Inhibition of myogenesis by transforming growth factor β is density-dependent and related to the translocation of transcription factor MEF2 to the cytoplasm

Transforming growth factor β (TGF-β) was found to inhibit differentiation of myogenic cells only when they were grown to high density. Inhibition also occurred when myogenic cells were cocultured with other types of mesenchymal cells but not when they were cocultured with epithelial cells. It is therefore possible that some density-dependent signaling mediates the intracellular response to TGF-β. Within 30 min of treatment, TGF-β induced translocation of MEF2, but not MyoD, myogenin, or p21, to the cytoplasm of myogenic cells grown to high density. Translocation was reversible on withdrawal of TGF-β. By using immune electron microscopy and Western blot analysis on subcellular fractions, MEF2 was shown to be tightly associated with cytoskeleton membrane components. To test whether MEF2 export from the nucleus was causally related to the inhibitory action of TGF-β, we transfected C2C12 myoblasts with MEF2C containing the nuclear localization signal of simian virus 40 large T antigen (nlsSV40). Myogenic cells expressing the chimerical MEF2C/nlsSV40, but not wild-type MEF2C, retained this transcription factor in the nucleus and were resistant to the inhibitory action of TGF-β. We propose a mechanism in which the inhibition of myogenesis by TGF-β is mediated through MEF2 localization to the cytoplasm, thus preventing it from participating in an active transcriptional complex.

Interaction of the human androgen receptor transactivation function with the general transcription factor TFIIF

The human androgen receptor (AR) is a ligand-activated transcription factor that regulates genes important for male sexual differentiation and development. To better understand the role of the receptor as a transcription factor we have studied the mechanism of action of the N-terminal transactivation function. In a protein–protein interaction assay the AR N terminus (amino acids 142–485) selectively bound to the basal transcription factors TFIIF and the TATA-box-binding protein (TBP). Reconstitution of the transactivation activity in vitro revealed that AR142–485 fused to the LexA protein DNA-binding domain was competent to activate a reporter gene in the presence of a competing DNA template lacking LexA binding sites. Furthermore, consistent with direct interaction with basal transcription factors, addition of recombinant TFIIF relieved squelching of basal transcription by AR142–485. Taken together these results suggest that one mechanism of transcriptional activation by the AR involves binding to TFIIF and recruitment of the transcriptional machinery.

CooA, a CO-sensing transcription factor from Rhodospirillum rubrum, is a CO-binding heme protein

Biological sensing of small molecules such as NO, O2, and CO is an important area of research; however, little is know about how CO is sensed biologically. The photosynthetic bacterium Rhodospirillum rubrum responds to CO by activating transcription of two operons that encode a CO-oxidizing system. A protein, CooA, has been identified as necessary for this response. CooA is a member of a family of transcriptional regulators similar to the cAMP receptor protein and fumavate nitrate reduction from Escherichia coli. In this study we report the purification of wild-type CooA from its native organism, R. rubrum, to greater than 95% purity. The purified protein is active in sequence-specific DNA binding in the presence of CO, but not in the absence of CO. Gel filtration experiments reveal the protein to be a dimer in the absence of CO. Purified CooA contains 1.6 mol heme per mol of dimer. Upon interacting with CO, the electronic spectrum of CooA is perturbed, indicating the direct binding of CO to the heme of CooA. A hypothesis for the mechanism of the protein’s response to CO is proposed.

Prospero is a panneural transcription factor that modulates homeodomain protein activity

The panneural protein Prospero is required for proper differentiation of neuronal lineages and proper expression of several genes in the nervous system of Drosophila. Prospero is an evolutionarily conserved, homeodomain-related protein with dual subcellular localization. Here we show that Prospero is a sequence-specific DNA-binding protein with novel sequence preferences that can act as a transcription factor. In this role, Prospero can interact with homeodomain proteins to differentially modulate their DNA-binding properties. The relevance of functional interactions between Prospero and homeodomain proteins is supported by the observation that Prospero, together with the homeodomain protein Deformed, is required for proper regulation of a Deformed-dependent neural-specific transcriptional enhancer. We have localized the DNA-binding and homeodomain protein-interacting activities of Prospero to its highly conserved C-terminal region, and we have shown that the two regulatory capacities are independent.

Cloning of the cDNA for the TATA-binding protein-associated factorII170 subunit of transcription factor B-TFIID reveals homology to global transcription regulators in yeast and Drosophila

The human transcription factor B-TFIID is comprised of TATA-binding protein (TBP) in complex with one TBP-associated factor (TAF) of 170 kDa. We report the isolation of the cDNA for TAFII170. By cofractionation and coprecipitation experiments, we show that the protein encoded by the cDNA encodes the TAF subunit of B-TFIID. Recombinant TAFII170 has (d)ATPase activity. Inspection of its primary structure reveals a striking homology with genes of other organisms, yeast MOT1, and Drosophila moira, which belongs to the Trithorax group. Both homologs were isolated in genetic screens as global regulators of pol II transcription. This supports our classification of B-TFIID as a pol II transcription factor and suggests that specific TBP–TAF complexes perform distinct functions during development.

Specificity in transforming growth factor β-induced transcription of the plasminogen activator inhibitor-1 gene: Interactions of promoter DNA, transcription factor μE3, and Smad proteins

Transforming growth factor β (TGF-β) regulates a broad range of biological processes, including cell growth, development, differentiation, and immunity. TGF-β signals through its cell surface receptor serine kinases that phosphorylate Smad2 or Smad3 proteins. Because Smad3 and its partner Smad4 bind to only 4-bp Smad binding elements (SBEs) in DNA, a central question is how specificity of TGF-β-induced transcription is achieved. We show that Smad3 selectively binds to two of the three SBEs in PE2.1, a TGF-β-inducible fragment of the plasminogen activator inhibitor-1 promoter, to mediate TGF-β-induced transcription; moreover, a precise 3-bp spacer between one SBE and the E-box, a binding site for transcription factor μE3 (TFE3), is essential for TGF-β-induced transcription. Whereas an isolated Smad3 MH1 domain binds to TFE3, TGF-β receptor-mediated phosphorylation of full-length Smad3 enhances its binding to TFE3. Together, these studies elucidate an important mechanism for specificity in TGF-β-induced transcription of the plasminogen activator inhibitor-1 gene.

Altered emotional and locomotor responses in mice deficient in the transcription factor CREM

Various transcription factors act as nuclear effectors of the cAMP-dependent signaling pathway. These are the products of three genes in the mouse, CREB, CRE modulator (CREM), and ATF-1. CREM proteins are thought to play important roles within the hypothalamic–pituitary axis and in the control of rhythmic functions in the pineal gland. We have generated CREM-mutant mice and investigated their response in a variety of behavioral tests. CREM-null mice show a drastic increase in locomotion. In contrast to normal mice, the CREM-deficient mice show equal locomotor activity during the circadian cycle. The anatomy of the hypothalamic suprachiasmatic nuclei, the center of the endogenous pacemaker, is normal in mutant mice. Remarkably, CREM mutant mice also elicit a different emotional state, revealed by a lower anxiety in two different behavioral models, but they preserve the conditioned reactiveness to stress. These results demonstrate the high degree of functional specificity of each cAMP-responsive transcription factor in behavioral control.

The maturity-onset diabetes of the young (MODY1) transcription factor HNF4α regulates expression of genes required for glucose transport and metabolism

Hepatocyte nuclear factor 4α (HNF4α) plays a critical role in regulating the expression of many genes essential for normal functioning of liver, gut, kidney, and pancreatic islets. A nonsense mutation (Q268X) in exon 7 of the HNF4α gene is responsible for an autosomal dominant, early-onset form of non-insulin-dependent diabetes mellitus (maturity-onset diabetes of the young; gene named MODY1). Although this mutation is predicted to delete 187 C-terminal amino acids of the HNF4α protein the molecular mechanism by which it causes diabetes is unknown. To address this, we first studied the functional properties of the MODY1 mutant protein. We show that it has lost its transcriptional transactivation activity, fails to dimerize and bind DNA, implying that the MODY1 phenotype is because of a loss of HNF4α function. The effect of loss of function on HNF4α target gene expression was investigated further in embryonic stem cells, which are amenable to genetic manipulation and can be induced to form visceral endoderm. Because the visceral endoderm shares many properties with the liver and pancreatic β-cells, including expression of genes for glucose transport and metabolism, it offers an ideal system to investigate HNF4-dependent gene regulation in glucose homeostasis. By exploiting this system we have identified several genes encoding components of the glucose-dependent insulin secretion pathway whose expression is dependent upon HNF4α. These include glucose transporter 2, and the glycolytic enzymes aldolase B and glyceraldehyde-3-phosphate dehydrogenase, and liver pyruvate kinase. In addition we have found that expression of the fatty acid binding proteins and cellular retinol binding protein also are down-regulated in the absence of HNF4α. These data provide direct evidence that HNF4α is critical for regulating glucose transport and glycolysis and in doing so is crucial for maintaining glucose homeostasis.

Phosphorylation of the rRNA transcription factor upstream binding factor promotes its association with TATA binding protein

rRNA synthesis by RNA polymerase I requires both the promoter selectivity factor 1, which is composed of TATA binding protein (TBP) and three TBP-associated factors, and the activator upstream binding factor (UBF). Whereas there is strong evidence implicating a role for phosphorylation of UBF in the control of growth-induced increases in rRNA transcription, the mechanism of this effect is not known. Results of immunoprecipitation studies with TBP antibodies showed increased recovery of phosphorylated UBF from growth-stimulated smooth muscle cells. Moreover, using an immobilized protein-binding assay, we found that phosphorylation of UBF in vivo in response to stimulation with different growth factors or in vitro with smooth muscle cell nuclear extract increased its binding to TBP. Finally, we demonstrated that UBF–TBP binding depended on the C-terminal ‘acidic tail’ of UBF that was hyperphosphorylated at multiple serine sites after growth factor stimulation. Results of these studies suggest that phosphorylation of UBF and subsequent binding to TBP represent a key regulatory step in control of growth-induced increases in rRNA synthesis.

Interference with gene regulation in living sea urchin embryos: Transcription factor Knock Out (TKO), a genetically controlled vector for blockade of specific transcription factors

“TKO” is an expression vector that knocks out the activity of a transcription factor in vivo under genetic control. We describe a successful test of this concept that used a sea urchin transcription factor of known function, P3A2, as the target. The TKO cassette employs modular cis-regulatory elements to express an encoded single-chain antibody that prevents the P3A2 protein from binding DNA in vivo. In normal development, one of the functions of the P3A2 transcription factor is to repress directly the expression of the CyIIIa cytoskeletal actin gene outside the aboral ectoderm of the embryo. Ectopic expression in oral ectoderm occurs if P3A2 sites are deleted from CyIIIa expression constructs, and we show here that introduction of an αP3A2⋅TKO expression cassette causes exactly the same ectopic oral expression of a coinjected wild-type CyIIIa construct. Furthermore, the αP3A2⋅TKO cassette derepresses the endogenous CyIIIa gene in the oral ectoderm and in the endoderm. αP3A2⋅TKO thus abrogates the function of the endogenous SpP3A2 transcription factor with respect to spatial repression of the CyIIIa gene. Widespread expression of αP3A2⋅TKO in the endoderm has the additional lethal effect of disrupting morphogenesis of the archenteron, revealing a previously unsuspected function of SpP3A2 in endoderm development. In principle, TKO technology could be utilized for spatially and temporally controlled blockade of any transcription factor in any biological system amenable to gene transfer.

The t(8;21) chromosomal translocation in acute myelogenous leukemia modifies intranuclear targeting of the AML1/CBFα2 transcription factor

Targeting of gene regulatory factors to specific intranuclear sites may be critical for the accurate control of gene expression. The acute myelogenous leukemia 8;21 (AML1/ETO) fusion protein is encoded by a rearranged gene created by the ETO chromosomal translocation. This protein lacks the nuclear matrix-targeting signal that directs the AML1 protein to appropriate gene regulatory sites within the nucleus. Here we report that substitution of the chromosome 8-derived ETO protein for the multifunctional C terminus of AML1 precludes targeting of the factor to AML1 subnuclear domains. Instead, the AML1/ETO fusion protein is redirected by the ETO component to alternate nuclear matrix-associated foci. Our results link the ETO chromosomal translocation in AML with modifications in the intranuclear trafficking of the key hematopoietic regulatory factor, AML1. We conclude that misrouting of gene regulatory factors as a consequence of chromosomal translocations is an important characteristic of acute leukemias.

MRG1, the product of a melanocyte-specific gene related gene, is a cytokine-inducible transcription factor with transformation activity

Identification of cytokine-inducible genes is imperative for determining the mechanisms of cytokine action. A cytokine-inducible gene, mrg1 [melanocyte-specific gene (msg1) related gene], was identified through mRNA differential display of interleukin (IL) 9-stimulated and unstimulated mouse helper T cells. In addition to IL-9, mrg1 can be induced by other cytokines and biological stimuli, including IL-1α, -2, -4, -6, and -11, granulocyte/macrophage colony-stimulating factor, interferon γ, platelet-derived growth factor, insulin, serum, and lipopolysaccharide in diverse cell types. The induction of mrg1 by these stimuli appears to be transient, with induction kinetics similar to other primary response genes, implicating its role in diverse biological processes. Deletion or point mutations of either the Box1 motif (binds Janus kinase 1) or the signal transducer and activator of transcription 3 binding site-containing region within the intracellular domain of the IL-9 receptor ligand binding subunit abolished or greatly reduced mrg1 induction by IL-9, suggesting that the Janus kinase/signal transducer and activator of transcription signaling pathway is required for mrg1 induction, at least in response to IL-9. Transfection of mrg1 cDNA into TS1, an IL-9-dependent mouse T cell line, converted these cells to IL-9-independent growth through a nonautocrine mechanism. Overexpression of mrg1 in Rat1 cells resulted in loss of cell contact inhibition, anchorage-independent growth in soft agar, and tumor formation in nude mice, demonstrating that mrg1 is a transforming gene. MRG1 is a transcriptional activator and may represent a founding member of an additional family of transcription factors.

T helper type 2 inflammatory disease in the absence of interleukin 4 and transcription factor STAT6

An important signaling pathway for the differentiation of T helper type 2 (TH2) cells from uncommitted CD4 T cell precursors is activation of the STAT6 transcription factor by interleukin 4 (IL-4). The protooncogene BCL-6 is also involved in TH2 differentiation, as BCL-6 −/− mice develop an inflammation of the heart and lungs associated with an overproduction of TH2 cells. Surprisingly, IL-4 −/− BCL-6 −/− and STAT6 −/− BCL-6 −/− double-mutant mice developed the same TH2-type inflammation of the heart and lungs as is characteristic of BCL-6 −/− mice. Furthermore, a TH2 cytokine response developed in STAT6 −/− BCL-6 −/− and IL-4 −/− BCL-6 −/− mice after immunization with a conventional antigen in adjuvant. In contrast to these in vivo findings, STAT6 was required for the in vitro differentiation of BCL-6 −/− T cells into TH2 cells. BCL-6, a transcriptional repressor that can bind to the same DNA binding motifs as STAT transcription factors, seems to regulate TH2 responses in vivo by a pathway independent of IL-4 and STAT6.

An essential component of a C-terminal domain phosphatase that interacts with transcription factor IIF in Saccharomyces cerevisiae

One of the essential components of a phosphatase that specifically dephosphorylates the Saccharomyces cerevisiae RNA polymerase II (RPII) large subunit C-terminal domain (CTD) is a novel polypeptide encoded by an essential gene termed FCP1. The Fcp1 protein is localized to the nucleus, and it binds the largest subunit of the yeast general transcription factor IIF (Tfg1). In vitro, transcription factor IIF stimulates phosphatase activity in the presence of Fcp1 and a second complementing fraction. Two distinct regions of Fcp1 are capable of binding to Tfg1, but the C-terminal Tfg1 binding domain is dispensable for activity in vivo and in vitro. Sequence comparison reveals that residues 173–357 of Fcp1 correspond to an amino acid motif present in proteins of unknown function predicted in many organisms.

Regulated expression of the diphtheria toxin A chain by a tumor-specific chimeric transcription factor results in selective toxicity for alveolar rhabdomyosarcoma cells

Alveolar rhabdomyosarcoma (ARMS) cells often harbor one of two unique chromosomal translocations, either t(2;13)(q35;q14) or t(1;13)(p36;q14). The chimeric proteins expressed from these rearrangements, PAX3-FKHR and PAX7-FKHR, respectively, are potent transcriptional activators. In an effort to exploit these unique cancer-specific molecules to achieve ARMS-specific expression of therapeutic genes, we have studied the expression of a minimal promoter linked to six copies of a PAX3 DNA binding site, prs-9. In transient transfections, expression of the prs-9-regulated reporter genes was ≈250-fold higher than expression of genes lacking the prs-9 sequences in cell lines derived from ARMS, but remained at or below baseline levels in other cells. High expression of these prs-9-regulated genes was also observed in a cancer cell line that lacks t(2;13) but was stably transfected with a plasmid expressing PAX3-FKHR. Transfection of a plasmid containing the diphtheria toxin A chain gene regulated by prs-9 sequences (pA3–6PED) was selectively cytotoxic for PAX3-FKHR-expressing cells. This was shown by inhibition of gene expression from cotransfected plasmids and by direct cytotoxicity after transfected cells were isolated by cell sorting. Gene transfer of pA3–6PED may thus be useful as a cancer-specific treatment strategy for t(2;13)- or t(1;13)-positive ARMS. Furthermore, gene transfer of fusion protein-regulated toxin genes might also be applied to the treatment of other cancers that harbor cancer-specific chromosomal translocations involving transcription factors.