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1
Nuchprayoon, I., S. Meyers, L. M. Scott, J. Suzow, S. Hiebert, and A. D. Friedman. "PEBP2/CBF, the murine homolog of the human myeloid AML1 and PEBP2 beta/CBF beta proto-oncoproteins, regulates the murine myeloperoxidase and neutrophil elastase genes in immature myeloid cells." Molecular and Cellular Biology 14, no. 8 (August 1994): 5558–68. http://dx.doi.org/10.1128/mcb.14.8.5558.
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The myeloperoxidase (MPO) and neutrophil elastase genes are expressed specifically in immature myeloid cells. The integrity of a polyomavirus enhancer core sequence, 5'-AACCACA-3', is critical to the activity of the murine MPO proximal enhancer. This element binds two species, myeloid nuclear factors 1 alpha and 1 beta (MyNF1 alpha and -beta), present in 32D cl3 myeloid cell nuclear extracts. The levels of the MyNF1s increase during early 32D cl3 cell granulocytic differentiation. Both MyNF1 alpha and -beta supershift with an antiserum raised by using a peptide derived from the N terminus of polyomavirus enhancer-binding protein 2/core-binding factor (PEBP2/CBF) alpha subunit. The specific peptide inhibits these supershifts. In vitro-translated PEBP2/CBF DNA-binding domain binds the murine MPO PEBP2/CBF site. An alternate PEBP2/CBF consensus site, 5'-GACCGCA-3', but not a simian virus 40 enhancer core sequence, 5'-TTCCACA-3', binds the MyNF1s in vitro and activates a minimal murine MPO-thymidine kinase promoter in vivo. The murine neutrophil elastase gene 100-bp 5'-flanking sequences contain several functional elements, including potential binding sites for PU.1, C/EBP, c-Myb, and PEBP2/CBF. The functional element 5'-GGCCACA-3' located at positions -66 to 72 differs from the PEBP2/CBF consensus (5'-PuACCPuCA-3') only by an A-to-G transition at position 2. This DNA element binds MyNF1 alpha and -beta weakly. The N terminis of two PEBP2/CBF alpha subunit family members, PEBP2 alpha A and PEBP2 alpha B (murine AML1), are nearly identical, and 32D c13 cl3 cells contain both corresponding mRNAs. Since t(8;21), t(3;21), and inv(16), associated with myeloid leukemias, disrupt subunits of PEBP2/CBF, we speculate that the resulting oncoproteins, AML1-ETO, AML1-EAP, AML1-Evi1, and CBF beta-MYH11, inhibit early myeloid differentiation.
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Nuchprayoon, I., S. Meyers, L. M. Scott, J. Suzow, S. Hiebert, and A. D. Friedman. "PEBP2/CBF, the murine homolog of the human myeloid AML1 and PEBP2 beta/CBF beta proto-oncoproteins, regulates the murine myeloperoxidase and neutrophil elastase genes in immature myeloid cells." Molecular and Cellular Biology 14, no. 8 (August 1994): 5558–68. http://dx.doi.org/10.1128/mcb.14.8.5558-5568.1994.
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The myeloperoxidase (MPO) and neutrophil elastase genes are expressed specifically in immature myeloid cells. The integrity of a polyomavirus enhancer core sequence, 5'-AACCACA-3', is critical to the activity of the murine MPO proximal enhancer. This element binds two species, myeloid nuclear factors 1 alpha and 1 beta (MyNF1 alpha and -beta), present in 32D cl3 myeloid cell nuclear extracts. The levels of the MyNF1s increase during early 32D cl3 cell granulocytic differentiation. Both MyNF1 alpha and -beta supershift with an antiserum raised by using a peptide derived from the N terminus of polyomavirus enhancer-binding protein 2/core-binding factor (PEBP2/CBF) alpha subunit. The specific peptide inhibits these supershifts. In vitro-translated PEBP2/CBF DNA-binding domain binds the murine MPO PEBP2/CBF site. An alternate PEBP2/CBF consensus site, 5'-GACCGCA-3', but not a simian virus 40 enhancer core sequence, 5'-TTCCACA-3', binds the MyNF1s in vitro and activates a minimal murine MPO-thymidine kinase promoter in vivo. The murine neutrophil elastase gene 100-bp 5'-flanking sequences contain several functional elements, including potential binding sites for PU.1, C/EBP, c-Myb, and PEBP2/CBF. The functional element 5'-GGCCACA-3' located at positions -66 to 72 differs from the PEBP2/CBF consensus (5'-PuACCPuCA-3') only by an A-to-G transition at position 2. This DNA element binds MyNF1 alpha and -beta weakly. The N terminis of two PEBP2/CBF alpha subunit family members, PEBP2 alpha A and PEBP2 alpha B (murine AML1), are nearly identical, and 32D c13 cl3 cells contain both corresponding mRNAs. Since t(8;21), t(3;21), and inv(16), associated with myeloid leukemias, disrupt subunits of PEBP2/CBF, we speculate that the resulting oncoproteins, AML1-ETO, AML1-EAP, AML1-Evi1, and CBF beta-MYH11, inhibit early myeloid differentiation.
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Wang, S., Q. Wang, B. E. Crute, I. N. Melnikova, S. R. Keller, and N. A. Speck. "Cloning and characterization of subunits of the T-cell receptor and murine leukemia virus enhancer core-binding factor." Molecular and Cellular Biology 13, no. 6 (June 1993): 3324–39. http://dx.doi.org/10.1128/mcb.13.6.3324.
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Moloney murine leukemia virus causes thymic leukemias when injected into newborn mice. A major determinant of the thymic disease specificity of Moloney virus genetically maps to the conserved viral core motif in the Moloney virus enhancer. Point mutations introduced into the core site significantly shifted the disease specificity of the Moloney virus from thymic leukemia to erythroid leukemia (N.A. Speck, B. Renjifo, E. Golemis, T.N. Fredrickson, J.W. Hartley, and N. Hopkins, Genes Dev. 4:233-242, 1990). We previously reported the purification of core-binding factors (CBF) from calf thymus nuclei (S. Wang and N.A. Speck, Mol. Cell. Biol. 12:89-102, 1992). CBF binds to core sites in murine leukemia virus and T-cell receptor enhancers. Affinity-purified CBF contains multiple polypeptides. In this study, we sequenced five tryptic peptides from two of the bovine CBF proteins and isolated three cDNA clones from a mouse thymus cDNA library encoding three of the tryptic peptides from the bovine proteins. The cDNA clones, which we call CBF beta p22.0, CBF beta p21.5, and CBF beta p17.6, encode three highly related but distinct proteins with deduced molecular sizes of 22.0, 21.5, and 17.6 kDa that appear to be translated from multiply spliced mRNAs transcribed from the same gene. CBF beta p22.0, CBF beta p21.5, and CBF beta p17.6 do not by themselves bind the core site. However, CBF beta p22.0 and CBF beta p21.5 form a complex with DNA-binding CBF alpha subunits and as a result decrease the rate of dissociation of the CBF protein-DNA complex. Association of the CBF beta subunits does not extend the phosphate contacts in the binding site. We propose that CBF beta is a non-DNA-binding subunit of CBF and does not contact DNA directly.
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Wang, S., Q. Wang, B. E. Crute, I. N. Melnikova, S. R. Keller, and N. A. Speck. "Cloning and characterization of subunits of the T-cell receptor and murine leukemia virus enhancer core-binding factor." Molecular and Cellular Biology 13, no. 6 (June 1993): 3324–39. http://dx.doi.org/10.1128/mcb.13.6.3324-3339.1993.
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Moloney murine leukemia virus causes thymic leukemias when injected into newborn mice. A major determinant of the thymic disease specificity of Moloney virus genetically maps to the conserved viral core motif in the Moloney virus enhancer. Point mutations introduced into the core site significantly shifted the disease specificity of the Moloney virus from thymic leukemia to erythroid leukemia (N.A. Speck, B. Renjifo, E. Golemis, T.N. Fredrickson, J.W. Hartley, and N. Hopkins, Genes Dev. 4:233-242, 1990). We previously reported the purification of core-binding factors (CBF) from calf thymus nuclei (S. Wang and N.A. Speck, Mol. Cell. Biol. 12:89-102, 1992). CBF binds to core sites in murine leukemia virus and T-cell receptor enhancers. Affinity-purified CBF contains multiple polypeptides. In this study, we sequenced five tryptic peptides from two of the bovine CBF proteins and isolated three cDNA clones from a mouse thymus cDNA library encoding three of the tryptic peptides from the bovine proteins. The cDNA clones, which we call CBF beta p22.0, CBF beta p21.5, and CBF beta p17.6, encode three highly related but distinct proteins with deduced molecular sizes of 22.0, 21.5, and 17.6 kDa that appear to be translated from multiply spliced mRNAs transcribed from the same gene. CBF beta p22.0, CBF beta p21.5, and CBF beta p17.6 do not by themselves bind the core site. However, CBF beta p22.0 and CBF beta p21.5 form a complex with DNA-binding CBF alpha subunits and as a result decrease the rate of dissociation of the CBF protein-DNA complex. Association of the CBF beta subunits does not extend the phosphate contacts in the binding site. We propose that CBF beta is a non-DNA-binding subunit of CBF and does not contact DNA directly.
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Zaiman, A. L., A. F. Lewis, B. E. Crute, N. A. Speck, and J. Lenz. "Transcriptional activity of core binding factor-alpha (AML1) and beta subunits on murine leukemia virus enhancer cores." Journal of virology 69, no. 5 (1995): 2898–906. http://dx.doi.org/10.1128/jvi.69.5.2898-2906.1995.
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Sun, Ge, Chunyu Wang, Shengli Wang, Hongmiao Sun, Kai Zeng, Renlong Zou, Lin Lin, et al. "An H3K4me3 reader, BAP18 as an adaptor of COMPASS-like core subunits co-activates ERα action and associates with the sensitivity of antiestrogen in breast cancer." Nucleic Acids Research 48, no. 19 (September 28, 2020): 10768–84. http://dx.doi.org/10.1093/nar/gkaa787.
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Abstract Estrogen receptor alpha (ERα) signaling pathway is essential for ERα-positive breast cancer progression and endocrine therapy resistance. Bromodomain PHD Finger Transcription Factor (BPTF) associated protein of 18kDa (BAP18) has been recognized as a crucial H3K4me3 reader. However, the whole genomic occupation of BAP18 and its biological function in breast cancer is still elusive. Here, we found that higher expression of BAP18 in ERα-positive breast cancer is positively correlated with poor prognosis. ChIP-seq analysis further demonstrated that the half estrogen response elements (EREs) and the CCCTC binding factor (CTCF) binding sites are the significant enrichment sites found in estrogen-induced BAP18 binding sites. Also, we provide the evidence to demonstrate that BAP18 as a novel co-activator of ERα is required for the recruitment of COMPASS-like core subunits to the cis-regulatory element of ERα target genes in breast cancer cells. BAP18 is recruited to the promoter regions of estrogen-induced genes, accompanied with the enrichment of the lysine 4-trimethylated histone H3 tail (H3K4me3) in the presence of E2. Furthermore, BAP18 promotes cell growth and associates the sensitivity of antiestrogen in ERα-positive breast cancer. Our data suggest that BAP18 facilitates the association between ERα and COMPASS-like core subunits, which might be an essential epigenetic therapeutic target for breast cancer.
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Beghini, Alessandro. "Core Binding Factor Leukemia: Chromatin Remodeling Moves Towards Oncogenic Transcription." Cancers 11, no. 12 (December 7, 2019): 1973. http://dx.doi.org/10.3390/cancers11121973.
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Acute myeloid leukemia (AML), the most common acute leukemia in adults, is a heterogeneous malignant clonal disorder arising from multipotent hematopoietic progenitor cells characterized by genetic and concerted epigenetic aberrations. Core binding factor-Leukemia (CBFL) is characterized by the recurrent reciprocal translocations t(8;21)(q22;q22) or inv(16)(p13;q22) that, expressing the distinctive RUNX1-RUNX1T1 (also known as Acute myeloid leukemia1-eight twenty-one, AML1-ETO or RUNX1/ETO) or CBFB-MYH11 (also known as CBFβ-SMMHC) translocation product respectively, disrupt the essential hematopoietic function of the CBF. In the past decade, remarkable progress has been achieved in understanding the structure, three-dimensional (3D) chromosomal topology, and disease-inducing genetic and epigenetic abnormalities of the fusion proteins that arise from disruption of the CBF subunit alpha and beta genes. Although CBFLs have a relatively good prognosis compared to other leukemia subtypes, 40–50% of patients still relapse, requiring intensive chemotherapy and allogenic hematopoietic cell transplantation (alloHCT). To provide a rationale for the CBFL-associated altered hematopoietic development, in this review, we summarize the current understanding on the various molecular mechanisms, including dysregulation of Wnt/β-catenin signaling as an early event that triggers the translocations, playing a pivotal role in the pathophysiology of CBFL. Translation of these findings into the clinical setting is just beginning by improvement in risk stratification, MRD assessment, and development of targeted therapies.
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Fischer, G., C. Schmidt, J. Opitz, Z. Cully, K. Kühn, and E. Pöschl. "Identification of a novel sequence element in the common promoter region of human collagen type IV genes, involved in the regulation of divergent transcription." Biochemical Journal 292, no. 3 (June 15, 1993): 687–95. http://dx.doi.org/10.1042/bj2920687.
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The expression of the heterotrimeric collagen IV molecule alpha 1(IV)2 alpha 2(IV) is essential for the structural integrity and functional properties of all basement membranes. The two genes COL4A1 and COL4A2 that code for the subunits are found closely linked on chromosome 13 in a head-to-head arrangement and are transcribed in divergent directions. We have identified a novel trans-acting factor that binds in vitro to a unique homopyrimidine/homopurine stretch within the shared promoter region of the two collagen IV genes. Additional binding sites have been identified within the first introns of both genes and the consensus sequence CCCTYCCCC for efficient binding has been deduced; the factor was named therefore ‘CTC-binding factor’ or ‘CTCBF’. Mutations in the binding site of CTC-binding factor within the promoter inhibited binding in vitro and resulted in reduced transcription from both genes. The effect of mutations on the transcription of COL4A2 is more pronounced than on the transcription of COL4A1. CTC-binding factor is a nuclear factor that binds dominantly in vitro to the collagen IV promoter and is involved in regulating the expression of both collagen IV genes.
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Park, Kyung-Ran, Joon-Yeop Lee, Myounglae Cho, Jin-Tae Hong, and Hyung-Mun Yun. "Paeonolide as a Novel Regulator of Core-Binding Factor Subunit Alpha-1 in Bone-Forming Cells." International Journal of Molecular Sciences 22, no. 9 (May 6, 2021): 4924. http://dx.doi.org/10.3390/ijms22094924.
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Paeonia suffruticosa has been extensively used as a traditional medicine with various beneficial effects; paeonolide (PALI) was isolated from its dried roots. This study aimed to investigate the novel effects and mechanisms of PALI in pre-osteoblasts. Here, cell viability was evaluated using an MTT assay. Early and late osteoblast differentiation was examined by analyzing the activity of alkaline phosphatase (ALP) and by staining it with Alizarin red S (ARS). Cell migration was assessed using wound healing and Boyden chamber assays. Western blot and immunofluorescence analyses were used to examine the intracellular signaling pathways and differentiation proteins. PALI (0.1, 1, 10, 30, and 100 μM) showed no cytotoxic or proliferative effects in pre-osteoblasts. In the absence of cytotoxicity, PALI (1, 10, and 30 μM) promoted wound healing and transmigration during osteoblast differentiation. ALP staining demonstrated that PALI (1, 10, and 30 μM) promoted early osteoblast differentiation in a dose-dependent manner, and ARS staining showed an enhanced mineralized nodule formation, a key indicator of late osteoblast differentiation. Additionally, low concentrations of PALI (1 and 10 μM) increased the bone morphogenetic protein (BMP)–Smad1/5/8 and Wnt–β-catenin pathways in osteoblast differentiation. Particularly, PALI (1 and 10 μM) increased the phosphorylation of ERK1/2 compared with BMP2 treatment, an FDA-approved drug for bone diseases. Furthermore, PALI-mediated early and late osteoblast differentiation was abolished in the presence of the ERK1/2 inhibitor U0126. PALI-induced RUNX2 (Cbfa1) expression and nuclear localization were also attenuated by blocking the ERK1/2 pathway during osteoblast differentiation. We suggest that PALI has biologically novel activities, such as enhanced osteoblast differentiation and bone mineralization mainly through the intracellular ERK1/2-RUNX2 signaling pathway, suggesting that PALI might have therapeutic action and aid the treatment and prevention of bone diseases, such as osteoporosis and periodontitis.
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Weiss, Andy, J. Antonio Ibarra, Jessica Paoletti, Ronan K. Carroll, and Lindsey N. Shaw. "The δ Subunit of RNA Polymerase Guides Promoter Selectivity and Virulence in Staphylococcus aureus." Infection and Immunity 82, no. 4 (February 3, 2014): 1424–35. http://dx.doi.org/10.1128/iai.01508-14.
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ABSTRACTIn Gram-positive bacteria, and particularly theFirmicutes, the DNA-dependent RNA polymerase (RNAP) complex contains an additional subunit, termed the δ factor, or RpoE. This enigmatic protein has been studied for more than 30 years for various organisms, but its function is still not well understood. In this study, we investigated its role in the major human pathogenStaphylococcus aureus. We showed conservation of important structural regions of RpoE inS. aureusand other species and demonstrated binding to core RNAP that is mediated by the β and/or β′ subunits. To identify the impact of the δ subunit on transcription, we performed transcriptome sequencing (RNA-seq) analysis and observed 191 differentially expressed genes in therpoEmutant. Ontological analysis revealed, quite strikingly, that many of the downregulated genes were known virulence factors, while several mobile genetic elements (SaPI5 and prophage ϕSA3usa) were strongly upregulated. Phenotypically, therpoEmutant had decreased accumulation and/or activity of a number of key virulence factors, including alpha toxin, secreted proteases, and Panton-Valentine leukocidin (PVL). We further observed significantly decreased survival of the mutant in whole human blood, increased phagocytosis by human leukocytes, and impaired virulence in a murine model of infection. Collectively, our results demonstrate that the δ subunit of RNAP is a critical component of theS. aureustranscription machinery and plays an important role during infection.
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Hajra, A., P. P. Liu, N. A. Speck, and F. S. Collins. "Overexpression of core-binding factor alpha (CBF alpha) reverses cellular transformation by the CBF beta-smooth muscle myosin heavy chain chimeric oncoprotein." Molecular and Cellular Biology 15, no. 9 (September 1995): 4980–89. http://dx.doi.org/10.1128/mcb.15.9.4980.
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A fusion between the transcription factor core-binding factor beta (CBF beta; also known as PEBP2 beta) and the tail region of smooth muscle myosin heavy chain (SMMHC) is generated by an inversion of chromosome 16 [inv(16) (p13q22)] associated with the M4Eo subtype of acute myeloid leukemia. We have previously shown that this CBF beta-SMMHC chimeric protein can transform NIH 3T3 cells and that this process requires regions of the chimeric protein necessary for association with the CBF alpha subunit. In this study, we show that NIH 3T3 cells overexpressing murine Cbf alpha 2 (also known as Aml1) cannot be transformed by CBF beta-SMMHC and that overexpression of Cbf alpha 2 in cells previously transformed by CBF beta-SMMHC reverts the cells to a less transformed phenotype. Cbf alpha 2 overexpression does not cause any gross morphological changes to NIH 3T3 cells but does result in increased CBF activity, as indicated by electrophoretic mobility shift assays and transactivation of reporter constructs. Cells transformed by CBF beta-SMMHC lack normal CBF-DNA complexes and have decreased levels of transactivation. Reversion of CBF beta-SMMHC transformation by Cbf alpha 2 is associated with a restoration of normal CBF-DNA complexes and transactivation activity. A Cbf alpha 2 mutant lacking transactivation properties does not transform cells when overexpressed, nor does it protect cells from CBF beta-SMMHC transformation. These results suggest that CBF beta-SMMHC interferes with the normal function of CBF and that this interference is necessary but not sufficient for cellular transformation.
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Jaffray, E., K. M. Wood, and R. T. Hay. "Domain organization of I kappa B alpha and sites of interaction with NF-kappa B p65." Molecular and Cellular Biology 15, no. 4 (April 1995): 2166–72. http://dx.doi.org/10.1128/mcb.15.4.2166.
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The DNA-binding activity and cellular distribution of the transcription factor NF-kappa B are regulated by the inhibitor protein I kappa B alpha. I kappa B alpha belongs to a family of proteins that contain multiple repeats of a 30- to 35-amino-acid sequence that was initially recognized in the erythrocyte protein ankyrin. Partial proteolysis has been used to study the domain structure of I kappa B alpha and to determine the sites at which it interacts with NF-kappa B. The data reveal a tripartite structure for I kappa B alpha in which a central, protease-resistant domain composed of five ankyrin repeats is flanked by an unstructured N-terminal extension and a compact, highly acidic C-terminal domain that is connected to the core of the protein by a flexible linker. Functional analysis of V8 cleavage products indicates that I kappa B alpha molecules lacking the N-terminal region can interact with and inhibit the DNA-binding activity of the p65 subunit of NF-kappa B, whereas I kappa B alpha molecules which lack both the N- and C-terminal regions are incapable of doing so. Protease cleavage of the N terminus of I kappa B alpha was unaffected by the presence of the p65 subunit of NF-kappa B, whereas bound p65 blocked cleavage of the flexible linker connecting the C-terminal domain to the ankyrin repeat-containing core of the protein. This linker region is highly conserved within the human, rat, pig, and chicken homologs of I kappa B alpha, and while it has been suggested that it represents a sixth ankyrin repeat, it is also likely that this is a flexible region of the protein that interacts with NF-kappa B.
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Steensma, David P., Richard J. Gibbons, Ruben A. Mesa, Ayalew Tefferi, and Douglas R. Higgs. "Somatic Point Mutations in RUNX1/CBFA2/AML1 Are Common in High-Risk Myelodysplastic Syndrome, but Not in Myelofibrosis with Myeloid Metaplasia." Blood 104, no. 11 (November 16, 2004): 2438. http://dx.doi.org/10.1182/blood.v104.11.2438.2438.
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Abstract Introduction: The core-binding factor subunit RUNX1/CBFA2/AML1 (Runt-related transcription factor 1, Core-binding factor alpha 2 subunit, Oncogene AML1) is critical for generation of hematopoietic stem cells during embryogenesis and for normal megakaryopoiesis in adults. RUNX1/CBFA2/AML1 has long been recognized as an oncogene, and is frequently involved in leukemia-associated translocations that create aberrant fusion proteins (e.g., AML1-ETO). Recently, acquired somatic point mutations in the RUNX1/CBFA2/AML1 gene have been described in a subset of patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). These point mutations are associated with the loss of normal RUNX1 trans-activation potential and/or altered binding to the non-DNA-binding subunit of core-binding factor, CBFB. Given the importance of core-binding factor in megakaryocytic differentiation and platelet production and the central role of megakaryocytes in the pathophysiology of myelofibrosis with myeloid metaplasia (MMM), and in light of the myelofibrotic phenotype of GATA-1low mice (GATA-1 and RUNX1 are co-expressed and co-operate in megakaryocytic differentiation), we hypothesized that RUNX1 gene mutations might be common in MMM. In addition, we wanted to know whether patients with MDS-associated acquired alpha thalassemia (i.e., ATMDS), a special MDS subgroup with a very high incidence of point mutations in the X-linked ATRX gene, have an especially high incidence of RUNX1 mutations that might account for the more severe hematopoietic defect found in these patients compared with boys with inherited ATR-X syndrome (detailed in Steensma et al, Blood2004;103:2019). Methods and Results: We analyzed samples from 26 well-characterized patients with MMM and from 52 with MDS (11 RA, 3 RARS, 8 RCMD, 3 RAEB-1, 7 RAEB-2, 2 MDS in transition to AML-M6, 14 atypical/unclassifable MDS, 4 MDS subtype unknown - including 18 with ATMDS) for RUNX1 point mutations by denaturing high-performance liquid chromatography (DHPLC) followed by subcloning and sequencing of samples exhibiting heteroduplex peaks. We found 5 RUNX1 mutations in MDS patients (9.6%), all of whom had RAEB-2 (4 patients) or a history of treated AML (1 patient), but detected no mutations in MMM patients. All patients with RUNX1 mutations progressed to overt leukemia within 1 year. ATMDS patients did not have an increased risk of RUNX1 point mutations (2/18 patients with mutations, 11.1%) when compared with MDS without thalassemia (3/34 patients, 8.8%; p=0.58). Additionally, 1 MDS patient had a clonally restricted mutation in GATA-1 (c.1066 C>T) (also screened by DHPLC), but this is not predicted to change the GATA-1 amino acid sequence and is likely a random event reflecting the genomic instability characteristic of MDS. Conclusion: RUNX1 point mutations are common in high-risk MDS, but not in MMM, and that ATMDS patients do not have a risk of RUNX1 mutations in excess of that expected for MDS in general.
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Wu, Kenneth, Serge Y. Fuchs, Angus Chen, Peilin Tan, Carlos Gomez, Ze'ev Ronai, and Zhen-Qiang Pan. "The SCFHOS/β-TRCP-ROC1 E3 Ubiquitin Ligase Utilizes Two Distinct Domains within CUL1 for Substrate Targeting and Ubiquitin Ligation." Molecular and Cellular Biology 20, no. 4 (February 15, 2000): 1382–93. http://dx.doi.org/10.1128/mcb.20.4.1382-1393.2000.
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ABSTRACT We describe a purified ubiquitination system capable of rapidly catalyzing the covalent linkage of polyubiquitin chains onto a model substrate, phosphorylated IκBα. The initial ubiquitin transfer and subsequent polymerization steps of this reaction require the coordinated action of Cdc34 and the SCFHOS/β-TRCP-ROC1 E3 ligase complex, comprised of four subunits (Skp1, cullin 1 [CUL1], HOS/β-TRCP, and ROC1). Deletion analysis reveals that the N terminus of CUL1 is both necessary and sufficient for binding Skp1 but is devoid of ROC1-binding activity and, hence, is inactive in catalyzing ubiquitin ligation. Consistent with this, introduction of the N-terminal CUL1 polypeptide into cells blocks the tumor necrosis factor alpha-induced and SCF-mediated degradation of IκB by forming catalytically inactive complexes lacking ROC1. In contrast, the C terminus of CUL1 alone interacts with ROC1 through a region containing the cullin consensus domain, to form a complex fully active in supporting ubiquitin polymerization. These results suggest the mode of action of SCF-ROC1, where CUL1 serves as a dual-function molecule that recruits an F-box protein for substrate targeting through Skp1 at its N terminus, while the C terminus of CUL1 binds ROC1 to assemble a core ubiquitin ligase.
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Hamey, Fiona K., Sonia Nestorowa, Sarah J. Kinston, David G. Kent, Nicola K. Wilson, and Berthold Göttgens. "Reconstructing blood stem cell regulatory network models from single-cell molecular profiles." Proceedings of the National Academy of Sciences 114, no. 23 (June 5, 2017): 5822–29. http://dx.doi.org/10.1073/pnas.1610609114.
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Adult blood contains a mixture of mature cell types, each with specialized functions. Single hematopoietic stem cells (HSCs) have been functionally shown to generate all mature cell types for the lifetime of the organism. Differentiation of HSCs toward alternative lineages must be balanced at the population level by the fate decisions made by individual cells. Transcription factors play a key role in regulating these decisions and operate within organized regulatory programs that can be modeled as transcriptional regulatory networks. As dysregulation of single HSC fate decisions is linked to fatal malignancies such as leukemia, it is important to understand how these decisions are controlled on a cell-by-cell basis. Here we developed and applied a network inference method, exploiting the ability to infer dynamic information from single-cell snapshot expression data based on expression profiles of 48 genes in 2,167 blood stem and progenitor cells. This approach allowed us to infer transcriptional regulatory network models that recapitulated differentiation of HSCs into progenitor cell types, focusing on trajectories toward megakaryocyte–erythrocyte progenitors and lymphoid-primed multipotent progenitors. By comparing these two models, we identified and subsequently experimentally validated a difference in the regulation of nuclear factor, erythroid 2 (Nfe2) and core-binding factor, runt domain, alpha subunit 2, translocated to, 3 homolog (Cbfa2t3h) by the transcription factor Gata2. Our approach confirms known aspects of hematopoiesis, provides hypotheses about regulation of HSC differentiation, and is widely applicable to other hierarchical biological systems to uncover regulatory relationships.
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Kaushal, Neelam, Divya Vohora, Rajinder K. Jalali, and Sujeet Jha. "Review of the Literature Examining the Association of Serum Uric Acid with Osteoporosis and Mechanistic Insights into Its Effect on Bone Metabolism." Endocrine, Metabolic & Immune Disorders - Drug Targets 19, no. 3 (April 15, 2019): 259–73. http://dx.doi.org/10.2174/1871530318666181102115106.
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Background And Objective:Osteoporosis is a common bone disorder that increases susceptibility to fragility bone fractures. The clinical and public health repercussions of osteoporosis are huge due to the morbidity, mortality, and cost of medical care linked with fragility fractures. Clinical assessment of osteoporotic risk factors can help to identify candidates at an early stage that will benefit from medical intervention and potentially lowering the morbidity and mortality seen with fractures and complications. Given this, research is ongoing to evaluate the association of osteoporosis with some novel or less well-studied risk factors/bio-markers such as uric acid (UA).Discussion:Uric acid’s antioxidant activity has been proposed to be one of the factors responsible for increasing longevity and lowering rates of age-related cancers during primate evolution, the level of which increased markedly due to loss of uricase enzyme activity (mutational silencing). Accumulated evidence shows that oxidative stress is the fundamental mechanism of age-related bone loss and acts via enhancing osteoclastic activity and increasing bone resorption. Antioxidant substances such as ascorbic acid scavenge free radicals are positively related to bone health. Thus, it is hypothesized that uric acid holds bone-protective potential owing to its potent antioxidative property. Several correlation studies have been conducted globally to investigate the relationship between serum uric acid with bone mineral density and osteoporosis. Few pre-clinical studies have tried to investigate the interaction between uric acid and bone mineral density and reported important role played via Runt-related transcription factor 2 (RUNX2)/core-binding factor subunit alpha-1 (CBF-alpha-1), Wingless-related integration site (Wnt)-3a/β-catenin signaling pathway and 11β Hydroxysteroid Dehydrogenase type 1.Conclusion:In this review, the authors provided a comprehensive summary of the literature related to association studies reported in humans as well work done until date to understand the potential cellular and molecular mechanisms that interplay between uric acid and bone metabolism.
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Cammenga, Jorg, Gabriela Putz, Birte Niebuhr, Stefan Horn, Ulla Bergholz, Frank Buchholz, and Carol Stocking. "The Role of RUNX1 DNA-Binding Mutations in Acute Myeloid Leukemia." Blood 106, no. 11 (November 16, 2005): 1373. http://dx.doi.org/10.1182/blood.v106.11.1373.1373.
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Abstract The RUNX1 gene encodes an alpha subunit of the core-binding factor (CBF), an important heterodimeric transcription factor in hematopoietic ontogeny and development, and is one of the most frequently disrupted genes in acute leukemia. In addition to its involvement in several translocations, the RUNX1 gene is often subject to deletions or point mutations in acute myelogenous leukemia (AML). Interestingly, in addition to complete loss-of-function mutations, many of the alterations involve missense point mutations within the Runt domain that disrupt DNA binding activity (DB-mutants). In vitro assays have suggested that these DB mutants have a dominant-negative (DN) activity, presumably due to their ability to bind and sequester CBF beta but inability to bind DNA. A strict correlation between the type of mutation and its monoallelic or biallelic incidence is not apparent even though a DN mutant should only affect one allele while a loss of function mutation should affect both alleles. It has been hypothesized that loss of one allele (haploinsufficiency) is sufficient for loss of tumor suppressor activity but the relative high incidence of specific DB mutations suggests a more complex scenario. We thus sought to determine if expression of DB mutants in murine bone marrow (BM) resulted in a similar phenotype as the loss of Runx1, or if these mutations are associated with a gain-of-function. Two RUNX1-DB mutants were thus evaluated using the established retroviral transduction/transplantation mouse model. Between 3 and 6 months after transplantation, peripheral blood, spleen and BM cells were analyzed. Long-term repopulating cells expressing RUNX1 DB-mutants were able to contribute normally to both myeloid and lymphoid compartments, although a disproportionate increase in the B-cell compartment was observed in 3 out of 10 mice. Surprisingly, and inconsistent with a DN activity, disruption of normal T-cell or megakaryocytic development was not observed in the mice, in contrast to Runx1−/+ mice. Significantly, however, replating assays in vitro demonstrated that RUNX1-DB mutants lead to a significant increase in self-renewal activity, in contrast to BM cells of floxed Runx1 mice expressing the Cre recombinase, which showed a less dramatic effect on self-renewal. Colonies derived from CFU-Cs expressing RUNX1-DB mutants were composed of dysplastic granulocytic and monocytic cells, with an increasing number of immature blasts after multiple replatings (>7), whereas residual colonies from Runx1fl/− BM receiving CRE showed a different morphology with more mature cells. Thus our data suggest that RUNX1-DB mutants do not act in a dominant negative fashion to inhibit normal RUNX1 function, but impart a gain-of-function that results in impaired myeloid differentiation and increased self-renewal potential, consistent with its association with AML.
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Burda, Pavel, Nikola Curik, Juraj Kokavec, Dana Mikulenkova, Arthur Skoultchi, Jiri Zavadil, and Tomas Stopka. "PU.1 Relieves Its GATA-1-Mediated Repression near Cebpa and Cbfb During Transdifferentiation of Murine Erythroleukemia - Tool of Inducing Leukemic Blasts to Differentiate." Blood 114, no. 22 (November 20, 2009): 547. http://dx.doi.org/10.1182/blood.v114.22.547.547.
Full textAbstract:
Abstract Abstract 547 Transcription factors GATA-1 and PU.1 interact on DNA to block transcriptional programs of undesired lineage during hematopoietic commitment. Murine erythroleukemia (MEL) cells that co-express GATA-1 and PU.1 are blocked at the blast stage but respond to down-regulation of PU.1 or up-regulation of GATA-1 by inducing terminal erythroid differentiation. To test whether GATA-1 blocks PU.1 in MEL cells we have conditionally activated a transgenic PU.1 protein fused with the estrogen receptor ligand-binding domain (PUER), resulting in activation of a myeloid transcriptional program. Gene expression arrays identified components of the PU.1-dependent transcriptome negatively regulated by GATA-1 in MEL cells, including CCAAT/enhancer binding protein (C/EBP) alpha (Cebpa) and Core-binding factor, beta subunit (Cbfb) that encode two key hematopoietic transcription factors. Inhibition of GATA-1 by siRNA resulted in derepression of PU.1 target genes. Chromatin immunoprecipitation and reporter assays identified PU.1 motif sequences near Cebpa and Cbfb co-occupied by PU.1 and GATA-1 in the leukemic blasts. Substantial derepression of Cebpa and Cbfb is achieved in MEL cells by either activation of PU.1 or knockdown of GATA-1. Furthermore, transcriptional regulation of these loci by manipulating the levels of PU.1 and GATA-1 involves quantitative increases in a transcriptionally active chromatin mark: acetylation of histone H3K9. Collectively, we demonstrate that either activation of PU.1 or inhibition of GATA-1 efficiently reverse the transcriptional block imposed by GATA-1 and lead to activation of a myeloid transcriptional program directed by PU.1. The mechanism of PU.1 and GATA-1 in leukemic state and upon leukemia differentiation involves the following putative steps: at myeloid genes such as Cebpa, PU.1 binds directly to DNA but is repressed by GATA1 that binds directly to PU.1 molecules on DNA. Activation of PU.1-ER and stable levels of GATA-1 create excess of availabel PU.1, which is not paired by availabel GATA-1 on DNA, allowing thus gene activation. Similarly, on erythroid genes such as Nfe2, GATA1 is bound to DNA, but is repressed by PU.1 that binds to this GATA-1 molecule. Activation of GATA1-ER creates an excess of availabel GATA-1 which is not paired on DNA by availabel PU.1, also allowing gene activation. Our mechanistic study implicates that transcription factor manipulation, such as inhibition of GATA-1 or activation of PU.1 in erythroleukemias, may represent an efficient tool of inducing leukemic blasts to differentiate. (Grants NR9021-4, 10310-3, 2B06077) Disclosures: No relevant conflicts of interest to declare.
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Qi, Xiangjun, Hongbin Xu, Peng Zhang, Guoming Chen, Zhiqiang Chen, Caishan Fang, and Lizhu Lin. "Investigating the Mechanism of Scutellariae barbata Herba in the Treatment of Colorectal Cancer by Network Pharmacology and Molecular Docking." Evidence-Based Complementary and Alternative Medicine 2021 (August 2, 2021): 1–18. http://dx.doi.org/10.1155/2021/3905367.
Full textAbstract:
Background. Colorectal cancer (CRC) is one of the most common gastrointestinal tumors, which accounts for approximately 10% of all diagnosed cancers and cancer deaths worldwide per year. Scutellariae barbatae Herba (SBH) is one of the most frequently used traditional Chinese medicine (TCM) in the treatment of CRC. Although many experiments have been carried out to explain the mechanisms of SBH, the mechanisms of SBH have not been illuminated fully. Thus, we constructed a network pharmacology and molecular docking to investigate the mechanisms of SBH. Methods. We adopted active constituent prescreening, target predicting, protein-protein interaction (PPI) analysis, Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, differentially expressed gene analysis, and molecular docking to establish a system pharmacology database of SBH against CRC. Results. A total of 64 active constituents of SBH were obtained and 377 targets were predicted, and the result indicated that quercetin, luteolin, wogonin, and apigenin were the main active constituents of SBH. Glucocorticoid receptor (NR3C1), pPhosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (PIK3CA), cellular tumor antigen p53 (TP53), transcription factor AP-1 (JUN), mitogen-activated protein kinase 1 (MAPK1), Myc protooncogene protein (MYC), cyclin-dependent kinase 1 (CDK1), and broad substrate specificity ATP-binding cassette transporter ABCG2 (ABCG2) were the major targets of SBH in the treatment of CRC. GO analysis illustrated that the core biological process regulated by SBH was the regulation of the cell cycle. Thirty pathways were presented and 8 pathways related to CRC were involved. Molecular docking presented the binding details of 3 key targets with 6 active constituents. Conclusions. The mechanisms of SBH against CRC depend on the synergistic effect of multiple active constituents, multiple targets, and multiple pathways.
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Stopka, Tomas, Pavel Burda, Petra Basova, Karin Vargova, Nikola Curik, Anna Jonasova, and Marek Trneny. "5-Azacytidine and G-CSF Derepressed Chromatin Structure of PU.1 and Its Targets Cebpa and Cbfb In Myelodysplastic Syndrome (MDS)." Blood 116, no. 21 (November 19, 2010): 124. http://dx.doi.org/10.1182/blood.v116.21.124.124.
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Abstract Abstract 124 The myelodysplastic syndrome (MDS) represents a heterogeneous disorder characterized by ineffective hematopoiesis and evolution to acute myelogenous leukemia that is strikingly refractory to current therapeutic approaches. Novel epigenetic drugs including DNA-methyltransferase inhibitor 5-Azacitidine (5-AZA, Vidaza) are currently considered to improve clinical response in patients with MDS. MDS is characterized by abnormal differentiation and blocked maturation responsive to 5-AZA, therefore we studied major regulator of hematopoietic differentiation, transcription factor PU.1 as a candidate target of the epigenetic therapy. Transcription factor PU.1 represents very important myelo-lymphoid regulator of differentiation. PU.1 expression is regulated by Upstream Regulatory Element (URE) and its deletion in mouse caused downregulation of PU.1 leading to acute leukemia (Rosenbauer 2004). Our laboratory recently demonstrated that PU.1 in murine acute leukemic cells binds and promotes derepression of CCAAT/enhancer binding protein (C/EBP) alpha (Cebpa) and Core-binding factor, beta subunit (Cbfb) (Burda 2009) that encode two key hematopoietic transcription factors involved in myeloid differentiation. Furthermore, transcriptional regulation through PU.1 binding sites of Cebpa and Cbfb loci involves quantitative increases in a transcriptionally active chromatin mark: acetylation of histone H3 lysine K9. Others reported that Cebpa expression is augmented by G-CSF (Dahl 2003). To determine if 5-AZA regulates PU.1 and its targets we determined their expression and chromatin structure following the 5-AZA treatment in MDS patient-derived blasts and in cell lines derived from MDS (MOLM-13, OCI-M2, SKM-1) and AML (K562). Our data provide evidence that in the chosen cell lines and in so far limited number of patients-derived cells (N=4) the gene expression of PU.1 and its direct targets Cebpa and Cbfb is stimulated by 5-AZA and this effect is further enhanced by G-CSF. Furthermore, marks of activated chromatin structure including histone H3K9 hyperacetylation and H3K4 hypermethylation are increased at the URE of the PU.1 gene again documenting its transcriptional activation. Conversely, levels of H3K9 methylation at URE are significantly reduced upon 5-AZA treatment documenting 5-AZA stimulates loss of repressive chromatin structure near PU.1 gene. These observations are currently compared with responsiveness of the patients to 5-AZA in vivo and expanded to larger set of patients. Our data collectively supports importance of the chromatin structure upstream of PU.1 gene and of its direct targets Cebpa and Cbfb in patients with MDS that may add to better understanding of effectiveness of epigenetic therapy in MDS. (Grants # IGA 10310-3, MSMT 2B06077, SVV-2010-254260507, MPO FR-TI2/509, GAUK 251135 82210). Disclosures: No relevant conflicts of interest to declare.
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Sangle, Nikhil A., and Sherrie L. Perkins. "Core-Binding Factor Acute Myeloid Leukemia." Archives of Pathology & Laboratory Medicine 135, no. 11 (November 1, 2011): 1504–9. http://dx.doi.org/10.5858/arpa.2010-0482-rs.
Full textAbstract:
Core-binding factor acute myeloid leukemia (AML) is cytogenetically defined by the presence of t(8;21)(q22;q22) or inv(16)(p13q22)/t(16;16)(p13;q22), commonly abbreviated as t(8;21) and inv(16), respectively. In both subtypes, the cytogenetic rearrangements disrupt genes that encode subunits of core-binding factor, a transcription factor that functions as an essential regulator of normal hematopoiesis. The rearrangements t(8;21) and inv(16) involve the RUNX1/RUNX1T1 (AML1-ETO) and CBFB/MYH11 genes, respectively. These 2 subtypes are categorized as AML with recurrent genetic abnormalities, and hence the cytogenetic fusion transcripts are considered diagnostic of acute leukemia even when the marrow blast count is less than 20%. The t(8;21) and inv(16) subtypes of AML have been usually grouped and reported together in clinical studies; however, recent studies have demonstrated genetic, clinical, and prognostic differences, supporting the notion that they represent 2 distinct biologic and clinical entities. This review summarizes the spectrum of this subset of AMLs, with particular emphasis on molecular genetics and pathologic findings.
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Fang, Yue, Xinquan Liu, and Jing Su. "Network Pharmacology Analysis of Traditional Chinese Medicine Formula Shuang Di Shou Zhen Tablets Treating Nonexudative Age-Related Macular Degeneration." Evidence-Based Complementary and Alternative Medicine 2021 (March 24, 2021): 1–14. http://dx.doi.org/10.1155/2021/6657521.
Full textAbstract:
Objective. To analyze the pharmacological mechanism of the treatment of dry age-related macular degeneration (dry AMD) based on a network pharmacological approach of Shuang Di Shou Zhen Tablets (SDSZT) and to provide a new reference for the current lack of effective treatment of dry AMD. Methods. The main chemical constituents and their targets of Rehmanniae Radix Praeparata, Ligustrum lucidum, Mori Fructus, Paeonia albiflora, Rhizoma Dioscoreae, Alisma orientale, Schisandra chinensis, Radix Polygoni Multiflori Preparata, Ophiopogon japonicus, and Radix Rehmanniae were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and Traditional Chinese Medicine Integrated Database (TCMID). The active ingredients of traditional Chinese medicine were screened according to Absorption, Distribution, Metabolism, and Excretion (ADME), the gene names of the targets of each active ingredient were obtained from the Uniprot database, the main targets of dry AMD were obtained from GeneCards and DisGeNET database, and the protein interaction analysis was performed on the String database. The Metascape database was used to analyze the “drug-component-target” and the biological processes and networks involved, and then, Cytoscape 3.8.1 was used to construct the “ SDSZT component-dry AMD target-pathway” network. Results. The main active ingredients of SDSZT for dry AMD treatment are quercetin, kaempferol, luteolin, β-glutamine, β-carotene, etc. And, the core targets are RAC-alpha serine/threonine-protein kinase (AKT1), prostaglandin G/H synthase 1 (PTGS1), tumor necrosis factor (TNF), transcription factor AP-1 (JUN), apoptosis regulator Bcl-2 (BCL2), caspase-3 (CASP3), phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (PIK3CG), androgen receptor (AR), apoptosis regulator BAX (BAX), etc. The biological pathways for the treatment of age-related macular degeneration by SDSZT mainly act on pathways in cancer, fluid shear stress and atherosclerosis, and TNF signaling pathway, and the main function of SDSZT is to regulate intracellular cytokine receptor binding. Conclusion. This study initially reveals the multiconstituent, multitarget, and multipathway mechanism of action of SDSZT in the treatment of dry AMD and provides the basis for the clinical application of SDSZT.
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Gugneja, S., J. V. Virbasius, and R. C. Scarpulla. "Four structurally distinct, non-DNA-binding subunits of human nuclear respiratory factor 2 share a conserved transcriptional activation domain." Molecular and Cellular Biology 15, no. 1 (January 1995): 102–11. http://dx.doi.org/10.1128/mcb.15.1.102.
Full textAbstract:
Nuclear respiratory factor 2 (NRF-2) was previously purified to near homogeneity from HeLa cells on the basis of its ability to bind tandem recognition sites in the rat cytochrome oxidase subunit IV (RCO4) promoter. It consisted of five subunits, alpha, beta 1, beta 2, gamma 1, and gamma 2. Sequencing of tryptic peptides from alpha and from mixtures of the two beta or two gamma subunits revealed sequence identities with subunits of the mouse GA-binding protein (GABP), a ubiquitously expressed ETS domain activator composed of three subunits, alpha, beta 1, and beta 2. To understand the precise relationship between NRF-2 and GABP, cDNAs for all five NRF-2 subunits have now been cloned and their products have been overexpressed. The results establish that the two additional NRF-2 subunits are molecular variants that differ from GABP beta 1 and beta 2 by having a 12-amino-acid insertion containing two serine doublets. PCR and RNase protection assays show that mRNAs for these variants are expressed in the human but not the rodent cells and tissues examined. The insertion did not alter the ability of the beta and gamma subunits to associate with alpha, the DNA-binding subunit, nor did it affect the ability of NRF-2 beta 1 or beta 2 to direct high-affinity binding of alpha to tandem sites in the RCO4 promoter. In addition, the four NRF-2 beta and gamma subunits were equally proficient in activating transcription in transfected cells when fused to a GAL4 DNA-binding domain. The domain responsible for this transcriptional activation was localized by deletion mapping to a region of approximately 70 amino acids that is conserved in all four NRF-2 beta and gamma subunits. The repeated glutamine-containing hydrophobic clusters within this region bear a strong resemblance to those recently implicated in protein-protein interactions within the transcriptional apparatus.
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Shen, Y., M. Igo, P. Yalamanchili, A. J. Berk, and A. Dasgupta. "DNA binding domain and subunit interactions of transcription factor IIIC revealed by dissection with poliovirus 3C protease." Molecular and Cellular Biology 16, no. 8 (August 1996): 4163–71. http://dx.doi.org/10.1128/mcb.16.8.4163.
Full textAbstract:
Transcription factor IIIC (TFIIIC) is a general RNA polymerase III transcription factor that binds the B-box internal promotor element of tRNA genes and the complex of TFIIIA with a 5S rRNA gene. TFIIIC then directs the binding of TFIIIB to DNA upstream of the transcription start site. TFIIIB in turn directs RNA polymerase III binding and initiation. Human TFIIIC contains five different subunits. The 243-kDa alpha subunit can be specifically cross-linked to B-box DNA, but its sequence does not reveal a known DNA binding domain. During poliovirus infection, TFIIIC is cleaved and inactivated by the poliovirus-encoded 3C protease (3Cpro). Here we analyzed the cleavage of TFIIIC subunits by 3Cpro in vitro and during poliovirus infection of HeLa cells. Analyses of the DNA binding activities of the resulting subcomplexes indicated that an N-terminal 83-kDa domain of the alpha subunit associates with the beta subunit to generate the TFIIIC DNA binding domain. Cleavage with 3Cpro also generated an approximately 125-kDa C-terminal fragment of the alpha subunit which remained associated with the gamma and epsilon subunits.
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Doshi, PD, and JF DiPersio. "Three conserved motifs in the extracellular domain of the human granulocyte-macrophage colony-stimulating factor receptor subunit are essential for ligand binding and surface expression." Blood 84, no. 8 (October 15, 1994): 2539–53. http://dx.doi.org/10.1182/blood.v84.8.2539.2539.
Full textAbstract:
Abstract The receptor for the human granulocyte-macrophage colony-stimulating factor (GM-CSF) (GM-R) is a heterodimeric complex consisting of two subunits, GM-R alpha and GM-R beta. Structural analyses have shown a number of highly conserved amino acid motifs present in both GM-R alpha and GM-R beta. These motifs include QYFLY, CXW, XW, and WSXWS motifs in the extracellular domain; a conserved cysteine in the transmembrane domain; and the entire cytoplasmic domain, including the LXVLX box in the carboxy terminal region of the cytoplasmic domain. We have investigated the role of these motifs in GM-R alpha by examining the effects of specific motif mutations on ligand binding and surface expression. Transient expression of these mutant GM-R alpha subunits in COS cells shows that these extracellular motis are essential for ligand binding. Alterations of the cytoplasmic region of GM-R alpha do not alter GM-CSF binding or the reconstitution of high-affinity receptors when coexpressed with GM-R beta. Permeabilization and immunostaining of cells transfected with mutant GM-R alpha subunits yields data suggesting that each of the mutant subunits is present in the cytoplasm. Immunostaining of both intact and permeabilized COS cells transiently transfected with wild-type or mutant GM-R alpha s showed that extracellular domain mutants accumulated in the cytoplasm and were not efficiently transported to the cell surface.
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Doshi, PD, and JF DiPersio. "Three conserved motifs in the extracellular domain of the human granulocyte-macrophage colony-stimulating factor receptor subunit are essential for ligand binding and surface expression." Blood 84, no. 8 (October 15, 1994): 2539–53. http://dx.doi.org/10.1182/blood.v84.8.2539.bloodjournal8482539.
Full textAbstract:
The receptor for the human granulocyte-macrophage colony-stimulating factor (GM-CSF) (GM-R) is a heterodimeric complex consisting of two subunits, GM-R alpha and GM-R beta. Structural analyses have shown a number of highly conserved amino acid motifs present in both GM-R alpha and GM-R beta. These motifs include QYFLY, CXW, XW, and WSXWS motifs in the extracellular domain; a conserved cysteine in the transmembrane domain; and the entire cytoplasmic domain, including the LXVLX box in the carboxy terminal region of the cytoplasmic domain. We have investigated the role of these motifs in GM-R alpha by examining the effects of specific motif mutations on ligand binding and surface expression. Transient expression of these mutant GM-R alpha subunits in COS cells shows that these extracellular motis are essential for ligand binding. Alterations of the cytoplasmic region of GM-R alpha do not alter GM-CSF binding or the reconstitution of high-affinity receptors when coexpressed with GM-R beta. Permeabilization and immunostaining of cells transfected with mutant GM-R alpha subunits yields data suggesting that each of the mutant subunits is present in the cytoplasm. Immunostaining of both intact and permeabilized COS cells transiently transfected with wild-type or mutant GM-R alpha s showed that extracellular domain mutants accumulated in the cytoplasm and were not efficiently transported to the cell surface.
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Coats, S. R., H. D. Love, and W. J. Pledger. "Platelet-derived growth factor (PDGF)-AB-mediated phosphorylation of PDGF β receptors." Biochemical Journal 297, no. 2 (January 15, 1994): 379–84. http://dx.doi.org/10.1042/bj2970379.
Full textAbstract:
Platelet-derived growth factor (PDGF) stimulates the proliferation of Balb/c-3T3 fibroblasts through binding and subsequent activation of PDGF receptors. Activation of the PDGF receptors has been proposed to involve receptor dimerization. PDGF-AB has been shown to bind PDGF alpha and beta receptor subunits to form PDGF alpha beta and alpha alpha receptor dimers. In this paper we demonstrate that, following the down-regulation of PDGF alpha receptors, the binding of PDGF-AB to beta receptors occurred at 37 degrees C but not at 4 degrees C. PDGF-AB stimulated the phosphorylation of PDGF beta receptor monomers in cells depleted of PDGF alpha receptors by prior exposure to PDGF-AA.
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Takahashi, A., M. Satake, Y. Yamaguchi-Iwai, SC Bae, J. Lu, M. Maruyama, YW Zhang, H. Oka, N. Arai, and K. Arai. "Positive and negative regulation of granulocyte-macrophage colony- stimulating factor promoter activity by AML1-related transcription factor, PEBP2." Blood 86, no. 2 (July 15, 1995): 607–16. http://dx.doi.org/10.1182/blood.v86.2.607.bloodjournal862607.
Full textAbstract:
The granulocyte-macrophage colony-stimulating factor (GM-CSF) gene promoter contains a consensus sequence for the polyomavirus enhancer binding-protein 2 (PEBP2) transcription factor, which consists of alpha and beta subunits. There are at least two genes, alpha A and alpha B, encoding the alpha subunit. alpha B is the mouse homologue of human AML1 gene detected at the breakpoints of t(8;21) and t(3;21) myeloid leukemias. We examined alpha A1 (an alpha A-gene product) and alpha B1 and alpha B2 (two alpha B-encoded isomers) for their effects on the GM- CSF promoter. PEBP2 alpha A1, alpha B1, and alpha B2 proteins bound the PEBP2 site within the mouse GM-CSF promoter. PEBP2 alpha A1 and alpha B1 enhanced the expression of the GM-CSF promoter-driven reporter plasmid in unstimulated and 12-O-tetradecanoylphorbol 13- acetate/phytohemagglutinin-stimulated human Jurkat T cells. In contrast, the promoter activity was suppressed by alpha B2. Coexpression of alpha B1 and alpha B2 showed that the promoter activity could be determined by the alpha B1/alpha B2 ratio. Jurkat cell extract contained PEBP2 site-binding protein(s) that cross-reacted with antimouse alpha A1 antibodies. Northern blot analysis indicated the expression of human PEBP2 alpha A, alpha B (AML1), and beta genes in Jurkat cells. Although further studies are required to determine the precise role of PEBP2 in the GM-CSF promoter activity, the present findings suggested the importance of the relative ratio of different PEBP2 isoforms in regulating the levels of the promoter activity.
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Weaver, Brian K., K. Prasanna Kumar, and Nancy C. Reich. "Interferon Regulatory Factor 3 and CREB-Binding Protein/p300 Are Subunits of Double-Stranded RNA-Activated Transcription Factor DRAF1." Molecular and Cellular Biology 18, no. 3 (March 1, 1998): 1359–68. http://dx.doi.org/10.1128/mcb.18.3.1359.
Full textAbstract:
ABSTRACT Cells respond to viral infection or double-stranded RNA with the transcriptional induction of a subset of alpha/beta interferon-stimulated genes by a pathway distinct from the interferon signal pathway. The transcriptional induction is mediated through a DNA sequence containing the alpha/beta interferon-stimulated response element (ISRE). We previously identified a novel transcription factor, designated double-stranded RNA-activated factor 1 (DRAF1), that recognizes this response element. The DNA-binding specificity of DRAF1 correlates with transcriptional induction, thereby distinguishing it as a positive regulator of alpha/beta interferon-stimulated genes. Two of the components of DRAF1 have now been identified as interferon regulatory factor 3 (IRF-3) and the transcriptional coactivator CREB-binding protein (CBP)/p300. We demonstrate that IRF-3 preexists in the cytoplasm of uninfected cells and translocates to the nucleus following viral infection. Translocation of IRF-3 is accompanied by an increase in serine and threonine phosphorylation. Coimmunoprecipitation analyses of endogenous proteins demonstrate an association of IRF-3 with the transcriptional coactivators CBP and p300 only subsequent to infection. In addition, antibodies to the IRF-3, CBP, and p300 molecules react with DRAF1 bound to the ISRE target site of induced genes. The cellular response that leads to DRAF1 activation and specific gene expression may serve to increase host survival during viral infection.
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Guo, Yalin, Laleh Talebian, Ivan Maillard, Caroline L. Speck, Warren S. Pear, Ellen V. Rothenberg, and Nancy A. Speck. "Reduction of Core Binding Factor beta (CBFβ) Dosage Blocks T Cell Development." Blood 106, no. 11 (November 16, 2005): 2714. http://dx.doi.org/10.1182/blood.v106.11.2714.2714.
Full textAbstract:
Abstract Core binding factors (CBFs) are heterodimers consisting of a DNA binding subunit (Runx1, Runx2, or Runx3) and a non-DNA binding CBFβ subunit. CBFβ increases the affinity of the Runx subunits for DNA. Embryos deficient for Runx1 or CBFβ die at midgestation with a complete failure of definitive hematopoiesis due to a block in hematopoietic stem cell (HSC) emergence. To examine the role of core binding factors at later stages of hematopoiesis, we generated a hypomorphic Cbfb allele (Cbfbrss), that when carried over a Cbfb null allele (Cbfbrss/−) results in a 3-4 fold reduction in CBFβ protein levels. Although HSCs emerge in Cbfbrss/− animals, fetal T cell development is severely impaired. Here we examined the T cell developmental block in more detail by culturing fetal liver cells from Cbfbrss/− animals on OP9 stromal cells that express the Notch ligand Delta-like-1 (DL1) (Schmitt and Zúñiga-Pflücker, Immunity17: 749, 2002). Fetal livers (E14.5) from Cbfbrss/− animals contained normal numbers of both c-kit+Sca-1+lin- cells and c-kit+IL7r+ lymphoid progenitors. Lin- fetal liver progenitors cultured on OP9-DL1 cells in the presence of IL-7 and Flt3L displayed a growth disadvantage relative to wild type cells, and a block at the double negative 1 (DN1, CD44+ CD25−) stage of T cell development. The T cell defect could be rescued by retroviral transduction of the CBFβ heterodimerization domain into lin- fetal liver cells, but not by a G61A/N104A mutant that cannot bind the Runx subunits. Genes whose expression was decreased in DN1 cells purified from the OP9-DL1 cultures included CD3 and the early T cell transcription factors GATA3 and TCF. Although expression of several Notch pathway genes (Notch1, Hes-1/5, Deltex-1) was mildly decreased, Notch signals were clearly transduced, suggesting that Notch signaling was intact. These data demonstrate that reduced CBFβ levels impair the differentiation of stem cells/progenitors into T cells at the earliest stage of T cell development. This in vitro model will be useful for characterizing the molecular circuitry involving CBFβ in T cell development, and for identifying CBFβ protein partners.
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Collart, M. A., P. Baeuerle, and P. Vassalli. "Regulation of tumor necrosis factor alpha transcription in macrophages: involvement of four kappa B-like motifs and of constitutive and inducible forms of NF-kappa B." Molecular and Cellular Biology 10, no. 4 (April 1990): 1498–506. http://dx.doi.org/10.1128/mcb.10.4.1498.
Full textAbstract:
This study characterizes the interaction of murine macrophage nuclear proteins with the tumor necrosis factor alpha (TNF-alpha) promoter. Gel retardation and methylation interference assays showed that stimulation of TNF-alpha gene transcription in peritoneal exudate macrophages was accompanied by induction of DNA-binding proteins that recognized with different affinities four elements related to the kappa B consensus motif and a Y-box motif. We suggest that the basal level of TNF-alpha expression in macrophages is due to the binding of a constitutive form of NF-kappa B, present at low levels in nuclei from resting thioglycolate exudate peritoneal macrophages, to some if not all of the kappa B motifs; we postulate that this constitutive form contains only the 50-kilodalton (kDa) DNA-binding protein subunits of NF-kappa B, not the 65-kDa protein subunits (P. Baeuerle and D. Baltimore, Genes Dev. 3:1689-1698, 1989). Agents such as glucocorticoids, which decrease TNF-alpha transcription, diminished the basal level of nuclear NF-kappa B. Stimulation of Stimulation of TNF-alpha transcription in macrophages by lipopolysaccharide, gamma interferon, or cycloheximide led to an increased content of nuclear NF-kappa B. This induced factor represents a different form of NF-kappa B, since it generated protein-DNA complexes of slower mobility; we propose that this induced form of NF-kappa B contains both the 50- and 65-kDa protein subunits, the latter ones being necessary to bind NF-kappa B to its cytoplasmic inhibitor in uninduced cells (Baeuerle and Baltimore, Genes Dev., 1989). In resting cells, this inducible form of NF-kappa B was indeed detectable in the cytosol after deoxycholate treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
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Collart, M. A., P. Baeuerle, and P. Vassalli. "Regulation of tumor necrosis factor alpha transcription in macrophages: involvement of four kappa B-like motifs and of constitutive and inducible forms of NF-kappa B." Molecular and Cellular Biology 10, no. 4 (April 1990): 1498–506. http://dx.doi.org/10.1128/mcb.10.4.1498-1506.1990.
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This study characterizes the interaction of murine macrophage nuclear proteins with the tumor necrosis factor alpha (TNF-alpha) promoter. Gel retardation and methylation interference assays showed that stimulation of TNF-alpha gene transcription in peritoneal exudate macrophages was accompanied by induction of DNA-binding proteins that recognized with different affinities four elements related to the kappa B consensus motif and a Y-box motif. We suggest that the basal level of TNF-alpha expression in macrophages is due to the binding of a constitutive form of NF-kappa B, present at low levels in nuclei from resting thioglycolate exudate peritoneal macrophages, to some if not all of the kappa B motifs; we postulate that this constitutive form contains only the 50-kilodalton (kDa) DNA-binding protein subunits of NF-kappa B, not the 65-kDa protein subunits (P. Baeuerle and D. Baltimore, Genes Dev. 3:1689-1698, 1989). Agents such as glucocorticoids, which decrease TNF-alpha transcription, diminished the basal level of nuclear NF-kappa B. Stimulation of Stimulation of TNF-alpha transcription in macrophages by lipopolysaccharide, gamma interferon, or cycloheximide led to an increased content of nuclear NF-kappa B. This induced factor represents a different form of NF-kappa B, since it generated protein-DNA complexes of slower mobility; we propose that this induced form of NF-kappa B contains both the 50- and 65-kDa protein subunits, the latter ones being necessary to bind NF-kappa B to its cytoplasmic inhibitor in uninduced cells (Baeuerle and Baltimore, Genes Dev., 1989). In resting cells, this inducible form of NF-kappa B was indeed detectable in the cytosol after deoxycholate treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
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Borthakur, Gautam, Nitin Jain, Hagop Kantarjian, E. Lin, Farhad Ravandi, Sherry Pierce, and Elihu Estey. "Survival Is Poorer in Patients with Secondary Core Binding Factor Acute Myelogenous Leukemia Compared with De Novo Core Binding Factor Leukemia." Blood 110, no. 11 (November 16, 2007): 2856. http://dx.doi.org/10.1182/blood.v110.11.2856.2856.
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Abstract Translocation (8;21)(q22;q22) and inversion of chromosome 16 [inv(16) (p13q22)] are considered good-risk cytogenetic abnormalities in acute myeloid leukemia (AML) accounting for 15% of primary AML cases (Blood. 2002 Dec 15;100(13):4325–36) and are characterized at the molecular level by disruption of genes encoding subunits of core binding factor (CBF). Increasing age, increasing peripheral blast percentage and complex cytogenetics are associated with poor overall survival in these patients (Br J Haematol. 2006 Oct;135(2):165–73). Here we assess differences in patient characteristics and outcomes between the primary and secondary core binding factor AML. One hundred eighty nine CBF AML patients treated at our institution were studied; 18 (9.5%) had secondary AML. Patients with secondary CBF AML were older (p= 0.02, median ages 57 vs. 44 years) and had lower WBC counts (p=0.03) (Table1). Overall survival (OS) was worse in the secondary AML patients (94 weeks versus 621 weeks, p=0.0016) (Figure 1). Age, hemoglobin, platelet count, and bilirubin were significantly associated with OS in the univariate analysis. In the multivariate analysis, after adjusting for age, hemoglobin, WBC, and bilirubin, secondary CBF AML was marginally significantly associated with worse OS (hazard ratio 1.884, CI95% 0.979–3.625, p=0.058) but not worse PFS (p= 0.15) (Table 2). These data suggest that the secondary CBF AML has much poorer prognosis than the primary CBF AML, further indicating that “CBF AML” is not a homogeneous entity with a uniformly good prognosis. Table 1: Summary statistics of patients’ continuous characteristics by abnormality status Patient characteristics Total patients Primary AML (n=171, male=96) Secondary AML (n=18, male=8) p-value (primary vs secondary AML) Median (range) Median (range) Median (range) Age (years) 44 (16–88) 44 (16–88) 57 (31–75) 0.02 WBC (103/mm3) 14.7 (0.6–387) 16.4 (0.6–387) 6.6 (0.8–103.8) 0.03 Hemoglobin (g/dl) 8.1 (2.5–14.3) 8.2 (2.5–14.3) 7.8 (4.8–10.8) 0.33 Platelet count (103/mm3) 38 (5–350) 38 (5–350) 39 (9–139) 0.88 Bilirubin (mg/dl) 0.6 (0.0–15.3) 0.6 (0.0–5.0) 0.6 (0.1–15.3) 0.94 Creatinine (mg/dl) 0.9 (0.5–2.9) 0.9 (0.5–2.9) 0.9 (0.5–1.8) 0.66 Table 2: Multivariate Cox proportional hazards model in estimating the association between covariates and patients’ survival Variable Hazard ratio (95% CI) p-value Abnormality (secondary vs primary) 1.884 (0.979–3.625) 0.058 Age (1 year increase) 1.028 (1.012–1.044) 0.0004 Hemoglobin (1gm/dl increase) 0.864 (0.769–0.970) 0.014 WBC (103/mm3 increase) 1.003 (1.000–1.007) 0.061 Bilirubin (1 mg/dl increase) 1.179 (1.044–1.332) 0.008 Figure Figure
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Hirst, S. J., P. J. Barnes, and C. H. Twort. "PDGF isoform-induced proliferation and receptor expression in human cultured airway smooth muscle cells." American Journal of Physiology-Lung Cellular and Molecular Physiology 270, no. 3 (March 1, 1996): L415—L428. http://dx.doi.org/10.1152/ajplung.1996.270.3.l415.
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The effect of recombinant platelet-derived growth factor (PDGF) isoforms (PDGF-AA, -BB and -AB) on mitogenesis of human cultured airway smooth muscle (ASM) was examined using the MTT-reduction assay and [3H]thymidine incorporation. Results were correlated with expression of PDGF receptor (PDGFR)-alpha and -beta subunits in the absence and presence of fetal calf serum (FCS). When FCS was absent PDGF-AB and -BB were potent mitogens, whereas PDGF-AA was weakly mitogenic, evoking <20% of the maximum response induced by the B-chain isoforms. When FCS (2.5%) was present, all PDGF isoforms stimulated marked ASM proliferation with similar efficacy and potency. Cross-competition binding analysis in FCS-deprived cells revealed that ASM cells in culture express mainly PDGFR-beta. Preincubation with PDGF-AA or PDGFR-alpha neutralizing antiserum abolished PDGF-AA binding and decreased total receptor number by approximately 15%. The ratio of PDGFR-alpha to beta subunits was approximately 1:8, supported by intense immunofluorescence staining for PDGFR-beta and weak staining for PDGFR-alpha. In parallel studies, uptake of [3H]thymidine stimulated by PDGF-AA, but not PDGF-AB or -BB, was inhibited by PDGFR-alpha immobilization. Western immunoblot analysis confirmed expression of mature PDGFR-alpha and -beta subunits in ASM cells. FCS did not cause any detectable increase in PDGFR-alpha expression or in PDGF-AA binding. These data support a role for PDGFR-beta mediating ASM mitogenesis during FCS-free conditions, but in the presence of FCS, both PDGFR-alpha and -beta subunits are linked to mitogenesis. The enhanced mitogenicity of PDGF-AA in the presence of FCS was independent of any detectable upregulation of PDGFR-alpha, suggesting that the inability of PDGF-AA to promote mitogenesis in the absence of FCS is not simply due to relative numbers of PDGFR-alpha and PDGFR-beta.
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Weiss, M., C. Yokoyama, Y. Shikama, C. Naugle, B. Druker, and CA Sieff. "Human granulocyte-macrophage colony-stimulating factor receptor signal transduction requires the proximal cytoplasmic domains of the alpha and beta subunits." Blood 82, no. 11 (December 1, 1993): 3298–306. http://dx.doi.org/10.1182/blood.v82.11.3298.3298.
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Abstract Human granulocyte-macrophage colony-stimulating factor (GM-CSF) controls the production, maturation, and function of cells in multiple hematopoietic lineages. These effects are mediated by a cell-surface receptor (GM-R) composed of alpha and beta subunits, each containing 378 and 881 amino acids, respectively. Whereas the alpha subunit exists as several isoforms that bind GM-CSF with low affinity, the beta common subunit (beta c) does not bind GM-CSF itself, but acts as a high- affinity converter for GM-CSF, interleukin-3 (IL-3), and IL-5 receptor alpha subunits. The cytoplasmic region of GM-R alpha consists of a membrane-proximal conserved region shared by the alpha 1 and alpha 2 isoforms and a C-terminal variable region that is divergent between alpha 1 and alpha 2. The cytoplasmic region of beta c contains membrane proximal serine and acidic domains. To investigate the amino acid sequences that influence signal transduction by this receptor complex, we constructed a series of cytoplasmic truncation mutants of the alpha 2 and beta subunits. To study these truncations, we stably transfected the IL-3-dependent murine cell line Ba/F3 with wild-type or mutant cDNAs. We found that the wild-type and mutant alpha subunits conferred similar low-affinity binding sites for human GM-CSF to Ba/F3, and the wild-type or mutant beta subunit converted some of these sites to high- affinity; the cytoplasmic domain of beta was unnecessary for this high- affinity conversion. Proliferation assays showed that the membrane- proximal conserved region of GM-R alpha and the serine-acidic domain of beta c are required for both cell proliferation and ligand-dependent phosphorylation of a 93-kD cytoplasmic protein. We suggest that these regions may represent an important signal transduction motif present in several cytokine receptors.
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Weiss, M., C. Yokoyama, Y. Shikama, C. Naugle, B. Druker, and CA Sieff. "Human granulocyte-macrophage colony-stimulating factor receptor signal transduction requires the proximal cytoplasmic domains of the alpha and beta subunits." Blood 82, no. 11 (December 1, 1993): 3298–306. http://dx.doi.org/10.1182/blood.v82.11.3298.bloodjournal82113298.
Full textAbstract:
Human granulocyte-macrophage colony-stimulating factor (GM-CSF) controls the production, maturation, and function of cells in multiple hematopoietic lineages. These effects are mediated by a cell-surface receptor (GM-R) composed of alpha and beta subunits, each containing 378 and 881 amino acids, respectively. Whereas the alpha subunit exists as several isoforms that bind GM-CSF with low affinity, the beta common subunit (beta c) does not bind GM-CSF itself, but acts as a high- affinity converter for GM-CSF, interleukin-3 (IL-3), and IL-5 receptor alpha subunits. The cytoplasmic region of GM-R alpha consists of a membrane-proximal conserved region shared by the alpha 1 and alpha 2 isoforms and a C-terminal variable region that is divergent between alpha 1 and alpha 2. The cytoplasmic region of beta c contains membrane proximal serine and acidic domains. To investigate the amino acid sequences that influence signal transduction by this receptor complex, we constructed a series of cytoplasmic truncation mutants of the alpha 2 and beta subunits. To study these truncations, we stably transfected the IL-3-dependent murine cell line Ba/F3 with wild-type or mutant cDNAs. We found that the wild-type and mutant alpha subunits conferred similar low-affinity binding sites for human GM-CSF to Ba/F3, and the wild-type or mutant beta subunit converted some of these sites to high- affinity; the cytoplasmic domain of beta was unnecessary for this high- affinity conversion. Proliferation assays showed that the membrane- proximal conserved region of GM-R alpha and the serine-acidic domain of beta c are required for both cell proliferation and ligand-dependent phosphorylation of a 93-kD cytoplasmic protein. We suggest that these regions may represent an important signal transduction motif present in several cytokine receptors.
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Tang, Yen-Yee, Barbara E. Crute, John J. Kelley, Xuemei Huang, Jiangli Yan, Jianxia Shi, Kari L. Hartman, Thomas M. Laue, Nancy A. Speck, and John H. Bushweller. "Biophysical characterization of interactions between the core binding factor α and β subunits and DNA." FEBS Letters 470, no. 2 (March 20, 2000): 167–72. http://dx.doi.org/10.1016/s0014-5793(00)01312-0.
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Lu, J., M. Maruyama, M. Satake, S. C. Bae, E. Ogawa, H. Kagoshima, K. Shigesada, and Y. Ito. "Subcellular localization of the alpha and beta subunits of the acute myeloid leukemia-linked transcription factor PEBP2/CBF." Molecular and Cellular Biology 15, no. 3 (March 1995): 1651–61. http://dx.doi.org/10.1128/mcb.15.3.1651.
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Each of the two human genes encoding the alpha and beta subunits of a heterodimeric transcription factor, PEBP2, has been found at the breakpoints of two characteristic chromosome translocations associated with acute myeloid leukemia, suggesting that they are candidate proto-oncogenes. Polyclonal antibodies against the alpha and beta subunits of PEBP2 were raised in rabbits and hamsters. Immunofluorescence labeling of NIH 3T3 cells transfected with PEBP2 alpha and -beta cDNAs revealed that the full-size alpha A1 and alpha B1 proteins, the products of two related but distinct genes, are located in the nucleus, while the beta subunit is localized to the cytoplasm. Deletion analysis demonstrated that there are two regions in alpha A1 responsible for nuclear accumulation of the protein: one mapped in the region between amino acids 221 and 513, and the other mapped in the Runt domain (amino acids 94 to 221) harboring the DNA-binding and the heterodimerizing activities. When the full-size alpha A1 and beta proteins are coexpressed in a single cell, the former is present in the nucleus and the latter still remains in the cytoplasm. However, the N- or C-terminally truncated alpha A1 proteins devoid of the region upstream or downstream of the Runt domain colocalized with the beta protein in the nucleus. In these cases, the beta protein appeared to be translocated into the nucleus passively by binding to alpha A1. The chimeric protein containing the beta protein at the N-terminal region generated as a result of the inversion of chromosome 16 colocalized with alpha A1 to the nucleus more readily than the normal beta protein. The implications of these results in relation to leukemogenesis are discussed.
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Veals, S. A., C. Schindler, D. Leonard, X. Y. Fu, R. Aebersold, J. E. Darnell, and D. E. Levy. "Subunit of an alpha-interferon-responsive transcription factor is related to interferon regulatory factor and Myb families of DNA-binding proteins." Molecular and Cellular Biology 12, no. 8 (August 1992): 3315–24. http://dx.doi.org/10.1128/mcb.12.8.3315.
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Alpha interferon stimulates transcription by converting the positive transcriptional regulator ISGF3 from a latent to an active form. This receptor-mediated event occurs in the cytoplasm, with subsequent translocation of the activated factor to the nucleus. ISGF3 has two components, termed ISGF3 alpha and ISGF3 gamma. ISGF3 gamma serves as the DNA recognition subunit, while ISGF3 alpha, which appears to consist of three polypeptides, is a target for alpha interferon signaling and serves as a regulatory component whose activation is required to form ISGF3. ISGF3 gamma DNA-binding activity was identified as a 48-kDa polypeptide, and partial amino acid sequence has allowed isolation of cDNA clones. ISGF3 gamma translated in vitro from recombinant clones bound DNA with a specificity indistinguishable from that of ISGF3 gamma purified from HeLa cells. Sequencing of ISGF3 gamma cDNA clones revealed significant similarity to the interferon regulatory factor (IRF) family of DNA binding proteins in the amino-terminal 117 residues of ISGF3 gamma. The other IRF family proteins bind DNA with a specificity related to but distinct from that of ISGF3 gamma. We note sequence similarities between the related regions of IRF family proteins and the imperfect tryptophan repeats which constitute the DNA-binding domain of the c-myb oncoprotein. These sequence similarities suggest that ISGF3 gamma and IRF proteins and the c-myb oncoprotein use a common structural motif for DNA recognition. Recombinant ISGF3 gamma, like the natural protein, interacted with HeLa cell ISGF3 alpha to form the mature ISGF3 DNA-binding complex. We suggest that other IRF family members may participate in signaling pathways by interacting with as yet unidentified regulatory subunits analogous to ISGF3 alpha.
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Veals, S. A., C. Schindler, D. Leonard, X. Y. Fu, R. Aebersold, J. E. Darnell, and D. E. Levy. "Subunit of an alpha-interferon-responsive transcription factor is related to interferon regulatory factor and Myb families of DNA-binding proteins." Molecular and Cellular Biology 12, no. 8 (August 1992): 3315–24. http://dx.doi.org/10.1128/mcb.12.8.3315-3324.1992.
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Alpha interferon stimulates transcription by converting the positive transcriptional regulator ISGF3 from a latent to an active form. This receptor-mediated event occurs in the cytoplasm, with subsequent translocation of the activated factor to the nucleus. ISGF3 has two components, termed ISGF3 alpha and ISGF3 gamma. ISGF3 gamma serves as the DNA recognition subunit, while ISGF3 alpha, which appears to consist of three polypeptides, is a target for alpha interferon signaling and serves as a regulatory component whose activation is required to form ISGF3. ISGF3 gamma DNA-binding activity was identified as a 48-kDa polypeptide, and partial amino acid sequence has allowed isolation of cDNA clones. ISGF3 gamma translated in vitro from recombinant clones bound DNA with a specificity indistinguishable from that of ISGF3 gamma purified from HeLa cells. Sequencing of ISGF3 gamma cDNA clones revealed significant similarity to the interferon regulatory factor (IRF) family of DNA binding proteins in the amino-terminal 117 residues of ISGF3 gamma. The other IRF family proteins bind DNA with a specificity related to but distinct from that of ISGF3 gamma. We note sequence similarities between the related regions of IRF family proteins and the imperfect tryptophan repeats which constitute the DNA-binding domain of the c-myb oncoprotein. These sequence similarities suggest that ISGF3 gamma and IRF proteins and the c-myb oncoprotein use a common structural motif for DNA recognition. Recombinant ISGF3 gamma, like the natural protein, interacted with HeLa cell ISGF3 alpha to form the mature ISGF3 DNA-binding complex. We suggest that other IRF family members may participate in signaling pathways by interacting with as yet unidentified regulatory subunits analogous to ISGF3 alpha.
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Larson, R. S., A. L. Corbi, L. Berman, and T. Springer. "Primary structure of the leukocyte function-associated molecule-1 alpha subunit: an integrin with an embedded domain defining a protein superfamily." Journal of Cell Biology 108, no. 2 (February 1, 1989): 703–12. http://dx.doi.org/10.1083/jcb.108.2.703.
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The leukocyte function-associated molecule 1 (LFA-1, CD11a/CD18) is a membrane glycoprotein which functions in cell-cell adhesion by heterophilic interaction with intercellular adhesion molecule 1 (ICAM-1). LFA-1 consists of an alpha subunit (Mr = 180,000) and a beta subunit (Mr = 95,000). We report the molecular biology and protein sequence of the alpha subunit. Overlapping cDNAs containing 5,139 nucleotides were isolated using an oligonucleotide specified by tryptic peptide sequence. The mRNA of 5.5 kb is expressed in lymphoid and myeloid cells but not in a bladder carcinoma cell line. The protein has a 1,063-amino acid extracellular domain, a 29-amino acid transmembrane region, and a 53-amino acid cytoplasmic tail. The extracellular domain contains seven repeats. Repeats V-VII are in tandem and contain putative divalent cation binding sites. LFA-1 has significant homology to the members of the integrin superfamily, having 36% identity with the Mac-1 and p150,95 alpha subunits and 28% identity with other integrin alpha subunits. An insertion of approximately 200 amino acids is present in the NH2-terminal region of LFA-1. This "inserted/interactive" or I domain is also present in the p150,95 and Mac-1 alpha subunits but is absent from other integrin alpha subunits sequenced to date. The I domain has striking homology to three repeats in human von Willebrand factor, two repeats in chicken cartilage matrix protein, and a region of complement factor B. These structural features indicate a bipartite evolution from the integrin family and from an I domain family. These features may also correspond to relevant functional domains.
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Brioschi, Matteo, John Fischer, Roberto Cairoli, Stefano Rossetti, Laura Pezzetti, Michele Nichelatti, Mauro Turrini, et al. "Down-regulation of MicroRNAs 222/221 in Acute Myelogenous Leukemia with Deranged Core-Binding Factor Subunits." Neoplasia 12, no. 11 (November 2010): 866—IN3. http://dx.doi.org/10.1593/neo.10482.
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Geering, K., P. Jaunin, F. Jaisser, A. M. Merillat, J. D. Horisberger, P. M. Mathews, V. Lemas, D. M. Fambrough, and B. C. Rossier. "Mutation of a conserved proline residue in the beta-subunit ectodomain prevents Na(+)-K(+)-ATPase oligomerization." American Journal of Physiology-Cell Physiology 265, no. 4 (October 1, 1993): C1169—C1174. http://dx.doi.org/10.1152/ajpcell.1993.265.4.c1169.
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A highly conserved sequence motif (4 tyrosines and 1 proline: YYPYY) of the Na(+)-K(+)-adenosinetriphosphatase (ATPase) beta 1-subunit ectodomain has been mutagenized to study its possible role in alpha/beta-assembly and sodium pump function. Single as well as double tyrosine mutants (tyrosine to phenylalanine: Y to F) of Xenopus laevis beta 1-subunits are able to associate with alpha 1-subunits and form functional Na-K pumps at the plasma membrane that are indistinguishable from wild-type alpha 1, beta 1-Na-K pumps (as assessed by measurements of ouabain binding, 86Rb flux, Na-K pump current, and activation by external potassium). In contrast, a single proline mutation (proline to glycine: P244G) reduced by > 90% the proper assembly and function of Na(+)-K(+)-ATPase, despite a normal rate of synthesis and core glycosylation. Our data indicate that proline-244 plays a critical role in the proper folding of the beta-subunit and its ability to associate efficiently with the alpha 1-subunit in the endoplasmic reticulum.
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Kallal, L., and J. Kurjan. "Analysis of the receptor binding domain of Gpa1p, the G(alpha) subunit involved in the yeast pheromone response pathway." Molecular and Cellular Biology 17, no. 5 (May 1997): 2897–907. http://dx.doi.org/10.1128/mcb.17.5.2897.
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The Saccharomyces cerevisiae G protein alpha subunit Gpa1p is involved in the response of both MATa and MAT alpha cells to pheromone. We mutagenized the GPA1 C terminus to characterize the receptor-interacting domain and to investigate the specificity of the interactions with the a- and alpha-factor receptors. The results are discussed with respect to a structural model of the Gpa1p C terminus that was based on the crystal structure of bovine transducin. Some mutants showed phenotypes different than the pheromone response and mating defects expected for mutations that affect receptor interactions, and therefore the mutations may affect other aspects of Gpa1p function. Most of the mutations that resulted in pheromone response and mating defects had similar effects in MATa and MAT alpha cells, suggesting that they affect the interactions with both receptors. Overexpression of the pheromone receptors increased the mating of some of the mutants tested but not the wild-type strain, consistent with defects in mutant Gpa1p-receptor interactions. The regions identified by the mating-defective mutants correlated well with the regions of mammalian G(alpha) subunits implicated in receptor interactions. The strongest mating type-specific effects were seen for mutations to proline and a mutation of a glycine residue predicted to form a C-terminal beta turn. The analogous beta turn in mammalian G(alpha) subunits undergoes a conformational change upon receptor interaction. We propose that the conformation of this region of Gpa1p differs during the interactions with the a- and alpha-factor receptors and that these mating type-specific mutations preclude the orientation necessary for interaction with one of the two receptors.
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Calvete, J. J., A. Henschen, and J. González-Rodríguez. "Assignment of disulphide bonds in human platelet GPIIIa. A disulphide pattern for the β-subunits of the integrin family." Biochemical Journal 274, no. 1 (February 15, 1991): 63–71. http://dx.doi.org/10.1042/bj2740063.
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Integrins are cell-surface heterodimers formed by the association of one alpha- and one beta-subunit. Glycoprotein IIIa (GPIIIa or beta 3 subunit) is the common beta-subunit of the beta 3 subfamily of integrins, which, when associated with glycoprotein IIb (GPIIb), constitutes the receptor for fibrinogen and other adhesive proteins at the platelet surface (the GPIIb-IIIa complex) and, when associated with the alpha v-subunit, constitutes the vitronectin receptor present in several cell types. Protein chemical analysis of GPIIIa allows us to define the following structural domains: the cysteine-rich and proteinase-resistant N-terminal domain (GPIIIa 1-62); the adhesive-protein-binding domain (GPIIIa 101-422); the cysteine-rich and proteinase-resistant core (GPIIIa 423-622); and the C-terminal domain comprising an extracellular subdomain (GPIIIa 623-692), a transmembrane subdomain (GPIIIa 693-721), and a cytoplasmic subdomain (GPIIIa 722-762). We also assign unambiguously the disulphide bonds within the N-terminal, the fibrinogen-binding and the C-terminal domains, and the two long-range disulphide bonds which join the N-terminus to the proteinase-resistant core (Cys5-Cys435) and the fibrinogen-binding domain to the extracellular side of the C-terminal domain (Cys406-Cys655). In addition, we propose three alternative models for the arrangement of the disulphide bonds within the core and of the disulphide bonds joining the core to the extracellular side of the C-terminal domain, consistent with our experimental findings, favouring temporarily that which imposes less steric hindrance for the formation of these disulphide bonds. On the basis of this information and on the highly conserved overall structure observed in the beta-subunits of the integrin family known so far, except in beta 4, we propose to extend the cysteine-pairing pattern and the structural domains outlined here for GPIIIa to all the beta-subunits of the integrin family.
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Sanders, J., M. Brandsma, G. M. Janssen, J. Dijk, and W. Moller. "Immunofluorescence studies of human fibroblasts demonstrate the presence of the complex of elongation factor-1 beta gamma delta in the endoplasmic reticulum." Journal of Cell Science 109, no. 5 (May 1, 1996): 1113–17. http://dx.doi.org/10.1242/jcs.109.5.1113.
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The eukaryotic elongation factor-1 (EF-1) consists of four subunits, EF-1 alpha, EF-1 beta, EF-1 gamma and EF-1 delta which induce efficient transfer of aminoacyl-tRNA to the ribosome. In this process EF-1 alpha.GTP acts as the carrier of the aminoacyl-tRNA on its way to the ribosome. After release of aminoacyl-tRNA to the ribosome under concomitant hydrolysis of GTP, the inactive EF-1 alpha.GDP form is recycled to EF-1 alpha.GTP by EF-1 beta gamma delta. In eukaryotic cells the concentration of EF-1 alpha exceeds that of the complex beta gamma delta by a factor of 5–10. In order to delineate the intracellular localization of the different subunits of EF-1, antibodies against the EF-1 subunits have been elicited and indirect immunofluorescence microscopy experiments were performed. In human fibroblasts, the guanine nucleotide exchange part of EF-1, EF-1 beta gamma delta, was found to co-localize with the endoplasmic reticulum (ER), displaying a distinct fine-structure in its staining pattern. The guanine nucleotide-binding subunit of EF-1, EF-1 alpha, shows a more diffuse distribution throughout the cytoplasm and is, in addition, associated with the nucleus.
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Hainaut, P., A. Kowalski, S. Giorgetti, V. Baron, and E. Van Obberghen. "Insulin and insulin-like-growth-factor-I (IGF-I) receptors in Xenopus laevis oocytes. Comparison with insulin receptors from liver and muscle." Biochemical Journal 273, no. 3 (February 1, 1991): 673–78. http://dx.doi.org/10.1042/bj2730673.
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Insulin and insulin-like-growth-factor-I (IGF-I) receptors were partially purified from full-grown (stages V-VI) Xenopus laevis oocytes by affinity chromatography on wheat-germ agglutinin-agarose. Competitive-binding assays revealed high-affinity binding sites for both insulin and IGF-I (Kd = 2.5 x 10(-10) M and 8 x 10(-10) M respectively). However, IGF-I receptors were about 15 times more abundant than insulin receptors (22.5 x 10(11) versus 1.5 x 10(11)/mg of protein). Moreover, comparison of intact and collagenase-treated oocytes showed that most of the insulin receptors were in the oocyte envelopes, whereas IGF-I receptors were essentially at the oocyte surface. Oocyte receptors were composed of alpha-subunits of approximately 130 kDa and a doublet of beta-subunits of 95 and 105 kDa, which both had ligand-induced phosphorylation patterns compatible with IGF-I receptor beta-subunits. Accordingly, the receptor tyrosine kinase was stimulated at low IGF-I concentrations [half-maximally effective concentration (EC50) approximately 0.5-1 nM], and at higher insulin concentrations (EC50 approximately 20-50 nM). Partially purified glycoproteins from Xenopus liver and muscle contained mainly receptors of the insulin-receptor type, with alpha-subunits of 140 kDa in liver and 125 kDa in muscle, and doublets of beta-subunits of 92-98 kDa in liver and 85-94 kDa in muscle. Immunoprecipitation of receptors from oocytes, liver and muscle by receptor-specific anti-peptide antibodies suggested that the beta-subunit heterogeneity resulted from the existence of two distinct IGF-I receptors in oocytes and of two distinct insulin receptors in both liver and muscle. In the different tissues, the two receptor subtypes differed at least by their beta-subunit C-terminal region.
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Suzuki-Yagawa, Y., K. Kawakami, and K. Nagano. "Housekeeping Na,K-ATPase alpha 1 subunit gene promoter is composed of multiple cis elements to which common and cell type-specific factors bind." Molecular and Cellular Biology 12, no. 9 (September 1992): 4046–55. http://dx.doi.org/10.1128/mcb.12.9.4046.
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Na,K-ATPase alpha 1 subunit gene (ATP1A1) is one of the housekeeping genes involved in homeostasis of Na+ and K+ in all animal cells. We identified and characterized the cis-acting elements that regulate the expression of ATP1A1. The region between -155 and -49 was determined as a positive regulatory region in five cultured cell lines of different tissue origins (MDCK, B103, L6, 3Y1, and HepG2). The region was divided into three subregions: from -120 to -106 (including the Sp1 binding site), from -102 to -61, and from -58 to -49 (including an Sp1 consensus sequence). Cell type-specific factors binding to the middle subregion (from -102 to -61) were detected by gel retardation analysis, using nuclear extracts prepared from MDCK and B103 cells. Two gel retardation complexes were formed in the B103 nuclear extract, and three were formed in the MDCK nuclear extract. DNA binding regions of these factors were located at -88 to -69 and differed from each other in DNase I footprinting experiments. These factors also showed different binding characteristics in gel retardation competition and methylation interference experiments. The identified cis element was named the ATP1A1 regulatory element. The core sequence of this element is found in several other genes involved in cellular energy metabolism, suggesting that the sequence is a common regulatory element responsive to the state of energy metabolism.
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Suzuki-Yagawa, Y., K. Kawakami, and K. Nagano. "Housekeeping Na,K-ATPase alpha 1 subunit gene promoter is composed of multiple cis elements to which common and cell type-specific factors bind." Molecular and Cellular Biology 12, no. 9 (September 1992): 4046–55. http://dx.doi.org/10.1128/mcb.12.9.4046-4055.1992.
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Na,K-ATPase alpha 1 subunit gene (ATP1A1) is one of the housekeeping genes involved in homeostasis of Na+ and K+ in all animal cells. We identified and characterized the cis-acting elements that regulate the expression of ATP1A1. The region between -155 and -49 was determined as a positive regulatory region in five cultured cell lines of different tissue origins (MDCK, B103, L6, 3Y1, and HepG2). The region was divided into three subregions: from -120 to -106 (including the Sp1 binding site), from -102 to -61, and from -58 to -49 (including an Sp1 consensus sequence). Cell type-specific factors binding to the middle subregion (from -102 to -61) were detected by gel retardation analysis, using nuclear extracts prepared from MDCK and B103 cells. Two gel retardation complexes were formed in the B103 nuclear extract, and three were formed in the MDCK nuclear extract. DNA binding regions of these factors were located at -88 to -69 and differed from each other in DNase I footprinting experiments. These factors also showed different binding characteristics in gel retardation competition and methylation interference experiments. The identified cis element was named the ATP1A1 regulatory element. The core sequence of this element is found in several other genes involved in cellular energy metabolism, suggesting that the sequence is a common regulatory element responsive to the state of energy metabolism.
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Bar-Zvi, D., I. Bar, M. Yoshida, and N. Shavit. "Covalent binding of 3‘-O-(4-benzoyl)benzoyl adenosine 5‘-triphosphate (BzATP) to the isolated alpha and beta subunits and the alpha 3 beta 3 core complex of TF1. Covalent binding of BzATP prevents association of alpha and beta subunits and induces dissociation of the alpha 3 beta 3 core complex." Journal of Biological Chemistry 267, no. 16 (June 1992): 11029–33. http://dx.doi.org/10.1016/s0021-9258(19)49870-0.
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