The DBP transcriptional activation domain is highly homologous to that of HLF and TEF and is not responsible for the tissue type-specific transcriptional activity of DBP

Abstract

DBP, HLF and TEF comprise a distinct subfamily of mammalian bZIP proteins that plays an important role in regulation of tissue-specific gene expression, particularly in the liver. In this report we demonstrate that DBP contains a 38 amino acid TAD which is highly homologous to the HLF and TEF TADs that we have delineated previously. Deletion of this domain completely abrogates transcriptional activity of native DBP and GAL4-DBP fusion proteins. This domain functions as a modular TAD that is a potent transcriptional activator when fused to the GAL4 DBD. While DBP itself is a liver-specific transactivator, the DBP TAD is active in a variety of cell types, indicating that liver-specific activity is not an intrinsic property of the TAD and must be conferred by other regions of the protein. Using GAL4-HLF fusion proteins, we further refine the core TAD of PAR proteins to a region of 13 amino acids. Recently described PAR-bZIP proteins from Drosophila and zebrafish also contain domains that share strong homology with the TAD of mammalian PAR proteins, making this one of the most highly evolutionarily conserved TADs identified to date.

Introduction

Transcription factors containing a bZIP DNA binding and protein dimerization motif comprise a large superfamily of proteins that play important roles in regulating growth and differentiation (Hurst, 1995). bZIP Proteins are commonly categorized into subfamilies based upon sequence homologies, dimerization specificities, and DNA-binding properties. One of the more recently recognized subfamilies of mammalian bZIP proteins is composed of DBP, TEF and HLF. DBP was initially isolated because of its ability to interact with the D-element of the albumin gene promoter (Mueller et al., 1990). Similarly, TEF was identified by screening an expression library with a regulatory element from the prolactin gene promoter (Drolet et al., 1991). In contrast, HLF was discovered because its bZIP domain is fused to the amino terminus of E2A proteins by the 17;19 translocation observed in a small subset of patients with acute lymphoblastic leukemia (Hunger et al., 1992, Inaba et al., 1992). DBP, HLF and TEF have been highly conserved during evolution and, unlike most other bZIP proteins, their similarity is not limited to the bZIP and includes large regions of the proteins outside of this domain (Fig. 1A). Members of this bZIP subfamily are often termed PAR proteins because they each contain a domain of unknown function amino terminal to the bZIP that is rich in proline and acidic amino acids (Drolet et al., 1991). Several non-mammalian PAR proteins have also been isolated. Vitellogenin binding protein is highly homologous to TEF and was identified independently by screening a chicken liver expression library with an important regulatory element of the chicken vitellogenin II promoter (Iyer et al., 1991). Drosophila and zebrafish cDNAs encoding proteins with bZIP and PAR domains highly homologous to those of DBP, HLF and TEF have recently been identified, indicating that these domains have been conserved throughout hundreds of millions of years of evolution (Lin et al., 1997, Xu et al., 1998).

The mammalian PAR proteins have important functional similarities. Each is expressed in tissue-and developmental stage-specific manners (Mueller et al., 1990, Drolet et al., 1991, Hunger et al., 1992, Inaba et al., 1992). All three PAR proteins are expressed at higher levels in the liver than in any other tissue, and the expression of each displays a strong circadian rhythm in rat liver; maximal levels occur in the evening and are 50–150-fold higher than trough levels observed in the morning, suggesting that they play an important role in regulation of hepatic-specific gene expression (Wuarin and Schibler, 1990, Lavery and Schibler, 1993, Falvey et al., 1995). Not surprisingly in light of the high homology between their respective basic regions (18/21 amino acid identity), DBP, HLF and TEF bind optimally in binding site selection assays to the consensus DNA sequence (G/A)TTA(C/T)GTAA(C/T) and their DNA-binding properties are nearly indistinguishable in gel shift assays (Hunger et al., 1994, Inaba et al., 1994, Haas et al., 1995, Falvey et al., 1996). Each of the PAR proteins can activate transcription of reporter genes under control of natural or synthetic promoters in transient transfection assays (Mueller et al., 1990, Drolet et al., 1991, Yano et al., 1992, Lavery and Schibler, 1993, Burch and Davis, 1994, Hunger et al., 1994, Inaba et al., 1994, Lee et al., 1994, Falvey et al., 1995, Fonjallaz et al., 1996). One major functional difference between the PAR proteins is that DBP activates transcription much more effectively when transfections are performed in hepatic as compared to non-hepatic cells, whereas TEF and HLF are transcriptionally active in a wider variety of cell types (Mueller et al., 1990, Yano et al., 1992, Lavery and Schibler, 1993, Lamprecht and Mueller, 1999).

To further understanding of PAR protein function and how they contribute to liver-specific gene expression, we have undertaken studies to delineate the portions of each protein that are responsible for transcriptional activation. We identified discrete ∼40 amino acid regions in the amino terminal portions of HLF and TEF that share 72% amino acid identity and 85% similarity and demonstrated that this domain, which we termed the THAD, is the TAD of these proteins (Hunger et al., 1996). Using GAL4 chimeras, we demonstrated that the THAD contained most, if not all, of the transcriptional activity present in TEF and HLF and was absolutely required for transcriptional activation (Hunger et al., 1996). We further demonstrated that native HLF and TEF strongly activated reporter gene transcription in diverse cell types while constructs from which the THAD was deleted (HLFΔTHAD and TEFΔTHAD) were completely inactive (Hunger et al., 1996). It is likely that the THAD interacts with a protein component of the general transcription machinery that has been conserved during evolution as, when fused to the LexA DBD, HLF and TEF were potent transcriptional activators in yeast and deletion of the THAD abolished this activity (Hunger et al., 1996).

In this report we demonstrate that DBP also contains a THAD that is responsible for transcriptional activation. The cell-type specific transcriptional activity of DBP is not an intrinsic property of this domain, but rather is conferred by other regions of DBP. We also present evidence that further delineates a minimal 13 amino acid region of the HLF THAD that contains all of its transcriptional activity.