PAR-domain protein 1


EVOLUTIONARY HOMOLOGS

PAR-domain proteins

Two cDNAs were cloned and sequenced, representing two isoforms of the zebrafish thyrotrophembryonic factor (TEF) gene products (tef alpha and beta); both are members of the PAR subfamily of bZIPtranscription 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 thePAR family of transcription factors of other species in the PAR domain, the DNA binding domain and theleucine zipper domain. Expression analysis by Northern blot and RT-PCR indicates that tef alpha andtef 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 oftranscription factors, thyrotroph embryonic factor (TEF) has been identified and characterized. Analysis of the ontogeny of TEF geneexpression reveals the presence of TEF transcripts, beginning on embryonic day 14, only in the regionof the rat anterior pituitary gland, in which thyrotrophic cells arise. This pattern of gene expressioncorresponds temporally and spatially to the onset of thyroid-stimulating hormone (TSH beta) geneexpression, which defines the thyrotroph phenotype. TEFcan bind to and trans-activate the TSH beta promoter. In contrast to this restricted pattern ofexpression during embryogenesis, TEF transcripts appear in several tissues in the mature organism.On the basis of the unique homologybetween 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 readilyform heterodimers. The DNA-binding and dimerization domains of TEF correspond to those found inother bZIP proteins. A cluster of basic amino acids, found only in TEF andDBP, has been identified as being necessary for the proper DNA-binding site specificity of TEF. A major trans-activationdomain of TEF resides outside the region of homology to other bZIP proteins. These data areconsistent with a role for a member of a new class of bZIP transcription factors in activating geneexpression in the developing thyrotroph (Drolet, 1991).

A chicken liver cDNA expression library was screened with a probe spanning the distal region of thechicken vitellogenin II (VTGII) gene promoter. A transcription factor termed VBP (for vitellogenin gene-binding protein) was isolated. VBP binds to one of the most importantpositive elements in the VTGII promoter and appears to play a pivotal role in the estrogen-dependentregulation of this gene. The protein sequence of VBP contains a basic/zipper (bZIP) motif. As expected for a bZIP factor, VBP bindsto its target DNA site as a dimer. VBP forms a stable dimer in solution. A data basesearch has revealed that VBP is related to rat DBP. However, despite the fact that the basic/hinge regionsof VBP and DBP differ at only three amino acid positions, the DBP binding site in the rat albuminpromoter is a relatively poor binding site for VBP. Thus, the optimal binding sites for VBP and DBPmay be distinct. Similarities between the VBP and DBP leucine zippers are largely confined to onlyfour of the seven helical spokes. Nevertheless, these leucine zippers are functionally compatible andappear to define a novel subfamily. In contrast to the bZIP regions, other portions of VBP and DBPare markedly different, as are the expression profiles for these two genes. In particular, expression ofthe 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 albuminpromoter. 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 ofthis 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 mRNAis present in all of the examined tissues. DBP as well as its mRNA accumulate to significant levelsonly in adult animals. During chemically induced liver regeneration, DBP expression is rapidlydown-regulated, suggesting that DBP may be involved in the proliferation control of hepatocytes. Thiscell growth-dependent expression of DBP, in contrast to its tissue specificity, appears to be controlledat the level of mRNA accumulation (Mueller, 1990).

Alternative splicing of PAR-domain proteins

An analysis of the chicken VBP gene reveals that thetwo different amino-terminal sequences map to alternative first exons and that the two differentcarboxyl-terminal sequences reflect an optional splicing event that can occur only on transcripts thatare polyadenylated at the more distal of two polyadenylation sites. An RT-PCR analysis furtherreveals that a total of four VBP isoforms are encoded by the combinatorial use of these two splicingoptions. The mRNAs for these four isoforms are differentially expressed in different tissues and celltypes. Evidence is provided that one function of the amino-terminal domains is to impose cell typespecificity on a core transactivation domain that is present in all four isoforms. Since it is known thatVBP can heterodimerize with other members of the PAR subfamily of bZIP factors, the evidence forfour VBP isoforms greatly expands the number of complexes that may be used to effect transcriptionalregulation 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 istranscribed from two alternative promoters, alpha and beta, with different circadian amplitudes andtissue specificities. The alpha RNA isoforms produce a 43 kDa protein, HLF43, abundant in brain, liverand kidney, as is human HLF RNA. The beta RNA HLF isoforms use a CUGcodon to initiate translation of a novel 36 kDa protein, HLF36, which is shorter at its N-terminusrelative to the 43 kDa form. HLF36 is expressed uniquely in the liver, where it is the most abundantHLF protein. Surprisingly, the two proteins accumulate in the liver with different circadian amplitudesand have distinct liver-specific promoter preferences in transfection experiments. Thus, HLF43stimulates transcription from the cholesterol 7 alpha-hydroxylase promoter much more efficiently thanfrom the albumin promoter, while the converse is true for HLF36 (Falvey, 1997).

PAR-1 phosphorylates Mind bomb to promote vertebrate neurogenesis

Generation of neurons in the vertebrate central nervous system requires a complex transcriptional regulatory network and signaling processes in polarized neuroepithelial progenitor cells. This study demonstrates that neurogenesis in the Xenopus neural plate in vivo and mammalian neural progenitors in vitro involves intrinsic antagonistic activities of the polarity proteins PAR-1 and aPKC. Furthermore, Mind bomb (Mib), a ubiquitin ligase that promotes Notch ligand trafficking and activity, is a crucial molecular substrate for PAR-1. The phosphorylation of Mib by PAR-1 results in Mib degradation, repression of Notch signaling, and stimulation of neuronal differentiation. These observations suggest a conserved mechanism for neuronal fate determination that might operate during asymmetric divisions of polarized neural progenitor cells (Ossipova, 2009).

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 intranscriptional regulation in the liver. Deletion analysis of the DBP proteinwas 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 DBPreporter construct when assayed in Hep G2 cells. These same sequences confersignificant transactivation to GAL4 DNA binding domain fusion proteins,indicating that this region acts as part of an independent activation domaincomprised 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 hepaticleukemia factor results in a dramatic increase in activation mediated by theGAL4-DBP fusion proteins, suggesting the involvement of a regulated coactivatorin this process. DBP transactivation appears to be a p300-dependent process, sincea 12 S E1A expression construct disrupts DBP-mediated transactivation, and ap300 expression vector, but not a CREB binding protein vector, is able torestore DBP transactivation. These results suggest that the PAR domain isrequired for DBP activation, which occurs through a regulated, p300-dependentprocess (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. VBPpreferentially binds to sequences that consist of abutted GTAAY half-sites (which are referred to as PARsites) as well as to sequences that contain either a C/EBP half-site (GCAAT) or a CREB/ATFhalf-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 ashigh-affinity binding sites for the E4BP4 transcriptional repressor, it is inferred that these sequences maybe targets for positive and negative regulation. Similarly, since the sequences described asPAR-C/EBP and PAR-CREB/ATF chimeric sites are known to be high-affinity binding sites forC/EBP and CREB/ATF factors, respectively, it is inferred that these sites may each be targets for multiplesubfamilies of bZIP factors. To gain insight regarding the molecular basis for the binding-sitespecificity of PAR factors, extensive mutational analysis of VBP was carried out. Bysubstituting five amino acid residues that differ between the Drosophila giant bZIP factor and thevertebrate PAR bZIP factors, it has been shown that the fork region, which bridges the basic and leucine zipperdomains, contributes to half-site sequence specificity. At least two domainsamino terminal to the core basic region are required for VBP to bind to the full spectrum of PARtarget sites. Thus, whereas direct base contacts may be restricted to basic-region residues (asindicated by GCN4-DNA crystal structures), several other domains also influence the DNA-bindingspecificity 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 andcholesterol 7 alpha hydroxylase genes. However, C/EBP proteins have a morerelaxed binding specificity than PAR proteins, in that they recognize many siteswithin promoter or randomly selected rat genomic DNA sequences that are ignoredby PAR proteins. Thus, DNAse I protection experiments suggest that C/EBPrecognizes a binding site every 200 to 300 bp with an affinity similar to that of the cholesterol7 alpha hydroxylase gene promoter. The frequency of PARprotein binding sites with comparable affinities is about 20-fold lower in therat genome. By using a PCR-based amplification assay, high affinityDNA-binding sites were selected for C/EBP beta and the PAR protein DBP from a pool ofoligonucleotides. Both proteins indeed recognize similar sequences with theoptimal core binding sequences 5'RTTAY.GTAAY3'. However, as expected, DBP, isconsiderably less tolerant to deviations from the consensus site. A single amino acid substitution mutant of C/EBP beta thatincreases its target site specificity has been characterized. This protein, C/EBP beta V to A, containsa valine to alanine substitution at position 13 of the basic domain (residue 216of C/EBP beta). C/EBP beta V to A selectively binds only the subset of C/EBPsites that are also DBP sites, both as oligonucleotides and within the naturalcontexts 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 accordingto a robust circadian rhythm in liver and kidney. In liver nuclei, the amplitude of daily oscillation hasbeen estimated to be 50-fold and 160-fold for TEF and DBP, respectively. While DBP mRNAexpression is the principal determinant of circadian DBP accumulation, the amplitude of TEF mRNAcycling is insufficient to explain circadian TEF fluctuation. Conceivably, daily variations in TEFdegradation or nuclear translocation efficiency may explain the discrepancy between mRNA andprotein accumulation. In vitro, TEF and DBP bind the same DNA sequences. Yet, in co-transfectionexperiments, 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 iscapable 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 DNAbinding/dimerization domain of DBP, enhances expression of the C7alphaH-CAT reporter gene asstrongly as wild-type DBP. These results suggest that the promoter environment, rather than the affinitywith which PAR proteins recognize their cognate DNA sequences in vitro, determines the promoterpreferences of TEF and DBP (Fonjallaz, 1996).

The human GH gene family includes the pituitary-specific hGH-1, placental-specific chorionicsomatomammotropin (hCS-5, hCS-2, and hCS-1), and hGH-2 genes. These duplicated, nearly identicalgenes 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 geneexpression. 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 enhancerthat is active in many cell types. A major feature of the enhancer is multiple CCAAT/enhancerelement motifs that could be important for the strong transcriptional activity of this unit. Thecontributions of the three CCAAT/enhancer elements to LTR function were examined in B cells, sincethis cell type is targeted for ALV tumor induction following integration of LTR sequences next to thec-myc proto-oncogene. One CCAAT/enhancer element, termed a3, is the most criticalfor LTR enhancement in transiently transfected B lymphoma cells, while in chicken embryo fibroblastsall three elements contribute equally to enhancement. Vitellogeningene-binding protein (VBP), a member of the PAR subfamily of C/EBP factors, is a major componentof the nuclear proteins binding to the a3 CCAAT/enhancer element. VBP activates transcriptionthrough the a3 CCAAT/enhancer element, supporting the idea that VBP is important for LTRenhancement in B cells. A member of the Rel family of proteins, RelA, is also identified as a component ofthe a3 protein binding complex in B cells. While RelA does not bind directly to the LTRCCAAT/enhancer elements, it does interact with VBP to potentiate VBP DNA binding activity. Thesynergistic 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 manycellular 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 leucinezipper (bZip) transcription factors, is expressed according to a robustdaily rhythm in the suprachiasmatic nucleus and several peripheral tissues. Other members of this familyinclude TEF (thyroid embryonicfactor), its avian ortholog VBP(vitellogenin promoter-bindingprotein), and HLF(hepatocyte leukemia factor). All of these proteins sharehigh amino acid sequence similarities within a amino-terminalactivation domain, a PAR domain rich in proline andacidic amino acid residues, and acarboxy-terminal moiety encompassing the bZip region necessary for DNAbinding and dimerization. In vitro all PAR bZip proteins avidly bindthe consensus DNA recognition sequence 5'-RTTAYGTAAY-3' ashomo- or hetero-dimers. In rat and mouse liver the expression of all three PAR bZip proteins issubject to strong circadian regulation, peak and trough levels beingreached in the early evening and morning, respectively. In the case of Dbp the amplitude of circadianmRNA oscillation can largely account for the daily amplitude in proteinoscillation. The mRNA accumulation oscillatesnot only in peripheral tissues such as liver, but also in neurons ofthe SCN, believed to harbor the central circadian pacemaker. Moreover, run-on experiments in isolated nuclei and physical mapping ofnascent RNA chains suggest that circadiantranscription plays a pivotal role in rhythmic DBP expression (Ripperger, 2000 and references therein).

Previous studies with mice that have been deleted for theDbp 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 thebasic helix-loop-helix-PAS protein CLOCK, an essential component of the negative-feedback circuitry generatingcircadian oscillations in mammals and fruit flies. Genetic and biochemical experiments suggest that CLOCK regulates Dbp expression by binding toE-box motifs within putative enhancer regions located in the first and second introns. Similar E-box motifs have been found previously in thepromoter sequence of the murine clock gene mPeriod1. Hence, the same molecular mechanisms generating circadian oscillations in the expressionof 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 transcriptionfactor family, exhibit a robust rhythm in suprachiasmatic nuclei, the mammaliancircadian center. DBP is able to activate the promoter of aputative clock oscillating gene, mPer1, by directly binding to the mPer1promoter. The mPer1 promoter is cooperatively activated by DBP and CLOCK-BMAL1.However, dbp transcription is activated by CLOCK-BMAL1 through E-boxesand inhibited by the mPER and mCRY proteins, as is the case for mPer1. Thus, aclock-controlled dbp gene may play an important role in central clockoscillation (Yamaguchi, 2000).

Albumin D-binding protein (DBP) is a PAR leucine zipper transcription factorthat is expressed according to a robust circadian rhythm in the suprachiasmaticnuclei, harboring the circadian master clock, and in most peripheral tissues.Mice lacking DBP display a shorter circadian period in locomotor activity andare less active. Thus, although DBP is not essential for circadian rhythmgeneration, it does modulate important clock outputs. The role of DBPin the circadian and homeostatic aspects of sleep regulation were studied by comparing DBPdeficient 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 thecircadian modulation of sleep time, as well as the consolidation of sleepepisodes, is reduced in dbp-/- under both LD and DD conditions. QuantitativeEEG analysis has demonstrated a marked reduction in the amplitude of thesleep-wake-dependent changes in slow-wave sleep delta power and an increase inhippocampal theta peak frequency in dbp-/- mice. The sleep deprivation-inducedcompensatory rebound of EEG delta power is similar in both genotypes. Incontrast, the rebound in paradoxical sleep is significant in dbp+/+ mice only.It is concluded that the transcriptional regulatory protein DBP modulatescircadian 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 thetranscription factor DBP (albumin D-site-binding protein). This PAR basicleucine zipper protein accumulates according to a robust circadian rhythm innuclei of hepatocytes and other cell types. The Cyp2a4gene, encoding the cytochrome P450 steroid 15alpha-hydroxylase, is a novelcircadian expression gene. This enzyme catalyzes one of the hydroxylationreactions leading to further metabolism of the sex hormones testosterone andestradiol in the liver. Accumulation of CYP2A4 mRNA in mouse liver displayscircadian kinetics indistinguishable from those of the highly related CYP2A5gene. Proteins encoded by both the Cyp2a4 and Cyp2a5 genes also display dailyvariation in accumulation, though this is more dramatic for CYP2A4 than forCYP2A5. Biochemical evidence, including in vitro DNase I footprinting on theCyp2a4 and Cyp2a5 promoters and cotransfection experiments with the humanhepatoma cell line HepG2, suggests that the Cyp2a4 and Cyp2a5 genes are indeedregulated by DBP. These conclusions are corroborated by genetic studies, inwhich the circadian amplitude of CYP2A4 and CYP2A5 mRNAs and protein expressionin the liver is significantly impaired in a mutant mouse strain homozygous fora dbp null allele. These experiments strongly suggest that DBP is a major factorcontrolling circadian expression of the Cyp2a4 and Cyp2a5 genes in the mouseliver (Lavery, 1999).

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

PAR-domain proteins and cancer

Oncogenic conversion of transcription factors by chromosomal translocations is implicated inleukemogenesis. The t(17;19) in acute lymphoblastic leukemia produces a chimerictranscription factor consisting of the amino-terminal portion of HLH proteins E12/E47 (products of theE2A gene) fused to the basic DNA-binding and leucine zipper dimerization motifs of a novel hepaticprotein 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 alsoincludes Dbp and Tef/Vbp. Wild-type Hlf is able to bind DNA specifically as a homodimer or as aheterodimer with other PAR factors. Structural alterations of the E2a-Hlf fusion protein markedlyimpair its ability to bind DNA as a homodimer, as compared with wild-type Hlf. However, E2a-Hlf canbind DNA as a heterodimer with other PAR proteins, suggesting a novel mechanism for leukemogenicconversion of a bZIP transcription factor (Hunger, 1992).

Genes encoding transcription factors are frequently altered by chromosomal translocations in acutelymphoblastic leukemia (ALL), suggesting that aberrant transcriptional regulation plays a prominentrole in leukemogenesis. E2A-hepatic leukemia factor (HLF), a chimeric transcription factor created bythe t(17;19), consists of the amino terminal portion of E2A proteins, including two experimentallydefined transcriptional activation domains (TADs), fused to the HLF DNA binding and proteindimerization basic leucine zipper (bZIP) domain. To understand the mechanisms by which E2A-HLFinduces leukemia and the crucial functions contributed by each constituent of the chimera, it is essentialto define the normal transcriptional regulatory properties of HLF and related bZIP proteins. To addressthese 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 sequence5'-GTTACGTAAT-3', which is identical to the previously determined HLF recognition site. TEF andHLF activate transcription of consensus site-containing reporter genes in several different cell typeswith similar potencies. Using GAL4 chimeric proteins, a TAD was mapped to an approximately40 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, butshares no sequence homology with previously reported TADs. The THAD contains most, if not all, ofthe transcriptional activation properties present in both TEF and HLF and its deletion completelyabrogates transcriptional activity of TEF and HLF in both mammalian cells and yeast. Thus, TEF andHLF share indistinguishable DNA-binding and transcriptional regulatory properties, whose alteration inleukemia may be pathogenetically important (Hunger, 1996).

The E2A-HLF fusion gene, created by the t(17;19)(q22;p13) chromosomal translocation in pro-Blymphocytes, encodes an oncogenic protein in which the E2A trans-activation domain is linked to theDNA-binding and protein dimerization domain of hepatic leukemia factor (HLF), a member of theproline- and acidic amino acid-rich (PAR) subfamily of bZIP transcription factors. This fusion productbinds to its DNA recognition site not only as a homodimer but also as a heterodimer with HLF and twoother members of the PAR bZIP subfamily: thyrotroph embryonic factor (TEF) and albumin promoterD-box binding protein (DBP). Thus, E2A-HLF could transform cells by direct regulation ofdownstream target genes, acting through homodimeric or heterodimeric complexes, or by sequesteringnormal PAR proteins into nonfunctional heterocomplexes (dominant-negative interference). Todistinguish among these models, mutant E2A-HLF proteins were constructed in which the leucine zipperdomain of HLF was extended by one helical turn or altered in essential charged amino acids, enabling thechimera 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 inducesanchorage-independent growth as efficiently as unaltered E2A-HLF, indicating that the chimericoncoprotein can transform cells in its homodimeric form. Transformation also depends on an intactE2A activator region, providing further support for a gain-of-function contribution to oncogenesis ratherthan one based on a dominant-interfering or dominant-negative mechanism. Thus, the tumorigeniceffects of E2A-HLF and its mutant forms in NIH 3T3 cells favor a straightforward model in whichE2A-HLF homodimers bind directly to promoter/enhancer elements of downstream target genes andalter the patterns of gene expression in early B-cell progenitors (Inukai, 1997).


PAR-domain protein 1: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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