Pathway-specific Transcription Factor Research Articles

Beyond microarrays: Finding key transcription factors controlling signal transduction pathways

BackgroundMassive gene expression changes in different cellular states measured by microarrays, in fact, reflect just an "echo" of real molecular processes in the cells. Transcription factors constitute a class of the regulatory molecules that typically require posttranscriptional modifications or ligand binding in order to exert their function. Therefore, such important functional changes of transcription factors are not directly visible in the microarray experiments.ResultsWe developed a novel approach to find key transcription factors that may explain concerted expression changes of specific components of the signal transduction network. The approach aims at revealing evidence of positive feedback loops in the signal transduction circuits through activation of pathway-specific transcription factors. We demonstrate that promoters of genes encoding components of many known signal transduction pathways are enriched by binding sites of those transcription factors that are endpoints of the considered pathways. Application of the approach to the microarray gene expression data on TNF-alpha stimulated primary human endothelial cells helped to reveal novel key transcription factors potentially involved in the regulation of the signal transduction pathways of the cells.ConclusionWe developed a novel computational approach for revealing key transcription factors by knowledge-based analysis of gene expression data with the help of databases on gene regulatory networks (TRANSFAC® and TRANSPATH®). The corresponding software and databases are available at .

HC-toxin

HC-toxin is a cyclic tetrapeptide of structure cyclo(D-Pro-L-Ala-D-Ala-L-Aeo), where Aeo stands for 2-amino-9,10-epoxi-8-oxodecanoic acid. It is a determinant of specificity and virulence in the interaction between the producing fungus, Cochliobolus carbonum, and its host, maize. HC-toxin qualifies as one of the few microbial secondary metabolites whose ecological function in nature is understood. Reaction to C. carbonum and to HC-toxin is controlled in maize by the Hm1 and Hm2 loci. These loci encode HC-toxin reductase, which detoxifies HC-toxin by reducing the 8-carbonyl group of Aeo. HC-toxin is an inhibitor of histone deacetylases (HDACs) in many organisms, including plants, insects, and mammals, but why inhibition of HDACs during infection by C. carbonum leads to disease is not understood. The genes for HC-toxin biosynthesis (collectively known as the TOX2 locus) are loosely clustered over >500 kb in C. carbonum. All of the known TOX2 genes are present in multiple, functional copies and are absent from natural toxin non-producing isolates. The central enzyme in HC-toxin biosynthesis is a 570-kDa non-ribosomal synthetase encoded by a 15.7-kb open reading frame. Other genes known to be required for HC-toxin encode alpha and beta subunits of fatty acid synthase, which are presumed to contribute to the synthesis of Aeo; a pathway-specific transcription factor; an efflux carrier; a predicted branched-chain amino acid aminotransferase; and an alanine racemase.

Regulatory elements in aflatoxin biosynthesis

This review provides a synopsis of factors involved in the regulation of aflatoxin inAspergillus species at the molecular level. Much of the knowledge available today on the regulation of secondary metabolite production in fungi has been gleaned from studies of the aflatoxin gene cluster inA. flavus andA. parasiticus and the sterigmatocystin gene cluster inA. nidulans. Regulation of these two gene clusters is under the control of both pathway-specific transcription factors such as AflR and AflJ and global or broad-domain transcription factors such as AreA and PacC. Study of secondary metabolite (sec-) mutants inA. parasiticus first identified an association between mycotoxin production and fungal development. This linkage has been extended at the molecular level by the characterization of a G-protein/cAMP/Protein kinase A signaling pathway that regulates sporulation via the transcription factor BrlA and aflatoxin/sterigmatocystin production via AflR. Another global regulator of mycotoxin production, VeA, mediates a developmental light-response inA. nidulans andA. flavus. Though not similar to any known fungal transcriptional regulators, VeA controls aflatoxin/sterigmatocystin production via transcriptional control of AflR and it also regulates development of sexual structures such as cleistothecia inA. nidulans and sclerotia inA. flavus.

Patterns of nucleosomal organization in the alc regulon of Aspergillus nidulans: roles of the AlcR transcriptional activator and the CreA global repressor

We have studied the chromatin organization of three promoters of the alc regulon of Aspergillus nidulans. No positioned nucleosomes are seen in the aldA (aldehyde dehydrogenase) promoter under any physiological condition tested by us. In the alcA (alcohol dehydrogenase I) and alcR (coding for the pathway-specific transcription factor) promoters, a pattern of positioned nucleosomes is seen under non-induced and non-induced repressed conditions. While each of these promoters shows a specific pattern of chromatin restructuring, in both cases induction results in loss of nucleosome positioning. Glucose repression in the presence of inducer results in a specific pattern of partial positioning in the alcA and alcR promoters. Loss of nucleosome positioning depends absolutely on the AlcR protein and it is very unlikely to be a passive result of the induction of transcription. In an alcR loss-of-function background and in strains carrying mutations of the respective AlcR binding sites of the alcA and alcR promoters, nucleosomes are fully positioned under all growth conditions. Analysis of mutant AlcR proteins establishes that all domains needed for transcriptional activation and chromatin restructuring are included within the first 241 residues. The results suggest a two-step process, one step resulting in chromatin restructuring, a second one in transcriptional activation. Partial positioning upon glucose repression shows a specific pattern that depends on the CreA global repressor. An alcR loss-of-function mutation is epistatic to a creA loss-of-function mutation, showing that AlcR does not act by negating a nucleosome positioning activity of CreA.

Endotoxin recognition and signal transduction by the TLR4/MD2-complex

Bacterial lipopolysaccharides are recognized in mammals by a receptor complex composed of CD14, Toll-like receptor (TLR)-4 and MD-2. Transduction of signaling is achieved following the recruitment of a combination of four Toll-interleukin-1 resistance (TIR)-domain-containing adapter molecules, which provide a structural platform enabling the recruitment and activation of downstream effectors essential for pathway-specific transcription factor activation and inflammatory gene expression.

Signaling Mucin in Yeast

In two separate screens for genes involved in the filamentous growth (FG) pathway in yeast, Cullen et al. identified MSB2 , which encodes a cell surface protein with a single transmembrane domain and a large extracellular domain with Ser-Thr-Pro-rich repeats typical of mammalian glycoproteins called mucins. MSB2 expression was stimulated by treatments that activated the FG pathway and was dependent on the FG pathway-specific transcription factor Ste12. Yeast with mutations in msb2 were defective in agar invasion and single-cell invasion assays, as well as pseudohyphal growth and activation of FG target genes, which confirmed the involvement of Msb2 as a component in the FG pathway. Sho1 is a plasma membrane protein involved in both the FG pathway and the osmosensing pathway. Overexpression of Msb2 or Sho1 causes hyperinvasive growth, and yeast with mutant sho1 or msb2 show the same phenotype. Cullen et al. observed an interaction between Sho1 and Msb2 using coimmunoprecipitation assays. In various assays, the cytoplasmic domain of Msb2 interacted with Cdc42, the guanosine triphosphatase that is downstream of Sho1 in the FG pathway. Deletion of the mucin domain or an N-terminal domain distinct from the mucin domain of Msb2 resulted in constitutive activation of the FG pathway. Glycosylation of Msb2 may be important for its negative effect on the FG pathway, and glycosylation-defective yeast show activation of the FG pathway that is Msb2- dependent. Yeast with deletion of the mucin domain of Msb2 (Msb2*) exhibit hyperactivity in the FG pathway, including increased phosphorylation and abundance of the FG-specific mitogen-activated protein kinase (MAPK) Kss1. Responsiveness to mating factor mediated by the mating pathway-specific MAPK Fus3 was not affected in yeast with mutant msb2 . Msb2 was also not required for activation of the osmosensing pathway. Thus, Msb2 appears to be a FG pathway-specific protein that controls the activity of the FG pathway by interacting with both Sho1 and Cdc42. P. J. Cullen, W. Sabbagh Jr., E. Graham, M. M. Irick, E. K. van Olden, C. Neal, J. Delrow, L. Bardwell, G. F. Sprague Jr., A signaling mucin at the head of the Cdc42- and MAPK-dependent filamentous growth pathway in yeast. Genes Dev. 18 , 1695-1708 (2004). [Abstract] [Full Text]

Identification of new genes positively regulated by Tri10 and a regulatory network for trichothecene mycotoxin production.

Tri10, a regulatory gene in trichothecene mycotoxin-producing Fusarium species, is required for trichothecene biosynthesis and the coordinated expression of four trichothecene pathway-specific genes (Tri4, Tri5, Tri6, and Tri101) and the isoprenoid biosynthetic gene for farnesyl pyrophosphate synthetase (FPPS). We showed that six more trichothecene genes (Tri3, Tri7, Tri8, Tri9, Tri11, and Tri12) are regulated by Tri10. We also constructed a cDNA library from a strain of Fusarium sporotrichioides that overexpresses Tri10 ( upward arrow Tri10) and used cDNA derived from the upward arrow Tri10 strain and a non-Tri10-expressing strain (DeltaTri10) to differentially screen macroarrays prepared from the cDNA library. This screen identified 15 additional Tri10-regulated transcripts. Four of these transcripts represent Tri1, Tri13, and Tri14 and a gene designated Tri15. Three other sequences are putative orthologs of genes for isoprenoid biosynthesis, the primary metabolic pathway preceding trichothecene biosynthesis. The remaining eight sequences have been designated Ibt (influenced by Tri10) genes. Of the 26 transcripts now known to be positively regulated by Tri10, 22 are positively coregulated by Tri6, a gene that encodes a previously characterized trichothecene pathway-specific transcription factor. These 22 Tri10- and Tri6-coregulated sequences include all of the known Tri genes (except for Tri10), the FPPS gene, and the other three putative isoprenoid biosynthetic genes. Tri6 also regulates a transcript that is not regulated by Tri10. Thus, Tri10 and Tri6 regulate overlapping sets of genes that include a common group of multiple genes for both primary and secondary metabolism.

The Amino Terminus of Smads Permits Transcriptional Specificity

Smad proteins transduce transforming growth factor-β signals from the cell surface to the nucleus, regulating a variety of physiologic processes. In the nucleus, Smads control gene expression by binding to both DNA and transcription factors. Individual Smads regulate distinct subsets of target genes. The key residues important for this specificity are thought to reside in the carboxyl-terminal MH2 domain. To further examine Smad specificity in vivo, we undertook structure-function studies in Xenopus laevis embryos and found that truncated Smads containing the MH2 domain activate gene transcription. A striking finding revealed by the in vivo analyses was that the functional truncated Smads all behaved identically and had lost wild-type specificity. For most Smads, wild-type activity required the presence of an MH1 domain, either in cis or in trans. Of note, even heterologous MH1 domains could restore wild-type signaling specificity to effector MH2 domains. We found a possible mechanism to account for these observations, as Smad MH1 domains altered the binding of pathway-specific transcription factors to the MH2 domain. Thus, Smad MH1 domains are important to the regulation of transcriptional specificity.

Transforming Growth Factor-β and Ciliary Neurotrophic Factor Synergistically Induce Vasoactive Intestinal Peptide Gene Expression through the Cooperation of Smad, STAT, and AP-1 Sites

The cytokine ciliary neurotrophic factor (CNTF) and transforming growth factor-beta (TGF-beta) both induce transcription of the vasoactive intestinal peptide (VIP) gene through a 180-base pair cytokine response element (CyRE) in the VIP promoter. While CNTF induces STAT and AP-1 proteins to bind to cognate sites in the VIP CyRE, the mechanism through which TGF-beta acts to induce VIP gene transcription is not known. Here we show that Smad3 and Smad4 proteins can bind to two distinct sites within the VIP CyRE. These sites are absolutely required for the induction of VIP CyRE transcription by TGF-beta. TGF-beta induces endogenous Smad-containing complexes to bind to these sites in human neuroblastoma cells. CNTF and TGF-beta synergize to induce VIP mRNA expression and transcription through the VIP CyRE. This synergy is dependent on the Smad, STAT, and AP-1 sites, suggesting that these two independent cytokine pathways synergize through the cooperation of pathway-specific transcription factors binding to distinct sites within the VIP CyRE.

Aspergillus nidulans mutants defective in stc gene cluster regulation.

The genes involved in the biosynthesis of sterigmatocystin (ST), a toxic secondary metabolite produced by Aspergillus nidulans and an aflatoxin (AF) precursor in other Aspergillus spp., are clustered on chromosome IV of A. nidulans. The sterigmatocystin gene cluster (stc gene cluster) is regulated by the pathway-specific transcription factor aflR. The function of aflR appears to be conserved between ST- and AF-producing aspergilli, as are most of the other genes in the cluster. We describe a novel screen for detecting mutants defective in stc gene cluster activity by use of a genetic block early in the ST biosynthetic pathway that results in the accumulation of the first stable intermediate, norsolorinic acid (NOR), an orange-colored compound visible with the unaided eye. We have mutagenized this NOR-accumulating strain and have isolated 176 Nor(-) mutants, 83 of which appear to be wild type in growth and development. Sixty of these 83 mutations are linked to the stc gene cluster and are likely defects in aflR or known stc biosynthetic genes. Of the 23 mutations not linked to the stc gene cluster, 3 prevent accumulation of NOR due to the loss of aflR expression.

Sequence-specific binding by Aspergillus nidulans AflR, a C6 zinc cluster protein regulating mycotoxin biosynthesis.

The Aspergillus nidulans aflR gene is found within a 60 kb gene cluster that includes approximately 24 other genes that putatively function in the production of the aflatoxin-related mycotoxin sterigmatocystin. Previous work showed that AflR is a C6 zinc binuclear cluster protein that is conserved across Aspergillus spp. and functions as a pathway-specific transcription factor in activating expression of other cluster genes. In this report, we demonstrate that A. nidulans AflR (AnAflR) is a 45kDa protein that binds to the palindromic sequence 5'-TCG(N5)CGA-3' found in the promoter regions of several aflatoxin and sterigmatocystin cluster genes (stc genes). The in vivo relevance of this AnAflR binding site was assessed by examining the contribution of the three TCG(N5)CGA elements in the 1.1 kb promoter region of stcU using gene fusions with the bacterial uidA gene encoding beta-glucuronidase (GUS). By mutating one, two or all three of the AnAflR-binding elements and examining GUS activity in wild-type aflR or delta aflR A. nidulans strains, we found that stc gene activation required both AnAflR and at least one TCG(N5)CGA AflR binding site.

Interference between pathway-specific transcription factors: glucocorticoids antagonize phorbol ester-induced AP-1 activity without altering AP-1 site occupation in vivo.

Phorbol esters stimulate and glucocorticoid hormones down-regulate a variety of promoters such as that of the collagenase gene through the transcription factor AP-1 (Fos/Jun). We now show by genomic footprinting of the collagenase promoter that phorbol ester treatment of cells results in the binding of AP-1 to its cognate DNA binding site in vivo. The DNA-protein contacts obtained in living cells are also found in vitro using cloned DNA and purified AP-1. Although in vitro synthesized glucocorticoid receptor can disturb the DNA binding of Jun homodimers, it does not interfere with the binding of Fos-Jun heterodimers or of purified AP-1 in vitro. Consistently, fully inhibitory doses of glucocorticoid hormone cause no change in apparent occupation of the AP-1 binding site in vivo. The hormone receptor acts without itself binding to DNA.