In eukaryotic cells, single regulatory elements are rarely sufficient to promote gene transcription during development and differentiation. Usually, activation of transcription by RNA polymerase II results from the synergistic effects of two or more cis
-acting DNA motifs which are arranged in distinct sets around the site of transcription initiation and which bind specific transcription factors. The enhanceosome model for transcriptional activation provides one of the best-understood examples of how combinatorial interactions between distinct regulatory proteins and the transcriptional apparatus can lead to a highly specific activation of gene transcription in higher eukaryotes (9
). Enhanceosome formation involves the creation of a stereospecific multiprotein-DNA complex that often includes the architectural transcription factor HMGI-Y and other transcription factors necessary for transcription initiation. Recently, we have shown that IR gene expression in eukaryotic cells is positively regulated by HMGI-Y (3
). In the present study, we performed experiments designed to explore the biochemical mechanisms involved in HMGI-Y activation of IR gene transcription in HepG2 human hepatoblastoma cells, a cell line readily expressing IRs. We provide compelling evidence that transcriptional activation of the IR gene in these cells requires the assembly of a transcriptionally active multiprotein-DNA complex involving, in addition to the HMGI-Y nuclear protein, the ubiquitously expressed transcription factor Sp1 and C/EBPβ. We demonstrate for the first time that HMGI-Y induces transcriptional activation of the IR gene by potentiating the recruitment and binding of Sp1 and C/EBPβ to the IR promoter. Mutational interference with the AT-rich HMGI-Y site in both C2 and E3 elements abolishes the binding of HMGI-Y, adversely affects interactions of Sp1 and C/EBPβ with DNA in vitro, and blunts the transactivation of C2- or E3-containing reporter constructs by Sp1 and C/EBPβ in vivo. Antisense oligonucleotide-mediated inhibition of HMGI-Y protein synthesis demonstrated that binding of Sp1 to the IR promoter was adversely affected and, as a consequence, constitutive IR promoter activity was very low. Similarly, knockout of Sp1 with antisense oligonucleotides markedly impaired activation of the IR promoter, indicating that HMGI-Y-Sp1 interactions may play a central role in regulating IR promoter activity.
However, Sp1 binding sites within the promoter region of the IR gene are unlikely to be the major determinant of the tissue-specific expression of the IR gene since the distribution of Sp1 in tissue does not reflect that of the IR and since Sp1 expression in the classical insulin target tissues, muscle, liver, and fat, is low (33
). Therefore, it appears likely that, in these tissues, additional factors are needed for transcriptional initiation even in the presence of low levels of Sp1. C/EBPβ plays an important role in the regulation of gene expression in the liver and other insulin-responsive tissues (7
). Studies of cell cultures and knockout mouse models indicate that C/EBPβ contributes to the regulation of hepatic glucose production and plays an important role in the regulation of metabolic processes (17
). Herein, we demonstrate that C/EBPβ is required for full activation of IR gene transcription in HepG2 cells and that this transactivation appears to be specifically supported and strongly potentiated by Sp1. Evidence for cooperative interactions between C/EBPβ and Sp1 in the transcriptional regulation of the rat CYP2D5 cytochrome P-450 gene has been previously reported (16
). In addition, a functional interplay among C/EBPβ and Sp1 family factors has been demonstrated in the context of the CD11c integrin gene promoter (19
). On the other hand, functional cooperation between HMGI-Y and C/EBPβ for the transcriptional activation of the leptin promoter has been recently reported (24
). However, cooperation between HMGI-Y and C/EBPβ occurs differently in the cases of the leptin gene and the IR gene. There are no HMGI-Y-DNA binding sites on the leptin promoter, and physical interaction between the two factors contributes to efficient functional cooperation in the transactivation of this gene. In contrast, the binding of HMGI-Y to the IR promoter is a prerequisite for the stimulation of the binding of C/EBPβ to its DNA binding site. Functional cooperation between HMGI-Y and C/EBPβ in the transactivation of the IR promoter could be mediated by HMGI-Y-induced changes in DNA structure that could enhance the affinity of C/EBPβ for its target DNA. In this scenario, HMGI-Y could facilitate the interaction between C/EBPβ and Sp1 and perhaps among other DNA-binding proteins that bind in the immediate vicinity, thereby promoting the formation of an active transcription complex. Mutations affecting either HMGI-Y or Sp1 DNA binding markedly impaired transactivation of the IR promoter by C/EBPβ, indicating that the binding of HMGI-Y and Sp1 to IR DNA is of crucial importance for both basal and induced IR gene expression.
Our observations consistently support the hypothesis that defects in the expression of these nuclear binding proteins may cause decreased IR expression and induce insulin resistance. Defects in a nuclear regulatory protein either identical to or highly related to the architectural transcription factor HMGI-Y in patients with insulin resistance and type 2 diabetes mellitus have been previously reported by our group (2
). We report herein having found a defect in a protein from one patient with the usual features of type 2 diabetes. In EBV-transformed lymphoblasts from this patient, IR gene transcription was significantly impaired despite the fact that the IR gene was normal. In this patient we found that the expression of HMGI-Y was markedly reduced, suggesting that this defect may induce insulin resistance and type 2 diabetes. Consistent with this, overexpression of HMGI-Y in transfected lymphoblasts from this patient significantly increased IR gene transcription and efficiently restored cell surface expression of the IR and insulin-binding capacity. The identification of this defect in a patient with a common form of diabetes is consistent with the notion that tissue-specific alterations of IR levels might be associated with insulin resistance, an early feature of type 2 diabetes.
There has been considerable progress over the past few years in unraveling the molecular defects that give rise to insulin resistance, an early feature of type 2 diabetes mellitus. Nevertheless, there are many gaps in our understanding of the pathophysiology underlying insulin resistance. Our studies demonstrate that the bulk of IR gene expression in liver cells depends on functional and physical interactions among HMGI-Y, Sp1, and C/EBPβ. Additionally, these observations consistently support the hypothesis that defects in the expression of these nuclear binding proteins may cause decreased IR expression and induce insulin resistance. Together, these new findings provide further insight into the molecular processes regulating IR gene expression and open up new avenues for understanding the causes of insulin resistance syndromes and other pathological states in humans with IR dysfunction and impairment of insulin signaling and action.