scholarly journals Functional synergism in the carbohydrate-induced activation of liver-type pyruvate kinase gene expression

1995 ◽  
Vol 308 (1) ◽  
pp. 105-111 ◽  
Author(s):  
Z Liu ◽  
H C Towle
Keyword(s):  
Pyruvate Kinase ◽  
Binding Sites ◽  
Transcriptional Level ◽  
Primary Hepatocytes ◽  
Kinase Gene ◽  
Glucose Response ◽  
Hepatic Expression ◽  
Elevated Glucose ◽  
Response Prompting ◽  
Late Transcription

Hepatic expression of the liver-type pyruvate kinase (L-PK) gene is induced at the transcriptional level by increased carbohydrate metabolism in the rat. The carbohydrate response of the L-PK gene requires sequences from -171 to -124, which encompass adjacent major late transcription factor (MLTF)-like and hepatic nuclear factor (HNF)-4 binding sites. Neither site alone is capable of conferring a response, prompting us to explore the mechanism of synergy between the MLTF-like factor and HNF-4. Spacing requirements between the two factor binding sites were tested by generating a series of mutations that altered the distance between these sites. Surprisingly, all of the constructs with spacing mutations were capable of responding to elevated glucose when introduced into primary hepatocytes. Thus the glucose response does not depend on the rigid phasing of the MLTF-like and HNF-4 factors, suggesting that the factors binding to these two sites do not interact directly with each other. Substitution or inversion of the PK HNF-4 site abrogated the response to glucose and also significantly suppressed the promoter activity under non-inducing conditions. We conclude that the MLTF-like factor and HNF-4 co-operate functionally to maintain the basal activity, as well as the carbohydrate responsiveness, of the L-PK gene. A mechanism other than co-operative DNA binding is responsible for the synergism.

Functional characterization of the L-type pyruvate kinase gene glucose response complex

1993 ◽  
Vol 13 (12) ◽  
pp. 7725-7733
Author(s):  
M J Diaz Guerra ◽  
M O Bergot ◽  
A Martinez ◽  
M H Cuif ◽  
A Kahn ◽  
...  
Keyword(s):  
Binding Site ◽  
Pyruvate Kinase ◽  
Binding Sites ◽  
Ex Vivo ◽  
Functional Characterization ◽  
Insulin Response ◽  
Kinase Gene ◽  
Glucose Response ◽  
E Boxes ◽  
Glucose Responsiveness

L-type pyruvate kinase (L-PK) gene expression is modulated by hormonal and nutritional conditions. We have previously shown that the glucose/insulin response element (GlRE) of the L-PK gene is built around two noncanonical E boxes (element L4) that cooperate closely with a contiguous binding site (element L3). We present in this report the identification of proteins that interact with both elements. The L3 site binds hepatocyte nuclear factor 4 (HNF4)- and COUP/TF-related proteins. In fibroblasts, the overexpression of HNF4 transactivates the L-PK promoter. On the contrary, COUP/TF strongly inhibits the active promoter in hepatocytes. The L4 site binds the major late transcription factor (MLTF) in vitro and ex vivo; mutations that suppress this binding activity also inactivated the GlRE function. Mutations transforming one or two noncanonical E boxes of element L4 into consensus MLTF/USF binding sites strongly increase the affinity for MLTF/USF and do not impair the glucose responsiveness. However, merely the ability to bind MLTF/USF does not seem to be sufficient to confer a GlRE activity: those elements in which one E box has been destroyed and the other has been transformed into a consensus MLTF/USF sequence bind MLTF/USF efficiently but do not confer a high glucose responsiveness on the L-PK gene promoter. Consequently, the full activity of the L-PK GlRE seems to require the cooperation between two putative MLTF/USF binding sites located in the vicinity of an HNF4 binding site.

scholarly journals Functional characterization of the L-type pyruvate kinase gene glucose response complex.

1993 ◽  
Vol 13 (12) ◽  
pp. 7725-7733 ◽  
Author(s):  
M J Diaz Guerra ◽  
M O Bergot ◽  
A Martinez ◽  
M H Cuif ◽  
A Kahn ◽  
...  
Keyword(s):  
Binding Site ◽  
Pyruvate Kinase ◽  
Binding Sites ◽  
Ex Vivo ◽  
Functional Characterization ◽  
Insulin Response ◽  
Kinase Gene ◽  
Glucose Response ◽  
E Boxes ◽  
Glucose Responsiveness

L-type pyruvate kinase (L-PK) gene expression is modulated by hormonal and nutritional conditions. We have previously shown that the glucose/insulin response element (GlRE) of the L-PK gene is built around two noncanonical E boxes (element L4) that cooperate closely with a contiguous binding site (element L3). We present in this report the identification of proteins that interact with both elements. The L3 site binds hepatocyte nuclear factor 4 (HNF4)- and COUP/TF-related proteins. In fibroblasts, the overexpression of HNF4 transactivates the L-PK promoter. On the contrary, COUP/TF strongly inhibits the active promoter in hepatocytes. The L4 site binds the major late transcription factor (MLTF) in vitro and ex vivo; mutations that suppress this binding activity also inactivated the GlRE function. Mutations transforming one or two noncanonical E boxes of element L4 into consensus MLTF/USF binding sites strongly increase the affinity for MLTF/USF and do not impair the glucose responsiveness. However, merely the ability to bind MLTF/USF does not seem to be sufficient to confer a GlRE activity: those elements in which one E box has been destroyed and the other has been transformed into a consensus MLTF/USF sequence bind MLTF/USF efficiently but do not confer a high glucose responsiveness on the L-PK gene promoter. Consequently, the full activity of the L-PK GlRE seems to require the cooperation between two putative MLTF/USF binding sites located in the vicinity of an HNF4 binding site.

c-Myc and ChREBP regulate glucose-mediated expression of the L-type pyruvate kinase gene in INS-1-derived 832/13 cells

2007 ◽  
Vol 293 (1) ◽  
pp. E48-E56 ◽  
Author(s):  
J. Jason Collier ◽  
Pili Zhang ◽  
Kim B. Pedersen ◽  
Susan J. Burke ◽  
John W. Haycock ◽  
...  
Keyword(s):  
Pyruvate Kinase ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Heterologous Promoter ◽  
Kinase Gene ◽  
Glucose Response ◽  
Glucose Flux ◽  
Element Binding Protein ◽  
Regulated Gene Expression

Increased glucose flux generates metabolic signals that control transcriptional programs through poorly understood mechanisms. Previously, we demonstrated a necessity in hepatocytes for c-Myc in the regulation of a prototypical glucose-responsive gene, L-type pyruvate kinase (L-PK) (Collier JJ, Doan TT, Daniels MC, Schurr JR, Kolls JK, Scott DK. J Biol Chem 278: 6588–6595, 2003). Pancreatic β-cells have many features in common with hepatocytes with respect to glucose-regulated gene expression, and in the present study we determined whether c-Myc was required for the L-PK glucose response in insulin-secreting (INS-1)-derived 832/13 cells. Glucose increased c-Myc abundance and association with its heterodimer partner, Max. Manipulations that prevented the formation of a functional c-Myc/Max heterodimer reduced the expression of the L-PK gene. In addition, glucose augmented the binding of carbohydrate response element binding protein (ChREBP), c-Myc, and Max to the promoter of the L-PK gene in situ. The transactivation of ChREBP, but not of c-Myc, was dependent on high glucose concentrations in the contexts of either the L-PK promoter or a heterologous promoter. The glucose-mediated transactivation of ChREBP was independent of mutations that alter phosphorylation sites thought to regulate the cellular location of ChREBP. We conclude that maximal glucose-induced expression of the L-PK gene in INS-1-derived 832/13 cells involves increased c-Myc abundance, recruitment of c-Myc, Max, and ChREBP to the promoter, and a glucose-stimulated increase in ChREBP transactivation.

Molecular analysis of the differential hepatic expression of rat kininogen family genes

1993 ◽  
Vol 13 (11) ◽  
pp. 6766-6777
Author(s):  
H M Chen ◽  
W S Liao
Keyword(s):  
Binding Sites ◽  
Gene Promoter ◽  
Transcriptional Level ◽  
Evolutionary Divergence ◽  
Binding Affinities ◽  
Promoter Regions ◽  
Hepatic Expression ◽  
Basal Expression ◽  
Promoter Swapping ◽  
Hepatic Synthesis

Serum concentration of rat T1 kininogen increases 20- to 30-fold in response to acute inflammation, an induced hepatic synthesis regulated primarily at the transcriptional level. We have demonstrated by transient transfection analyses that rat T1 kininogen gene/chloramphenicol acetyltransferase (T1K/CAT) constructs are highly responsive to interleukin-6 and dexamethasone. In these studies we examined the regulation of a highly homologous K kininogen gene promoter and showed that it is minimally induced under identical conditions. The basal expression of the KK/CAT construct was, however, five- to sevenfold higher than that of the analogous T1K/CAT construct. Promoter-swapping experiments to examine the molecular basis of this differentially regulated basal expression showed that at least two K kininogen promoter regions are important for conferring its high basal expression: a distal 19-bp region (C box) constituted a binding site for C/EBP family proteins, and a proximal 66-bp region contained two adjacent binding sites for hepatocyte nuclear factor 3 (HNF-3). While the C box in the K kininogen promoter was able to interact with C/EBP transcription factors, the T1 kininogen promoter C box could not. In addition, HNF-3 binding sites of the K kininogen promoter demonstrated stronger affinities than those of the T1 kininogen promoter. Since C/EBP and HNF-3 are highly enriched in the liver and are known to enhance transcription of liver-specific genes, these differences in their binding activities thus accounted for the K kininogen gene's higher basal expression. Our studies demonstrated that evolutionary divergence of a few critical nucleotides may lead to subtle changes in the binding affinities of a transcription factor to its recognition site, profoundly altering expression of the downstream gene.

scholarly journals Upstream Stimulatory Factor Proteins Are Major Components of the Glucose Response Complex of the L-type Pyruvate Kinase Gene Promoter

1995 ◽  
Vol 270 (6) ◽  
pp. 2640-2643 ◽  
Author(s):  
Anne-Marie Lefran¸ois-Martinez ◽  
Antoine Martinez ◽  
Bénédicte Antoine ◽  
Michel Raymondjean ◽  
Axel Kahn
Keyword(s):  
Pyruvate Kinase ◽  
Gene Promoter ◽  
Upstream Stimulatory Factor ◽  
Kinase Gene ◽  
Glucose Response ◽  
Stimulatory Factor ◽  
Response Complex

scholarly journals Molecular analysis of the differential hepatic expression of rat kininogen family genes.

1993 ◽  
Vol 13 (11) ◽  
pp. 6766-6777 ◽  
Author(s):  
H M Chen ◽  
W S Liao
Keyword(s):  
Binding Sites ◽  
Gene Promoter ◽  
Transcriptional Level ◽  
Evolutionary Divergence ◽  
Binding Affinities ◽  
Promoter Regions ◽  
Hepatic Expression ◽  
Basal Expression ◽  
Promoter Swapping ◽  
Hepatic Synthesis

Serum concentration of rat T1 kininogen increases 20- to 30-fold in response to acute inflammation, an induced hepatic synthesis regulated primarily at the transcriptional level. We have demonstrated by transient transfection analyses that rat T1 kininogen gene/chloramphenicol acetyltransferase (T1K/CAT) constructs are highly responsive to interleukin-6 and dexamethasone. In these studies we examined the regulation of a highly homologous K kininogen gene promoter and showed that it is minimally induced under identical conditions. The basal expression of the KK/CAT construct was, however, five- to sevenfold higher than that of the analogous T1K/CAT construct. Promoter-swapping experiments to examine the molecular basis of this differentially regulated basal expression showed that at least two K kininogen promoter regions are important for conferring its high basal expression: a distal 19-bp region (C box) constituted a binding site for C/EBP family proteins, and a proximal 66-bp region contained two adjacent binding sites for hepatocyte nuclear factor 3 (HNF-3). While the C box in the K kininogen promoter was able to interact with C/EBP transcription factors, the T1 kininogen promoter C box could not. In addition, HNF-3 binding sites of the K kininogen promoter demonstrated stronger affinities than those of the T1 kininogen promoter. Since C/EBP and HNF-3 are highly enriched in the liver and are known to enhance transcription of liver-specific genes, these differences in their binding activities thus accounted for the K kininogen gene's higher basal expression. Our studies demonstrated that evolutionary divergence of a few critical nucleotides may lead to subtle changes in the binding affinities of a transcription factor to its recognition site, profoundly altering expression of the downstream gene.

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