Biomedical subjects

M Yaniv

Publications and source records attributed to M Yaniv.

At least 91 records · Page 5Linked to original sources

PubMed1993

The transactivation potential of variant hepatocyte nuclear factor 1 is modified by alternative splicing.

Two forms of the transcription factor vHNF1 (HNF1 beta or LFB3) have been previously described, derived by alternative splicing from a common premessenger RNA, and have been called vHNF1-A and vHNF1-B. vHNF1 proteins share a homologous homeo-related DNA-binding domain with the HNF1 protein, initially characterized as a liver-restricted transcription factor, and bind to a similar sequence motif. Here we demonstrate that vHNF1-A is a stronger transactivator than vHNF1-B when assayed in transient transfections using two different promoters. vHNF1-A also binds DNA with a higher affinity suggesting that a region of the protein located immediately upstream of the homeodomain can modulate the protein/DNA interaction and transactivation. Both vHNF1 transcripts were found at a constant ratio in every tissue where vHNF1 expression could be detected, using a quantitative reverse transcriptase-polymerase chain reaction.

Animals↗

PubMed1993

The PML-retinoic acid receptor alpha translocation converts the receptor from an inhibitor to a retinoic acid-dependent activator of transcription factor AP-1.

We report here that the fusion of PML, a nuclear protein defined by the t(15;17) chromosomal translocation in acute promyelocytic leukemia, with retinoic acid receptor alpha (RAR alpha) changes the RAR alpha from a retinoic acid (RA)-dependent inhibitor to a RA-dependent activator of AP-1 transcriptional activity. The PML-RAR alpha chimera cooperates with c-Jun and, strikingly, with c-Fos to stimulate the transcription of both synthetic and natural reporter genes containing an AP-1 site. Stimulation is dependent on the concentration of RA and its dose-response curve is comparable to that for activation by RAR alpha of transcription on RA-responsive genes. Further, in the absence of RA, a circumstance in which RAR alpha has no effect on AP-1 activity, PML-RAR alpha is an inhibitor. Deletion of the dimerization, transactivation, or DNA-binding domains of c-Jun and removal of the PML dimerization domain in the PML-RAR alpha hybrid abrogates their transcriptional cooperatively. In view of the association between AP-1 activity and hemopoietic differentiation, we suggest that these properties of PML-RAR alpha could contribute to the leukemic phenotype and its response to RA.

Animals↗

PubMed1993

More potent transcriptional activators or a transdominant inhibitor of the HNF1 homeoprotein family are generated by alternative RNA processing.

We report the isolation of cDNAs from human liver encoding several isoforms of the hepatocyte nuclear factor homeoproteins HNF1 and vHNF1 generated by the differential use of polyadenylation sites and by alternative splicing. In the novel isoforms intron sequences that are excised in the previously described forms are translated in the same frame as exon sequences until the first termination codon is encountered. Hence, the newly found isoforms all contain different C-terminal domains. For HNF1 it has been shown that its C-terminal region is responsible for the activation of transcription. In transient transfection assays the two novel HNF1 isoforms, HNF1-B and -C, transactivate 5-fold better than the previously described HNF1 protein (HNF1-A). The newly isolated isoform of vHNF1, designated vHNF1-C, is unable to transactivate and behaves as a transdominant repressor when cotransfected with HNF1-A, -B or -C. All of the different isoforms of HNF1 and vHNF1 can form homo- and heterodimers and their mRNAs are differentially expressed in fetal and adult human liver, kidney and intestine, suggesting distinct roles during development. Our studies show that the transactivation domain of the members of the HNF1 homeoprotein family is organized in modules which can be exchanged to generate either more potent transcriptional activators or a transdominant repressor.

Alternative Splicing↗

PubMed1993

A human homologue of Saccharomyces cerevisiae SNF2/SWI2 and Drosophila brm genes potentiates transcriptional activation by the glucocorticoid receptor.

Several of the SNF and SWI genes of Saccharomyces cerevisiae code for proteins believed to assist transcriptional activators by relieving nucleosome repression. One of these proteins, SNF2/SWI2, has a homologue in Drosophila, a regulator of homeotic genes known as brahma or brm. In this report, we show that a counterpart of SNF2/SWI2 also exists in mice and humans. The human protein, designated hbrm, is a 180 kDa nuclear factor that can function as a transcriptional activator when fused to a heterologous DNA binding domain. The mouse homologue of hbrm is expressed in all mouse organs tested while hbrm was detected in some but not all investigated human cell lines. In cells failing to express the endogenous gene, transfected hbrm cooperates with the glucocorticoid receptor (GR) in transcriptional activation. However, hbrm had no effect on the activity of several other transcription factors, including the homeoprotein HNF-1. The co-operation between hbrm and GR required the DNA binding domain of GR and two separated regions of the hbrm protein, including a domain with homology to known helicases.

Amino Acid Sequence↗

PubMed1993

In vitro transforming capacities of mouse c-jun:junD chimeric genes.

Among the murine Jun family of transcription factors, c-Jun and JunD are closely-related proteins with similar dimerization, DNA binding and transactivating properties. However, when expressed from a self-replicating retroviral RCAS vector, c-jun, but not junD, transforms chick embryo fibroblasts. We attempted to map the regions of c-jun which are important for transformation by constructing hybrids between c-jun and junD. Using common restriction sites, we prepared six different chimeric molecules. All of these c-jun:junD hybrids code for transactivators of AP1-containing promoters. An N-terminal segment of 79 amino acids of c-Jun converts JunD into a strong transforming protein, while other segments of c-Jun contribute to a lesser extent. Contrary to what has been reported with rat embryo fibroblasts, a c-Jun derivative with serines substituted by alanines in positions 63 and 73 still transforms CEFs efficiently.

Amino Acid Sequence↗

PubMed1993

Interleukin 6 induces DNA binding activity of AP1 in M1 myeloblastic cells but not in a growth resistant cell derivative.

The effects that three different growth inhibitory cytokines exert on expression and function of members of the Jun family were studied in this work. M1 myeloblastic cells were chosen for this purpose because of their high growth sensitivity to interleukin 6 (IL-6), transforming growth factor beta 1 and alpha- and beta-interferons. It is reported here that IL-6 elevated the junB and c-jun mRNA levels and induced the formation of a novel DNA-protein complex with high sequence specificity to 12-O-tetradecanoylphorbol-13-acetate response element (TRE) oligonucleotides. This IL-6 induced TRE binding complex was abolished by anti-Jun specific antibodies and was efficiently competed by an oligonucleotide that comprises the mouse homologue of a previously described human c-myc negative DNA element. It persisted in cells for at least 48 h after IL-6 treatment and failed to be induced by alpha- and beta-interferons or by transforming growth factor beta 1, which affected differently the pattern of jun mRNA expression. To further explore regulatory and functional aspects of this induced TRE binding activity, an IL-6 resistant M1 clone was isolated and further analyzed. This clone carried a postreceptor deficiency that abrogated completely the growth inhibitory responses to IL-6 but did not interfere with the induction of two differentiation related cell surface markers. Interestingly, the IL-6 resistant clone had lost two molecular responses to IL-6, induction of TRE binding activity and suppression of the c-myc gene. The data correlate the IL-6 induced AP1 activity with the suppression of c-myc and growth inhibition.

Animals↗

PubMed1993

The HNF1 C-terminal domain contributes to transcriptional activity and modulates nuclear localisation.

HNF1 is a homeoprotein that regulates the expression of a large number of liver specific genes. By performing transient expression assays with a series of C-terminal deletion mutants and with a LexA-HNF1 fusion protein, we show that the C-terminal half of HNF1 is necessary and sufficient for in vivo transcriptional activity, and we map the residues essential for this activity. However, since our data for some mutants showed discrepancies with previous in vitro studies, we undertook a more careful analysis of the mutant proteins using gel retardation assays and immunoblots made with nuclear extracts from transfected cells. We show that progressive C-terminal deletions drastically increase the amount of protein that accumulates in transfected cells. Immunofluorescence microscopy reveals that mutants containing between 348 and 416 residues accumulate outside the nuclear membrane, while longer mutants are nuclear like the 628 amino acid long wild type HNF1. A mutant with 289 residues is predominantly nuclear. Since the only obvious candidate for a nuclear localisation signal is located within this last mutant, we suggest that certain C-terminal deletions expose a sequence that blocks nuclear transport.

Blotting, Western↗

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↗