Showing papers by "Michael Karin published in 1990"

Regulation of the pituitary-specific homeobox gene GHF1 by cell-autonomous and environmental cues.

Abstract: Homeodomain proteins function in determination of mating type in yeast, segmentation in fruit flies and cell-type specific gene expression in mammals. In Drosophila, expression of homeobox genes is controlled by cell-autonomous interactions between regulatory proteins and environmental clues. Similar controls may operate during mammalian limb development and frog embryogenesis. But, the exact way in which expression of homeodomain proteins is regulated in these systems is not clear and requires biochemical analysis of homeobox gene transcription. We now describe such an analysis of the GHF1 gene, which encodes a mammalian homeodomain protein specifying expression of the growth hormone (GH) gene in anterior pituitary somatotrophs. GHF1 is transcribed in a highly restricted manner and the presence of GHF1 protein is correlated both temporally and spatially with activation of the GH gene during pituitary development. Analysis of the GHF1 promoter indicates that transcription is also controlled by cell-autonomous interactions involving positive autoregulation by GHF1, and environmental cues that modulate the intracellular level of cyclic AMP and thereby the activity of cAMP response element binding protein (CREB), a ubiquitous transactivator that binds to the GHF1 promoter.

Interleukin-1 stimulates and all-trans-retinoic acid inhibits collagenase gene expression through its 5' activator protein-1-binding site.

Abstract: Collagenase production by synovial fibroblast-like cells (synoviocytes) plays a major role in cartilage and bone destruction in rheumatoid arthritis. Interleukin-1 (IL-1) increases collagenase secretion by elevating the steady state levels of collagenase mRNA in cultured rheumatoid synoviocytes, while all-trans-retinoic acid (RA) has the opposite effect. We have studied the regulation of collagenase gene transcription by IL-1 and RA in synoviocytes by transient transfection of plasmid constructs containing deletion mutants of the 5'-flanking region of the collagenase gene or the isolated phorbol ester-responsive element ligated to a chloramphenicol acetyltransferase reporter gene. We show that the phorbol ester-responsive element of the collagenase gene mediates both positive and negative regulatory effects, respectively, of IL-1 and RA on transcription. In addition, we show that IL-1 and 12-O-tetradecanoyl-phorbol-13-acetate transiently induce c-jun and c-fos expression and that retinoic acid inhibits IL-1 and 12-O-tetradecanoyl-phorbol-13-acetate induction of c-fos, but not c-jun. These results suggest that RA inhibits collagenase transcription at least in part through inhibition of c-fos.

Too many transcription factors: positive and negative interactions.

Abstract: Eukaryotic transcription factors can be classified into several families on the basis of conserved sequences among their DNA-binding domains. Because of such structural conservation, several different trans-acting factors can often interact with a common binding site. Recent findings reviewed herein indicate that the interaction of different factors with a common target site does not necessarily result in equivalent transcriptional responses. While some factors activate transcription, others that bind to the same site repress this process. The examples described here illustrate an emerging new mode of cellular regulation mediated by closely related but functionally distinct transcription factors that appear to compete for common binding sites.

A Simplified Method for the Preparation of Transcriptionally Active Liver Nuclear Extracts

Abstract: We have developed a simplified method for the preparation of liver nuclear extracts to study gene regulation and protein-DNA interactions. This protocol uses conventional laboratory equipment and standard reagents. The liver tissue is homogenized in a low-salt solution at physiological molarity with subsequent adjustment of the molarity and purification of nuclei by density sedimentation. The nuclear extracts are transcriptionally active in a validated cell-free transcription assay and contain functional DNA-binding proteins. This protocol results in the rapid preparation of highly reproducible and active liver nuclear extracts.

Overexpressed human metallothionein IIA gene protects Chinese hamster ovary cells from killing by alkylating agents.

Abstract: Experiments were designed to detect survival advantages that cells gain by overexpressing metallothionein (MT). Chinese hamster ovary K1-2 cells and an x-ray-sensitive derivative were transfected with a bovine papillomavirus (BPV)-linked construct carrying the human metallothionein IIA (hMT-IIA) gene. Transfectants survived 40-fold higher levels of cadmium chloride, harbored at least 30 copies of hMT-IIA, and contained 25- to 166-fold more MT than the parent cells. Even under conditions of reduced glutathione synthesis, the transfectants were not more resistant to the lethal effects of ionizing radiation and bleomycin than the parent cells. Thus free radicals generated by these agents cannot be scavenged efficiently by MT in vivo. The hMT-IIA transfectants, however, but not control transfectants harboring a BPV-MT promoter-neo construct, tolerated significantly higher doses of the alkylating agents N-methyl-N-nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine. Resistance and MT overexpression occurred irrespective of selection and cultivation in cadmium and zinc. There was no increase in resistance to methyl methanesulfonate and N-hydroxyethyl-N-chloroethylnitrosourea. MT did not affect the degree of overall DNA methylation after N-methyl-N-nitrosourea treatment nor the level of O6-methylguanine-DNA methyltransferase. The results suggest that MT participates as a cofactor or regulatory element in repair or tolerance of toxic alkylation lesions.

Elevation of AP1 activity during F9 cell differentiation is due to increased c-jun transcription.

Abstract: The regulation of jun family genes and AP1 activity during the course of differentiation of F9 embryonal carcinoma stem cells was investigated. The induction of differentiation by retinoic acid (RA) leads to an accumulation of c-jun mRNA caused by increased c-jun transcription. This induction is an indirect response to RA and requires a functional AP1 binding site within the c-jun promoter. Expression of jun-B mRNA, however, is transiently induced but at a later time point is repressed by RA. The third member of the family, jun-D, is already active in undifferentiated cells and is only slightly induced after differentiation. Differentiation also converts c-jun from being refractory to phorbol esters to a highly inducible state. The development of this response is correlated with increased AP1 activity in RA-treated cells. By contrast, the induction of c-fos by phorbol esters or cAMP is greatly diminished after RA treatment. Transfection experiments indicate that, in the absence of c-Fos, only c-Jun is an effective transactivator. Hence, the major increase in AP1 activity is due to elevated c-jun expression and probably involves positive autoregulation by the c-Jun protein. Furthermore, these results demonstrate that AP1 activity can be stimulated by phorbol ester without concomitant c-fos induction. Forced expression of c-Jun and v-Jun results in activation of at least two differentiation marker genes, EndoB and tissue plasminogen activator, whose regulatory regions contain AP1 binding sites. Thus, the induction of c-jun transcription by RA, although indirect, can have an important role in the differentiation process.

Regulation of collagenase gene expression by okadaic acid, an inhibitor of protein phosphatases

Abstract: Human collagenase gene expression is regulated transcriptionally and is inducible by various mitogens in many cell types. To investigate the molecular mechanisms of this response, we examined the effects on collagenase gene expression of okadaic acid, a non-12-O-tetradecanoyl-phorbol-13-acetate (TPA)-type tumor promoter, which induces apparent "activation" of protein kinases by inhibition of protein phosphatases. Steady state levels of collagenase mRNA were markedly increased by okadaic acid treatment. We show that the AP-1 consensus sequence in the collagenase promoter is required for the induction of collagenase gene expression by okadaic acid, even though sequences upstream of the AP-1 consensus site have an additive effect. We also examined the regulation by okadaic acid of expression of the components of the AP-1 complex, c-fos and c-jun. c-fos expression is dramatically stimulated by okadaic acid, whereas c-jun expression is stimulated to a lesser extent. Induction of c-fos gene mRNA occurs through a region known to contain multiple regulatory elements. These results suggest that phosphorylation regulates collagenase gene expression mediated by an AP-1 binding site.

A single amino acid change in CUP2 alters its mode of DNA binding.

Abstract: CUP2 is a copper-dependent transcriptional activator of the yeast CUP1 metallothionein gene. In the presence of Cu+ and Ag+) ions its DNA-binding domain is thought to fold as a cysteine-coordinated Cu cluster which recognizes the palindromic CUP1 upstream activation sequence (UASc). Using mobility shift, methylation interference, and DNase I and hydroxyl radical footprinting assays, we examined the interaction of wild-type and variant CUP2 proteins produced in Escherichia coli with the UASc. Our results suggest that CUP2 has a complex Cu-coordinated DNA-binding domain containing different parts that function as DNA-binding elements recognizing distinct sequence motifs embedded within the UASc. A single-amino-acid substitution of cysteine 11 with a tyrosine results in decreased Cu binding, apparent inactivation of one of the DNA-binding elements and a dramatic change in the recognition properties of CUP2. This variant protein interacts with only one part of the wild-type site and prefers to bind to a different half-site from the wild-type protein. Although the variant has about 10% of wild-type DNA-binding activity, it appears to be completely incapable of activating transcription.

DNA-binding activity of Jun is increased through its interaction with Fos.

Abstract: Transcription factor AP-1 mediates induction of a set of genes in response to the phorbol ester tumor promoter TPA Recently, AP-1 preparations from HeLa cells were shown to contain a product of the c-JUN protooncogene (Jun/AP-1) which forms a tight complex with the Fos protein In this paper, we examine the role of the Fos protein in the DNA-binding activity of the AP-1 complex We show that the DNA-binding activity of bacterially expressed trpE-Jun fusion proteins is increased many-fold upon their interaction with Fos (or a Fos-related antigen) expressed from a baculovirus vector The site of Fos interaction is within the DNA-binding domain of Jun/AP-1, and anti-Fos antibodies interfere with the binding of affinity purified AP-1 to DNA These results suggest that, by associating with Jun/AP-1, Fos is responsible for the formation of a multimeric protein complex that has greater affinity for the target sequence than does Jun/AP-1 alone

Tissue-specific expression of the growth hormone gene and its control by growth hormone factor-1.

Abstract: Publisher Summary This chapter discusses the tissue-specific expression of the growth hormone gene and its control by growth hormone. The GH gene family represents an excellent system for studying the mechanisms responsible for cell-type-specific gene expression. Rodents contain a single GH gene, and primates contain five tandemly linked GH genes. In addition to GH, this family includes several other genes coding for hormones of physiological and clinical importance such as prolactin (PRL) and the various placental lactogens (PLs). The GH and PRL genes arose by duplication of a common precursor. The function of the various PLs is not clear, but they are thought to have a role in lactogenesis, fetal growth, and stimulation of the corpus luteum and to affect carbohydrate and lipid metabolism. All of these hormones function in the control of growth and development through fetal and adult life. Insufficient production of GH leads to growth retardation or dwarfism, while its overproduction before puberty leads to gigantism and to acromegaly after puberty. Insufficient production of PRL causes a failure in lactation. Overproduction of PRL leads to amenorrhea or impotence, and low levels of PLs seem to correlate with fetal-growth retardation. Several different protein factors binding to the GH promoter are detected, of which one is unique to GH-expressing cells. A restricted distribution of the protein—known as GHF-1—suggests that it plays a major role in the pituitary-specific expression of the GH gene. The chapter discusses various features of GHF-1. GHF-1 has a novel type of activation domain that interacts with the components of the transcriptional machinery.

A novel T-cell trans-activator that recognizes a phorbol ester-inducible element of the interleukin-2 promoter.

Abstract: The interleukin 2 (IL-2) gene promoter is recognized by several cell-type-specific and ubiquitous transcriptional regulators that integrate information transmitted by various signaling systems leading to IL-2 production and T-cell activation. Using a combination of transfection, protein-DNA binding, and in vitro transcription methods, we have discovered the novel T-cell-specific transcriptional activator TCF-1 (for T-Cell Factor-1), which recognizes a T-cell-specific response element (TCE) located within the IL-2 promoter. Although the TCE is similar in sequence to a consensus NF kappa B site, several criteria indicate that TCF-1 is distinct from NF kappa B. However, like NF kappa B, TCF-1 activity is induced by phorbol esters and other T-cell activators.

Transcriptional Activation of Human and Yeast Metallothionein Genes by Heavy Metal Ions

Abstract: Organisms from yeast to mammals express low molecular weight metal binding, cysteine-rich proteins, the metallotheineins (MTs). The metal-dependent expression of MTs is controlled transcriptionally. Up to now no transacting factor conferring metal-inducibility upon mammalian MT-genes was isolated. Recently, however, the product of the yeast CUP2 gene was identified as a sequence specific Cu-dependent activator of the yeast MT, CUP1 gene. The N-terminal DNA-binding domain of CUP2 resembles CUP1 in its cystein-content and arrangement. Study of in vitro synthesized wild-type and mutant CUP2 proteins demonstrated the effect of metal-binding on specific DNA-affinity and the importance of single amino acid residues in this process.

Cell type specific expression of the growth hormone gene and its control by GHF-1.

Abstract: One of the major challenges in molecular genetics is to determine the mechanisms which control the utilization of genetic information in time and space dependent manners. Differential gene activation is the basis for the processes of cellular differentiation and specialization. Through genetic analysis, a great deal has been learned about the general mechanisms which control these processes in organisms such as D. melanogaster and C . elegans . Although mammals, on the other hand, are refractory to genetic analysis, significant progress has been made in understanding the mechanisms involved in determining tissue specific gene expression in these organisms. This can be mostly attributed to a biochemical approach employing in vitro transcription systems in which differential promoter utilization could be observed, resulting in identification of transcription factors mediating cell-type specific gene expression (Scheidereit et al. 1987; Lichtsteiner et al. 1989; Bodner and Karin 1987) . Several of these factors were purified to homogeneity and their cDNAs were cloned (Bodner et al. 1988; Scheidereit et al. 1988) . In this review we will describe our studies on the control of growth hormone (GH) gene expression. A major emphasis will be placed on the mechanisms responsible for the cell type specific expression of this gene. The GH gene family represents an excellent system for studying the mechanisms responsible for cell type specific gene expression (Karin, 1989) . In addition to GH, this family includes several other genes coding for hormones of physiological and clinical importance, such as prolactin (Prl) and the various placental lactogens (PL) (Miller and Eberhardt, 1983) . GH is specifically expressed in specialized cells, the somatotrophs, of the anterior pituitary. It is required for postnatal growth and maintenance of nitrogen, mineral, lipid and carbohydrate metabolism (Martin, 1973) . Its most important function is probably the stimulation of protein synthesis,