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M M Zakin

Publications and source records attributed to M M Zakin.

At least 37 records · Page 2Linked to original sources
PubMed1997

The apolipoprotein A-I/C-III/A-IV gene cluster: ApoC-III and ApoA-IV expression is regulated by two common enhancers.

Genetic, epidemiological and clinical evidence have clearly demonstrated the importance of the human apolipoprotein (apo) A-I/C-III/A-IV gene cluster in lipid metabolism and heart attack. The transcriptional regulation of these genes determines the level of the encoded proteins and therefore influences the concentration of triglycerides and cholesterol. Here, we analyze the existence of transcription control elements in the 6.6 kb apoC-III/A-IV intergenic region and their influence on the expression of both genes. Two main positive common control elements were found to modulate apoC-III and apoA-IV expression in HepG2 and in Caco-2 cells: the previously described apoC-III enhancer, located 0.8 kb upstream from the cap site of the gene, and a newly detected activating region located in the center of the intergenic sequence. The activity of both elements is highly increased by the hepatic and intestinal transcription factor HNF-4. Analysis of a 641 bp fragment containing the central element showed that it has the properties of a tissue-specific enhancer. Liver nuclear proteins interact with seven DNA binding sites present in this enhancer and HNF-4 specifically interacts with one of these sites. A third positive element, situated immediately upstream from the apoA-IV minimal promoter, is also activated by HNF-4; however, this element is not involved in apoC-III expression. In addition, two negative regions were identified, one located near the apoA-IV gene and the other one between the apoC-III enhancer and the newly identified central enhancer. In conclusion, negative and positive control elements are located in the apoC-III/A-IV intergenic region, including two enhancers important for the expression of the two genes. These results add new evidence that common regulatory elements for the expression of the apoA-I, apoC-III and apoA-IV genes are interspersed throughout the cluster.

Apolipoprotein A-I↗
PubMed1997

Alpha-difluoromethylornithine-resistant cell lines obtained after one-step selection of Leishmania mexicana promastigote cultures.

Proliferation of Leishmania mexicana promastigotes in synthetic medium can be blocked by the depletion of intracellular polyamine pools induced by the presence of D,L-alpha-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of ornithine decarboxylase (ODC). Here we report that DFMO-resistant cell lines growing normally at DFMO levels of 10 mM have been obtained from non-proliferating cultures after a single-step selection in the presence of high concentrations of the drug. The DFMO-resistant promastigotes underwent a morphological transformation into an 'amastigote-like' form after incubation for several hours at gradually increasing temperatures up to 35 degrees C. The uptake of DFMO was not significantly altered in the drug-resistant cell lines but in both cases (promastigote and 'amastigote-like' forms) the ODC specific activity was increased approx. 15-fold over the normal enzymic levels found in the wild-type Leishmania. The enzyme affinities for its substrate and for DFMO gave very similar values in the drug-resistant promastigotes and the wild-type parasites. In contrast, ODC from the 'amastigote-like' Leishmania showed a higher affinity for ornithine and a decreased capacity for the binding of DFMO. An 80-fold amplification of the ODC gene and a corresponding increase in its transcripts have been detected in both DFMO-resistant Leishmania cell lines. The drug-resistant phenotypes with their characteristic morphologies, the increased levels of ODC activity and the amplification of the ODC gene have been stable for at least 6 months in the absence of selective pressure.

Animals↗
PubMed1997

Single intracranial injection of apotransferrin in young rats increases the expression of specific myelin protein mRNA.

Transferrin (Tf) is a possible regulator of oligodendrocyte development in vitro (Espinosa de los Monteros et al., 1989). At least two different mechanisms may account for the effects of Tf on myelin synthesis. It may act as a trophic factor and enhance the formation of new myelin sheaths. Tf may also induce the synthesis of myelin proteins in the central nervous system. We recently demonstrated that a single intracranial injection of apotransferrin (aTf) in young rats induces an increased myelination (Escobar Cabrera et al., 1994). In the present study, we investigated the in vivo effect of aTf on the expression of mRNAs of specific myelin genes. Three-day-old rats were injected intracranially with aTf and killed at different ages after injection. Total brain RNA was isolated, and the expression of different mRNAs was analyzed by Northern blot. The amount of mRNAs of myelin basic protein and of 2'-3' cyclic nucleotide 3'-phosphohydrolase were markedly increased in the experimental animals, whereas myelin proteolipid protein mRNA did not show differences relative to controls. These results indicate that in the animals treated with aTf, there is a differential effect on the expression of certain specific myelin protein genes. They also suggest that aTf might exert its action at the posttranscriptional level and/or by direct transcriptional regulation of the genes.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1996

Melanotransferrin gene expression in melanoma cells is correlated with high levels of Jun/Fos family transcripts and with the presence of a specific AP1-dependent ternary complex.

The involvement of the transcription factor AP1 in the regulation of melanotransferrin (MTf) gene expression was investigated. MTf, also known as p97, is a tumour-associated antigen that is overproduced in most melanomas. Its gene expression is under the control of an enhancer element containing two AP1 binding sites. By Northern analysis, we demonstrate that MTf mRNA is detected at various levels in melanoma SK-MEL-28 cells and that its greatest expression coincides with the presence of large amounts of jun and fos transcripts. Gel retardation assays revealed that the induction of expression of these proto-oncogenes is correlated with increased AP1 binding activity and that a region of the MTf enhancer is involved in the formation of a ternary AP1-dependent complex, implicating a second nuclear factor whose binding characteristics are similar to those of nuclear factor of activated T cells (NF-AT). In transient expression experiments, the activity resulting from ternary complex formation was high and specific to melanoma cells. These data provide a possible explanation for the mechanisms of AP1 factor family involvement in MTf up-regulation in melanoma cells.

Antigens, Neoplasm↗
PubMed1996

The polypyrimidine tract binding (PTB) protein interacts with single-stranded DNA in a sequence-specific manner.

Polypyrimidine tract binding (PTB) protein is a cellular factor whose function is unknown. Various RNA or single-stranded DNA sequences have been shown to interact with PTB. In this paper, using laser UV crosslinking and electrophoretic mobility shift assays to probe DNA-protein interactions, we demonstrate that PTB binding at a single-stranded DNA target is highly sequence-specific. We provide data showing that PTB interacts with the top strand of the adenovirus major late promoter transcriptional initiator, a sequence rich in pyrimidine residues. We also demonstrate that PTB is organised into at least two different binding domains.

Adenoviridae↗
PubMed1996

Transcriptional suppression of the transferrin gene by hypolipidemic peroxisome proliferators.

Activation of gene expression by hypolipidemic peroxisome proliferators (e.g. native and substituted long chain fatty acids, aryloxyalkanoic fibrate drugs) is accompanied by transcriptional suppression of liver transferrin gene in treated animals or human hepatoma cell line. Transcriptional suppression of liver transferrin by hypolipidemic peroxisome proliferators results from (a) displacement of hepatic nuclear factor (HNF)-4 from the transferrin promoter by nonproductive binding of the peroxisome proliferator-activated receptor-retinoic acid X receptor heterodimer to the (-76/-52) PRI promoter element of the human transferrin gene and (b) suppression of liver HNF-4 gene expression by hypolipidemic peroxisome proliferators with a concomitant decrease in its availability for binding to the transferrin PRI promoter element. HNF-4 gene suppression and its displacement from the transferrin promoter result in eliminating HNF-4-enhanced transcription of transferrin. Liver transferrin suppression by hypolipidemic peroxisome proliferators may result in reduced iron availability as well as modulation of transferrin-induced differentiation processes. Transcriptional suppression of HNF-4-enhanced liver genes (e.g. apolipoprotein C-III, transferrin) may complement the pleiotropic biological effect exerted by hypolipidemic peroxisome proliferators.

Animals↗
PubMed1996

Expression, purification, and functional analysis of the DNA binding domain of the nuclear receptor Rev-erb beta.

Rev-erb beta is a member of the nuclear receptor superfamily, which includes a group of transcription factors involved in the response to steroids, vitamin D, retinoic acids, and other lipophilic molecules. The Rev-erb nuclear receptors exist at least in two forms, namely alpha and beta, with a high degree of evolutionary conservation at the level of the DNA binding domain. Nevertheless, the exact type of DNA binding of these proteins is not fully understood. In order to get insight into this DNA binding mechanism we obtained a pure and functional homogeneous recombinant protein in bacteria corresponding to the DNA binding domain of Rev-erb beta (REDBD). REDBD interacts with oligonucleotides containing an A/T-rich sequence preceding a single AGG-TCA site (Rev-RE) or with an AGGTCA direct repeat separated by 2 bp (Rev-DR2). These results, and the affinity parameters of the interaction between REDBD and Rev-RE or Rev-DR2, indicate that our REDBD preparation is capable of specific and tight binding to DNA targets.

Amino Acid Sequence↗
PubMed1995

NOR-2 (neuron-derived orphan receptor), a brain zinc finger protein, is highly induced during liver regeneration.

Zinc-finger proteins are involved in several cellular processes. Some of these proteins are implicated in the primary cellular response in regenerating liver and mitogen-stimulated cells. Using a rat cDNA brain library, we have isolated a clone designated NOR-2, encoding a protein containing two zinc-finger motifs and whose expression is highly induced during G0/G1 transition. We analysed the expression of NOR-2 mRNAs during early growth in regenerating liver and in both insulin-stimulated H4-II cells and pheochromocytoma-derived cell line PC12 treated by NGF. In these systems, there is an early, rapid and transient accumulation of NOR-2 mRNAs. The induction of NOR-2 mRNAs does not require de novo protein synthesis, since it is not prevented by cycloheximide treatment. Mobility shift assays show that NOR-2 protein binds to NBRE, a target sequence for r-NGFI-B family. Structurally, NOR-2 is closely related to the recently identified NOR-1 factor. Therefore, like NOR-1, NOR-2 belongs to the r-NGFI-B sub-family of nuclear receptors superfamily.

Amino Acid Sequence↗
PubMed1995

Interaction of DNA binding domain of HNF-3 alpha with its transferrin enhancer DNA specific target site.

Transferrin hepato-specific gene enhancer, associated with the liver-enriched HNF-3 alpha transcriptional factor and ubiquitous proteins, is a complex molecular edifice maintained through DNA-protein and protein-protein interactions. As a first step to understand the mechanisms responsible for its organization and activity, we have analyzed the interaction of the DNA binding domain of HNF-3 alpha (HDBD) with a specific DNA segment present in the transferrin enhancer by different biophysical techniques. The kinetic constants of this interaction were measured using surface plasmon resonance. The HDBD-DNA interaction was also characterized by circular dichroism and fluorescence spectroscopy. HDBD binds to its specific DNA site with high affinity (Kd approximately equal to 10(-8) M). The affinity is reduced after sequence modification of the target DNA. Size exclusion chromatography and binding stoichiometry determined by fluorescence measurements indicate that the protein is present in a monomeric form before and after interaction with the DNA. The secondary structure of the protein was not significantly altered upon binding to specific DNA. By contrast, a structural change of DNA by interaction with HDBD seems to occur.

Base Sequence↗
PubMed1995

The apolipoprotein AICIII-AIV gene cluster: sequence of the ApoCIII-ApoAIV intergenic region.

The genes coding for human apolipoproteins AI, CIII and AIV are tandemly organised in a cluster on chromosome 11. The sequence of 4 kb of the 6.6-kb ApoCIII-ApoAIV intergenic region was unknown until now. Since different elements involved in the transcriptional regulation of the three genes of the cluster were previously identified in this region, we decided to sequence it. We present here the complete sequence of the region. Its availability will allow study of the transcriptional regulation of ApoCIII and ApoAIV at the molecular level. In addition, the use of PCR to study the polymorphic sites detected in the ApoCIII-ApoAIV intergenic region will now be possible. Some of these sites have been associated with lipid disorders and coronary heart disease.

Apolipoprotein C-III↗
PubMed1995

Regulation of melanotransferrin gene in melanoma cells. Analysis of the enhancer region.

The human iron-binding protein melanotransferrin is up-regulated in most skin melanomas. With the goal to understand the mechanisms controlling the expression of the gene in these tumor cells, we previously reported the identification of an enhancer exhibiting melanoma specificity. We show here that, in the highly expressing SK-MEL-28 melanoma cell line, the chromatin structure in the enhancer region is in an open configuration and that the transcription factors governing its activity belong to the helix-loop-helix and the Jun/Fos leucine zipper families.

Antigens, Neoplasm↗
PubMed1995

Rev-erb beta 2, a novel isoform of the Rev-erb family of orphan nuclear receptors.

We have isolated a rat complementary DNA clone corresponding to a novel isoform of the hormone nuclear receptor superfamily. This clone encodes a 383 amino acid residue protein designated as Rev-erb beta 2. This protein is identical until residue 382 to the Rev-erb beta 1 protein, which is 195 amino acids longer. Several arguments pointed out that the Rev-erb beta 2 cDNA may originate from the same gene as Rev-erb beta 1 by alternative splicing and using a different polyadenylation site. Our results indicate that Rev-erb beta 2 is a new isoform of the Rev-erb family of orphan nuclear receptors.

Amino Acid Sequence↗
PubMed1994

Brain-specific expression of the human transferrin gene. Similar elements govern transcription in oligodendrocytes and in a neuronal cell line.

We have identified the regulatory sequences that govern the expression of the human transferrin gene in cultured brain cells and compared them with the data obtained with the neuronal cell line B103. Oligodendrocytes and epithelial choroid plexus cells from rat brain were cultured and used for transient expression experiments. Deletion analysis of 1.8 kilobase pairs of the 5' regulatory sequences revealed a -1530/-1140 positive-acting region in oligodendrocytes. The -164/+1 promoter region was sufficient to confer cell type-specific transcription in oligodendrocytes, epithelial choroid plexus cells, and B103 cells. DNase I footprinting experiments revealed three protected sequences, the proximal regions I and II, and the central region I. Gel retardation and antibody reactivity data allowed us to identify most of the nuclear factors present in oligodendrocytes interacting with the promoter sequences. Chicken ovalbumin upstream promoter transcription factor, a CAAT/enhancer-binding protein, and a cAMP response element-binding protein called CRI-BP interact with the proximal regions I and II and central region I sites, respectively. These data confirm the results obtained with the neuronal cell line and emphasize the importance of the three promoter elements for the transferrin gene-specific expression in the central nervous system compared with only two elements required for liver- and testis-specific expression.

Animals↗
PubMed1994

Fenofibric acid modulates the human apolipoprotein A-IV gene expression in HepG2 cells.

The influence of the hypolipidemic drug, fenofibric acid, on the regulation of apolipoprotein A-IV (apoA-IV) gene expression was investigated in two human cell lines, HepG2 and Caco-2. As shown in the present report, fenofibric acid induces a strong dose-dependent increase of the apoA-IV mRNA level in HepG2 cells, while other apolipoproteins mRNA levels are only slightly modified. In Caco-2 cells, no modification is observed, except for a 2-fold increase of the apoE mRNA level. The increase of the apoA-IV mRNA level could be correlated with a clear enhancement of DNase I hypersensitive sites in the 5' flanking region of the gene in nuclei of HepG2 cells treated with fenofibric acid. Thus, fenofibric acid may act by facilitating the interaction of nuclear regulatory proteins with the DNA in the control regions of the apoA-IV gene.

Apolipoproteins↗
PubMed1993

Human apolipoprotein A-IV gene expression is modulated by members of the nuclear hormone receptor superfamily.

Human apolipoprotein A-IV (apo A-IV) is essentially synthesized in intestine and to a lower extent in liver. The presence of detectable amounts of apo A-IV messenger in HepG2 and Caco-2 cells enables us to study the transcription regulation of the gene in these cells. A main hypersensitive site to DNase I is detected in the DNA of HepG2 nuclei, in a region situated about 3400 base pairs upstream from the cap site. Transient expression experiments in HepG2, Caco-2 and HeLa cells indicate that HNF-4 activates the transcription of a reporter gene through the -3500/+67 sequence of the apo A-IV gene. This activation is repressed by another transcription factor, Ear3/COUP-TF. On the contrary, HNF-1, whose expression is controlled by HNF-4, is not involved in the regulation of the reporter gene transcription through the -6700/+67 genome sequence. These results indicate that the apo A-IV gene expression is modulated by orphan ligand members of the superfamily of nuclear hormone receptors.

Apolipoproteins A↗
PubMed1993

A different combination of transcription factors modulates the expression of the human transferrin promoter in liver and Sertoli cells.

We have previously identified the functional regions involved in the regulation of human transferrin (Tf) gene expression in the liver and in Sertoli cells of the testis. Here, we show that a different cellular distribution of transcription factors, interacting with the same proximal promoter regions (PRI and PRII), modulates cell type-specific transcription. In the liver, hepatocyte nuclear factor 4 (HNF-4) and the chicken ovalbumin upstream promoter transcription factor (COUP-TF) act at the PRI site, while CCAAT/enhancer-binding proteins (C/EBPs) act at the PRII site. In the testis, distinct combinations of Sertoli proteins SP-A and SP-D and COUP-TF bind to the PRI site, while SP-alpha and SP-beta bind to the PRII site. Cotransfection experiments in Hep3B cells revealed that mostly HNF-4, C/EBP-alpha, C/EBP-delta, and, to a lesser extent, COUP-TF stimulated transcription driven by the -125/+39 region. In Sertoli cells, HNF-4 and COUP-TF appeared to repress, while the C/EBP factors were able to stimulate transcription driven by the -100/+39 region. However, the specific activating combination remains to be defined among the Sertoli proteins. In the non-Tf-expressing HeLa cells, the Tf promoter could be activated by C/EBP-delta. Our data revealed functional antagonism between HNF-4 and COUP-TF, binding to PRI, as well as cross-coupling interactions between HNF-4 and C/EBP, binding to adjacent sites. Thus, cell type-specific DNA-protein interactions, together with protein-protein interactions, may explain the transcriptional regulation of the Tf gene in different cell types.

Base Sequence↗