Preexisting Transcription Factors Research Articles

Distinct ecological fitness factors coordinated by a conserved Escherichia coli regulator during systemic bloodstream infection

The ability of bacterial pathogens to adapt to host niches is driven by the carriage and regulation of genes that benefit pathogenic lifestyles. Genes that encode virulence or fitness-enhancing factors must be regulated in response to changing host environments to allow rapid response to challenges presented by the host. Furthermore, this process can be controlled by preexisting transcription factors (TFs) that acquire new roles in tailoring regulatory networks, specifically in pathogens. However, the mechanisms underlying this process are poorly understood. The highly conserved Escherichia coli TF YhaJ exhibits distinct genome-binding dynamics and transcriptome control in pathotypes that occupy different host niches, such as uropathogenic E. coli (UPEC). Here, we report that this important regulator is required for UPEC systemic survival during murine bloodstream infection (BSI). This advantage is gained through the coordinated regulation of a small regulon comprised of both virulence and metabolic genes. YhaJ coordinates activation of both Type 1 and F1C fimbriae, as well as biosynthesis of the amino acid tryptophan, by both direct and indirect mechanisms. Deletion of yhaJ or the individual genes under its control leads to attenuated survival during BSI. Furthermore, all three systems are up-regulated in response to signals derived from serum or systemic host tissue, but not urine, suggesting a niche-specific regulatory trigger that enhances UPEC fitness via pleiotropic mechanisms. Collectively, our results identify YhaJ as a pathotype-specific regulatory aide, enhancing the expression of key genes that are collectively required for UPEC bloodstream pathogenesis.

Control of resistance against bacteriophage killing by a metabolic regulator in meningitis-associated Escherichia coli

Ecologically beneficial traits in bacteria are encoded by intrinsic and horizontally acquired genes. However, such traits are not universal, and the highly mosaic nature of bacterial genomes requires control at the transcriptional level to drive these processes. It has emerged that regulatory flexibility is widespread in the Escherichia coli species, whereby preexisting transcription factors can acquire new and unrelated roles in regulating beneficial traits. DsdC is the regulator of D-serine tolerance in E. coli, is essential for D-serine catabolism, and is often encoded by two copies in neonatal meningitis-associated E. coli (NMEC). Here, we reveal that DsdC is a global regulator of transcription in NMEC and does not require D-serine for the control of novel beneficial traits. We show that DsdC binds the chromosome in an unusual manner, with many binding sites arranged in clusters spanning entire operons and within gene coding sequences, such as neuO. Importantly, we identify neuO as the most significantly down-regulated gene in a strain deleted for both dsdC copies, in both the presence and absence of D-serine. NeuO is prophage encoded in several NMEC K1 isolates and mediates capsule O-acetylation but has no effect on attachment to or invasion of human brain endothelial cells. Instead, we demonstrate that NeuO provides resistance against K1 bacteriophage attack and that this critical function is regulated by DsdC. This work highlights how a horizontally acquired enzyme that functions in cell-surface modulation can be controlled by an intrinsic regulator to provide a key ecological benefit to an E. coli pathotype.

Abstract B37: Genomic view of YAP1 dependent transcription

Abstract YAP1 is a transcriptional coactivator regulated by the Hippo pathway, mechano-transduction, G-protein coupled receptors, and WNT signaling. YAP1 activity depends on its stabilization and its nuclear localization. Once in the nucleus, YAP1 can regulate gene expression by interacting with transcription factors with DNA binding capability. While YAP1 main transcriptional partners belong to the TEAD family, several reports indicate that YAP1 can also associate with SMADs, RUNX, and other transcription factors. YAP1 acts as a broad regulator of cell growth and differentiation, thus playing a pivotal role in regeneration, reprogramming, and cancer. How YAP1 may control such a large number of cellular processes is still to be fully understood. In the adult mouse liver, activation of YAP1 supports proficient cell proliferation and triggers dedifferentiation of hepatocytes towards a bipotent progenitor-like state. Overall, this results in abnormal liver growth, which, upon prolonged YAP1 activation, may lead to the emergence of focal hepatocellular carcinomas. In order to gain further insight into the role of YAP1 in these processes, we conducted a genome-wide investigation in the mouse liver, to identify how YAP1 controls transcription and to define gene expression programs directly regulated by YAP1. YAP1 binding to the genome was exclusively mediated by TEAD, yet given the preponderance of TEAD bound loci, only a subset of these sites was bound by YAP1. Such loci showed a defined pattern of epigenetic bookmarking and selective binding of transcription factors, thus indicating that pre-existing chromatin modifications and transcription factors are pivotal in biasing YAP1 binding to a defined set of TEAD bound genomic regions. Integrated analyses of ChIP-seq and RNA-seq data indicated that YAP1 can act both as an activator and as a repressor in a context-dependent way and allowed the dissection of transcriptional mechanisms. Longitudinal analysis by ChIP-seq and RNA-seq highlighted differences in genome binding and transcriptional control, which allowed the identification of acute and tonic YAP1 dependent responses. While during acute activation YAP1 triggered cell cycle and signal transduction programs, its prolonged activation sustained a feed-forward loop that potentiated and expanded YAP1-dependent transcription, thus leading to the direct engagement of cell identity programs. Overall this study provides a genome-scale understanding of how YAP1 controls gene expression and reveals principles in gene regulation that account for YAP1 ability to orchestrate development, regeneration, and cell reprogramming. Citation Format: Francesca Biagioni, Ottavio Croci, Elisa Donato, Silvia Sberna, Serena De Fazio, Arianna Sabò, Bruno Amati, Stefano Campaner. Genomic view of YAP1 dependent transcription [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B37.

Plastic Circuits: Regulatory Flexibility in Fine Tuning Pathogen Success.

Bacterial pathogens employ diverse fitness and virulence mechanisms to gain an advantage in competitive niches. These lifestyle-specific traits require integration into the regulatory network of the cell and are often controlled by pre-existing transcription factors. In this review, we highlight recent advances that have been made in characterizing this regulatory flexibility in prominent members of the Enterobacteriaceae. We focus on the direct global interactions between transcription factors and their target genes in pathogenic Escherichia coli and Salmonella revealed using chromatin immunoprecipitation coupled with next-generation sequencing. Furthermore, the implications and advantages of such regulatory adaptations in benefiting distinct pathogenic lifestyles are discussed.

Two Types of Etiological Mutation in the Limb-Specific Enhancer of Shh.

An enhancer named MFCS1 regulates Sonic hedgehog (Shh) expression in the posterior mesenchyme of limb buds. Several mutations in MFCS1 induce ectopic Shh expression in the anterior limb bud, and these result in preaxial polydactyly (PPD). However, the molecular basis of ectopic Shh expression remains elusive, although some mutations are known to disrupt the negative regulation of Shh expression in the anterior limb bud. Here, we analyzed the molecular mechanism of ectopic Shh expression in PPD including in a mouse mutation—hemimelic extra toes (Hx)—and in other MFCS1 mutations in different species. First, we generated transgenic mouse lines with a LacZ reporter cassette flanked with tandem repeats of 40 bp MFCS1 fragments harboring a mutation. The transgenic mouse line with the Hx-type fragment showed reporter expression exclusively in the anterior, but not in the posterior margins of limb buds. In contrast, no specific LacZ expression was observed in lines carrying the MFCS1 fragment with other mutations. Yeast one-hybrid assays revealed that the msh-like homeodomain protein, MSX1, bound specifically to the Hx sequence of MFCS1. Thus, PPD caused by mutations in MFCS1 has two major types of molecular etiology: loss of a cis-motif for negative regulation of Shh, and acquisition of a new cis-motif binding to a preexisting transcription factor, as represented by the Hx mutation.

Chromatin priming elements establish immunological memory in T cells without activating transcription: T cell memory is maintained by DNA elements which stably prime inducible genes without activating steady state transcription.

We have identified a simple epigenetic mechanism underlying the establishment and maintenance of immunological memory in T cells. By studying the transcriptional regulation of inducible genes we found that a single cycle of activation of inducible factors is sufficient to initiate stable binding of pre-existing transcription factors to thousands of newly activated distal regulatory elements within inducible genes. These events lead to the creation of islands of active chromatin encompassing nearby enhancers, thereby supporting the accelerated activation of inducible genes, without changing steady state levels of transcription in memory T cells. These studies also highlighted the need for more sophisticated definitions of gene regulatory elements. The chromatin priming elements defined here are distinct from classical enhancers because they function by maintaining chromatin accessibility rather than directly activating transcription. We propose that these priming elements are members of a wider class of genomic elements that support correct developmentally regulated gene expression.

DeepCAGE transcriptomics identify HOXD10 as a transcription factor regulating lymphatic endothelial responses to VEGF-C.

Lymphangiogenesis plays a crucial role during development, in cancer metastasis and in inflammation. Activation of VEGFR-3 (also known as FLT4) by VEGF-C is one of the main drivers of lymphangiogenesis, but the transcriptional events downstream of VEGFR-3 activation are largely unknown. Recently, we identified a wave of immediate early transcription factors that are upregulated in human lymphatic endothelial cells (LECs) within the first 30 to 80 min after VEGFR-3 activation. Expression of these transcription factors must be regulated by additional pre-existing transcription factors that are rapidly activated by VEGFR-3 signaling. Using transcription factor activity analysis, we identified the homeobox transcription factor HOXD10 to be specifically activated at early time points after VEGFR-3 stimulation, and to regulate expression of immediate early transcription factors, including NR4A1. Gain- and loss-of-function studies revealed that HOXD10 is involved in LECs migration and formation of cord-like structures. Furthermore, HOXD10 regulates expression of VE-cadherin, claudin-5 and NOS3 (also known as e-NOS), and promotes lymphatic endothelial permeability. Taken together, these results reveal an important and unanticipated role of HOXD10 in the regulation of VEGFR-3 signaling in lymphatic endothelial cells, and in the control of lymphangiogenesis and permeability.

Combinatorial Rules of Precursor Specification Underlying Olfactory Neuron Diversity

Sensory neuron diversity ensures optimal detection of the external world and is a hallmark of sensory systems. An extreme example is the olfactory system, as individual olfactory receptor neurons (ORNs) adopt unique sensory identities by typically expressing a single receptor gene from a large genomic repertoire. In Drosophila, about 50 different ORN classes are generated from a field of precursor cells, giving rise to spatially restricted and distinct clusters of ORNs on the olfactory appendages. Developmental strategies spawning ORN diversity from an initially homogeneous population of precursors are largely unknown. Here we unravel the nested and binary logic of the combinatorial code that patterns the decision landscape of precursor states underlying ORN diversity in the Drosophila olfactory system. The transcription factor Rotund (Rn) is a critical component of this code that is expressed in a subset of ORN precursors. Addition of Rn to preexisting transcription factors that assign zonal identities to precursors on the antenna subdivides each zone and almost exponentially increases ORN diversity by branching off novel precursor fates from default ones within each zone. In rn mutants, rn-positive ORN classes are converted to rn-negative ones in a zone-specific manner. We provide a model describing how nested and binary changes in combinations of transcription factors could coordinate and pattern a large number of distinct precursor identities within a population to modulate the level of ORN diversity during development and evolution.

The Increase in the Number of Subunits in Eukaryotic RNA Polymerase III Relative to RNA Polymerase II Is due to the Permanent Recruitment of General Transcription Factors

The number of subunits of RNA polymerases (RNAPs) increases during evolution from 5 in eubacteria to 12 in archaea. In eukaryotes, which have at least three RNAPs, the number of subunits has expanded from 12 in RNA polymerase II (RNAPII) to 14 in RNA polymerase I (RNAPI) and to 17 in RNA polymerase III (RNAPIII). It was recently demonstrated that the two additional subunits found in RNAPI relative to RNAPII are homologous to TFIIF, a dimeric general transcription factor of RNAPII. Here, we extend this finding by demonstrating that four of the five RNAPIII-specific subunits are also homologous to transcription factors of RNAPII. We use the available evidence to propose an evolutionary history of the eukaryotic RNAPs and argue that the increases in the number of subunits that occurred in RNAPs I and III are due to the permanent recruitment of preexisting transcription factors.

Differential roles of MAPKs and MSK1 signalling pathways in the regulation of c-Jun during phenylephrine-induced cardiac myocyte hypertrophy

Gq-protein-coupled receptor (GqPCR) signalling is associated with the induction of cardiac myocyte hypertrophy, which is characterized by an increase in expression of immediate early genes via activation of pre-existing transcription factors. Here, we explore the role of MSK1 and MAPK signalling pathways in the regulation of the immediate early gene c-jun. The results provide further support for the role of MSK1 in cardiac myocyte hypertrophy and indicate that PE activates distinct signalling mechanisms which culminate with a complex activation of c-jun. ERK1/2 and JNKs are the principal kinases responsible for phosphorylation of c-Jun, whereas c-jun mRNA and protein up-regulation by PE is mediated by multiple signalling pathways that include MSK1, ERK1/2, p38-MAPK and JNKs. These signalling mechanisms seem to be critical to the phenotypic changes of cardiac myocytes in response to hypertrophic stimulation.

14-3-3 Mediates Transcriptional Regulation by Modulating Nucleocytoplasmic Shuttling of Tobacco DNA-binding Protein Phosphatase-1

Tobacco DBP1 is the founding member of a novel class of plant transcription factors featuring sequence-specific DNA binding and protein phosphatase activity. To understand the mechanisms underlying the function of this family of transcriptional regulators, we have identified the tobacco 14-3-3 isoform G as the first protein interacting with a DBP factor. 14-3-3 recognition involves the N-terminal region of DBP1, which also supports the DNA binding activity attributed to DBP1. The relevance of this interaction is reinforced by its conservation in Arabidopsis plants, where the closest relative of DBP1 in this species also interacts with a homologous 14-3-3 protein through its N-terminal region. Furthermore, we show that in planta 14-3-3 G is directly involved in regulating DBP1 function by promoting nuclear export and subsequent cytoplasmic retention of DBP1 under conditions that in turn alleviate DBP1-mediated repression of target gene expression.

Heterotopic expression of MPF2 is the key to the evolution of the Chinese lantern of Physalis, a morphological novelty in Solanaceae.

Morphological novelties arise through changes in development, but the underlying causes of such changes are largely unknown. In the genus Physalis, sepals resume growth after pollination to encapsulate the mature fruit, forming the "Chinese lantern," a trait also termed inflated-calyx syndrome (ICS). STMADS16, which encodes a MADS-box transcription factor, is expressed only in vegetative tissues in Solanum tuberosum. Its ortholog in Physalis pubescens, MPF2, is expressed in floral tissues. Knockdown of MPF2 function in Physalis by RNA interference (RNAi) reveals that MPF2 function is essential for the development of the ICS. The phenotypes of transgenic S. tuberosum plants that overexpress MPF2 or STMADS16 corroborate these findings: these plants display enlarged sepals. Although heterotopic expression of MPF2 is crucial for ICS, remarkably, fertilization is also required. Although the ICS is less prominent or absent in the knockdown transgenic plants, epidermal cells are larger, suggesting that MPF2 exerts its function by inhibiting cell elongation and promoting cell division. In addition, severely affected Physalis knockdown lines are male sterile. Thus, heterotopic expression of MPF2 in floral tissues is involved in two novel traits: expression of the ICS and control of male fertility. Sequence differences between the promoter regions of the MPF2 and STMADS16 genes perhaps reflect exposure to different selection pressures during evolution, and correlate with the observed differences in their expression patterns. In any case, the effects of heterotopic expression of MPF2 underline the importance of recruitment of preexisting transcription factors in the evolution of novel floral traits.

Endothelin-1 promotes phosphorylation of CREB transcription factor in primary cultures of neonatal rat cardiac myocytes: implications for the regulation of c- jun expression

Cardiac myocyte hypertrophy is associated with an increase in expression of immediate early genes (e.g. c- jun) via activation of pre-existing transcription factors. The activity of CREB transcription factor is regulated through phosphorylation of Ser-133 by one of several protein kinases (e.g. protein kinase A (PKA), p90 ribosomal S6 kinases (RSKs) and the related kinase, MSK1). A cell-permeable form of cAMP, hypertrophic agonists (endothelin-1 (ET-1), phenylephrine (PE)) and hyperosmotic shock all promoted phosphorylation of CREB(Ser-133) in rat neonatal cardiac myocytes. The response to endothelin-1 required the extracellular signal-regulated kinase cascade which stimulates both RSKs and MSK1. Phosphorylation of CREB(Ser-133) in response to ET-1 was not associated with any increase in DNA binding to a consensus cAMP-response element (CRE). The rat c- jun promoter contains elements which may bind either c-Jun/ATF2 or CREB/ATF1 dimers. Using extracts from rat cardiac myocytes, we identified at least two complexes which bind to the most proximal of these elements, one of which contained CREB and the other c-Jun. Thus, phosphorylation and activation of CREB in cardiac myocytes may be effected by a range of different stimuli to influence the expression of immediate early genes such as c- jun.

Intermediate and long-term memories of associative learning are differentially affected by transcription versus translation blockers in Lymnaea.

Aerial respiratory behaviour in the pond snail, Lymnaea stagnalis, can be operantly conditioned. This associative learning then undergoes consolidation into a long-lasting memory which, depending on the training procedure used, causes intermediate-term memory (ITM; lasting 3 h) or long-term memory (LTM; lasting >6 h) to be formed. We determined the differential susceptibility of these two forms of memory to translation and transcription blockers. The injection of a translation blocker, Anisomycin, 2.5 h before training prevents the establishment of both ITM and LTM. On the other hand, injection of the transcription blocker Actinomycin D, 2.5 h before training, did not prevent the establishment of ITM, but did, however, prevent LTM formation. Thus in Lymnaea, following associative learning, both ITM and LTM are dependent on new protein synthesis. ITM appears to be dependent on protein synthesis from preexisting transcription factors, whilst LTM is dependent on protein synthesis from new transcription messages.

Melphalan-induced expression of IFN-beta in MOPC-315 tumor-bearing mice and its importance for the up-regulation of TNF-alpha expression.

We have previously shown that administration of a low-dose of melphalan (L-phenylalanine mustard; L-PAM) to mice bearing a large s.c. MOPC-315 tumor leads to up-regulation of TNF-alpha expression, which is first evident at the mRNA level at 24 h after the chemotherapy. In this study, we show accumulation of IFN-beta mRNA in the spleen and tumor nodule of such mice as early as 1 h after the chemotherapy followed by elevated production of IFN-beta protein. IFN-beta protein in turn was found to be important for the L-PAM-induced up-regulation of TNF-alpha expression, as neutralization of IFN-beta inhibited the L-PAM-induced up-regulation of TNF-alpha mRNA expression in MOPC-315 tumor cells. In addition, L-PAM failed to up-regulate TNF-alpha expression in spleen cells from mice in which signaling by IFN-beta is deficient. Studies into the mechanism through which L-PAM leads to rapid accumulation of IFN-beta mRNA revealed that it requires de novo RNA synthesis, indicating that the regulation is at the transcriptional level. However, it did not require de novo protein synthesis, indicating that activation of pre-existing transcription factors is sufficient for IFN-beta gene expression. The L-PAM-induced accumulation of IFN-beta mRNA was mimicked with H(2)O(2) and was prevented with the antioxidant N-acetyl-L-cysteine, indicating that reactive oxygen species are involved in the transcriptional regulation of L-PAM-induced IFN-beta gene expression. Thus, the IFN-beta gene is an early response gene that is activated in response to L-PAM via a pathway that involves reactive oxygen species, and IFN-beta in turn plays an important role in L-PAM-induced TNF-alpha up-regulation.

Cell type-specific regulation of fibrinogen expression in lung epithelial cells by dexamethasone and interleukin-1beta.

Our recent studies demonstrating the expression of fibrinogen (FBG) by an alveolar type II cell line stimulated with proinflammatory mediators and also in the inflamed pulmonary epithelium of animals with Pneumocystis carinii pneumonia suggest that extrahepatic FBG participates in the local acute phase response (APR) to infection and subsequent wound repair. However, the mechanisms that regulate extrahepatic FBG expression are poorly understood. This study compares the regulation of hepatic and pulmonary FBG expression by mediators of the APR, interleukin (IL)-6, IL-1beta, and dexamethasone (DEX), a synthetic glucocorticoid. Northern blotting and metabolic labeling studies revealed that IL-6 with or without DEX upregulates gammaFBG messenger RNA and protein, whereas IL-1beta inhibits gammaFBG expression in human lung (A549) and liver (HepG2) epithelial cells. In contrast, the addition of DEX relieved the IL-1beta-mediated inhibition of FBG expression in lung epithelial cells only; this response is termed "DEX rescue." Studies with cycloheximide indicate that only DEX rescue required de novo protein synthesis. Nuclear run-on analysis revealed no increase in gammaFBG transcription by DEX treatment. Although DEX treatment alone increased the stability of gammaFBG transcripts in lung cells, this effect was not observed in the presence of IL-1beta. Together, these results suggest that pre-existing transcription factors mediate the effects of IL-6 with or without DEX, DEX, and IL-1beta on gammaFBG gene expression in lung and liver cells. Also, the data suggest that DEX induces new protein synthesis of an inhibitor of IL-1beta signal transduction to effectively "rescue" FBG production in lung but not liver epithelial cells. This cell type-specific stimulation of FBG production by glucocorticoids to overcome IL-1beta inhibition may promote pulmonary wound repair mechanisms.

Transcriptional activation of the proopiomelanocortin gene by cyclic AMP-responsive element binding protein.

The proopiomelanocortin (POMC) gene expressed in corticotrophs of the anterior pituitary encodes several biologically active peptides and is primarily under the positive control of hypophysiotropic factors (e.g. corticotropin releasing hormone). Using AtT20 cells as a model, we show that these factors increase levels of POMC primary RNA transcripts representative of a transcriptional activation of the gene. This effect is mimicked by several activators of the cAMP signaling pathway. Inhibition of protein synthesis with cycloheximide did not modify the CRH-induced increase in POMC hnRNA suggesting that these early effects are mediated by preexisting transcription factors. Using a reporter gene containing 706 bp of the POMC promoter region, we observe transcriptional activation with the same compounds, their effects being abolished when protein kinase A (PKA) is inactivated by a dominant inhibitory mutant. Promoter deletion analyses mapped an essential cAMP inducible element within the first exon of the POMC gene. This element (PTRE: TGACTAA) located at nucleotides +41/+47 was shown to bind the cAMP responsive element binding protein (CREB) by gel shift analyses and confers strong transcriptional activation by an expression vector coding a CREB-VP16 activator domain fusion protein. Further, expression of a dominant inhibitory mutant of CREB reduced cAMP stimulated transcription of the full length POMC promoter and the PTRE. Taken together, these results show that the major hypophysiotropic factors stimulate POMC transcription through a signaling cascade that involves PKA and CREB.

Differential time course of angiotensin-induced AP-1 and Krox proteins in the rat lamina terminalis and hypothalamus

We studied the time course of expression of the inducible transcription factors (ITF) c-Fos, FosB, c-Jun, JunB, JunD, Krox-20 and Krox-24, induced by a single intracerebroventricular injection of angiotensin II, in the subfornical organ (SFO), median preoptic nucleus (MnPO) paraventricular nucleus (PVN) and supraoptic nucleus (SON). c-Fos and Krox-24 were expressed rapidly in neurons of all four areas but completely disappeared after 4 h. FosB showed a delayed but persistent expression between 4 h and 24 h in the MnPO and PVN. c-Jun was induced in the MnPO, SFO and PVN after 1.5 h and in the SON after 4 h. JunB was selectively expressed in the MnPO and SFO and the level of JunD did not change. The expression of the pre-existing transcription factors SRF, CREB and ATF-2 which contribute to the transcriptional control of jun, fos and krox genes, was not affected by Ang II. Thus, we could show for the first time that an acute stimulation of AT receptors results in continual changes in ITF expression over 24 h.

Fatty Acids Rapidly Induce the Carnitine Palmitoyltransferase I Gene in the Pancreatic β-Cell Line INS-1

Fatty acids are important metabolic substrates for the pancreatic beta-cell, and long term exposure of pancreatic islets to elevated concentrations of fatty acids results in an alteration of glucose-induced insulin secretion. Previous work suggested that exaggerated fatty acid oxidation may be implicated in this process by a mechanism requiring changes in metabolic enzyme expression. We have therefore studied the regulation of carnitine palmitoyltransferase I (CPT I) gene expression by fatty acids in the pancreatic beta-cell line INS-1 since this enzyme catalyzes the limiting step of fatty acid oxidation in various tissues. Palmitate, oleate, and linoleate (0.35 mM) elicited a 4-6-fold increase in CPT I mRNA. The effect was dose-dependent and was similar for saturated and unsaturated fatty acids. It was detectable after 1 h and reached a maximum after 3 h. The induction of CPT I mRNA by fatty acids did not require their oxidation, and 2-bromopalmitate, a nonoxidizable fatty acid, increased CPT I mRNA to the same extent as palmitate. The induction was not prevented by cycloheximide treatment of cells indicating that it was mediated by pre-existing transcription factors. Neither glucose nor pyruvate and various secretagogues had a significant effect except glutamine (7 mM) which slightly induced CPT I mRNA. The half-life of the CPT I transcript was unchanged by fatty acids, and nuclear run-on analysis showed a rapid (less than 45 min) and pronounced transcriptional activation of the CPT I gene by fatty acids. The increase in CPT I mRNA was followed by a 2-3-fold increase in CPT I enzymatic activity measured in isolated mitochondria. The increase in activity was time-dependent, detectable after 4 h, and close to maximal after 24 h. Fatty acid oxidation by INS-1 cells, measured at low glucose, was also 2-3-fold higher in cells cultured with fatty acid in comparison with control cells. Long term exposure of INS-1 cells to fatty acid was associated with elevated secretion of insulin at a low (5 mM) concentration of glucose and a decreased effect of higher glucose concentrations. It also resulted in a decreased oxidation of glucose. The results indicate that the CPT I gene is an early response gene induced by fatty acids at the transcriptional level in beta- (INS-1) cells. It is suggested that exaggerated fatty acid oxidation caused by CPT-1 induction is implicated in the process whereby fatty acids alter glucose-induced insulin secretion.

REVIEW ■ : Jun, Fos, and CREB/ATF Transcription Factors in the Brain: Control of Gene Expression under Normal and Pathophysiological Conditions

The expression and activation of transcription factors and the control of gene transcription in the nervous system is a recent and rapidly expanding field in neurosciences. This research area may provide insights concerning the information transfer that arises from postsynaptic potentials or ligand-coupling of membrane receptors and terminates in gene expression. Visualization of both de novo synthesis of inducible transcription factors (ITFs) and phosphorylation of preexisting transcription factors have been used to mark neurons, pathways, and networks excited by various stimuli. This article summarizes basics of the transcription process and the complex functions of Jun, Fos, and CREB/ATF proteins, as well as the use of ITFs as experimental instruments in neurophysiology and neurobiology. The major focus is on the alterations in ITF expression following acute or chronic pathophysiological stimuli as mirrors of alterations in neuronal programs underlying adaptation, dysfunctions, or the development of diseases affecting the nervous system. NEUROSCIENTIST 2:153-161, 1996