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M L Short

Publications and source records attributed to M L Short.

12 recordsLinked to original sources
PubMed2000

Structural determinants for post-transcriptional stabilization of lactate dehydrogenase A mRNA by the protein kinase C signal pathway.

Activation of protein kinase C (PKC) and protein kinase A (PKA) in rat C6 glioma cells increases the half-life of short-lived lactate dehydrogenase (LDH)-A mRNA about 5- and 8-fold, respectively. PKA and PKC act synergistically and prolong LDH-A mRNA half-life more than 21-fold. Similar effects were observed after transfection and transcription of a globin/lactate dehydrogenase minigene consisting of a beta-globin expression vector in which the 3'-untranslated region (UTR) of beta-globin had been replaced with the LDH-A 3'-UTR. Synergism was only obtained by transcription of minigenes containing the entire 3'-UTR and did not occur when truncated 3'-UTR fragments were analyzed. Additional mutational analyses showed that a 20-nucleotide region, named PKC-stabilizing region (PCSR), is responsible for mediating the stabilizing effect of PKC. Previous studies (Tian, D., Huang, D., Short, S., Short, M. L., and Jungmann, R. A. (1998) J. Biol. Chem. 273, 24861-24866) have demonstrated the existence of a cAMP-stabilizing region in LDH-A 3'-UTR. Sequence analysis of PCSR identified a 13-nucleotide AU-rich region that is common to both cAMP-stabilizing region and PCSR. These studies identify a specific PKC-responsive stabilizing element and indicate that interaction of PKA and PKC results in a potentiating effect on LDH-A mRNA stabilization.

3' Untranslated Regions↗
PubMed1998

Protein kinase A-regulated instability site in the 3'-untranslated region of lactate dehydrogenase-A subunit mRNA.

Expression of the lactate dehydrogenase A subunit (LDH-A) gene can be controlled by transcriptional as well as posttranscriptional mechanisms. In rat C6 glioma cells, LDH-A mRNA is stabilized by activation and synergistic interaction of protein kinases A and C. In the present study, we aimed to identify the sequence domain which determines and regulates mRNA stability/instability by protein kinase A and focused our attention on the 3'-untranslated region (3'-UTR) of LDH-A mRNA. We have constructed various chimeric globin/lactate dehydrogenase (ldh) genes linked to the c-fos promoter and stably transfected them into rat C6 glioma cells. After their transfection, we determined the half-life of transcribed chimeric globin/ldh mRNAs. The results showed that at least three sequence domains within the LDH-A 3'-UTR consisting of nucleotides 1286-1351, 1453-1471, and 1471-1502 are responsible for the relatively rapid rate of LDH-A mRNA turnover in the cytoplasm. Whereas chimeric globin/ldh mRNAs containing the base sequences 1286-1351 and 1453-1471 were not stabilized by (Sp)-cAMPS, an activator of protein kinase A, instability caused by the 1471-1502 domain was significantly reversed. Additional deletion and mutational analyses demonstrated that the 3'-UTR fragment consisting of the 22 bases 1478-1499 is a critical determinant for the (Sp)-cAMPS-mediated LDH-A mRNA stabilizing activity. Because of its functional characteristics, we named the 22-base region "cAMP-stabilizing region."

Animals↗
PubMed1997

Complex protein binding to the mouse M-lysozyme gene downstream enhancer involves single-stranded DNA binding.

The mouse M-lysozyme downstream enhancer has been previously characterized on several levels of gene regulation. The enhancer was co-localized with a DNase I hypersensitive site in the chromatin of mature macrophages, the in vivo interaction of transcription factor GABP with the enhancer core (MLDE) demonstrated binding being restricted to mature macrophage cells, and analysis of the MLDE methylation state revealed a correlation between demethylation of CpG dinucleotides and the in vivo GABP binding. Here, we analyzed in detail the full-length enhancer in addition to the core element. We identified a total of nine binding sites for nuclear factors. Most of these factors are found ubiquitously in all cell types tested. These factors include several unknown proteins as well as the transcription factor NF-Y. In addition, three binding sites for a new single-stranded DNA binding protein were found. The presence of this factor in mature macrophages correlates with the in vivo DNA melting of one of the binding sites and with the enhancer strength.

Animals↗
PubMed1996

Lysozyme gene expression and regulation.

Analysis of lysozyme gene expression in chicken and mouse identified two evolutionarily different mechanisms of lysozyme gene regulation. The lysozyme gene in chicken is expressed in the oviduct and macrophage cells with expression regulated by different, partially overlapping sets of tissue specific cis-acting elements. In contrast to chicken, the mouse genome contains two lysozyme genes generated by a gene duplication event allowing each gene to be regulated by its own regulatory region. One gene is expressed in macrophages, the other in Paneth cells of the small intestine. The macrophage-specific gene contains a single strong enhancer in the 3'-flanking sequences that interacts with ubiquitously factors. Cytosine methylation of the core enhancer sequence has been implicated in the regulation of the enhancer activity. In spite of these evolutionary regulatory differences, the chicken lysozyme transgene retains macrophage-specific expression in mice.

Animals↗
PubMed1996

In vivo protein interaction with the mouse M-lysozyme gene downstream enhancer correlates with demethylation and gene expression.

Differentiation of myeloid precursor cells results in transcriptional activation of the myeloid-specific murine M-lysozyme gene. M-lysozyme gene expression depends on the differentiation state of the myeloid cells and provides a marker for myeloid leukemias. The mouse lysozyme downstream enhancer (MLDE) was colocalized previously with the DNase I hypersensitive site in the chromatin of mature macrophages and shown to be macrophage differentiation-dependent. The correlation of the hypersensitive site appearance with expression of the M-lysozyme gene suggests that the enhancer becomes activated during macrophage differentiation. However, the predominant MLDE-binding protein GABP is ubiquitously expressed, indicating that additional regulatory mechanisms are required for restricting the tissue-specific activity of the enhancer. To demonstrate the specificity of the enhancer in vivo, we examined the in vivo interaction of factors with the MLDE in T cells, immature macrophage cells, and in macrophage cells. Although identical DNase I protection activity is present in extracts from all tested cell lines in vitro, the in vivo interaction of proteins is restricted to mature macrophage cells. The presence of factors capable of interacting with the enhancer is not sufficient for enhancer activity, suggesting that the process of differentiation results in factor accessibility for the MLDE. Analysis of the MLDE methylation state revealed a correlation between demethylation of the single CpG dinucleotide within the MLDE sequence and the in vivo interaction of proteins.

Animals↗
PubMed1995

Methylation of the mouse M-lysozyme downstream enhancer inhibits heterotetrameric GABP binding.

Expression of the mouse M-lysozyme gene is a specific marker for the differentiation of macrophage/granulocyte cell lineages. Analysis of the mechanisms regulating M-lysozyme gene expression revealed an enhancer element in the 3'-flanking region of the gene, termed the M-lysozyme downstream enhancer (MLDE). Here we demonstrate that the nuclear factors binding to MLDE are present in all tested myeloid and non-myeloid mouse cell lines. Sequence analysis of MLDE identified two different sequences, CAGGAAGT and CCGGAAGT, which match the consensus binding sequences for proteins of the ets gene superfamily. The two sites are oriented palindromicly and separated by 10 bp. DMS/DEPC interference assays revealed different patterns of DNA-protein contacts on the two sites. Mutation of each consensus sequence leads to an individual change in protein binding in vitro. Despite these differences, both sequences are bound by GABP, forming a heterotetrameric complex. Tissue specificity is correlated with demethylation of a single CpG dinucleotide located in one of the two Ets motifs. This site when methylated inhibits GABP binding to both sequences in non-macrophage cell types.

Animals↗
PubMed1995

Identification of a silencer module which selectively represses cyclic AMP-responsive element-dependent gene expression.

The cyclic AMP (cAMP)-inducible promoter from the rat lactate dehydrogenase A subunit gene (LDH A) is associated with a distal negative regulatory element (LDH-NRE) that represses inherent basal and cAMP-inducible promoter activity. The element is of dyad symmetry, consisting of a palindromic sequence with two half-sites, 5'-TCTTG-3'. It represses the expression of an LDH A/chloramphenicol acetyltransferase (CAT) reporter gene in a dose-dependent, orientation- and position-independent fashion, suggesting that it is a true silencer element. Uniquely, it selectively represses cAMP-responsive element (CRE)-dependent transcription but has no effect on promoters lacking a CRE sequence. The repressing action of LDH-NRE could be overcome by cotransfection with LDH A/CAT vector oligonucleotides containing either the LDH-NRE or CRE sequence. This suggests that the reversal of repression was caused by the removal of functional active, limiting transacting factors which associate with LDH-NRE as well as with CRE. Gel mobility shift, footprinting, and Southwestern blotting assays demonstrated the presence of a 69-kDa protein with specific binding activity for LDH-NRE. Additionally, gel supershift assays with anti-CREB and anti-Fos antibodies indicate the presence of CREB and Fos or antigenically closely related proteins with the LDH-NRE/protein complex. We suggest that the LDH-NRE and CRE modules functionally interact to achieve negative modulation of cAMP-responsive LDH A transcriptional activity.

Animals↗
PubMed1994

Analysis of the rat lactate dehydrogenase A subunit gene promoter/regulatory region.

The rat lactate dehydrogenase (LDH) A subunit gene promoter contains a putative AP-1 binding site at -295/-289 bp, two consensus Sp1 binding sites at -141/-136 bp and -103/-98 bp, and a single copy of a consensus cyclic AMP-responsive element (CRE) at -48 to -41 bp upstream of the transcription initiation site. Additionally, an as yet unidentified silencer element is located within the -1173/-830 bp 5'-flanking region. Transient transfection analyses of a -1173/+25 bp LDH A-chLoramphenicol acetyltransferase fusion gene has indicated a complete inability of the promoter fragment to direct basal or forskolin-induced transcription. Deletion of the -1173/-830 bp sequence restored basal and cyclic AMP (cAMP)-inducible activity. Point mutations in the Sp1 binding sites of a -830/+25 bp promoter fragment reduced basal but not the relative degree of cAMP-inducible activity. cAMP-regulated transcriptional activity was dependent upon an 8 bp CRE, -TGACGTCA-, located at the -48/-41 bp upstream region. Mutations in the CRE abolished cAMP-mediated induction and reduced basal activity by about 65%. The CRE binds a 47 kDa protein which has previously been identified as CRE binding protein (CREB)-327, an isoform of the activating transcription factor/CREB transcription factor gene family. Co-transfection of a vector that expresses the catalytic subunit of cAMP-dependent protein kinase stimulates LDH A subunit promoter activity suggesting that cAMP induces LDH A subunit gene expression through phosphorylative modification of CREB-327. This study emphasizes a fundamental role of several modules including Sp1 and CREB binding sites in regulating basal and cAMP-mediated transcriptional activity of the LDH A gene.

Animals↗
PubMed1992

Glucocorticoid induction of CRE-binding protein isoform mRNAs in rat C6 glioma cells.

Mammalian cells express several distinct isoforms of transcription factor CREB (cAMP-responsive element binding protein). At least two forms, alpha- and delta CREB, arise through alternative splicing of the CREB gene transcript. In this communication we demonstrate that the mRNAs of several CREB isoforms are expressed in rat C6 glioma cells and that the intracellular levels of these mRNAs are markedly induced by the synthetic glucocorticoid dexamethasone. Nuclear run-off assays show that the induction occurs, at least in part, through a transcriptional mechanism. The enhanced cellular levels of CREB mRNAs are accompanied by increased CREB protein and CRE-binding activity of nuclear extracts as evaluated by immunoblot and Southwestern blot assays.

Animals↗
PubMed1991

Functional analysis of cis- and trans-regulatory elements of the lactate dehydrogenase A subunit promoter by in vitro transcription.

Using a transcription system from nuclear extracts of rat C6 glioma cells we have investigated the mechanism by which transcription from the lactate dehydrogenase A subunit (LDH) promoter is regulated via the cAMP-activated pathway. We demonstrated that the system accurately initiates transcription from the LDH promoter. Analysis of the competitive effects of linker-scanning mutants showed that the wild-type LDH promoter exhibited the highest competitive effect and reduced the rate of basal transcription, whereas LDH promoter fragments with a mutated cAMP-responsive element had little competitive activity. Cyclic AMP and the catalytic subunit of cAMP-dependent protein kinase stimulated the rate of transcription from the wild-type promoter, an effect which was inhibited by the catalytic subunit inhibitor protein. A beta-galactosidase-cAMP-responsive element binding protein fusion protein had no effect on the basal rate of transcription. Addition of beta-galactosidase-cAMP-responsive element binding protein together with cAMP or the catalytic subunit, however, enhanced the rate of transcription. The demonstrated regulatory effects indicate that the sensitivity of the transcription system makes it suitable for the functional analysis of homologous LDH and possibly heterologous transcription regulatory elements.

Animals↗
PubMed1989

Identification of rat ovarian nuclear factors that interact with the cAMP-inducible lactate dehydrogenase A subunit promoter.

Utilizing the gel electrophoresis/DNA binding assay and a new technique of direct binding of radioactive DNA to protein blots, we have investigated putative factors selective for the cAMP-responsive element (CRE) of the lactate dehydrogenase A subunit promoter in rat ovary nuclear extracts. Analysis of linker-scanning mutants of lactate dehydrogenase A subunit promoter fragments by DNA binding assay identified DNA binding activity selective for the 11-nucleotide sequence 5' TCTGACGTCAG 3' located between positions -51 and -41 relative to the transcription initiation site. This sequence contains the previously identified CRE 5' TGACGTCA 3'. Probing of protein blots with radioactive promoter fragments containing the CRE demonstrated that ovarian nuclear extracts contain a protein of relative molecular mass 47,000 (Mr 47,000) which exhibits selective binding affinity for the CRE. The 47-kDa CRE binding protein was found to be present in comparable levels in the ovaries of normal and hypophysectomized rats. Furthermore, our data suggest the presence of a 37,000-dalton (Mr 37,000) protein which possesses selective binding affinity for part of the CRE sequence. The binding activity/level of the 37-kDa CRE binding protein appeared to be modulated by short-term hypophysectomy/follicle-stimulating hormone administration. These results provide evidence for the presence of CRE binding factors in rat ovarian nuclei, which may be involved in the molecular events responsible for transcriptional regulation of ovarian cAMP-inducible genes.

Animals↗