Basic DNA Binding Domain Research Articles

Cloning, purification and preliminary X-ray data analysis of the human ID2 homodimer.

The ID proteins are named for their role as inhibitors of DNA binding and differentiation. They contain a helix-loop-helix (HLH) domain but lack a basic DNA-binding domain. In complex with basic HLH (bHLH) transcription factors, gene expression is regulated by DNA-binding inactivation. Although the HLH domain is highly conserved and shares a similar topology, the IDs preferentially bind class I bHLH-group members such as E47 (TCF3) but not the class III bHLH member Myc. A structure of an ID protein could potentially shed light on its mechanism. Owing to their short half-lives in vivo and reported in vitro instability, this paper describes the strategies that went into expressing sufficient soluble and stable ID2 to finally obtain diffraction-quality crystals. A 2.1 Å resolution data set was collected from a crystal belonging to space group P3(1)21 with unit-cell parameters a=b=51.622, c=111.474 Å, α=β=90, γ=120° that was obtained by hanging-drop vapour diffusion in a precipitant solution consisting of 0.1 M MES pH 6.5, 2.0 M potassium acetate. The solvent content was consistent with the presence of one or two molecules in the asymmetric unit.

Basic leucine zipper transcription factor Hac1 binds DNA in two distinct modes as revealed by microfluidic analyses

A quantitative understanding of how transcription factors interact with genomic target sites is crucial for reconstructing transcriptional networks in vivo. Here, we use Hac1, a well-characterized basic leucine zipper (bZIP) transcription factor involved in the unfolded protein response (UPR) as a model to investigate interactions between bZIP transcription factors and their target sites. During the UPR, the accumulation of unfolded proteins leads to unconventional splicing and subsequent translation of HAC1 mRNA, followed by transcription of UPR target genes. Initial candidate-based approaches identified a canonical cis-acting unfolded protein response element (UPRE-1) within target gene promoters; however, subsequent studies identified a large set of Hac1 target genes lacking this UPRE-1 and containing a different motif (UPRE-2). Using a combination of unbiased and directed microfluidic DNA binding assays, we established that Hac1 binds in two distinct modes: (i) to short (6-7bp) UPRE-2-like motifs and (ii) to significantly longer (11-13bp) extended UPRE-1-like motifs. Using a genetic screen, we demonstrate that a region of extended homology N-terminal to the basic DNA binding domain is required for this dual site recognition. These results establish Hac1 as the first bZIP transcription factor known to adopt more than one binding mode and unify previously conflicting and discrepant observations of Hac1 function into a cohesive model of UPR target gene activation. Our results also suggest that even structurally simple transcription factors can recognize multiple divergent target sites of very different lengths, potentially enriching their downstream target repertoire.

A “Twist box” Code of p53 Inactivation: Twist box:p53 Interaction Promotes p53 Degradation

Twist proteins have been shown to contribute to cancer development and progression by impinging on different regulatory pathways, but their mechanism of action is poorly defined. By investigating the role of Twist in sarcomas, we found that Twist1 acts as a mechanism alternative to TP53 mutation and MDM2 overexpression to inactivate p53 in mesenchymal tumors. We provide evidence that Twist1 binds p53 C terminus through the Twist box. This interaction hinders key posttranslational modifications of p53 and facilitates itsMDM2-mediated degradation. Our study suggests the existence of a Twist box code of p53 inactivation and provides the proof of principle that targeting the Twist box:p53 interaction might offer additional avenues for cancer treatment.

Abstract 63: Id4 acts as a tumor suppressor by inducing apoptosis and sensitizing prostate cancer cells to chemotherapeutic agent doxorubicin

Abstract PURPOSE:Id proteins (Id1, Id2, Id3, and Id4) function as dominant negative regulators of basic helix loop helix transcription factor family. They share the conserved HLH domain but lack the DNA binding basic domain. This domain configuration allows the Id proteins to dimerize with bHLH proteins. The Id-bHLH dimer therefore fails to bind the E box consensus sequence and activate transcription. In general Id1 and Id3 promote proliferation and inhibit differentiation and expression is generally high in many cancers. The function of Id4 appears to be unique as compared to Id1-Id3. Recent studies have demonstrated that Id4 is epigenetically silenced in many cancers including prostate cancer, suggesting that Id4 may in fact act as a tumor suppressor. The broad focus of this study is to investigate how Id4 acts as a tumor suppressor using prostate cancer cell line DU145, in which Id4 is epigenetically silenced. We report that at least two distinct mechanisms by which Id4 acts as a tumor suppressor is by inducing apoptosis and senescence in DU145 cells. Based on these observations, we hypothesize that Id4 will also sensitize prostate cancer cells to chemotherapeutic agents, such as doxorubicin. DESIGN METHODS: Apoptosis was quantitated using Annexin V staining in DU145 cells in which Id4 was ectopically expressed. We also used Mitocasp, which measures Caspase 3/7 activity and mitochondrial membrane potential for cytochrome c release. ηeta-galactosidase staining was used to confirm the senescent cells. Western blotting and other proteomics based approaches were used to determine the levels of apoptotic and senescence markers (Vimentin, Id1, MYC, MDM2, p16, and p21). Lastly, we treated prostate cancer cells with doxorubicin to investigate if Id4 promotes sensitivity to doxorubicin induced apoptosis. RESULTS Apoptosis assay indicated that over-expression of Id4 induced senescence and apoptosis in DU145 cells. At mRNA and protein levels, p21 and vimentin expression was increased. The expression of Id1, c-MYC, and MDM2 were decreased in DU145+Id4 cell line as compared to DU145. Our results also demonstrated that Id4 sensitizes DU145+Id4 cells to doxorubicin induced apoptosis that was Caspase 3/7 and cytochrome c dependent. CONCLUSIONS: Id4 may act as a tumor suppressor by inducing senescence and apoptosis in DU145 cells. Id4 not only induces apoptosis in prostate cancer cell line DU145, but also sensitizes these cells to doxorubicin. Id4 expression can thus be used as a clinical biomarker for prostate cancer progression and determining sensitivity to chemotherapeutic treatments. NIH/NCRR/RCMI grant #G12RR03062, NIH grant #R01CA128914 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 63. doi:1538-7445.AM2012-63

Abstract 4026: Id4 acts as a tumor suppressor by regulating c-Myc and MDM2 in prostate cancer

Abstract PURPOSE: Id proteins (Id1, Id2, Id3, and Id4) function as dominant negative regulators of basic helix loop helix transcription factor family. They share the conserved HLH domain but lack the DNA binding basic domain. This domain configuration allows the Id proteins to dimerize with bHLH proteins. The Id-bHLH dimer therefore fails to bind the E box consensus sequence and activate transcription. In general Id1 and Id3 promote proliferation and inhibit differentiation and thus their expression is generally high in many cancers. The function of Id4 appears to be unique as compared to Id1-Id3. Recent studies have demonstrated that Id4 is epigenetically silenced in many cancers suggesting that Id4 may in fact act as a tumor suppressor. The present study was carried out to determine the molecular mechanism by which Id4 acts as a tumor suppressor in prostate cancer. DESIGN METHODS: Initial studies were carried out on DU145 cells in which Id4 is epigenetically silenced. Over-expression of Id4 decreased proliferation and promoted epithelial like morphology in DU145 cells. A proteomics based approach suggested that MDM2 is a potential downstream target of Id4. The upstream regulators and downstream targets of MDM2 were then evaluated to understand the mechanism and functional significance of Id4-MDM2 cross talk. RESULTS: A combination of expression and immuno-localization studies demonstrated that c-Myc and p14ARF, the upstream regulators of MDM2 expression are down-regulated in the presence of Id4. Id4 also significantly up-regulated the expression and nuclear localization of CDKNIs p21and p27. Unexpectedly, the expression of p53 a known target of MDM2 was decreased both at the transcript and protein level. CONCLUSIONS: Although MDM2 was discovered as a potential downstream target, our pathway analysis suggests that c-Myc could also be a downstream target of Id4. The de-repression of CDKNI appears to be c-Myc dependent but p53 independent primarily because in DU145 cells, p53 is mutated. Alternatively, decreased MDM2 could promote p21 stability resulting in increased nuclear localization and expression. Taken together our data support the role of Id4 as a potential tumor suppressor that is unique and opposite to the oncogenic properties of other HLH members Id1, Id2 and Id3. Supported by: NIH/NCI R01CA128914 and NIH/NCRR/RCMI G12RR03062. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4026. doi:10.1158/1538-7445.AM2011-4026

Abstract LB-141: Expression and localization of Id3 in human prostate cancer

Abstract Purpose: The inhibitor of differentiation (Id) proteins are dominant negative basic helix-loop-helix family of transcription factors that lack the DNA-binding basic domain but have intact HLH domain. The four members of the Id protein family (Id1-Id4) are known to play a critical role in the coordinated regulation of gene expression during cell growth, differentiation and tumorigenesis. Although in vitro studies have demonstrated the role of Id1 in regulating prostate cancer (PCa) cell proliferation, apoptosis and androgen independence, its clinical significance in PCa remains controversial. On the other hand there is lack of evidence on the expression of Id2 and Id3 in PCa progression. Studies have demonstrated that silencing of Id3 is significantly more effective in blocking proliferation of PCa cells as compared to Id1 whereas Id2 silencing has been primarily shown to be involved in apoptotic pathway. Hence in this study we re-evaluated the role of Id1 in PCa and extended our study to show that Id3 protein expression has better clinical relevance compared to Id1 and Id2 in PCa. Experimental design: The expression of Id-1, -2 and -3 was determined by real time and Western blotting along with their sub-cellular localization by ICC in PCa cell lines LNCaP, DU145 and PC3. To assess their significance in prostate cancer progression, the protein expression was investigated in clinical prostate tumor samples on high density tissue microarray by IHC. Results: The expression of Id1 and Id3 mRNA was low in LNCaP and DU145 as compared to PC3 whereas the level of Id2 expression was similar between all the PCa cell lines. The protein expression data was consistent with the mRNA levels in the PCa cell lines. All three Id's were primarily cytoplasmic with some nuclear presence. The IHC data showed a significant increase in the expression of Id1 and Id3 in clinical prostate tumor tissue samples compared to normal prostate tissue whereas Id2 showed elevated levels but was not significant as compared to Id1 and Id3. The expression of Id3 protein correlated with the Gleason grading and stage of PCa. Conclusions: This is the first study demonstrating a correlation between increased expression of Id3 and prostate cancer progression. It also reconfirms and establishes the association of Id1with PCa. The results warrant further investigation into isoform specific effects of Id1and Id3 on prostate tumorigenesis. (Research Support: NIH/NCI RO1 CA128914 and NIH/NCRR/RCMI G12RR03062. The first two authors contributed equally to this work. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-141. doi:10.1158/1538-7445.AM2011-LB-141

Olig1 and ID4 interactions in living cells visualized by bimolecular fluorescence complementation technique

Olig1, a member of class B basic-helix-loop-helix (bHLH), plays key roles in early oligodendrocyte specification. Inhibitors of DNA binding (Id) is another sub-class of HLH proteins, act as dominant-negative regulators of bHLH proteins, which can form heterodimers with class A or B bHLH proteins, but lack the critical basic DNA binding domain. Id4 was recently found to interact with olig1 and inhibit oligodendrocyte differentiation. However, there still no direct evidence to reveal the spatial and temporal interaction of olig1 and ID4 in living cells. In this study, we performed bimolecular fluorescence complementation (BiFC) analysis to further characterize the distinct subcellular localization of olig1, ID4 and their dimer in living SW1116 cells. To examine the subcellular localization of olig1 and ID4 by themselves, the olig1-EGFP or ID4-DsRed2 fusion proteins were also expressed in SW1116 cells, respectively. As predicted, the olig1-EGFP fusion proteins were located in the nucleus, and ID4-DsRed2 fusion proteins were located in the cytoplasm. When olig1-EGFP and ID4-DsRed2 fusion proteins were co-expressed, the green and red signals were co-located in the cytoplasm. Using BiFC, the strong BiFC signals could be detected in pBiFC-olig1VN173 and pBiFC-ID4VC155 co-transfected cells and the fluorescence signal was located in the cytoplasm. These results collectively confirmed that olig1 and ID4 could interact and form dimer in living cells, and ID4 could block the transport of olig1 from cytoplasm to nucleus.

Inhibition of Id proteins by a peptide aptamer induces cell-cycle arrest and apoptosis in ovarian cancer cells

Background:Inhibitors of DNA-binding proteins (Id1-4), lacking the basic DNA-binding domain, function as dominant inhibitors of cell-cycle regulators. Overexpression of Id proteins promotes cancer cell proliferation and resistance against apoptosis. Level of Id protein expression, especially of Id1, correlates with poor differentiation, enhanced malignant potential and more aggressive clinical behaviour of ovarian tumours. Although overexpression of Ids has been found and shown to correlate with poor clinical outcome, their inhibition at protein level has never been studied.Methods:A peptide aptamer, Id1/3-PA7, targeting Id1 and Id3, was isolated from a randomised combinatorial expression library using yeast and mammalian two-hybrid systems. Id1/3-PA7 was fused, expressed and purified with a cell-penetrating protein transduction domain.Results:Intracellular-delivered Id1/3-PA7 colocalised to Id1 and Id3. It induced cell-cycle arrest and apoptosis in ovarian cancer cells ES-2 and PA-1. It activated the E-box promoter and increased the expression level of cyclin-dependent kinase inhibitor (CDKN2A) in a dose-dependent manner that is paralleled by the cleavage of poly-ADP ribose polymerase. These effects were counteracted by ectopically overexpressed Id1 and Id3.Conclusion:Id1/3-PA7 could represent an exogenous anti-tumour agent that can significantly trigger cell-cycle arrest and apoptosis in ovarian cancer.

FIAT, the factor‐inhibiting ATF4‐mediated transcription, also represses the transcriptional activity of the bZIP factor FRA‐1

FIAT is a leucine zipper protein whose name was coined for its interaction with ATF4 and subsequent blockage of ATF4-directed osteocalcin gene transcription. FIAT lacks a basic DNA-binding domain but contains three leucine zippers; it heterodimerizes with ATF4 to prohibit binding to DNA. FIAT could also potentially interact with additional basic domain-leucine zipper transcriptional regulators of osteoblast activity, such as FRA-1. We have found that FIAT inhibits transcriptional activation by a FRA-1/c-JUN heterodimer without affecting transcription mediated by a c-JUN homodimer. The repressor effect of FIAT on FRA-1-dependent transcription was measured using reporter constructs for the natural FRA-1 targets, Mmp-9 and Mgp. The FIAT-FRA-1 interaction is mediated through the second leucine zipper of FIAT. These data confirm an additional target of the FIAT transcriptional repressor activity and suggest that FIAT can both modulate early osteoblast activity by interacting with ATF4 and regulate later osteoblast function through inhibition of FRA-1.

PNA zipper as a dimerization tool: Development of a bZip mimic

The article describes the use of a PNA duplex (PNA zipper) as a tool to dimerize or bring in close proximity two polypeptides or protein domains. The amino acid sequence to be dimerized is covalently bound to complementary PNA sequences. Annealing of the PNA strands results in dimer formation. To test the ability of the "PNA-zipper" as a dimerization tool, we designed a GCN4 mimetic, where the leucine-zipper dimerization domain was replaced by the PNA zipper, whereas the basic DNA-binding domain was covalently attached to the PNA. The molecule was assembled by chemical ligation of the peptide corresponding to the DNA-binding domain of GCN4 modified with a succinyl thioester with two complementary PNAs harboring a cysteine residue. Electromobility-shift experiments show the ability of the PNA zipper-GCN4 to bind selected DNA duplexes. The PNA zipper-GCN4 binds both the TRE and CRE DNA sites, but it does not bind TRE and CRE mutants containing even a single base mutation, as the native GCN4. The ability to fold upon complexation with DNA was investigated by CD. A good correlation between the ability of the PNA zipper-GCN4 to fold into alpha helices and the ability to bind DNA was found.

Defective co‐activator recruitment in osteoclasts from microphthalmia‐oak ridge mutant mice

The three basic DNA-binding domain mutations of the microphthalmia-associated transcription factor (Mitf), Mitf(mi/mi), Mitf(or/or), and Mitf(wh/wh) affect osteoclast differentiation with variable penetrance while completely impairing melanocyte development. Mitf(or/or) mice exhibit osteopetrosis that improves with age and their osteoclasts form functional multinuclear osteoclasts, raising the question as to why the Mitf(or/or) mutation results in osteopetrosis. Here we show that Mitf(or/or) osteoclasts express normal levels of acid phosphatase 5 (Acp5) mRNA and significantly lower levels of Cathepsin K (Ctsk) mRNA during receptor activator of nuclear factor kappa B (NFkappaB) ligand (RANKL)-mediated differentiation. Studies using chromatin immunoprecipitation (ChIP) analysis indicate that low levels of Mitf(or/or) protein are recruited to the Ctsk promoter. However, enrichment of Mitf-transcriptional co-activators PU.1 and Brahma-related gene 1 (Brg1) are severely impaired at the Ctsk promoter of Mitf(or/or) osteoclast precursors, indicating that defective recruitment of co-activators by the mutant Mitf(or/or) results in impaired Ctsk expression in osteoclasts. Cathepsin K may thus represent a unique class of Mitf-regulated osteoclast-specific genes that are important for osteoclast function.

A complexTFAP2Aallele is associated with branchio‐oculo‐facial syndrome and inner ear malformation in a deaf child

We present a 4-year-old girl with congenital profound sensorineural deafness associated with inner ear malformation (incomplete partition type II, enlarged vestibule, and enlarged vestibular aqueduct). The proposita also had pseudocleft lips, skin defects, auricle abnormalities, and unilateral multicystic dysplastic kidney, leading to the diagnosis of branchio-oculo-facial (BOF) syndrome. Mutation analysis of the TFAP2A gene showed a de novo deletion of 18 and insertion of 6 nucleotides, resulting in deletion of amino acids LPGARR and insertion of RI between amino acids 276 and 281. Altered amino acids are located within the basic DNA binding and dimerization domains of TFAP2A. Previously reported amino acid substitutions in TFAP2A involved only DNA binding domain in four patients with BOF syndrome who were not reported to have profound sensorineural deafness. Our report implies that the localization of mutations in TFAP2A might be responsible with the phenotypic findings in BOF syndrome.

High mobility group B2 is secreted by myeloid cells and has mitogenic and chemoattractant activities similar to high mobility group B1

High mobility group B box (HMGB) proteins are a family of chromatin proteins made up of two basic DNA binding domains, HMG box A and B, and a C-terminal acidic tail. HMGB have a highly conserved sequence, but different expression pattern: HMGB1 is almost ubiquitous, whereas the others are highly expressed in only a few tissues in adults. We previously demonstrated that HMGB1 is released by necrotic cells and has chemoattractant activity for inflammatory and stem cells, via binding to receptor for advanced glycation endproducts (RAGE). HMGB1 can be actively secreted by inflammatory cells. Here, we report that also HMGB2 can be secreted by THP-1 cells, and promotes proliferation and migration of endothelial cells. These functions of HMGB2 are exerted via engagement of RAGE, whose blockade completely abrogates cell responses. Since extracellular HMGB2 has been detected in the blood and other biological fluids, it might be necessary to target HMGB2 at the same time as HMGB1 for therapeutical efficacy.

An Id-like molecule, HHM, is a synexpression group-restricted regulator of TGF-β signalling

Transforming growth factor (TGF)-β induces various cellular responses principally through Smad-dependent transcriptional regulation. Activated Smad complexes cooperate with transcription factors in regulating a group of target genes. The target genes controlled by the same Smad-cofactor complexes are denoted a synexpression group. We found that an Id-like helix-loop-helix protein, human homologue of Maid (HHM), is a synexpression group-restricted regulator of TGF-β signalling. HHM suppressed TGF-β-induced growth inhibition and cell migration but not epithelial–mesenchymal transition. In addition, HHM inhibited TGF-β-induced expression of plasminogen activator inhibitor-type 1 (PAI-1), PDGF-B, and p21WAF, but not Snail. We identified a basic-helix-loop-helix protein, Olig1, as one of the Smad-binding transcription factors affected by HHM. Olig1 interacted with Smad2/3 in response to TGF-β stimulation, and was involved in transcriptional activation of PAI-1 and PDGF-B. HHM, but not Id proteins, inhibited TGF-β signalling-dependent association of Olig1 with Smad2/3 through physical interaction with Olig1. HHM thus appears to regulate a subset of TGF-β target genes including the Olig1-Smad synexpression group. HHM is the first example of a cellular response-selective regulator of TGF-β signalling with clearly determined mechanisms.

A male‐specific effect of dominant‐negative Fos

The transcription factor Fos contains a basic DNA binding domain combined with a leucine zipper (bZip). Expression of a truncated form of Fos in Drosophila that contains only the bZip region (Fos bZip) elicits phenotypes resembling fos mutations. These effects presumably derive from competition between wild-type and truncated forms for dimerization partners, with the truncation acting in a dominant-negative manner. We found that expression of Fos bZip elicits male-specific phenotypes. Moreover, genetic interactions occur between Fos bZip and mutations in loci encoding the X chromosome dosage compensation complex. Fos bZip effects are correlated with aberrant male X chromosome structure and depressed signaling through the X-linked Notch locus. Unexpectedly, the male-specific effects are not reproduced with Fos RNAi, suggesting that Fos bZip can be neomorphic in nature. These results provide insight into how mutations in bZip proteins can exhibit gain of function activity.

GATA-4 modulates C/EBP-dependent transcriptional activation of acute phase protein genes

C/EBP transcription factors are involved in the regulation of the intestinal epithelial cell response to inflammatory stimuli. GATA transcription factors modulate C/EBP-dependent transcriptional activation in various cell types. We thus determined whether GATA-4 whose expression is restricted to epithelial cells, modulate C/EBP transcriptional activity and C/EBP-dependent acute phase protein expression in intestinal epithelial cells. Interaction between C/EBPδ and GATA-4 required both C/EBPδ leucine zipper and basic DNA-binding domains, and the GATA-4 C-terminal region. C/EBP isoforms and GATA-4 synergistically activated the α-acid glycoprotein gene while GATA-4 repressed thiostatin and haptoglobin C/EBP-dependent transactivation. In GATA-4 expressing intestinal epithelial cells, GATA-4 led to decreased C/EBPβ and C/EBPδ protein levels, and decreased thiostatin expression in response to IL-1β and dexamethasone. This correlated with decreased C/EBPδ recruitment to the thiostatin promoter, as assessed by chromatin immunoprecipitation assays. In contrast, increased AGP expression in response to dexamethasone correlated with increased basal histone H4 acetylation. Thus, GATA-4 may be involved in the establishment of specific intestinal epithelial cell C/EBP-dependent and C/EBP-independent transcriptional responses.

Characterization of SBZ1, a soybean bZIP protein that binds to the chalcone synthase gene promoter

Several plant basic leucine zipper (bZIP) proteins have been shown to play a role in chalcone synthase (CHS) gene expression, and some are regulated by phosphorylation/dephosphorylation. We isolated SBZ1 (Soybean bZIP protein 1) and showed that the recombinant protein binds in vitro to the 5� region of soybean CHS1, at a sequence that confers the elicitor-inducible expression of CHS genes. The deduced amino acid sequence of SBZ1 has features characteristic of bZIP transcription factors, including a highly basic putative DNA-binding domain containing a nuclear localization sequence, as well as four domains designated D1-D4 that are highly conserved among the subfamily of bZIP factors, which includes tobacco BZI-1 and parsley CPRF2. The presence of these regions indicates that SBZ1 is a CPRF2-related bZIP transcription factor. The protein kinase inhibitor K252a blocks CHS induction in elicited soybean cells, suggesting that protein phosphorylation is involved in induction of the CHS signal pathway. Phosphorylation assays indicated that SBZ1 is phosphorylated in vitro in a soybean cell extract, and that this phosphorylation depends on Ca 2� . Furthermore, recombinant soybean CDPK and the a subunit of CKII phosphorylate SBZ1 in vitro. However, unlike other related bZIP proteins, phosphorylation had no effect on either the DNA-binding activity of SBZ1. Therefore, we conclude that SBZ1 is regulated by phosphorylation, but in a different manner than are related bZIP factors.

Identification of Additional Dimerization Partners of FIAT, the Factor Inhibiting ATF4‐Mediated Transcription

FIAT is a leucine zipper protein whose name was coined for its interaction with ATF4 and the subsequent blockage of ATF4-directed osteocalcin gene transcription. FIAT is a nuclear protein that lacks a basic DNA-binding domain but contains three identifiable leucine zipper domains. FIAT heterodimerizes with ATF4 through one of these zippers and thereby prohibits ATF4 from binding to its cognate DNA sequence. We tested whether FIAT also interacts with additional basic domain-leucine zipper transcriptional regulators of osteoblast activity, such as the Fos family member Fra-1 or one of its dimerization partners, c-Jun. Transient transfection assays in osteoblastic MC3T3-E1 cells with the heterologous AP-1-tk-luciferase reporter revealed that FIAT does not affect c-Jun-mediated transcription, even in the presence of the c-Jun coactivator alphaNAC. However, FIAT inhibited transcriptional activation by a c-Jun~Fra-1 heterodimer. Thus FIAT specifically inhibits Fra-1 transcriptional activity. These data identify a second target of the FIAT transcriptional repressor activity and suggest that FIAT can modulate early osteoblast activity by interacting with ATF4, as well as regulate later osteoblast function through inhibition of Fra-1.

Heterodimerization with Jun Family Members Regulates c-Fos Nucleocytoplasmic Traffic

c-Fos proto-oncoprotein forms AP-1 transcription complexes with heterodimerization partners such as c-Jun, JunB, and JunD. Thereby, it controls essential cell functions and exerts tumorigenic actions. The dynamics of c-Fos intracellular distribution is poorly understood. Hence, we have combined genetic, cell biology, and microscopic approaches to investigate this issue. In addition to a previously characterized basic nuclear localization signal (NLS) located within the central DNA-binding domain, we identified a second NLS within the c-Fos N-terminal region. This NLS is non-classic and its activity depends on transportin 1 in vivo. Under conditions of prominent nuclear localization, c-Fos can undergo nucleocytoplasmic shuttling through an active Crm-1 exportin-independent mechanism. Dimerization with the Jun proteins inhibits c-Fos nuclear exit. The strongest effect is observed with c-Jun probably in accordance with the relative stabilities of the different c-Fos:Jun dimers. Retrotransport inhibition is not caused by binding of dimers to DNA and, therefore, is not induced by indirect effects linked to activation of c-Fos target genes. Monomeric, but not dimeric, Jun proteins also shuttle actively. Thus, our work unveils a novel regulation operating on AP-1 by demonstrating that dimerization is crucial, not only for active transcription complex formation, but also for keeping them in the compartment where they exert their transcriptional function.

Effect of phosphorylation and S–S bond-induced dimerization on DNA binding and transcriptional activation by C/EBPβ

CCAAT enhancer binding protein beta (C/EBPbeta) plays an essential role in the cascade that triggers adipocyte differentiation. C/EBPbeta activates transcription of C/EBPalpha and peroxisome proliferator-activated receptor-gamma, transcriptional activators of genes that give rise to the adipocyte phenotype. Sequential phosphorylation of C/EBPbeta/liver activating protein (LAP) on Thr(188) by MAPK and on Ser(184) or Thr(179) by glycogen synthase kinase beta (GSK3beta) is required for acquisition of DNA binding activity and transcriptional activation. To investigate how phosphorylation and dimerization of C/EBPbeta/LAP alter these activities, wild-type (Wt) and mutant rC/EBPbetas were prepared and purified to assess DNA binding and transcription in cell-free systems. rC/EBPbeta/LAP, phosphorylated by MAPK and GSK3beta in vitro, produced a >100-fold increase in DNA binding activity. Mutation of the phosphorylation to Glu increased DNA binding activity. Using a cell-free transcription system with nuclear extract from 3T3-L1 preadipocytes and rC/EBPbeta/LAP, only doubly phosphorylated rC/EBPbeta/LAP (by MAPK and GSK3beta) activated transcription driven by Wt C/EBPalpha, 422/aP2, and SCD1 promoters. Oxidation-induced dimerization of doubly phosphorylated Wt rC/EBPbeta/LAP increased DNA binding, whereas unphosphorylated Wt rC/EBPbeta/LAP lacked DNA binding activity. Mutation of the C-terminal Cys(296) adjacent to the leucine zipper and Cys(143) just upstream of the DNA binding domain eliminated phosphorylation-, oxidation-, and dimerization-dependent DNA binding activity, whereas mutation of Cys(201) within the basic DNA binding domain had little effect on DNA binding. These findings indicate that dual phosphorylation of C/EBPbeta/LAP caused a conformational change that facilitates S-S bond formation and dimerization, rendering the basic region accessible to the C/EBP regulatory element.