CCAAT Box-binding Factor Research Articles

Identification of a module of HAP transcription factors for seed development in rice

Heme activator protein (HAP) is a CCAAT box-binding factor family conserved in all higher eukaryotes. HAP transcription factors (TFs) consist of three subunits: HAP2, HAP3, and HAP5. Using recent rice genome information, we identified 11 HAP2 genes, 11 HAP3 genes, and 12 HAP5 genes. A HAP2 and five HAP5 genes were acknowledged more in this study than in the previous studies. Due to the functional association of HAP subunits, we carried out clustering analysis for expression patterns of all HAP members using anatomical meta-expression data of 983 rice Affymetrix arrays collected from the public database. Subsequently, we identified HAP members with endosperm and seed-preferred expression and focused on nine HAP family members associated with this expression pattern. We confirmed their expression patterns by RT-PCR analyses. The nine members were OsHAP2G, OsHAP2I, OsHAP3D, OsHAP3K, and five HAP5 genes (LOC_Os05g23910, LOC_Os10g11580, LOC_Os01g24460, LOC_Os01g01290, and LOC_Os01g39850). Promoter analyses using GUS activity again demonstrated that OsHAP3K is preferentially expressed in the aleurone layer of the seed and the LOC_Os05g23910 gene-encoding HAP5 showed endosperm-preferred expression. Network analysis using RiceNet suggests a functional gene network mediated by four rice HAP genes, further helping in the prediction of the biological function of OsHAP2G and OsHAP5 (LOC_Os10g11580). Our data provide new insight into HAP family-mediated regulatory pathways for endosperm or seed development in rice.

Functional analysis of the promoters of the small GTPases RhoA and RhoB in embryonic stem cells

Small GTPases of the Rho subfamily have been implicated in many physiological and pathological processes in various cell types including embryonic stem cells (ESCs). In the present study we performed a functional analysis of the promoters of the RhoA and the RhoB genes in order to identify regulatory elements that are important for their transcriptional control in ESCs. We show that RhoA mRNA levels were significantly higher compared with the RhoB mRNA levels in ESCs as well in various cancer cell lines and this difference could be accounted for by differences in the activities of the corresponding promoters. Deletion analysis of the RhoA and RhoB promoters in ESCs revealed that the proximal regions contain regulatory elements that are critical for their activity. Both proximal promoters contain CCAAT boxes and mutagenesis of these elements decreased significantly the activity of both promoters suggesting a coordinated regulation of the two genes by CCAAT box binding factors. Finally, we show that both genes are subjects to autoregulation in ESCs and in the case of RhoB, this autoregulation requires the GTPase activity of the Rho proteins. Understanding the mechanisms that control the transcription of Rho GTPases in ESCs may shed new light into the still unknown roles of these proteins in stem cell functions.

Identification and characterization of NF-YB family genes in tung tree.

The NF-YB transcription factor gene family encodes a subunit of the CCAAT box-binding factor (CBF), a highly conserved trimeric activator that strongly binds to the CCAAT box promoter element. Studies on model plants have shown that NF-YB proteins participate in important developmental and physiological processes, but little is known about NF-YB proteins in trees. Here, we identified seven NF-YB transcription factor-encoding genes in Vernicia fordii, an important oilseed tree in China. A phylogenetic analysis separated the genes into two groups; non-LEC1 type (VfNF-YB1, 5, 7, 9, 11, 13) and LEC1-type (VfNF-YB 14). A gene structure analysis showed that VfNF-YB 5 has three introns and the other genes have no introns. The seven VfNF-YB sequences contain highly conserved domains, a disordered region at the N terminus, and two long helix structures at the C terminus. Phylogenetic analyses showed that VfNF-YB family genes are highly homologous to GmNF-YB genes, and many of them are closely related to functionally characterized NF-YBs. In expression analyses of various tissues (root, stem, leaf, and kernel) and the root during pathogen infection, VfNF-YB1, 5, and 11 were dominantly expressed in kernels, and VfNF-YB7 and 9 were expressed only in the root. Different VfNF-YB family genes showed different responses to pathogen infection, suggesting that they play different roles in the pathogen response. Together, these findings represent the first extensive evaluation of the NF-YB family in tung tree and provide a foundation for dissecting the functions of VfNF-YB genes in seed development, stress adaption, fatty acid synthesis, and pathogen response.

Isolation and characterization of LEAFY COTYLEDON 1-LIKE gene related to embryogenic competence in Citrus sinensis

A member of the LEAFY COTYLEDON gene family encoding a HAP3 (heme activated protein 3) subunit of the CCAAT box-binding factor was isolated and termed as Citrus sinensis LEAFY COTYLEDON 1-LIKE (CsL1L). The deduced amino acid sequence shared a high similarity with LEAFY COTYLEDON 1-LIKE (L1L) in Arabidopsis thaliana, Phaseolus coccineus, Theobroma cacao, and Helianthus annuus. Quantitative RT-PCR results indicated that CsLIL was highly expressed in embryogenic callus, somatic embryos and immature seeds, but was rarely detected in non-embryogenic callus, vegetative and floral tissues. Ectopic expression of CsL1L in vegetative tissues could induce embryo-like structures, suggesting that CsL1L has the capability to transit cells from vegetative to embryogenic phase. Comparison of CsL1L expression in the newly formed and long-term subcultured embryogenic calli of W. Murcott tangor (C. sinensis × C. reticulata) and Hongkong kumquat (Fortunella hindsii Swingle) revealed that the potency of embryogenesis was related to the level of CsL1L expression. Sub-cellular localization analysis indicated that CsL1L was a nuclear protein in plant. A microsatellite in CsL1L was verified with polymorphism among the citrus species.

Erratum to: ‘CCAAT-box binding transcription factors in plants: Y so many?’

In the review article ‘CCAAT-box binding transcription factors in plants: Y so many’ by Tom Laloum, Stéphane De Mita, Pascal Gamas, Maël Baudin, and Andreas Niebel, which was published in the March 2013 issue of Trends in Plant Science, the procedure used for reconstructing the phylogenetic trees in Figure 2 was incorrectly described. The sentence ‘The tree has been reconstructed using maximum likelihood under the general time-reversible model as implemented in the PhyML software’ should read ‘The tree has been reconstructed using maximum likelihood under the LG model as implemented in the PhyML software’. This change to the figure legend does not affect the conclusions mentioned in the paper. Below is the corrected version of Figure 2. CCAAT-box binding transcription factors in plants: Y so many?Laloum et al.Trends in Plant ScienceAugust 30, 2012In BriefTranscription factors belonging to the CCAAT-box binding factor family (also known as the Nuclear Factor Y) are present in all higher eukaryotes. Studies in plants have revealed that each subunit of this heterotrimeric transcription factor is encoded by a gene belonging to a multigene family allowing a considerable modularity. In this review, we focus on recent findings concerning the expression patterns and potential functions of different members of these NF-Y protein families using a phylogenetic approach. Full-Text PDF

ABSCISIC ACID INSENSITIVE3 regulates abscisic acid‐responsive gene expression with the nuclear factor Y complex through the ACTT‐core element in Physcomitrella patens

The phytohormone ABA and the transcription factor ABSCISIC ACID INSENSITIVE 3 (ABI3)/VIVIPAROUS 1 (VP1) function in protecting embryos during the desiccation stage of seed development. In a similar signaling pathway, vegetative tissue of the moss Physcomitrella patens survives desiccation by activating downstream genes (e.g. LEA1) in response to ABA and ABI3. We show that the PpLEA1 promoter responds to PpABI3 primarily through the ACTT-core element (5'-TCCACTTGTC-3'), while the ACGT-core ABA-responsive element (ABRE) appears to respond to ABA alone. We also found by yeast-two-hybrid screening that PpABI3A interacts with PpNF-YC1, a subunit of CCAAT box binding factor (CBF)/nuclear factor Y (NF-Y). PpNF-YC1 increased the activation of the PpLEA1 promoter when incubated with PpABI3A, as did NF-YB, NF-YC, and ABI3 from Arabidopsis. This new response element (ACTT) is responsible for activating the ABI3-dependent ABA response pathway cooperatively with the nuclear factor Y (NF-Y) complex. These results further define the regulatory interactions at the transcriptional level for the expression of this network of genes required for drought/desiccation tolerance. This gene regulatory set is in large part conserved between vegetative tissue of bryophytes and seeds of angiosperms and will shed light on the evolution of this pathway in the green plant lineage.

Identification of Direct Targets of FUSCA3, a Key Regulator of Arabidopsis Seed Development

FUSCA3 (FUS3) is a B3 domain transcription factor that is a member of the LEAFY COTYLEDON (LEC) group of genes. The LEC genes encode proteins that also include LEC2, a B3 domain factor related to FUS3, and LEC1, a CCAAT box-binding factor. LEC1, LEC2, and FUS3 are essential for plant embryo development. All three loss-of-function mutants in Arabidopsis (Arabidopsis thaliana) prematurely exit embryogenesis and enter seedling developmental programs. When ectopically expressed, these genes promote embryo programs in seedlings. We report on chromatin immunoprecipitation-tiling array experiments to globally map binding sites for FUS3 that, along with other published work to assess transcriptomes in response to FUS3, allow us to determine direct from indirect targets. Many transcription factors associated with embryogenesis are direct targets of FUS3, as are genes involved in the seed maturation program. FUS3 regulates genes encoding microRNAs that, in turn, control transcripts encoding transcription factors involved in developmental phase changes. Examination of direct targets of FUS3 reveals that FUS3 acts primarily or exclusively as a transcriptional activator. Regulation of microRNA-encoding genes is one mechanism by which FUS3 may repress indirect target genes. FUS3 also directly up-regulates VP1/ABI3-LIKE1 (VAL1), encoding a B3 domain protein that functions as a repressor of transcription. VAL1, along with VAL2 and VAL3, is involved in the transition from embryo to seedling development. Many genes are responsive to FUS3 and to VAL1/VAL2 but with opposite regulatory consequences. The emerging picture is one of complex cross talk and interactions among embryo transcription factors and their target genes.

CCAAT-box binding transcription factors in plants: Y so many?

Transcription factors belonging to the CCAAT-box binding factor family (also known as the Nuclear Factor Y) are present in all higher eukaryotes. Studies in plants have revealed that each subunit of this heterotrimeric transcription factor is encoded by a gene belonging to a multigene family allowing a considerable modularity. In this review, we focus on recent findings concerning the expression patterns and potential functions of different members of these NF-Y protein families using a phylogenetic approach. During the course of evolution plant CCAAT-box binding factors seem to have diversified into at least two main groups. The first group has more general expression patterns and/or functions whereas the second group has acquired more specific expression patterns and/or functions and could play key roles in specific pathways.

Molecular analysis of a sunflower gene encoding an homologous of the B subunit of a CAAT binding factor

A genomic DNA fragment containing the complete LEAFY COTYLEDON1-LIKE (HaL1L) gene was retrieved by chromosome walking. Its sequence was confirmed and elongated by screening a sunflower genomic DNA BAC Library. HaL1L, whose cDNA had already been sequenced and characterized, encodes a NF-YB subunit of a CCAAT box-binding factor (NF-Y) involved in the early stages of zygotic and somatic embryogenesis in the Helianthus genus. In the HaL1L 5'-flanking region, elements specific to a putative TATA-box promoter and two "CG isles" were identified. An investigation of the methylation status of these CG rich DNA regions showed that differentially methylated cytosines were recognizable in the DNA of embryos on the fifth day after pollination in comparison to leaf DNA suggesting that during plant development epigenetic regulation of HaL1L transcription was achieved by methylating cytosine residues. We also searched the HaL1L nucleotide sequence for cis-regulatory elements able to interact with other transcription factors (TFs) involved in the HaL1L regulation. Of the elements identified, one of the most intriguing is WUSATA, the target sequence for the WUSCHEL (WUS) TF, which may be part of a complex regulation network controlling embryo development. In this article, we show that the WUSATA target site, located in the intron of HaL1L, is able to bind the TF WUS. Interestingly, we found auxin and abscisic acid responsive motifs in the HaL1L promoter region suggesting that this gene may additionally by under hormonal control. Finally, the presence of a cytoplasmic polyadenylation signal downstream to the coding region indicates that this gene may also be controlled at the translation level by a temporarily making the pre-synthesized HaL1L mRNA unavailable for protein synthesis.

BZIP28 and NF-Y Transcription Factors Are Activated by ER Stress and Assemble into a Transcriptional Complex to Regulate Stress Response Genes in Arabidopsis

Stress agents known to elicit the unfolded protein response in Arabidopsis thaliana upregulate the expression of a constellation of genes dependent on the membrane-associated basic domain/leucine zipper (bZIP) transcription factor, bZIP28. Among the stress-activated genes, a consensus promoter sequence corresponding to the endoplasmic reticulum (ER) stress-responsive element I (ERSE-I), CCAAT-N10-CACG, was identified. Disruption of either the CCAAT or CACG subelement in ERSE-I resulted in reduction of the transcriptional response to ER stress. bZIP28 forms homo- and heterodimers with other bZIP TF family members (in subgroup D) and interacts with CCAAT box binding factors, heterotrimeric factors composed of NF-Y subunits. Arabidopsis encodes 36 NF-Y subunits, and it was found that subunits NF-YB3 and -YC2 interact with bZIP28 and NF-YA4, respectively, in a yeast three-hybrid system. A transcriptional complex containing bZIP28 and the above-mentioned three NF-Y subunits was assembled in vitro on DNA containing ERSE-I. bZIP28, on its own, binds to the CACG subelement in ERSE-I to form a smaller complex I, and in combination with the NF-Y subunits above, bZIP28 assembles into a larger transcriptional complex (complex II). bZIP28 was shown to interact with NF-Y subunits in vivo in bimolecular fluorescence complementation analyses and in coimmunoprecipitation assays. Treatment of seedlings with ER stress agents led to the upregulation of NF-YC2 and the relocation of NF-YB3 from the cytoplasm to the nucleus. Thus, in response to ER stress, bZIP28 is mobilized by proteolysis and recruits NF-Y subunits to form a transcriptional complex that upregulates the expression of ER stress-induced genes.

Cloning and functional characterization of the 5′ regulatory region of ovine Hormone Sensitive Lipase ( HSL) gene

Hormone Sensitive Lipase (HSL) catalyzes the rate-limiting step in the mobilization of fatty acids from adipose tissue, thus determining the supply of energy substrates in the body. HSL enzymatic activity is increased by adrenergic agonists, such as catecholamines and glucagons, which induce cyclic AMP (cAMP) intracellular production, subsequently followed by the activation of Protein Kinase A (PKA) and its downstream signaling cascade reactions. HSL constitutes the critical enzyme in the modulation of lipid stores and the only component being subjected to hormonal control in terms of the recently identified Adipose Triglyceride Lipase (ATGL). In order to acquire detailed knowledge with regard to the mechanisms regulating ovine HSL ( ovHSL) gene transcription activity, we initially isolated and cloned the 5′ proximal and distal promoter regions through a genome walking approach, with the utilization of the already characterized ovHSL cDNAs. As evinced by BLAST analysis and a multiple alignment procedure, the isolated genomic fragment of 2.744 kb appeared to contain the already specified 5′-untranslated region (5′-UTR), which was interrupted by a relatively large intron of 1.448 kb. Regarding the upstream remaining part of 1.224 kb, it was demonstrated to represent a TATA-less promoter area, harboring several cis-regulatory elements that could be putatively recognized by relatively more general transcription factors, mainly including Stimulating protein 1 (Sp1), CCAAT-box Binding Factors (CBFs), Activator Protein 2 (AP2) and Glucocorticoid Receptor (GR), as well as other cis-acting regions denominated as Insulin Response Element (IRE), Glucose Response Element (GRE), Fat Specific Element (FSE) and cAMP Response Element (CRE), which could likely function in a nourishment (i.e. glucose)-/hormone-dependent fashion. When different genomic fragments were directionally (5′ to 3′) cloned into a suitable reporter vector upstream of a promoter-less luciferase gene and transiently transfected into 3T3-L1 (mouse fibroblasts) as well as T24 (human bladder cancer) cell lines, strong promoter activities were unambiguously detected, with the − 140/+ 18 nucleotide sequence bearing the highest transcriptional response, thus indicating that the 1.224 kb 5′ flanking region, isolated by genome walking, veritably contains the ovHSL gene promoter. Of particular significance are the observations that the functional promoter fragments could trigger the transcriptional activity of luciferase gene only under high concentration of glucose conditions in both cell lines.

Duplication and Functional Diversification of HAP3 Genes Leading to the Origin of the Seed-Developmental Regulatory Gene, LEAFY COTYLEDON1 (LEC1), in Nonseed Plant Genomes

The HAP3 gene encodes a subunit of the CCAAT-box-binding factor (CBF), a highly conserved trimeric activator that recognizes and binds the ubiquitous CCAAT promoter element with high affinity. Two types of HAP3 gene have been identified in plant genomes. The LEAFY COTYLEDON1 (LEC1)-type HAP3 genes encode a functionally specialized subunit of CBF, which is expressed specifically in developing seeds. In contrast, most non-LEC1-type HAP3 genes are expressed in various tissues. It has been proposed that the LEC1-type HAP3 genes originated from the duplication and functional divergence of non-LEC1-type HAP3 genes. However, it is not yet known when this duplication event took place or whether the LEC1-type HAP3 genes appeared at the same time as the origin of seed plants. Here we describe a comprehensive comparison of the duplication patterns of HAP3 genes in different plant genomes. We recognize a major expansion of the HAP3 gene family accompanying the origin and early diversification of land plants and postulate that retrotransposition and other mechanisms of gene duplication have been involved in the expansion of the plant HAP3 gene family. We provide evidence that the LEC1-type HAP3 genes originated in nonseed vascular plant genomes and demonstrate that they are inductively expressed under drought stress in nonseed plants. These genes, however, were recruited to a novel regulatory network in the early stages of seed plant evolution and steadily expressed during seed development and maturation.

Characterization of leafy cotyledon1-like during embryogenesis in Theobroma cacao L.

Theobroma cacao L., an economically important crop for developing countries, can be experimentally propagated by somatic embryogenesis. Because of their potential roles in embryogenesis, a gene candidate strategy was initiated to find gene homologues of the members of the leafy cotyledon family of transcription factors. A homologue of the leafy cotyledon1-like gene, that encodes the HAP 3 subunit of the CCAAT box-binding factor, was found in the cocoa genome (TcL1L). The translated peptide shared a high amino acid sequence identity with the homologous genes of Arabidopsis thaliana, Phaseolus coccineus and Helianthus annuus. TcL1L transcripts mainly accumulated in young and immature zygotic embryos, and, to a lesser extent, in young and immature somatic embryos. In situ hybridization specified the localization of the transcripts as being mainly in embryonic cells of young embryos, the meristematic cells of the shoot and root apex of immature embryos, and in the protoderm and epidermis of young and immature embryos, either zygotic or somatic. Non-embryogenic explants did not show TcL1L expression. Ectopic expression of the TcL1L gene could partially rescue the Arabidopsis lec1 mutant phenotype, suggesting a similarity of function in zygotic embryogenesis.

The Induction Levels of Heat Shock Protein 70 Differentiate the Vulnerabilities to Mutant Huntingtin among Neuronal Subtypes

The reason why vulnerabilities to mutant polyglutamine (polyQ) proteins are different among neuronal subtypes is mostly unknown. In this study, we compared the gene expression profiles of three types of primary neurons expressing huntingtin (htt) or ataxin-1. We found that heat shock protein 70 (hsp70), a well known chaperone molecule protecting neurons in the polyQ pathology, was dramatically upregulated only by mutant htt and selectively in the granule cells of the cerebellum. Granule cells, which are insensitive to degeneration in the human Huntington's disease (HD) pathology, lost their resistance by suppressing hsp70 with siRNA, whereas cortical neurons, affected in human HD, gained resistance by overexpressing hsp70. This indicates that induction levels of hsp70 are a critical factor for determining vulnerabilities to mutant htt among neuronal subtypes. CAT (chloramphenicol acetyltransferase) assays showed that CBF (CCAAT box binding factor, CCAAT/enhancer binding protein zeta) activated, but p53 repressed transcription of the hsp70 gene in granule cells. Basal and mutant htt-induced expression levels of p53 were remarkably lower in granule cells than in cortical neurons, suggesting that different magnitudes of p53 are linked to distinct induction levels of hsp70. Surprisingly, however, heat shock factor 1 was not activated in granule cells by mutant htt. Collectively, different levels of hsp70 among neuronal subtypes might be involved in selective neuronal death in the HD pathology.

Inhibition of systemic sclerosis dermal fibroblast type I collagen production and gene expression by simvastatin

To examine whether statins are capable of modulating collagen gene expression in cultured systemic sclerosis dermal fibroblasts. Cultured dermal fibroblasts from 3 patients with diffuse systemic sclerosis of recent onset were treated with 5 microM and 10 microM of simvastatin for 3 or 4 days. Morphologic features, cytotoxicity, and type I collagen production and messenger RNA (mRNA) levels in the fibroblasts were examined. The effects of mevalonate, geranylgeranyl pyrophosphate (GGPP), and farnesyl pyrophosphate (FPP), which are lipids downstream from the hydroxymethylglutaryl-coenzyme A block, were also examined. Transient transfections with COL1A1 promoter-reporter constructs and electrophoretic gel mobility shift assays were utilized to examine COL1A1 transcription and Sp1 and CCAAT-box binding factor (CBF) binding. Simvastatin did not cause morphologic changes or cytotoxicity in the fibroblasts, even after 4 days of treatment. Type I collagen production and mRNA levels showed a potent and dose-related inhibition following 3 and 4 days of treatment. The inhibition of collagen gene expression by simvastatin was completely reversed by mevalonate and GGPP, but not by FPP. The statin effects occurred at the transcriptional level and involved the proximal COL1A1 promoter region encompassing -174 bp. A significant reduction in Sp1 and CBF binding activity was also found in simvastatin-treated cells. Simvastatin is a powerful inhibitor of type I collagen gene expression in normal and systemic sclerosis fibroblasts. The pleiotropic protective effects of statins on various endothelial and immune cell functions in conjunction with their potent inhibitory effects on type I collagen gene expression suggest that statins may be effective therapeutic agents in systemic sclerosis.

Retinoic acids and trichostatin A (TSA), a histone deacetylase inhibitor, induce human pyruvate dehydrogenase kinase 4 (PDK4) gene expression

Induction of pyruvate dehydrogenase kinase 4 (PDK4) conserves glucose and substrates for gluconeogenesis and thereby helps regulate blood glucose levels during starvation. We report here that retinoic acids (RA) as well as Trichostatin A (TSA), an inhibitor of histone deacetylase (HDAC), regulate PDK4 gene expression. Two retinoic acid response elements (RAREs) to which retinoid X receptor α (RXRα) and retinoic acid receptor α (RARα) bind and activate transcription are present in the human PDK4 (hPDK4) proximal promoter. Sp1 and CCAAT box binding factor (CBF) bind to the region between two RAREs. Mutation of either the Sp1 or the CBF site significantly decreases basal expression, transactivation by RXRα/RARα/RA, and the ability of TSA to stimulate hPDK4 gene transcription. By the chromatin immunoprecipitation assay, RA and TSA increase acetylation of histones bound to the proximal promoter as well as occupancy of CBP and Sp1. Interaction of p300/CBP with E1A completely prevented hPDK4 gene activation by RXRα/RARα/RA and TSA. The p300/CBP may enhance acetylation of histones bound to the hPDK4 promoter and cooperate with Sp1 and CBF to stimulate transcription of the hPDK4 gene in response to RA and TSA.

Characterization of LEAFY COTYLEDON1-LIKE gene in Helianthus annuus and its relationship with zygotic and somatic embryogenesis

The Helianthus annuus LEAFY COTYLEDON1-LIKE (HaL1L) gene encodes a heme-activated protein 3 subunit of the CCAAT box-binding factor. The phylogenetic analysis indicates that HaL1L is closely related to LEAFY COTYLEDON1 (LEC1)-type of Arabidopsis thaliana. In particular, the peptide results homologous to the LEC1-LIKE gene of A. thaliana, with which it shares a high amino acid sequence identity (56%). HaL1L transcripts are accumulated primarily at an early stage of sunflower embryogenesis. High levels of HaL1L messenger RNA (mRNA) have been detected in the developing embryo proper, suspensor, endosperm, integument, and integumentary tapetum cells, while in unfertilized ovules, HaL1L mRNA was present at rather low levels. In an attempt to examine the involvement of HaL1L on somatic embryogenesis, a somaclonal variant of H. annuus x H. tuberosus (EMB-2) that produces ectopic embryo- and shoot-like structures, arranged in clusters along leaf veins, was used. We found that the epiphyllous proliferation of ectopic embryos on EMB-2 leaves was associated to HaL1L mRNA accumulation. The detection of HaL1L transcripts was evident in somatic embryos at the heart- and early cotyledon-stage. On the contrary, no signal related to HaL1L transcript accumulation was observed in EMB-2 leaves characterized by the presence of shoot-like structures. Together, these results support the conclusion that the transcription of the HaL1L gene is maintained both in zygotic and in somatic embryogenesis. In addition, the ectopic accumulation of HaL1L mRNA in parenchymal cells around the vascular bundles of epiphyllous leaves opens the possibility that HaL1L could also be involved in switching somatic cell fate towards embryogenic competence.

Novel Autoregulatory Function of Hepatitis B Virus M Protein on Surface Gene Expression

The hepatitis B virus surface gene consists of a single open reading frame divided into three coding regions: pre-S1, pre-S2, and S. By alternate translation at each of the three initiation codons, L, M, and S proteins can be synthesized. Studies have shown that M protein is not essential for viral replication, virion morphogenesis, or in vitro infectivity. In this study, we show that native M protein can regulate surface gene expression at the transcriptional level. The regulatory effect of M protein is mediated through the CCAAT box within the S promoter. Deletion mapping analysis indicated that the transactivating effect of M protein is mediated through amino acids 1-57 of M protein (the MHBs(au) domain), although its maximal transactivation activity coincides with that of the pre-S2 domain. This conclusion is supported by the fact that disruption of the putative V8 protease site at the pre-S2/S domain junction not only rendered M protein incapable of transactivating the S promoter but also inactivated its nuclear translocation potential. Immunoprecipitation and immunoblot experiments demonstrated that pre-S2 interacts with the three subunits of the CCAAT box-binding factor/nuclear factor Y, the cognate binding protein of the CCAAT box. These results demonstrate and define a novel regulatory role of M protein, which, under natural conditions, may undergo a proteolytic process to generate an MHBs(au) species that will be translocated inside the nucleus, where it will interact with the CCAAT box-binding factor to regulate surface gene expression. Because the CCAAT box is located at a fixed position within numerous promoters, these observations might provide a plausible explanation for hepatitis B virus-associated hepatocarcinogenesis.

Transactivation of the cyclin A promoter by bovine papillomavirus type 4 E5 protein

Bovine papillomavirus type 4 (BPV-4) E5 (formerly E8) is a 42-residue hydrophobic, membrane-localised protein that can transform NIH-3T3 cells by a poorly defined mechanism. In E5-expressing cells, the observed up-regulation of cyclin A is underpinned by transactivation of the cyclin A promoter. Here we show that E5 transactivates the minimal cell cycle-regulated cyclin A promoter in cells both stably and acutely expressing the viral protein. There are no detectable differences between control and E5 cells in protein complexes binding the E2F-like cell cycle-dependent element (CDE)/cell cycle-regulated element (CCRE) of the cyclin A promoter and E5 does not transactivate E2F reporter plasmids in an E2F-dependent manner in vivo. CCAAT box integrity and functional NF-Y complexes are required for E5-mediated transactivation and a M r ∼110 K CCAAT-box binding factor (p110 CBF) associates with NF-YA only in E5 cells. This suggests that E5 sets the extent of cyclin A promoter activation by a mechanism similar to other, structurally unrelated, DNA tumour virus oncoproteins but distinct from the action of serum factors and so is inconsistent with E5 acting through constitutive activation of tyrosine kinase growth factor receptors.

Global Gene Expression Profiling of End-Stage Dilated Cardiomyopathy Using a Human Cardiovascular-Based cDNA Microarray

To obtain a genomic portrait of heart failure derived from end-stage dilated cardiomyopathy (DCM), we explored expression analysis using the CardioChip, a nonredundant 10,848-element human cardiovascular-based expressed sequence tag glass slide cDNA microarray constructed in-house. RNA was extracted from the left ventricular free wall of seven patients undergoing transplantation, and five nonfailing heart samples. Cy3- and Cy5-labeled (and reverse dye-labeled) cDNA probes were synthesized from individual diseased or nonfailing adult heart RNA, and hybridized to the array. More than 100 transcripts were consistently differentially expressed in DCM >1.5-fold (versus pooled nonfailing heart, P < 0.05). Atrial natriuretic peptide was found to be up-regulated in DCM (19-fold compared to nonfailing, P < 0.05), as well as numerous sarcomeric and cytoskeletal proteins (eg, cardiac troponin, tropomyosin), stress response proteins (eg, HSP 40, HSP 70), and transcription/translation regulators (eg, CCAAT box binding factor, eIF-1AY). Down-regulation was most prominently observed with cell-signaling channels and mediators, particularly those involved in Ca(2+) pathways (Ca(2+)/calmodulin-dependent kinase, inositol 1,4,5-trisphosphate receptor, SERCA). Most intriguing was the co-expression of several novel, cardiac-enriched expressed sequence tags. Quantitative real-time reverse transcriptase-polymerase chain reaction of a selection of these clones verified expression. Our study provides a preliminary molecular profile of DCM using the largest human heart-specific cDNA microarray to date.