scholarly journals cDNA cloning and expression of rat homeobox gene, Hex, and functional characterization of the protein

1999 ◽  
Vol 339 (1) ◽  
pp. 111-117 ◽  
Author(s):  
Takashi TANAKA ◽  
Tetsuya INAZU ◽  
Kazuya YAMADA ◽  
Zaw MYINT ◽  
Vincent W. KENG ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Rat Liver ◽  
Functional Characterization ◽  
Homeobox Gene ◽  
Hepatoma Cells ◽  
Cloning And Expression ◽  
Expression Vectors ◽  
Luciferase Reporter ◽  
Cdna Clones

We isolated two cDNA clones of rat Hex, a homeobox protein, studied its expression in rat liver and various cells, and characterized the protein. The levels of Hex mRNA were only slightly increased in liver of rats refed with a high-carbohydrate diet or after partial hepatectomy. Whereas the expression of Hex mRNA was detected in hepatocytes isolated from adult rat liver and also in highly differentiated hepatoma cells, no Hex mRNA was detected in poorly differentiated hepatoma cells. Hex mRNA was also detected in liver from embryo aged 15 days. Expression of Hex was increased in F9 cells during differentiation into visceral endoderm cells by treatment with retinoic acid. This stimulation occurred prior to an increase in the level of α-fetoprotein mRNA. When fusion-protein expression vectors of GAL4 DNA-binding domain and Hex were co-transfected with luciferase reporter plasmid, with or without five copies of the GAL4-binding site, into HepG2 cells, the luciferase activities were decreased in concentration- and GAL4-binding site-dependent manners. This repression did not require the presence of the homeodomain, which is located between the amino acid residues 137 and 196. Its repression domain was mapped between the residues 45 and 136 in the proline-rich N-terminal region. In addition, the homeodomain was responsible for DNA-binding of Hex. These results indicate that Hex functions as a transcriptional repressor and may be involved in the differentiation and/or maintenance of the differentiated state in hepatocytes.

DNA-binding peptides from rat liver and Novikoff hepatoma cells: quantitative level and possible biochemical differences

1983 ◽  
Vol 9 (3) ◽  
pp. 169-174 ◽  
Author(s):  
G. L. Gianfranceschi ◽  
D. Barra ◽  
S. Coderoni ◽  
M. Paparelli ◽  
F. Venanzi ◽  
...  
Keyword(s):  
Dna Binding ◽  
Rat Liver ◽  
Hepatoma Cells ◽  
Novikoff Hepatoma ◽  
Quantitative Level ◽  
Binding Peptides

scholarly journals The monomer-binding orphan receptor Rev-Erb represses transcription as a dimer on a novel direct repeat.

1995 ◽  
Vol 15 (9) ◽  
pp. 4791-4802 ◽  
Author(s):  
H P Harding ◽  
M A Lazar
Keyword(s):  
Retinoic Acid ◽  
Dna Binding ◽  
Binding Site ◽  
Binding Domain ◽  
Luciferase Reporter ◽  
Dna Binding Domain ◽  
Transactivation Domain ◽  
Direct Repeats ◽  
Basal Transcription ◽  
C Terminus

Rev-Erb is an orphan nuclear receptor which binds as a monomer to the thyroid/retinoic acid receptor half-site AGGTCA flanked 5' by an A/T-rich sequence, referred to here as a Rev monomer site. Fusion of Rev-Erb to the DNA binding domain of yeast GAL4 strongly repressed basal transcription of a GAL4-luciferase reporter gene as a result of the presence of a C-terminal domain containing both the hinge and heptad repeat regions. Nevertheless, wild-type Rev-Erb did not repress basal transcription from the Rev monomer binding site. Therefore, a DNA binding site selection strategy was devised to test the hypothesis that Rev-Erb may function on a different site as a dimer. This approach identified sequences containing two Rev monomer sites arranged as direct repeats with the AGGTCA motifs separated by 2 bp (Rev-DR2). Remarkably, Rev-Erb bound as a homodimer to Rev-DR2 but not to other direct repeats or to a standard DR2 sequence. The DNA binding domain contained all of the determinants for Rev-DR2-specific homodimerization. Rev-Erb bound cooperatively as a homodimer to Rev-DR2, and this interaction was 5 to 10 times more stable than Rev-Erb monomer binding to the Rev monomer site. Functionally, Rev-Erb markedly repressed the basal activity of a variety of promoters with a strong Rev-DR2 specificity. The C terminus was required for this repression, consistent with the GAL4 results. However, the Rev-DR2 specificity did not require the C terminus in vivo, since fusion of C-terminally truncated Rev-Erb to a heterologous transactivation domain created a transcriptional activator specific for Rev-DR2. In addition to idealized Rev-DR2 sites, Rev-Erb also repressed basal as well as retinoic acid-induced transcription from a naturally occurring Rev-DR2 in the CRBPI gene. Thus, although Rev-Erb is distinguished from other thyroid/steroid receptor superfamily members by its ability to bind DNA as a monomer, it functions as a homodimer to repress transcription of genes containing a novel DR2 element.

scholarly journals An exceptionally conserved transcriptional repressor, CTCF, employs different combinations of zinc fingers to bind diverged promoter sequences of avian and mammalian c-myc oncogenes.

1996 ◽  
Vol 16 (6) ◽  
pp. 2802-2813 ◽  
Author(s):  
G N Filippova ◽  
S Fagerlie ◽  
E M Klenova ◽  
C Myers ◽  
Y Dehner ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Expression Patterns ◽  
Transcriptional Repressor ◽  
Ctcf Binding ◽  
Ctcf Binding Site ◽  
Cdna Clones ◽  
Regulatory Sequences ◽  
P2 Promoter ◽  
Gel Shift

We have isolated and analyzed human CTCF cDNA clones and show here that the ubiquitously expressed 11-zinc-finger factor CTCF is an exceptionally highly conserved protein displaying 93% identity between avian and human amino acid sequences. It binds specifically to regulatory sequences in the promoter-proximal regions of chicken, mouse, and human c-myc oncogenes. CTCF contains two transcription repressor domains transferable to a heterologous DNA binding domain. One CTCF binding site, conserved in mouse and human c-myc genes, is found immediately downstream of the major P2 promoter at a sequence which maps precisely within the region of RNA polymerase II pausing and release. Gel shift assays of nuclear extracts from mouse and human cells show that CTCF is the predominant factor binding to this sequence. Mutational analysis of the P2-proximal CTCF binding site and transient-cotransfection experiments demonstrate that CTCF is a transcriptional repressor of the human c-myc gene. Although there is 100% sequence identity in the DNA binding domains of the avian and human CTCF proteins, the regulatory sequences recognized by CTCF in chicken and human c-myc promoters are clearly diverged. Mutating the contact nucleotides confirms that CTCF binding to the human c-myc P2 promoter requires a number of unique contact DNA bases that are absent in the chicken c-myc CTCF binding site. Moreover, proteolytic-protection assays indicate that several more CTCF Zn fingers are involved in contacting the human CTCF binding site than the chicken site. Gel shift assays utilizing successively deleted Zn finger domains indicate that CTCF Zn fingers 2 to 7 are involved in binding to the chicken c-myc promoter, while fingers 3 to 11 mediate CTCF binding to the human promoter. This flexibility in Zn finger usage reveals CTCF to be a unique "multivalent" transcriptional factor and provides the first feasible explanation of how certain homologous genes (i.e., c-myc) of different vertebrate species are regulated by the same factor and maintain similar expression patterns despite significant promoter sequence divergence.

Mechanisms of Leukemogenesis by MLL-CALM: Characterization of a CALM-Derived Transcriptional Regulatory Domain.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3387-3387
Author(s):  
Mwe Mwe Chao ◽  
Emily J. Fox ◽  
Daniel S. Wechsler
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Nuclear Export ◽  
Transcriptional Activity ◽  
Hox Genes ◽  
Luciferase Activity ◽  
Expression Vectors ◽  
Luciferase Reporter ◽  
Fusion Partner ◽  
Cytoplasmic Localization

Abstract Background: MLL translocations are common in infant leukemias, and >50 distinct translocation partners have been described. We recently identified the CALM gene as a novel MLL partner in an infant with aggressive AML. Interestingly, CALM was first discovered as a translocation partner for AF10, which had previously been identified as an MLL fusion partner in aggressive leukemias and lymphomas. The native CALM protein exhibits predominantly cytoplasmic localization, and participates in clathrin-dependent endocytosis and intracellular vesicle transport. We have previously shown that expression of MLL-CALM immortalizes murine hematopoietic progenitors, and that fusion of the carboxy terminus of CALM to MLL alters MLL transcriptional activity. We hypothesize that CALM possesses a specific transcriptional activation domain (TAD) which modulates MLL transcriptional activity of HOX genes, thereby contributing to leukemogenesis. Objectives: 1) To determine whether native CALM localizes to the nucleus, 2) To delineate specific CALM domains which constitute the CALM TAD, and 3) To determine whether MLL-CALM activates transcription through the murine HOXA7 promoter. Methods: Human fibroblast cells were treated with Leptomycin B (an antifungal antibiotic which specifically inhibits nuclear export) and stained with an anti-CALM antibody. We prepared a set of expression vectors in which various portions of CALM are fused to a GAL4 DNA-binding domain. These vectors were co-transfected with a GAL4-luciferase reporter plasmid into COS7 cells, and luciferase activity was measured 48 hours after transient transfection. Luciferase assays were also performed using MSCV-MLL-CALM or MSCV-CALM plasmids co-transfected with a HOXA7 promoter-luciferase reporter construct. Results: After inhibition of nuclear export, native CALM localized to both the nucleus and cytoplasm. Significant luciferase activity was only observed with constructs containing distal CALM carboxy amino acids (aa 436–660). Mutation of an NR (Nuclear Receptor) Box motif (aa 510–514) did not affect CALM-dependent transcription. We found that two endocytosis-related NPF domains play opposite roles: deletion of NPF#1 (aa 437–439) dramatically reduced, while mutation of NPF#2 (aa 639–641) increased transcriptional activity. Expression constructs lacking GAL4 DNA binding domains had no effect on transcription, and GAL4 binding sites were required for luciferase activity in this system. Finally, MLL-CALM activated transcription of the murine HOXA7 promoter in comparison with native CALM or empty vector. Conclusions: We have confirmed that native CALM is able to localize to the nucleus, and we have begun to identify specific critical residues in the CALM TAD. The presence of a CALM TAD in MLL-CALM suggests that altered transcriptional regulation of MLL-dependent HOX genes may play an important role in MLL-CALM dependent transformation. Our observations raise the possibility that other MLL partners with native cytoplasmic localization may possess unrecognized transcriptional activity, and provide new insight into both MLL-CALM and CALM-AF10 mediated leukemogenesis.

NF-κB Is Active in Mcl-1 Promoter Regulation in Human CLL.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2265-2265
Author(s):  
Erin K Hertlein ◽  
Derek A. West ◽  
Ruth W Craig ◽  
David M Lucas ◽  
John C. Byrd
Keyword(s):  
Transcription Factor ◽  
Transcriptional Regulation ◽  
Dna Binding ◽  
Cell Survival ◽  
Binding Site ◽  
Nuclear Localization ◽  
Lymphocytic Leukemia ◽  
Luciferase Reporter ◽  
Critical Event ◽  
Direct Link

Abstract The NF-κB family of transcription factors is linked to neoplasia due to its role in increasing cell proliferation as well as inhibiting apoptosis. NF-κB has been reported to be constitutively active in chronic lymphocytic leukemia (CLL), but the mechanism underlying this this activation is not fully understood and the critical target genes involved have not been identified. To further define the mechanism(s) by which NF-κB promotes survival in CLL, we investigated the role of this transcription factor in the regulation of the myeloid cell leukemia 1 (Mcl-1) gene. The promoter region of Mcl-1 contains a putative NF-κB binding site, and increased expression of the Mcl-1 protein has been related to rapid disease progression and resistance to apoptosis in CLL. However, a direct link between NF-κB and Mcl-1 transcriptional regulation has not yet been established in B-cells or human CLL. We demonstrate here that NF-κB binding is a critical event in the transcriptional regulation of Mcl-1, as deletion of the NF-κB binding site in the promoter results in decreased activity of an Mcl-1-luciferase reporter construct. In addition, pharmacological inhibition of NF-κB reduced p65 nuclear localization and binding of NF-κB to the Mcl-1 promoter. Interestingly, when CLL patient samples were exposed to an IKK inhibitor, Bay-11, the extent of Mcl-1 inhibition varied in patients. We therefore examined whether the effect on Mcl-1 correlated with in vitro cell survival, a result that might be expected given that NF-κB expression has been previously reported to correlate with CLL cell survival. We found that CLL patient cells more sensitive to Bay-11-induced apoptosis also showed a larger decrease in Mcl-1 mRNA, suggesting that Mcl-1 message level may be a useful diagnostic to predict patients that will respond to NF-κB targeted therapy. Furthermore, different inducers of the NF-κB signaling pathway (CD40L, immune stimulatory CpG-ODN and TNF-α), promote differential effects on Mcl-1 regulation in CLL patient cells. Although all three treatments increase NF-κB nuclear localization and DNA binding, Mcl-1 RNA and protein increased with CpG and CD40L treatment, but not TNFα. This result provides evidence of stimulus-specific regulation of Mcl-1 by NF-κB. Based on these observations, we hypothesize that there is a direct link between NF-κB DNA binding and transcriptional control of Mcl-1, and that this gene could serve as a pharmacodynamic endpoint to monitor the efficacy of NF-κB inhibitors in CLL cells. Additionally, a direct link of NF-κB activity to Mcl-1 expression and cell survival provide further justification for targeting this transcription factor for treatment in CLL.

scholarly journals cDNA cloning and expression of rat homeobox gene, Hex, and functional characterization of the protein

1999 ◽  
Vol 339 (1) ◽  
pp. 111 ◽  
Author(s):  
Takashi TANAKA ◽  
Tetsuya INAZU ◽  
Kazuya YAMADA ◽  
Zaw MYINT ◽  
Vincent W. KENG ◽  
...  
Keyword(s):  
Cdna Cloning ◽  
Functional Characterization ◽  
Homeobox Gene ◽  
Cloning And Expression

scholarly journals Hypoxic up-regulation of erythroid 5-aminolevulinate synthase

Blood ◽  
2003 ◽  
Vol 101 (1) ◽  
pp. 348-350 ◽  
Author(s):  
Thomas Hofer ◽  
Roland H. Wenger ◽  
Marianne F. Kramer ◽  
Gloria C. Ferreira ◽  
Max Gassmann
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Hypoxia Inducible Factor ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Binding Assays ◽  
Luciferase Reporter Gene ◽  
Aminolevulinate Synthase ◽  
Erythroleukemia Cells ◽  
Murine Erythroleukemia

Abstract The erythroid-specific isoform of 5-aminolevulinate synthase (ALAS2) catalyzes the rate-limiting step in heme biosynthesis. The hypoxia-inducible factor–1 (HIF-1) transcriptionally up-regulates erythropoietin, transferrin, and transferrin receptor, leading to increased erythropoiesis and hematopoietic iron supply. To test the hypothesis that ALAS2 expression might be regulated by a similar mechanism, we exposed murine erythroleukemia cells to hypoxia (1% O2) and found an up to 3-fold up-regulation of ALAS2 mRNA levels and an increase in cellular heme content. A fragment of the ALAS2 promoter ranging from −716 to +1 conveyed hypoxia responsiveness to a heterologous luciferase reporter gene construct in transiently transfected HeLa cells. In contrast, iron depletion, known to induce HIF-1 activity but inhibit ALAS2 translation, did not increase ALAS2 promoter activity. Mutation of a previously predicted HIF-1–binding site (−323/−318) within this promoter fragment and DNA-binding assays revealed that hypoxic up-regulation is independent of this putative HIF-1 DNA-binding site.

Proteomic Identification of TAL1/SCL-Interacting Proteins: ETO-2 and MTGR1 Interact with TAL1 in Erythroid Progenitors.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 357-357
Author(s):  
Ying Cai ◽  
Zhixiong Xu ◽  
Jingping Xie ◽  
Mark J. Koury ◽  
Scott W. Hiebert ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Expression Vectors ◽  
Luciferase Reporter ◽  
Bhlh Transcription Factor ◽  
Interacting Proteins ◽  
Erythroid Cells ◽  
Erythroid Progenitors ◽  
Murine Erythroleukemia ◽  
In Erythroid Cells

Abstract The TAL1/SCL gene, originally identified from its involvement by a recurrent chromosomal translocation in T-cel acute lymphoblastic leukemia, encodes a basic helix-loop-helix (bHLH) transcription factor essential for hematopoietic and vascular development. Although TAL1 is believed to regulate transcription of specific sets of target genes, the mechanisms underlying TAL1-directed gene expression are poorly understood. Previous studies have shown, in fact, that it can act as either an activator or repressor depending on the coregulator(s) with which it interacts. To comprehensively identify TAL1’s interaction partners in erythroid cells, we stably expressed a tandem epitope-tagged mouse TAL1 protein in murine erythroleukemia (MEL) cells and determined the composition of affinity-purified TAL1-containing complexes by multidimensional mass spectrometry. From this analysis, we identified all known members of a TAL1-containing DNA binding complex previously characterized in erythroid cells, including TAL1, its E protein DNA-binding partners, the zinc finger transcription factor GATA-1, the LIM-only protein LMO2, and the LIM domain-binding protein Ldb1, as well as proteins described to interact with GATA-1 (FOG-1), LMO2 (ELF2A2), and Ldb1 (SSDP2 and SSDP3). In addition, we identified a number of other DNA binding proteins, chromatin modifying proteins, and transcriptional regulators, including the ETO family members ETO-2 and MTGR1. TAL1 interaction with ETO-2 and MTGR1 was verified by coimmunoprecipitation analysis in MEL cells expressing these proteins at endogenous levels, in MEL cells stably expressing an epitope-tagged TAL1 protein, and in COS cells transiently transfected with TAL1 and ETO-2 or MTGR1 expression vectors. Mapping analysis with GAL4 fusion proteins identified the bHLH domain as the region in TAL1 responsible for interaction with these ETO family proteins. Significantly, expression of MTGR1 enhanced ETO-2 interaction with TAL1-GAL4 protein. Finally, transient transfection analysis with a luciferase reporter construct linked to multiple GAL4 DNA binding sites showed greater than additive augmentation of TAL1-directed gene repression with coexpression of the two ETO-related proteins compared to that observed with ETO-2 or MTGR1 transfected individually. These results identify ETO-2 and MTGR1 as authentic TAL1 interacting proteins and suggest that a hetero-oligomeric complex of the two contributes to TAL1-directed repression in erythroid progenitors.

scholarly journals Identification of Transcriptional Enhancer Factor-4 as a Transcriptional Modulator of CTP:Phosphocholine Cytidylyltransferase α

2001 ◽  
Vol 276 (15) ◽  
pp. 12338-12344 ◽  
Author(s):  
Hiroyuki Sugimoto ◽  
Marica Bakovic ◽  
Satoshi Yamashita ◽  
Dennis E. Vance
Keyword(s):  
Dna Binding ◽  
Promoter Activity ◽  
Binding Domain ◽  
Luciferase Reporter ◽  
Cdna Clones ◽  
Mrna Levels ◽  
Dna Binding Domain ◽  
Transcriptional Enhancer ◽  
Ctp:Phosphocholine Cytidylyltransferase ◽  
Enhancer Factor

CTP:phosphocholine cytidylyltransferase (CCT) is the rate-limiting and regulated enzyme of mammalian phosphatidylcholine biosynthesis. There are three isoforms, CCTα, CCTβ1, and CCTβ2. The mouse CCTα gene promoter is regulated by an enhancer element (Eb) located between −103 and −82 base pairs (5′-GTTTTCAGGAATGCGGAGGTGG-3′) upstream from the transcriptional start site (Bakovic, M., Waite, K., Tang, W., Tabas, I., and Vance, D. E. (1999)Biochim. Biophys. Acta1436, 147–165). To identify the Eb-binding protein(s), we screened a mouse embryo cDNA library by the yeast one-hybrid system and obtained 19 positive clones. Ten cDNA clones were identified as transcriptional enhancer factor-4 (TEF-4). The TEF-binding consensus sequence, 5′-(A/T)(A/G)(A/G)(A/T)ATG(C/T)(G/A)-3′, was identified within the Eb binding region. Gel-shift analysis using radiolabeled Eb fragment as a probe showed that cell extracts from yeast expressing hemagglutinin-tagged TEF-4 caused a marked band retardation that could be prevented with an anti-hemagglutinin antibody. When COS-7 cells were transfected with TEF-4, CCTα promoter-luciferase reporter activity and CCTα mRNA levels increased. A TEF-4 deletion mutant containing a DNA-binding domain, mTEA(+), stimulated the CCTα promoter activity, whereas protein lacking the DNA binding domain, mTEA(−), did not. Unexpectedly, when the ATG core of the TEF-4 binding consensus within the Eb region was mutated, promoter activity was enhanced rather than decreased. Thus, TEF-4 might act as a dual transcriptional modulator as follows: as a suppressor via its direct binding to the Eb element and as an activator via its interactions with the basal transcriptional machinery. These results provide the first evidence that TEF-4 is an important regulator of CCTα gene expression.

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