PAR-domain proteins

Two cDNAs were cloned and sequenced, representing two isoforms of the zebrafish thyrotroph embryonic factor (TEF) gene products (tef alpha and beta); both are members of the PAR subfamily of bZIP transcription factors. The two isoforms encode two potential proteins of 300 and 293 amino acids, respectively. Sequence comparison analysis indicates that the zebrafish TEFs show high homology to the PAR family of transcription factors of other species in the PAR domain, the DNA binding domain and the leucine zipper domain. Expression analysis by Northern blot and RT-PCR indicates that tef alpha and tef beta are expressed throughout the zebrafish embryonic development and in some, but not all, adult tissues (Xu, 1998).

A new member of the leucine zipper (bZIP) gene family of transcription factors, thyrotroph embryonic factor (TEF) has been identified and characterized. Analysis of the ontogeny of TEF gene expression reveals the presence of TEF transcripts, beginning on embryonic day 14, only in the region of the rat anterior pituitary gland, in which thyrotrophic cells arise. This pattern of gene expression corresponds temporally and spatially to the onset of thyroid-stimulating hormone (TSH beta) gene expression, which defines the thyrotroph phenotype. TEF can bind to and trans-activate the TSH beta promoter. In contrast to this restricted pattern of expression during embryogenesis, TEF transcripts appear in several tissues in the mature organism. On the basis of the unique homology between TEF and another member of the bZIP gene family, it has been proposed that TEF belongs to a new class of bZIP proteins, the albumin D box-binding protein (DBP). TEF and DBP transcripts are coexpressed in a pituitary cell line, and these two proteins can readily form heterodimers. The DNA-binding and dimerization domains of TEF correspond to those found in other bZIP proteins. A cluster of basic amino acids, found only in TEF and DBP, has been identified as being necessary for the proper DNA-binding site specificity of TEF. A major trans-activation domain of TEF resides outside the region of homology to other bZIP proteins. These data are consistent with a role for a member of a new class of bZIP transcription factors in activating gene expression in the developing thyrotroph (Drolet, 1991).

A chicken liver cDNA expression library was screened with a probe spanning the distal region of the chicken vitellogenin II (VTGII) gene promoter. A transcription factor termed VBP (for vitellogenin gene-binding protein) was isolated. VBP binds to one of the most important positive elements in the VTGII promoter and appears to play a pivotal role in the estrogen-dependent regulation of this gene. The protein sequence of VBP contains a basic/zipper (bZIP) motif. As expected for a bZIP factor, VBP binds to its target DNA site as a dimer. VBP forms a stable dimer in solution. A data base search has revealed that VBP is related to rat DBP. However, despite the fact that the basic/hinge regions of VBP and DBP differ at only three amino acid positions, the DBP binding site in the rat albumin promoter is a relatively poor binding site for VBP. Thus, the optimal binding sites for VBP and DBP may be distinct. Similarities between the VBP and DBP leucine zippers are largely confined to only four of the seven helical spokes. Nevertheless, these leucine zippers are functionally compatible and appear to define a novel subfamily. In contrast to the bZIP regions, other portions of VBP and DBP are markedly different, as are the expression profiles for these two genes. In particular, expression of the VBP gene commences early in liver ontogeny and is not subject to circadian control (Iyer, 1991).

The full-length cDNA for a transcriptional activator, DBP, has been cloned; it binds to the D site of the albumin promoter. DBP belongs to a family of related transcription factors including Fos, Jun, CREB, and C/EBP, which share a conserved basic domain. However, unlike most other members of this family, DBP does not contain a "leucine zipper" structure. Among several rat tissues tested, significant levels of its protein are only observed in liver; yet, with the exception of testis, DBP mRNA is present in all of the examined tissues. DBP as well as its mRNA accumulate to significant levels only in adult animals. During chemically induced liver regeneration, DBP expression is rapidly down-regulated, suggesting that DBP may be involved in the proliferation control of hepatocytes. This cell growth-dependent expression of DBP, in contrast to its tissue specificity, appears to be controlled at the level of mRNA accumulation (Mueller, 1990).

Alternative splicing of PAR-domain proteins

An analysis of the chicken VBP gene reveals that the two different amino-terminal sequences map to alternative first exons and that the two different carboxyl-terminal sequences reflect an optional splicing event that can occur only on transcripts that are polyadenylated at the more distal of two polyadenylation sites. An RT-PCR analysis further reveals that a total of four VBP isoforms are encoded by the combinatorial use of these two splicing options. The mRNAs for these four isoforms are differentially expressed in different tissues and cell types. Evidence is provided that one function of the amino-terminal domains is to impose cell type specificity on a core transactivation domain that is present in all four isoforms. Since it is known that VBP can heterodimerize with other members of the PAR subfamily of bZIP factors, the evidence for four VBP isoforms greatly expands the number of complexes that may be used to effect transcriptional regulation through PAR-factor binding sites (Burch, 1994).

Hepatic leukemia factor (HLF) is a member of the PAR family of transcription regulatory proteins. The rat HLF gene is transcribed from two alternative promoters, alpha and beta, with different circadian amplitudes and tissue specificities. The alpha RNA isoforms produce a 43 kDa protein, HLF43, abundant in brain, liver and kidney, as is human HLF RNA. The beta RNA HLF isoforms use a CUG codon to initiate translation of a novel 36 kDa protein, HLF36, which is shorter at its N-terminus relative to the 43 kDa form. HLF36 is expressed uniquely in the liver, where it is the most abundant HLF protein. Surprisingly, the two proteins accumulate in the liver with different circadian amplitudes and have distinct liver-specific promoter preferences in transfection experiments. Thus, HLF43 stimulates transcription from the cholesterol 7 alpha-hydroxylase promoter much more efficiently than from the albumin promoter, while the converse is true for HLF36 (Falvey, 1997).

Transcriptional regulation of PAR-domain proteins

The D-site binding protein (DBP) is a member of the PAR domain subfamily of b/ZIP proteins, whose expression in the liver is highly sensitive to the growth state of that organ. This paper examines the regulation of the DBP promoter by C/EBP alpha and examines the role of autoregulation in DBP expression. Of four previously characterized proximal promoter sites, sites I and III have been shown to bind C/EBP alpha, but cotransfection in Hep G2 cells of a C/EBP alpha expression vector is unable to transactivate the promoter. In contrast, the expression of DBP, particularly in conjunction with the related protein HLF, is able to dramatically upregulate expression directed by the proximal promoter. Deletion analysis and the use of single site reporter constructs demonstrate that sites II and IV are highly responsive to transactivation by DBP and HLF. The DBP promoter is active in the UOC-B1 cell line, which bears a 17:19 translocation resulting in the creation of an E2A:HLF fusion protein. The proteins binding to site IV are elevated in this line, suggesting that upregulation of DBP expression in response to inappropriate HLF activity may be mediated through this site (Newcombe, 1998).

The D-site binding protein (DBP) is a member of the proline- and acid-rich (PAR) domain subfamily of basic/leucine zipper proteins and is involved in transcriptional regulation in the liver. Deletion analysis of the DBP protein was carried out in an effort to define the function of the conserved PAR domain. Internal deletions of the protein, i.e. removing portions of the PAR domain, result in a substantial loss in transactivation of a high affinity DBP reporter construct when assayed in Hep G2 cells. These same sequences confer significant transactivation to GAL4 DNA binding domain fusion proteins, indicating that this region acts as part of an independent activation domain comprised of sequences in both the amino terminus and in the PAR domain of DBP. The coexpression of full-length expression constructs for both DBP and hepatic leukemia factor results in a dramatic increase in activation mediated by the GAL4-DBP fusion proteins, suggesting the involvement of a regulated coactivator in this process. DBP transactivation appears to be a p300-dependent process, since a 12 S E1A expression construct disrupts DBP-mediated transactivation, and a p300 expression vector, but not a CREB binding protein vector, is able to restore DBP transactivation. These results suggest that the PAR domain is required for DBP activation, which occurs through a regulated, p300-dependent process (Lamprecht, 1999).

Binding site specificity of PAR-domain proteins

The PAR subfamily of basic leucine zipper (bZIP) factors comprises three proteins (VBP/TEF, DBP, and HLF) that have conserved basic regions flanked by proline- and acidic-amino-acid-rich (PAR) domains and functionally compatible leucine zipper dimerization domains. VBP preferentially binds to sequences that consist of abutted GTAAY half-sites (which are referred to as PAR sites) as well as to sequences that contain either a C/EBP half-site (GCAAT) or a CREB/ATF half-site (GTCAT) in place of one of the PAR half-sites. Since the sequences that describe PAR sites and PAR-CREB/ATF chimeric sites, respectively, have both been described as high-affinity binding sites for the E4BP4 transcriptional repressor, it is inferred that these sequences may be targets for positive and negative regulation. Similarly, since the sequences described as PAR-C/EBP and PAR-CREB/ATF chimeric sites are known to be high-affinity binding sites for C/EBP and CREB/ATF factors, respectively, it is inferred that these sites may each be targets for multiple subfamilies of bZIP factors. To gain insight regarding the molecular basis for the binding-site specificity of PAR factors, extensive mutational analysis of VBP was carried out. By substituting five amino acid residues that differ between the Drosophila giant bZIP factor and the vertebrate PAR bZIP factors, it has been shown that the fork region, which bridges the basic and leucine zipper domains, contributes to half-site sequence specificity. At least two domains amino terminal to the core basic region are required for VBP to bind to the full spectrum of PAR target sites. Thus, whereas direct base contacts may be restricted to basic-region residues (as indicated by GCN4-DNA crystal structures), several other domains also influence the DNA-binding specificity of PAR bZIP proteins (Haas, 1995).

PAR and C/EBP family proteins are liver-enriched basic leucine zipper (bZip) transcription factors that bind similar sites on the promoters of albumin and cholesterol 7 alpha hydroxylase genes. However, C/EBP proteins have a more relaxed binding specificity than PAR proteins, in that they recognize many sites within promoter or randomly selected rat genomic DNA sequences that are ignored by PAR proteins. Thus, DNAse I protection experiments suggest that C/EBP recognizes a binding site every 200 to 300 bp with an affinity similar to that of the cholesterol 7 alpha hydroxylase gene promoter. The frequency of PAR protein binding sites with comparable affinities is about 20-fold lower in the rat genome. By using a PCR-based amplification assay, high affinity DNA-binding sites were selected for C/EBP beta and the PAR protein DBP from a pool of oligonucleotides. Both proteins indeed recognize similar sequences with the optimal core binding sequences 5'RTTAY.GTAAY3'. However, as expected, DBP, is considerably less tolerant to deviations from the consensus site. A single amino acid substitution mutant of C/EBP beta that increases its target site specificity has been characterized. This protein, C/EBP beta V to A, contains a valine to alanine substitution at position 13 of the basic domain (residue 216 of C/EBP beta). C/EBP beta V to A selectively binds only the subset of C/EBP sites that are also DBP sites, both as oligonucleotides and within the natural contexts of the albumin and cholesterol hydroxylase promoters (Falvey, 1996).

Transcriptional targets of PAR-domain proteins

The two highly related PAR basic region leucine zipper proteins TEF and DBP accumulate according to a robust circadian rhythm in liver and kidney. In liver nuclei, the amplitude of daily oscillation has been estimated to be 50-fold and 160-fold for TEF and DBP, respectively. While DBP mRNA expression is the principal determinant of circadian DBP accumulation, the amplitude of TEF mRNA cycling is insufficient to explain circadian TEF fluctuation. Conceivably, daily variations in TEF degradation or nuclear translocation efficiency may explain the discrepancy between mRNA and protein accumulation. In vitro, TEF and DBP bind the same DNA sequences. Yet, in co-transfection experiments, these two proteins exhibit different activation potentials for the two reporter genes examined. While TEF stimulates transcription from the albumin promoter more potently than DBP, only DBP is capable of activating transcription efficiently from the cholesterol 7 alpha hydroxylase (C7alphaH) promoter. However, a TEF-DBP fusion protein, carrying N-terminal TEF sequences and the DNA binding/dimerization domain of DBP, enhances expression of the C7alphaH-CAT reporter gene as strongly as wild-type DBP. These results suggest that the promoter environment, rather than the affinity with which PAR proteins recognize their cognate DNA sequences in vitro, determines the promoter preferences of TEF and DBP (Fonjallaz, 1996).

The human GH gene family includes the pituitary-specific hGH-1, placental-specific chorionic somatomammotropin (hCS-5, hCS-2, and hCS-1), and hGH-2 genes. These duplicated, nearly identical genes are localized on approximately 50 kb of DNA on chromosome 17q23-q24. An enhancer (CSEn2), located downstream of the hCS-2 gene, participates in mediating placental-specific hCS gene expression. CSEn2 activity derives from the cooperative binding of transcription factor-1, TEF-1, and a placental-specific factor CSEF-1 to multiple enhansons (Enh1-Enh5) that are related to the SV40 GT-IIC and SphI/SphII enhansons. Two copies of CSEn2 or a single copy of CSEn2 linked to either of the other two enhancers in the hGH/hCS locus (CSEn1 and CSEn5) act cooperatively to enhance hCS promoter activity in choriocarcinoma (BeWo) cells, but silence the promoter in pituitary GC cells. Mutation of Enh4, an essential GT-IIC-like enhanson in the context of the intact enhancer, abolishes silencer activity, and multimerized GT-IIC enhansons mimic the intact CSEn enhancer/silencer activities in BeWo and GC cells, respectively. TEF-1 has been identified as the GT-IIC-binding factor in pituitary cells. The data suggest that TEF-1 may be involved in pituitary-specific repression of placental GH/CS gene transcription through long-range interactions between the multiple CS enhancers present on the GH/CS gene locus (Jiang, 1997).

The avian leukosis virus (ALV) long terminal repeat (LTR) contains a compact transcription enhancer that is active in many cell types. A major feature of the enhancer is multiple CCAAT/enhancer element motifs that could be important for the strong transcriptional activity of this unit. The contributions of the three CCAAT/enhancer elements to LTR function were examined in B cells, since this cell type is targeted for ALV tumor induction following integration of LTR sequences next to the c-myc proto-oncogene. One CCAAT/enhancer element, termed a3, is the most critical for LTR enhancement in transiently transfected B lymphoma cells, while in chicken embryo fibroblasts all three elements contribute equally to enhancement. Vitellogenin gene-binding protein (VBP), a member of the PAR subfamily of C/EBP factors, is a major component of the nuclear proteins binding to the a3 CCAAT/enhancer element. VBP activates transcription through the a3 CCAAT/enhancer element, supporting the idea that VBP is important for LTR enhancement in B cells. A member of the Rel family of proteins, RelA, is also identified as a component of the a3 protein binding complex in B cells. While RelA does not bind directly to the LTR CCAAT/enhancer elements, it does interact with VBP to potentiate VBP DNA binding activity. The synergistic interaction of VBP and RelA increases CCAAT/enhancer element-mediated transcription, indicating that both factors may be important for viral LTR regulation and also for expression of many cellular genes (Curristin, 1997).

The regulatory regions of the genes for coagulation Factors VIII and IX contain binding sites for both liver-enriched and ubiquitous transcriptional regulators. The role of the liver-enriched protein, hepatic leukemia factor (HLF), in mediating transcriptional regulation of the Factor VIII and IX genes was examined. Using transient transfection assays in HepG2 hepatoma cells, the ability of HLF alone and in synergistic combination with the D-box binding protein (DBP), another proline and acidic-rich (PAR) protein family member, to transactivate these promoters was examined. HLF is capable of binding to multiple sites in both the Factor VIII and Factor IX promoters. At least some of the synergistic activation of the Factor VIII promoter seen with HLF and DBP cotransfection can be attributed to increased binding of HLF-DBP heterodimers to two Factor VIII promoter sites. An E2A-HLF chimera, derived from a t(17;19) translocation in pre-B acute lymphoblastic leukemia (ALL) cells, is capable of mediating expression from the Factor VIII and Factor IX promoters in both hepatoma cells and pre-B ALL cells. These observations indicate that the PAR family of transcription factors plays an important and complex role in regulating expression of the Factor VIII and Factor IX genes, involving the binding of both homodimeric and heterodimeric complexes of HLF and DBP to several sites in the promoters. Finally, these studies reaffirm the potential role of dimeric transcription factor complexes in mediating interactions with specific promoter elements, which, in the case of the Factor VIII promoter, results in dramatically enhanced binding of HLF-DBP heterodimers to two cis-acting sequences. These observations further an understanding of the role played by members of the PAR family of transcription factors in regulating expression of the Factor VIII and Factor IX genes (Begbie, 1999).

PAR-domain proteins and circadian rhythms

D-element binding protein (DBP), the founding member of the PAR family of basic leucine zipper (bZip) transcription factors, is expressed according to a robust daily rhythm in the suprachiasmatic nucleus and several peripheral tissues. Other members of this family include TEF (thyroid embryonic factor), its avian ortholog VBP (vitellogenin promoter-binding protein), and HLF (hepatocyte leukemia factor). All of these proteins share high amino acid sequence similarities within a amino-terminal activation domain, a PAR domain rich in proline and acidic amino acid residues, and a carboxy-terminal moiety encompassing the bZip region necessary for DNA binding and dimerization. In vitro all PAR bZip proteins avidly bind the consensus DNA recognition sequence 5'-RTTAYGTAAY-3' as homo- or hetero-dimers. In rat and mouse liver the expression of all three PAR bZip proteins is subject to strong circadian regulation, peak and trough levels being reached in the early evening and morning, respectively. In the case of Dbp the amplitude of circadian mRNA oscillation can largely account for the daily amplitude in protein oscillation. The mRNA accumulation oscillates not only in peripheral tissues such as liver, but also in neurons of the SCN, believed to harbor the central circadian pacemaker. Moreover, run-on experiments in isolated nuclei and physical mapping of nascent RNA chains suggest that circadian transcription plays a pivotal role in rhythmic DBP expression (Ripperger, 2000 and references therein).

Previous studies with mice that have been deleted for the Dbp gene have established that DBP participates in the regulation of several clock outputs, including locomotor activity, sleep distribution, and liver gene expression. Evidence that circadian Dbp transcription requires the basic helix-loop-helix-PAS protein CLOCK, an essential component of the negative-feedback circuitry generating circadian oscillations in mammals and fruit flies. Genetic and biochemical experiments suggest that CLOCK regulates Dbp expression by binding to E-box motifs within putative enhancer regions located in the first and second introns. Similar E-box motifs have been found previously in the promoter sequence of the murine clock gene mPeriod1. Hence, the same molecular mechanisms generating circadian oscillations in the expression of clock genes may directly control the rhythmic transcription of clock output regulators such as Dbp (Ripperger, 2000).

Transcript levels of DBP, a member of the PAR leucine zipper transcription factor family, exhibit a robust rhythm in suprachiasmatic nuclei, the mammalian circadian center. DBP is able to activate the promoter of a putative clock oscillating gene, mPer1, by directly binding to the mPer1 promoter. The mPer1 promoter is cooperatively activated by DBP and CLOCK-BMAL1. However, dbp transcription is activated by CLOCK-BMAL1 through E-boxes and inhibited by the mPER and mCRY proteins, as is the case for mPer1. Thus, a clock-controlled dbp gene may play an important role in central clock oscillation (Yamaguchi, 2000).

Albumin D-binding protein (DBP) is a PAR leucine zipper transcription factor that is expressed according to a robust circadian rhythm in the suprachiasmatic nuclei, harboring the circadian master clock, and in most peripheral tissues. Mice lacking DBP display a shorter circadian period in locomotor activity and are less active. Thus, although DBP is not essential for circadian rhythm generation, it does modulate important clock outputs. The role of DBP in the circadian and homeostatic aspects of sleep regulation were studied by comparing DBP deficient mice (dbp-/-) with their isogenic controls (dbp+/+) under light-dark (LD) and constant-dark (DD) baseline conditions, as well as after sleep loss. Whereas total sleep duration is similar in both genotypes, the amplitude of the circadian modulation of sleep time, as well as the consolidation of sleep episodes, is reduced in dbp-/- under both LD and DD conditions. Quantitative EEG analysis has demonstrated a marked reduction in the amplitude of the sleep-wake-dependent changes in slow-wave sleep delta power and an increase in hippocampal theta peak frequency in dbp-/- mice. The sleep deprivation-induced compensatory rebound of EEG delta power is similar in both genotypes. In contrast, the rebound in paradoxical sleep is significant in dbp+/+ mice only. It is concluded that the transcriptional regulatory protein DBP modulates circadian and homeostatic aspects of sleep regulation (Franken, 2000).

To study the molecular mechanisms of circadian gene expression, attempts have been made to identify genes whose expression in mouse liver is regulated by the transcription factor DBP (albumin D-site-binding protein). This PAR basic leucine zipper protein accumulates according to a robust circadian rhythm in nuclei of hepatocytes and other cell types. The Cyp2a4 gene, encoding the cytochrome P450 steroid 15alpha-hydroxylase, is a novel circadian expression gene. This enzyme catalyzes one of the hydroxylation reactions leading to further metabolism of the sex hormones testosterone and estradiol in the liver. Accumulation of CYP2A4 mRNA in mouse liver displays circadian kinetics indistinguishable from those of the highly related CYP2A5 gene. Proteins encoded by both the Cyp2a4 and Cyp2a5 genes also display daily variation in accumulation, though this is more dramatic for CYP2A4 than for CYP2A5. Biochemical evidence, including in vitro DNase I footprinting on the Cyp2a4 and Cyp2a5 promoters and cotransfection experiments with the human hepatoma cell line HepG2, suggests that the Cyp2a4 and Cyp2a5 genes are indeed regulated by DBP. These conclusions are corroborated by genetic studies, in which the circadian amplitude of CYP2A4 and CYP2A5 mRNAs and protein expression in the liver is significantly impaired in a mutant mouse strain homozygous for a dbp null allele. These experiments strongly suggest that DBP is a major factor controlling circadian expression of the Cyp2a4 and Cyp2a5 genes in the mouse liver (Lavery, 1999).

DBP, a PAR leucine zipper transcription factor, accumulates according to a robust circadian rhythm in liver and several other tissues of mouse and rat. DBP mRNA levels also oscillate strongly in the suprachiasmatic nucleus (SCN) of the hypothalamus, believed to harbor the central mammalian pacemaker. However, peak and minimum levels of DBP mRNA are reached about 4 h earlier in the SCN than in liver, suggesting that circadian DBP expression is controlled by different mechanisms in SCN and in peripheral tissues. Mice homozygous for a DBP-null allele display less locomotor activity and free-run with a shorter period than otherwise isogenic wild-type animals. The altered locomotor activity in DBP mutant mice and the highly rhythmic expression of the DBP gene in SCN neurons suggest that DBP is involved in controlling circadian behavior. However, since DBP-/- mice are still rhythmic and since DBP protein is not required for the circadian expression of its own gene, dbp is more likely to be a component of the circadian output pathway than a master gene of the clock (Lopez-Molina, 2000).

PAR-domain proteins and cancer

Oncogenic conversion of transcription factors by chromosomal translocations is implicated in leukemogenesis. The t(17;19) in acute lymphoblastic leukemia produces a chimeric transcription factor consisting of the amino-terminal portion of HLH proteins E12/E47 (products of the E2A gene) fused to the basic DNA-binding and leucine zipper dimerization motifs of a novel hepatic protein called hepatic leukemia factor (Hlf). Hlf, which is not normally transcribed in lymphoid cells, belongs to the recently described PAR subfamily of basic leucine zipper (bZIP) proteins, which also includes Dbp and Tef/Vbp. Wild-type Hlf is able to bind DNA specifically as a homodimer or as a heterodimer with other PAR factors. Structural alterations of the E2a-Hlf fusion protein markedly impair its ability to bind DNA as a homodimer, as compared with wild-type Hlf. However, E2a-Hlf can bind DNA as a heterodimer with other PAR proteins, suggesting a novel mechanism for leukemogenic conversion of a bZIP transcription factor (Hunger, 1992).

Genes encoding transcription factors are frequently altered by chromosomal translocations in acute lymphoblastic leukemia (ALL), suggesting that aberrant transcriptional regulation plays a prominent role in leukemogenesis. E2A-hepatic leukemia factor (HLF), a chimeric transcription factor created by the t(17;19), consists of the amino terminal portion of E2A proteins, including two experimentally defined transcriptional activation domains (TADs), fused to the HLF DNA binding and protein dimerization basic leucine zipper (bZIP) domain. To understand the mechanisms by which E2A-HLF induces leukemia and the crucial functions contributed by each constituent of the chimera, it is essential to define the normal transcriptional regulatory properties of HLF and related bZIP proteins. To address these questions, the human homologue of TEF/VBP, a bZIP protein closely related to HLF was cloned. Using a binding site selection assay, it was found that TEF bound preferentially to the consensus sequence 5'-GTTACGTAAT-3', which is identical to the previously determined HLF recognition site. TEF and HLF activate transcription of consensus site-containing reporter genes in several different cell types with similar potencies. Using GAL4 chimeric proteins, a TAD was mapped to an approximately 40 amino acid region of TEF and HLF within which the two proteins share 72% amino acid identity and 85% similarity. The TEF/HLF activation domain (THAD) has a predicted helical secondary structure, but shares no sequence homology with previously reported TADs. The THAD contains most, if not all, of the transcriptional activation properties present in both TEF and HLF and its deletion completely abrogates transcriptional activity of TEF and HLF in both mammalian cells and yeast. Thus, TEF and HLF share indistinguishable DNA-binding and transcriptional regulatory properties, whose alteration in leukemia may be pathogenetically important (Hunger, 1996).

The E2A-HLF fusion gene, created by the t(17;19)(q22;p13) chromosomal translocation in pro-B lymphocytes, encodes an oncogenic protein in which the E2A trans-activation domain is linked to the DNA-binding and protein dimerization domain of hepatic leukemia factor (HLF), a member of the proline- and acidic amino acid-rich (PAR) subfamily of bZIP transcription factors. This fusion product binds to its DNA recognition site not only as a homodimer but also as a heterodimer with HLF and two other members of the PAR bZIP subfamily: thyrotroph embryonic factor (TEF) and albumin promoter D-box binding protein (DBP). Thus, E2A-HLF could transform cells by direct regulation of downstream target genes, acting through homodimeric or heterodimeric complexes, or by sequestering normal PAR proteins into nonfunctional heterocomplexes (dominant-negative interference). To distinguish among these models, mutant E2A-HLF proteins were constructed in which the leucine zipper domain of HLF was extended by one helical turn or altered in essential charged amino acids, enabling the chimera to bind to DNA as a homodimer but not as a heterodimer with HLF or other PAR proteins. When introduced into NIH 3T3 cells in a zinc-inducible vector, each of these mutants induces anchorage-independent growth as efficiently as unaltered E2A-HLF, indicating that the chimeric oncoprotein can transform cells in its homodimeric form. Transformation also depends on an intact E2A activator region, providing further support for a gain-of-function contribution to oncogenesis rather than one based on a dominant-interfering or dominant-negative mechanism. Thus, the tumorigenic effects of E2A-HLF and its mutant forms in NIH 3T3 cells favor a straightforward model in which E2A-HLF homodimers bind directly to promoter/enhancer elements of downstream target genes and alter the patterns of gene expression in early B-cell progenitors (Inukai, 1997).