4284-4290 Nucleic Acids Research, 1994, Vol. 22, No. 20 LFB1/HNF1 acts as a repressor of its own transcription Giulia Piaggio*, Licia Tomei1, Carlo Toniattil, Raffaele De Francesco1, Jutta Gerstner2,+ and Riccardo Cortese1 Istituto 'Regina Elena' Centro Ricerca Sperimentale, Laboratorio Oncogenesi Molecolare, via Delle Messi d'Oro 156, 00158 Rome, 1IRBM- Istituto di Ricerche di Biologia Molecolare P.Angeletti, via Pontina Km 40.600, 00040 Pomezia, Rome, Italy and 2EMBL, European Molecular Biology Laboratory, MeyerhofstraBe 1, 6200 Heidelberg, Germany Received May 27, 1994; Revised and Accepted September 5, 1994 ABSTRACT LFB1/HNF1 is a hepatocyte-enriched trans-activator involved in the regulation of many liver-specific genes. We report the cloning and characterization of a rat genomic DNA fragment containing about 3.5 kb of the LFB1/HNF1 gene 5'-flanking region. This DNA segment is capable of directing the liver-specific expression of a reporter gene in transfection assays. More interestingly, the basal activity of the LFB1/HNF1 promoter in cultured hepatoma cell lines is down- regulated by exogenously added LFB1/HNF1 protein itself. The ability to repress transcription starting from its own promoter requires the integrity of the N-terminal LFB1/HNF1 DNA-binding domain. Contrary to the expectations, in vitro binding experiments failed to demonstrate any specific and functional interaction of purified LFB1/HNF1 with the -3.5 kb promoter sequence. In addition to the DNA-binding domain, a 60 aa region contained in the C-terminus of the protein and distinct from the previously characterized activation domains, is also required for the repressing function. INTRODUCTION The liver provides a useful model system in which to study the mechanisms that govern the differential expression of tissue- specific genes (1). Several cDNAs coding for factors that play an important role in controlling the hepatocyte-specific expression have recently been cloned. They include the Liver Factor BI/Hepatocyte Nuclear Factor 1 (LFB1/HNFl; 2,3) and the related factor LFB3/vHNFI (4,5), the Drosophila fork head homologue HNF3 (6), HNF4, a member of the steroid hormone receptor superfamily (7) and the leucine-zipper containing factors C/EBP (8) and DBP (9). The interaction of these transcription factors with a given array of cis-acting elements on the promoter region of liver-specific genes modulates their expression rate. This interaction may in fact activate or repress promoter activity according to the stage of development, hormonal induction or pathological condition (10). The comprehension of the molecular EMBL accession no. X67649 mechanisms underlying hepatocyte-specific transcriptional regulation may provide insight into the events leading to liver development and differentiation. LFB1/HNF1 was initially identified as a hepatocyte factor interacting with the promoter regions of many liver-specific genes (10,11). Although its expression was shown not to be limited to hepatic cells, both in vivo and in vitro studies have confirmed this protein to be one of the major determinants of the liver phenotype (5,12-14). LFBI/HNF1 binds as a dimer to specific palindromic sites (2,11,15). The functional architecture of the LFB1/HNF1 protein has been extensively studied in our laboratory. A protein fragment that contains the N-terminal 281 residues of LFBI/HNF1 binds to DNA with affinity and specificity comparable to those of the native protein (2). Further dissection of this minimal DNA- binding domain revealed a unique tripartite DNA binding structure that includes an unusually long homeodomain, a region related to the POU-specific A-box, and a short N-terminal dimerization domain (15,16). The C-terminus of the molecule (aa 282-628) contains at least three regions that have been shown to be indispensable for the transcription activation function: the serine-rich ADI (Activation Domain I, aa 547-628), the proline- rich ADIH (aa 282-318) and the glutamine-rich ADHI (aa 440-506) (15,17,18). The cloning and functional characterization of the rat and mouse LFBI/HNFI promoter region was reported (19, 20). It has been shown that the activity of this promoter in cultured hepatoma cell lines can be enhanced by co-transfection with cDNAs coding for the hepatocyte-enriched trans-activators HNF4 and HNF3 (19, 20). Autoregulation is a common mechanism through which the correct balance between transcription factors is established and mantained during the developmental stages of the cells or in response to external stimuli (21-23). Conflicting results have been reported about the role of LFBI/HNF1 in regulating the transcriptional activity of the promoter of its gene (24-26). In this paper we show evidences of a negative autoregulatory loop, through which LFBI/HNFI negatively controls transcription * To whom correspondence should be addressed 'Present address: Leica Vertrieb GmbH, Lilienthalstral3e 39-45, D-6140 Bensheim, Germany 1994 Oxford University Press