scholarly journals The N-Terminal CCHC Zinc Finger Motif Mediates Homodimerization of Transcription Factor BCL11B

2017 ◽  
Vol 38 (5) ◽  
Author(s):  
Piotr Grabarczyk ◽  
Passorn Winkler ◽  
Martin Delin ◽  
Praveen K. Sappa ◽  
Sander Bekeschus ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Zinc Finger ◽  
De Novo ◽  
Germ Line ◽  
Affinity Purification ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dominant Negative ◽  
Zinc Finger Motif ◽  
Cchc Zinc Finger

ABSTRACT The BCL11B gene encodes a Krüppel-like, sequence-specific zinc finger (ZF) transcription factor that acts as either a repressor or an activator, depending on its posttranslational modifications. The importance of BCL11B in numerous biological processes in multiple organs has been well established in mouse knockout models. The phenotype of the first de novo monoallelic germ line missense mutation in the BCL11B gene (encoding N441K) strongly implies that the mutant protein acts in a dominant-negative manner by neutralizing the unaffected protein through the formation of a nonfunctional dimer. Using a Förster resonance energy transfer-assisted fluorescence-activated cell sorting (FACS-FRET) assay and affinity purification followed by mass spectrometry (AP-MS), we show that the N-terminal CCHC zinc finger motif is necessary and sufficient for the formation of the BCL11B dimer. Mutation of the CCHC ZF in BCL11B abolishes its transcription-regulatory activity. In addition, unlike wild-type BCL11B, this mutant is incapable of inducing cell cycle arrest and protecting against DNA damage-driven apoptosis. Our results confirm the BCL11B dimerization hypothesis and prove its importance for BCL11B function. By mapping the relevant regions to the CCHC domain, we describe a previously unidentified mechanism of transcription factor homodimerization.

scholarly journals PKC-α mediates flow-stimulated superoxide production in thick ascending limbs

2010 ◽  
Vol 298 (4) ◽  
pp. F885-F891 ◽  
Author(s):  
Nancy J. Hong ◽  
Guillermo B. Silva ◽  
Jeffrey L. Garvin
Keyword(s):  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Mesangial Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Oxidase Inhibitor ◽  
Dominant Negative ◽  
Aortic Endothelial Cells ◽  
Nadph Oxidase Activity ◽  
Measured Flow

We showed that luminal flow increases net superoxide (O2−) production via NADPH oxidase in thick ascending limbs. Protein kinase C (PKC) activates NADPH oxidase activity in phagocytes, cardiomyocytes, aortic endothelial cells, vascular smooth muscle cells, and renal mesangial cells. However, the flow-activated pathway that induces NADPH oxidase activity in thick ascending limbs is unclear. We hypothesized that PKC mediates flow-stimulated net O2− production by thick ascending limbs. Initiation of flow (20 nl/min) increased net O2− production from 4 ± 1 to 61 ± 12 AU/s ( P < 0.007; n = 5). The NADPH oxidase inhibitor apocynin completely blocked the flow-induced increase in net O2− production (2 ± 1 vs. 1 ± 1 AU/s; P > 0.05; n = 5). Flow-stimulated O2− was also blocked in p47phox-deficient mice. We measured flow-stimulated PKC activity with a fluorescence resonance energy transfer (FRET)-based membrane-targeted PKC activity reporter and found that the FRET ratio increased from 0.87 ± 0.02 to 0.96 ± 0.04 AU ( P < 0.05; n = 6). In the absence of flow, the PKC activator phorbol 12-myristate 13-acetate (200 nM) enhanced net O2− production from 5 ± 2 to 92 ± 6 AU/s ( P < 0.001; n = 6). The PKC-α- and βI-selective inhibitor Gö 6976 (100 nM) decreased flow-stimulated net O2− production from 54 ± 15 to 2 ± 1 AU/s ( P < 0.04; n = 5). Flow-induced net O2− production was inhibited in thick ascending limbs transduced with dominant-negative (dn)PKC-α but not dnPKCβI or LacZ (Δ = 11 ± 3 AU/s for dnPKCα, 55 ± 7 AU/s for dnPKCβI, and 63 ± 7 AU/s for LacZ; P < 0.001; n = 6). We concluded that flow stimulates net O2− production in thick ascending limbs via PKC-α-mediated activation of NADPH oxidase.

A Pedigree Harboring a Germ-Line N-Terminal C/EBPα Mutation and Development of Acute Myeloblastic Leukemia with a Somatic C-Terminal C/EBPα Mutation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1916-1916 ◽  
Author(s):  
Tomoko Nanri ◽  
Naokuni Uike ◽  
Toshiro Kawakita ◽  
Eisaku Iwanaga ◽  
Koyu Hoshino ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Germ Line ◽  
Terminal Region ◽  
Dominant Negative ◽  
Acute Myeloblastic Leukemia ◽  
Peripheral Blood Cells ◽  
Insertion Mutations

Abstract Transcription factors involved in myeloid cell differentiation are frequent targets of chromosomal translocations and point mutations in patients with acute myeloblastic leukemia (AML). Familial AML harboring a mutation in a transcription factor should provide an association of clinical features with functions of the transcription factor. Recently, two pedigrees of AML carrying a germ-line mutation in the CEBPA, a gene encoding transcription factor C/EBPα have been reported. As C/EBPα-null mice lack of mature neutrophil and eosinophil granulocytes, C/EBPα is thought to play a central role in regulating the differentiation of granulocytes. We here present clinical and molecular features of a Japanese family in which two individuals developing AML and one healthy individual had an identical CEBPA mutation. Father had received a diagnosis of AML M2 in 1988 at the age of 39 years. Following a relapse 7 years after his first complete remission (CR), he received autologous stem cell transplantation, after which he has been in a lasting CR. His elderly son was diagnosed to have M2Eo in 2004 at the age of 26 years. He has achieved a continuous CR. Bone marrow cells at the time of diagnosis and peripheral blood cells obtained during CR in both patients showed a 4-base pair insertion in the N-terminal region of the CEBPA (350_351insCTAC). The corresponding protein is predicted to terminate prematurely at codon 107 (I68fsX107). Therefore, this heterozygous mutation causes truncation of the 42-kD C/EBPα protein and overproduction of a 30-kD isoform, which lacks a transactivation domain and functions in a dominant negative fashion, causing a decrease in C/EBPα activity. His younger son unaffected by AML aged 21 years also had the same mutation. The N-terminal C/EBPα mutations in sporadic AML patients are associated with FAB M1/M2 subtypes, presence of Auer rods, CD7 expression, normal karyotype, and a favorable prognosis. Familial AMLs with an N-terminal C/EBPα mutation demonstrate links of these unique features to a dominant negative C/EBPα mutation. It is of note that different in-frame insertion mutations in the C-terminal region of C/EBPα were identified in both patients at the time of diagnosis. As these C-terminal CEBPA mutations were not found in peripheral blood cells during CR in both patients, these in-frame insertion mutations in the C/EBPα basic region leucine zipper DNA-binding domain should be somatic mutations. Interestingly, father showed different in-frame insertion mutations in the C-terminal CEBPA at the time of diagnosis and relapse. These N- and C-terminal biallelic mutations in the CEBPA are thought to result in complete loss-of-function of C/EBPα. A recent study using conditional Cebpa knockout mice showed that C/EBPα deficiency in adult mice leads to persistence of immature myeloid blasts in the bone marrow without developing AML. Although the mechanism underlying the development of AML is yet unclear, this study indicates that the N-terminal CEBPA mutation appears to confer an increased risk occurring C-terminal C/EBPα mutations and other mutations as a second and further genetic hits, generating undifferentiated myeloid cells and eventually leading to AML over a long latency.

scholarly journals Sequence of the HAP3 transcription factor of Kluyveromyces lactis predicts the presence of a novel 4-cysteine zinc-finger motif

1994 ◽  
Vol 245 (1) ◽  
pp. 96-106 ◽  
Author(s):  
Wietse Mulder ◽  
Inge H. J. M. Scholten ◽  
René W. de Boer ◽  
Leslie A. Grivell
Keyword(s):  
Transcription Factor ◽  
Zinc Finger ◽  
Kluyveromyces Lactis ◽  
Zinc Finger Motif

scholarly journals A High-Throughput Cell-Based Assay to Identify Specific Inhibitors of Transcription Factor AP-1

2006 ◽  
Vol 12 (1) ◽  
pp. 133-139 ◽  
Author(s):  
Katie M. Ruocco ◽  
Ekaterina I. Goncharova ◽  
Matthew R. Young ◽  
Nancy H. Colburn ◽  
James B. McMahon ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Natural Products ◽  
Natural Product ◽  
High Throughput ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Nordihydroguaiaretic Acid ◽  
Activator Protein 1 ◽  
Taxonomic Groups ◽  
High Throughput Assay

The oncogenic transcription factor AP-1 (activator protein–1) is required for tumor promotion and progression. Identification of novel and specific AP-1 inhibitors would be beneficial for cancer prevention and therapy. The authors have developed a high-throughput assay to screen synthetic and natural product libraries for noncytotoxic inhibitors of mitogen-activated AP-1 activity. The cell-based high-throughput screen is conducted in a 384-well format using a fluorescent resonance energy transfer (FRET) substrate to quantify the activity of a β-lactamase reporter under the control of an AP-1-dependent promoter. The ratiometric FRET readout makes this assay extremely robust and reproducible, particularly for use with natural product extracts. To eliminate false positives due to cell killing, a cytotoxicity assay was incorporated. The AP-1 β-lactamase reporter was validated with inhibitors of kinases located upstream of AP-1 and with known natural product inhibitors of AP-1 (nordihydroguaiaretic acid and curcumin). The assay was able to identify other known AP-1 inhibitors and protein kinase C modulators, as well as a number of chemically diverse compounds with unknown mechanisms of action from natural products libraries. Application to natural product extracts identified hits from a range of taxonomic groups. Screening of synthetic compounds and natural products should identify novel AP-1 inhibitors that may be useful in the prevention and treatment of cancers.

scholarly journals Nonphosphorylated Human La Antigen Interacts with Nucleolin at Nucleolar Sites Involved in rRNA Biogenesis

2004 ◽  
Vol 24 (24) ◽  
pp. 10894-10904 ◽  
Author(s):  
Robert V. Intine ◽  
Miroslav Dundr ◽  
Alex Vassilev ◽  
Elena Schwartz ◽  
Yingmin Zhao ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Rna Binding ◽  
Affinity Purification ◽  
Close Association ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Rna Recognition Motif ◽  
Translational Machinery ◽  
Terminal Domain

ABSTRACT La is a RNA-binding protein implicated in multiple pathways related to the production of tRNAs, ribosomal proteins, and other components of the translational machinery (D. J. Kenan and J. D. Keene, Nat. Struct. Mol. Biol. 11 :303-305, 2004). While most La is phosphorylated and resides in the nucleoplasm, a fraction is in the nucleolus, the site of ribosome production, although the determinants of this localization are incompletely known. In addition to its conserved N-terminal domain, human La harbors a C-terminal domain that contains an atypical RNA recognition motif and a short basic motif (SBM) adjacent to phosphoserine-366. We report that nonphosphorylated La (npLa) is concentrated in nucleolar sites that correspond to the dense fibrillar component that harbors nascent pol I transcripts as well as fibrillarin and nucleolin, which function in early phases of rRNA maturation. Affinity purification and native immunoprecipitation of La and fluorescence resonance energy transfer in the nucleolus reveal close association with nucleolin. Moreover, La lacking the SBM does not localize to nucleoli. Lastly, La exhibits SBM-dependent, phosphorylation-sensitive interaction with nucleolin in a yeast two-hybrid assay. The data suggest that interaction with nucleolin is, at least in part, responsible for nucleolar accumulation of La and that npLa may be involved in ribosome biogenesis.

Essential Role of Kindlin 3 in Zebrafish Thrombopoiesis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1166-1166
Author(s):  
James J. Mann ◽  
Nathaniel B. Langer ◽  
Andrew Woo ◽  
Tyler B. Moran ◽  
Yocheved Schindler ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Zinc Finger ◽  
Essential Role ◽  
Molecular Mechanisms ◽  
Fetal Liver ◽  
Affinity Purification ◽  
Transgenic Zebrafish ◽  
Zinc Finger Transcription Factor ◽  
Stage Of Development

Abstract The zinc finger transcription factor GATA-1 is required for proliferative inhibition and terminal maturation of megakaryocytes, and is mutated in Down Syndrome Transient Myeloproliferative Disorder (TMD) and Acute Megakaryoblastic Leukemia (DS-AMKL). Yet the molecular mechanisms that regulate GATA-1 activity in megakaryopoiesis remain incompletely understood. Many transcription factors, in addition to binding DNA, make important protein-protein interactions that modulate their activity. In order to further understand GATA-1’s function, and possibly identify new factors involved in megakaryopoiesis, we purified GATA-1 containing multiprotein complexes from the murine L8057 megakaryocytic cell line. We generated stable L8057 cell lines expressing metabolically biotinylated and FLAG epitope tagged GATA-1, and then performed a tandem anti-FLAG immunoaffinity and streptavidin affinity purification. Using mass spectrometry (LC/MS/MS), we identified the known GATA-1 associated proteins Friend of GATA-1 (FOG-1), SCL, Ldb1, Runx-1/Cbf-β. SP1 and all components of the NuRD complex (which binds FOG-1) as co-purifying proteins. In addition, we reproducibly obtained several novel proteins. We previously reported the identification of the kruppel-type zinc finger transcription factor zfp148 (also called ZBP-89), and showed that it plays an essential role in megakaryopoiesis and definitive erythropoiesis. Here we report the identification of Kindlin 3 (also called URP2 for UNC-112 related protein 2), a member of a family of PH and FERM domain containing proteins that are thought to play a role in integrin-mediated processes. Expression of Kindlin 3 is restricted to hematopoietic cells, principally megakaryocytes and lymphocytes. It is first expressed at ~E9.5 during murine embryogenesis, and is abundant in fetal liver megakaryocytes by day E14.5. In order to begin to assess the role of Kindlin 3 in megakaryopoiesis in vivo, we performed morpholino-mediated knockdown of Kindlin 3 expression in CD41-GFP transgenic zebrafish embryos. In contrast to control embryos, embryos injected with Kindlin 3 specific morpholinos exhibited nearly complete loss of GFP+ thrombocytes (equivalent to mammalian megakaryocyte/platelets). Erythroid development (equivalent to mammalian primitive erythropoiesis at this stage of development) was not significantly affected, similar to embryos injected with zfp148-specific morpholinos. Given the role of integrin outside-to-inside signaling in megakaryopoiesis, we propose that Kindlin 3 may play a role linking extracellular signals to megakaryocyte maturation and growth control via GATA-1 transcription complexes. Further analysis in murine systems is underway to test this hypothesis.

scholarly journals Fluorescence Resonance Energy Transfer Microscopy of the Helicobacter pylori Vacuolating Cytotoxin within Mammalian Cells

2002 ◽  
Vol 70 (7) ◽  
pp. 3824-3832 ◽  
Author(s):  
David C. Willhite ◽  
Dan Ye ◽  
Steven R. Blanke
Keyword(s):  
Helicobacter Pylori ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dominant Negative ◽  
Wild Type ◽  
Vacuolating Cytotoxin ◽  
Vacuolated Cells ◽  
Mutant Forms

ABSTRACT The Helicobacter pylori vacuolating cytotoxin (VacA) binds and enters mammalian cells to induce cellular vacuolation. To investigate the quaternary structure of VacA within the intracellular environment where toxin cytotoxicity is elaborated, we employed fluorescence resonance energy transfer (FRET) microscopy. HeLa cells coexpressing full-length and truncated forms of VacA fused to cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP) were analyzed for FRET to indicate direct associations. These studies revealed that VacA-CFP and VacA-YFP interact within vacuolated cells, supporting the belief that monomer associations at an intracellular site are important for the toxin's vacuolating activity. In addition, the two fragments of proteolytically nicked VacA, p37 and p58, interact when coexpressed within mammalian cells. Because p37 and p58 function in trans when expressed separately within mammalian cells, these data suggest that the mechanism by which these two fragments induce vacuolation requires direct association. FRET microscopy also demonstrated interactions between mutant forms of VacA, as well as wild-type VacA with mutant forms of the toxin within vacuolated cells. Finally, a dominant-negative form of the toxin directly associates with wild-type VacA in cells where vacuolation was not detectable, suggesting that the formation of complexes comprising wild-type and dominant-negative forms of toxin acts to block intracellular toxin function.

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