Normal Myeloid Development Requires Both the Glutamine-Rich Transactivation Domain and the PEST Region of Transcription Factor PU.1 but Not the Potent Acidic Transactivation Domain

ABSTRACT Gene targeting of transcription factor PU.1 results in an early block to fetal hematopoiesis, with no detectable lymphoid or myeloid cells produced in mouse embryos. Furthermore,PU.1 −/− embryonic stem (ES) cells fail to differentiate into Mac-1+ and F4/80+macrophages in vitro. We have previously shown that a PU.1 transgene under the control of its own promoter restores the ability ofPU.1 −/− ES cells to differentiate into macrophages. In this study, we take advantage of ourPU.1 −/− ES cell rescue system to genetically test which previously identified PU.1 functional domains are necessary for the development of mature macrophages. PU.1 functional domains include multiple N-terminal acidic and glutamine-rich transactivation domains, a PEST domain, several serine phosphorylation sites, and a C-terminal Ets DNA binding domain, all delineated and characterized by using standard biochemical and transactivational assays. By using the production of mature macrophages as a functional readout in our assay system, we have established that the glutamine-rich transactivation domain, a portion of the PEST domain, and the DNA binding domain are required for myelopoiesis. Deletion of three acidic domains, which exhibit potent transactivation potential in vitro, had no effect on the ability of PU.1 to promote macrophage development. Furthermore, mutagenesis of four independent sites of serine phosphorylation also had no effect on myelopoiesis. Collectively, our results indicate that PU.1 interacts with important regulatory proteins during macrophage development via the glutamine-rich and PEST domains. ThePU.1 −/− ES cell rescue system represents a powerful, in vitro strategy to functionally map domains of PU.1 essential for normal hematopoiesis and the generation of mature macrophages.

Download Full-text

Erythroid-cell-specific properties of transcription factor GATA-1 revealed by phenotypic rescue of a gene-targeted cell line.

Molecular and Cellular Biology ◽

10.1128/mcb.17.3.1642 ◽

1997 ◽

Vol 17 (3) ◽

pp. 1642-1651 ◽

Cited By ~ 240

Author(s):

M J Weiss ◽

C Yu ◽

S H Orkin

Keyword(s):

Transcription Factor ◽

Cell Line ◽

Zinc Finger ◽

Es Cells ◽

Embryonic Stem ◽

Erythroid Cell ◽

Transactivation Domain ◽

Stage Of Development ◽

Erythroid Maturation

The zinc finger transcription factor GATA-1 is essential for erythropoiesis. In its absence, committed erythroid precursors arrest at the proerythroblast stage of development and undergo apoptosis. To study the function of GATA-1 in an erythroid cell environment, we generated an erythroid cell line from in vitro-differentiated GATA-1- murine embryonic stem (ES) cells. These cells, termed G1E for GATA-1- erythroid, proliferate as immature erythroblasts yet complete differentiation upon restoration of GATA-1 function. We used rescue of terminal erythroid maturation in G1E cells as a stringent cellular assay system in which to evaluate the functional relevance of domains of GATA-1 previously characterized in nonhematopoietic cells. At least two major differences were established between domains required in G1E cells and those required in nonhematopoietic cells. First, an obligatory transactivation domain defined in conventional nonhematopoietic cell transfection assays is dispensable for terminal erythroid maturation. Second, the amino (N) zinc finger, which is nonessential for binding to the vast majority of GATA DNA motifs, is strictly required for GATA-1-mediated erythroid differentiation. Our data lead us to propose a model in which a nuclear cofactor(s) interacting with the N-finger facilitates transcriptional action by GATA-1 in erythroid cells. More generally, our experimental approach highlights critical differences in the action of cell-specific transcription proteins in different cellular environments and the power of cell lines derived from genetically modified ES cells to elucidate gene function.

Download Full-text

Methylation of RUNX1 by the Nuclear PRMT5/COPR5 Complex Regulates Its Cellular Location in Leukemia Cells,

Blood ◽

10.1182/blood.v118.21.3403.3403 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3403-3403

Author(s):

Xinyang Zhao ◽

Ly P. Vu ◽

Fabiana Perna ◽

Fan Liu ◽

Hao Xu ◽

...

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Arginine Methylation ◽

Binding Domain ◽

Dependent Manner ◽

Dna Binding Domain ◽

Sm Proteins ◽

Differentiation Potential ◽

Hematopoietic Stem

Abstract Abstract 3403 RUNX1 is a transcription factor that is required for definitive hematopoietic development, and helps regulate long term hematopoietic stem cell self-renewal, platelet production, and lymphocyte development during adult hematopoiesis. RUNX1 is known to be modified via phosphorylation, acetylation, ubiquitination and methylation, for example on R208 and R210 by PRMT1, which activates its activating function. We continue to investigate how the methylation of RUNX1 by other protein arginine methyl transferases (PRMTs) regulates its function. Loop 9 of the DNA binding domain (the Runt domain) of RUNX1 contains an SGRGK sequence that is also present on the tails of histone H2A and H4. The histone tails of H4 and H2A can be methylated by a purified PRMT5 complex in vitro. An enzymatically active in vitro PRMT5 complex capable of methylating histones and SM proteins requires two subunits: both PRMT5 and MEP50, a WD 40 repeat domain protein. Nevertheless, this purified PRMT5/MEP50 complex cannot methylate the DNA binding domain of the RUNX1 protein in vitro. We show that RUNX1 also can be symmetrically methylated at R142 within the SGRGK motif in vitro by a nuclear PRMT5/MEP50 complex which also contains COPR5. We show after RUNX1 is methylated on R142 within the nucleus of HEL cells, RUNX1 is exported to the cytoplasm in a CRM1 dependent manner, as the export of methylated RUNX1 is blocked by lemptomycin B. CRM1 interacts with PRMT5, supporting that PRMT5 mediated arginine methylation tags protein for nuclear export. Therefore, PRMT5 not only involves in epigenetic regulation by methylation of histones but also it can directly controls the level of transcription factor proteins within the nucleus. Polycytocemia Vera patients who express the Jak2V617F mutation have low PRMT5 activity due to JAK2V617F mediated PRMT5 phosphorylation (Liu et al 2011). How Jak2 signaling affects RUNX1 methylation and RUNX1 localization within the nucleus is still under investigation. By controlling the amount of RUNX1 available within the cell nucleus, PRMT5 may regulate lineage differentiation potential and growth potential of hematopoietic stem and progenitor cells. The nuclear localization of RUNX1 can be changed through post translational modification such as arginine methylation in addition to point mutations and translocations involving RUNX1 found patients with leukemia and pre-leukemic diseases. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Balance between mutually exclusive traits shifted by variants of a yeast transcription factor

10.1101/117911 ◽

2017 ◽

Cited By ~ 1

Author(s):

Michael W. Dorrity ◽

Josh T. Cuperus ◽

Jolie A. Carlisle ◽

Stanley Fields ◽

Christine Queitsch

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcription Factor ◽

Amino Acid ◽

Dna Binding ◽

Single Amino Acid ◽

Environmental Cues ◽

Dna Binding Domain ◽

Independent Manner ◽

Half Site

AbstractIn Saccharomyces cerevisiae, the decision to mate or invade relies on environmental cues that converge on a shared transcription factor, Ste12. Specificity toward invasion occurs via Ste12 binding cooperatively with the co-factor Tec1. Here, we characterize the in vitro binding preferences of Ste12 to identify a defined spacing and orientation of dimeric sites, one that is common in pheromone-regulated genes. We find that single amino acid changes in the DNA-binding domain of Ste12 can shift the preference of yeast toward either mating or invasion. These mutations define two distinct regions of this domain, suggesting alternative modes of DNA binding for each trait. Some exceptional Ste12 mutants promote hyperinvasion in a Tec1-independent manner; these fail to bind cooperative sites with Tec1 and bind to unusual dimeric Ste12 sites that contain one highly degenerate half site. We propose a model for how activation of invasion genes could have evolved with Ste12 alone.

Download Full-text

Functional and conserved domains of the Drosophila transcription factor encoded by the segmentation gene knirps

Molecular and Cellular Biology ◽

10.1128/mcb.14.12.7899-7908.1994 ◽

1994 ◽

Vol 14 (12) ◽

pp. 7899-7908

Author(s):

N Gerwin ◽

A La Rosée ◽

F Sauer ◽

H P Halbritter ◽

M Neumann ◽

...

Keyword(s):

Transcription Factor ◽

Amino Acid ◽

Dna Binding ◽

Broad Band ◽

Binding Domain ◽

Orphan Receptor ◽

Dna Binding Domain ◽

Coding Region ◽

Amino Acid Region

The Drosophila gap gene knirps (kni) is required for abdominal segmentation. It encodes a steroid/thyroid orphan receptor-type transcription factor which is distributed in a broad band of nuclei in the posterior region of the blastoderm. To identify essential domains of the kni protein (KNI), we cloned and sequenced the DNA encompassing the coding region of nine kni mutant alleles of different strength and kni-homologous genes of related insect species. We also examined in vitro-modified versions of KNI in various assay systems both in vitro and in tissue culture. The results show that KNI contains several functional domains which are arranged in a modular fashion. The N-terminal 185-amino-acid region which includes the DNA-binding domain and a functional nuclear location signal fails to provide kni activity to the embryo. However, a truncated KNI protein that contains additional 47 amino acids exerts rather strong kni activity which is functionally defined by a weak kni mutant phenotype of the embryo. The additional 47-amino-acid stretch includes a transcriptional repressor domain which acts in the context of a heterologous DNA-binding domain of the yeast transcriptional activator GAL4. The different domains of KNI as defined by functional studies are conserved during insect evolution.

Download Full-text

VWRPY motif–dependent and –independent roles of AML1/Runx1 transcription factor in murine hematopoietic development

Blood ◽

10.1182/blood-2003-06-2109 ◽

2004 ◽

Vol 103 (2) ◽

pp. 562-570 ◽

Cited By ~ 37

Author(s):

Motohiro Nishimura ◽

Yoko Fukushima-Nakase ◽

Yasuko Fujita ◽

Mitsushige Nakao ◽

Shogo Toda ◽

...

Keyword(s):

Transcription Factor ◽

Es Cells ◽

Embryonic Stem ◽

Es Cell ◽

Thymocyte Development ◽

Hematopoietic Development ◽

Cell Clones ◽

Definitive Hematopoiesis

Abstract AML1/Runx1 is a frequent target of leukemia-associated gene aberration, and it encodes a transcription factor essential for definitive hematopoiesis. We previously reported that the AML1 molecules with trans-activation subdomains retained can rescue in vitro hematopoietic defects of AML1-deficient mouse embryonic stem (ES) cells when expressed by using a knock-in approach. Extending this notion to in vivo conditions, we found that the knock-in ES cell clones with AML1 mutants, which retain trans-activation subdomains but lack C-terminal repression subdomains including the conserved VWRPY motif, contribute to hematopoietic tissues in chimera mice. We also found that germline mice homozygous for the mutated AML1 allele, which lacks the VWRPY motif, exhibit a minimal effect on hematopoietic development, as was observed in control knock-in mice with full-length AML1. On the other hand, reduced cell numbers and deviant CD4 expression were observed during early T-lymphoid ontogeny in the VWRPY-deficient mice, whereas the contribution to the thymus by the corresponding ES cell clones was inadequate. These findings demonstrate that AML1 with its trans-activating subdomains is essential and sufficient for hematopoietic development in the context of the entire mouse. In addition, its trans-repression activity, depending on the C-terminal VWRPY motif, plays a role in early thymocyte development.

Download Full-text

Effects of mutations within two hydrophilic regions of the bovine papillomavirus type 1 E1 DNA-binding domain on E1–E2 interaction

Journal of General Virology ◽

10.1099/0022-1317-82-10-2341 ◽

2001 ◽

Vol 82 (10) ◽

pp. 2341-2351 ◽

Cited By ~ 7

Author(s):

Kelly J. Woytek ◽

Dhandapani Rangasamy ◽

Cynthia Bazaldua-Hernandez ◽

Mike West ◽

Van G. Wilson

Keyword(s):

Dna Binding ◽

Binding Domain ◽

Genome Replication ◽

Bovine Papillomavirus ◽

Dna Binding Domain ◽

Transactivation Domain ◽

Recognition Element ◽

C Terminus

The interaction between papillomavirus E1 and E2 proteins is essential for viral genome replication. Using both in vivo and in vitro assays to evaluate the regions of the two proteins necessary for the E1–E2 interaction, three independent interactions were identified for bovine papillomavirus E1: the N terminus of E1 (E1N, residues 1–311) interacts with the E2 transactivation domain (E2TAD) and the E2 DNA-binding domain (E2DBD) and the C terminus of E1 (E1C, residues 315–605) interacts with E2. Nine mutations within E1N were evaluated for their effects on E2 interaction. Five mutations eliminated interaction with the E2TAD; four of these were located within two previously identified conserved, hydrophilic regions, HR1 and HR3. Since HR1 and HR3 residues appear to comprise the origin of replication recognition element for E1, simultaneous interaction with the E2TAD during initiation complex formation would seem unlikely. Consistent with this inference is the fact that three of the five mutants defective for E2TAD binding exhibited wild-type levels of replication. The replication-positive phenotype of these mutants suggests that the E1N–E2TAD interaction is not essential for replication function and is probably involved in some other E1–E2 function, such as regulating transcription. Only one of the five mutations defective for E2TAD binding also prevented E2DBD interaction, indicating that the regions of E1N that interact with the E2TAD and the E2DBD are not identical. The ability of E1N to cooperatively interact with E2 bound to E2-binding site (E2BS) 11 versus E2BS12 was also examined, and cooperative binding was only observed when E2 was bound to E2BS12.

Download Full-text

GATA-1 associates with and inhibits p53

Blood ◽

10.1182/blood-2008-10-180489 ◽

2009 ◽

Vol 114 (1) ◽

pp. 165-173 ◽

Cited By ~ 24

Author(s):

Cecelia D. Trainor ◽

Caroline Mas ◽

Patrick Archambault ◽

Paola Di Lello ◽

James G. Omichinski

Keyword(s):

Dna Binding ◽

Mutational Analysis ◽

Binding Domain ◽

Erythroid Cell ◽

Dna Binding Domain ◽

Transactivation Domain ◽

Nuclear Condensation ◽

P53 Pathway ◽

In Vitro Interaction

Abstract In addition to orchestrating the expression of all erythroid-specific genes, GATA-1 controls the growth, differentiation, and survival of the erythroid lineage through the regulation of genes that manipulate the cell cycle and apoptosis. The stages of mammalian erythropoiesis include global gene inactivation, nuclear condensation, and enucleation to yield circulating erythrocytes, and some of the genes whose expression are altered by GATA-1 during this process are members of the p53 pathway. In this study, we demonstrate a specific in vitro interaction between the transactivation domain of p53 (p53TAD) and a segment of the GATA-1 DNA-binding domain that includes the carboxyl-terminal zinc-finger domain. We also show by immunoprecipitation that the native GATA-1 and p53 interact in erythroid cells and that activation of p53-responsive promoters in an erythroid cell line can be inhibited by the overexpression of GATA-1. Mutational analysis reveals that GATA-1 inhibition of p53 minimally requires the segment of the GATA-1 DNA-binding domain that interacts with p53TAD. This inhibition is reciprocal, as the activation of a GATA-1–responsive promoter can be inhibited by p53. Based on these findings, we conclude that inhibition of the p53 pathway by GATA-1 may be essential for erythroid cell development and survival.

Download Full-text

Functionally distinct roles for T and Tbx6 during mouse development

Biology Open ◽

10.1242/bio.054692 ◽

2020 ◽

Vol 9 (8) ◽

pp. bio054692

Author(s):

Amy K. Wehn ◽

Deborah R. Farkas ◽

Carly E. Sedlock ◽

Dibya Subedi ◽

Deborah L. Chapman

Keyword(s):

Dna Binding ◽

Binding Sites ◽

Transcriptional Activity ◽

Es Cells ◽

Binding Domain ◽

Dna Binding Domain ◽

Mouse Development ◽

T Box

ABSTRACTThe mouse T-box transcription factors T and Tbx6 are co-expressed in the primitive streak and have unique domains of expression; T is expressed in the notochord, while Tbx6 is expressed in the presomitic mesoderm. T-box factors are related through a shared DNA binding domain, the T-domain, and can therefore bind to similar DNA sequences at least in vitro. We investigated the functional similarities and differences of T and Tbx6 DNA binding and transcriptional activity in vitro and their interaction genetically in vivo. We show that at one target, Dll1, the T-domains of T and Tbx6 have different affinities for the binding sites present in the mesoderm enhancer. We further show using in vitro assays that T and Tbx6 differentially affect transcription with Tbx6 activating expression tenfold higher than T, that T and Tbx6 can compete at target gene enhancers, and that this competition requires a functional DNA binding domain. Next, we addressed whether T and Tbx6 can compete in vivo. First, we generated embryos that express Tbx6 at greater than wild-type levels embryos and show that these embryos have short tails, resembling the T heterozygous phenotype. Next, using the dominant-negative TWis allele, we show that Tbx6+/− TWis/+ embryos share similarities with embryos homozygous for the Tbx6 hypomorphic allele rib-vertebrae, specifically fusions of several ribs and malformation of some vertebrae. Finally, we tested whether Tbx6 can functionally replace T using a knockin approach, which resulted in severe T null-like phenotypes in chimeric embryos generated with ES cells heterozygous for a Tbx6 knockin at the T locus. Altogether, our results of differences in affinity for DNA binding sites and transcriptional activity for T and Tbx6 provide a potential mechanism for the failure of Tbx6 to functionally replace T and possible competition phenotypes in vivo.

Download Full-text

Crystal structure of the DNA binding domain of the transcription factor T-bet suggests simultaneous recognition of distant genome sites

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1613914113 ◽

2016 ◽

Vol 113 (43) ◽

pp. E6572-E6581 ◽

Cited By ~ 7

Author(s):

Ce Feng Liu ◽

Gabriel S. Brandt ◽

Quyen Q. Hoang ◽

Natalia Naumova ◽

Vanja Lazarevic ◽

...

Keyword(s):

Crystal Structure ◽

Transcription Factor ◽

Dna Binding ◽

Quaternary Structure ◽

Regulatory Element ◽

Binding Domain ◽

Dna Binding Domain ◽

Chromosome Conformation

The transcription factor T-bet (Tbox protein expressed in T cells) is one of the master regulators of both the innate and adaptive immune responses. It plays a central role in T-cell lineage commitment, where it controls the TH1 response, and in gene regulation in plasma B-cells and dendritic cells. T-bet is a member of the Tbox family of transcription factors; however, T-bet coordinately regulates the expression of many more genes than other Tbox proteins. A central unresolved question is how T-bet is able to simultaneously recognize distant Tbox binding sites, which may be located thousands of base pairs away. We have determined the crystal structure of the Tbox DNA binding domain (DBD) of T-bet in complex with a palindromic DNA. The structure shows a quaternary structure in which the T-bet dimer has its DNA binding regions splayed far apart, making it impossible for a single dimer to bind both sites of the DNA palindrome. In contrast to most other Tbox proteins, a single T-bet DBD dimer binds simultaneously to identical half-sites on two independent DNA. A fluorescence-based assay confirms that T-bet dimers are able to bring two independent DNA molecules into close juxtaposition. Furthermore, chromosome conformation capture assays confirm that T-bet functions in the direct formation of chromatin loops in vitro and in vivo. The data are consistent with a looping/synapsing model for transcriptional regulation by T-bet in which a single dimer of the transcription factor can recognize and coalesce distinct genetic elements, either a promoter plus a distant regulatory element, or promoters on two different genes.

Download Full-text

The Yeast Heat Shock Transcription Factor Changes Conformation in Response to Superoxide and Temperature

Molecular Biology of the Cell ◽

10.1091/mbc.11.5.1753 ◽

2000 ◽

Vol 11 (5) ◽

pp. 1753-1764 ◽

Cited By ~ 44

Author(s):

Sengyong Lee ◽

Tage Carlson ◽

Noah Christian ◽

Kristi Lea ◽

Jennifer Kedzie ◽

...

Keyword(s):

Transcription Factor ◽

Heat Shock ◽

Dna Binding ◽

Conformational Change ◽

Mutational Analysis ◽

Binding Domain ◽

Heat Shock Transcription Factor ◽

Dna Binding Domain ◽

Yeast Saccharomyces Cerevisiae

In vitro DNA-binding assays demonstrate that the heat shock transcription factor (HSF) from the yeast Saccharomyces cerevisiae can adopt an altered conformation when stressed. This conformation, reflected in a change in electrophoretic mobility, requires that two HSF trimers be bound to DNA. Single trimers do not show this change, which appears to represent an alteration in the cooperative interactions between trimers. HSF isolated from stressed cells displays a higher propensity to adopt this altered conformation. Purified HSF can be stimulated in vitro to undergo the conformational change by elevating the temperature or by exposing HSF to superoxide anion. Mutational analysis maps a region critical for this conformational change to the flexible loop between the minimal DNA-binding domain and the flexible linker that joins the DNA-binding domain to the trimerization domain. The significance of these findings is discussed in the context of the induction of the heat shock response by ischemic stroke, hypoxia, and recovery from anoxia, all known to stimulate the production of superoxide.

Download Full-text