TRM1, a YY1-like suppressor of rbcS-m3 expression in maize mesophyll cells

The genes rbcS and rbcL encode, respectively, the small and large subunits of the photosynthetic carbon dioxide fixation enzyme ribulose bisphosphate carboxylase/oxygenase. There is a single rbcL gene in each chloroplast chromosome; a family of rbcS genes is located in the nuclear genome. These two genes are not expressed in mesophyll cells but are in adjacent bundle-sheath cells of leaves of the C4 plant Zea mays. Two regions of the maize gene rbcS-m3 are required for suppressing expression in mesophyll cells. One region is just beyond the translation termination site in the 3′ region, and the other is several hundred base pairs upstream of the transcription start site. A binding site for a protein with limited homology to the viral, yeast, and mammalian transcription repressor-activator YY1 (Yin-Yang I), has now been identified in the 3′ region. A maize gene for a protein with zinc fingers homologous to those of YY1 has been isolated, characterized, and expressed in Escherichia coli. The gene is designated trm1 (transcription repressor-maize 1). The protein TRM1 binds to the YY1-like site and, in addition, TRM1 binds to two sequence regions in the 5′ region of the gene that have no homology to the YY1 site. Mutagenesis or deletion of any of these three sequences eliminates repression of rbcS-m3 reporter genes in mesophyll cells.

QscR, a modulator of quorum-sensing signal synthesis and virulence in Pseudomonas aeruginosa

The opportunistic pathogenic bacterium Pseudomonas aeruginosa uses quorum-sensing signaling systems as global regulators of virulence genes. There are two quorum-sensing signal receptor and signal generator pairs, LasR–LasI and RhlR–RhlI. The recently completed P. aeruginosa genome-sequencing project revealed a gene coding for a homolog of the signal receptors, LasR and RhlR. Here we describe a role for this gene, which we call qscR. The qscR gene product governs the timing of quorum-sensing-controlled gene expression and it dampens virulence in an insect model. We present evidence that suggests the primary role of QscR is repression of lasI. A qscR mutant produces the LasI-generated signal prematurely, and this results in premature transcription of a number of quorum-sensing-regulated genes. When fed to Drosophila melanogaster, the qscR mutant kills the animals more rapidly than the parental P. aeruginosa. The repression of lasI by QscR could serve to ensure that quorum-sensing-controlled genes are not activated in environments where they are not useful.

Multifaceted Physiological Response Allows Yeast to Adapt to the Loss of the Signal Recognition Particle-dependent Protein-targeting PathwayD⃞

Translational control has recently been recognized as an important facet of adaptive responses to various stress conditions. We describe the adaptation response of the yeast Saccharomyces cerevisiae to the loss of one of two mechanisms to target proteins to the secretory pathway. Using inducible mutants that block the signal recognition particle (SRP) pathway, we find that cells demonstrate a physiological response to the loss of the SRP pathway that includes specific changes in global gene expression. Upon inducing the loss of the SRP pathway, SRP-dependent protein translocation is initially blocked, and cell growth is considerably slowed. Concomitantly, gene expression changes include the induction of heat shock genes and the repression of protein synthesis genes. Remarkably, within hours, the efficiency of protein sorting improves while cell growth remains slow in agreement with the persistent repression of protein synthesis genes. Our results suggest that heat shock gene induction serves to protect cells from mislocalized precursor proteins in the cytosol, whereas reduced protein synthesis helps to regain efficiency in protein sorting by reducing the load on the protein translocation apparatus. Thus, we suggest that cells trade speed in cell growth for fidelity in protein sorting to adjust to life without SRP.

Insulin inhibits transcription of IRS-2 gene in rat liver through an insulin response element (IRE) that resembles IREs of other insulin-repressed genes

Recent data indicate that sustained elevations in plasma insulin suppress the mRNA for IRS-2, a component of the insulin signaling pathway in liver, and that this deficiency contributes to hepatic insulin resistance and inappropriate gluconeogenesis. Here, we use nuclear run-on assays to show that insulin inhibits transcription of the IRS-2 gene in the livers of intact rats. Insulin also inhibited transcription of a reporter gene driven by the human IRS-2 promoter that was transfected into freshly isolated rat hepatocytes. The human promoter contains a heptanucleotide sequence, TGTTTTG, that is identical to the insulin response element (IRE) identified previously in the promoters of insulin-repressed genes. Single base pair substitutions in this IRE decreased transcription of the IRS-2-driven reporter in the absence of insulin and abolished insulin-mediated repression. We conclude that insulin represses transcription of the IRS-2 gene by blocking the action of a positive factor that binds to the IRE. Sustained repression of IRS-2, as occurs in chronic hyperinsulinemia, contributes to hepatic insulin resistance and accelerates the development of the diabetic state.

Switch from Myc/Max to Mad1/Max binding and decrease in histone acetylation at the telomerase reverse transcriptase promoter during differentiation of HL60 cells

Recent evidence suggests that the Myc and Mad1 proteins are implicated in the regulation of the gene encoding the human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase. We have analyzed the in vivo interaction between endogenous c-Myc and Mad1 proteins and the hTERT promoter in HL60 cells with the use of the chromatin immunoprecipitation assay. The E-boxes at the hTERT proximal promoter were occupied in vivo by c-Myc in exponentially proliferating HL60 cells but not in cells induced to differentiate by DMSO. In contrast, Mad1 protein was induced and bound to the hTERT promoter in differentiated HL60 cells. Concomitantly, the acetylation of the histones at the promoter was significantly reduced. These data suggest that the reciprocal E-box occupancy by c-Myc and Mad1 is responsible for activation and repression of the hTERT gene in proliferating and differentiated HL60 cells, respectively. Furthermore, the histone deacetylase inhibitor trichostatin A inhibited deacetylation of histones at the hTERT promoter and attenuated the repression of hTERT transcription during HL60 cell differentiation. In addition, trichostatin A treatment activated hTERT transcription in resting human lymphocytes and fibroblasts. Taken together, these results indicate that acetylation/deacetylation of histones is operative in the regulation of hTERT expression.

The dual role of ultraspiracle, the Drosophila retinoid X receptor, in the ecdysone response

The Drosophila homolog of the retinoid X receptor, ultraspiracle (USP), heterodimerizes with the ecdysone receptor (EcR) to form a functional complex that mediates the effects of the steroid molting hormone ecdysone by activating and repressing expression of ecdysone response genes. As with other retinoid X receptor heterodimers, EcR/USP affects gene transcription in a ligand-modulated manner. We used in vivo, cell culture, and biochemical approaches to analyze the functions of two usp alleles, usp3 and usp4, which encode stable proteins with defective DNA-binding domains. We observed that USP is able to activate as well as repress the Z1 isoform of the ecdysone-responsive broad complex (BrC-Z1). Activation of BrC-Z1 as well as EcR, itself an ecdysone response gene, can be mediated by both the USP3 and USP4 mutant proteins. USP3 and USP4 also activate an ecdysone-responsive element, hsp27EcRE, in cultured cells. These results differ from the protein null allele, usp2, which is unable to mediate activation [Schubiger, M. & Truman, J. W. (2000) Development 127, 1151–1159]. BrC-Z1 repression is compromised in all three usp alleles, suggesting that repression involves the association of USP with DNA. Our results distinguish two mechanisms by which USP modulates the properties of EcR: one that involves the USP DNA-binding domain and one that can be achieved solely through the ligand-binding domain. These newly revealed properties of USP might implicate similar properties for retinoid X receptor.

Mutations in Drosophila heat shock cognate 4 are enhancers of Polycomb

The homeotic genes controlling segment identity in Drosophila are repressed by the Polycomb group of genes (PcG) and are activated by genes of the trithorax group (trxG). An F1 screen for dominant enhancers of Polycomb yielded a point mutation in the heat shock cognate gene, hsc4, along with mutations corresponding to several known PcG loci. The new mutation is a more potent enhancer of Polycomb phenotypes than an apparent null allele of hsc4 is, although even the null allele occasionally displays homeotic phenotypes associated with the PcG. Previous biochemical results had suggested that HSC4 might interact with BRAHMA, a trxG member. Further analyses now show that there is no physical or genetic interaction between HSC4 and the Brahma complex. HSC4 might be needed for the proper folding of a component of the Polycomb repression complex, or it may be a functional member of that complex.

Mammalian Scratch: A neural-specific Snail family transcriptional repressor

Members of the Snail family of zinc finger transcription factors are known to play critical roles in neurogenesis in invertebrates, but none of these factors has been linked to vertebrate neuronal differentiation. We report the isolation of a gene encoding a mammalian Snail family member that is restricted to the nervous system. Human and murine Scratch (Scrt) share 81% and 69% identity to Drosophila Scrt and the Caenorhabditis elegans neuronal antiapoptotic protein, CES-1, respectively, across the five zinc finger domain. Expression of mammalian Scrt is predominantly confined to the brain and spinal cord, appearing in newly differentiating, postmitotic neurons and persisting into postnatal life. Additional expression is seen in the retina and, significantly, in neuroendocrine (NE) cells of the lung. In a parallel fashion, we detect hScrt expression in lung cancers with NE features, especially small cell lung cancer. hScrt shares the capacity of other Snail family members to bind to E-box enhancer motifs, which are targets of basic helix–loop–helix (bHLH) transcription factors. We show that hScrt directly antagonizes the function of heterodimers of the proneural bHLH protein achaete-scute homolog-1 and E12, leading to active transcriptional repression at E-box motifs. Thus, Scrt has the potential to function in newly differentiating, postmitotic neurons and in cancers with NE features by modulating the action of bHLH transcription factors critical for neuronal differentiation.

HDA2 and HDA3 are related proteins that interact with and are essential for the activity of the yeast histone deacetylase HDA1

Histone deacetylase HDA1, the prototype for the class II mammalian deacetylases, is likely the catalytic subunit of the HDA1-containing complex that is involved in TUP1-specific repression and global deacetylation in yeast. Although the class I RPD3-like enzymatic complexes have been well characterized, little is known about the identity and interactions of the factors that associate to form the HDA1 complex. In this paper, we identify related HDA2 and HDA3 proteins that are found in the HDA1 complex and show that HDA1 interacts with itself and with the HDA2-HDA3 subcomplex to form a likely tetramer. These interactions are necessary for catalytic activity because mutations in any of the three components disrupt activity both in vitro and in vivo. In this respect the HDA1 complex differs from yeast RPD3, which has components such as SIN3 that are not essential for activity in vitro, and yeast HOS3, which has intrinsic in vitro activity as a homodimer in the absence of other subunits.

Myc represses the p21(WAF1/CIP1) promoter and interacts with Sp1/Sp3

The cyclin-dependent kinase inhibitor p21(WAF1/CIP1) inhibits proliferation both in vitro and in vivo, and overexpression of p21 in normal and tumor cell lines results in cell cycle arrest. In contrast, ectopic expression of Myc alleviates G1 cell cycle arrest. Recent studies showed that Myc can repress p21 transcription, thereby overriding a p21-mediated cell cycle checkpoint. We found that activation of a Myc-estrogen receptor fusion protein by 4-hydroxytamoxifen in mouse cells resulted in suppression of endogenous p21 transcription. This effect was observed in the absence of de novo protein synthesis and was independent of histone deacetylase activity. In transient transfection studies, Myc effectively repressed p21 promoter constructs containing only 119 bp of sequence upstream of the transcription start site. This region contains multiple Sp1-binding sites and a potential initiator element, but no canonical Myc DNA-binding sites. Deletion of the potential initiator element does not affect repression of the p21 promoter by c-Myc. Coimmunoprecipitation and glutathione S-transferase pull-down experiments demonstrate that c-Myc may form complexes with Sp1/Sp3. We found that the central region of c-Myc interacts with the zinc finger domain of Sp1. Because Sp1 is required for p21 transcription, it is possible that Myc may down-regulate p21 transcription, at least in part, by sequestering Sp1. Repression of the p21 promoter may contribute to the ability of c-Myc to promote cell proliferation.

Cloning and Characterization of a Gibberellin-Induced RNase Expressed in Barley Aleurone Cells1

We cloned a cDNA for a gibberellin-induced ribonuclease (RNase) expressed in barley (Hordeum vulgare) aleurone and the gene for a second barley RNase expressed in leaf tissue. The protein encoded by the cDNA is unique among RNases described to date in that it contains a novel 23-amino acid insert between the C2 and C3 conserved sequences. Expression of the recombinant protein in tobacco (Nicotiana tabacum) suspension-cultured protoplasts gave an active RNase of the expected size, confirming the enzymatic activity of the protein. Analyses of hormone regulation of expression of mRNA for the aleurone RNase revealed that, like the pattern for α-amylase, mRNA levels increased in the presence of gibberellic acid, and its antagonist abscisic acid prevented this effect. Quantitative studies at early times demonstrated that cycloheximide treatment of aleurone layers increased mRNA levels 4-fold, whereas a combination of gibberellin plus cycloheximide treatment was required to increase α-amylase mRNA levels to the same extent. These results are consistent with loss of repression as an initial effect of gibberellic acid on transcription of those genes, although the regulatory pathways for the two genes may differ.

Mannose Inhibits Arabidopsis Germination via a Hexokinase-Mediated Step1

Low concentrations of the glucose (Glc) analog mannose (Man) inhibit germination of Arabidopsis seeds. Man is phosphorylated by hexokinase (HXK), but the absence of germination was not due to ATP or phosphate depletion. The addition of metabolizable sugars reversed the Man-mediated inhibition of germination. Carbohydrate-mediated regulation of gene expression involving a HXK-mediated pathway is known to be activated by Glc, Man, and other monosaccharides. Therefore, we investigated whether Man blocks germination through this system. By testing other Glc analogs, we found that 2-deoxyglucose, which, like Man, is phosphorylated by HXK, also blocked germination; no inhibition was observed with 6-deoxyglucose or 3-O-methylglucose, which are not substrates for HXK. Since these latter two sugars are taken up at a rate similar to that of Man, uptake is unlikely to be involved in the inhibition of germination. Furthermore, we show that mannoheptulose, a specific HXK inhibitor, restores germination of seeds grown in the presence of Man. We conclude that HXK is involved in the Man-mediated repression of germination of Arabidopsis seeds, possibly via energy depletion.

Myo-Inositol-Dependent Sodium Uptake in Ice Plant1

In salt-stressed ice plants (Mesembryanthemum crystallinum), sodium accumulates to high concentrations in vacuoles, and polyols (myo-inositol, d-ononitol, and d-pinitol) accumulate in the cytosol. Polyol synthesis is regulated by NaCl and involves induction and repression of gene expression (D.E. Nelson, B. Shen, and H.J. Bohnert [1998] Plant Cell 10: 753–764). In the study reported here we found increased phloem transport of myo-inositol and reciprocal increased transport of sodium and inositol to leaves under stress. To determine the relationship between increased translocation and sodium uptake, we analyzed the effects of exogenous application of myo-inositol: The NaCl-inducible ice plant myo-inositol 1-phosphate synthase is repressed in roots, and sodium uptake from root to shoot increases without stimulating growth. Sodium uptake and transport through the xylem was coupled to a 10-fold increase of myo-inositol and ononitol in the xylem. Seedlings of the ice plant are not salt-tolerant, and yet the addition of exogenous myo-inositol conferred upon them patterns of gene expression and polyol accumulation observed in mature, salt-tolerant plants. Sodium uptake and transport through the xylem was enhanced in the presence of myo-inositol. The results indicate an interdependence of sodium uptake and alterations in the distribution of myo-inositol. We hypothesize that myo-inositol could serve not only as a substrate for the production of compatible solutes but also as a leaf-to-root signal that promotes sodium uptake.

c-Jun interacts with the corepressor TG-interacting factor (TGIF) to suppress Smad2 transcriptional activity

The Sma and Mad related (Smad) family proteins are critical mediators of the transforming growth factor-β (TGF-β) superfamily signaling. After TGF-β-mediated phosphorylation and association with Smad4, Smad2 moves to the nucleus and activates expression of specific genes through cooperative interactions with DNA-binding proteins, including members of the winged-helix family of transcription factors, forkhead activin signal transducer (FAST)-1 and FAST2. TGF-β has also been described to activate other signaling pathways, such as the c-Jun N-terminal Kinase (JNK) pathway. Here, we show that activation of JNK cascade blocked the ability of Smad2 to mediate TGF-β-dependent activation of the FAST proteins. This inhibitory activity is mediated through the transcriptional factor c-Jun, which enhances the association of Smad2 with the nuclear transcriptional corepressor TG-interacting factor (TGIF), thereby interfering with the assembly of Smad2 and the coactivator p300 in response to TGF-β signaling. Interestingly, c-Jun directly binds to the nuclear transcriptional corepressor TGIF and is required for TGIF-mediated repression of Smad2 transcriptional activity. These studies thus reveal a mechanism for suppression of Smad2 signaling pathway by JNK cascade through transcriptional repression.

CtBP-dependent activities of the short-range Giant repressor in the Drosophila embryo

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.