scholarly journals HOXA9 Forms Triple Complexes with PBX2 and MEIS1 in Myeloid Cells

1999 ◽  
Vol 19 (4) ◽  
pp. 3051-3061 ◽  
Author(s):  
Wei-Fang Shen ◽  
Sophia Rozenfeld ◽  
Angela Kwong ◽  
Laszlo G. Kömüves ◽  
H. Jeffrey Lawrence ◽  
...  
Keyword(s):  
Dna Binding ◽  
Myeloid Leukemia ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Homeodomain Protein ◽  
Nuclear Speckles ◽  
Mobility Shift ◽  
Dna Binding Complexes

ABSTRACT Aberrant activation of the HOX, MEIS, and PBX homeodomain protein families is associated with leukemias, and retrovirally driven coexpression of HOXA9 and MEIS1 is sufficient to induce myeloid leukemia in mice. Previous studies have demonstrated that HOX-9 and HOX-10 paralog proteins are unique among HOX homeodomain proteins in their capacity to form in vitro cooperative DNA binding complexes with either the PBX or MEIS protein. Furthermore, PBX and MEIS proteins have been shown to form in vivo heterodimeric DNA binding complexes with each other. We now show that in vitro DNA site selection for MEIS1 in the presence of HOXA9 and PBX yields a consensus PBX-HOXA9 site. MEIS1 enhances in vitro HOXA9-PBX protein complex formation in the absence of DNA and forms a trimeric electrophoretic mobility shift assay (EMSA) complex with these proteins on an oligonucleotide containing a PBX-HOXA9 site. Myeloid cell nuclear extracts produce EMSA complexes which appear to contain HOXA9, PBX2, and MEIS1, while immunoprecipitation of HOXA9 from these extracts results in coprecipitation of PBX2 and MEIS1. In myeloid cells, HOXA9, MEIS1, and PBX2 are all strongly expressed in the nucleus, where a portion of their signals are colocalized within nuclear speckles. However, cotransfection of HOXA9 and PBX2 with or without MEIS1 minimally influences transcription of a reporter gene containing multiple PBX-HOXA9 binding sites. Taken together, these data suggest that in myeloid leukemia cells MEIS1 forms trimeric complexes with PBX and HOXA9, which in turn can bind to consensus PBX-HOXA9 DNA targets.

scholarly journals Macromolecular Synthesis Shutoff Resistance by Myeloid Cells Is Critical to IRF7-Dependent Systemic Interferon Alpha/Beta Induction after Alphavirus Infection

2019 ◽  
Vol 93 (24) ◽  
Author(s):  
Nishank Bhalla ◽  
Christina L. Gardner ◽  
Sierra N. Downs ◽  
Matthew Dunn ◽  
Chengqun Sun ◽  
...  
Keyword(s):  
Host Cell ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Cell Types ◽  
Macromolecular Synthesis ◽  
Transcription Inhibition ◽  
Antiviral Responses ◽  
Alphavirus Infection

ABSTRACT Alphavirus infection of fibroblastic cell types in vitro inhibits host cell translation and transcription, leading to suppression of interferon alpha/beta (IFN-α/β) production. However, the effect of infection upon myeloid cells, which are often the first cells encountered by alphaviruses in vivo, is unclear. Previous studies demonstrated an association of systemic IFN-α/β production with myeloid cell infection efficiency. Murine infection with wild-type Venezuelan equine encephalitis virus (VEEV), a highly myeloid-cell-tropic alphavirus, results in secretion of very high systemic levels of IFN-α/β, suggesting that stress responses in responding cells are active. Here, we infected myeloid cell cultures with VEEV to identify the cellular source of IFN-α/β, the timing and extent of translation and/or transcription inhibition in infected cells, and the transcription factors responsible for IFN-α/β induction. In contrast to fibroblast infection, myeloid cell cultures infected with VEEV secreted IFN-α/β that increased until cell death was observed. VEEV inhibited translation in most cells early after infection (<6 h postinfection [p.i.]), while transcription inhibition occurred later (>6 h p.i.). Furthermore, the interferon regulatory factor 7 (IRF7), but not IRF3, transcription factor was critical for IFN-α/β induction in vitro and in sera of mice. We identified a subset of infected Raw 264.7 myeloid cells that resisted VEEV-induced translation inhibition and secreted IFN-α/β despite virus infection. However, in the absence of IFN receptor signaling, the size of this cell population was diminished. These results indicate that IFN-α/β induction in vivo is IRF7 dependent and arises in part from a subset of myeloid cells that are resistant, in an IFN-α/β-dependent manner, to VEEV-induced macromolecular synthesis inhibition. IMPORTANCE Most previous research exploring the interaction of alphaviruses with host cell antiviral responses has been conducted using fibroblast lineage cell lines. Previous studies have led to the discovery of virus-mediated activities that antagonize host cell antiviral defense pathways, such as host cell translation and transcription inhibition and suppression of STAT1 signaling. However, their relevance and impact upon myeloid lineage cell types, which are key responders during the initial stages of alphavirus infection in vivo, have not been well studied. Here, we demonstrate the different abilities of myeloid cells to resist VEEV infection compared to nonmyeloid cell types and begin to elucidate the mechanisms by which host antiviral responses are upregulated in myeloid cells despite the actions of virus-encoded antagonists.

Mcl-1 in Transgenic Mice Promotes Survival in a Spectrum of Hematopoietic Cell Types and Immortalization in the Myeloid Lineage

Blood ◽  
1998 ◽  
Vol 92 (9) ◽  
pp. 3226-3239 ◽  
Author(s):  
Ping Zhou ◽  
Liping Qian ◽  
Christine K. Bieszczad ◽  
Randolph Noelle ◽  
Michael Binder ◽  
...  
Keyword(s):  
Myeloid Cells ◽  
Myeloid Cell ◽  
Cell Types ◽  
Cell Number ◽  
Hematopoietic Cell ◽  
Viable Cell Number ◽  
Wide Range ◽  
Transgenic Cells

Abstract Mcl-1 is a member of the Bcl-2 family that is expressed in early monocyte differentiation and that can promote viability on transfection into immature myeloid cells. However, the effects of Mcl-1 are generally short lived compared with those of Bcl-2 and are not obvious in some transfectants. To further explore the effects of this gene, mice were produced that expressed Mcl-1 as a transgene in hematolymphoid tissues. The Mcl-1 transgene was found to cause moderate viability enhancement in a wide range of hematopoietic cell types, including lymphoid (B and T) as well as myeloid cells at both immature and mature stages of differentiation. However, enhanced hematopoietic capacity in transgenic bone marrow and spleen was not reflected in any change in pool sizes in the peripheral blood. In addition, among transgenic cells, mature T cells remained long lived compared with B cells and macrophages could live longer than either of these. Interestingly, when hematopoietic cells were maintained in tissue culture in the presence of interleukin-3, Mcl-1 enhanced the probability of outgrowth of continuously proliferating myeloid cell lines. Thus, Mcl-1 transgenic cells remained subject to normal in vivo homeostatic mechanisms controlling viable cell number, but these constraints could be overridden under specific conditions in vitro. Within the organism, Bcl-2 family members may act at “viability gates” along the differentiation continuum, functioning as part of a system for controlled hematopoietic cell amplification. Enforced expression of even a moderate viability-promoting member of this family such as Mcl-1, within a conducive intra- and extracellular environment in isolation from normal homeostatic constraints, can substantially increase the probability of cell immortalization. © 1998 by The American Society of Hematology.

GABP Transcription Factor Is Required for Myeloid Cell Development In Vivo.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 374-374 ◽  
Author(s):  
Zhong-fa Yang ◽  
Karen Drumea ◽  
Alan G. Rosmarin
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
T Cell ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Cell Development ◽  
Wild Type ◽  
Ets Domain

Abstract GABP is an ets transcription factor that regulates genes that are required for innate immunity, including CD18 (β2 leukocyte integrin), lysozyme, and neutrophil elastase. GABP consists of two distinct and unrelated proteins. GABPα binds to DNA through its ets domain and recruits GABPβ, which contains the transactivation domain; together, they form a functional tetrameric transcription factor complex. We recently showed that GABP is required for entry into S phase of the cell cycle through its regulation of genes that are required for DNA synthesis and cyclin dependent kinase inhibitors (Yang, et al. Nature Cell Biol9:339, 2007). Furthermore, GABP is an essential component of a retinoic acid responsive myeloid enhanceosome (Resendes and Rosmarin Mol Cell Biol26:3060, 2006). We cloned Gabpa (the gene that encodes mouse Gabpα) from a mouse genomic BAC library and prepared a targeting vector in which the ets domain is flanked by loxP recombination sites (floxed allele). Deletion of both floxed Gabpa alleles causes an early embryonic lethal defect. In order to define the role of Gabpα in myelopoiesis, we bred floxed Gabpa mice to mice that bear the Mx1-Cre transgene, which drives expression of Cre recombinase in response to injection of the synthetic polynucleotide, poly I-C. Deletion of Gabpa dramatically reduced granulocytes and monocytes in the peripheral blood, spleen, and bone marrow, but myeloid cells recovered within weeks. In vitro colony forming assays indicated that myeloid cells in these mice were derived only from Gabpa replete myeloid precursors (that failed to delete both Gabpa alleles), suggesting strong pressure to retain Gabpα in vivo. We used a novel competitive bone marrow transplantation approach to determine if Gabp is required for myeloid cell development in vivo. Sub-lethally irradiated wild-type recipient mice bearing leukocyte marker CD45.1 received equal proportions of bone marrow from wild type CD45.1 donor mice and floxed-Mx1-Cre donor mice that bear CD45.2. Both the CD45.2 (floxed-Mx1-Cre) and CD45.1 (wild type) bone marrow engrafted well. Mice were then injected with pI-pC to induce Cre-mediated deletion of floxed Gabpa. The mature myeloid and T cell compartments were derived almost entirely from wild type CD45.1 cells. This indicates that the proliferation and/or differentiation of myeloid and T cell lineages requires Gabp. In contrast, B cell development was not impaired. We conclude that Gabpa disruption causes a striking loss of myeloid cells in vivo and corroborates prior in vitro data that GABP plays a crucial role in proliferation of myeloid progenitor cells.

Mcl-1 in Transgenic Mice Promotes Survival in a Spectrum of Hematopoietic Cell Types and Immortalization in the Myeloid Lineage

Blood ◽  
1998 ◽  
Vol 92 (9) ◽  
pp. 3226-3239 ◽  
Author(s):  
Ping Zhou ◽  
Liping Qian ◽  
Christine K. Bieszczad ◽  
Randolph Noelle ◽  
Michael Binder ◽  
...  
Keyword(s):  
Myeloid Cells ◽  
Myeloid Cell ◽  
Cell Types ◽  
Cell Number ◽  
Hematopoietic Cell ◽  
Viable Cell Number ◽  
Wide Range ◽  
Transgenic Cells

Mcl-1 is a member of the Bcl-2 family that is expressed in early monocyte differentiation and that can promote viability on transfection into immature myeloid cells. However, the effects of Mcl-1 are generally short lived compared with those of Bcl-2 and are not obvious in some transfectants. To further explore the effects of this gene, mice were produced that expressed Mcl-1 as a transgene in hematolymphoid tissues. The Mcl-1 transgene was found to cause moderate viability enhancement in a wide range of hematopoietic cell types, including lymphoid (B and T) as well as myeloid cells at both immature and mature stages of differentiation. However, enhanced hematopoietic capacity in transgenic bone marrow and spleen was not reflected in any change in pool sizes in the peripheral blood. In addition, among transgenic cells, mature T cells remained long lived compared with B cells and macrophages could live longer than either of these. Interestingly, when hematopoietic cells were maintained in tissue culture in the presence of interleukin-3, Mcl-1 enhanced the probability of outgrowth of continuously proliferating myeloid cell lines. Thus, Mcl-1 transgenic cells remained subject to normal in vivo homeostatic mechanisms controlling viable cell number, but these constraints could be overridden under specific conditions in vitro. Within the organism, Bcl-2 family members may act at “viability gates” along the differentiation continuum, functioning as part of a system for controlled hematopoietic cell amplification. Enforced expression of even a moderate viability-promoting member of this family such as Mcl-1, within a conducive intra- and extracellular environment in isolation from normal homeostatic constraints, can substantially increase the probability of cell immortalization. © 1998 by The American Society of Hematology.

scholarly journals The Pseudomonas Quorum-Sensing Regulator RsaL Belongs to the Tetrahelical Superclass of H-T-H Proteins

2006 ◽  
Vol 189 (5) ◽  
pp. 1922-1930 ◽  
Author(s):  
Giordano Rampioni ◽  
Fabio Polticelli ◽  
Iris Bertani ◽  
Karima Righetti ◽  
Vittorio Venturi ◽  
...  
Keyword(s):  
Dna Binding ◽  
Quorum Sensing ◽  
In Silico ◽  
Mobility Shift ◽  
Specific Domain ◽  
Signal Molecule ◽  
In Silico Model ◽  
Opportunistic Human Pathogen

ABSTRACT In the opportunistic human pathogen Pseudomonas aeruginosa, quorum sensing (QS) is crucial for virulence. The RsaL protein directly represses the transcription of lasI, the synthase gene of the main QS signal molecule. On the basis of sequence homology, RsaL cannot be predicted to belong to any class of characterized DNA-binding proteins. In this study, an in silico model of the RsaL structure was inferred showing that RsaL belongs to the tetrahelical superclass of helix-turn-helix proteins. The overall structure of RsaL is very similar to the N-terminal domain of the lambda cI repressor and to the POU-specific domain of the mammalian transcription factor Oct-1 (Oct-1 POUs). Moreover, residues of Oct-1 POUs important for structural stability and/or DNA binding are conserved in the same positions in RsaL and in its homologs found in GenBank. These residues were independently replaced with Ala, and the activities of the mutated variants of RsaL were compared to that of the wild-type counterpart in vivo by complementation assays and in vitro by electrophoretic mobility shift assays. The results validated the RsaL in silico model and showed that residues Arg 20, Gln 38, Ser 42, Arg 43, and Glu 45 are important for RsaL function. Our data indicate that RsaL could be the founding member of a new protein family within the tetrahelical superclass of helix-turn-helix proteins. Finally, the minimum DNA sequence required for RsaL binding on the lasI promoter was determined, and our data support the hypothesis that RsaL binds DNA as a dimer.

scholarly journals BCL11A promotes myeloid leukemogenesis by repressing PU.1 target genes

2021 ◽  
Author(s):  
Yoshitaka Sunami ◽  
Takashi Yokoyama ◽  
Seiko Yoshino ◽  
Tomoko Takahara ◽  
Yukari Yamazaki ◽  
...  
Keyword(s):  
Dna Binding ◽  
Myeloid Leukemia ◽  
Target Genes ◽  
Cell Interaction ◽  
Binding Activity ◽  
Hemoglobin Switching ◽  
Dna Binding Activity ◽  
Adult Hemoglobin

The transcriptional repressor, BCL11A, is involved in hematological malignancies, B-cell development, and fetal-to-adult hemoglobin switching. However, the molecular mechanism by which it promotes the development of myeloid leukemia remains largely unknown. We find that Bcl11a cooperates with the pseudokinase, Trib1, in the development of acute myeloid leukemia (AML). Bcl11a promotes the proliferation and engraftment of Trib1-expressing AML cells both in vitro and in vivo. ChIP-seq analysis showed that upon DNA-binding, Bcl11a is significantly associated with PU.1, an inducer of myeloid differentiation, and that Bcl11a represses several PU.1 target genes, such as Asb2, Clec5a, and Fcgr3. Asb2, as a Bcl11a target gene that modulates cytoskeleton and cell-cell interaction, plays a key role in Bcl11a-induced malignant progression. The repression of PU.1 target genes by Bcl11a is achieved by both sequence-specific DNA-binding activity and recruitment of corepressors by Bcl11a. Suppression of the corepressor components, HDAC and LSD1, reverses the repressive activity. Moreover, treatment of AML cells with the HDAC inhibitor, pracinostat, and LSD1 inhibitor, GSK2879552, resulted in growth inhibition both in vitro and in vivo. High BCL11A expression is associated with worse prognosis in human AML patients. Blocking of BCL11A expression upregulates the expression of PU.1 target genes, and inhibits the growth of HL-60 cells and their engraftment to the bone marrow, suggesting that BCL11A is involved in human myeloid malignancies via the suppression of PU.1 transcriptional activity.

The Saccharomyces cerevisiae PUT3 activator protein associates with proline-specific upstream activation sequences

1989 ◽  
Vol 9 (11) ◽  
pp. 4706-4712
Author(s):  
A H Siddiqui ◽  
M C Brandriss
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Complex Formation ◽  
Binding Activity ◽  
Lacz Gene ◽  
Wild Type ◽  
Mobility Shift ◽  
Proline Utilization

The PUT1 and PUT2 genes encoding the enzymes of the proline utilization pathway of Saccharomyces cerevisiae are induced by proline and activated by the product of the PUT3 gene. Two upstream activation sequences (UASs) in the PUT1 promoter were identified by homology to the PUT2 UAS. Deletion analysis of the two PUT1 UASs showed that they were functionally independent and additive in producing maximal levels of gene expression. The consensus PUT UAS is a 21-base-pair partially palindromic sequence required in vivo for induction of both genes. The results of a gel mobility shift assay demonstrated that the proline-specific UAS is the binding site of a protein factor. In vitro complex formation was observed in crude extracts of yeast strains carrying either a single genomic copy of the PUT3 gene or the cloned PUT3 gene on a 2 microns plasmid, and the binding was dosage dependent. DNA-binding activity was not observed in extracts of strains carrying either a put3 mutation that caused a noninducible (Put-) phenotype or a deletion of the gene. Wild-type levels of complex formation were observed in an extract of a strain carrying an allele of PUT3 that resulted in a constitutive (Put+) phenotype. Extracts from a strain carrying a PUT3-lacZ gene fusion formed two complexes of slower mobility than the wild-type complex. We conclude that the PUT3 product is either a DNA-binding protein or part of a DNA-binding complex that recognizes the UASs of both PUT1 and PUT2. Binding was observed in extracts of a strain grown in the presence or absence of proline, demonstrating the constitutive nature of the DNA-protein interaction.

scholarly journals The Sequence-specific DNA Binding of NF-κB Is Reversibly Regulated by the Automodification Reaction of Poly (ADP-ribose) Polymerase 1

2001 ◽  
Vol 276 (50) ◽  
pp. 47664-47670 ◽  
Author(s):  
Woo-Jin Chang ◽  
Rafael Alvarez-Gonzalez
Keyword(s):  
Dna Binding ◽  
Hela Cells ◽  
Specific Binding ◽  
High Specificity ◽  
Immunoblot Analysis ◽  
Mobility Shift ◽  
Electrophoretic Mobility Shift Assays ◽  
Parp 1

Recent studies suggest that the synthesis of protein-bound ADP-ribose polymers catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1) regulates eucaryotic gene expression, including the NF-κB-dependent pathway. Here, we report the molecular mechanism by which PARP-1 activates the sequence-specific binding of NF-κB to its oligodeoxynucleotide. We co-incubated pure recombinant human PARP-1 and the p50 subunit of NF-κB (NF-κB-p50) in the presence or absence of βNAD+in vitro.Electrophoretic mobility shift assays showed that, when PARP-1 was present, NF-κB-p50 DNA binding was dependent on the presence of βNAD+. DNA binding by NF-κB-p50 was not efficient in the absence of βNAD+. In fact, the binding was not efficient in the presence of 3-aminobenzamide (3-AB) either. Thus, we conclude that NF-κB-p50 DNA binding is protein-poly(ADP-ribosyl)ation dependent. Co-immunoprecipitation and immunoblot analysis revealed that PARP-1 physically interacts with NF-κB-p50 with high specificity in the absence of βNAD+. Because NF-kB-p50 was not an efficient covalent target for poly(ADP-ribosyl)ation, our results are consistent with the conclusion that the auto-poly(ADP-ribosyl)ation reaction catalyzed by PARP-1 facilitates the binding of NF-κB-p50 to its DNA by inhibiting the specific protein·protein interactions between NF-κB-p50 and PARP-1. We also report the activation of NF-κB DNA binding by the automodification reaction of PARP-1 in cultured HeLa cells following exposure to H2O2. In these experiments, preincubation of HeLa cells with 3-AB, prior to oxidative damage, strongly inhibited NF-κB activationin vivoas well.

scholarly journals The Acute Myeloid Leukemia-Associated Protein, Dek, Forms a Splicing-Dependent Interaction with Exon-Product Complexes

2000 ◽  
Vol 150 (2) ◽  
pp. 309-320 ◽  
Author(s):  
Tim McGarvey ◽  
Emanuel Rosonina ◽  
Susan McCracken ◽  
Qiyu Li ◽  
Ramy Arnaout ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Chromosomal Translocation ◽  
Sr Proteins ◽  
Splicing Factor ◽  
Nuclear Speckles ◽  
Myeloid Leukemias ◽  
Acute Myeloid ◽  
Dependent Interaction

DEK is an ∼45-kD phosphoprotein that is fused to the nucleoporin CAN as a result of a (6;9) chromosomal translocation in a subset of acute myeloid leukemias (AMLs). It has also been identified as an autoimmune antigen in juvenile rheumatoid arthritis and other rheumatic diseases. Despite the association of DEK with several human diseases, its function is not known. In this study, we demonstrate that DEK, together with SR proteins, associates with the SRm160 splicing coactivator in vitro. DEK is recruited to splicing factor-containing nuclear speckles upon concentration of SRm160 in these structures, indicating that DEK and SRm160 associate in vivo. We further demonstrate that DEK associates with splicing complexes through interactions mediated by SR proteins. Significantly, DEK remains bound to the exon-product RNA after splicing, and this association requires the prior formation of a spliceosome. Thus, DEK is a candidate factor for controlling postsplicing steps in gene expression that are influenced by the prior removal of an intron from pre-mRNA.

scholarly journals Modulation of DNA binding properties of CCAAT/enhancer binding protein epsilon by heterodimer formation and interactions with NFkappaB pathway

Blood ◽  
2007 ◽  
Vol 109 (10) ◽  
pp. 4209-4219 ◽  
Author(s):  
Alexey M. Chumakov ◽  
Agnes Silla ◽  
Elizabeth A. Williamson ◽  
H. Phillip Koeffler
Keyword(s):  
Dna Binding ◽  
Myeloid Cell ◽  
Enhancer Binding Protein ◽  
Dna Binding Site ◽  
Zip Family ◽  
Family Protein ◽  
The Family ◽  
Dna Binding Properties

Abstract C/EBP epsilon is a transcription factor involved in myeloid cell differentiation. Along with C/EBP-α, -β, -γ, -δ, and -ζ, C/EBP-ϵ belongs to the family of CCAAT/enhancer binding proteins that are implicated in control of growth and differentiation of several cell lineages in inflammation and stress response. We have previously shown that C/EBP-ϵ preferentially binds DNA as a heterodimer with other C/EBP family members such as C/EBP-δ, CHOP (C/EBP-ζ), and the b-zip family protein ATF4. In this study, we define the consensus binding sites for C/EBP-ϵ dimers and C/EBP-ϵ–ATF4 heterodimers. We show that the activated NFkappaB pathway promotes interaction of the C/EBP-ϵ subunit with its cognate DNA binding site via interaction with RelA. RelA-C/EBP interaction is enhanced by phosphorylation of threonine at amino acid 75 and results in increased DNA binding compared with the wild-type nonphosphorylated C/EBP both in vitro and in vivo. We suggest that interaction of the activated NFkappaB pathway and C/EBP-ϵ may be important in selective activation of a subset of C/EBP-ϵ–responsive genes.

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