Biomedical subjects

M Yaniv

Publications and source records attributed to M Yaniv.

At least 19 recordsLinked to original sources

PubMed1992

A potent enhancer made of clustered liver-specific elements in the transcription control sequences of human alpha 1-microglobulin/bikunin gene.

alpha 1-Microglobulin (A1M) and bikunin are plasma proteins which are present both as free molecules and as complexes with either IgA heavy chains for A1M or the H1, H2, and H3 heavy chains of the inter-alpha-inhibitor family for bikunin. Mature A1M and bikunin originate from the cleavage of an A1M/bikunin precursor (ABP) synthesized from a single gene with liver-specific expression. Five kilobases of the 5'-flanking region of the human ABP gene were sequenced. Deletion mutants of this region subcloned upstream of a CAT reporter gene were transfected into HepG2 hepatoma cells. A segment covering the -2.7- to -2.8-kb area is required for full activity of the ABP gene. This segment contains a cluster of six elements (boxes 1-6, 5' to 3') which are potential binding sites for the liver-enriched trans-acting factors HNF-1, HNF-4, HNF-3, HNF-1, HNF-3, and HNF-4, respectively. This cluster enhances the activity of heterologous minimal promoters in a position- and distance-independent fashion in HepG2 cells. This enhancer activity is restricted to liver cells as the cluster is unable to activate promoters in Chinese hamster ovary (CHO) or HeLa cells. By band-shift experiments we have shown that the liver-enriched transcription factors HNF-1, or HNF-3, do bind to boxes 1 and 4, or 3, respectively. The combination of a weak promoter and a strong distant and liver-specific enhancer distinguishes the ABP gene from most other plasma protein genes expressed in hepatocytes.

Alpha-Globulins↗

PubMed1992

Structure of the gene encoding hepatocyte nuclear factor 1 (HNF1).

Genomic clones have been isolated that cover the entire gene for the transcription factor HNF1 (hepatocyte nuclear factor 1). This protein governs the expression of many genes, synthesized in the liver in a tissue-specific manner. We have determined the intron/exon structure of the HNF1 gene, which is strictly conserved between rat and mouse and estimate that it spans not more than 40kb in the rat genome. Whereas most homeoprotein genes do not contain introns within the homeodomain, HNF1 displays an intron between the regions encoding the second and the third helices. We discuss possible evolutionary mechanisms leading to this homeobox intron/exon pattern.

Amino Acid Sequence↗

PubMed1992

How do eukaryotic activator proteins stimulate the rate of transcription by RNA polymerase II?

A large number of activator proteins have now been identified in higher and lower eukaryotes, which bind to the regulatory regions of protein-encoding genes and increase the rate at which they are transcribed by RNA polymerase II. The mechanism by which activators function is being intensively studied and some of the targets of transcriptional activation domains have now been identified. These studies have also revealed novel classes of regulatory factors, which were not anticipated by extrapolating from the principles obtained with prokaryotic promoters.

Animals↗

PubMed1992

HNF1, a homeoprotein member of the hepatic transcription regulatory network.

Numerous liver specific genes are transcriptionally activated by the binding to their promoter or enhancer of Hepatic Nuclear Factor 1 (HNF1). HNF1 contains a variant homeo-domain and binds to DNA as either a homodimer or a heterodimer with the vHNF1 protein. Surprisingly, HNF1 is not restricted to hepatocytes but is expressed in epithelial cells of several endoderm derived organs and in mesoderm derived kidney tubules. Hence, HNF1 alone can not account for the differentiated state of the hepatic cells. In fact, several other liver-enriched transcription factors have been cloned. The hepatic phenotype could result from the combinatorial expression of these regulators. Possible involvement of these trans-acting factors in liver organogenesis and hepatic differentiation is discussed.

Amino Acid Sequence↗

PubMed1992

Two AP1 sites binding JunB are essential for human papillomavirus type 18 transcription in keratinocytes.

The activity and epithelial tropism of the human papillomavirus type 18 P105 early promoter, which directs the synthesis of the E6 and E7 transforming genes, are controlled by cis elements included in the viral long control region. To identify potential cellular regulators of this promoter, we mutagenized one or both of the 5'-TGACTAA-3' cis elements capable of interacting with the AP1 transcription factor, which is composed either of homodimers or heterodimers of the Jun products or of heterodimers of Jun and Fos. Mutation of both elements completely abolished P105 promoter activity in human keratinocytes. We show that either AP1 site can interact efficiently in vitro with any of the three different Jun products as heterodimers with c-Fos. However, in nuclear extracts prepared from human keratinocytes, JunB was the predominant Jun component bound to the DNA probe containing this cis element. These results implicate JunB as an important factor in human papillomavirus type 18 transcription in keratinocytes and strongly suggest a potential role of this Jun gene product in the tissue-specific transcription of the genital papillomaviruses.

Base Sequence↗

PubMed1991

Wild-type p53 can down-modulate the activity of various promoters.

The wild-type (wt) p53 protein is the product of a tumor suppressor gene that is a frequent target for inactivation in many types of tumors. The nuclear localization of the protein, as well as additional features, suggest that it may be involved in the regulation of gene expression. To explore this possibility, the effects of overproduced wt p53 were investigated in a number of systems. Induction of growth arrest via the antiproliferative effect of wt p53 greatly impaired the ability of cells to exhibit an increase in c-fos mRNA upon serum stimulation. Experiments in which cells were cotransfected with p53 expression plasmids together with a reporter gene linked to various promoters revealed that wt p53 could effectively reduce transcription from a series of promoters derived from serum-inducible genes, but not from a major histocompatibility complex gene. The p53-mediated repression of c-fos gene expression occurred even in the presence of cycloheximide. Kinetic studies indicate that the effect of wt p53 is rapid, rather than representing a secondary consequence of growth arrest. These findings support a role for p53 in transcriptional regulation, perhaps by reducing the expression of genes that are needed for ongoing cell proliferation.

Animals↗

PubMed1991

NFY or a related CCAAT binding factor can be replaced by other transcriptional activators for co-operation with HNF1 in driving the rat albumin promoter in vivo.

Like many eukaryotic genes, the rat albumin promoter contains a CCAAT consensus motif at position -80. In transfected H4II hepatoma cells the strength of this promoter depends to a large extent on the integrity of a hepatic nuclear factor 1 (HNF1) binding site located at position -60 and to a lesser extent on the CCAAT element. However, if the affinity for HNF1 is reduced, the CCAAT-box becomes essential for high, and tissue specific, promoter activity. We wished to determine which, among the different CCAAT binding factors co-existing in eukaryotic cells, was responsible for this co-operativity with HNF1. To this end we prepared a series of mutants of the CCAAT sequence and compared their effects on albumin promoter activity in vivo and on the binding of different CCAAT binding factors in vitro. Our results strongly suggest that a ubiquitous factor NFY (also designated CBF, ACF, CP1) interacts with this CCAAT element in vivo. We propose that during development NFY could facilitate transcription of the albumin gene in hepatocytes when the concentration of HNF1 is limiting. This co-operativity in transcriptional activation is not due to strict co-operativity in DNA binding between the two proteins and is not limited to NFY or a closely related factor, as the CCAAT-box can be replaced by AP1, SP1 or E2 target sites without significantly affecting the final activity.

Adenoviridae↗

PubMed1991

Overexpression of c-jun, junB, or junD affects cell growth differently.

The coding sequences of murine c-jun, junB, or junD, which code for proteins with practically identical dimerization and DNA binding properties, were introduced into a nondefective retroviral vector, and the phenotype of primary avian fibroblasts chronically infected with each of these viruses was studied. Cells expressing c-jun grew in low-serum medium and developed into colonies in agar, two properties characteristic of in vitro transformation. Cells expressing junB grew in agar, with a reduced efficiency as compared to c-jun, but did not grow in low-serum medium. Finally, no effect of junD expression on cell growth was observed. These different phenotypes suggest that these three closely related transcription factors play distinct roles during normal cell growth. Analysis of c-jun deletion mutants and of c-jun/junB and c-jun/junD chimeric genes showed that the N-terminal portion (amino acids 2-168) of the c-Jun protein that is involved in transcriptional activation is required for efficient transformation. On the contrary, cells expressing a truncated mouse c-Jun lacking this N-terminal domain grew slower than normal embryo fibroblasts. The reduced growth rate may be related to the finding that expression of the intact or the truncated mouse c-jun repressed the endogenous avian c-Jun homologue, suggesting that functional c-Jun product is required for normal cell growth.

Amino Acid Sequence↗

PubMed1991

Degradation of transcription factors, c-Jun and c-Fos, by calpain.

c-Jun protein, and AP1/PEA1 transcription factor component, is a typical short-lived protein, and like other short-lived proteins such as c-Fos, contains PEST regions. Calcium-dependent neutral protease (calpain), a candidate for the degradation of PEST-containing proteins, digests c-Jun and c-Fos efficiently in vitro. This is the first demonstration that transcription factors are substrates for calpain. The C-terminal portion of c-Jun is relatively resistant to calpain such that an 18kDa fragment, which includes the DNA binding domain, accumulates under moderate digestion conditions. The activity of c-Jun in cultured cells can be modified by changing the level of calpastatin, an endogenous calpain inhibitor, indicating that c-Jun is also a substrate for calpain in vivo.

Amino Acid Sequence↗

PubMed1991

Two members of an HNF1 homeoprotein family are expressed in human liver.

HNF1 is a transcriptional activator, required for the liver-specific expression of a variety of genes, that binds to DNA as a dimer via the most diverged homeodomain known so far. We were interested to examine whether HNF1 is a unique homeoprotein example or whether it is the prototype of a new subfamily of homeodomain containing proteins. In this work we describe the isolation of a cDNA clone from a human liver library encoding a protein, highly homologous to HNF1 in three regions, including the homeo- and dimerization domains. We show that this protein can heterodimerize with human HNF1 in vitro. Sequence comparison of our clone with a rat variant HNF1 (vHNF1) clone, isolated in parallel in our laboratory from the dedifferentiated H5 hepatoma cell line, identified our cDNA as human vHNF1. vHNF1 is a nuclear protein recognizing the same binding site as HNF1 and previously thought to occur only in dedifferentiated hepatoma cells that fail to express most liver specific genes. Nevertheless, we show by Northern blot analysis that vHNF1 transcripts are present in differentiated human HepG2 hepatoma cells as well as in rat liver and that this transcript level is 10-20 fold lower than that of HNF1. We assigned the vHNF-1 gene to human chromosome 17 and murine chromosome 11. These chromosomal localizations differ from that of the HNF-1 gene indicating that both genes are not clustered on the genome.

Amino Acid Sequence↗

PubMed1991

vHNF1 is a homeoprotein that activates transcription and forms heterodimers with HNF1.

vHNF1 and HNF1 are two nuclear proteins that bind to an essential element in the promoter proximal sequences of albumin and of many other liver-specific genes. HNF1 predominates in hepatocytes but is absent in dedifferentiated hepatoma cells. These cells contain vHNF1 but fail to express most of the liver traits. In the present work we have isolated cDNA clones for vHNF1 and found that it is a homeoprotein homologous to HNF1 in regions important for DNA binding. Unexpectedly, vHNF1 transactivated the albumin promoter in transfection experiments. Like the HNF1 mRNA, the vHNF1 message was found in kidney, liver and intestine although in different proportions. The fact that vHNF1 and HNF1 readily form heterodimers in vitro and the biochemical characterization of vHNF1/HNF1 heterodimers in nuclear extracts of kidney, liver and several cell lines, strongly argue that such heterodimers exist in vivo. Our results raise the possibility that heterodimerization between homeoproteins could be a common phenomenon in higher eukaryotes, which may have implications in the regulatory network sustained between these factors.

Albumins↗

PubMed1991

Unregulated expression of c-Jun or c-Fos proteins but not Jun D inhibits oestrogen receptor activity in human breast cancer derived cells.

We present evidence that oestrogen receptor activity in human MCF-7 breast cancer cells is reduced by over-expression of c-Jun or c-Fos proteins and to a lesser extent by Jun B overexpression. In contrast, overexpression of Jun D protein does not affect the activity of the oestrogen receptor. A region of c-Jun found to be required for repression of oestrogen receptor activity is located outside the DNA binding domain and is not conserved among the three Jun proteins. Finally, we suggest that c-Jun and c-Fos act independently to inactivate the oestrogen receptor.

Base Sequence↗

PubMed1991

Several different upstream promoter elements can potentiate transactivation by the BPV-1 E2 protein.

The enhancer and upstream promoter regions of RNA polymerase II transcribed genes modulate the rate of transcription initiation and establish specific patterns of gene expression. Both types of region consist of clusters of DNA binding sites for nuclear proteins. To determine how efficiently the same factor can activate transcription when acting as an enhancer or promoter factor, we have studied transactivation by the BPV-1 E2 protein, a papillomavirus transcriptional regulator. By cotransfecting a BPV-1 E2 expression vector and a series of reporter plasmids containing well-defined chimeric promoters we have found that whilst E2 can strongly stimulate complex promoters such as that of the HSV tk gene, it does not efficiently activate constructions containing only a TATA box and initiation site. We show that insertion of upstream promoter elements, but not of spacer DNA, between E2 binding sites and the TATA box greatly increases E2 activation. This effect was observed with more than one type of upstream promoter element, is not related to the strength of the promoter and is unlikely to result from co-operative DNA binding by E2 and the transcription factors tested. These results would suggest that E2 has the properties of an enhancer rather than promoter factor and that in certain cases promoter and enhancer factors may affect different steps in the process of transcriptional activation.

Base Sequence↗

PubMed1991

vHNF1 is expressed in epithelial cells of distinct embryonic origin during development and precedes HNF1 expression.

HNF1 (Hepatic Nuclear Factor 1) and vHNF1 are transcriptional regulators containing a highly divergent homeodomain. The first was initially found in liver nuclear extracts and is crucial for the transcription of albumin and many other hepatocyte specific genes, while the second was found in dedifferentiated hepatoma cells. Both recognize the same DNA binding site and can form homo and heterodimers in vitro and in vivo. In situ hybridization analyses have been performed to delineate the spatial and temporal pattern of expression of vHNF1 relative to HNF1 during mouse embryogenesis. The results show that accumulation of vHNF1 mRNAs expression is detected in several tissues of the embryo of both endodermal and mesodermal origin. Expression occurs in the yolk sac, the primitive gut, the liver primordium, and at different stages of kidney development in polarized epithelial structures and usually precedes that of HNF1. vHNF1 expression seems particularly prevalent with morphogenetic events in the kidney and may be a marker for certain polarized epithelium.

Animals↗

PubMed1991

Nuclear oncogenes.

Ample evidence has accumulated in recent years to establish that most, if not all, nuclear proto-oncogenes are in fact sequence-specific DNA-binding proteins that modulate gene expression. Their synthesis or activity is modulated by extracellular signals or by cross talk between different classes of transcription factors.

Animals↗

PubMed1991

The papillomavirus E2 protein: a factor with many talents.

The products of the papillomavirus E2 open reading frame play a key role in the regulation of the viral cycle. E2 proteins can activate or repress viral promoters by several distinct mechanisms and viral DNA replication requires the expression of the full-length E2 protein together with the product of the E1 open reading frame. This is an interesting example of how a single eukaryotic DNA-binding protein has evolved to perform several different functions and it provides a valuable model system for studying the regulation of eukaryotic transcription and DNA replication.

Animals↗

PubMed1991

Structural analysis of the human papillomavirus type 16-E2 transactivator with antipeptide antibodies reveals a high mobility region linking the transactivation and the DNA-binding domains.

In order to probe the structure of the transcription factor encoded by the E2 Open Reading Frame of papillomaviruses, we raised polyclonal antibodies against a series of synthetic peptides that cover the HPV16-E2 protein. In gel shift experiments with the native form of the protein, we detected supershifts (caused by the binding of antibodies to the E2-DNA complex) with antibodies synthesized against peptides covering a central region 50 residues long in the E2 protein. On the contrary, antibodies raised against peptides from the NH2- and COOH-termini did not give any supershifted band. Western blot experiments showed that several of these non reacting antibodies did however interact with the denatured protein. These results suggest that the central region that connects the NH2-terminal domain responsible for transcriptional activation and the COOH-domain involved in DNA-binding is exposed and maintained in a conformation resembling the peptide, indicating a high mobility region. In contrast, the DNA-binding and transactivation domains were not recognized by the antipeptide antibodies, in line with secondary structure predictions and sequence comparisons indicating that the E2 protein consists of structured and conserved NH2 and COOH-terminal regions separated by a non-conserved and unstructured region. This flexible 'hinge' region may facilitate contacts between E2 dimers at distance in mechanisms of transcriptional activation steps that involve homosynergy or DNA-looping.

Amino Acid Sequence↗

PubMed1991

The functional BPV-1 E2 trans-activating protein can act as a repressor by preventing formation of the initiation complex.

The products encoded by the E2 open reading frame of the papillomaviruses are DNA-binding transcription factors involved in the positive or negative regulation of multiple viral promoters. To further understand the mechanisms by which the same transcription factor may act differentially, the full-length BPV-1 E2 protein was expressed and purified from yeast and assayed in vitro for its capacity to modulate transcription. E2 stimulated transcription of the HSV thymidine kinase (TK) promoter when E2-binding sites were positioned in an enhancer configuration approximately 100 bp upstream of the promoter start site. In contrast, the same full-length E2 protein repressed transcription of the HPV-18 E6/E7 P105 promoter. This repression was mediated through binding to the E2 DNA-binding site immediately upstream of the P105 promoter TATA box and could be abrogated by preincubation of the HPV-18 P105 promoter template with the nuclear extract allowing the formation of the preinitiation complex. In vitro DNA-binding experiments with purified E2 and TFIID showed that binding of E2 to its DNA target placed at different positions with respect to the TATA box differentially affects binding of TFIID to its cognate site. In these respects, E2 is similar to the bacteriophage lambda repressor, which can act either as a repressor or an activator of transcription depending on the position of its binding sites relative to the promoter sequences.

Animals↗