PU.1 inhibits GATA-1 function and erythroid differentiation by blocking GATA-1 DNA binding

Blood ◽  
2000 ◽  
Vol 96 (8) ◽  
pp. 2641-2648 ◽  
Author(s):  
Pu Zhang ◽  
Xiaobo Zhang ◽  
Atsushi Iwama ◽  
Channing Yu ◽  
Kent A. Smith ◽  
...  
Keyword(s):  
Dna Binding ◽  
Fusion Protein ◽  
Cell Line ◽  
Protein Interactions ◽  
Messenger Rna ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Protein Protein Interactions ◽  
C Terminus ◽  
N Terminus

The lineage-specific transcription factors GATA-1 and PU.1 can physically interact to inhibit each other's function, but the mechanism of repression of GATA-1 function by PU.1 has not been elucidated. Both the N terminus and the C terminus of PU.1 can physically interact with the C-terminal zinc finger of GATA-1. It is demonstrated that the PU.1 N terminus, but not the C terminus, is required for inhibiting GATA-1 function. Induced overexpression of PU.1 in K562 erythroleukemia cells blocks hemin-induced erythroid differentiation. In this system, PU.1 does not affect the expression of GATA-1 messenger RNA, protein, or nuclear localization. However, GATA-1 DNA binding decreases dramatically. By means of electrophoretic mobility shift assays with purified proteins, it is demonstrated that the N-terminal 70 amino acids of PU.1 can specifically block GATA-1 DNA binding. In addition, PU.1 had a similar effect in the G1ER cell line, in which the GATA-1 null erythroid cell line G1E has been transduced with a GATA-1–estrogen receptor fusion gene, which is directly dependent on induction of the GATA-1 fusion protein to effect erythroid maturation. Consistent with in vitro binding assays, overexpression of PU.1 blocked DNA binding of the GATA-1 fusion protein as well as GATA-1–mediated erythroid differentiation of these G1ER cells. These results demonstrate a novel mechanism by which function of a lineage-specific transcription factor is inhibited by another lineage-restricted factor through direct protein–protein interactions. These findings contribute to understanding how protein–protein interactions participate in hematopoietic differentiation and leukemogenesis.

PU.1 inhibits GATA-1 function and erythroid differentiation by blocking GATA-1 DNA binding

Blood ◽  
2000 ◽  
Vol 96 (8) ◽  
pp. 2641-2648 ◽  
Author(s):  
Pu Zhang ◽  
Xiaobo Zhang ◽  
Atsushi Iwama ◽  
Channing Yu ◽  
Kent A. Smith ◽  
...  
Keyword(s):  
Dna Binding ◽  
Fusion Protein ◽  
Cell Line ◽  
Protein Interactions ◽  
Messenger Rna ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Protein Protein Interactions ◽  
C Terminus ◽  
N Terminus

Abstract The lineage-specific transcription factors GATA-1 and PU.1 can physically interact to inhibit each other's function, but the mechanism of repression of GATA-1 function by PU.1 has not been elucidated. Both the N terminus and the C terminus of PU.1 can physically interact with the C-terminal zinc finger of GATA-1. It is demonstrated that the PU.1 N terminus, but not the C terminus, is required for inhibiting GATA-1 function. Induced overexpression of PU.1 in K562 erythroleukemia cells blocks hemin-induced erythroid differentiation. In this system, PU.1 does not affect the expression of GATA-1 messenger RNA, protein, or nuclear localization. However, GATA-1 DNA binding decreases dramatically. By means of electrophoretic mobility shift assays with purified proteins, it is demonstrated that the N-terminal 70 amino acids of PU.1 can specifically block GATA-1 DNA binding. In addition, PU.1 had a similar effect in the G1ER cell line, in which the GATA-1 null erythroid cell line G1E has been transduced with a GATA-1–estrogen receptor fusion gene, which is directly dependent on induction of the GATA-1 fusion protein to effect erythroid maturation. Consistent with in vitro binding assays, overexpression of PU.1 blocked DNA binding of the GATA-1 fusion protein as well as GATA-1–mediated erythroid differentiation of these G1ER cells. These results demonstrate a novel mechanism by which function of a lineage-specific transcription factor is inhibited by another lineage-restricted factor through direct protein–protein interactions. These findings contribute to understanding how protein–protein interactions participate in hematopoietic differentiation and leukemogenesis.

The Drosophila pipsqueak gene encodes a nuclear BTB-domain-containing protein required early in oogenesis

Development ◽  
1996 ◽  
Vol 122 (6) ◽  
pp. 1859-1871 ◽  
Author(s):  
H. Horowitz ◽  
C.A. Berg
Keyword(s):  
Fusion Protein ◽  
Protein Interactions ◽  
Protein Isoforms ◽  
Follicle Cells ◽  
Protein Protein Interactions ◽  
Gene Encoding ◽  
Btb Domain ◽  
Multiple Transcripts ◽  
C Terminus ◽  
Evolutionarily Conserved

Mutations at the pipsqueak locus affect early patterning in the Drosophila egg and embryo. We have cloned pipsqueak and found that it is a large and complex gene, encoding multiple transcripts and protein isoforms. One protein, PsqA, is absent in all of the mutants that we have examined. We show that PsqA is a nuclear protein present in the germ cells and somatically derived follicle cells throughout oogenesis and that it is required prior to stage one of oogenesis. PsqA contains a BTB (POZ) domain at its amino terminus; additionally, we have identified an evolutionarily conserved motif of unknown function present four times in tandem at the C terminus of the protein. PZ pipsqueak mutants produce a putative fusion protein containing the pipsqueak BTB domain fused to sequences resident on the PZ element (H. Horowitz and C. Berg, 1995 Genetics 139, 327–335). We demonstrate here that expression of this fusion protein in wild-type flies has a dominant effect, resulting in infertility and eggshell defects. These dominant phenotypes are discussed in light of current theories on the role of the BTB domain in protein-protein interactions.

scholarly journals Domains of alpha-SNAP required for the stimulation of exocytosis and for N-ethylmalemide-sensitive fusion protein (NSF) binding and activation.

1996 ◽  
Vol 7 (5) ◽  
pp. 693-701 ◽  
Author(s):  
R J Barnard ◽  
A Morgan ◽  
R D Burgoyne
Keyword(s):  
Membrane Proteins ◽  
Fusion Protein ◽  
Conformational Changes ◽  
Protein Interactions ◽  
Atp Hydrolysis ◽  
Coiled Coil ◽  
Protein Protein Interactions ◽  
Permeabilized Cells ◽  
C Terminus ◽  
Adrenal Chromaffin

The binding of alpha-SNAP to the membrane proteins syntaxin, SNAP-25, and synaptobrevin leads to the recruitment of the N-ethylmaleimide-sensitive fusion protein (NSF). ATP hydrolysis by NSF has been suggested to drive conformational changes in one or more of these membrane proteins that are essential for regulated exocytosis. Functional evidence for a role of alpha-SNAP in exocytosis in adrenal chromaffin cells comes from the ability of this protein to stimulate Ca(2+)-dependent exocytosis in digitonin-permeabilized cells. Here we examine the effect of a series of deletion mutants of alpha-SNAP on exocytosis, and on the ability of alpha-SNAP to interact with NSF, to define essential domains involved in protein-protein interactions in exocytosis. Deletion of extreme N- or C-terminal regions of alpha-SNAP produced proteins unable to bind to syntaxin or to stimulate exocytosis, suggesting that these domains participate in essential interactions. Deletion of C-terminal residues abolished the ability of alpha-SNAP to bind NSF. In contrast, deletion of up to 120 N-terminal residues did not prevent the binding of NSF to immobilized alpha-SNAP and such mutants were also able to stimulate the ATPase activity of NSF. These results suggest that the C-terminus, but not the N-terminus, of alpha-SNAP is crucial for interactions with NSF. The involvement of the C-terminus of alpha-SNAP, which contains a predicted coiled-coil domain, in the binding of both syntaxin and NSF would place the latter two proteins in proximity in a ternary complex whereupon the energy derived from ATP hydrolysis by NSF could induce a conformational change in syntaxin required for exocytosis to proceed.

scholarly journals Analysis of the SWI4/SWI6 protein complex, which directs G1/S-specific transcription in Saccharomyces cerevisiae.

1993 ◽  
Vol 13 (2) ◽  
pp. 1069-1077 ◽  
Author(s):  
J Sidorova ◽  
L Breeden
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
C Terminus ◽  
Specific Complex ◽  
Dependent Cell ◽  
The Stability

SWI4 and SWI6 play a crucial role in START-specific transcription in Saccharomyces cerevisiae. SWI4 and SWI6 form a specific complex on the SCB (SWI4/6-dependent cell cycle box) sequences which have been found in the promoters of HO and G1 cyclin genes. Overproduction of SWI4 eliminates the SWI6 dependency of HO transcription in vivo and results in a new SWI6-independent, SCB-specific complex in vitro, which is heterogeneous and reacts with SWI4 antibodies. The C terminus of SWI4 is not required for SWI6-independent binding of SWI4 to SCB sequences, but it is necessary and sufficient for association with SWI6. Both SWI4 and SWI6 contain two copies of a 33-amino-acid TPLH repeat, which has been implicated in protein-protein interactions in other proteins. These repeats are not required for the SWI4-SWI6 association. Alanine substitutions in both TPLH repeats of SWI6 reduce its activity but do not affect the stability of the protein or its association with SWI4. However, these mutations reduce the ability of the SWI4/6 complex to bind DNA. Deletion of the lucine zipper motif in SWI6 also allows SWI4/6 complex formation, but it eliminates the DNA-binding ability of the SWI4/6 complex. This indicates that the integrity of two different regions of SWI6 is required for DNA binding by the SWI4/6 complex. From these data, we propose that the sequence-specific DNA-binding domain resides in SWI4 but that SWI6 controls the accessibility of this domain in the SWI4/6 complex.

scholarly journals DNA Binding Induces a cis to trans Switch in Cre Recombinase to Enable Intasome Assembly

2020 ◽  
Author(s):  
Aparna Unnikrishnan ◽  
Carlos D. Amero ◽  
Deepak Kumar Yadav ◽  
Kye Stachowski ◽  
Devante Potter ◽  
...  
Keyword(s):  
Dna Binding ◽  
Protein Interactions ◽  
Dna Cleavage ◽  
Gene Editing ◽  
Dna Recombination ◽  
Protein Protein Interactions ◽  
C Terminus ◽  
Dna Cleavage Activity ◽  
Binding Surface ◽  
Synaptic Complexes

ABSTRACTMechanistic understanding of DNA recombination in the Cre-loxP system has largely been guided by crystallographic structures of tetrameric synaptic complexes. Those studies have suggested a role for protein conformational dynamics that has not been well characterized at the atomic level. We used solution NMR to discover the link between intrinsic flexibility and function in Cre recombinase. TROSY-NMR spectra show the N-terminal and C-terminal catalytic domains (CreNTD, CreCat) to be structurally independent. Amide 15N relaxation measurements of the CreCat domain reveal fast time scale dynamics in most regions that exhibit conformational differences in active and inactive Cre protomers in crystallographic tetramers. However, the C-terminal helix αN, implicated in assembly of synaptic complexes and regulation of DNA cleavage activity via trans protein-protein interactions, is unexpectedly rigid in free Cre. Chemical shift perturbations and intra- and inter-molecular paramagnetic relaxation enhancement (PRE) NMR data reveal an alternative auto-inhibitory conformation for the αN region of free Cre, wherein it packs in cis over the protein DNA binding surface and active site. Moreover, binding to loxP DNA induces a conformational change that dislodge the C-terminus, resulting in a cis to trans switch that is likely to enable protein-protein interactions required for assembly of recombinogenic Cre intasomes. These findings necessitate a re-examination of the mechanisms by which this widely-utilized gene editing tool selects target sites, avoids spurious DNA cleavage activity, and controls DNA recombination efficiency.SIGNIFICANCE STATEMENTThe Cre-loxP system is a widely used gene editing tool that has enabled transformative advances in immunology, neuroscience and cardiovascular research. Still, off-target activities confound research results and present obstacles to biomedical applications. Overcoming those limitations requires understanding the steps leading to assembly of recombination complexes, intasomes. We measured the magnetic properties of nitrogen nuclei in the backbone of the enzyme to correlate its intrinsic dynamics with its function in DNA recognition and cleavage. Remarkably, we found that in the absence of DNA the C-terminus of Cre appears to block the DNA binding surface and active site of the enzyme. Binding to loxP DNA induces a conformational switch that would enable the intermolecular protein-protein interactions required for assembly of recombinogenic Cre intasomes.

Analysis of the SWI4/SWI6 protein complex, which directs G1/S-specific transcription in Saccharomyces cerevisiae

1993 ◽  
Vol 13 (2) ◽  
pp. 1069-1077
Author(s):  
J Sidorova ◽  
L Breeden
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
C Terminus ◽  
Specific Complex ◽  
Dependent Cell ◽  
The Stability

SWI4 and SWI6 play a crucial role in START-specific transcription in Saccharomyces cerevisiae. SWI4 and SWI6 form a specific complex on the SCB (SWI4/6-dependent cell cycle box) sequences which have been found in the promoters of HO and G1 cyclin genes. Overproduction of SWI4 eliminates the SWI6 dependency of HO transcription in vivo and results in a new SWI6-independent, SCB-specific complex in vitro, which is heterogeneous and reacts with SWI4 antibodies. The C terminus of SWI4 is not required for SWI6-independent binding of SWI4 to SCB sequences, but it is necessary and sufficient for association with SWI6. Both SWI4 and SWI6 contain two copies of a 33-amino-acid TPLH repeat, which has been implicated in protein-protein interactions in other proteins. These repeats are not required for the SWI4-SWI6 association. Alanine substitutions in both TPLH repeats of SWI6 reduce its activity but do not affect the stability of the protein or its association with SWI4. However, these mutations reduce the ability of the SWI4/6 complex to bind DNA. Deletion of the lucine zipper motif in SWI6 also allows SWI4/6 complex formation, but it eliminates the DNA-binding ability of the SWI4/6 complex. This indicates that the integrity of two different regions of SWI6 is required for DNA binding by the SWI4/6 complex. From these data, we propose that the sequence-specific DNA-binding domain resides in SWI4 but that SWI6 controls the accessibility of this domain in the SWI4/6 complex.

scholarly journals E. coli primase and DNA polymerase III holoenzyme are able to bind concurrently to a primed template during DNA replication

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Andrea Bogutzki ◽  
Natalie Naue ◽  
Lidia Litz ◽  
Andreas Pich ◽  
Ute Curth
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Protein Interactions ◽  
Dna Polymerase Iii ◽  
Protein Protein Interactions ◽  
Single Stranded Dna ◽  
E Coli ◽  
Polymerase Iii ◽  
C Terminus ◽  
Pol Iii

Abstract During DNA replication in E. coli, a switch between DnaG primase and DNA polymerase III holoenzyme (pol III) activities has to occur every time when the synthesis of a new Okazaki fragment starts. As both primase and the χ subunit of pol III interact with the highly conserved C-terminus of single-stranded DNA-binding protein (SSB), it had been proposed that the binding of both proteins to SSB is mutually exclusive. Using a replication system containing the origin of replication of the single-stranded DNA phage G4 (G4ori) saturated with SSB, we tested whether DnaG and pol III can bind concurrently to the primed template. We found that the addition of pol III does not lead to a displacement of primase, but to the formation of higher complexes. Even pol III-mediated primer elongation by one or several DNA nucleotides does not result in the dissociation of DnaG. About 10 nucleotides have to be added in order to displace one of the two primase molecules bound to SSB-saturated G4ori. The concurrent binding of primase and pol III is highly plausible, since even the SSB tetramer situated directly next to the 3′-terminus of the primer provides four C-termini for protein-protein interactions.

scholarly journals Mechanism of NanR gene repression and allosteric induction of bacterial sialic acid metabolism

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Christopher R. Horne ◽  
Hariprasad Venugopal ◽  
Santosh Panjikar ◽  
David M. Wood ◽  
Amy Henrickson ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Dna Binding ◽  
Protein Interactions ◽  
Acid Metabolism ◽  
Environmental Changes ◽  
Dna Interaction ◽  
Protein Protein Interactions ◽  
Nutrient Source ◽  
Cryo Electron Microscopy ◽  
Close Proximity

AbstractBacteria respond to environmental changes by inducing transcription of some genes and repressing others. Sialic acids, which coat human cell surfaces, are a nutrient source for pathogenic and commensal bacteria. The Escherichia coli GntR-type transcriptional repressor, NanR, regulates sialic acid metabolism, but the mechanism is unclear. Here, we demonstrate that three NanR dimers bind a (GGTATA)3-repeat operator cooperatively and with high affinity. Single-particle cryo-electron microscopy structures reveal the DNA-binding domain is reorganized to engage DNA, while three dimers assemble in close proximity across the (GGTATA)3-repeat operator. Such an interaction allows cooperative protein-protein interactions between NanR dimers via their N-terminal extensions. The effector, N-acetylneuraminate, binds NanR and attenuates the NanR-DNA interaction. The crystal structure of NanR in complex with N-acetylneuraminate reveals a domain rearrangement upon N-acetylneuraminate binding to lock NanR in a conformation that weakens DNA binding. Our data provide a molecular basis for the regulation of bacterial sialic acid metabolism.

scholarly journals Transactivation Functions of the N-Terminal Domains of Nuclear Hormone Receptors: Protein Folding and Coactivator Interactions

2003 ◽  
Vol 17 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Raj Kumar ◽  
E. Brad Thompson
Keyword(s):  
Dna Binding ◽  
Protein Interactions ◽  
Hormone Receptors ◽  
Nuclear Hormone Receptor ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Protein Protein Interactions ◽  
Carboxy Terminal ◽  
And Function ◽  
Terminal Domains

Abstract The N-terminal domains (NTDs) of many members of the nuclear hormone receptor (NHR) family contain potent transcription-activating functions (AFs). Knowledge of the mechanisms of action of the NTD AFs has lagged, compared with that concerning other important domains of the NHRs. In part, this is because the NTD AFs appear to be unfolded when expressed as recombinant proteins. Recent studies have begun to shed light on the structure and function of the NTD AFs. Recombinant NTD AFs can be made to fold by application of certain osmolytes or when expressed in conjunction with a DNA-binding domain by binding that DNA-binding domain to a DNA response element. The sequence of the DNA binding site may affect the functional state of the AFs domain. If properly folded, NTD AFs can bind certain cofactors and primary transcription factors. Through these, and/or by direct interactions, the NTD AFs may interact with the AF2 domain in the ligand binding, carboxy-terminal portion of the NHRs. We propose models for the folding of the NTD AFs and their protein-protein interactions.

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