The ubiquitous upstream stimulatory factor (USF) transcription factors encoded by two distinct genes (USF1 and USF2) exist under the form of various dimers able to bind E-boxes. We report the molecular cloning and functional characterization of USF2 isoforms, corresponding to a 44-kDa subunit, USF2a, and a new 38-kDa subunit, USF2b, generated by differential splicing. Using specific anti-USF antibodies, we define the different binding complexes in various nuclear extracts. In vivo, the USF1/USF2a heterodimer represents over 66% of the USF binding activity whereas the USF1 and USF2a homodimers represent less than 10%, which strongly suggests an in vivo preferential association in heterodimers. In particular, an USF1/USF2b heterodimer accounted for almost 15% of the USF species in some cells. The preferential heterodimerization of USF subunits was reproduced ex vivo, while the in vitro association of cotranslated subunits, or recombinant USF proteins, appeared to be random. In transiently transfected HeLa or hepatoma cells, USF2a and USF1 homodimers transactivated a minimal promoter with similar efficiency, whereas USF2b, which lacks an internal 67-amino acid domain, was a poor transactivator. Additionally, USF2b was as efficient as USF1 and USF2a homodimers in transactivating the liver-specific pyruvate kinase gene promoter.

No abstract

The glucose/insulin response element of the L-pyruvate kinase gene is a perfect palindrome located from nt -168 to -144 with respect to the cap site. This element (L4) is partially homologous to MLTF binding sites. Its full efficiency requires cooperation with a contiguous binding site for HNF4, termed L3 and located from nt -145 to -125. In the presence of the L4 element contiguous to L3, cyclic AMP inhibits activity of the L-PK promoter while in its absence, or when the normal L4-L3 contiguity is modified, cyclic AMP behaves as a transcriptional activator that does not seem to be sequence-specific. Therefore, we propose that the mechanism of inhibition of the L-PK gene by cyclic AMP requires precise interactions between the nucleoprotein complex built up at sites L4 and L3 and other components of the L-PK transcription initiation complex.

Hepatocyte nuclear factor 4 (HNF4), a liver-enriched transcription factor of the nuclear receptor superfamily, is critical for development and liver-specific gene expression. Here, we demonstrate that its DNAbinding activity is modulated posttranslationally by phosphorylation in vivo, ex vivo, and in vitro. In vivo, HNF4 DNA-binding activity is reduced by fasting and by inducers of intracellular cyclic AMP (cAMP) accumulation. A consensus protein kinase A (PKA) phosphorylation site located within the A box of its DNA-binding domain has been identified, and its role in phosphorylation-dependent inhibition of HNF4 DNA-binding activity has been investigated. Mutants of HNF4 in which two potentially phosphorylatable serines have been replaced by either neutral or charged amino acids were able to bind DNA in vitro with affinity similar to that of the wild-type protein. However, phosphorylation by PKA strongly repressed the binding affinity of the wild-type factor but not that of HNF4 mutants. Accordingly, in transfection assays, expression vectors for the mutated HNF4 proteins activated transcription more efficiently than that for the wild-type protein when cotransfected with the PKA catalytic subunit expression vector. Therefore, HNF4 is a direct target of PKA which might be involved in the transcriptional inhibition of liver genes by cAMP inducers.The liver-enriched transcription factor hepatocyte nuclear factor 4 (HNF4) (54) is involved in close association with other factors in hormonal and dietary control of liver and intestine genes. In addition, HNF4 has a potential role as a developmental regulator that has been conserved during evolution from invertebrates to vertebrates. HNF4 expression is restricted to the liver, kidney, and intestine (60) and, in Drosophila melanogaster, to the malpighian tubules (61). HNF4 exists as various isoforms. At least five different cDNAs are generated by differential splicing from a single gene, the HNF4␣ gene, in the amino-and carboxy-terminal regions; in all isoforms the DNA-binding domain remains unchanged (6,14,20,33,53,54). Recently, novel human and Xenopus HNF4 isoforms (HNF4␥ and HNF4␤, respectively) have been identified and shown to be derived from two distinct and differentially expressed genes (14,24). HNF4 is expressed very early during embryo development and has been found to be a crucial positive-acting factor for the expression of the HNF1 gene (55), placing HNF4 at the top of a transactivator hierarchy in hepatic cells (23,36). Disruption of the HNF4␣ gene led to early embryonic death due to malfunction of the yolk sac (7).HNF4 is a member of the nuclear receptor superfamily that binds to DNA at direct repeats separated by one nucleotide (DR1). This transactivator preexists as very stable homodimers in solution, and no HNF4-specific ligand has been identified so far (27). Therefore, HNF4 is classified as an orphan nuclear receptor, characterized by two zinc finger DNA-binding motifs, a large conserved hydrophobic domain containing the dimerization, and a putative ...

No abstract

Even though a "CCAAT" box element is localized -80 base pairs upstream of the transcription initiation site in many eukaryotic promoters, it is not recognized by a single ubiquitous transcription factor. We show here that such a sequence present in the rat albumin promoter binds a protein distinct from the CCAAT-binding transcription factor CTF/NFI or from the CCAAT-binding protein (CBP) previously described. The protein binding to the albumin CCAAT sequence (ACF or albumin CCAAT factor) is not exclusive to liver, since we found a protein with identical properties in spleen.Albumin is the most abundant protein in plasma. It is synthesized mainly in liver; only trace amounts of specific mRNA are detected in some kidney cells and in brain (1, 2). The tissue specificity of albumin synthesis appears to be regulated at the level of transcription, since only liver cells or hepatoma cells in culture actively transcribe this gene (3,4). Recent studies have shown that =150 base pairs (bp) preceding the albumin mRNA initiation site are sufficient to direct tissue-specific expression of linked receptor genes in vivo or in vitro (2, 5-7). Additional sequences may be essential for transcriptional control during development, since a far upstream element present 10 kilobases (kb) 5' to the transcription start site is required for efficient expression of the albumin gene in transgenic mice (8).Comparison of the promoter sequences of albumin genes from mouse, rat, human, and chicken revealed several conserved segments in the 150 bp preceding the cap site (7, 9, 10). These elements include the widespread TATA and CCAAT consensus sequences as well as a proximal element and three distal elements (DEI, DEII, and DEIII) (Fig. 1). Analysis of deletions and linker scan mutants indicates that all these conserved elements contribute to the strength of the albumin promoter (ref. 7; P. Herbomel, F. Tronche, M. 0. Ott, A. Mottura-Rollier, M.Y., and M. Weiss, unpublished observations). Conservation of sequence motifs in the albumin promoters and their importance for the biological activity of this DNA fragment strongly suggest that these elements are binding sites for trans-acting factors. We have shown by DNase I "footprinting" that at least four distinct liver nuclear proteins interact with the proximal element and DEI, -II, . Surprisingly, the conserved -80 "CCAAT" box was not protected in these experiments. Furthermore, the protein interacting with albumin DEII -120 element was identified as nuclear factor I (NFI), shown recently to be identical to the CCAAT-binding transcription factor (CTF) (11). In fact, the conserved DEII includes the sequence TTGGCA (TGCCAA in the other strand) that is a good half-recognition site for NFL. It binds this factor with an affinity slightly lower than that of the symmetrical adenovirus NFI binding site (10). In the course of our DNase I footprinting studies, we realized that mutation of DEI, which abolished binding to this element, promoted partial protection of the CCAAT box sequences, suggestin...

USF1 and USF2 are ubiquitous transcription factors of the basic helix-loop-helix leucine zipper family. They form homo- and heterodimers and recognize a CACGTG motif termed E box. In the liver, USF binding activity is mainly accounted for by the USF1/USF2 heterodimer, which binds in vitro the glucose/carbohydrate response elements (GlRE/ChoRE) of glucose-responsive genes. To assign a physiological role of USFs in vivo, we have undertaken the disruption of USF1 and USF2 genes in mice. We present here the generation of USF1-deficient mice. In the liver of these mice, we demonstrate that USF2 remaining dimers can compensate for glucose responsiveness, even though the level of total USF binding activity is reduced by half as compared with wild type mice. The residual USF1 binding activity was similarly reduced in the previously reported USF2 -/- mice in which an impaired glucose responsiveness was observed (Vallet, V. S., Henrion, A. A., Bucchini, D., Casado, M. , Raymondjean, M., Kahn, A., and Vaulont, S. (1997) J. Biol. Chem. 272, 21944-21949). Taken together, these results clearly suggest differential transactivating efficiencies of USF1 and USF2 in promoting the glucose response. Furthermore, they support the view that USF2 is the functional transactivator of the glucose-responsive complex.

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 (GIRE) of the L-PK gene is built around two noncanonical E boxes (element IA) 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 COUPITF-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 GIRE function. Mutations transforming one or two noncanonical E boxes of element LA 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 GIRE 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 GIRE seems to require the cooperation between two putative MLTF/USF binding sites located in the vicinity of an HNF4 binding site.The L-type pyruvate kinase (L-PK) is a key enzyme of the glycolytic pathway. It is coordinately regulated at the transcriptional and posttranscriptional levels, positively by carbohydrates in the presence of insulin and negatively by glucagon via cyclic AMP (8,32). The regulatory region of the L-PK gene, responsible for its transcriptional response to carbohydrates and hormones, has been ascribed to a -120/ -183-bp proximal promoter fragment (2, 30), ex vivo by transient expression assays in hepatocytes in primary culture, and in vivo in transgenic mice (7). We had previously characterized the important cis-acting DNA elements in the L-PK gene promoter: in the 3'-5' direction upstream from the TATA box, we found, respectively, box Li, a binding site for hepatocyte nuclear factor 1 (HNF1); box L2, a binding site for nuclear factor 1; box L3, a binding site for HNF4; and box LA, a weak in vitro binding site for major late transcription factor (MLTF)/USF ( Fig. 1) (33). Box IA contains the L-PK gene glucose/insulin response element (GlRE) and is also indispensable for the action of glucagon and cyclic AMP. When L4 is oligomerized, it is able to confer glucose/insulin and cyclic AMP responsiveness on a