AP2 DNA-binding Domain Research Articles

Isolation and Characterization of a Cold-Induced DREB Gene from Aloe Vera L.

A new cold-induced dehydration-responsive element-binding (DREB) gene encoding an AP2/ethylene response element binding protein transcription factor was isolated from Aloe vera L. by rapid amplification of complementary DNA ends (RACE). The deduced protein contained a putative acidic activation domain and an AP2 DNA binding domain of 64 amino acids. The expression analysis showed that the transcripts accumulated rapidly under cold stress and peaked at 12 h, then decreased to the original level. Yeast one-hybrid analysis indicated that Aloe DREB1 can bind to DRE cis-elements and activate the expressions of reporter genes, but the binding and activation abilities were weak. Aloe DREB1 localized in nucleus, although it lacks typical nuclear localization signal. These results implied that Aloe DREB1 might function as a transcription activator involved in the regulation of cold-responsive genes via signal transduction pathway dependent or independent of the DREB transcription factor.

An ERF transcription factor in Medicago truncatula that is essential for Nod factor signal transduction.

Rhizobial bacteria activate the formation of nodules on the appropriate host legume plant, and this requires the bacterial signaling molecule Nod factor. Perception of Nod factor in the plant leads to the activation of a number of rhizobial-induced genes. Putative transcriptional regulators in the GRAS family are known to function in Nod factor signaling, but these proteins have not been shown to be capable of direct DNA binding. Here, we identify an ERF transcription factor, ERF Required for Nodulation (ERN), which contains a highly conserved AP2 DNA binding domain, that is necessary for nodulation. Mutations in this gene block the initiation and development of rhizobial invasion structures, termed infection threads, and thus block nodule invasion by the bacteria. We show that ERN is necessary for Nod factor-induced gene expression and for spontaneous nodulation activated by the calcium- and calmodulin-dependent protein kinase, DMI3, which is a component of the Nod factor signaling pathway. We propose that ERN is a component of the Nod factor signal transduction pathway and functions downstream of DMI3 to activate nodulation gene expression.

Homing into the origin of the AP2 DNA binding domain

The AP2 DNA binding domain was thought to be plant specific because of its presence in plant, but not animal, transcriptional regulators, particularly members of the AP2/ERF family. Two recent studies have identified the AP2 domain in bacteria, bacteriophage and a ciliate as part of proteins that also encode site-specific endonucleases. The association of AP2 with an enzyme known to catalyze its own movement within populations and between species explains the unusual distribution of AP2 and, as such, adds to a growing list of phenomena where mobile DNA has promoted evolutionary novelty.

From endonucleases to transcription factors: evolution of the AP2 DNA binding domain in plants.

All members of the AP2/ERF family of plant transcription regulators contain at least one copy of a DNA binding domain called the AP2 domain. The AP2 domain has been considered plant specific. Here, we show that homologs are present in the cyanobacterium Trichodesmium erythraeum, the ciliate Tetrahymena thermophila, and the viruses Enterobacteria phage Rb49 and Bacteriophage Felix 01. We demonstrate that the T. erythraeum AP2 domain selectively binds stretches of poly(dG)/poly(dC) showing functional conservation with plant AP2/ERF proteins. The newly discovered nonplant proteins bearing an AP2 domain are predicted to be HNH endonucleases. Sequence conservation extends outside the AP2 domain to include part of the endonuclease domain for the T. erythraeum protein and some plant AP2/ERF proteins. Our phylogenetic analysis of the broader family of AP2 domains supports the possibility of lateral gene transfer. We hypothesize that a horizontal transfer of an HNH-AP2 endonuclease from bacteria or viruses into plants may have led to the origin of the AP2/ERF family of transcription factors via transposition and homing processes.

An AP2/EREBP-type transcription-factor gene from rice is cold-inducible and encodes a nuclear-localized protein.

We cloned an AP2/EREBP gene by dot blotting and named it OsDREBL. Analysis of its deduced amino-acid sequence indicated that this protein had a potential nuclear-localization signal, a possible acidic-activation domain and an AP2 DNA binding domain. Northern analysis showed that the transcripts of OsDREBL accumulated rapidly (within 30 min) in response to low temperature, but not in response to ABA, NaCl and dehydration treatments. Southern analysis indicated the presence of a single-copy of the OsDREBL gene in the Oryza sativa genome. Our research also demonstrated that OsDREBL was localized to the nucleus but did not bind effectively to the C-repeat/dehydration responsive element (CRT/DRE). These results suggested that OsDREBL may function as a transcription factor in the cold-stress response, independent of the DREB signal-transduction pathway.

The DNA-binding activity of an AP2 transcriptional activator HvCBF2 involved in regulation of low-temperature responsive genes in barley is modulated by temperature.

An HvCBF2 cDNA was isolated from barley leaves. It encoded a protein containing an AP2 DNA-binding domain homologous to C-repeat (CRT)/dehydration-responsive element (DRE) binding factors (CBF/DREB1). In contrast to the previously reported cold-inducible CBF/DREB1 genes, HvCBF2 was expressed in barley leaves under non-stress conditions. Only a transient increase in the HvCBF2 transcript level was observed during cold treatment. Transactivation analysis showed that HvCBF2 was a transcriptional activator, capable of activating expression of a reporter gene driven by a low-temperature and drought-responsive HVA1s promoter in barley leaves. The activity of HvCBF2 as a transcriptional activator was upregulated by low temperature. DNA-binding analysis revealed that HvCBF2 did not bind to the CRT/DRE motif at 30 degrees C. A low, but detectable, binding activity was observed at 25 degrees C and the binding activity gradually increased as the temperature decreased. The binding activity at 0 degrees C was the highest and more than 10 times higher than that at 25 degrees C. The activation and inactivation of HvCBF2 activity were reversible and were achieved in a cell-free system simply by temperature change. Analysis of the binding sequence showed that HvCBF2 bound to a (G/a)(T/c)CGAC core motif, where the lower-case letters are less efficient bases. These data suggest that HvCBF2 is a transcription factor interacting with the core CRT/DRE motif containing a preferred sequence of GTCGAC and its DNA-binding activity is regulated by temperature. This represents a new type of activation mechanism for transcriptional activators.

Characterisation of the DNA-binding profile of barley HvCBF1 using an enzymatic method for rapid, quantitative and high-throughput analysis of the DNA-binding activity.

A rapid and quantitative DNA-binding assay was developed based on the translational fusion of a DNA-binding protein (DBP) with a Neocallimastix patriciarum beta-1,4-D-glucanase, CelD. CelD releases a fluorescent 4-methylumbelliferyl product from 4-methylumbelliferyl cellobioside. This hydrolysis activity was used to quantify the amount of DBP-CelD bound to an immobilised biotin-labelled target sequence. The DNA-binding assay can be performed in a 96-well plate format for high- throughput analysis of putative DBPs. This method was applied to analysis of the binding properties and sequence selectivity of a cold-inducible transcription factor HvCBF1 from barley containing an AP2 DNA-binding domain. A base-scanning approach using degenerate oligonucleotide probes was employed for rapid identification of the conserved core motif of the HvCBF1 binding site. Quantitative analysis of the binding site of HvCBF1 using systematic base substitution revealed that a (G/a) (C/t)CGAC sequence was sufficient to constitute a functional motif, where the lower-case letters represent less efficient bases. The method enables us to provide accurate and quantitative data on a comprehensive DNA-binding profile for a cold-inducible AP2 transcription factor as well as information on environmental parameters potentially influencing the DNA-binding activity. The accurate binding sequence data facilitate identification of candidate genes regulated by HvCBF1 from genome sequence databases.

Regulation and characterization of four CBF transcription factors from Brassica napus.

Four orthologues of the Arabidopsis CBF/Dreb transcriptional activator genes were isolated from the winter Brassica napus, cv. Jet neuf. All four BNCBF clones encode a putative DRE/CRT (LTRE)-binding protein with an AP2 DNA-binding domain, a putative nuclear localization signal and a possible acidic activation domain. Deduced amino acid sequences suggested that BNCBFs 5, 7and 16 are very similar to the Arabidopsis CBFI whereas BNCBF17 is different in that it contains two extra regions of 16 and 21 amino acids in the acidic domain. Transcripts hybridizing specifically to BNCBF17 and to one or more of the other BNCBFs accumulated in leaves within 30 min of cold exposure of the Brassica seedlings and preceded transcript accumulation of the cold-inducible BN28 gene, a Brassica orthologue of the cor6.6 or KIN gene from Arabidopsis. Cold-induced accumulation of BNCBF17 mRNA was rapid but was short-lived compared to transcripts hybridizing to BNCBF5/7/16. Transcripts hybridizing to one or more of BNCBF5/7/16 accumulated at low levels after the plants were subjected to prolonged exposure to salt stress. BNCBF17 was not responsive to salt stress. BNCBF transcript accumulation was similar in both spring and winter Brassica but the persistence of the transcripts in the cold were generally shorter in the spring than in the winter type. BNCBF5 and 17 proteins bind in vitro to the LTRE domains of the cold-inducible BN115 (cor15a orthologue) or BN28 promoters. Differential binding preferences, however, to LTREs between BNI 15 and BN28 were observed. Mutation of the core CCGAC sequence of the LTRE indicated that BNCBF17 had a lower sequence binding specificity than BNCBF5. Furthermore, experiments indicated that the LTREs were able to drive BNCBF5 and 17 trans-activation of the Lac-Z reporter gene in yeast. We conclude that the BNCBFs reported here could function as trans-acting factors in low-temperature responses in Brassica, controlling the expression of cold-induced genes through an ABA-independent pathway.

RAV1, a novel DNA-binding protein, binds to bipartite recognition sequence through two distinct DNA-binding domains uniquely found in higher plants.

We have cloned and characterized two novel DNA binding proteins designated RAV1 and RAV2 from Arabidopsis thaliana. RAV1 and RAV2 contain two distinct amino acid sequence domains found only in higher plant species. The N-terminal regions of RAV1 and RAV2 are homologous to the AP2 DNA-binding domain present in a family of transcription factors represented by the Arabidopsis APETALA2 and tobacco EREBP proteins, while the C-terminal region exhibits homology to the highly conserved C-terminal domain, designated B3, of VP1/ABI3 transcription factors. Binding site selection assays using a recombinant glutathione S-transferase fusion protein have revealed that RAV1 binds specifically to bipartite recognition sequences composed of two unrelated motifs, 5'-CAACA-3' and 5'-CACCTG-3', separated by various spacings in two different relative orientations. Analyses using various deletion derivatives of the RAV1 fusion protein show that the AP2 and B3-like domains of RAV1 bind autonomously to the CAACA and CACCTG motifs, respectively, and together achieve a high affinity and specificity of binding. From these results, we suggest that the AP2 and B3-like domains of RAV1 are connected by a highly flexible structure enabling the two domains to bind to the CAACA and CACCTG motifs in various spacings and orientations.

Redox Modulation of AP-2 DNA Binding Activityin Vitro

Transcription factor AP-2 plays a critical role in regulating gene expression during vertebrate development and cellular differentiation. We report here that AP-2 DNA bindingin vitrocan be reversibly modulated by redox conditions and that thioredoxin is a potent stimulator of AP-2 DNA binding. Our studies indicate that oxidation of recombinant human AP-2 with diamide or hydrogen peroxide inhibits its DNA binding activity to synthetic AP-2 oligodeoxynucleotides. The inhibitory effect of diamide on AP-2 DNA binding was dose-dependent and was reversible by addition of reducing agents β-mercaptoethanol, dithiothreitol, and thioredoxin. Immunoblot assays indicated that treatment with oxidants caused a change in migration of the AP-2 protein in nonreducing gels and that this effect was also reversible by treatment of the oxidized AP-2 with reducing agents. These data suggest a mechanism by which the oxidation state of conserved cysteine residues in the AP-2 DNA binding domain may contribute to its DNA binding activity. AP-2 joins a group of other transcription factors whose functions are regulated in part by their redox states.

AP-2.2: A Novel AP-2-Related Transcription Factor Induced by Retinoic Acid during Differentiation of P19 Embryonal Carcinoma Cells

A 2.8-kb cDNA encoding a new transcription factor (AP-2.2) has been cloned from mouse P19 embryonal carcinoma cells, in which the corresponding mRNA begins to accumulate 30 min after retinoic acid (RA) addition. The predicted protein is 449 amino acids long and exhibits approximately 65% overall identity with other AP-2-related proteins (human AP-2, mouse AP-2α and β). A 96-amino-acid-long sequence, which is almost fully conserved between all these proteins, corresponds to the previously characterized human AP-2 DNA binding domain. Expression of AP-2.2 inEscherichia coligenerated a protein that formed a specific complex with the AP-2 recognition site GCCN3GGC. AP-2.2 activated transcription from a reporter gene containing an AP-2 DNA binding site and acted synergistically with RARα to activate transcription from the CRABPII gene promoter. Transcriptional activation required the AP-2.2 amino-terminal region that contains a domain rich in proline and glutamine residues. The pattern of AP-2.2 expression in adult tissues, which is distinct from that of AP-2α, is essentially restricted to male and female gonads, to most if not all the squamous epithelia, and to several exocrine glands.