Genetic analysis of theSalmonellatranscription factor HilA

2008 ◽  
Vol 54 (10) ◽  
pp. 854-860 ◽  
Author(s):  
Rebecca A. Daly ◽  
C. Phoebe Lostroh
Keyword(s):  
Amino Acids ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Dna Binding ◽  
Genetic Analysis ◽  
Transcription Activation ◽  
Winged Helix ◽  
Activation Domains

HilA, a Salmonella transcription factor, activates the invF-1 and prgH promoters through binding to the HilA box, which contains 2 copies of a TTKHAT motif separated by a T centered at –45 relative to the start sites of transcription. The N-terminal 112 amino acids of HilA are similar to winged helix-turn-helix DNA binding/transcription activation domains (wHTH DBDs). The remaining 441 amino acids are not similar in sequence to any other well-characterized transcription factors. Here, we report that the wHTH DBD is essential for activation of both promoters, but amino acids 113–554 are only required for normal activation of invF-1. Some alanine substitutions in the putative α loop, which connects the recognition and positioning helices in wHTH DBDs, cause a loss-of-activation phenotype. A hilA allele encoding a protein with an alanine substituted for arginine at position 71 in the α loop has a loss-of-activation defect exclusively at the prgH promoter. The results suggest distinct roles for one or more domains formed by amino acids 113–554 and for arginine 71 in activation of the 2 promoters.

Differentiation-Dependent Interactions Between Runx-1 and Fli-1 during Megakaryocyte Development

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 279-279
Author(s):  
Hui Huang ◽  
Ming Yu ◽  
Tyler B Moran ◽  
Nathan Tu ◽  
Thomas E Akie ◽  
...  
Keyword(s):  
Amino Acids ◽  
Transcription Factor ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Gel Filtration ◽  
Cell Types ◽  
Myelogenous Leukemia ◽  
Activation Domains ◽  
Platelet Disorder ◽  
Familial Platelet Disorder

Abstract The transcription factor Runx-1 is required for the ontogeny of all definitive hematopoiesis, and plays a specific role in megakaryopoiesis during later stages of development. Germline mutations in Runx-1 cause Familial Platelet Disorder with Propensity to Develop AML (FPD/AML), and acquired mutations occur in a subset of patients with myelodysplastic syndrome (MDS) and acute myelogenous leukemia. Although many of the reported Runx-1 mutations affect DNA binding and/or its interaction with the cofactor CBF-beta, other mutations occur outside of these binding regions and have unknown mechanistic effects. In this study, we purified Runx-1 containing multiprotein complexes from murine megakaryocytic cells in order to identify potential novel Runx-1 associated factors whose interaction may be altered by Runx-1 mutations. Here we report the identification of the key megakaryocyte ets transcription factor Fli- 1 as a direct Runx-1 binding partner. This interaction involves the negative regulatory DNA binding and activation domains of Runx-1 (amino acids 179–370), and a region around the Ets DNA binding region of Fli-1 (amino acids 281–361). The interaction is lost in the MDS- associated Y254X Runx-1 mutation. We also show that Runx-1 and Fli-1 co-occupy the c-mpl promoter in primary megakaryocytes and act synergistically in transcriptional reporter assays. Interestingly, the interaction between Runx-1 and Fli- 1 occurs in murine L8057 megakaryoblastic cells only after they have been induced to differentiate, even though both proteins are expressed abundantly in uninduced cells. The interaction correlates with assembly of a large multiprotein complex that also includes the key megakaryocyte transcription factor GATA-1 and its cofactor Friend of GATA-1 (FOG- 1) based on gel filtration chromatography experiments. Furthermore, we show that Fli-1 from this large complex lacks phosphorylation of a specific residue that is phosphorylated on non-complexed Fli-1. Mutation of this site to aspartic acid, which mimics constitutive phosphorylation, disrupts the interaction between Fli-1 and Runx-1 and abrogates their transcriptional synergy. We propose that dephosphorylation of Fli-1 is a key event in the transcriptional activation of megakaryocyte terminal maturation by facilitating the assembly of a RUNX-1/FLI-1/GATA-1/FOG-1 enhancesome complex. These findings have implications for the differentiation of other cell types where interactions between Runx and ets family proteins occur.

Specific transcription factors stimulate simian virus 40 and polyomavirus origins of DNA replication

1992 ◽  
Vol 12 (6) ◽  
pp. 2514-2524 ◽  
Author(s):  
Z S Guo ◽  
M L DePamphilis
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Dna Replication ◽  
Binding Sites ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Activation Domains ◽  
Auxiliary Component ◽  
Cell Extracts

The origins of DNA replication (ori) in simian virus 40 (SV40) and polyomavirus (Py) contain an auxiliary component (aux-2) composed of multiple transcription factor binding sites. To determine whether this component stimulated replication by binding specific transcription factors, aux-2 was replaced by synthetic oligonucleotides that bound a single transcription factor. Sp1 and T-antigen (T-ag) sites, which exist in the natural SV40 aux-2 sequence, provided approximately 75 and approximately 20%, respectively, of aux-2 activity when transfected into monkey cells. In cell extracts, only T-ag sites were active. AP1 binding sites could replace completely either SV40 or Py aux-2. Mutations that eliminated AP1 binding also eliminated AP1 stimulation of replication. Yeast GAL4 binding sites that strongly stimulated transcription in the presence of GAL4 proteins failed to stimulate SV40 DNA replication, although they did partially replace Py aux-2. Stimulation required the presence of proteins consisting of the GAL4 DNA binding domain fused to specific activation domains such as VP16 or c-Jun. These data demonstrate a clear role for transcription factors with specific activation domains in activating both SV40 and Py ori. However, no correlation was observed between the ability of specific proteins to stimulate promoter activity and their ability to stimulate origin activity. We propose that only transcription factors whose specific activation domains can interact with the T-ag initiation complex can stimulate SV40 and Py ori-core activity.

Dimeric transcription factor families: it takes two to tango but who decides on partners and the venue?

1992 ◽  
Vol 103 (1) ◽  
pp. 9-14 ◽  
Author(s):  
K.A. Lee
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Dna Binding ◽  
Experimental Evidence ◽  
Cdna Cloning ◽  
Transcriptional Activation ◽  
Effector Functions ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Dna Elements

Dimeric transcription factors that bind to DNA are often grouped into families on the basis of dimerization and DNA-binding specificities. cDNA cloning studies have established that members of the same family have structurally related dimerisation and DNA-binding domains but diverge in other regions that are important for transcriptional activation. These features lead to the straightforward suggestion that although all members of a family bind to similar DNA elements, individual members exhibit distinct transcriptional effector functions. This simple view is now supported by experimental evidence from those systems that have proved amenable to study. There are however some largely unaddressed questions that concern the mechanisms that allow family members to go about their business without interference from their highly related siblings. Here I will discuss some insights from studies of the bZIP class of transcription factors.

Transcriptional activity of the zinc finger protein NGFI-A is influenced by its interaction with a cellular factor

1993 ◽  
Vol 13 (11) ◽  
pp. 6858-6865
Author(s):  
M W Russo ◽  
C Matheny ◽  
J Milbrandt
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Transcriptional Activity ◽  
Zinc Fingers ◽  
Fold Increase ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Activation Domains ◽  
Cellular Factor ◽  
Inhibitory Domain

NGFI-A is an immediate-early gene that encodes a transcription factor whose DNA-binding domain is composed of three zinc fingers. To define the domains responsible for its transcriptional activity, a mutational analysis was conducted with an NGFI-A molecule in which the zinc fingers were replaced by the GAL4 DNA-binding domain. In a cotransfection assay, four activation domains were found within NGFI-A. Three of the activation domains are similar to those characterized previously: one contains a large number of acidic residues, another is enriched in proline and glutamine residues, and another has some sequence homology to a domain found in Krox-20. The fourth bears no resemblance to previously described activation domains. NGFI-A also contains an inhibitory domain whose removal resulted in a 15-fold increase in NGFI-A activity. This increase in activity occurred in all mammalian cell types tested but not in Drosophila S2 cells. Competition experiments in which increasing amounts of the inhibitory domain were cotransfected along with NGFI-A demonstrated a dose-dependent increase in NGFI-A activity. A point mutation within the inhibitory domain of the competitor (I293F) abolished this property. When the analogous mutation was introduced into native NGFI-A, a 17-fold increase in activity was observed. The inhibitory effect therefore appears to be the result of an interaction between this domain and a titratable cellular factor which is weakened by this mutation. Downmodulation of transcription factor activity through interaction with a cellular factor has been observed in several other systems, including the regulation of transcription factor E2F by retinoblastoma protein, and in studies of c-Jun.

scholarly journals The Prodomain of Ssy5 Protease Controls Receptor-Activated Proteolysis of Transcription Factor Stp1

2010 ◽  
Vol 30 (13) ◽  
pp. 3299-3309 ◽  
Author(s):  
Thorsten Pfirrmann ◽  
Stijn Heessen ◽  
Deike J. Omnus ◽  
Claes Andréasson ◽  
Per O. Ljungdahl
Keyword(s):  
Amino Acids ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Amino Acid ◽  
Signaling Pathway ◽  
Temperature Control ◽  
Regulatory Mechanism ◽  
Potent Inhibitor ◽  
Large N

ABSTRACT Extracellular amino acids induce the yeast SPS sensor to endoproteolytically cleave transcription factors Stp1 and Stp2 in a process termed receptor-activated proteolysis (RAP). Ssy5, the activating endoprotease, is synthesized with a large N-terminal prodomain and a C-terminal chymotrypsin-like catalytic (Cat) domain. During biogenesis, Ssy5 cleaves itself and the prodomain and Cat domain remain associated, forming an inactive primed protease. Here we show that the prodomain is a potent inhibitor of Cat domain activity and that its inactivation is a requisite for RAP. Accordingly, amino acid-induced signals trigger proteasome-dependent degradation of the prodomain. A mutation that stabilizes the prodomain prevents Stp1 processing, whereas destabilizing mutations lead to constitutive RAP-independent Stp1 processing. We fused a conditional degron to the prodomain to synthetically reprogram the amino acid-responsive SPS signaling pathway, placing it under temperature control. Our results define a regulatory mechanism that is novel for eukaryotic proteases functioning within cells.

scholarly journals Transcriptional interference between the EBV transcription factors EB1 and R: both DNA-binding and activation domains of EB1 are required

1991 ◽  
Vol 19 (6) ◽  
pp. 1251-1258 ◽  
Author(s):  
Jean-francois Giot ◽  
Ivan Mikaelian ◽  
Monique Buisson ◽  
Evelyne Manet ◽  
Irene Joab ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Transcriptional Interference ◽  
Activation Domains

Can Fibroblasts Be Reprogrammed into Macrophages?.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 443-443
Author(s):  
Ru Feng ◽  
Thomas Graf
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Cell Lines ◽  
Fibroblast Cell ◽  
Bzip Transcription Factor ◽  
Nih3t3 Cells ◽  
Activation Domains ◽  
Gm Csf ◽  
Infected Cells ◽  
Factor C

Abstract Previous work showed that B cell precursors can be reprogrammed into functional macrophages by the enforced expression of the bZip transcription factor C/EBPalpha. The efficient activation of myelomonocytic genes, such as Mac-1, required the co-operation with endogenous PU.1 (Xie et al. 2004), reflecting the fact that many myelomonocytic genes are regulated by a combination of the two transcription factors. We therefore asked: Is C/EBPa and PU.1 sufficient to convert non-hematopoietic cells into macrophages? To test this, NIH-3T3 cells were co-infected with PU.1-GFP and C/EBPa-hCD4 retrovirusesor control vectors encoding the indicators GFP and hCD4 only. Uninfected cells in the retrovirus treated cultures served as additional controls. Our results showed that ~25% of the PU.1 only infected cells express Mac-1 and that this percentage could be increased ~3 fold by co-expression with C/EBPa. In addition, most cells also expressed CD45 and some expressed F4/80 antigen. The PU.1 infected and the double infected cells, but not the C/EBPa only infected cells, also expressed a number of other myelomonocytic genes as detected by RT-PCR. These included CSF-1R (M-CSFR), GM-CSF Ralpha, Lysozyme, CD32, PYK2 as well as endogenous PU.1. The PU.1 induced reprogramming of fibroblasts required the DNA binding and transcription activation domains, but not the PEST domain of the transcription factor. To test whether the reprogrammed cells have functional macrophage properties, we generated two stable cell lines co-expressing C/EBPa and PU.1 delta PEST (wild type PU.1 is toxic in long-term cultures). These cells were morphologically altered, ingested carboxylated particles, and expressed functional Fc-gamma receptors but were unable to phagocytize antibody coated red blood cells. Remarkably, the two cells lines acquired CSF-1 dependence for growth. In accordance with this finding they exhibited a 10–15 fold reduction of CSF-1 production compared to NIH3T3 cells. The response observed was not restricted to fibroblast cell lines since both embryonic and adult fibroblasts could also be partially reprogrammed by co-infection with PU.1 and C/EBPa in that they expressed Mac-1, CD45, F4/80 and IA MHC antigens. In conclusion, enforced expression of PU.1 and C/EBPa converts fibroblasts into macrophage like cells, indicating that the combination of these two transcription factors is sufficient to regulate the majority of genes that define the myelomonocytic phenotype.

scholarly journals Two Domains Unique to Osteoblast-Specific Transcription Factor Osf2/Cbfa1 Contribute to Its Transactivation Function and Its Inability To Heterodimerize with Cbfβ

1998 ◽  
Vol 18 (7) ◽  
pp. 4197-4208 ◽  
Author(s):  
Kannan Thirunavukkarasu ◽  
Muktar Mahajan ◽  
Keith W. McLarren ◽  
Stefano Stifani ◽  
Gerard Karsenty
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Critical Role ◽  
Transcription Activation ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Amino Terminal ◽  
Related Proteins ◽  
Repression Domain

ABSTRACT Osf2/Cbfa1, hereafter called Osf2, is a member of the Runt-related family of transcription factors that plays a critical role during osteoblast differentiation. Like all Runt-related proteins, it contains a runt domain, which is the DNA-binding domain, and a C-terminal proline-serine-threonine-rich (PST) domain thought to be the transcription activation domain. Additionally, Osf2 has two amino-terminal domains distinct from any other Runt-related protein. To understand the mechanisms of osteoblast gene regulation by Osf2, we performed an extensive structure-function analysis. After defining a short Myc-related nuclear localization signal, a deletion analysis revealed the existence of three transcription activation domains and one repression domain. AD1 (for activation domain 1) comprises the first 19 amino acids of the molecule, which form the first domain unique to Osf2, AD2 is formed by the glutamine-alanine (QA) domain, the second domain unique to Osf2, and AD3 is located in the N-terminal half of the PST domain and also contains sequences unique to Osf2. The transcription repression domain comprises the C-terminal 154 amino acids of Osf2. DNA-binding, domain-swapping, and protein interaction experiments demonstrated that full-length Osf2 does not interact with Cbfβ, a known partner of Runt-related proteins, whereas a deletion mutant of Osf2 containing only the runt and PST domains does. The QA domain appears to be responsible for preventing this heterodimerization. Thus, our results uncover the unique functional organization of Osf2 by identifying functional domains not shared with other Runt-related proteins that largely control its transactivation and heterodimerization abilities.

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