DOCSLIB.ORG

A Synthetic Leucine Zipper-Based Dimerization System for Combining Multiple Promoter Specificities

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
A Synthetic Leucine Zipper-Based Dimerization System for Combining Multiple Promoter Specificities Gene Therapy (2001) 8, 725–729 2001 Nature Publishing Group All rights reserved 0969-7128/01 $15.00 www.nature.com/gt BRIEF COMMUNICATION A synthetic leucine zipper-based dimerization system for combining multiple promoter specificities VJe´roˆme and R Mu¨ ller Institute of Molecular Biology and Tumor Research (IMT), Philipps University, Emil-Mannkopff-Strasse 2, 35033 Marburg, Germany One of the biggest challenges facing gene therapy is the with different selectivity. A crucial determinant of the DCTF development of vectors that direct the activity of therapeutic system is the heterodimerization interface that should pro- genes specifically to the sites of disease. To achieve this vide for a high affinity interaction without interference by goal, the restriction of transgene transcription via synthetic endogenous proteins. Here, we describe such a dimerization promoters that are endowed with multiple specificities rep- system based on engineered Fos and Jun leucine zippers. resents a particularly promising strategy. Towards this end, We show the usefulness of this system for the combination we have developed a generally applicable strategy (DCTF of cell type-specific and cell cycle-regulated transcription system) where a synthetic promoter is driven by an artificial and demonstrate its functionality in an in vivo setting. Gene heterodimeric transcription factor whose DNA-binding and Therapy (2001) 8, 725–729. transactivating subunits are expressed from two promoters Keywords: cell cycle regulation; DCTF system; leucine zipper; tissue-specific promoter; transcriptional targeting The dual specificity chimeric transcription factor (DCTF) acids in the Jun LZ and the negatively charged residues system has been established for the targeting of prolifer- in the Fos counterpart leading to strong self-repulsion of ating melanoma cells.1 Two subunits harboring the DNA- both proteins.11,12 Both, Jun and Fos family members are binding domain of the yeast transcription factor GAL4 or expressed to variable extents in all cells. To make the the transactivation domain of herpes simplex virus VP16 Jun/Fos LZs suitable for the DCTF system it was there- were expressed from the tyrosinase and cyclin A pro- fore necessary to prevent interactions between the chim- moter, respectively. Heterodimerization was achieved eric transcription factors of the DCTF system and the through the interaction surfaces of the CD4 and the Lck endogenous Jun and Fos proteins. We hypothesized that protein kinase. Although proof of principle could be this could be possible by swapping between the two LZs demonstrated by this system, it was clear that the overall several of the oppositely charged amino acids involved transcriptional activity had to be improved for a potential in heterodimerization. application in cancer gene therapy.1 Since the CD4–Lck Based on the resolved crystal structure12 we introduced interaction is weak and potential interactions with three acidic amino acids from c-Fos into the c-Jun LZ endogenous molecules might result in the sequestration (mJun) and three basic amino acids from c-Jun into the c- of the chimeric transcription factor used in the DCTF sys- Fos LZ (mFos), as depicted in Figure 1a, thereby creating tem, we reasoned that the replacement of the hetero- mixed-charged structures. To analyze the oligomeriz- dimerization interface would lead to a substantial ation properties of transcription factors carrying such improvement. engineered LZs in the context of the herpes simplex virus The leucine zippers (LZs) represent particularly strong VP16 transactivation domain13 (VP16-mJun) or the yeast interaction domains commonly frequent in transcription GAL4 DNA-binding domain14 (GAL4-mFos) we investi- factors2 and have been shown to be functional in a heter- gated the association of in vitro translated proteins by ologous context.3–5 Dimerization through LZs is mediated radioimmunoprecipitation (RIP). 35S-methionine-labeled by regularly spaced leucines (heptad repeats) in parallel VP16-Jun proteins (VP16-mJun, VP16-wtJun with a wild- ␣-helices through hydrophobic interactions, while the type Jun LZ, and VP16-⌬Jun lacking a dimerization choice of the dimerization partner is determined by other domain; Figure 1b) were mixed with unlabeled GAL4- amino acids, mainly charged residues forming salt mFos proteins and immunoprecipitated with an antibody bridges.6–11 This is exemplified by the Fos and Jun pro- directed against GAL4. Figure 1c shows that the proteins teins which preferentially form heterodimers and only containing engineered LZs formed heterodimers, but that have a very limited ability to homodimerize. This speci- complex formation between VP16-wtJun and GAL4-mFos ficity is brought about by the positively charged amino or VP16-⌬Jun and GAL4-wtFos was prevented. Likewise, VP16-mJun did not interact with GAL4-wtFos in an analogous experiment (not shown). Correspondence: R Mu¨ller These observations suggest that the mJun and mFos LZ Received 8 December 2000; accepted 30 January 2001 domains could be suitable for mediating specific intra- In vivo transcriptional targeting VJe´roˆme and R Mu¨ller 726 Figure 1 (a) Structure of the chimeric transcription factors. Schematic representation of the GAL4-fos and VP16-jun fusion proteins. Electrostatic interactions between Fos and Jun LZ are shown by arrows.12 The amino acids swapped between the leucine zippers are indicated by black arrows. (b) SDS-PAGE of 35S-methionine-labeled in vitro translated Fos and Jun LZ fusion proteins. (c) Complex formation between VP16-mJun and GAL4-mFos LZ fusion proteins. Wild-type VP16-wtJun and mutated VP16-mJun were translated in the presence of 35S-methionine, complexed with unlabeled mutated GAL4-mFos and immunoprecipitated with a GAL4-specific antibody. The precipitated proteins were analyzed by SDS-PAGE followed by autoradiography. As a control an empty pGEM4 vector was translated in the presence of 35S-methionine and complexed with unlabeled GAL4-mFos; as a second control the labeled VP16-⌬Jun was incubated with unlabelled GAL4-mFos. For in vitro transcription/translation assay, the chimeric transcrip- tion factors were placed under the control of the T7 promoter. They were excised from the pGAL-wtFos, pGAL-mFos, pVP⌬Jun, pVP-wtJun, and pVP- mJun constructs (see legend to Figure 2) as HindIII/XbaI fragments and cloned in the same sites into the pGEM4 vector (Promega, Mannheim, Germany) to yield GAL4-wtFos, GAL4-mFos, VP16-⌬Jun, VP16-wtJun andVP16-mJun. In vitro translation was performed with the TNT T7 quick kit (Promega) according to the manufacturer protocol. Briefly, 1 ␮g of DNA template was mixed with 40 ␮l of TNT T7 master mix and 2 ␮l 35S-methionine (1000 Ci/mmol, 10 mCi/ml; Amersham, Freiburg, Germany) and incubated at 30°C for 90 min. To assess the quality of the translated products, 5 ␮l of the translation reaction was analyzed on SDS-PAGE 12.5%. To avoid nonspecific interactions of the rabbit reticulocyte lysate with the rabbit anti- serum used later on to perform the immunoprecipitation, the in vitro translated products were pre-cleared. Twenty ␮l of proteins were incubated with 5 ␮l of normal rabbit serum (Vector Laboratories, Gru¨nberg, Germany) at 4°C for 1 h before adding 20 ␮l of protein-A sepharose (Pharmacia, Freiburg, Germany) and incubating for 30 min. After centrifugation at 4°C for 5 min at 13 000 g the pre-cleared supernatant was ready for the in vitro association assay. For in vitro protein-binding assays, equal volumes of pre-cleared supernatant (5 ␮l each) containing 35S-methionine-labeled VP16-⌬Jun, VP16- wtJun or VP16-mJun proteins in 50 mm Tris pH 7.2 were combined and incubated at 30°C for 90 min as described.20 Protein complexes were mixed with 2 ␮l rabbit polyclonal IgG GAL4 (DBD) (Santa Cruz, Heidelberg, Germany). The immune complexes were collected by the addition of sepharose- A, washed in RIPA buffer (10 mm Tris-Cl (pH 7.5), 150 mm NaCl, 1% NP40, 1% deoxycholate, 0.25% SDS 1 mm DTT) and prepared for SDS- PAGE. The immunoprecipitated proteins were separated on 12% gels followed by autoradiography. cellular protein–protein interactions. We therefore ana- wild-type LZs, nor the combination of one wild-type and lyzed in the next step the functionality of the VP16-mJun one mutant LZ, was able to enable heterodimerization. and GAL4-mFos proteins in a cellular environment using The former is presumably due to the sequestration of the the DCTF system. For this purpose, we designed recombinant factors by endogenous Fos and Jun proteins, expression constructs where VP16-wtJun, VP16-mJun the latter due to structural reasons, as supported by the and VP16-⌬Jun were under the control of the cyclin A in vitro association data shown in Figure 1b. promoter, and GAL4-wtFos and GAL4-mFos were driven We next attempted to optimize the transcriptional by a tyrosinase promoter.1 An SV40 promoter-driven activity achievable by the Jun/Fos DCTF system by clon- luciferase gene with 10 GAL4 binding sites (10xgal4RE- ing both expression cassettes into one plasmid in a head- SVpGL3) served as reporter construct. Cotransfections of to-tail configuration, rationing that this would lead to melanoma cells (MeWo) in various combinations clearly equimolar concentrations of both subunits in the trans- showed that only the coexpression of the VP16-mJun fected cell. As shown in Figure 2b, this strategy lead to (pVP-mJun) and GAL4-mFos (pGAL-mFos) proteins an ෂ24-fold increase (36:1.5) in luciferase activity in mela- gave rise to any significant activity (ෂ1.5-fold SV40 pro- noma cells (36-fold SV40 promoter). We also compared moter; Figure 2a). Cotransfection of GAL4-mFos with the activity of the Jun/Fos DCTF system with that of the either VP16-wtJun (pVP-wtJun) or VP16-⌬Jun (pVP- previously described CD4-LCK dimerization system.1 ⌬Jun) gave only marginal signals (in the range of the pro- The transcriptional activity of the Jun/Fos ‘one-plasmid’ moterless luciferase plasmid pGL3), as did the coex- system was found to be ෂ7-fold greater relative to the pression of GAL4-wtFos (pGAL-wtFos) with any of the corresponding CD4-LCK system (36:5.5).
Load more

Recommended publications
  • Amplitude Modulation of Androgen Signaling by C-MYC
    Downloaded from genesdev.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press Amplitude modulation of androgen signaling by c-MYC Min Ni,1,2 Yiwen Chen,3,4 Teng Fei,1,2 Dan Li,1,2 Elgene Lim,1,2 X. Shirley Liu,3,4,5 and Myles Brown1,2,5 1Division of Molecular and Cellular Oncology, Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA; 2Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02215, USA; 3Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA; 4Harvard School of Public Health, Boston, Massachusetts 02215, USA Androgen-stimulated growth of the molecular apocrine breast cancer subtype is mediated by an androgen receptor (AR)-regulated transcriptional program. However, the molecular details of this AR-centered regulatory network and the roles of other transcription factors that cooperate with AR in the network remain elusive. Here we report a positive feed-forward loop that enhances breast cancer growth involving AR, AR coregulators, and downstream target genes. In the absence of an androgen signal, TCF7L2 interacts with FOXA1 at AR-binding sites and represses the basal expression of AR target genes, including MYC. Direct AR regulation of MYC cooperates with AR-mediated activation of HER2/HER3 signaling. HER2/HER3 signaling increases the transcriptional activity of MYC through phosphorylation of MAD1, leading to increased levels of MYC/MAX heterodimers. MYC in turn reinforces the transcriptional activation of androgen-responsive genes. These results reveal a novel regulatory network in molecular apocrine breast cancers regulated by androgen and AR in which MYC plays a central role as both a key target and a cooperating transcription factor to drive oncogenic growth.
    [Show full text]
  • Dimerization Specificity of Myogenic Helix-Loop-Helix DNA-Binding Factors Directed by Nonconserved Hydrophilic Residues
    Downloaded from genesdev.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press Dimerization specificity of myogenic helix-loop-helix DNA-binding factors directed by nonconserved hydrophilic residues Masaki Shirakata, Fred K. Friedman, 1 Qin Wei, and Bruce M. Paterson Laboratory of Biochemistry, ~Laboratory of Molecular Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 USA The myogenic regulatory factor MyoD dimerizes with other positive and negative regulatory factors through a conserved region called the helix-loop-helix (HLH) domain. Using a non-DNA-binding MyoD mutant with a normal HLH domain as a dimerization competitor in gel mobility shift assays in conjunction with various MyoD HLH mutants, nonhydrophobic amino acids were identified in the HLH domain that contribute to dimerization specificity with El2. The assay detected subtle differences in dimerization activity among the mutant MyoD proteins that correlated with their ability to activate transcription in vivo, but this correlation was not apparent in the absence of competitor. The identification of such nonhydrophobic residues enabled us to predict the differences in dimerization affinity among the four vertebrate myogenic factors with El2. The experiments confirmed the prediction. Furthermore, a high-affinity homodimerizing analog of MyoD was designed by a single substitution at one of these residue positions. These experimental results were strengthened when they were analyzed in terms of the crystal structure for the Max bHLHZip domain homodimer. This analysis has allowed us to identify those residues that form charged residue pairs between the two HLH domains of MyoD and El2 and determine the dimerization specificity of the bHLH proteins.
    [Show full text]
  • KLF6 Depletion Promotes NF-Κb Signaling in Glioblastoma
    OPEN Oncogene (2017) 36, 3562–3575 www.nature.com/onc ORIGINAL ARTICLE KLF6 depletion promotes NF-κB signaling in glioblastoma AP Masilamani1,2, R Ferrarese1,2, E Kling1,2, NK Thudi3, H Kim4, DM Scholtens5, F Dai1,2, M Hadler1,2, T Unterkircher1,2, L Platania1,2, A Weyerbrock1,2, M Prinz6,7, GY Gillespie8, GR Harsh IV9, M Bredel3,10 and MS Carro1,2,10 Dysregulation of the NF-κB transcription factor occurs in many cancer types. Krüppel-like family of transcription factors (KLFs) regulate the expression of genes involved in cell proliferation, differentiation and survival. Here, we report a new mechanism of NF- κB activation in glioblastoma through depletion of the KLF6 tumor suppressor. We show that KLF6 transactivates multiple genes negatively controlling the NF-κB pathway and consequently reduces NF-κB nuclear localization and downregulates NF-κB targets. Reconstitution of KLF6 attenuates their malignant phenotype and induces neural-like differentiation and senescence, consistent with NF-κB pathway inhibition. KLF6 is heterozygously deleted in 74.5% of the analyzed glioblastomas and predicts unfavorable patient prognosis suggesting that haploinsufficiency is a clinically relevant means of evading KLF6-dependent regulation of NF-κB. Together, our study identifies a new mechanism by which KLF6 regulates NF-κB signaling, and how this mechanism is circumvented in glioblastoma through KLF6 loss. Oncogene (2017) 36, 3562–3575; doi:10.1038/onc.2016.507; published online 6 February 2017 INTRODUCTION coding region have been controversial.16,18–22 KLF6 has been The NF-κB transcription factor family is oncogenic through proposed to perform its tumor suppression function by promoting suppression of programmed cell death, and promotion of tumor G1 cell cycle arrest mainly through cyclin-dependent kinase 15 growth and invasion.1 In tumors, NF-κB can be activated by inhibitor 1A (CDKN1A) promoter transactivation.
    [Show full text]
  • KLF6-SV1 Overexpression Accelerates Human and Mouse Prostate Cancer Progression and Metastasis
    KLF6-SV1 overexpression accelerates human and mouse prostate cancer progression and metastasis Goutham Narla, … , Mark A. Rubin, John A. Martignetti J Clin Invest. 2008;118(8):2711-2721. https://doi.org/10.1172/JCI34780. Research Article Oncology Metastatic prostate cancer (PCa) is one of the leading causes of death from cancer in men. The molecular mechanisms underlying the transition from localized tumor to hormone-refractory metastatic PCa remain largely unknown, and their identification is key for predicting prognosis and targeted therapy. Here we demonstrated that increased expression of a splice variant of the Kruppel-like factor 6 (KLF6) tumor suppressor gene, known as KLF6-SV1, in tumors from men after prostatectomy predicted markedly poorer survival and disease recurrence profiles. Analysis of tumor samples revealed that KLF6-SV1 levels were specifically upregulated in hormone-refractory metastatic PCa. In 2 complementary mouse models of metastatic PCa, KLF6-SV1–overexpressing PCa cells were shown by in vivo and ex vivo bioluminescent imaging to metastasize more rapidly and to disseminate to lymph nodes, bone, and brain more often. Interestingly, while KLF6-SV1 overexpression increased metastasis, it did not affect localized tumor growth. KLF6-SV1 inhibition using RNAi induced spontaneous apoptosis in cultured PCa cell lines and suppressed tumor growth in mice. Together, these findings demonstrate that KLF6-SV1 expression levels in PCa tumors at the time of diagnosis can predict the metastatic behavior of the tumor; thus, KLF-SV1 may represent a novel therapeutic target. Find the latest version: https://jci.me/34780/pdf Research article KLF6-SV1 overexpression accelerates human and mouse prostate cancer progression and metastasis Goutham Narla,1,2 Analisa DiFeo,1 Yolanda Fernandez,1 Saravana Dhanasekaran,3 Fei Huang,1 Jaya Sangodkar,1,2 Eldad Hod,2 Devin Leake,4 Scott L.
    [Show full text]
  • Leucine Zippers
    Leucine Zippers Leucine Zippers Advanced article Toshio Hakoshima, Nara Institute of Science and Technology, Nara, Japan Article contents Introduction The leucine zipper (ZIP) motif consists of a periodic repetition of a leucine residue at every Structural Basis of ZIP seventh position and forms an a-helical conformation, which facilitates dimerization and in Occurrence of ZIP and Coiled-coil Motifs some cases higher oligomerization of proteins. In many eukaryotic gene regulatory proteins, Dimerization Specificity of ZIP the ZIP motif is flanked at its N-terminus by a basic region containing characteristic residues DNA-binding Specificity of bZIP that facilitate DNA binding. doi: 10.1038/npg.els.0005049 Introduction protein modules for protein–protein interactions. Knowing the structure and function of these motifs A structure referred to as the leucine zipper or enables us to understand the molecular recognition simply as ZIP has been proposed to explain how a system in several biological processes. class of eukaryotic gene regulatory proteins works (Landschulz et al., 1988). A segment of the mammalian CCAAT/enhancer binding protein (C/EBP) of 30 Structural Basis of ZIP amino acids shares notable sequence similarity with a segment of the cellular Myc transforming protein. The The a helix is a secondary structure element that segments have been found to contain a periodic occurs frequently in proteins. Alpha helices are repetition of a leucine residue at every seventh stabilized in proteins by being packed into the position. A periodic array of at least four leucines hydrophobic core of a protein through hydrophobic has also been noted in the sequences of the Fos and side chains.
    [Show full text]
  • The Function and Evolution of C2H2 Zinc Finger Proteins and Transposons
    The function and evolution of C2H2 zinc finger proteins and transposons by Laura Francesca Campitelli A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Molecular Genetics University of Toronto © Copyright by Laura Francesca Campitelli 2020 The function and evolution of C2H2 zinc finger proteins and transposons Laura Francesca Campitelli Doctor of Philosophy Department of Molecular Genetics University of Toronto 2020 Abstract Transcription factors (TFs) confer specificity to transcriptional regulation by binding specific DNA sequences and ultimately affecting the ability of RNA polymerase to transcribe a locus. The C2H2 zinc finger proteins (C2H2 ZFPs) are a TF class with the unique ability to diversify their DNA-binding specificities in a short evolutionary time. C2H2 ZFPs comprise the largest class of TFs in Mammalian genomes, including nearly half of all Human TFs (747/1,639). Positive selection on the DNA-binding specificities of C2H2 ZFPs is explained by an evolutionary arms race with endogenous retroelements (EREs; copy-and-paste transposable elements), where the C2H2 ZFPs containing a KRAB repressor domain (KZFPs; 344/747 Human C2H2 ZFPs) are thought to diversify to bind new EREs and repress deleterious transposition events. However, evidence of the gain and loss of KZFP binding sites on the ERE sequence is sparse due to poor resolution of ERE sequence evolution, despite the recent publication of binding preferences for 242/344 Human KZFPs. The goal of my doctoral work has been to characterize the Human C2H2 ZFPs, with specific interest in their evolutionary history, functional diversity, and coevolution with LINE EREs.
    [Show full text]
  • Tgfβ-Regulated Gene Expression by Smads and Sp1/KLF-Like Transcription Factors in Cancer VOLKER ELLENRIEDER
    ANTICANCER RESEARCH 28 : 1531-1540 (2008) Review TGFβ-regulated Gene Expression by Smads and Sp1/KLF-like Transcription Factors in Cancer VOLKER ELLENRIEDER Signal Transduction Laboratory, Internal Medicine, Department of Gastroenterology and Endocrinology, University of Marburg, Marburg, Germany Abstract. Transforming growth factor beta (TGF β) controls complex induces the canonical Smad signaling molecules which vital cellular functions through its ability to regulate gene then translocate into the nucleus to regulate transcription (2). The expression. TGFβ binding to its transmembrane receptor cellular response to TGF β can be extremely variable depending kinases initiates distinct intracellular signalling cascades on the cell type and the activation status of a cell at a given time. including the Smad signalling and transcription factors and also For instance, TGF β induces growth arrest and apoptosis in Smad-independent pathways. In normal epithelial cells, TGF β healthy epithelial cells, whereas it can also promote tumor stimulation induces a cytostatic program which includes the progression through stimulation of cell proliferation and the transcriptional repression of the c-Myc oncogene and the later induction of an epithelial-to-mesenchymal transition of tumor induction of the cell cycle inhibitors p15 INK4b and p21 Cip1 . cells (1, 3). In the last decade it has become clear that both the During carcinogenesis, however, many tumor cells lose their tumor suppressing and the tumor promoting functions of TGF β ability to respond to TGF β with growth inhibition, and instead, are primarily regulated on the level of gene expression through activate genes involved in cell proliferation, invasion and Smad-dependent and -independent mechanisms (1, 2, 4).
    [Show full text]
  • Mechanism of Nucleosome Targeting by Pioneer Transcription Factors
    University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2019 Mechanism Of Nucleosome Targeting By Pioneer Transcription Factors Meilin Mary Fernandez Garcia University of Pennsylvania Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biochemistry Commons, and the Biophysics Commons Recommended Citation Fernandez Garcia, Meilin Mary, "Mechanism Of Nucleosome Targeting By Pioneer Transcription Factors" (2019). Publicly Accessible Penn Dissertations. 3624. https://repository.upenn.edu/edissertations/3624 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/3624 For more information, please contact [email protected]. Mechanism Of Nucleosome Targeting By Pioneer Transcription Factors Abstract Transcription factors (TFs) forage the genome to instruct cell plasticity, identity, and differentiation. These developmental processes are elicited through TF engagement with chromatin. Yet, how and which TFs can engage with chromatin and thus, nucleosomes, remains largely unexplored. Pioneer TFs are TF that display a high affinity for nucleosomes. Extensive genetic and biochemical studies on the pioneer TF FOXA, a driver of fibroblast to hepatocyte reprogramming, revealed its nucleosome binding ability and chromatin targeting lead to chromatin accessibility and subsequent cooperative binding of TFs. Similarly, a number of reprogramming TFs have been suggested to have pioneering activity due to their ability to target compact chromatin and increase accessibility and enhancer formation in vivo. But whether these factors directly interact with nucleosomes remains to be assessed. Here we test the nucleosome binding ability of the cell reprogramming TFs, Oct4, Sox2, Klf4 and cMyc, that are required for the generation of induced pluripotent stem cells. In addition, we also test neuronal and macrophage reprogramming TFs.
    [Show full text]
  • Expression Is a New Potential Biomarker for Adverse
    www.nature.com/scientificreports OPEN Decreased MYC‑associated factor X (MAX) expression is a new potential biomarker for adverse prognosis in anaplastic large cell lymphoma Takahisa Yamashita1, Morihiro Higashi1, Shuji Momose1, Akiko Adachi2, Toshiki Watanabe3, Yuka Tanaka4, Michihide Tokuhira4, Masahiro Kizaki4 & Jun‑ichi Tamaru1* MYC-associated factor X (MAX) is a protein in the basic helix‑loop‑helix leucine zipper family, which is ubiquitously and constitutively expressed in various normal tissues and tumors. MAX protein mediates various cellular functions such as proliferation, diferentiation, and apoptosis through the MYC-MAX protein complex. Recently, it has been reported that MYC regulates the proliferation of anaplastic large cell lymphoma. However, the expression and function of MAX in anaplastic large cell lymphoma remain to be elucidated. We herein investigated MAX expression in anaplastic large cell lymphoma (ALCL) and peripheral T-cell lymphoma, not otherwise specifed (PTCL-NOS) and found 11 of 37 patients (30%) with ALCL lacked MAX expression, whereas 15 of 15 patients (100%) with PTCL-NOS expressed MAX protein. ALCL patients lacking MAX expression had a signifcantly inferior prognosis compared with patients having MAX expression. Moreover, patients without MAX expression signifcantly had histological non-common variants, which were mainly detected in aggressive ALCL cases. Immunohistochemical analysis showed that MAX expression was related to the expression of MYC and cytotoxic molecules. These fndings demonstrate that lack of MAX expression is a potential poor prognostic biomarker in ALCL and a candidate marker for diferential diagnosis of ALCL and PTCL‑noS. Anaplastic large cell lymphoma (ALCL) is an aggressive mature T-cell lymphoma that usually expresses the lymphocyte activation marker CD30 and ofen lacks expression of T-cell antigens, such as CD3, CD5, and CD71.
    [Show full text]
  • AP-1 in Cell Proliferation and Survival
    Oncogene (2001) 20, 2390 ± 2400 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc AP-1 in cell proliferation and survival Eitan Shaulian1 and Michael Karin*,1 1Laboratory of Gene Regulation and Signal Transduction, Department of Pharmacology, University of California San Diego, 9500 Gilman Drive, La Jolla, California, CA 92093-0636, USA A plethora of physiological and pathological stimuli extensively discussed previously (Angel and Karin, induce and activate a group of DNA binding proteins 1991; Karin, 1995). that form AP-1 dimers. These proteins include the Jun, The mammalian AP-1 proteins are homodimers and Fos and ATF subgroups of transcription factors. Recent heterodimers composed of basic region-leucine zipper studies using cells and mice de®cient in individual AP-1 (bZIP) proteins that belong to the Jun (c-Jun, JunB proteins have begun to shed light on their physiological and JunD), Fos (c-Fos, FosB, Fra-1 and Fra-2), Jun functions in the control of cell proliferation, neoplastic dimerization partners (JDP1 and JDP2) and the closely transformation and apoptosis. Above all such studies related activating transcription factors (ATF2, LRF1/ have identi®ed some of the target genes that mediate the ATF3 and B-ATF) subfamilies (reviewed by (Angel eects of AP-1 proteins on cell proliferation and death. and Karin, 1991; Aronheim et al., 1997; Karin et al., There is evidence that AP-1 proteins, mostly those that 1997; Liebermann et al., 1998; Wisdom, 1999). In belong to the Jun group, control cell life and death addition, some of the Maf proteins (v-Maf, c-Maf and through their ability to regulate the expression and Nrl) can heterodimerize with c-Jun or c-Fos (Nishiza- function of cell cycle regulators such as Cyclin D1, p53, wa et al., 1989; Swaroop et al., 1992), whereas other p21cip1/waf1, p19ARF and p16.
    [Show full text]
  • Physical Interaction of the Retinoblastoma Protein with Human D Cyclins
    Cell, Vol. 73, 499-511, May 7, 1993, Copyright 0 1993 by Cell Press Physical Interaction of the Retinoblastoma Protein with Human D Cyclins Steven F. Dowdy,* Philip W. Hinds,’ Kenway Louie,’ into Rb- tumor cells by microinjection, viral infection, or Steven I. Reed,t Andrew Arnold,* transfection can lead to the growth arrest of these recipient and Robert A. Weinberg” cells (Huang et al., 1988; Goodrich et al., 1991; Templeton *The Whitehead Institute for Biomedical Research et al., 1991; Hinds et al., 1992). and Department of Biology Oncoproteins specified by the SV40, adenovirus, and Massachusetts Institute of Technology papilloma DNA tumor viruses have been shown to associ- Cambridge, Massachusetts 02142 ate with pRb in virus-transformed cells (Whyte et al., 1988; tThe Scripps Research Institute DeCaprio et al., 1988; Dyson et al., 1989). Oncoprotein Department of Molecular Biology binding of pRb is presumed to lead to its sequestration 10666 North Torrey Pines Road and functional inactivation. Conserved region II mutants La Jolla, California 92037 of adenovirus ElA, SV40 large T antigen, human papil- *Endocrine Unit loma E7 viral oncoproteins that have lost their ability to and Massachusetts General Hospital Cancer Center bind pflb, and other pRb-related proteins exhibit signifi- Massachusetts General Hospital cantly reduced transforming potential (Moran et al., 1986; and Harvard Medical School Lillie et al., 1987; Cherington et al., 1988; DeCaprio et al., Boston, Massachusetts 02114 1988; Moran, 1988; Smith and Ziff, 1988; Whyte et al., 1989). This suggests that binding of pRb and related pro- teins by these oncoproteins is critical to their transforming abilities.
    [Show full text]
  • Mechanistic Insights Into the Effect of RUNX1/ETO Knockdown in T(8;21) AML
    Mechanistic insights into the effect of RUNX1/ETO knockdown in t(8;21) AML Anna Pickin A thesis submitted to the University of Birmingham for the degree of DOCTOR OF PHILOSOPHY Institute of Biomedical Research Birmingham Centre for Genomic Biology College of Medical and Dental Sciences University of Birmingham September 2016 1 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. ABSTRACT The mutation of transcription factor genes is a main cause for acute myeloid leukaemia. RUNX1/ETO, the product of the t(8;21) chromosomal translocation, subverts normal blood cell development by impairing myeloid differentiation. RUNX1/ETO knockdown alleviates this block, with a global reprogramming of transcription factor binding and initiation of myeloid differentiation. Co-depletion of the myeloid transcription factor C/EBPα with RUNX1/ETO suppressed this differentiation response. Furthermore, C/EBPα overexpression largely phenocopied the effect of RUNX1/ETO knockdown. Our data show that low levels of C/EBPα are critical to the maintenance of t(8;21) AML and that C/EBPα drives the response to RUNX1/ETO depletion. To examine how changes in transcription factor binding impact on the activity of cis- regulatory elements we mapped genome wide promoter-distal-element interactions in a t(8;21) AML cell line, via Capture HiC, and found that hundreds of interactions were altered by RUNX1/ETO knockdown.
    [Show full text]