Functional Characterization of the L-Type Pyruvate Kinase Gene Glucose Response Complex

Abstract

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.

Complex Interactions between SP1 Bound to Multiple Distal Regulatory Sites and HNF-4 Bound to the Proximal Promoter Lead to Transcriptional Activation of Liver-Specific Human APOCIII Gene

“…This finding is interesting in light of previous observations showing that ARP-1 and EAR-3, when bound to proximal element B, repress the apoCIII promoter (Ladias et al, 1992;Myetus-Snyder et al, 1992). In addition, studies on several other promoters, including apoB, apoA-I, apoA-II, apoA-IV (Ladias et al, 1992;Ktistaki et al, 1994), L-type pyruvate kinase (Guerra et al, 1993), ornithine transcarbamylase (Kimura et al, 1993), and the cellular retinol binding protein (Nakshatri et al, 1994), showed that these factors modulate transcription in a negative direction. Although repressor activity of a transcription factor can be achieved by several different mechanisms, it is well-documented that in all cases when ARP-1 and EAR-3 act as repressors the negative effect is being exerted by competition with HNF-4 or other nuclear hormone receptors for the same binding site (Ladias et al, 1992).…”

“…Recent studies indicate that HNF-4 can synergize with a wide variety of transcription factors including C/EBP (Metzger et al, 1993), CREB (Nitsch et al, 1993), and HNF-1 (Guerra et al, 1993). This is reminiscent of the derivatives of the yeast activator GAL4, which can stimulate transcription in synergy with various mammalian transcription factors such as the glucocorticoid receptor, ATF, and USF (Carey et al, 1990;Lin et al, 1988Lin et al, , 1990.…”