Cell cycle-dependent regulation of RNA polymerase I transcription: The nucleolar transcription factor UBF is inactive in mitosis and early G1

Transcription of ribosomal RNA genes by RNA polymerase (pol) I oscillates during the cell cycle, being maximal in S and G2 phase, repressed during mitosis, and gradually recovering during G1 progression. We have shown that transcription initiation factor (TIF)-IB/SL1 is inactivated during mitosis by cdc2/cyclin B-directed phosphorylation of TAFI110. In this study, we have monitored reactivation of transcription after exit from mitosis. We demonstrate that the pol I factor UBF is also inactivated by phosphorylation but recovers with different kinetics than TIF-IB/SL1. Whereas TIF-IB/SL1 activity is rapidly regained on entry into G1, UBF is reactivated later in G1, concomitant with the onset of pol I transcription. Repression of pol I transcription in mitosis and early G1 can be reproduced with either extracts from cells synchronized in M or G1 phase or with purified TIF-IB/SL1 and UBF isolated in the presence of phosphatase inhibitors. The results suggest that two basal transcription factors, e.g., TIF-IB/SL1 and UBF, are inactivated at mitosis and reactivated by dephosphorylation at the exit from mitosis and during G1 progression, respectively.

Regulation of the OxyR transcription factor by hydrogen peroxide and the cellular thiol—disulfide status

The Escherichia coli transcription factor OxyR is activated by the formation of an intramolecular disulfide bond and subsequently is deactivated by enzymatic reduction of the disulfide bond. Here we show that OxyR can be activated by two possible pathways. In mutants defective in the cellular disulfide-reducing systems, OxyR is constitutively activated by a change in the thiol—disulfide redox status in the absence of added oxidants. In wild-type cells, OxyR is activated by hydrogen peroxide. By monitoring the presence of the OxyR disulfide bond after exposure to hydrogen peroxide in vivo and in vitro, we also show that the kinetics of OxyR oxidation by low concentrations of hydrogen peroxide is significantly faster than the kinetics of OxyR reduction, allowing for transient activation in an overall reducing environment. We propose that the activity of OxyR in vivo is determined by the balance between hydrogen peroxide levels and the cellular redox environment.

Dynamic regulation of neuronal NO synthase transcription by calcium influx through a CREB family transcription factor-dependent mechanism

Neuronal nitric oxide (NO) synthase (nNOS) is dynamically regulated in response to a variety of physiologic and pathologic stimuli. Although the dynamic regulation of nNOS is well established, the molecular mechanisms by which such diverse stimuli regulate nNOS expression have not yet been identified. We describe experiments demonstrating that Ca2+ entry through voltage-sensitive Ca2+ channels regulates nNOS expression through alternate promoter usage in cortical neurons and that nNOS exon 2 contains the regulatory sequences that respond to Ca2+. Deletion and mutational analysis of the nNOS exon 2 promoter reveals two critical cAMP/Ca2+ response elements (CREs) that are immediately upstream of the transcription start site. CREB binds to the CREs within the nNOS gene. Mutation of the nNOS CREs as well as blockade of CREB function results in a dramatic loss of nNOS transcription. These findings suggest that nNOS is a Ca2+-regulated gene through the interactions of CREB on the CREs within the nNOS exon 2 promoter and that these interactions are likely to be centrally involved in the regulation of nNOS in response to neuronal injury and activity-dependent plasticity.

CIITA stimulation of transcription factor binding to major histocompatibility complex class II and associated promoters in vivo

CIITA is a master transactivator of the major histocompatibility complex class II genes, which are involved in antigen presentation. Defects in CIITA result in fatal immunodeficiencies. CIITA activation is also the control point for the induction of major histocompatibility complex class II and associated genes by interferon-γ, but CIITA does not bind directly to DNA. Expression of CIITA in G3A cells, which lack endogenous CIITA, followed by in vivo genomic footprinting, now reveals that CIITA is required for the assembly of transcription factor complexes on the promoters of this gene family, including DRA, Ii, and DMB. CIITA-dependent promoter assembly occurs in interferon-γ-inducible cell types, but not in B lymphocytes. Dissection of the CIITA protein indicates that transactivation and promoter loading are inseparable and reveal a requirement for a GTP binding motif. These findings suggest that CIITA may be a new class of transactivator.

Altering the anaerobic transcription factor FNR confers a hemolytic phenotype on Escherichia coli K12

The recent outbreaks of Escherichia coli 0157-associated food poisoning have focused attention on the virulence determinants of E. coli. Here, it is reported that single base substitutions in the fnr gene encoding the oxygen-responsive transcription regulator FNR (fumarate and nitrate reduction regulator) are sufficient to confer a hemolytic phenotype on E. coli K12, the widely used laboratory strain. The mechanism involves enhancing the expression of a normally dormant hemolysin gene (hlyE) located in the E. coli chromosome. The mutations direct single amino acid substitutions in the activating regions (AR1 and AR3) of FNR that contact RNA polymerase. It is concluded that altering a resident transcription regulator, or acquisition of a competent heterologous regulator, could generate a pool of hemolytic, and therefore more virulent, strains of E. coli in nature.

Transcription factor Mts1/Mts2 (Atf1/Pcr1, Gad7/Pcr1) activates the M26 meiotic recombination hotspot in Schizosaccharomyces pombe

Homologous recombination hotspots increase the frequency of recombination in nearby DNA. The M26 hotspot in the ade6 gene of Schizosaccharomyces pombe is a meiotic hotspot with a discrete, cis-acting nucleotide sequence (5′-ATGACGT-3′) defined by extensive mutagenesis. A heterodimeric M26 DNA binding protein, composed of subunits Mts1 and Mts2, has been identified and purified 40,000-fold. Cloning, disruption, and genetic analyses of the mts genes demonstrate that the Mts1/Mts2 heterodimer is essential for hotspot activity. This provides direct evidence that a specific trans-acting factor, binding to a cis-acting site with a unique nucleotide sequence, is required to activate this meiotic hotspot. Intriguingly, the Mts1/Mts2 protein subunits are identical to the recently described transcription factors Atf1 (Gad7) and Pcr1, which are required for a variety of stress responses. However, we report differential dependence on the Mts proteins for hotspot activation and stress response, suggesting that these proteins are multifunctional and have distinct activities. Furthermore, ade6 mRNA levels are equivalent in hotspot and nonhotspot meioses and do not change in mts mutants, indicating that hotspot activation is not a consequence of elevated transcription levels. These findings suggest an intimate but separable link between the regulation of transcription and meiotic recombination. Other studies have recently shown that the Mts1/Mts2 protein and M26 sites are involved in meiotic recombination elsewhere in the S. pombe genome, suggesting that these factors help regulate the timing and distribution of homologous recombination.

The E2F6 transcription factor is a component of the mammalian Bmi1-containing polycomb complex

The E2F transcription factors play a key role in the regulation of cellular proliferation and terminal differentiation. E2F6 is the most recently identified and the least well understood member of the E2F family. It is only distantly related to the other E2Fs and lacks the sequences responsible for both transactivation and binding to the retinoblastoma protein. Consistent with this finding, E2F6 can behave as a dominant negative inhibitor of the other E2F family members. In this study, we continue to investigate the possible role(s) of E2F6 in vivo. We report the isolation of RYBP, a recently identified member of the mammalian polycomb complex, as an E2F6-interacting protein. Mapping studies indicate that RYBP binds within the known “repression domain” of E2F6. Moreover, we demonstrate that endogenous E2F6 and polycomb group proteins, including RYBP, Ring1, MEL-18, mph1, and the oncoprotein Bmi1, associate with one another. These findings suggest that the biological properties of E2F6 are mediated through its ability to recruit the polycomb transcriptional repressor complex.

The Ath5 proneural genes function upstream of Brn3 POU domain transcription factor genes to promote retinal ganglion cell development

During retinogenesis, the Xenopus basic helix–loop–helix transcription factor Xath5 has been shown to promote a ganglion cell fate. In the developing mouse and chicken retinas, gene targeting and overexpression studies have demonstrated critical roles for the Brn3 POU domain transcription factor genes in the promotion of ganglion cell differentiation. However, the genetic relationship between Ath5 and Brn3 genes is unknown. To understand the genetic regulatory network(s) that controls retinal ganglion cell development, we analyzed the relationship between Ath5 and Brn3 genes by using a gain-of-function approach in the chicken embryo. We found that during retinogenesis, the chicken Ath5 gene (Cath5) is expressed in retinal progenitors and in differentiating ganglion cells but is absent in terminally differentiated ganglion cells. Forced expression of both Cath5 and the mouse Ath5 gene (Math5) in retinal progenitors activates the expression of cBrn3c following central-to-peripheral and temporal-to-nasal gradients. As a result, similar to the Xath5 protein, both Cath5 and Math5 proteins have the ability to promote the development of ganglion cells. Moreover, we found that forced expression of all three Brn3 genes also can stimulate the expression of cBrn3c. We further found that Ath5 and Brn3 proteins are capable of transactivating a Brn3b promoter. Thus, these data suggest that the expression of cBrn3c in the chicken and Brn3b in the mouse is initially activated by Ath5 factors in newly generated ganglion cells and later maintained by a feedback loop of Brn3 factors in the differentiated ganglion cells.

rSNP_Guide, a database system for analysis of transcription factor binding to target sequences: application to SNPs and site-directed mutations

rSNP_Guide is a novel curated database system for analysis of transcription factor (TF) binding to target sequences in regulatory gene regions altered by mutations. It accumulates experimental data on naturally occurring site variants in regulatory gene regions and site-directed mutations. This database system also contains the web tools for SNP analysis, i.e., active applet applying weight matrices to predict the regulatory site candidates altered by a mutation. The current version of the rSNP_Guide is supplemented by six sub-databases: (i) rSNP_DB, on DNA–protein interaction caused by mutation; (ii) SYSTEM, on experimental systems; (iii) rSNP_BIB, on citations to original publications; (iv) SAMPLES, on experimentally identified sequences of known regulatory sites; (v) MATRIX, on weight matrices of known TF sites; (vi) rSNP_Report, on characteristic examples of successful rSNP_Tools implementation. These databases are useful for the analysis of natural SNPs and site-directed mutations. The databases are available through the Web, http://wwwmgs.bionet.nsc.ru/mgs/systems/rsnp/.

Phylogenetic footprinting of transcription factor binding sites in proteobacterial genomes

Toward the goal of identifying complete sets of transcription factor (TF)-binding sites in the genomes of several gamma proteobacteria, and hence describing their transcription regulatory networks, we present a phylogenetic footprinting method for identifying these sites. Probable transcription regulatory sites upstream of Escherichia coli genes were identified by cross-species comparison using an extended Gibbs sampling algorithm. Close examination of a study set of 184 genes with documented transcription regulatory sites revealed that when orthologous data were available from at least two other gamma proteobacterial species, 81% of our predictions corresponded with the documented sites, and 67% corresponded when data from only one other species were available. That the remaining predictions included bona fide TF-binding sites was proven by affinity purification of a putative transcription factor (YijC) bound to such a site upstream of the fabA gene. Predicted regulatory sites for 2097 E.coli genes are available at http://www.wadsworth.org/resnres/bioinfo/.

Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice

Induction of phase 2 enzymes, which neutralize reactive electrophiles and act as indirect antioxidants, appears to be an effective means for achieving protection against a variety of carcinogens in animals and humans. Transcriptional control of the expression of these enzymes is mediated, at least in part, through the antioxidant response element (ARE) found in the regulatory regions of their genes. The transcription factor Nrf2, which binds to the ARE, appears to be essential for the induction of prototypical phase 2 enzymes such as glutathione S-transferases (GSTs) and NAD(P)H:quinone oxidoreductase (NQO1). Constitutive hepatic and gastric activities of GST and NQO1 were reduced by 50–80% in nrf2-deficient mice compared with wild-type mice. Moreover, the 2- to 5-fold induction of these enzymes in wild-type mice by the chemoprotective agent oltipraz, which is currently in clinical trials, was almost completely abrogated in the nrf2-deficient mice. In parallel with the enzymatic changes, nrf2-deficient mice had a significantly higher burden of gastric neoplasia after treatment with benzo[a]pyrene than did wild-type mice. Oltipraz significantly reduced multiplicity of gastric neoplasia in wild-type mice by 55%, but had no effect on tumor burden in nrf2-deficient mice. Thus, Nrf2 plays a central role in the regulation of constitutive and inducible expression of phase 2 enzymes in vivo and dramatically influences susceptibility to carcinogenesis. Moreover, the total loss of anticarcinogenic efficacy of oltipraz in the nrf2-disrupted mice highlights the prime importance of elevated phase 2 gene expression in chemoprotection by this and similar enzyme inducers.

Different dynamics in nuclear entry of subunits of the repair/transcription factor TFIIH

We report here the different ways in which four subunits of the basal transcription/repair factor TFIIH (XPB, XPD, p62 and p44) and the damage recognition XPC repair protein can enter the nucleus. We examined their nuclear localization by transiently expressing the gene products tagged with the enhanced green fluorescent protein (EGFP) in transfected 3T3 cells. In agreement with the identification of more than one putative nuclear localization signal (NLS) in their protein sequences, XPB, XPC, p62 and p44 chimeras were rapidly sorted to the nucleus. In contrast, the XPD–EGFP chimeras appeared mainly localized in the cytoplasm, with a minor fraction of transfectants showing the EGFP-based fluorescence also in the nucleus. The ability of the XPD chimeras to enter the nucleus was confirmed by western blotting on fractionated cell extracts and by functional complementation of the repair defect in the UV5 rodent cells, mutated in the XPD homologous gene. By deletion mutagenesis, we were unable to identify any sequence specific for nuclear localization. In particular, deletion of the putative NLS failed to affect subcellular localization and, conversely, the C-terminal part of XPD containing the putative NLS showed no specific nuclear accumulation. These findings suggest that the nuclear entry of XPD depends on its complexation with other proteins in the cytoplasm, possibly other components of the TFIIH complex.

The wing in yeast heat shock transcription factor (HSF) DNA-binding domain is required for full activity

The yeast heat shock transcription factor (HSF) belongs to the winged helix family of proteins. HSF binds DNA as a trimer, and additional trimers can bind DNA co-operatively. Unlike other winged helix–turn–helix proteins, HSF’s wing does not appear to contact DNA, as based on a previously solved crystal structure. Instead, the structure implies that the wing is involved in protein–protein interactions, possibly within a trimer or between adjacent trimers. To understand the function of the wing in the HSF DNA-binding domain, a Saccharomyces cerevisiae strain was created that expresses a wingless HSF protein. This strain grows normally at 30°C, but shows a decrease in reporter gene expression during constitutive and heat-shocked conditions. Removal of the wing does not affect the stability or trimeric nature of a protein fragment containing the DNA-binding and trimerization domains. Removal of the wing does result in a decrease in DNA-binding affinity. This defect was mainly observed in the ability to form the first trimer-bound complex, as the formation of larger complexes is unaffected by the deletion. Our results suggest that the wing is not involved in the highly co-operative nature of HSF binding, but may be important in stabilizing the first trimer bound to DNA.

A postgermination developmental arrest checkpoint is mediated by abscisic acid and requires the ABI5 transcription factor in Arabidopsis

Seed dormancy is a trait of considerable adaptive significance because it maximizes seedling survival by preventing premature germination under unfavorable conditions. Understanding how seeds break dormancy and initiate growth is also of great agricultural and biotechnological interest. Abscisic acid (ABA) plays primary regulatory roles in the initiation and maintenance of seed dormancy. Here we report that the basic leucine zipper transcription factor ABI5 confers an enhanced response to exogenous ABA during germination, and seedling establishment, as well as subsequent vegetative growth. These responses correlate with total ABI5 levels. We show that ABI5 expression defines a narrow developmental window following germination, during which plants monitor the environmental osmotic status before initiating vegetative growth. ABI5 is necessary to maintain germinated embryos in a quiescent state thereby protecting plants from drought. As expected for a key player in ABA-triggered processes, ABI5 protein accumulation, phosphorylation, stability, and activity are highly regulated by ABA during germination and early seedling growth.