Differential roles of upstream stimulatory factors 1 and 2 in the transcriptional response of liver genes to glucose.

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.

Glucose-dependent liver gene expression in upstream stimulatory factor 2 -/- mice.

Upstream stimulatory factors (USF) 1 and 2 belong to the Myc family of transcription factors characterized by a basic/helix loop helix/leucine zipper domain responsible for dimerization and DNA binding. These ubiquitous factors form homo- and heterodimers and recognize in vitro a CACGTG core sequence termed E box. Through binding to E boxes of target genes, USF factors have been demonstrated to activate gene transcription and to enhance expression of some genes in response to various stimuli. In particular, in the liver USF1 and USF2 have been shown to bind in vitro glucose/carbohydrate response elements of glycolytic and lipogenic genes and have been proposed, from ex vivo experiments, to be involved in their transcriptional activation by glucose. However, the direct involvement of these factors in gene expression and nutrient gene regulation in vivo has not yet been demonstrated. Therefore, to gain insight into the specific role of USF1 and USF2 in vivo, and in particular to determine whether the USF products are required for the response of genes to glucose, we have created, by homologous recombination, USF2 -/- mice. In this paper, we provide the first evidence that USF2 proteins are required in vivo for a normal transcriptional response of L-type pyruvate kinase and Spot 14 genes to glucose in the liver.

Mouse USF1 gene cloning: comparative organization within the c-myc gene family.

Upstream stimulatory factors (USF/MLTF) belong to the c-myc family of transcription factors. Through binding to target DNA as dimers, the ubiquitous USF proteins regulate a variety of genes. USF proteins are encoded by two genes, USF1 and USF2. Protein sequences of USF1 and 2 are highly homologous across species, suggesting functional conservation. To determine whether the genomic organization was conserved between USF1 and USF2, we isolated the murine USF1 gene and characterized its genomic structure. Both genes are similarly organized in 10 exons spanning over 10 kbp. By the 5'-rapid amplification of cDNA ends and S1 nuclease mapping methods, exon 1 was defined and the transcription initiation sites were mapped. The sequence of 8 kb of the gene, including 1.75 kb of 5'-flanking DNA, was determined. The promoter region is GC rich and lacks a typical TATA or CCAAT element. Strikingly, a comparison of the murine and human untranslated sequences reveals regions that exhibit greater than 73% sequence identity. A genomic alignment of the dimerization and DNA binding domains is presented for five genes of the c-myc family, suggesting a hypothetical common ancestor gene.

Structure, sequence, and chromosomal location of the gene for USF2 transcription factors in mouse.

The ubiquitously expressed upstream stimulatory factor (USF) involved in the transcription of a wide variety of cellular genes is defined as dimers of c-myc-related proteins, composed of a basic helix-loop-helix/leucine zipper region. The USF family consists of different members that split into two groups: MLTF or USF1 and USF2 or FIP. We present here evidence that USF1 and USF2 are distinct closely related genes in human, rat, and mouse. Based on the recent cloning of rat and human new cDNAs, we have isolated genomic clones encompassing the murine USF2 gene, which consists of at least 10 exons spanning a minimum of 10 kb of genomic DNA. Unexpectedly, the organization of USF2 appears very split up by introns (0.08 to over 6 kb in size), compared to the myc gene structure. The entire gene (but the larger intron) and 1.6 kb of the 5' flanking region were sequenced. This 5' flanking region is GC-rich, contains several putative transcription binding sites, and has no apparent TATA box. Gene mapping of murine USF2 and USF1 has been determined by in situ hybridization, indicating the localization of USF2 on chromosome 7 and of USF1 on chromosomes 1 and 11.