Biomedical subjects

R Chalkley

Publications and source records attributed to R Chalkley.

At least 19 recordsLinked to original sources

PubMed2000

Phosphoenolpyruvate carboxykinase is necessary for the integration of hepatic energy metabolism.

We used an allelogenic Cre/loxP gene targeting strategy in mice to determine the role of cytosolic phosphoenolpyruvate carboxykinase (PEPCK) in hepatic energy metabolism. Mice that lack this enzyme die within 3 days of birth, while mice with at least a 90% global reduction of PEPCK, or a liver-specific knockout of PEPCK, are viable. Surprisingly, in both cases these animals remain euglycemic after a 24-h fast. However, mice without hepatic PEPCK develop hepatic steatosis after fasting despite up-regulation of a variety of genes encoding free fatty acid-oxidizing enzymes. Also, marked alterations in the expression of hepatic genes involved in energy metabolism occur in the absence of any changes in plasma hormone concentrations. Given that a ninefold elevation of the hepatic malate concentration occurs in the liver-specific PEPCK knockout mice, we suggest that one or more intermediary metabolites may directly regulate expression of the affected genes. Thus, hepatic PEPCK may function more as an integrator of hepatic energy metabolism than as a determinant of gluconeogenesis.

Alleles↗

PubMed1999

Characterization of a kidney-specific pattern of chromatin structure in the rat phosphoenolpyruvate carboxykinase gene.

The kidney-specific chromatin structure of the phosphoenolpyruvate carboxykinase (PEPCK) gene was examined and compared to that of the liver. Kidney nuclear extracts were found to lack a liver-enriched factor, pepA, that binds to HSS A, a distal enhancer of the PEPCK gene that may be involved in opening the chromatin domain of the PEPCK gene in the liver. To begin the characterization of the kidney-specific chromatin structure of the PEPCK gene, nuclease hypersensitive sites (HSS) were mapped by indirect end-labeling analysis in proximal tubules from control rats, proximal tubules from acidotic rats which express induced levels of PEPCK, and NRK52E cells, a rat kidney epithelial cell line which does not express the PEPCK gene. A subset of HSS, at -400/+1 over the proximal promoter and at +1900 within the coding region, correlate with kidney-specific PEPCK expression. Two other HSS, at -3.1 kb and +6.2 kb, are detected in kidney cells regardless of PEPCK expression. The HSS at -4800, -1240, and +4650, previously identified in PEPCK-expressing liver cells, were not observed in the kidney. As in the liver, the pattern of hypersensitivity in the kidney does not change by altering the rate of transcription.

Acidosis↗

PubMed1999

Scanning a DRB3*0101 (DR52a)-restricted epitope cross-presented by DR3: overlapping natural and artificial determinants in the human acetylcholine receptor.

A recurring epitope in the human acetylcholine receptor (AChR) alpha subunit (alpha146-160) is presented to specific T cells from myasthenia gravis patients by HLA-DRB3*0101-"DR52a"-or by DR4. Here we first map residues critical for DR52a in this epitope by serial Ala substitution. For two somewhat similar T cells, this confirms the recently deduced importance of hydrophobic "anchor" residues at peptide p1 and p9; also of Asp at p4, which complements this allele's distinctive Arg74 in DRbeta. Surprisingly, despite the 9 sequence differences in DRbeta between DR52a and DR3, merely reducing the bulk of the peptide's p1 anchor residue (Trp149-->Phe) allowed maximal cross-presentation to both T cells by DR3 (which has Val86 instead of Gly). The shared K71G73R74N77 motif in the alpha helices of DR52a and DR3 thus outweighs the five differences in the floor of the peptide-binding groove. A second issue is that T cells selected in vitro with synthetic AChR peptides rarely respond to longer Ag preparations, whereas those raised with recombinant subunits consistently recognize epitopes processed naturally even from whole AChR. Here we compared one T cell of each kind, which both respond to many overlapping alpha140-160 region peptides (in proliferation assays). Even though both use Vbeta2 to recognize peptides bound to the same HLA-DR52a in the same register, the peptide-selected line nevertheless proved to depend on a recurring synthetic artifact-a widely underestimated problem. Unlike these contaminant-responsive T cells, those that are truly specific for natural AChR epitopes appear less heterogeneous and therefore more suitable targets for selective immunotherapy.

Alleles↗

PubMed1999

Isolation and characterization of the mouse cytosolic phosphoenolpyruvate carboxykinase (GTP) gene: evidence for tissue-specific hypersensitive sites.

A 72 kilobase pair DNA fragment that contains the mouse phosphoenolpyruvate carboxykinase (PEPCK) gene locus, pck1, was isolated from a genomic bacterial artificial chromosome library. The region from approximately -5.5 to +6.6 kilobase pairs relative to the pck1 transcription start site was sequenced and exhibits a high degree of homology to the rat and human genes. Additionally, the chromatin structure of the PEPCK gene in mouse liver resembles that seen in rat. Backcross panel analysis of a microsatellite sequence confirms that the gene is located on chromosome 2. Hypersensitive site analysis was performed on nuclei isolated from the adipocyte cell line 3T3-F442A in the preadipose and adipose states. Several hypersensitive sites are present in the undifferentiated 3T3-F442A cells, before PEPCK mRNA is detected. The same sites are present after differentiation, however, the sensitivity of mHS 3 increases relative to the others. We conclude that the chromatin is open in 3T3-F442A cells and that factors are able to bind in the undifferentiated state but that something else is required for transcription.

3T3 Cells↗

PubMed1998

Multiple domains for initiator binding proteins TFII-I and YY-1 are present in the initiator and upstream regions of the rat XDH/XO TATA-less promoter.

We previously reported that the TATA-less rat xanthine dehydrogenase/oxidase (XDH/XO) promoter is organized with multiple initiator elements (Inr 1, 2, 3 and 4). Additionally, we identified six factor binding footprints in the upstream region of this promoter (FP 1-FP 6), two of which (FP 2 and FP 4) we showed to be C/EBP binding sites. In this report we continue our characterization of the XDH/XO promoter, detailing other cis elements which comprise the Inr and upstream binding factors. Interestingly, multiple binding domains for known initiator binding proteins, YY-1 and USF-related factor/TFII-I, have been identified which potentially play an important role in transcription initiation.

Animals↗

PubMed1998

Correct usage of multiple transcription initiation sites and C/EBP-dependent transcription activation of the rat XDH/XO TATA-less promoter requires downstream elements located in the coding region of the gene.

In the present study, we have shown that a downstream element located in the coding region of the TATA-less rat xanthine dehydrogenase/oxidase (XDH/XO) gene (-7 to +42) plays an important role in transcription initiation and C/EBP transcriptional activation. Previous work from our laboratory has shown that the promoter is organized with multiple initiator elements (Inr 1, 2, 3 and 4) which are important for transcription initiation. Additionally, we had identified two C/EBP binding sites upstream of this promoter. Deletional and mutational studies revealed that C/EBP binding was not essential for the basal level of transcriptional initation. However when XO-luciferase constructs include downstream sequence extending to +42 there is development of C/EBP sensitivity as well as a shift in the initiator usage. In the absence of the downstream element, primer extension analyses reveals Inr 3 and 4 to be the major start sites but in the presence of this additional sequence the usage is shifted to Inr 1 and 2. This shift in Inr usage more closely resembles that seen in intact macrophages or liver cells. Gel mobility shift assays indicate the presence of several binding factors located in this downstream region, one of which has been identified as YY-1. We postulate that YY-1 allows DNA bending which permits the upstream C/EBP elements to exhibit a transcriptional activation which is not seen when the downstream element is absent. This study presents a potential model for regulation of the XDH/XO promoter.

Animals↗

PubMed1996

In vitro transcription in the presence of DNA oligonucleotides can generate strong anomalous initiation sites.

In the present study, we have explored an unexpected observation in transcription initiation that is mediated by single-stranded oligonucleotides. Initially, our goal was to understand the function of different upstream regulatory elements/initiation sites in the rat xanthine dehydrogenase/oxidase (XDH/XO) promoter. We performed in vitro transcription with HeLa nuclear extracts in the presence of different double-stranded oligonucleotides against upstream elements as competitors. A new and unusual transcription initiation site was detected by primer extension. This new initiation site maps to the downstream region of the corresponding competitor. Subsequent analyses have indicated that the induction of a new transcription initiation site is anomalous which is due to the presence of a small amount of single-stranded oligonucleotide in the competitor. We found that this anomalous initiation site is insensitive to the orientation of the promoter and requires only a small amount of single-stranded oligonucleotide (< 2-fold molar excess relative to template). We surmise that a complementary interaction between the single-stranded oligonucleotide and transiently denatured promoter template may be responsible for this sequence-specific transcription initiation artifact. To study the regulation of transcription initiation by in vitro transcription approaches, we propose that one should probe the effect of removing transacting factors by adding an excess of a cognate oligonucleotide which does not bear exact sequence identity to the template.

Animals↗

PubMed1995

Cell specific differences in DNase I hypersensitivity between the two promoters of the rat glucokinase gene.

Glucokinase (GK) gene transcription occurs in the liver and the beta cell of the endocrine pancreas where it is subject to different modes of regulation. This is accomplished largely through the use of two linked, cell-specific promoters separated by at least 12 kbp. We have used DNase I hypersensitivity to explore the chromatin structure surrounding the two promoters in cells that express either the liver or beta cell form of the GK gene, as well as cells that do not express GK. In RIN38 cells, a beta-cell-derived cell line, hypersensitive sites are detected over both the proximal and distal promoters. In liver, hypersensitive sites are present in the proximal promoter but not the distal promoter. Interestingly, in H4IIEC3 cells, a hepatoma cell line that has lost the ability to express GK, hypersensitive sites are also found in the proximal promoter but not the distal promoter.

Animals↗

PubMed1995

Multiple initiators and C/EBP binding sites are involved in transcription from the TATA-less rat XDH/XO basal promoter.

In the present study, we have explored further the organization of the TATA-less rat xanthine dehydrogenase/oxidase gene (XDH/XO). A DNase I hypersensitive site has been identified which it colocalizes with the basal promoter reported previously [Chow et al. (1994) Nucleic Acids Res., 22, 1846-1854]. Gel mobility shift assays indicate the presence of multiple binding factors located in the promoter. At least six footprints were detected of which two have been shown to be C/EBP binding sites. Members of the C/EBP-alpha and C/EBP-beta, but not C/EBP-delta, family are able to bind to these two sites. Deletional and mutational studies revealed that C/EBP binding is not essential for the basal level of transcription initiation of this promoter. Much of the transcriptional activity resides in the -102 to -7 DNA fragment, which contains all initiator activity which acts unidirectionally. Within this fragment, four putative initiator elements could be identified; interestingly, the linear integrity of these initiators is important for efficient transcription of the XDH/XO gene. Separation of the initiators leads to a complete loss of transcription activity; however, this loss could be partially restored by the introduction of an Sp1 binding site upstream of the separated initiators. Despite a difference in usage/frequency of initiation at the various initiators, primer extension analyses reveal similar positions for transcription initiations in both XDH/XO reporter constructs and in the endogenous XDH/XO gene. The differential usage of initiators may imply a possible post-transcriptional regulation for the XDH/XO gene.

Animals↗

PubMed1994

Extent of in vivo binding by an upstream activation factor and the role of multiple binding sites in synergistic transcriptional activation.

We have analyzed site occupancy in vivo with constructs containing one or more copies of the binding site for the yeast trans-activator, yIBF. The data indicate only a modest difference in site occupancy (at most 2-fold) even though multiple copies activate transcription several hundredfold more than one copy. Using studies in which we have mutated the IES2 sequence and slightly decreased its affinity for yIBF, we have also shown that synergistic activation of transcription is dependent on overall site occupancy. Finally, synergy declines as yIBF-binding sites are separated, and loss of synergy appears to be correlated with loss of a linking surface. These results are consistent with the simultaneous contact model of synergistic activation.

Base Sequence↗

PubMed1994

Identification of the rat xanthine dehydrogenase/oxidase promoter.

Inflammation and ischemia--reperfusion tissue injury are important pathophysiologic processes with a wide spectrum of clinical presentations; the enzyme xanthine dehydrogenase/oxidase (XDH/XO) is thought to play a key role in ischemia--reperfusion injury. Recent studies have shown the transcriptional regulation of XDH/XO by cytokines (Dupont et al., 1992, J. Clin. Invest. 89, 197-202). In the present study, the 5' structure of the XDH/XO gene and characterization of its promoter are undertaken providing an initial step to further elucidate the regulatory mechanism(s) of this enzyme. XDH/XO cDNA from rat bone marrow macrophage has been isolated and used to screen a rat genomic library in order to identify and characterize the promoter of the XDH/XO gene. By Southern analysis, XDH/XO was found to be a single copy gene in the rat genome. Primer extension, RNase protection, and anchor-PCR studies indicate the presence of multiple start sites within a 65 bp window located some 20-85 bp upstream of the translation initiator (ATG). Functional studies of the sequences up to 116 nt upstream of the translational start site, which encompasses the several transcriptional start sites, indicate that this region is sufficient to drive the expression of a luciferase reporter gene and is presumed to represent the promoter. Neither a TATA box nor a GC-rich region are present in close proximity to any of the transcriptional start sites; however, sequences with homology to known initiator elements are found within this 116 bp fragment. Several possible regulatory elements, including a NF-IL6 motif, are also located upstream of the transcriptional start site. This study represents the first description of the XDH/XO promoter from a vertebrate system.

Animals↗

PubMed1994

Analysis of the competition between nucleosome formation and transcription factor binding.

We have studied the competitive binding of histones and the Rous sarcoma virus internal enhancer binding factor (IBF) factor (which recent studies indicate is almost certainly cEBP beta). We find that histones and IBF are incapable of forming a ternary complex with a 159-base pair (bp) fragment of DNA containing a single IBF binding site and that histones and factor are mutually exclusive in binding. We have analyzed the various physical parameters of binding, in an attempt to understand how the factor might establish an exclusive binding in the cell. The stability of the nucleosome and the factor-DNA complex have been determined, and in addition a minimum value for the affinity of the histone octamer has been computed. We find that in simple competition the IBF can successfully compete, only if the substrate DNA is shorter than 140 bp. The relevance of these results is discussed in terms of a kinetic model for successful factor competition during the replication of the factor binding site in the cell.

Avian Sarcoma Viruses↗

PubMed1993

Dynamic aspects of DNA/protein interactions in the transcriptional initiation complex and the hormone-responsive domains of the phosphoenolpyruvate carboxykinase promoter in vivo.

Transcription initiation of the gene encoding phosphoenolpyruvate carboxykinase (PEPCK) is stimulated by glucocorticoids and glucagon, via cAMP, and dominantly inhibited by insulin in rat liver and H4IIE cells. Lysolecithin-permeabilized H4IIE cells recover completely and continue to multiply, yet are transiently penetrable by macromolecules. These cells, after various hormonal treatments, were utilized for in situ DNase I protection studies of the PEPCK promoter. Nearly all of the sites of protein interaction observed in vitro are protected in vivo as well as several additional sites. The DNase I protection pattern is the same in cells without or with any of the hormone treatments, suggesting that hormonal modulation of transcription does not involve addition or removal of factors from the hormone response elements of the promoter. We focused on the organization and stability of the transcription initiation complex as well as the dynamic nature of distal promoter factors in their interaction with DNA. The transcription initiation complex was detected, and it appears to be co-existent with a short region of naked single-stranded DNA over the TATA box on the template strand, as determined by potassium permanganate reactivity. This complex is quite stable, even under conditions of much reduced RNA synthesis, which suggests that the complex is not broken down and reformed with each round of initiation by RNA polymerase II. Other factors bind to the PEPCK promoter with half-lives ranging from a few minutes to more than 40 min. The cAMP response element apparently involves transcriptional modulation achieved through modification of a bound factor (presumably cAMP response element-binding protein), whereas the glucocorticoid/insulin-responsive region of the promoter functions through factors which are involved in a rapid exchange, suggesting quite different modes of transcriptional regulation.

Animals↗

PubMed1993

Characterization of rat pseudogenes for enhancer factor I subunit A: ripping provides clues to the evolution of the EFIA/dbpB/YB-1 multigene family.

Genomic Southern blot analysis of rat EFIA (gene encoding enhancer factor I subunit A) reveals a complex band pattern when cDNA subfragment probes are used. Screening a rat genomic library with a rat EFIA cDNA probe yields two different processed EFIA pseudogenes, designated rat psi EFIA#(2/3) and #(4/7), in addition to two other different, but less extensively characterized clones. psi EFIA#(4/7) has no open reading frame (ORF) sequences. psi EFIA#(2/3) contains two ORFs (83 and 178 codons), the products of which (if expressed) might be negative-acting EFIA transcription factors. Located nearly 0.6 kb upstream from psi EFIA#(2/3) is a perfect 69-bp dinucleotide (CT) tandem repeat, a sequence element associated with other isolated pseudogenes. Additionally, the 3' end of this processed gene is interrupted by an unusual retroposon, an inverted dimeric B1-like short interspersed repetitive element (SINE). The isolation of several independent clones of the same EFIA processed pseudogenes indicates that they comprise a significant component of the rat EFIA copy multiplicity. The phenomenon of repeat induced point mutagenesis (ripping) at rat EFIA pseudogene CpG doublets occurs at a frequency at least 6.5 times higher than predicted from random mutagenesis. This is consonant with the proposal that ripping may be the mechanism which inactivates the ectopic recombination potential of the rat EFIA pseudogenes.

Amino Acid Sequence↗

PubMed1993

Isolation of the CCAAT transcription factor subunit EFIA cDNA and a potentially functional EFIA processed pseudogene from Bos taurus: insights into the evolution of the EFIA/dbpB/YB-1 gene family.

The genomic copy multiplicity of the CCAAT transcription complex component enhancer factor I subunit A (EFIA) has been examined. When a mammalian genomic Southern blot was hybridized to a rat EFIA cDNA, a complex pattern consisting of numerous related sequences was found in all the species examined, with Bos taurus being the least complex. An EFIA#1 cDNA from Bos taurus was isolated from a primary lung endothelial cell cDNA library by screening with the 1489-bp rat EFIA cDNA. The deduced bovine EFIA#1 amino acid (aa) sequence is 98% identical to rat EFIA and 100% identical to human EFIA/DbpB/YB-1 family member DNA-binding protein B (DbpB). In addition, a processed EFIA pseudogene from Bos taurus, designated bovine psi EFIA#1, was obtained from a genomic library by screening with a rat EFIA cDNA probe. The bovine psi EFIA#1 gene has an ORF which, if expressed, would encode a 140-aa sequence, with aa 31-140 having 84% identity to bovine EFIA#1. The genomic cloning data indicate that processed pseudogenes are partially responsible for the complexity of the EFIA genomic Southern blots. The phenomenon of 'repeat induced point mutation' (ripping) at bovine psi EFIA#1 gene CpG dinucleotides occurs at a 6.5-fold higher frequency than expected from random mutagenesis. Therefore, ripping is likely to be the mechanism by which the bovine EFIA#1 pseudogene's ectopic recombination potential was inactivated.

Amino Acid Sequence↗

PubMed1992

Characterization of the factors binding to a PEPCK gene upstream hypersensitive site with LCR activity.

A previously described upstream hypersensitive site (HS) in the PEPCK gene at -4800 bp, termed HS A (1), has been characterized and determined to bind at least two factors. One of these is a member of the ubiquitous CREB/ATF family, and the second is a novel tissue specific protein, pep A. A construct carrying HS A and the PEPCK proximal promoter was tested in transgenic mice and its CAT activity compared to the proximal promoter alone. The HS A was shown to drive tissue-specific, position-independent transcription of the CAT reporter gene 2-3 fold more effectively than the proximal promoter alone, with a concommitant 4-5 fold higher expression of CAT. Protein binding activity has been localized to a 33 bp region. This region contains a CRE (2) which is shown to bind a member of the CREB/ATF family through competition assays with an oligo containing a CRE from the proximal promoter and by the appearance of a supershift when the factor/oligo complex was exposed to CREB polyclonal antibody. Through restriction enzyme digests and competition of protein binding with an oligonucleotide homologous to HS A with a mutated CRE we have characterized a putative binding site for a liver-specific factor. In vitro and 'in vivo' footprinting studies complement each other, as well as, mobility shift assay data in designating the binding site of the proteins. The CREB/ATF factor and Pep A bind independently of each other during short term incubations, however, both factors can be accomodated on the DNA substrate as a function of extended time of incubation. Preliminary biochemical analysis defines the subunit molecular mass of the CREB/ATF like proteins at 55, 42, and 35 kD, while the tissue specific material exists as a single homogeneous subunit polypeptide in SDS of molecular mass = 49 kD.

Animals↗

PubMed1991

The interplay of ubiquitous DNA-binding factors, availability of binding sites in the chromatin, and DNA methylation in the differential regulation of phosphoenolpyruvate carboxykinase gene expression.

We have identified DNA elements in the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter which are bound 'in vivo' by proteins under conditions of basal level gene expression and have evaluated several hypothesis to account for the tissue specific expression of the gene. In vitro DNase I footprinting demonstrated that factors which bind to basal expression elements of the PEPCK promoter, the BSE/CRE and NFI/CCAAT sites, are also present in HTC and XC cells which do not express the PEPCK gene. 'In vivo' DNase I footprinting demonstrated that the BSE/CRE, NFI/CCAAT, and three additional sites are bound by protein in H4IIE cells which express the PEPCK gene but not in the HTC or XC cells. No evidence for a repressor protein or for phased nucleosome binding to the PEPCK promoter in HTC or XC cells could be detected. Genomic sequencing was used to determine if differential methylation of the PEPCK promoter could account for the lack of factor binding in HTC and XC nuclei. None of the 14 cytosine residues in CpG dinucleotides was methylated in H4IIE or rat liver DNA, all were methylated in rat sperm DNA, and 6 were methylated in HTC DNA; including the cytosine at position--90 within the BSE/CRE. Only one cytosine residue, at position--90, was methylated in XC DNA. Treatment of XC cells with 5-azacytidine resulted in loss of methylation at the--90 position yet this was insufficient to allow synthesis of a detectable amount of PEPCK mRNA.

Animals↗

PubMed1990

Isolation and characterization of a cDNA clone for the CCAAT transcription factor EFIA reveals a novel structural motif.

Enhancer factor I (EFI) is a trans-acting factor which binds to the Rous sarcoma virus long terminal repeat enhancer and promoter at two inverted CCAAT-box motifs. We demonstrate that two forms of EFI DNA binding activity exist in nuclear extracts of avian cells. One form requires two heterologous components (EFIA)(EFIB) for high affinity, specific DNA binding activity, whereas a second form is not dependent on EFIB for binding and may be composed solely of EFIA, perhaps as a multimer. Both forms give rise to the same mobility shift in gel retardation assays, but the two forms can be separated chromatographically under buffer conditions which stabilize the two DNA binding activities. A cDNA for EFIA has been isolated from a rat liver cDNA expression library. The 1489-base pair EFIA cDNA encodes a 322-amino acid protein which is nearly identical to two previously described human DNA binding proteins. These are dbpB, a DNA binding protein of unknown specificity which binds to the epidermal growth factor receptor enhancer and c-erbB-2 gene promoter (Sakura, H., Maekawa, T., Imamoto, F., Yasuda, K., and Ishii, S. (1988) Gene (Amst.) 73, 499-507), and YB-1, a protein which recognizes the Y-box (inverted CCAAT motif) of the HLA-DR alpha chain gene (Didier, D. K., Schiffenbauer, J., Woulfe, S. L., Zacheis, M., and Schwartz, B. D. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 7322-7326). EFIA/dbpB/YB-1 share a highly conserved region of 100 amino acids with dbpA, another protein identified by Sakura et al. (1988) which binds to the epidermal growth factor receptor enhancer and c-erbB-2 gene promoter, and with two Xenopus CCAAT binding proteins, FRG Y1 and FRG Y2 (Tafuri, S. R., and Wolffe, A. P. (1990) Proc. Natl. Acad. Sci. U. S. A., in press). This highly conserved domain among all six proteins is presumed to represent or contain a DNA binding domain for the CCAAT motif. In addition, we note that the EFIA/dbpB/YB-1 polypeptide contains a novel arrangement of alternating clusters of positively and negatively charged amino acids not yet reported for any trans-acting factor. The functional significance of this novel structural motif, which is also conserved in dbpA, FRG Y1, and FRG Y2, will be discussed.

Amino Acid Sequence↗