scholarly journals Cell-Specific Expression of Glucose-Dependent-Insulinotropic Polypeptide Is Regulated by the Transcription Factor PDX-1

Endocrinology ◽  
2005 ◽  
Vol 146 (1) ◽  
pp. 383-391 ◽  
Author(s):  
Lisa I. Jepeal ◽  
Yoshio Fujitani ◽  
Michael O. Boylan ◽  
Cherrell N. Wilson ◽  
Christopher V. Wright ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Small Intestine ◽  
Specific Binding ◽  
Critical Role ◽  
Regulatory Region ◽  
Specific Expression ◽  
Intact Cells ◽  
K Cells ◽  
Nuclear Extracts ◽  
Specific Increase

Glucose-dependent insulinotropic polypeptide (GIP) is a potent stimulator of insulin secretion and comprises an important component of the enteroinsular axis. GIP is synthesized in enteroendocrine K-cells located principally in the upper small intestine. The homeobox-containing gene PDX-1 is also expressed in the small intestine and plays a critical role in pancreatic development and in the expression of pancreatic-specific genes. Previous studies determined that the transcription factors GATA-4 and ISL-1 are important for GIP expression. In this study, we demonstrate that PDX-1 is also involved in regulating GIP expression in K-cells. Using immunohistochemistry, we verified the expression of PDX-1 protein in the nucleus of GIP-expressing mouse K-cells and evaluated the expression of PDX-1, serotonin, and GIP in wild-type and PDX-1−/− mice at 18.5 d after conception. Although we demonstrated a 97.8% reduction in the number of GIP-expressing cells in PDX-1−/− mice; there was no statistical difference in the number of serotonin-positive cells. Additionally, PDX-1 transcripts and protein were detected in a GIP-expressing neuroendocrine cell line, STC-1. Electromobility shift assays using STC-1 nuclear extracts demonstrated the specific binding of PDX-1 protein to a specific regulatory region in the GIP promoter. Using chromatin immunoprecipitation analysis, we demonstrated binding of PDX-1 to this same region of the GIP promoter in intact cells. Lastly, overexpression of PDX-1 in transient transfection assays led to a specific increase in the activity of GIP/Luc reporter constructs. The results of these studies indicate that the transcription factor PDX-1 plays a critical role in the cell-specific expression of the GIP gene.

scholarly journals Involvement of an AP-1 complex in zone-specific expression of the CYP11B1 gene in the rat adrenal cortex.

1995 ◽  
Vol 15 (11) ◽  
pp. 6003-6012 ◽  
Author(s):  
K Mukai ◽  
F Mitani ◽  
H Shimada ◽  
Y Ishimura
Keyword(s):  
Transcription Factor ◽  
Adrenal Cortex ◽  
Transcriptional Activation ◽  
Expression System ◽  
Regulatory Region ◽  
Gene Promoter ◽  
Suppressive Effect ◽  
Zona Fasciculata ◽  
Specific Expression ◽  
Nuclear Extracts

The CYP11B1 gene, which encodes steroid 11 beta-monooxygenase, which is responsible for the synthesis of cortisol and corticosterone, the major glucocorticoids in mammals, is expressed specifically in the zona fasciculata of the adrenal cortex. We have analyzed the promoter region of the rat CYP11B1 gene by using a transient-expression system with adrenocortical Y1 cells and have identified a positive regulatory region. The region contained two adjacent sites for the binding of Y1-cell nuclear proteins: the binding site for an AP-1 transcription factor composed of JunD and a Fos-related protein, and the site for Ad4-binding protein (Ad4BP). The binding of the AP-1 factor to the regulatory region had a suppressive effect on that of Ad4BP in the nuclear extracts. Mutational analyses revealed that the transcriptional activation of the CYP11B1 gene promoter in Y1 cells was attributable to the AP-1 site but not to the Ad4 site. Subsequently, nuclear extracts of the zona fasciculata cells from the rat adrenal cortex were found to contain both AP-1 factor and Ad4BP, whose binding properties to the regulatory region were almost identical to those of the two factors in the Y1-cell nuclear extracts. Moreover, immunohistochemical analyses of rat adrenal cortices showed that the AP-1 factor was present in the nuclei of CYP11B1-expressing cells in the zona fasciculata but not in the nuclei of cells in the other zones. From these results, we propose that the AP-1 transcription factor found in this study plays an important role in the zone-specific expression of the CYP11B1 gene in rat adrenal cortex.

scholarly journals A novel cis-acting element controlling the rat CYP2D5 gene and requiring cooperativity between C/EBP beta and an Sp1 factor.

1994 ◽  
Vol 14 (2) ◽  
pp. 1383-1394 ◽  
Author(s):  
Y H Lee ◽  
M Yano ◽  
S Y Liu ◽  
E Matsunaga ◽  
P F Johnson ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Ternary Complex ◽  
Sequence Similarity ◽  
Consensus Sequence ◽  
Regulatory Region ◽  
Mrna Levels ◽  
Binding Studies ◽  
Mobility Shift ◽  
Specific Expression ◽  
Nuclear Extracts

The rat CYP2D5 gene encodes a cytochrome P450 and is expressed in liver cells. Its expression commences a few days after birth, and maximal mRNA levels are achieved when animals reach puberty. Transfection and DNA binding studies were performed to investigate the mechanism controlling developmentally programmed, liver-specific expression of CYP2D5. Transfection studies using a series of CYP2D5 upstream DNA chloramphenicol acetyltransferase gene fusion constructs identified a segment of DNA between nucleotides -55 and -156 that conferred transcriptional activity in HepG2 cells. Activity was markedly increased by cotransfection with a vector expressing C/EBP beta but was unaffected by vectors producing other liver-enriched transcription factors (C/EBP alpha, HNF-1 alpha, and DBP). DNase I footprinting revealed a region protected by both HepG2 and liver cell nuclear extracts between nucleotides -83 and -112. This region displayed some sequence similarity to the Sp1 consensus sequence and was able to bind the Sp1 protein, as assessed by a gel mobility shift assay. The role of Sp1 in CYP2D5 transcription was confirmed by trans activation of the 2D5-CAT construct in Drosophila melanogaster cells by using an Sp1 expression vector. C/EBP beta alone was unable to directly bind the -83 to -112 region of the promoter but was able to produce a ternary complex when combined with HepG2 nuclear extracts or recombinant human Sp1. C/EBP alpha was unable to substitute for C/EBP beta in forming this ternary complex. A poor C/EBP binding site is present adjacent to the Sp1 site, and mutagenesis of this site abolished formation of the ternary complex with the CYP2D5 regulatory region. These result establish that two transcription factors can work in conjunction, possibly by protein-protein interaction, to activate the CYP2D5 gene.

scholarly journals Krüppel-like factor 4 regulates adaptive expression of the zinc transporter Zip4 in mouse small intestine

2009 ◽  
Vol 296 (3) ◽  
pp. G517-G523 ◽  
Author(s):  
Juan P. Liuzzi ◽  
Liang Guo ◽  
Shou-Mei Chang ◽  
Robert J. Cousins
Keyword(s):  
Transcription Factor ◽  
Small Intestine ◽  
Zinc Transporter ◽  
Intestinal Epithelial ◽  
Rna Transfection ◽  
Nuclear Extracts ◽  
Zinc Depletion ◽  
Mouse Small Intestine ◽  
Nuclear Rna ◽  
Interfering Rna

Epithelial cells of the small intestine are the site of zinc absorption. Intestinal uptake of zinc is inversely proportional to the dietary supply of this essential micronutrient. The mechanism responsible for this adaptive differential in apical zinc transport is not known. The zinc transporter Zip4 ( Slc39a4) is essential for adequate enteric zinc uptake. In mice, Zip4 expression is upregulated at low zinc intakes with a concomitant ZIP4 localization to the apical enterocyte plasma membrane. With the present experiments, we show that the zinc finger transcription factor Krüppel-like factor 4 (KLF4), produced in high abundance in the intestine, is expressed at elevated levels in mice fed a low-zinc diet. In the murine intestinal epithelial cell (IEC) line MODE-K, zinc depletion of culture medium with cell-permeant and cell-impermeant chelators increased Zip4 and Klf4 mRNA and Zip4 heterogeneous nuclear RNA expression. Zinc depletion led to increased KLF4 in nuclear extracts. Knockdown of KLF4 using small interfering RNA transfection drastically limited ZIP4 induction upon zinc depletion and reduced 65Zn uptake by depleted IECs. EMSAs with nuclear extracts of IECs showed KLF4 binding to cis elements of the mouse Zip4 promoter, with increased binding under zinc-limited conditions. Reporter constructs with the Zip4 promoter and mutation studies further demonstrated that Zip4 is regulated through a KLF4 response element. These data from experiments with mice and murine IECs demonstrate that KLF4 is induced during zinc restriction and is a transcription factor involved in adaptive regulation of the zinc transporter ZIP4.

scholarly journals Potential Autoregulation of Transcription Factor PU.1 by an Upstream Regulatory Element

2005 ◽  
Vol 25 (7) ◽  
pp. 2832-2845 ◽  
Author(s):  
Yutaka Okuno ◽  
Gang Huang ◽  
Frank Rosenbauer ◽  
Erica K. Evans ◽  
Hanna S. Radomska ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Reporter Gene ◽  
Regulatory Element ◽  
Mammalian Species ◽  
Critical Role ◽  
Homologous Region ◽  
Specific Expression ◽  
Upstream Regulatory Element

ABSTRACT Regulation of the hematopoietic transcription factor PU.1 (Spi-1) plays a critical role in the development of white cells, and abnormal expression of PU.1 can lead to leukemia. We previously reported that the PU.1 promoter cannot induce expression of a reporter gene in vivo, and cell-type-specific expression of PU.1 in stable lines was conferred by a 3.4-kb DNA fragment including a DNase I hypersensitive site located 14 kb upstream of the transcription start site. Here we demonstrate that this kb −14 site confers lineage-specific reporter gene expression in vivo. This kb −14 upstream regulatory element contains two 300-bp regions which are highly conserved in five mammalian species. In Friend virus-induced erythroleukemia, the spleen focus-forming virus integrates into the PU.1 locus between these two conserved regions. DNA binding experiments demonstrated that PU.1 itself and Elf-1 bind to a highly conserved site within the proximal homologous region in vivo. A mutation of this site abolishing binding of PU.1 and Elf-1 led to a marked decrease in the ability of this upstream element to direct activity of reporter gene in myelomonocytic cell lines. These data suggest that a potential positive autoregulatory loop mediated through an upstream regulatory element is essential for proper PU.1 gene expression.

scholarly journals Evolution is in the details: Regulatory differences in modern human and Neanderthal

2020 ◽  
Author(s):  
Harlan R. Barker ◽  
Seppo Parkkila ◽  
Martti E.E. Tolvanen
Keyword(s):  
Transcription Factor ◽  
Specific Binding ◽  
Critical Role ◽  
Modern Human ◽  
Genomic Variation ◽  
Complete Analysis ◽  
Functional Sites ◽  
Neural Tissues ◽  
Study Sites ◽  
Genomic Locations

AbstractTranscription factor (TF) proteins play a critical role in the regulation of eukaryote gene expression by sequence-specific binding to genomic locations known as transcription factor binding sites.Here we present the TFBSFootprinter tool which has been created to combine transcription-relevant data from six large empirical datasets: Ensembl, JASPAR, FANTOM5, ENCODE, GTEX, and GTRD to more accurately predict functional sites. A complete analysis integrating all experimental datasets can be performed on genes in the human genome, and a limited analysis can be done on a total of 125 vertebrate species.As a use-case, we have used TFBSFootprinter to study sites of genomic variation between modern humans and Neanderthal promoters. We found significant differences in binding affinity for 86 transcription factors, groups of which are both highly expressed, and show correlation of expression, in immune cells and adult and developing neural tissues.

The cell-specific elastase I enhancer comprises two domains

1988 ◽  
Vol 8 (2) ◽  
pp. 893-902 ◽  
Author(s):  
F Kruse ◽  
C T Komro ◽  
C H Michnoff ◽  
R J MacDonald
Keyword(s):  
Base Pair ◽  
Regulatory Region ◽  
Acinar Cells ◽  
Chloramphenicol Acetyltransferase ◽  
Pancreatic Tissue ◽  
Pancreatic Acinar Cells ◽  
Specific Expression ◽  
Nuclear Extracts ◽  
Dna Strands ◽  
Rat Elastase

Two separate domains within the 134-base-pair rat elastase I enhancer and a third domain at the enhancer-promoter boundary are required for selective expression in pancreatic acinar cells. The domains were detected by a series of 10-base-pair substitution mutations across the elastase I gene regulatory region from positions -200 to -61. The effect of each mutant on the pancreas-specific expression of a linked chloramphenicol acetyltransferase gene was assayed by transfection into pancreatic 266-6 acinar cells and control NIH/3T3 cells. The two enhancer domains are nonredundant, because mutations in either eliminated (greater than 100-fold reduction) expression in 266-6 cells. DNase I protection studies of the elastase I enhancer-promoter region with partially purified nuclear extracts from pancreatic tissue and 266-6 cells revealed nine discrete protected regions (footprints) on both DNA strands. One of three footprints that lie within the two functional domains of the enhancer contained a sequence, conserved among several pancreas-specific genes, which when mutated decreased linked chloramphenicol acetyltransferase expression up to 170-fold in 266-6 cells. This footprint may represent a binding site for one or more pancreas-specific regulatory proteins.

scholarly journals Transcription factor Sp1 activates involucrin promoter activity in non-epithelial cell types

1999 ◽  
Vol 337 (3) ◽  
pp. 507-512 ◽  
Author(s):  
Eric B. BANKS ◽  
James F. CRISH ◽  
Richard L. ECKERT
Keyword(s):  
Transcription Factor ◽  
Promoter Activity ◽  
Regulatory Region ◽  
Epidermal Keratinocytes ◽  
Specific Expression ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Transcription Factor Sp1 ◽  
293 Cells ◽  
Strong Activator

The gene for human involucrin (hINV) is selectively expressed in stratifying epithelial cells lining external body surfaces. Previously, we characterized the hINV promoter 5´ distal regulatory region (DRR) located between nt -2473 and -2088 upstream of the transcription start site. This region is required for optimal hINV gene expression. The DRR contains weak and strong activator elements. The strong activator comprises AP1- and Sp1-binding sites that combine to drive high-level promoter expression in human keratinocytes. Here we show that the hINV promoter is expressed in a cell-specific manner in vitro and that the DRR contains elements that are partly responsible for cell-type-specific expression of hINV. hINV promoter activity is barely detectable in 3T3 fibroblasts or HEK-293 human embryonic kidney cells. Reporter plasmids containing the full-length promoter or the isolated DRR can, however, be activated in 3T3 and HEK-293 cells by co-transfection with a plasmid encoding the transcription factor Sp1. Consistently with the lower hINV promoter activity, immunoblotting studies indicate that Sp1 protein levels are lower in 3T3 and HEK-293 cells than in human epidermal keratinocytes. Increased Sp1 protein in transfected 3T3 cells and HEK-293 cells correlates with increased promoter activity. In addition, Sp1 transfection activates the expression of the endogenous gene for hINV in HEK-293 cells. These studies suggest that Sp1 might have a role in cell-specific expression of hINV.

scholarly journals The cell-specific elastase I enhancer comprises two domains.

1988 ◽  
Vol 8 (2) ◽  
pp. 893-902 ◽  
Author(s):  
F Kruse ◽  
C T Komro ◽  
C H Michnoff ◽  
R J MacDonald
Keyword(s):  
Base Pair ◽  
Regulatory Region ◽  
Acinar Cells ◽  
Chloramphenicol Acetyltransferase ◽  
Pancreatic Tissue ◽  
Pancreatic Acinar Cells ◽  
Specific Expression ◽  
Nuclear Extracts ◽  
Dna Strands ◽  
Rat Elastase

Two separate domains within the 134-base-pair rat elastase I enhancer and a third domain at the enhancer-promoter boundary are required for selective expression in pancreatic acinar cells. The domains were detected by a series of 10-base-pair substitution mutations across the elastase I gene regulatory region from positions -200 to -61. The effect of each mutant on the pancreas-specific expression of a linked chloramphenicol acetyltransferase gene was assayed by transfection into pancreatic 266-6 acinar cells and control NIH/3T3 cells. The two enhancer domains are nonredundant, because mutations in either eliminated (greater than 100-fold reduction) expression in 266-6 cells. DNase I protection studies of the elastase I enhancer-promoter region with partially purified nuclear extracts from pancreatic tissue and 266-6 cells revealed nine discrete protected regions (footprints) on both DNA strands. One of three footprints that lie within the two functional domains of the enhancer contained a sequence, conserved among several pancreas-specific genes, which when mutated decreased linked chloramphenicol acetyltransferase expression up to 170-fold in 266-6 cells. This footprint may represent a binding site for one or more pancreas-specific regulatory proteins.

scholarly journals The Transcriptional Repressor REST Determines the Cell-Specific Expression of the Human MAPK8IP1 Gene Encoding IB1 (JIP-1)

2001 ◽  
Vol 21 (21) ◽  
pp. 7256-7267 ◽  
Author(s):  
Amar Abderrahmani ◽  
Myriam Steinmann ◽  
Valérie Plaisance ◽  
Guy Niederhauser ◽  
Jacques-Antoine Haefliger ◽  
...  
Keyword(s):  
Cell Lines ◽  
Transcriptional Activity ◽  
Expression Vector ◽  
Zinc Finger Protein ◽  
Trichostatin A ◽  
Critical Role ◽  
Regulatory Region ◽  
Luciferase Reporter ◽  
Specific Expression ◽  
Amino Terminal

ABSTRACT Islet-brain 1 (IB1) is the human and rat homologue of JIP-1, a scaffold protein interacting with the c-Jun amino-terminal kinase (JNK). IB1 expression is mostly restricted to the endocrine pancreas and to the central nervous system. Herein, we explored the transcriptional mechanism responsible for this preferential islet and neuronal expression of IB1. A 731-bp fragment of the 5′ regulatory region of the human MAPK8IP1 gene was isolated from a human BAC library and cloned upstream of a luciferase reporter gene. This construct drove high transcriptional activity in both insulin-secreting and neuron-like cells but not in unrelated cell lines. Sequence analysis of this promoter region revealed the presence of a neuron-restrictive silencer element (NRSE) known to bind repressor zinc finger protein REST. This factor is not expressed in insulin-secreting and neuron-like cells. By mobility shift assay, we confirmed that REST binds to the NRSE present in the IB1 promoter. Once transiently transfected in β-cell lines, the expression vector encoding REST repressed IB1 transcriptional activity. The introduction of a mutated NRSE in the 5′ regulating region of the IB1 gene abolished the repression activity driven by REST in insulin-secreting β cells and relieved the low transcriptional activity of IB1 observed in unrelated cells. Moreover, transfection in non-β and nonneuronal cell lines of an expression vector encoding REST lacking its transcriptional repression domain relieved IB1 promoter activity. Last, the REST-mediated repression of IB1 could be abolished by trichostatin A, indicating that deacetylase activity is required to allow REST repression. Taken together, these data establish a critical role for REST in the control of the tissue-specific expression of the humanIB1 gene.

A novel cis-acting element controlling the rat CYP2D5 gene and requiring cooperativity between C/EBP beta and an Sp1 factor

1994 ◽  
Vol 14 (2) ◽  
pp. 1383-1394
Author(s):  
Y H Lee ◽  
M Yano ◽  
S Y Liu ◽  
E Matsunaga ◽  
P F Johnson ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Ternary Complex ◽  
Sequence Similarity ◽  
Consensus Sequence ◽  
Regulatory Region ◽  
Mrna Levels ◽  
Binding Studies ◽  
Mobility Shift ◽  
Specific Expression ◽  
Nuclear Extracts

The rat CYP2D5 gene encodes a cytochrome P450 and is expressed in liver cells. Its expression commences a few days after birth, and maximal mRNA levels are achieved when animals reach puberty. Transfection and DNA binding studies were performed to investigate the mechanism controlling developmentally programmed, liver-specific expression of CYP2D5. Transfection studies using a series of CYP2D5 upstream DNA chloramphenicol acetyltransferase gene fusion constructs identified a segment of DNA between nucleotides -55 and -156 that conferred transcriptional activity in HepG2 cells. Activity was markedly increased by cotransfection with a vector expressing C/EBP beta but was unaffected by vectors producing other liver-enriched transcription factors (C/EBP alpha, HNF-1 alpha, and DBP). DNase I footprinting revealed a region protected by both HepG2 and liver cell nuclear extracts between nucleotides -83 and -112. This region displayed some sequence similarity to the Sp1 consensus sequence and was able to bind the Sp1 protein, as assessed by a gel mobility shift assay. The role of Sp1 in CYP2D5 transcription was confirmed by trans activation of the 2D5-CAT construct in Drosophila melanogaster cells by using an Sp1 expression vector. C/EBP beta alone was unable to directly bind the -83 to -112 region of the promoter but was able to produce a ternary complex when combined with HepG2 nuclear extracts or recombinant human Sp1. C/EBP alpha was unable to substitute for C/EBP beta in forming this ternary complex. A poor C/EBP binding site is present adjacent to the Sp1 site, and mutagenesis of this site abolished formation of the ternary complex with the CYP2D5 regulatory region. These result establish that two transcription factors can work in conjunction, possibly by protein-protein interaction, to activate the CYP2D5 gene.

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