes are pivotal regulators in floral developmental processes. Although the evolutionary diversification and functional dynamics of MADS-box genes have been extensively characterized in model plants such as Arabidopsis thaliana and Oryza sativa, their evolutionary relationships and functional profiles in Lavandula angustifolia, an economically significant aromatic plant, remain poorly understood. RESULTS: Genome-wide analysis identified 173 MADS-box genes in L. angustifolia, categorized into type I (Mα: 26; Mβ: 0; Mγ: 10) and type II (MIKCC: 125; MIKC*: 12) based on phylogenetic comparisons with A. thaliana. The MIKCC subgroup was further subdivided into 12 subclasses, including genes central to the ABCDE model of floral organ specification. Structural analyses revealed distinct conserved motifs and exon-intron configurations specific to each subgroup, indicative of functional divergence. Synteny analysis demonstrated Whole Genome Duplication (WGD) and segmental duplications as major contributors to MIKCC gene family expansion, notably among genes linked to floral organ development. Expression profiling via RNA-seq and quantitative real-time PCR (qPCR) showed type II MADS-box genes exhibited higher expression levels with pronounced tissue-specific and developmental stage-specific expression patterns compared to type I genes. Many type II genes displayed significant associations with floral organogenesis, floral transition, and abiotic stress responses, underscoring their essential roles in reproductive development and environmental adaptability in L. angustifolia. CONCLUSIONS: The identification and comprehensive characterization of 173 MADS-box genes in L. angustifolia highlight the significant expansion of the MIKCC subgroup driven primarily by WGD and segmental duplications. The distinct structural features and specific expression patterns observed provide insights into the functional divergence and complexity of these genes, particularly regarding floral organogenesis and adaptation to environmental stress. This study establishes a robust molecular basis for further functional analysis and genetic improvement of aromatic plants.

MADS Domain Proteins↗

PubMed1995

Role of the TATA box in transcription of the mouse mammary tumor virus long terminal repeat.

An in vitro transcription system from mammary cells was established to study transcription of the long terminal repeat (LTR) of the mouse mammary tumor virus (MMTV). Experiments with progressive 5'-deletion constructs of the MMTV LTR revealed that a 19-base pair (bp) region from -41 to -23 bp, encompassing the TATA box and flanking DNA sequence, was as transcriptionally active as larger promoter constructs, both in nuclear extracts from human mammary cell lines (T47D and MCF7) and a nonmammary cell line (HeLa). The cell-free system was capable of supporting transcriptional induction by factors binding upstream of the TATA box, however, since purified glucocorticoid receptor-induced transcription in larger promoter constructs encompassing the MMTV hormone-responsive elements. Transcription from two other promoters, the adenovirus major late promoter and the human immunodeficiency virus LTR, also revealed a significant transcriptional contribution of upstream elements. The 19-bp TATA region from the MMTV LTR was shown to have considerably more activity in this transcription system than comparable TATA regions from other promoters. Sequences critical to the MMTV TATA region were evaluated by single base pair mutagenesis and found to comprise a consensus TATA box sequence, TATAAAA, as well as a single A just upstream of the TATAAAA sequence. Thus, the high level of basal transcription observed with the TATA region from MMTV is due to a perfect consensus TATA box sequence and a single base immediately 5' adjacent. It is likely that the high basal rate of transcription observed with this TATA box region on histone-free templates represents an inappropriate level of basal expression and that a complete evaluation of transactivation mechanisms in this system will require the recapitulation in vitro of the chromatin-mediated repressive state that exists in vivo.

Base Sequence↗

PubMed2002

Epithelial monolayer wounding stimulates binding of USF-1 to an E-box motif in the plasminogen activator inhibitor type 1 gene.

Several proteases and their co-expressed inhibitors modulate the interdependent processes of cell migration and matrix proteolysis during wound repair. Transcription of the gene encoding plasminogen activator inhibitor type 1 (PAI-1), a serine protease inhibitor important in the control of barrier proteolysis and cell-to-matrix adhesion, is spatially-temporally regulated following epithelial denudation injury in vitro as well as in vivo. Using a well-defined culture model of acute epidermal wounding and reepithelialization, PAI-1 mRNA/protein synthesis was induced early after monolayer scraping and restricted to cells comprising the motile cohort. PAI-1 levels in locomoting cells remained elevated (relative to the distal, contact-inhibited monolayer regions) throughout the time course of trauma repair. Targeted PAI-1 downregulation by transfection of antisense PAI-1 expression constructs significantly impaired keratinocyte migration and monolayer scrape wound closure. Injury-induced PAI-1 transcription closely paralleled growth state-dependent controls on the PAI-1 gene. An E-box motif (CACGTG) in the PAI-1 proximal promoter (located at nucleotides -160 to -165), previously shown to be necessary for serum-induced PAI-1 expression, was bound by nuclear factors from wound-stimulated but not quiescent, contact-inhibited, keratinocytes. UV crosslinking approaches to identify E-box-binding factors coupled with deoxyoligonucleotide affinity chromatography and gel retardation assays confirmed at least one major E-box-binding protein in both serum- and wound-activated cells to be USF-1, a member of the helix-loop-helix family of transcription factors. An intact hexanucleotide E-box motif was necessary and sufficient for USF-1 binding using nuclear extracts from both serum- and wound-simulated cells. Two species of immunoreactive USF-1 were identified by western blotting of total cellular lysates that corresponded to the previously characterized phosphorylated and non-phosphorylated forms of the protein. USF-1 isolated by PAI-1 promoter-DNA affinity chromatography was almost exclusively phosphorylated. Only a fraction of the total cellular USF-1 in proliferating cultures, by comparison, was phosphorylated at any given time. PAI-1 E-box binding activity, assessed by probe mobility shift criteria, increased within 2 hours of monolayer scrape injury, a time frame consistent with wound-stimulated increases in PAI-1 transcription. Relative to intact cultures, scrape site-juxtaposed cells had significantly greater cytoplasmic and nuclear USF-1 immunoreactivity correlating with the specific in situ-restricted expression of PAI-1 transcripts/protein in the wound-edge cohort. USF-1 immunocytochemical staining declined significantly with increasing distance from the denudation site. These data are the first to indicate that binding of USF-1 to its target motif can be induced by 'tissue' injury in vitro and implicate USF-1 as a transcriptional regulator of genes (e.g. PAI-1) involved in wound repair.

Animals↗

PubMed2001

The roles of two TATA boxes and 3'-flanking region of soybean beta-tubulin gene (tubB1) in light-sensitive expression.

The soybean tubB1 gene is expressed primarily in the germinating seedling and is strongly down regulated in response to light in the upper hypocotyl. Previous studies demonstrate that the 1 kb 5'-flanking region of this gene is sufficient for its appropriate expression in etiolated seedlings. Transient expression studies demonstrated that the presence of the tubB1 3'-flanking sequence element decreased reporter gene expression as compared to the nopaline synthase (NOS) 3'-flanking sequence element. In this study we investigated the ability of the 3' flanking region to influence the expression of a beta-glucuronidase (GUS) reporter gene in transgenic tobacco and Arabidopsis. The presence of the tubB1 3'-flanking sequence element in chimera constructs reduced reporter gene expression specifically in the hypocotyl and petioles of light-grown, transgenic seedlings. Additionally, site-directed mutagenesis of the two TATA sequences in the 1 kb tubB1 5'-flanking sequence element (TATA box A in the -122 to -117 bp region and TATA box B in the -35 to -30 region) showed that both elements are functional and additive in controlling tubB1 gene expression in seedling tissues. While transcription from TATA box A was predominant regardless of lighting conditions, the relative usage of TATA box B increased in the dark. We conclude that both TATA box sequences are utilized to direct expression of the tubB1 gene to the cotyledons, hypocotyl and root tip of germinating seedlings that are regions of cell expansion and that the 3'-flanking sequence element down-regulates expression in the hypocotyl in response to light. Thus, it is plausible that the tubB1 protein may play an important role in cell expansion in seedling development requiring its regulated expression by light.

Arabidopsis↗

PubMed2002

The E-box motif in the proximal ABCA1 promoter mediates transcriptional repression of the ABCA1 gene.

To identify regulatory elements in the proximal human ATP-binding cassette transporter A1 (hABCA1) gene promoter we transfected RAW cells with plasmids containing mutations in the E-box, AP1, and liver X receptor (LXR) elements as well as the two Sp1 motifs. Point mutations in either Sp1 site or in the AP1 site had only a minor effect whereas mutation of the LXR element decreased promoter activity. In contrast, mutation or deletion of the E-box motif caused a 3-fold increase in transcriptional activity under basal conditions. Gel shift and DNaseI footprint analysis showed binding of a protein or protein complex to this region. Preincubation of nuclear extracts with antibodies established that USF1, USF2, and fos related antigen (Fra) 2 bind to DNA sequences in the human ABCA1 promoter that contains the intact E-box but not the mutant or deleted E-box. Co-transfection of USF1 and USF2 enhanced, but Fra2 repressed, ABCA1 promoter activity. Thus, a complex consisting of USF1, USF2, and Fra2 binds the E-box motif 147 bp upstream of the transcriptional start site and facilitates repression of the human ABCA1 promoter. These combined studies identify a novel site in the human ABCA1 promoter involved in the regulation of ABCA1 gene expression.

ATP Binding Cassette Transporter 1↗

PubMed2002

Recognition of substrate and Skp1 by the Homologue of Slimb (HOS) ubiquitin ligase receptor D role of the F-box.

BACKGROUND: SCFHOS-Roc1 E3 ubiquitin ligase is an enzymatic complex, which mediates ubiquitination and subsequent proteasome-dependent degradation of phosphorylated inhibitor of NF-kB (IkB) and b-catenin. HOS is a WD40 repeats/F-box-containing protein that actually associates with the substrates and binds to Skp1 via the F-box. MATERIAL/METHODS: Here, we have studied the structural determinants of the substrate recognition and ligase recruitment by HOS. The binding (pull-down and immunoprecipitation assays) and ubiquitination assays were performed in vitro with purified or partially purified recombinant proteins obtained via expression in bacteria or mammalian cells or by in vitro translation. RESULTS: We identified specific amino acid residues (I143 and L152) within the F-box of HOS that play a critical role in maintaining the hydrophobic interface of HOS-Skp1 interaction and found substantial similarity between interaction of Skp1 with HOS and with another F-box protein Skp2. Binding of Skp1 augments the ability of HOS to recognize the phosphorylated IkBa. CONCLUSIONS: These observations indicate the role of the F-box of HOS in both recruitment of ubiquitin ligase activity and substrate recognition as well as identify the structural elements that are important for both functions of HOS F-box domain.

Amino Acid Motifs↗

PubMed2005

[Experimental study of E-box, a transcriptional suppressor element in gamma-GCS gene].

OBJECTIVE: To validate that the E-box element in the -745-705 region of rat gamma-GCS catalytic subunit gene (GCLC gene), already found to be a negative regulatory region, is an important transcriptional suppressor element. METHODS: Electrophoretic mobility shift assays (EMSA) and antibody supershift assay were used to confirm the specific binding of USF, a transcription factor that binds the regulatory sequence -745 - 705, and E-box element. Rat alveolar epidermal cells of the line CCL-149 were cultured and then divided into 4 groups: group 1 to be transfected with GCLC-Luc, group 2 to be co-transfected with pGCLC-Luc and pCMV-USF1, group 3 to be co-transfected with pGCLC-Luc and pCMV-USF2, and group 4 to be co-transfected with pGCLC-Luc and blank vector pCMV. Recombinant retrovirus vector PLXSN-USF1 and recombinant retrovirus vector PLXSN-USF2 were constructed. Another CCL-149 cells were divided into 4 groups: group A to be infected with recombinant USF1 virus, group B to be infected with recombinant USF2 virus, group C to be infected with blank vector, and group D not to be infected with any plasmid. Western blotting was used to detect the concentrations of GCLC protein. RESULTS: EMSA showed that only the probes with complete E-box element could be bound by the USFs. Supershift assay showed that the transcription factor binding the probe was USF. The luciferase activity of the CCL-149 cells co-transfected with pCMV-USF1/USF2 and GCLC-Luc were decreased by 66.4% and 63.2% respectively in comparison with those transfected with GCLC-Luc only (both P < 0.05) and decreased by 54.5% and 61.1% respectively in comparison with those of the cells of the blank vector group (both P < 0.05). Western blotting showed that the expression of GCLC protein of the CCL-149 cells transfected with PLXSN-USF1 and PLXSN-USF2 were decreased in comparison with that of the control cells. CONCLUSION: The interaction between E-box and USF suppresses the expression of GCLC gene. E-box is an important transcriptional suppressor element of gamma-GCS gene.

Animals↗

PubMed1992

Kinetic analysis of yeast TFIID-TATA box complex formation suggests a multi-step pathway.

The eukaryotic transcription factor TFIID recognizes and binds a promoter sequence element called the TATA box. We have analyzed the interaction of yeast TFIID with the consensus TATA box sequence of the adenovirus major late promoter. To facilitate this detailed characterization, we developed a method for obtaining quantitative information from a gel retardation (bandshift) assay, allowing measurement of the rate and extent of TFIID-TATA box complex formation. Using this assay and DNase I protection assays, we determined that the association rate constant for TFIID binding to the major late promoter was too low to be consistent with a simple diffusion-limited association, suggesting that the binding proceeds by a multi-step pathway. Furthermore, we found that the slow rate of TFIID binding reported by other research groups was not the consequence of a rate-limiting conformational change, as has been previously suggested. Instead, we observed that the formation of a stable TFIID-TATA box complex was relatively rapid (complete in less than 1 min) at saturating concentrations of TFIID. We have proposed a two-step pathway consistent with the observed kinetics and have considered the possible contributions of each step to the overall rate of TFIID binding. This study lays the groundwork for a systematic characterization of the interaction of TFIID with additional TATA box sequences, including an experimental test of the possibility that different steps in the binding reaction are rate-limiting for different promoters.

DNA Fingerprinting↗

PubMed1991

Boxing: the preparticipation evaluation.

Boxing is a controversial sport that has drawn public and physician criticism since its inception in 900 BC. The American Medical Association has recently taken a formal position to ban boxing because of the alleged dangers, including the possibility of the long-term effect of dementia pugilistica. Proponents of amateur boxing are working diligently to document this claim both as premature and false. They cite amateur boxing's purpose as one boxer's attempt to outpoint or outscore his opponent by utilization of superior strength and skill. Despite this medical and political controversy, thousands of primary care patients are boxing. This report offers the physician guidelines for the preparticipation evaluation. Specific disqualifying conditions are addressed.

Boxing↗

PubMed1990

The progesterone receptor can regulate transcription in the absence of a functional TATA box element.

We have investigated the importance of the TATA box element in the induction of transcription by the progesterone receptor. Transcription was analyzed from promoters containing a steroid response element upstream of a wild-type or mutated TATA box. Mutation of the TATA box resulted in a loss of correctly initiated transcripts and abolished binding of TATA factor to the TATA box in vitro but did not inhibit transcriptional activation by the progesterone receptor. Thus we conclude that the receptor is able to stimulate the rate of transcription in the absence of a functional TATA box.

Animals↗

PubMed2003

Successful treatment of collagen-induced arthritis in mice and rats by targeting extracellular high mobility group box chromosomal protein 1 activity.

OBJECTIVE: Extracellular high mobility group box chromosomal protein 1 (HMGB-1) is a recently identified, endogenous, potent tumor necrosis factor- and interleukin-1 (IL-1)-inducing protein detectable in inflamed synovia in both human and experimental disease. In the present study, we examined clinical effects in collagen-induced arthritis (CIA) using therapeutic administration of neutralizing HMGB-1 antibodies or truncated HMGB-1-derived A-box protein, a specific, competitive antagonist of HMGB-1. METHODS: CIA was induced in DBA/1j mice or dark agouti rats, and animals were examined daily for signs of arthritis. Treatment with polyclonal anti-HMGB-1 antibodies or the A-box protein was initiated at the onset of disease and was administered intraperitoneally twice daily for 7 days. Animals were killed 8 days after initiation of therapy, and immunohistochemical analysis of synovial tissue specimens was performed. RESULTS: Systemic administration of anti-HMGB-1 antibodies or A-box protein significantly reduced the mean arthritis score, the disease-induced weight loss, and the histologic severity of arthritis. Beneficial effects were observed in both mice and rats. Immunohistochemical analysis revealed pronounced synovial IL-1beta expression and articular cartilage destruction in vehicle-treated mice. Both these features were significantly less manifested in animals treated with anti-HMGB-1 antibodies or A-box protein. CONCLUSION: Counteracting extracellular HMGB-1 with either neutralizing antibodies or a specific HMGB-1 antagonist may offer a new method for the successful treatment of arthritis. Inflammation and tissue destruction were suppressed in CIA after HMGB-1 blockade.

Animals↗

PubMed1987

Titration of DnaA protein by oriC DnaA-boxes increases dnaA gene expression in Escherichia coli.

Binding of the DnaA protein to its binding sites, the DnaA-boxes (TTATCCACA), was measured by a simple physiological approach. The presence of extra DnaA-boxes in growing cells leads to a derepression of dnaA gene expression, measured as beta-galactosidase activity of a dnaA-lacZ fusion polypeptide. Different DnaA-boxes caused different degrees of derepression indicating that the DnaA protein requires sequences in addition to the DnaA-box for efficient binding. The DnaA-boxes in oriC might act cooperatively in binding of the DnaA protein. The derepressed levels of DnaA protein obtained in a strain carrying an oriC+-pBR322 chimera were very high and sufficient to activate oriC on the chimeric plasmid, which was maintained at a copy number more than three times that of pBR322.

Bacterial Proteins↗

PubMed1989

Directionality of DnaA protein/DNA interaction. Active orientation of the DnaA protein/dnaA box complex in transcription termination.

The complex of DnaA protein with its 9 bp consensus binding site, the dnaA box 5'-TT(A/T)T(A/C)CA(A/C)A, blocks transcribing RNA polymerase. In a model system, the rate of transcription was monitored distal to the dnaA box 5'-TTTTCCACA by the expression of a reporter gene. DnaA-dependent transcription termination occurred irrespective of whether the dnaA box region was or was not translated. Only the dnaA box orientation 5'-TTTTCCACA on the non-coding strand, but not the reverse orientation, was active in termination. This suggests that DnaA protein contacts only one strand of the DNA duplex. Oligonucleotide-directed mutation of a dnaA box present within the dnaA coding region resulted in increased expression of dnaA. This demonstrates that DnaA protein-directed transcription termination is an element of the autoregulation of the dnaA gene.

Amino Acid Sequence↗

PubMed1992

Sequence-specific interaction of the HMG box proteins TCF-1 and SRY occurs within the minor groove of a Watson-Crick double helix.

The high mobility group I (HMG) box is proposed to mediate DNA binding in a novel group of transcription-regulating proteins. Two of these, the proteins encoded by the T cell-specific TCF-1 and the mammalian sex-determining gene SRY, carry a single HMG box with specificity for the heptamer motif A/T A/T C A A A G. We have now analysed the mode of interaction of the HMG boxes of TCF-1 and SRY with this motif. Methylation interference footprinting revealed that both HMG boxes contacted adenines on both strands in the minor groove, whereas no major groove guanine contacts were discerned. Diethylpyrocarbonate (DEPC) carbethoxylation interference footprinting of TCF-1 indicated the absence of major groove contacts on positions 5, 6 and 7 of the motif. Carbethoxylation interference was observed, however, on positions 2, 3 and 4 and to a lesser extent on position 1 in the major groove. Combined T----C and A----I substitution, which changes the surface of the major groove but leaves the minor groove intact, did not interfere with sequence-specific binding by TCF-1 and SRY. These observations indicate that recognition of the heptamer motif by the HMG boxes of the distantly related TCF-1 and SRY proteins predominantly occurs through nucleotide contacts in the minor groove.

Alkylation↗

PubMed1994

The E box is essential for activity of the cardiac actin promoter in skeletal but not in cardiac muscle.

The sequence CANNTG (E box) is frequently found in the promoters of muscle-specific genes and binds members of the basic helix-loop-helix (bHLH) family of transcription factors. We compared the need for the E box in the expression of a muscle-specific promoter normally expressed in both cardiac and skeletal muscle. The E box was mutated in a construct of the cardiac alpha-actin promoter driving the Escherichia coli lacZ gene. The wild-type and mutant constructs were transfected and stably integrated into the genomes of P19 embryonal carcinoma cells. The wild-type promoter was expressed in both cardiac and skeletal myocytes. The promoter lacking an E box was expressed in cardiac but not in skeletal muscle. Neither promoter was active in nonmuscle cells. Thus the E box is not necessary for the cardiac actin promoter activity in P19-derived cardiac muscle but is essential for its activity in skeletal muscle. This result is consistent with our inability to detect cardiac muscle-specific members of the MyoD family of bHLH transcription factors.

Actins↗

PubMed1993

Interactions of NodD at the nod Box: NodD binds to two distinct sites on the same face of the helix and induces a bend in the DNA.

The Rhizobium meliloti nodD gene products are positive transcriptional activators of genes required for early stages of nodule morphogenesis in the R. meliloti-alfalfa symbiosis (nod genes). The regulatory activity of NodD, a member of the LysR family of activator proteins, is mediated in part through its binding to conserved DNA sequences termed nod boxes which lie upstream of the inducible nod genes. Here we use interference footprinting to identify two NodD binding sites in the nodA, nodF and nodH nod boxes. These two binding sites are located on the same face of the DNA helix and can be separated by an additional 10 bp with retention of activity. By systematic alteration of the phasing of the two binding sites on the DNA helix, we showed that only constructs which contain both sites on the same side of the helix are recognized by NodD as determined by migration retardation assay and by in vivo activation of nod box-lacZ fusions. Moreover, NodD apparently induces a bend in the DNA upon binding at the nod box as shown by migration retardation behavior of circularly permuted nod box fragments.

Acyltransferases↗

PubMed1996

Polymorphism in both X and Y box motifs controls level of expression of HLA-DRB1 genes.

The HLA class II antigens of the human major histocompatibility complex play an important role in immune response. The quality of the immune response is determined not only by polymorphisms in their coding region, but also by the level of their cell-surface expression which affects, for example, the extent of T-cell activation. We have previously described allelic polymorphisms in the upstream regulatory regions of HLA-DRB genes, which affected DNA-protein interactions and resulted in significantly different promoter strengths. In the present study, we investigated the effect of polymorphisms in the X and Y box motifs on the transcriptional activity of DRB1 gene promoters in the DR1, DR51, and DR53 haplotype groups. We used normal, chimeric, and mutated DRB promoters and compared their relative abilities to initiate transcription of the CAT reporter gene in human B-cell lines. The results show that polymorphisms in both the X1 and Y box motifs play a dominant role in the promoter strength. In the gel mobility shift assay, we observed differential ability of nuclear proteins that bind to the polymorphic X1 and Y box elements. The results in the present study confirm earlier data in that the nucleotide variation in the X1 box affects the level of expression of DRB1 genes. In addition, the present data demonstrate that polymorphism in the Y box, which affects the inverted CCAAT sequence, also plays a dominant role in the transcriptional activity of DRB1 promoters.

Base Sequence↗

PubMed1992

Synergistic effect of upstream sequences, CCAAT box elements, and HSE sequences for enhanced expression of chimaeric heat shock genes in transgenic tobacco.

The thermoregulated expression of the soybean heat shock (hs) gene Gmhsp17.3-B is regulated via the heat shock promoter elements (HSEs), but full promoter activity requires additional sequences located upstream of the HSE-containing region. Structural features within this putative enhancer region include a run of simple sequences which are also present upstream of HSE-like sequences of other soybean hs genes, and three perfect CCAAT box sequences located immediately upstream from the most distal HSE of the promoter. A series of heterologous and homologous promoter fusions linked to the chloramphenicol acetyl transferase (CAT) gene was constructed and examined in transgenic tobacco plants. The region containing the AT-rich domain of the 5' flanking region was unable to direct transcription from the TATA box of a truncated delta CaMV35S promoter. Heat-inducible CAT activity was detectable when additional sequences from the native promoter containing three CCAAT boxes and a single HSE were present in the constructions. Complete reconstitution of the native hs promoter/enhancer region increased hs specific CAT activities only very little, but deletion of CCAAT box sequences reduced CAT expression five-fold. Our results suggest that AT-rich sequences have a moderate effect on thermoinducible expression levels of the soybean heat shock gene and that CCAAT box sequences act cooperatively with HSEs to increase the hs promoter activity.

Base Sequence↗