Biomedical subjects

M Kornuc

Publications and source records attributed to M Kornuc.

7 recordsLinked to original sources

PubMed2000

APRIL and TALL-I and receptors BCMA and TACI: system for regulating humoral immunity.

We report that the tumor neurosis factor homolog APRIL (a proliferation-inducing ligand) stimulates in vitro proliferation of primary B and T cells and increases spleen weight due to accumulation of B cells in vivo. APRIL functions via binding to BCMA (B cell maturation antigen) and TACI (transmembrane activator and CAML-interactor) and competes with TALL-I (also called BLyS or BAFF) for receptor binding. Soluble BCMA and TACI specifically prevent binding of APRIL and block APRIL-stimulated proliferation of primary B cells. BCMA-Fc also inhibits production of antibodies against keyhole limpet hemocyanin and Pneumovax in mice, indicating that APRIL and/or TALL-I signaling via BCMA and/or TACI are required for generation of humoral immunity. Thus, APRIL-TALL-I and BCMA-TACI form a two ligands-two receptors pathway involved in stimulation of B and T cell function.

Animals↗

PubMed1998

Hippocampal plasticity involves extensive gene induction and multiple cellular mechanisms.

Long-term plasticity of the central nervous system (CNS) involves induction of a set of genes whose identity is incompletely characterized. To identify candidate plasticity-related genes (CPGs), we conducted an exhaustive screen for genes that undergo induction or downregulation in the hippocampus dentate gyrus (DG) following animal treatment with the potent glutamate analog, kainate. The screen yielded 362 upregulated CPGs and 41 downregulated transcripts (dCPGs). Of these, 66 CPGs and 5 dCPGs are known genes that encode for a variety of signal transduction proteins, transcription factors, and structural proteins. Seven novel CPGs predict the following putative functions: cpg2--a dystrophin-like cytoskeletal protein; cpg4--a heat-shock protein: cpg16--a protein kinase; cpg20--a transcription factor; cpg21--a dual-specificity MAP-kinase phosphatase; and cpg30 and cpg38--two new seven-transmembrane domain receptors. Experiments performed in vitro and with cultured hippocampal cells confirmed the ability of the cpg-21 product to inactivate the MAP-kinase. To test relevance to neural plasticity, 66 CPGs were tested for induction by stimuli producing long-term potentiation (LTP). Approximately one-fourth of the genes examined were upregulated by LTP. These results indicate that an extensive genetic response is induced in mammalian brain after glutamate receptor activation, and imply that a significant proportion of this activity is coinduced by LTP. Based on the identified CPGs, it is conceivable that multiple cellular mechanisms underlie long-term plasticity of the nervous system.

Amino Acid Sequence↗

PubMed1995

Molecular cloning and characterization of NF-IL3A, a transcriptional activator of the human interleukin-3 promoter.

To isolate transcription factors important in the regulation of the human interleukin-3 (IL-3) gene, we screened a lambda gt11 cDNA library, constructed from phytohemagglutinin-stimulated human T-cell RNA, with a probe containing regulatory sequences in the upstream region of the IL-3 gene (located from bp -165 to -128 and referred to as the DNase I footprint A region). We isolated a 0.96-kb cDNA that encoded a basic amino acid domain and a leucine zipper domain and used the "rapid amplification and cloning of 3' ends" technique to isolate the 3' half of the cDNA clone, generating a 1.9-kb full-length cDNA clone. Using in vitro-translated protein, which we call NF-IL3A, we defined the IL-3 promoter sequences bound by NF-IL3A in DNase I footprinting assays as TAATTACGTCTG and, using gel shift assays, defined ATTACG as the minimal sequence required for binding of NF-IL3A in vitro. Proteins that bind to the NF-IL3A binding site are found in both unstimulated and stimulated T-cell lines in similar amounts, although the level of NF-IL3A mRNA increases after T-cell activation in several mature T-cell lines. The NF-IL3A protein is nearly identical to a recently identified transcriptional repressor protein, E4BP4, and NF-IL3A binds specifically to regulatory sequences in both the adenovirus E4 promoter and the human gamma interferon promoter. Cotransfection experiments demonstrate that introduction of an expression vector containing the NF-IL3A cDNA into resting T cells transactivates IL-3 promoter-chloramphenicol acetyltransferase gene plasmids that contain the A region; this effect requires the presence of an intact NF-IL3A binding site. One or more copies of the A region also confer NF-IL3A responsiveness on a heterologous promoter in T cells. NF-IL3A appears to play an important role in the expression of IL-3 by T cells.

Amino Acid Sequence↗

PubMed1993

Differential effect of HTLV infection and HTLV Tax on interleukin 3 expression.

To characterize the interactions between human T-cell leukemia virus (HTLV) infection and cellular gene expression, we examined the expression of the lymphokine interleukin 3 (IL-3) in the presence and absence of HTLV infection. IL-3, like granulocyte-macrophage colony-stimulating factor (GM-CSF), is produced by activated but not resting T cells, but although GM-CSF is constitutively expressed in HTLV-infected T cells IL-3 mRNA cannot be detected in either unstimulated or mitogen-stimulated HTLV-infected cells by polymerase chain reaction (PCR) analysis. In contrast, transient co-transfection studies with an IL-3 promoter-CAT reporter gene and an HTLV-II Tax expression construct demonstrate that Tax can transactivate the IL-3 promoter in HTLV-uninfected T cells. To determine whether differences in IL-3 promoter-binding proteins present in HTLV-infected and uninfected T cells account for this discrepancy, DNAase I footprinting of the IL-3 promoter was performed. Although crude nuclear extracts from both cell types protected the IL-3 sequences located between base pairs -168 and -125, the sequences between -125 and -103, which contain the lymphokine consensus sequences CK-1 and CK-2, were protected by extracts from HTLV-infected but not HTLV-uninfected T cells. Deletion of the region containing the CK-1 and CK-2 sequences from an IL-3 promoter CAT construct resulted in a sixfold rise in promoter activity in HTLV-infected but not uninfected T-cell lines, indicating that this region participates in the repression of IL-3 gene expression in HTLV-infected T cells.

Base Sequence↗

PubMed1990

Adenovirus early region 3 promoter regulation by E1A/E1B is independent of alterations in DNA binding and gene activation of CREB/ATF and AP1.

Transcription of the adenovirus early region 3 promoter is strongly induced by the adenovirus E1A protein. Previous DNase I footprinting has indicated that four regions in this promoter serve as binding sites for HeLa nuclear proteins. These include binding sites for NF-1 (site IV), AP1 (site III), CREB/activating transcription factor (ATF) (site II), and TATA (site I). To determine the relative importance of these sites in both the in vivo and in vitro transcriptional regulation of the E3 promoter, oligonucleotide-directed mutagenesis of these sites was performed. Each of these constructs was assayed by transfection onto HeLa cells in the presence of either dl434, an E1A/E1B deletion mutant, or wild-type adenovirus. Mutations of either the ATF- or AP1-binding sites but not the TATA- and NF1-binding sites resulted in severe decreases in both basal and E1A/E1B-induced transcriptional levels. These constructs were also assayed in in vitro transcription assays with cellular extracts prepared from dl434-infected or wild-type-adenovirus-infected HeLa cells. The wild-type E3 promoter was transcribed approximately 30 times more efficiently in extracts containing the E1A/E1B proteins compared with extracts lacking these proteins. Mutations of either the TATA element, the ATF site, or the AP1-binding site decreased both basal and E1A/E1B-induced transcriptional levels. Gel retardation analysis using these extracts indicated that the binding to ATF, AP1, or NF1 oligonucleotides was not altered in the presence of the E1A/E1B proteins compared with extracts lacking these proteins. Northern (RNA) blot analysis of c-jun and CREB RNA prepared from wild-type adenovirus and dl434-infected cells indicated that the levels of these RNAs were not altered by the E1A/E1B proteins. Immunoprecipitation of AP1 and CREB from both dl434- and wild-type-adenovirus-infected cells indicated that the amounts of these proteins were not significantly altered. These results suggest that E1A/E1B-induced activation of the E3 promoter does not involve activation of transcription factor genes nor a change in the DNA binding activity of important promoter-binding components. Our results are consistent with a model in which the E1A/E1B proteins either directly or indirectly alter the interactions of factors that bind to the basal E3 promoter transcription complex, thereby inducing transcription.

Adenovirus Early Proteins↗

PubMed1990

CREB regulation of cellular cyclic AMP-responsive and adenovirus early promoters.

The cyclic AMP (cAMP) response element-binding protein (CREB) has been demonstrated to be a key mediator of cellular promoter response to cAMP. The binding site for this protein in many cellular cAMP inducible promoters (CRE) contains the palindrome sequence TGACGTCA, which contains two half-sites for CREB binding. A related promoter element, with the core sequence TGACG, has significant homology to an AP1-binding site and contains only one half-site for CREB binding. A group of factors known as activating transcription factors (ATF) have been found to bind to the latter and related sequences found upstream of early adenovirus promoters induced by E1A, and these factors are highly homologous to the CREB protein. We wished to characterize CREB, c-jun, and c-fos binding to these sites in the somatostatin gene (CRE) and in the adenovirus early region 3 promoter (E3/ATF). Oligonucleotides complementary to each of these sites were used in gel retardation assays with in vitro-translated CREB protein. These studies indicated that CREB bound primarily as a dimer to both a single and two half-sites, though there was increased affinity to the double compared with the single half-site. The c-jun and c-fos proteins also bound to both the somatostatin CRE- and E3/ATF-binding sites, but CREB did not bind to AP1 recognition sites nor was it capable of forming heterodimers with either c-jun or c-fos. Truncations of the CREB protein, which eliminated regions of the protein containing consensus sites for phosphorylation by protein kinase A, protein kinase C, and casein kinase II, bound to both the CRE and ATF sites, indicating that these consensus sites were not essential for DNA binding or dimer formation. Transfection of CREB and protein kinase A expression constructs into F9 cells with promoters containing either a single or two half-sites for CREB binding indicated that CREB was capable of similar levels of activation of these constructs. However, the fold activation by CREB was higher for constructs containing a single half-site compared with those containing two half-sites. These results demonstrate that multiple mechanisms may regulate CREB binding, including variations in the sequences in the promoter-binding site and the presence of related DNA-binding proteins.

Adenoviridae↗

PubMed1988

Adenovirus transcriptional regulatory regions are conserved in mammalian cells and Saccharomyces cerevisiae.

The adenovirus early region 3 (E3) promoter is an early viral promoter which is strongly induced by the adenovirus transactivator protein E1A. DNase I footprinting with HeLa cell extracts has identified four factor-binding domains which appear to be involved in basal and E1A-induced transcriptional regulation. These binding domains may bind TATA region-binding factors (site I), the CREB/ATF protein (site II), the AP-1 protein (site III), and nuclear factor I/CTF (site IV). Recently, it has been shown that the DNA-binding domain of transcription factor AP-1 has homology with the yeast transcription factor GCN4 and that the yeast transactivator protein GAL4 is able to stimulate transcription in HeLa cells from promoters containing GAL4-binding sites. These results suggest an evolutionary conservation of both transcription factors and the mechanisms responsible for transcriptional activation in Saccharomyces cerevisiae and higher eucaryotic organisms. To determine whether similar patterns of transcriptional regulation were seen with the E3 promoter in HeLa and yeast cells, the E3 promoter fused to the chloramphenicol acetyltransferase (cat) gene was cloned into a high-copy-number plasmid and stably introduced into yeast cells. S1 analysis revealed that similar E3 promoter mRNA start sites were found in yeast and HeLa cells. DNase I footprinting with partially purified yeast extracts revealed that four regions of the E3 promoter were protected. Several of these regions were similar to binding sites determined by using HeLa cell extracts. Oligonucleotide mutagenesis of these binding domains indicated their importance in the transcriptional regulation of the E3 promoter in yeast cells. These results suggest that similar cellular transcription factor-binding sites may be involved in the regulation of promoters in both yeast and mammalian cells.

Adenoviridae↗