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Natto, a traditional Japanese fermented soybean food, is well known to be nutritious and beneficial for health. In this study, we examined whether natto impairs infection by viruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as well as bovine herpesvirus 1 (BHV-1). Interestingly, our results show that both SARS-CoV-2 and BHV-1 treated with a natto extract were fully inhibited infection to the cells. We also found that the glycoprotein D of BHV-1 was shown to be degraded by Western blot analysis and that a recombinant SARS-CoV-2 receptor-binding domain (RBD) was proteolytically degraded when incubated with the natto extract. In addition, RBD protein carrying a point mutation (UK variant N501Y) was also degraded by the natto extract. When the natto extract was heated at 100 °C for 10 min, the ability of both SARS-CoV-2 and BHV-1 to infect to the cells was restored. Consistent with the results of the heat inactivation, a serine protease inhibitor inhibited anti-BHV-1 activity caused by the natto extract. Thus, our findings provide the first evidence that the natto extract contains a protease(s) that inhibits viral infection through the proteolysis of the viral proteins.

C-terminal binding protein (CtBP) is an evolutionarily and functionally conserved transcriptional corepressor known to integrate diverse signals to regulate transcription. Drosophila CtBP (dCtBP) regulates tissue specification and segmentation during early embryogenesis. Here, we investigated the roles of dCtBP during development of the peripheral nervous system (PNS). Our study includes a detailed quantitative analysis of how altered dCtBP activity affects the formation of adult mechanosensory bristles. We found that dCtBP loss-of-function resulted in a series of phenotypes with the most prevalent being supernumerary bristles. These dCtBP phenotypes are more complex than those caused by Hairless, a known dCtBP-interacting factor that regulates bristle formation. The emergence of additional bristles correlated with the appearance of extra sensory organ precursor (SOP) cells in earlier stages, suggesting that dCtBP may directly or indirectly inhibit SOP cell fates. We also found that development of a subset of bristles was regulated by dCtBP associated with U-shaped through the PxDLS dCtBP-interacting motif. Furthermore, the double bristle with sockets phenotype induced by dCtBP mutations suggests the involvement of this corepressor in additional molecular pathways independent of both Hairless and U-shaped. We therefore propose that dCtBP is part of a gene circuitry that controls the patterning and differentiation of the fly PNS via multiple mechanisms.

The Drosophila Snail protein is a transcriptional repressor that is necessary for mesoderm formation. Here, we identify the Ebi protein as an essential Snail co-repressor. In ebi mutant embryos, Snail target genes are derepressed in the presumptive mesoderm. Ebi and Snail interact both genetically and physically. We identify a Snail domain that is sufficient for Ebi binding, and which functions independently of another Snail co-repressor, Drosophila CtBP. This Ebi interaction domain is conserved among all insect Snail-related proteins, is a potent repression domain and is required for Snail function in transgenic embryos. In mammalian cells, the Ebi homologue TBL1 is part of the NCoR/SMRT-HDAC3 (histone deacetylase 3) co-repressor complex. We found that Ebi interacts with Drosophila HDAC3, and that HDAC3 knockdown or addition of a HDAC inhibitor impairs Snail-mediated repression in cells. In the early embryo, Ebi is recruited to a Snail target gene in a Snail-dependent manner, which coincides with histone hypoacetylation. Our results demonstrate that Snail requires the combined activities of Ebi and CtBP, and indicate that histone deacetylation is a repression mechanism in early Drosophila development.

The CBP protein is a transcriptional co-activator and histone acetyltransferase. Reduced expression of Drosophila CBP (dCBP) in the early embryo specifically impairs signaling by the TGF-β molecules Dpp and Screw (Scw). This occurs by a failure to activate transcription of the tolloid ( tld ) gene, which codes for a protease that generates active Dpp and Scw ligands. We show that dCBP directly regulates this gene by binding to the tld enhancer, and that tld expression can be partially rescued with a dCBP transgene. At a slightly later stage of development, Dpp/Scw signaling recovers in mutant embryos, but is unable to turn on expression of the Dpp/Scw-target gene rhomboid ( rho ). Interestingly, an acetyltransferase (AT)-defective dCBP transgene rescued tld and rho gene expression to an extent comparable to the wild-type transgene, whereas a transgene containing a 130 amino acid deletion rescued tld but not late rho expression. A tracheal phenotype caused by the reduced dCBP levels was also rescued more efficiently with the wild-type dCBP transgene than with this mutant transgene. Our results indicate that separate parts of the dCBP protein are required on different promoters, and that the AT activity of dCBP is dispensable for certain aspects of Dpp signaling. We discuss the similarity of these results to the role of p300/CBP in TGF-β signaling in the mouse.

A main challenge of modern biology is to understand how specific constellations of genes are activated to differentiate cells and give rise to distinct tissues. This study focuses on elucidating how gene expression is initiated in the notochord, an axial structure that provides support and patterning signals to embryos of humans and all other chordates. Although numerous notochord genes have been identified, the regulatory DNAs that orchestrate development and propel evolution of this structure by eliciting notochord gene expression remain mostly uncharted, and the information on their configuration and recurrence is still quite fragmentary. Here we used the simple chordate Ciona for a systematic analysis of notochord cis-regulatory modules (CRMs), and investigated their composition, architectural constraints, predictive ability and evolutionary conservation. We found that most Ciona notochord CRMs relied upon variable combinations of binding sites for the transcription factors Brachyury and/or Foxa2, which can act either synergistically or independently from one another. Notably, one of these CRMs contains a Brachyury binding site juxtaposed to an (AC) microsatellite, an unusual arrangement also found in Brachyury-bound regulatory regions in mouse. In contrast, different subsets of CRMs relied upon binding sites for transcription factors of widely diverse families. Surprisingly, we found that neither intra-genomic nor interspecific conservation of binding sites were reliably predictive hallmarks of notochord CRMs. We propose that rather than obeying a rigid sequence-based cis-regulatory code, most notochord CRMs are rather unique. Yet, this study uncovered essential elements recurrently used by divergent chordates as basic building blocks for notochord CRMs., Author Summary Transcription factors control the spatial and temporal expression of a multitude of genes by binding their cis-regulatory modules (CRMs). In this study, we investigated the architecture and composition of CRMs that direct gene expression in the notochord, a structure necessary for the support and patterning of the embryonic body plan of all chordates. We used the simple chordate Ciona to carry out a comparative study of notochord CRMs and we identified the sequences necessary for their function. These sequences, in turn, highlighted the existence of multiple mechanisms that enable gene expression in the notochord. Surprisingly, combinations of binding sites identical to those found in active CRMs were not necessarily able to direct notochord gene expression and were often poorly conserved among cogener species. These results challenge the concept of a notochord-specific cis-regulatory “code”, and outline the limitations of methods for CRM identification that rely upon interspecific conservation of non-coding sequences. Nevertheless, a broad comparison of the structure of the Ciona CRMs with that of the notochord CRMs characterized thus far from all chordates outlines the existence of essential evolutionarily conserved building blocks, such as binding sites for the transcription factors Brachyury and Foxa2, that are shared by subsets of these regulatory modules.

A major challenge in interpreting genome sequences is understanding how the genome encodes the information that specifies when and where a gene will be expressed. The first step in this process is the identification of regions of the genome that contain regulatory information. In higher eukaryotes, this cis-regulatory information is organized into modular units [cis-regulatory modules (CRMs)] of a few hundred base pairs. A common feature of these cis-regulatory modules is the presence of multiple binding sites for multiple transcription factors. Here, we evaluate the extent to which the tendency for transcription factor binding sites to be clustered can be used as the basis for the computational identification of cis-regulatory modules. By using published DNA binding specificity data for five transcription factors active in the early Drosophila embryo, we identified genomic regions containing unusually high concentrations of predicted binding sites for these factors. A significant fraction of these binding site clusters overlap known CRMs that are regulated by these factors. In addition, many of the remaining clusters are adjacent to genes expressed in a pattern characteristic of genes regulated by these factors. We tested one of the newly identified clusters, mapping upstream of the gap gene giant (gt) , and show that it acts as an enhancer that recapitulates the posterior expression pattern of gt.

The coordination of growth with nutritional status is essential for proper development and physiology. Nutritional information is mostly perceived by peripheral organs before being relayed to the brain, which modulates physiological responses. Hormonal signaling ensures this organ-to-organ communication, and the failure of endocrine regulation in humans can cause diseases including obesity and diabetes. In Drosophila melanogaster, the fat body (adipose tissue) has been suggested to play an important role in coupling growth with nutritional status. Here, we show that the peripheral tissue-derived peptide hormone CCHamide-2 (CCHa2) acts as a nutrient-dependent regulator of Drosophila insulin-like peptides (Dilps). A BAC-based transgenic reporter revealed strong expression of CCHa2 receptor (CCHa2-R) in insulin-producing cells (IPCs) in the brain. Calcium imaging of brain explants and IPC-specific CCHa2-R knockdown demonstrated that peripheral-tissue derived CCHa2 directly activates IPCs. Interestingly, genetic disruption of either CCHa2 or CCHa2-R caused almost identical defects in larval growth and developmental timing. Consistent with these phenotypes, the expression of dilp5, and the release of both Dilp2 and Dilp5, were severely reduced. Furthermore, transcription of CCHa2 is altered in response to nutritional levels, particularly of glucose. These findings demonstrate that CCHa2 and CCHa2-R form a direct link between peripheral tissues and the brain, and that this pathway is essential for the coordination of systemic growth with nutritional availability. A mammalian homologue of CCHa2-R, Bombesin receptor subtype-3 (Brs3), is an orphan receptor that is expressed in the islet β-cells; however, the role of Brs3 in insulin regulation remains elusive. Our genetic approach in Drosophila melanogaster provides the first evidence, to our knowledge, that bombesin receptor signaling with its endogenous ligand promotes insulin production., Author Summary Animals need to couple growth with nutritional availability for proper development and physiology, which leads to better survival. Nutritional information is mostly perceived by peripheral organs, particularly metabolic organs such as adipose tissue and gut, before being relayed to the brain, which modulates physiological responses. Hormonal signaling ensures this organ-to-organ communication, and defects in this endocrine regulation in humans often cause diseases including obesity and diabetes. In the fruit fly Drosophila melanogaster, adipose tissue (the “fat body”) has been suggested to play an important role in coordinating growth with metabolism. Here, we show that the Drosophila CCHamide-2 (CCHa2) gene, expressed in the fat body and gut, encodes a nutrient-sensitive peptide hormone. The CCHa2 peptide signals to neuroendocrine cells in the brain that produce Drosophila insulin-like peptides (Dilps) through its receptor (CCHa2-R) and promotes the production of Dilps. Mutants of both CCHa2 and CCHa2-R display severe growth retardation during larval stages. These results suggest that CCHa2 and CCHa2-R functionally connect peripheral tissues with the brain, and that CCHa2/CCHa2-R signaling coordinates the animal’s growth with its nutritional conditions by regulating its production of insulin-like peptides.

There are at least three short-range gap repressors in the precellular Drosophila embryo: Krüppel, Knirps, and Giant. Krüppel and Knirps contain related repression motifs, PxDLSxH and PxDLSxK, respectively, which mediate interactions with the dCtBP corepressor protein. Here, we present evidence that Giant might also interact with dCtBP. The misexpression of Giant in ventral regions of transgenic embryos results in the selective repression of eve stripe 5. A stripe5-lacZ transgene exhibits an abnormal staining pattern in dCtBP mutants that is consistent with attenuated repression by Giant. The analysis of Gal4-Giant fusion proteins identified a minimal repression domain that contains a sequence motif, VLDLS, which is conserved in at least two other sequence-specific repressors. Removal of this sequence from the native Giant protein does not impair its repression activity in transgenic embryos. We propose that Giant-dCtBP interactions might be indirect and mediated by an unknown bZIP subunit that forms a heteromeric complex with Giant. We also suggest that the VLDLS motif recruits an as yet unidentified corepressor protein.

We previously identified various upstream and downstream regulatory elements and factors important for hepatic expression of the human angiotensinogen (ANG) gene, the precursor of vasoactive octapeptide angiotensin II. In the present study, to further investigate the molecular mechanism of human ANG transcriptional regulation, we generated transgenic mice carrying the fusion gene composed of the 1. 3-kilobase promoter of the human ANG gene, its downstream enhancer, and the chloramphenicol acetyltransferase reporter gene. Because expression of the chloramphenicol acetyltransferase gene was observed strongly in the liver and weakly in the kidney, we suspected that hepatocyte nuclear factor (HNF) 4 with a tissue expression pattern similar to that of the reporter gene would regulate ANG transcription. In vitro assays indicated that HNF4 bound to the promoter elements and strongly activated the ANG transcription, but that chicken ovalbumin upstream promoter transcription factor (COUP-TF), a transcriptional repressor, dramatically repressed human ANG transcription through the promoter elements and the downstream enhancer core elements. Furthermore, COUP-TF dramatically decreased the human ANG transcription in the mouse liver by the Helios Gene Gun system in vivo. These results suggest that an interplay between HNF4 and COUP-TF could be important in hepatic human ANG transcription.

Small differences in the levels of an extracellular signaling molecule can specify cell fate during development. Threshold responses are often determined at the level of transcription. Cell-specific and spatially localized patterns of gene expression depend on combinations of sequence-specific activators and repressors that bind to extensive cis-regulatory regions. Different mechanisms for integrating this complex regulatory information are discussed, particularly the role of coregulatory proteins, which are recruited to the DNA template by sequence-specific transcription factors. Recent studies suggest that a growing set of coactivators and corepressors mediate communication between diverse upstream regulatory proteins and the core RNA polymerase II transcription complex.

The pre-cellular Drosophila embryo contains 10 well characterized sequence-specific transcriptional repressors, which represent a broad spectrum of DNA-binding proteins. Previous studies have shown that two of the repressors, Hairy and Dorsal, recruit a common co-repressor protein, Groucho. Here we present evidence that three different repressors, Knirps, Krüppel and Snail, recruit a different co-repressor, dCtBP. Mutant embryos containing diminished levels of maternal dCtBP products exhibit both segmentation and dorsoventral patterning defects, which can be attributed to loss of Krüppel, Knirps and Snail activity. In contrast, the Dorsal and Hairy repressors retain at least some activity in dCtBP mutant embryos. dCtBP interacts with Krüppel, Knirps and Snail through a related sequence motif, PXDLSXK/H. This motif is essential for the repression activity of these proteins in transgenic embryos. We propose that dCtBP represents a major form of transcriptional repression in development, and that the Groucho and dCtBP co-repressors mediate separate pathways of repression.

Human CtBP attenuates transcriptional activation and tumorigenesis mediated by the adenovirus E1A protein. The E1A sequence motif that interacts with CtBP, Pro-X-Asp-Leu-Ser-X-Lys (P-DLS-K), is present in the repression domains of two unrelated short-range repressors in Drosophila , Knirps and Snail, and is essential for the interaction of these proteins with Drosophila CtBP (dCtBP). A P-element–induced mutation in dCtBP exhibits gene-dosage interactions with a null mutation in knirps , which is consistent with the occurrence of Knirps-dCtBP interactions in vivo. These observations suggest that CtBP and dCtBP are engaged in an evolutionarily conserved mechanism of transcriptional repression, which is used in both Drosophila and mammals.

An alginate lyase was purified from an extracellular enzyme (commercial preparation) of Flavobacterium multivolum K-11 by successive column chromatographies, such as cation exchange, chromatofocusing, and gel filtration. The purified enzyme migrated as a single band on SDS-PAGE and analytical isoelectric focusing. The molecular weight of the enzyme was 32,000 by SDS-PAGE and 33,000 by HPLC gel filtration chromatography, and the pI of the enzyme was 8.2 on isoelectric focusing. The enzyme exhibited maximum activity at pH 7.5 and 40°C, and was stable between pH 6.0 and 9.0, and at temperatures up to 20°C. The enzyme activity was remarkably inhibited by chemical compounds such as SDS, MIA, TNBS, and N-bromosuccinimide, while EDTA and PCMB had no effect on the enzyme activity. The enzyme decomposed both the G-block (guluronic acid content; 89%) and the M-block (mannuronic content; 92%) at nearly equal rates, and produced several kinds of unsaturated oligomers. Because such activity of alginate lyase has not been reported, we believe that this is a novel alginate lyase.

An alginate lyase was purified from a crude enzyme of Flavobacterium multivolum K-11 by successive column chromatographies, such as cation exchange, chromatofocusing, and gel filtration. The enzyme thus obtained migrated as a single band on SDS-PAGE. The relative molecular mass of the enzyme was 43-kDa by SDS-PAGE and 41-kDa by HPLC gel filtration chromatography. The isoelectric point of the enzyme was 8.7. The enzyme exhibited maximum activity at pH 8.0 and 40°C, and was stable in the pH range of 6.0 to 9.0 and at temperatures up to 30°C. The enzyme activity was remarkably inhibited by chemical compounds such as EDTA, PCMB, MIA, TNBS, and N-bromosuccinimide. The enzyme was specific for poly-guluronate and produced several kinds of oligomers. Thus, the results suggested that the enzyme was classified as endo poly (α-L-guluronate) lyase (EC 4.2.2.11).

During notochord formation in chordate embryos, the transcription factor Brachyury employs different regulatory strategies to ensure the sequential activation of downstream genes and thereby the deployment of a specific developmental program at the right time and place., The appearance of the notochord represented a milestone in Deuterostome evolution. The notochord is necessary for the development of the chordate body plan and for the formation of the vertebral column and numerous organs. It is known that the transcription factor Brachyury is required for notochord formation in all chordates, and that it controls transcription of a large number of target genes. However, studies of the structure of the cis-regulatory modules (CRMs) through which this control is exerted are complicated in vertebrates by the genomic complexity and the pan-mesodermal expression territory of Brachyury. We used the ascidian Ciona, in which the single-copy Brachyury is notochord-specific and CRMs are easily identifiable, to carry out a systematic characterization of Brachyury-downstream notochord CRMs. We found that Ciona Brachyury (Ci-Bra) controls most of its targets directly, through non-palindromic binding sites that function either synergistically or individually to activate early- and middle-onset genes, respectively, while late-onset target CRMs are controlled indirectly, via transcriptional intermediaries. These results illustrate how a transcriptional regulator can efficiently shape a shallow gene regulatory network into a multi-tiered transcriptional output, and provide insights into the mechanisms that establish temporal read-outs of gene expression in a fast-developing chordate embryo., Author Summary Transcription factors control where and when gene expression is switched on by binding to specific stretches of DNA known as cis-regulatory modules (CRMs). In this study, we investigated the architecture and composition of CRMs that direct gene expression in the notochord—a transient rod-like structure found in all embryos that belong to the phylum chordata, which includes humans. Here we used the sea squirt Ciona, a simple chordate, and analyzed how the transcription factor Brachyury ensures the appropriate deployment of its target genes at specific times during the sequential steps of notochord formation. We compared CRMs found in different notochord genes downstream of Brachyury, expecting to find genes associated with greater numbers of Brachyury binding sites to be expressed at higher levels. To our surprise, we found instead that a higher number of functional Brachyury binding sites is typical of CRMs associated with genes that are expressed early in notochord development, while single-site CRMs are characteristic of genes that are turned on during the intermediate stages of this process. Finally, CRMs associated with genes expressed late in notochord development do not contain functional Brachyury binding sites but are controlled by Brachyury indirectly, through the action of intermediary transcription factors. These differences explain how a transcription factor that is present at all stages in a certain cell type can generate a sequential transcriptional output of gene expression.

T-box genes are potent regulators of mesoderm development in many metazoans. In chordate embryos, the T-box transcription factor Brachyury (Bra) is required for specification and differentiation of the notochord. In some chordates, including the ascidian Ciona, members of the Tbx2 subfamily of T-box genes are also expressed in this tissue; however, their regulatory relationships with Bra and their contributions to the development of the notochord remain uncharacterized. We determined that the notochord expression of Ciona Tbx2/3 (Ci-Tbx2/3) requires Ci-Bra, and identified a Ci-Tbx2/3 notochord CRM that necessitates multiple Ci-Bra binding sites for its activity. Expression of mutant forms of Ci-Tbx2/3 in the developing notochord revealed a role for this transcription factor primarily in convergent extension. Through microarray screens, we uncovered numerous Ci-Tbx2/3 targets, some of which overlap with known Ci-Bra-downstream notochord genes. Among the Ci-Tbx2/3 notochord targets are evolutionarily conserved genes, including caspases, lineage-specific genes, such as Noto4, and newly identified genes, such as MLKL. This work sheds light on a large section of the notochord regulatory circuitry controlled by T-box factors, and reveals new components of the complement of genes required for the proper formation of this structure.

Chordoma is a rare, but often malignant, bone cancer that preferentially affects the axial skeleton and the skull base. These tumors are both sporadic and hereditary and appear to occur more frequently after the fourth decade of life; however, modern technologies have increased the detection of pediatric chordomas. Chordomas originate from remnants of the notochord, the main embryonic axial structure that precedes the backbone, and share with notochord cells both histological features and the expression of characteristic genes. One such gene isBrachyury, which encodes for a sequence-specific transcription factor. Known for decades as a main regulator of notochord formation,Brachyuryhas recently gained interest as a biomarker and causative agent of chordoma, and therefore as a promising therapeutic target. Here, we review the main characteristics of chordoma, the molecular markers, and the clinical approaches currently available for the early detection and possible treatment of this cancer. In particular, we report on the current knowledge of the role ofBrachyuryand of its possible mechanisms of action in both notochord formation and chordoma etiogenesis.

On the basis of cDNA cloning, primer extension, and transfection experiments, we identified a novel 5′ exon of the human activin βA subunit gene, and found its enhancer and promoter regions as well as multiple transcription start sites. A series of deletion and mutation analyses of the enhancer sequences defined the 45-base pair core region (DR-1 core) containing two short elements with similarity to AP-1 (12-O-tetradecanoylphorbol-13-acetate response element; TRE) and CREB/ATF (cyclic AMP response element; CRE) binding sites, both of which were necessary for full enhancer activity. Gel shift and antibody supershift assays using DR-1 core region revealed the formation of two specific DNA-protein complexes, one of which could be partially dissociated by a competing oligonucleotide containing a single copy of the consensus TRE, but the other of which contained neither CREB/ATF nor AP-1 as major components. Although 12-O-tetradecanoylphorbol-13-acetate and cAMP induced the activin enhancer/promoter-driven CAT activity, such drug induction was obscured when either the TRE- or CRE-like elements were mutated in the native promoter context. Our results demonstrate that the promoter and enhancer regions identified here are essential for maintaining the efficient promoter activity of the human activin βA subunit gene.

Chromatin immunoprecipitation (ChIP) assays allow the efficient characterization of the in vivo occupancy of genomic regions by DNA-binding proteins and thus facilitate the prediction of cis-regulatory sequences in silico and guide their validation in vivo. For these reasons, these assays and their permutations (e.g., ChIP-on-chip and ChIP-sequencing) are currently being extended to several non-mainstream model organisms, as the availability of specific antibodies increases. Here, we describe the development of a polyclonal antibody against the Brachyury protein of the marine invertebrate chordate Ciona intestinalis and provide a detailed ChIP protocol that should be easily adaptable to other marine organisms.

Transcriptional repression is essential for patterning gene expression in the early Drosophila embryo. Biochemical and genetic studies on Drosophila C-terminal binding protein (dCtBP) have provided solid evidence that dCtBP acts as a corepressor for several transcriptional repressors. Similarly to mammalian CtBPs, dCtBP interacts with a short peptide motif, PxDLS, or related motifs. It appears that dCtBP is essential for short-range transcriptional repression in the early embryo. In contrast, it has been recendy reported that dCtBP participates in Polycomb-mediated long-range repression. In this chapter, we will review how the dCtBP corepressor functions, from the biochemical, developmental, and genetic point of views.

A harmonious balance between transcriptional activation and repression in eukaryotes is necessary for a variety of biological phenomena, such as pattern formation, tissue differentiation, and normal development. In this chapter, we will use well-understood cases to provide an overview of the molecular mechanisms by which transcription factors mediate repression.

ANGUSTIFOLIA (AN) controls leaf morphology in the plant Arabidopsis thaliana. Previous studies on sequence similarity demonstrated that the closest proteins to AN are members of animal C-terminal-binding proteins (CtBPs) found in nematodes, arthropods, and vertebrates. Drosophila CtBP (dCtBP) functions as a transcriptional corepressor for deoxyribonucleic acid (DNA)-binding repressors containing the short amino acid motif, PXDLS, to regulate tissue specification and segmentation during early embryogenesis. It has previously been shown that AN was thought to repress transcription similar to the function of CtBPs; however, AN lacks some of the structural features that are conserved in animal CtBPs. In this paper, we examined whether AN is functionally related to dCtBP. Firstly, we re-examined sequence similarity among AN and various CtBPs from several representative species in the plant and animal kingdoms. Secondly, yeast two-hybrid assays demonstrated that AN failed to interact with an authentic CtBP-interacting factor, adenovirus E1A oncoprotein bearing the PXDLS motif. Thirdly, AN tethered to DNA was unable to repress the expression of reporter genes in transgenic Drosophila embryos. Fourthly, overexpression assays suggested that dCtBP and AN function differently in Drosophila tissues. Finally, AN failed to rescue the zygotic lethality caused by dCtBP loss-of-function. These data, taken together, suggest that AN is functionally distinct from dCtBP. Likely, ancestral CtBPs acquired corepressor function (capability of both repression and binding to repressors containing the PXDLS motif) after the animal–plant divergence but before the protostome–deuterostome split. We therefore propose to categorize AN as a subfamily member within the CtBP/BARS/RIBEYE/AN superfamily.

We previously identified a regulatory element at the 3'-downstream region of the human angiotensinogen (hANG) gene. Using this element as a probe by the Southwestern screening, we isolated a cDNA clone, encoding Finb, a transcriptional activator with multiple zinc finger domains. The N-terminal zinc finger domain of Finb bound to the GGATGG sequence within the regulatory element. Unexpectedly, Finb repressed transcription dependent on the regulatory element. Inspection of the 5'-flanking region in the hANG promoter identified the GGATGG-like elements, which prompted us to examine the effect of Finb on the hANG promoter activity. We also found the two Finb binding elements in the 5'-flanking region of the hANG gene by the gel shift assay, both of which were necessary for transcriptional repression of the hANG promoter. These findings suggest that Finb functions as a sequence-specific transcriptional repressor of the hANG gene.

There are three mechanisms of transcriptional repression in eukaryotes. The first is quenching, whereby repressors and activators co-occupy closely linked sites and then the repressor inhibits adjacent activators. The second is direct repression, in which repressors block the function of the core transcription complex. The third is competition, in which repressors compete with activators for a common DNA-binding site. Previous studies have shown that the Drosophila melanogaster CtBP corepressor (dCtBP) is essential for the quenching activity of three short-range sequence-specific repressors in the early Drosophila embryo: Kruppel, Knirps, and Snail. Here we demonstrate that dCtBP is dispensable for target enhancers that contain overlapping activator and repressor binding sites. However, it is essential when Kruppel and Knirps repressor sites do not overlap activator sites but are instead located adjacent to either activators or the core promoter. These findings provide evidence that competition is distinct from quenching and direct repression. Quenching and direct repression depend on dCtBP, whereas competition does not.

Previous studies have identified two corepressors in the early Drosophila embryo: Groucho and dCtBP. Both proteins are recruited to the DNA template by interacting with short peptide motifs conserved in a variety of sequence‐specific transcriptional repressors. Once bound to DNA, Groucho appears to mediate long‐range repression, while dCtBP directs short‐range repression. The short‐range Kruppel repressor was converted into a long‐range repressor by replacing the dCtBP interaction motif (PxDLSxH) with a Groucho motif (WRPW). The resulting chimeric repressor causes a different mutant phenotype from that of the native Kruppel protein when misexpressed in transgenic embryos. The different patterning activities can be explained on the basis of long‐range silencing within the hairy 5′ regulatory region. The analysis of a variety of synthetic transgenes provides evidence that Groucho‐dependent long‐range repressors do not always cause the dominant silencing of linked enhancers within a complex cis ‐regulatory region. We suggest a ‘hot chromatin’ model, whereby repressors require activators to bind DNA.

The promoter of the human angiotensinogen (hAG) gene functioned in its own core promoter context but not when replaced with simian virus 40 (SV40) core promoter, suggesting the presence of a transcriptionally important cis-acting sequence. Electrophoretic mobility shift assays demonstrated that a ubiquitously expressed nuclear factor, AGCF1, bound to AGCE1 (hore promoter lement 1; positions −25 to −1) located between the TATA box and transcription initiation site. Substitution mutation in AGCE1 which disrupted AGCF1 binding affected the promoter activity more severely than a nonsense mutation of the hAG TATA sequences did. When AGCE1 was placed at the downstream of SV40 core promoter, the responsiveness to hAG upstream region was significantly restored. Furthermore, mutation and in vivo competition experiments suggested that AGCF1 acts as a critical regulator of hAG transcription by mediating the activity of the hAG upstream and downstream enhancer elements. DNase I footprinting and UV cross-linking analyses showed that AGCF1 with apparent molecular masses of 31, 33, and 43 kDa as the components protected the region from −26 to −9 which partially overlapped with the TATA box consensus sequences. These findings indicate that AGCE1 in addition to the TATA box plays a key role in mediating the hAG regulatory elements.

We have cloned the rat angiotensin II receptor type 2 (AT2) gene, whose physiological function remains unclear. Sequence analysis indicated that exons 1 and 2 exist in the 5′-untranslated region and the initiation codon ATG is located in exon 3. The 1.6-kb genomic fragment at positions − 1567 to + 26 relative to the putative transcription start site was found to contain a functional promoter region using transient chloramphenicol acetyltransferase assay. This is the first report demonstrating the nucleotide sequence of the promoter region of this gene.

An alginate lyase from the culture supernatant of Enterobacter cloacae M-1 was purified by ammonium sulfate precipitation, cation-exchange chromatography (SP-Toyopearl), and gel filtration (Ultrogel AcA44). The final preparation thus obtained showed a single band on SDS-PAGE. The purified enzyme had the molecular weight of 38,000 and 32,000 by SDS-PAGE and gel filtration, respectively. The pI of the enzyme was 8.9. The optimum pH and temperature for the enzyme reaction were around 7.8 and 30 degrees C, respectively. The enzyme was unstable on heating. EDTA completely inhibited the enzyme activity, but the activity was completely restored by the treatment with CaCl2. The enzyme was specific for poly-guluronate and produced several kinds of unsaturated oligomers from the gluluronate. This suggested that the enzyme could be classified as an endo poly-guluronate lyase.

The activities of developmentally critical transcription factors are regulated via interactions with cofactors. Such interactions influence transcription factor activity either directly through protein–protein interactions or indirectly by altering the local chromatin environment. Using a yeast double-interaction screen, we identified a highly conserved nuclear protein, Akirin, as a novel cofactor of the key Drosophila melanogaster mesoderm and muscle transcription factor Twist. We find that Akirin interacts genetically and physically with Twist to facilitate expression of some, but not all, Twist-regulated genes during embryonic myogenesis. akirin mutant embryos have muscle defects consistent with altered regulation of a subset of Twist-regulated genes. To regulate transcription, Akirin colocalizes and genetically interacts with subunits of the Brahma SWI/SNF-class chromatin remodeling complex. Our results suggest that, mechanistically, Akirin mediates a novel connection between Twist and a chromatin remodeling complex to facilitate changes in the chromatin environment, leading to the optimal expression of some Twist-regulated genes during Drosophila myogenesis. We propose that this Akirin-mediated link between transcription factors and the Brahma complex represents a novel paradigm for providing tissue and target specificity for transcription factor interactions with the chromatin remodeling machinery., Author Summary The proper development of the diverse array of cell types in an organism depends upon the induction and repression of specific genes at particular times and places. This gene regulation requires both the activity of tissue-specific transcriptional regulators and the modulation of the chromatin environment. To date, a complete picture of the interplay between these two processes remains unclear. To address this, we examined the activity of the evolutionarily conserved transcription factor Twist during embryogenesis of Drosophila melanogaster. While Twist has multiple activities and roles during development, a direct link between Twist and chromatin remodeling is unknown. We identified a highly conserved protein, Akirin, as a link between Twist and chromatin remodeling factors. Akirin is required for optimal expression of a Twist-dependent target during muscle development via interactions with the Drosophila SWI/SNF chromatin remodeling complex. Interestingly, Akirin is not required for activation of all Twist-dependent enhancers, suggesting that Akirin refines Twist activity outputs and that different Twist-dependent targets have different requirements for chromatin remodeling during development. Our data further suggests that Akirin similarly links the SWI/SNF chromatin remodeling complex with other transcription factors during development. This work has important ramifications for understanding both normal development and diseases such as cancer.

We have recently characterized a cell type-dependent downstream enhancer that is contained within an 832-bp B2 sequences spanning the exon 5 and 3′-flanking region of the human angiotensinogen gene and have localized one of core elements to a 24-bp region in the 3′-flanking region. In the present study, we functionally dissected the 5′-half of B2 region and identified an additional cell type-dependent enhancer core element composed of the 80-bp sequences (+1399 to +1478) in the exon 5. Electrophoretic mobility shift assays demonstrated that the element interacts with two HepG2 cell-specific and several ubiquitous nuclear factors. These results, together with our previous results, suggest that the 80-bp exon 5 enhancer core element as well as 24-bp region plays an important role in the downstream enhancer effect.

We previously demonstrated that the 3.8-kilobase DNA fragment containing exons 4 and 5 of the human angiotensinogen (hAG) gene enhances the expression of chloramphenicol acetyltransferase gene, under control of the hAG promoter, in human hepatoma HepG2 cells. In the present study, to define regulatory elements of the hAG gene, we have functionally dissected this downstream region and localized a cell type-dependent enhancer to the 832-base pair sequence containing the exon 5 and 3'-flanking region. Further deletion analyses revealed that the 24-base pair core DNA fragment present in the 3'-flanking region was responsible for this enhancement. Electrophoretic mobility shift assay demonstrated that the 3'-enhancer core element interacts specifically with two nuclear factors from the HepG2 cells, one of which is an uncharacterized factor (human angiotensinogen enhancer factor-1: hAEF-1), the other is an AP-3-related factor. Mutation analyses indicated that the disruption of hAEF-1 binding alone completely impaired the enhancer activity of the core element. These results suggested that the downstream enhancer core element interacting with hAEF-1 plays an important role in activating the angiotensinogen promoter in a cell type-dependent manner.