Biomedical subjects

E Falvey

Publications and source records attributed to E Falvey.

11 recordsLinked to original sources

PubMed2001

The activation function-1 of hepatocyte nuclear factor-4 is an acidic activator that mediates interactions through bulky hydrophobic residues.

The hepatocyte nuclear factor-4 (HNF-4) contains two transcription activation domains. One domain, activation function-1 (AF-1), consists of the extreme N-terminal 24 amino acids and functions as a constitutive autonomous activator of transcription. This short transactivator belongs to the class of acidic activators, and it is predicted to adopt an amphipathic alpha-helical structure. Transcriptional analysis of sequential point mutations of the negatively charged residues (Asp and Glu) revealed a stepwise decrease in activity, while mutation of all acidic residues resulted in complete loss of transcriptional activity. Mutations of aromatic and hydrophobic amino acids surrounding the negatively charged residues had a much more profound effect than mutations of acidic amino acids, since even a single mutation of these residues resulted in a dramatic decrease in transactivation, thus demonstrating the importance of hydrophobic residues in AF-1 activity. Like other acidic activators, the AF-1 of HNF-4 binds the transcription factor IIB and the TATA-binding protein directly in vitro. In addition, the cAMP-response-element-binding-protein, a transcriptional adapter involved in the transactivation of a plethora of transcription factors, interacts with the AF-1 of HNF-4 and co-operates in the process of transactivation by HNF-4. The different protein targets of AF-1 suggest that the AF-1 of HNF-4 may be involved in recruiting both general transcription factors and chromatin remodelling proteins during activation of gene expression.

Amino Acid Sequence↗

PubMed1996

DNA-binding specificity of PAR and C/EBP leucine zipper proteins: a single amino acid substitution in the C/EBP DNA-binding domain confers PAR-like specificity to C/EBP.

PAR and C/EBP family proteins are liver-enriched basic leucine zipper (bZip) transcription factors that bind similar sites on the promoters of albumin and cholesterol 7 alpha hydroxylase genes. However, C/EBP proteins have a more relaxed binding specificity than PAR proteins, in that they recognize many sites within promoter or randomly selected rat genomic DNA sequences that are ignored by PAR proteins. Thus, DNAse I protection experiments suggest that C/EBP recognizes a binding site with an affinity similar to the one of the cholesterol 7 alpha hydroxylase gene promoter every 200 to 300 bp. The frequency of PAR protein binding sites with comparable affinities is about 20-fold lower in the rat genome. By using a PCR-based amplification assay we selected high affinity DNA-binding sites for C/EBP beta and the PAR protein DBP from a pool of oligonucleotides. Both proteins indeed recognize similar sequences with the optimal core binding sequences 5'RTTAY.GTAAY3'. However, as expected, DBP, is considerably less tolerant to deviations from the consensus site. Here we have characterized a single amino acid substitution mutant of C/EBP beta that increases its target site specificity. This protein, C/EBP beta V > A, contains a valine to alanine substitution at position 13 of the basic domain (residue 216 of C/EBP beta). C/EBP beta V > A selectively binds only the subset of C/EBP sites that are also DBP sites, both as oligonucleotides and within the natural contexts of the albumin and cholesterol hydroxylase promoters.

Animals↗

PubMed1995

The rat hepatic leukemia factor (HLF) gene encodes two transcriptional activators with distinct circadian rhythms, tissue distributions and target preferences.

Hepatic leukemia factor (HLF) is a member of the PAR family of transcription regulatory proteins. We have characterized the rat HLF gene and studied its expression and activity. The rat HLF gene is transcribed from two alternative promoters, alpha and beta, with different circadian amplitudes and tissue specificities. The alpha RNA isoforms produce a 43 kDa protein, HLF43, abundant in brain, liver and kidney, like the previously described human HLF RNA. The beta RNA HLF isoforms use a CUG codon to initiate translation of a novel 36 kDa protein, HLF36, which is shorter at its N-terminus relative to the 43 kDa form. HLF36 is expressed uniquely in the liver, where it is the most abundant HLF protein. Surprisingly, the two proteins accumulate in the liver with different circadian amplitudes and have distinct liver-specific promoter preferences in transfection experiments. Thus, HLF43 stimulates transcription from the cholesterol 7 alpha-hydroxylase promoter much more efficiently than from the albumin promoter, while the converse is true for HLF36.

Amino Acid Sequence↗

PubMed1992

Role of the liver-enriched transcription factor DBP in expression of the cytochrome P450 CYP2C6 gene.

The CYP2C6 gene becomes maximally transcriptionally activated in livers of postpubertal rats. We examined the role of upstream DNA and liver-specific transcription factors in regulation of this promoter by use of transient transfection of heterologous chloramphenicol acetyltransferase gene constructs and vectors containing cDNAs encoding the liver-enriched transcription factors HNF-1 alpha, C/EBP, and DBP. Only DBP was able to activate the CYP2C6 promoter in HepG2 cells. Transactivation was not observed in one mouse and two human nonhepatic origin cell lines tested. Analysis of various constructs in which CYP2C6 upstream DNA was deleted revealed that DNA between -38 to -103 was involved in DBP-mediated activation. A partially purified preparation of DBP produced a footprint between -43 and -64 bp upstream of the transcription start site. A 32P-labeled double-stranded oligonucleotide, containing sequence information corresponding to -40 to -65, bound to both partially pure DBP and extracts from livers of rats as young as 1 week and as old as 25 weeks of age, as assessed by gel mobility shift analysis. This binding was eliminated by coincubation with excess unlabeled -40/-65 double-stranded oligonucleotide and by an oligonucleotide corresponding to the D site of the rat albumin gene. A gel mobility shift-Western immunoblot analysis revealed that the -40/-65 sequence bound to DBP only in liver nuclear extracts from rats older than 3 weeks; maximal binding was observed by 7 weeks of age, and no binding was detected from 1-week-old rat liver extracts. Interestingly, the DBP-binding regions of both CYP2C6 and albumin bind to C/EBP, but this factor is capable of transactivating only the latter gene. Although the DBP-binding regions in these two genes share no obvious sequence similarities, the CYP2C6 region contains consensus palindromic half sites for DBP-related binding proteins and affinity for recombinant DBP of 17-fold greater than that of the D site of albumin. This difference in affinity is probably responsible for the markedly lower amounts of DBP required for half-maximal activation of the CYP2C6 promoter, as compared with the albumin promoter, in transactivation transfection assays. These data indicate that the CYP2C6 gene may be regulated, at least in part, by DBP, a liver transcription factor produced when rats reach puberty that may also be involved in maintenance of albumin gene transcription.

Age Factors↗

PubMed1992

The role of the transcriptional activator protein DBP in circadian liver gene expression.

DBP, a liver-enriched transcriptional activator protein of the leucine zipper protein family, accumulates according to a very strong circadian rhythm (amplitude approx. 1000-fold). In rat parenchymal hepatocytes, the protein is barely detectable during the morning hours. At about 2 p.m., DBP levels begin to rise, reach maximal levels at 8 p.m. and decline sharply during the night. This rhythm is free-running: it persists with regard to both its amplitude and phase in the absence of external time cues, such as daily dark/light switches. Also, fasting of rats for several days influences neither the amplitude nor the phase of circadian DBP expression. Since the levels of DBP mRNA and nascent transcripts also oscillate with a strong amplitude, circadian DBP expression is transcriptionally controlled. While DBP mRNA fluctuates with a similar phase and amplitude in most tissues examined, DBP protein accumulates to high concentrations only in liver nuclei. Hence, at least in nonhepatic tissues, cyclic DBP transcription is unlikely to be controlled by a positive and/or negative feedback mechanism involving DBP itself. More likely, the circadian DBP expression is governed by hormones whose peripheral concentrations also oscillate during the day. Several lines of evidence suggest a pivotal role of glucocorticoid hormones in establishing the DBP cycle. Two genes whose mRNAs and protein products accumulate according to a strong circadian rhythm with a phase compatible with regulation by DBP encode enzymes with key functions in cholesterol metabolism: HMG-coA reductase is the rate-limiting enzyme in cholesterol synthesis; cholesterol 7-alpha hydroxylase performs the rate-limiting step in the conversion of cholesterol to bile acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1991

How are the regulators regulated?

The cell-type-specific expression of many genes is determined at the level of transcription. Transcription factors, acting at promoter and enhancer elements, are involved in the control of this process. To understand the basis of this regulation it has become important to analyze the control of transcription factors themselves. A variety of transcriptional, translational, and posttranslational mechanisms have been described, and are discussed in this review article.

Animals↗

PubMed1990

LAP, a novel member of the C/EBP gene family, encodes a liver-enriched transcriptional activator protein.

A gene, encoding a liver-enriched transcriptional activator protein (LAP) has been isolated. LAP is a 32-kD protein that stimulates the transcription of chimeric genes containing albumin D-promoter elements both in vivo and in vitro. LAP shares extensive sequence homology (71%) in its DNA-binding and leucine zipper domains with C/EBP. As a consequence, these two proteins show an indistinguishable DNA-binding specificity and readily heterodimerize. In addition, both genes, lap and cebp, are devoid of intervening sequences. Although correctly initiated transcripts from the LAP gene accumulate in the six examined tissues--liver, lung, spleen, kidney, brain, and testis--LAP protein is highly enriched in liver nuclei. Thus, the preferential accumulation of LAP protein in liver appears to be regulated post-transcriptionally.

Albumins↗

PubMed1988

Uncoupling of the recombination and topoisomerase activities of the gamma delta resolvase by a mutation at the crossover point.

In several well-characterized site-specific recombination systems it has been shown that, for efficient recombination, the two recombining sites must have identical DNA sequences across the region between the staggered points of exchange. The precise DNA sequence of this overlap region, however, appears to be of little importance (with the exception of one position in the loxP site of bacteriophage P1 (ref. 6]. In this report we characterize a mutant recombination site for the site-specific recombination enzyme gamma delta resolvase (encoded by the gamma delta transposon), in which the dinucleotide at the crossover point is changed from AT to CT. Our results indicate that identity of the two overlap regions is not sufficient for recombination. Although resolvase binds normally to the mutant site and induces the structural deformation characteristic of the wild-type recombination site, catalysis at the crossover point (cutting and rejoining of DNA strands) is effectively limited to just one of the two strands, allowing resolvase to act as a topoisomerase but not as a recombinational enzyme.

Base Sequence↗

PubMed1987

Contacts between gamma delta resolvase and the gamma delta res site.

We have investigated the interaction between resolvase and the res site of the transposon gamma delta by methylation and ethylation interference experiments. We have examined the effect of these DNA modifications both on binding and resolution in vitro. Major groove methylations within a 9 bp sequence that borders each site inhibit binding of resolvase to that site. Ethylation of certain phosphates within, and adjacent to, this border sequence inhibits binding. Together, these interference points define a contact region, present at all three res sites. In vitro resolution is inhibited only by modifications within site I. Inhibition of resolution by methylation of adenines at the center of site I suggests that minor groove contacts near the crossover may be required for resolution activity.

Alkylation↗

PubMed1983

A sequence related to 4.5 S RNA and the B1 family of repeated DNA in the 5' flanking region of the mouse beta-globin gene.

A beta-globin gene probe containing 5' flanking DNA hybridizes to a small nuclear RNA from Friend erythroleukemia cells, which was subsequently identified as 4.5 S RNA. This hybridization is shown to reflect complementarity between the Alu consensus region of 4.5 S RNA and the DNA sequence between nucleotides -33 and -48 from the beta-globin gene transcription initiation site.

Animals↗

PubMed1983

Sequence arrangement and protein coding capacity of the adenovirus type 2 "i" leader.

The adenovirus type 2 (Ad-2) "i" leader is an RNA segment which is preserved in some mRNA species from the Ad-2 late transcription unit. It maps between the second and third segments of the standard tripartite leader. We located the boundaries of the i leader in genomic Ad-2 DNA and determined its nucleotide sequence. The leader contains an ATG initiator near its 5' boundary, followed by a reading frame which is open for translation. We suggest that the i leader constitutes an Ad-2 coding sequence whose novel position within the leader of major late transcription unit messengers allows it to be translated in preference to coding sequences in mRNA main bodies. The i leader potentially contributes to the coding sequences of a family of proteins. Also, a Northern blot analysis of late mRNAs containing the i leader suggests that it may be retained in the leaders of many different late transcription unit mRNAs. We compare the i leader to the simian virus 40 agnogene.

Adenoviruses, Human↗