Conserved interaction between distinct Krüppel-associated box domains and the transcriptional intermediary factor 1 β

The Krüppel-associated box (KRAB) domain, originally identified as a 75-aa sequence present in numerous Krüppel-type zinc-finger proteins, is a potent DNA-binding-dependent transcriptional repression domain that is believed to function through interaction with the transcriptional intermediary factor 1 (TIF1) β. On the basis of sequence comparison and phylogenetic analysis, we have recently defined three distinct subfamilies of KRAB domains. In the present study, individual members of each subfamily were tested for transcriptional repression and interaction with TIF1β and two other closely related family members (TIF1α and TIF1γ). All KRAB variants were shown, (i) to repress transcription when targeted to DNA through fusion to a heterologous DNA-binding domain in mammalian cells, and (ii) to interact specifically with TIF1β, but not with TIF1α or TIF1γ. Taken together, these results implicate TIF1β as a common transcriptional corepressor for the three distinct subfamilies of KRAB zinc-finger proteins and suggest a high degree of conservation in the molecular mechanism underlying their transcriptional repression activity.

Transcriptional activation of the small GTPase gene rhoB by genotoxic stress is regulated via a CCAAT element

The gene encoding the Ras-related GTPase RhoB-specific is immediate-early inducible by genotoxic treatments. Regulation of transcriptional activation of rhoB is still unclear. Here we show that cells lacking either p53 or c-Fos are not different from wild-type cells with respect to the level of rhoB induction upon UV irradiation, indicating that these transcription factors are not crucial for stimulation of rhoB mRNA expression. Extracts from UV-irradiated and non-irradiated cells revealed similar DNA-binding activities to a 0.17 kb rhoB promoter fragment harboring the functional element(s) necessary for stimulation of rhoB by UV light. By means of immunoprecipitation we found that an ATF-2-specific antibody co-precipitates the 32P-labeled 0.17 kb rhoB fragment, whereas an anti-AP1 antibody did not. Since no consensus sequence for binding of ATF-2 is present within the rhoB promoter, ATF-2 is likely to be associated with another factor that binds to the minimal promoter. Deletion analysis and site-directed mutagenesis of the 0.17 kb rhoB fragment revealed a CCAAT box to be an essential requirement for stimulation of rhoB by UV light and methyl methanesulfonate. Moreover, immunoprecipitation experiments showed that the CCAAT-binding factor NF-YA is complexed with ATF-2. Overall, the data strongly indicate that transcriptional activation of the rhoB gene by genotoxic stress is regulated via a CCAAT box and that interaction of CCAAT-binding factor and ATF-2 triggers the stress-inducible expression of rhoB.

Smad proteins function as co-modulators for MEF2 transcriptional regulatory proteins

An emerging theme in transforming growth factor-β (TGF-β) signalling is the association of the Smad proteins with diverse groups of transcriptional regulatory proteins. Several Smad cofactors have been identified to date but the diversity of TGF-β effects on gene transcription suggests that interactions with other co-regulators must occur. In these studies we addressed the possible interaction of Smad proteins with the myocyte enhancer-binding factor 2 (MEF2) transcriptional regulators. Our studies indicate that Smad2 and 4 (Smad2/4) complexes cooperate with MEF2 regulatory proteins in a GAL4-based one-hybrid reporter gene assay. We have also observed in vivo interactions between Smad2 and MEF2A using co-immunoprecipitation assays. This interaction is confirmed by glutathione S-transferase pull-down analysis. Immunofluorescence studies in C2C12 myotubes show that Smad2 and MEF2A co-localise in the nucleus of multinuclear myotubes during differentiation. Interestingly, phospho-acceptor site mutations of MEF2 that render it unresponsive to p38 MAP kinase signalling abrogate the cooperativity with the Smads suggesting that p38 MAP Kinase-catalysed phosphorylation of MEF2 is a prerequisite for the Smad–MEF2 interaction. Thus, the association between Smad2 and MEF2A may subserve a physical link between TGF-β signalling and a diverse array of genes controlled by the MEF2 cis element.

DNA sequencing and genotyping by transcriptional synthesis of chain-terminated RNA ladders and MALDI-TOF mass spectrometry

Sets of RNA ladders can be synthesized by transcription of a bacteriophage-encoded RNA polymerase using 3′-deoxynucleotides as chain terminators. These ladders can be used for sequencing of DNA. Using a nicked form of phage SP6 RNA polymerase in this study substantially enhanced yields of transcriptional sequencing ladders. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of chain-terminated RNA ladders allowed DNA sequence determination of up to 56 nt. It is also demonstrated that A→G and C→T variations in heterozygous and homozygous samples can be unambiguously identified by the mass spectrometric analysis. As a step towards single-tube sequencing reactions, α-thiotriphosphate nucleotide analogs were used to overcome problems caused by chain terminator-independent, premature termination and by the small mass difference between natural pyrimidine nucleotides.

Analysis of transforming activity of human synovial sarcoma-associated chimeric protein SYT-SSX1 bound to chromatin remodeling factor hBRM/hSNF2α

Human synovial sarcoma has been shown to exclusively harbor the chromosomal translocation t(X;18) that produces the chimeric gene SYT-SSX. However, the role of SYT-SSX in cellular transformation remains unclear. In this study, we have established 3Y1 rat fibroblast cell lines that constitutively express SYT, SSX1, and SYT-SSX1 and found that SYT-SSX1 promoted growth rate in culture, anchorage-independent growth in soft agar, and tumor formation in nude mice. Deletion of the N-terminal 181 amino acids of SYT-SSX1 caused loss of its transforming activity. Furthermore, association of SYT-SSX1 with the chromatin remodeling factor hBRM/hSNF2α, which regulates transcription, was demonstrated in both SYT-SSX1-expressing 3Y1 cells and in the human synovial sarcoma cell line HS-SY-II. The binding region between the two molecules was shown to reside within the N-terminal 181 amino acids stretch (aa 1–181) of SYT-SSX1 and 50 amino acids (aa 156–205) of hBRM/hSNF2α and we found that the overexpression of this binding region of hBRM/hSNF2α significantly suppressed the anchorage-independent growth of SYT-SSX1-expressing 3Y1 cells. To analyze the transcriptional regulation by SYT-SSX1, we established conditional expression system of SYT-SSX1 and examined the gene expression profiles. The down-regulation of potential tumor suppressor DCC was observed among 1,176 genes analyzed by microarray analysis, and semi-quantitative reverse transcription–PCR confirmed this finding. These data clearly demonstrate transforming activity of human oncogene SYT-SSX1 and also involvement of chromatin remodeling factor hBRM/hSNF2α in human cancer.

Extracellular matrix composition determines the transcriptional response to epidermal growth factor receptor activation

The transcriptional response to epidermal growth factor (EGF) was examined in a cultured cell model of adhesion. Gene expression was monitored in human embryonic kidney cells (HEK293) after attachment of cells to the extracellular matrix (ECM) proteins, laminin, and fibronectin, by using complementary DNA micorarrays printed with 1,718 individual human genes. Cluster analysis revealed that the influence of EGF on gene expression, either positive or negative, was largely independent of ECM composition. However, clusters of EGF-regulated genes were identified that were diagnostic of the type of ECM proteins to which cells were attached. In these clusters, attachment of cells to a laminin or fibronectin substrata specifically modified the direction of gene expression changes in response to EGF stimulation. For example, in HEK293 cells attached to fibronectin, EGF stimulated an increase in the expression of some genes; however, genes in the same group were nonresponsive or even suppressed in cells attached to laminin. Many of the genes regulated by EGF and ECM proteins in this manner are involved in ECM and cytoskeletal architecture, protein synthesis, and cell cycle control, indicating that cell responses to EGF stimulation can be dramatically affected by ECM composition.

Influences of aging and caloric restriction on the transcriptional profile of skeletal muscle from rhesus monkeys

In laboratory rodents, caloric restriction (CR) retards several age-dependent physiological and biochemical changes in skeletal muscle, including increased steady-state levels of oxidative damage to lipids, DNA, and proteins. We have previously used high-density oligonucleotide arrays to show that CR can prevent or delay most of the major age-related transcriptional alterations in the gastrocnemius muscle of C57BL/6 mice. Here we report the effects of aging and adult-onset CR on the gene expression profile of 7,070 genes in the vastus lateralis muscle from rhesus monkeys. Gene expression analysis of aged rhesus monkeys (mean age of 26 years) was compared with that of young animals (mean age of 8 years). Aging resulted in a selective up-regulation of transcripts involved in inflammation and oxidative stress, and a down-regulation of genes involved in mitochondrial electron transport and oxidative phosphorylation. Middle-aged monkeys (mean age of 20 years) subjected to CR since early adulthood (mean age of 11 years) were studied to determine the gene expression profile induced by CR. CR resulted in an up-regulation of cytoskeletal protein-encoding genes, and also a decrease in the expression of genes involved in mitochondrial bioenergetics. Surprisingly, we did not observe any evidence for an inhibitory effect of adult-onset CR on age-related changes in gene expression. These results indicate that the induction of an oxidative stress-induced transcriptional response may be a common feature of aging in skeletal muscle of rodents and primates, but the extent to which CR modifies these responses may be species-specific.

Significance analysis of microarrays applied to the ionizing radiation response

Microarrays can measure the expression of thousands of genes to identify changes in expression between different biological states. Methods are needed to determine the significance of these changes while accounting for the enormous number of genes. We describe a method, Significance Analysis of Microarrays (SAM), that assigns a score to each gene on the basis of change in gene expression relative to the standard deviation of repeated measurements. For genes with scores greater than an adjustable threshold, SAM uses permutations of the repeated measurements to estimate the percentage of genes identified by chance, the false discovery rate (FDR). When the transcriptional response of human cells to ionizing radiation was measured by microarrays, SAM identified 34 genes that changed at least 1.5-fold with an estimated FDR of 12%, compared with FDRs of 60 and 84% by using conventional methods of analysis. Of the 34 genes, 19 were involved in cell cycle regulation and 3 in apoptosis. Surprisingly, four nucleotide excision repair genes were induced, suggesting that this repair pathway for UV-damaged DNA might play a previously unrecognized role in repairing DNA damaged by ionizing radiation.

Analysis of gene expression during myc oncogene-induced lymphomagenesis in the bursa of Fabricius

The transcriptional effects of deregulated myc gene overexpression are implicated in tumorigenesis in a spectrum of experimental and naturally occurring neoplasms. In follicles of the chicken bursa of Fabricius, myc induction of B-cell neoplasia requires a target cell population present during early bursal development and progresses through preneoplastic transformed follicles to metastatic lymphomas. We developed a chicken immune system cDNA microarray to analyze broad changes in gene expression that occur during normal embryonic B-cell development and during myc-induced neoplastic transformation in the bursa. The number of mRNAs showing at least 3-fold change was greater during myc-induced lymphomagenesis than during normal development, and hierarchical cluster analysis of expression patterns revealed that levels of several hundred mRNAs varied in concert with levels of myc overexpression. A set of 41 mRNAs were most consistently elevated in myc-overexpressing preneoplastic and neoplastic cells, most involved in processes thought to be subject to regulation by Myc. The mRNAs for another cluster of genes were overexpressed in neoplasia independent of myc expression level, including a small subset with the expression signature of embryonic bursal lymphocytes. Overexpression of myc, and some of the genes overexpressed with myc, may be important for generation of preneoplastic transformed follicles. However, expression profiles of late metastatic tumors showed a large variation in concert with myc expression levels, and some showed minimal myc overexpression. Therefore, high-level myc overexpression may be more important in the early induction of these lymphomas than in maintenance of late-stage metastases.

Genomic analysis of orthologous mouse and human olfactory receptor loci

Olfactory receptor (OR) genes represent ≈1% of genomic coding sequence in mammals, and these genes are clustered on multiple chromosomes in both the mouse and human genomes. We have taken a comparative genomics approach to identify features that may be involved in the dynamic evolution of this gene family and in the transcriptional control that results in a single OR gene expressed per olfactory neuron. We sequenced ≈350 kb of the murine P2 OR cluster and used synteny, gene linkage, and phylogenetic analysis to identify and sequence ≈111 kb of an orthologous cluster in the human genome. In total, 18 mouse and 8 human OR genes were identified, including 7 orthologs that appear to be functional in both species. Noncoding homology is evident between orthologs and generally is confined within the transcriptional unit. We find no evidence for common regulatory features shared among paralogs, and promoter regions generally do not contain strong promoter motifs. We discuss these observations, as well as OR clustering, in the context of evolutionary expansion and transcriptional regulation of OR repertoires.

Comparative Analysis of the Regulation of Expression and Structures of Two Evolutionarily Divergent Genes for Δ1-Pyrroline-5-Carboxylate Synthetase from Tomato1

We isolated two tomato (Lycopersicon esculentum) cDNA clones, tomPRO1 and tomPRO2, specifying Δ1-pyrroline-5-carboxylate synthetase (P5CS), the first enzyme of proline (Pro) biosynthesis. tomPRO1 is unusual because it resembles prokaryotic polycistronic operons (M.G. García-Ríos, T. Fujita, P.C. LaRosa, R.D. Locy, J.M. Clithero, R.A. Bressan, L.N. Csonka [1997] Proc Natl Acad Sci USA 94: 8249–8254), whereas tomPRO2 encodes a full-length P5CS. We analyzed the accumulation of Pro and the tomPRO1 and tomPRO2 messages in response to NaCl stress and developmental signals. Treatment with 200 mm NaCl resulted in a >60-fold increase in Pro levels in roots and leaves. However, there was a <3-fold increase in the accumulation of the tomPRO2 message and no detectable induction in the level of the tomPRO1 message in response to NaCl stress. Although pollen contained approximately 100-fold higher levels of Pro than other plant tissues, there was no detectable increase in the level of either message in pollen. We conclude that transcriptional regulation of these genes for P5CS is probably not important for the osmotic or pollen-specific regulation of Pro synthesis in tomato. Using restriction fragment-length polymorphism mapping, we determined the locations of tomPRO1 and tomPRO2 loci in the tomato nuclear genome. Sequence comparison suggested that tomPRO1 is similar to prokaryotic P5CS loci, whereas tomPRO2 is closely related to other eukaryotic P5CS genes.

Transcriptional Down-Regulation by Abscisic Acid of Pathogenesis-Related β-1,3-Glucanase Genes in Tobacco Cell Cultures1

Class I isoforms of β-1,3-glucanases (βGLU I) and chitinases (CHN I) are antifungal, vacuolar proteins implicated in plant defense. Tobacco (Nicotiana tabacum L.) βGLU I and CHN I usually exhibit tightly coordinated developmental, hormonal, and pathogenesis-related regulation. Both enzymes are induced in cultured cells and tissues of cultivar Havana 425 tobacco by ethylene and are down-regulated by combinations of the growth hormones auxin and cytokinin. We report a novel pattern of βGLU I and CHN I regulation in cultivar Havana 425 tobacco pith-cell suspensions and cultured leaf explants. Abscisic acid (ABA) at a concentration of 10 μm markedly inhibited the induction of βGLU I but not of CHN I. RNA-blot hybridization and immunoblot analysis showed that only class I isoforms of βGLU and CHN are induced in cell culture and that ABA inhibits steady-state βGLU I mRNA accumulation. Comparable inhibition of β-glucuronidase expression by ABA was observed for cells transformed with a tobacco βGLU I gene promoter/β-glucuronidase reporter gene fusion. Taken together, the results strongly suggest that ABA down-regulates transcription of βGLU I genes. This raises the possibility that some of the ABA effects on plant-defense responses might involve βGLU I.

Differential Regulation of Sugar-Sensitive Sucrose Synthases by Hypoxia and Anoxia Indicate Complementary Transcriptional and Posttranscriptional Responses1

The goal of this research was to resolve the hypoxic and anoxic responses of maize (Zea mays) sucrose (Suc) synthases known to differ in their sugar regulation. The two maize Suc synthase genes, Sus1 and Sh1, both respond to sugar and O2, and recent work suggests commonalities between these signaling systems. Maize seedlings (NK508 hybrid, W22 inbred, and an isogenic sh1-null mutant) were exposed to anoxic, hypoxic, and aerobic conditions (0, 3, and 21% O2, respectively), when primary roots had reached approximately 5 cm. One-centimeter tips were excised for analysis during the 48-h treatments. At the mRNA level, Sus1 was rapidly up-regulated by hypoxia (approximately 5-fold in 6 h), whereas anoxia had less effect. In contrast, Sh1 mRNA abundance increased strongly under anoxia (approximately 5-fold in 24 h) and was much less affected by hypoxia. At the enzyme level, total Suc synthase activity rose rapidly under hypoxia but showed little significant change during anoxia. The contributions of SUS1 and SH1 activities to these responses were dissected over time by comparing the sh1-null mutant with the isogenic wild type (Sus+, Sh1+). Sh1-dependent activity contributed most markedly to a rapid protein-level response consistently observed in the first 3 h, and, subsequently, to a long-term change mediated at the level of mRNA accumulation at 48 h. A complementary midterm rise in SUS1 activity varied in duration with genetic background. These data highlight the involvement of distinctly different genes and probable signal mechanisms under hypoxia and anoxia, and together with earlier work, show parallel induction of “feast and famine” Suc synthase genes by hypoxia and anoxia, respectively. In addition, complementary modes of transcriptional and posttranscriptional regulation are implicated by these data, and provide a mechanism for sequential contributions from the Sus1 and Sh1 genes during progressive onset of naturally occurring low-O2 events.

Molecular cloning and sequence analysis of the complestatin biosynthetic gene cluster

Streptomyces lavendulae produces complestatin, a cyclic peptide natural product that antagonizes pharmacologically relevant protein–protein interactions including formation of the C4b,2b complex in the complement cascade and gp120-CD4 binding in the HIV life cycle. Complestatin, a member of the vancomycin group of natural products, consists of an α-ketoacyl hexapeptide backbone modified by oxidative phenolic couplings and halogenations. The entire complestatin biosynthetic and regulatory gene cluster spanning ca. 50 kb was cloned and sequenced. It consisted of 16 ORFs, encoding proteins homologous to nonribosomal peptide synthetases, cytochrome P450-related oxidases, ferredoxins, nonheme halogenases, four enzymes involved in 4-hydroxyphenylglycine (Hpg) biosynthesis, transcriptional regulators, and ABC transporters. The nonribosomal peptide synthetase consisted of a priming module, six extending modules, and a terminal thioesterase; their arrangement and domain content was entirely consistent with functions required for the biosynthesis of a heptapeptide or α-ketoacyl hexapeptide backbone. Two oxidase genes were proposed to be responsible for the construction of the unique aryl-ether-aryl-aryl linkage on the linear heptapeptide intermediate. Hpg, 3,5-dichloro-Hpg, and 3,5-dichloro-hydroxybenzoylformate are unusual building blocks that repesent five of the seven requisite monomers in the complestatin peptide. Heterologous expression and biochemical analysis of 4-hydroxyphenylglycine transaminon confirmed its role as an aminotransferase responsible for formation of all three precursors. The close similarity but functional divergence between complestatin and chloroeremomycin biosynthetic genes also presents a unique opportunity for the construction of hybrid vancomycin-type antibiotics.

Transcriptional regulation of the Rhodococcus rhodochrous J1 nitA gene encoding a nitrilase.

The 1.4-kb downstream region from a nitrilase gene (nitA) of an actinomycete Rhodococcus rhodochrous J1, which is industrially in use, was found to be required for the isovaleronitrile-dependent induction of nitrilase synthesis in experiments using a Rhodococcus-Escherichia coli shuttle vector pK4 in a Rhodococcus strain. Sequence analysis of the 1.4-kb region revealed the existence of an open reading frame (nitR) of 957 bp, which would encode a protein with a molecular mass of 35,100. Deletion of the central and 3'-terminal portion of nitR resulted in the complete loss of nitrilase activity, demonstrating that nitR codes for a transcriptional positive regulator in nitA expression. The deduced amino acid sequence of nitR showed similarity to a positive regulator family including XylS from Pseudomonas putida and AraC from E. coli. By Northern blot analysis, the 1.4-kb transcripts for nitA were detected in R. rhodochrous J1 cells cultured in the presence of isovaleronitrile, but not those cultured in the absence of isovaleronitrile. The transcriptional start site for nitA was mapped to a C residue located 26 bp upstream of its translational start site. Deletion analysis to define the nitA promoter region suggested the possible participation of an inverted repeat sequence, centered on base pair -52, in induction of nitA transcription.