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

Preferences in a trait decision determined by transcription factor variants

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1805882115 ◽

2018 ◽

Vol 115 (34) ◽

pp. E7997-E8006 ◽

Cited By ~ 8

Author(s):

Michael W. Dorrity ◽

Josh T. Cuperus ◽

Jolie A. Carlisle ◽

Stanley Fields ◽

Christine Queitsch

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Fungal Pathogens ◽

Binding Domain ◽

Single Amino Acid ◽

Environmental Cues ◽

Dna Binding Domain ◽

Independent Manner ◽

Dna Binding Specificity ◽

The Many

Few mechanisms are known that explain how transcription factors can adjust phenotypic outputs to accommodate differing environments. In 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 cofactor Tec1. Here, we determine the range of phenotypic outputs (mating vs. invasion) of thousands of DNA-binding domain variants in Ste12 to understand how preference for invasion may arise. We find that single amino acid changes in the DNA-binding domain 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. We characterize the DNA-binding specificity of wild-type Ste12 to identify a strong preference for spacing and orientation of both homodimeric and heterodimeric sites. Ste12 mutants that promote hyperinvasion in a Tec1-independent manner fail to bind cooperative sites with Tec1 and bind to unusual dimeric Ste12 sites composed of one near-perfect and one highly degenerate site. We propose a model in which Ste12 alone may have evolved to activate invasion genes, which could explain how preference for invasion arose in the many fungal pathogens that lack Tec1.

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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.

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Two Regulators of Ste12p Inhibit Pheromone-Responsive Transcription by Separate Mechanisms

Molecular and Cellular Biology ◽

10.1128/mcb.20.12.4199-4209.2000 ◽

2000 ◽

Vol 20 (12) ◽

pp. 4199-4209 ◽

Cited By ~ 62

Author(s):

K. Amy Olson ◽

Chris Nelson ◽

Georgia Tai ◽

Wesley Hung ◽

Carl Yong ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcription Factor ◽

Dna Binding ◽

Transcriptional Activation ◽

Dna Binding Domain ◽

Wild Type ◽

C Terminus ◽

Multiple Regions ◽

Vp16 Fusion

ABSTRACT The yeast Saccharomyces cerevisiae transcription factor Ste12p is responsible for activating genes in response to MAP kinase cascades controlling mating and filamentous growth. Ste12p is negatively regulated by two inhibitor proteins, Dig1p (also called Rst1p) and Dig2p (also called Rst2p). The expression of a C-terminal Ste12p fragment (residues 216 to 688) [Ste12p(216–688)] from aGAL promoter causes FUS1 induction in a strain expressing wild-type STE12, suggesting that this region can cause the activation of endogenous Ste12p. Residues 262 to 594 are sufficient to cause STE12-dependent FUS1induction when overexpressed, and this region of Ste12p was found to bind Dig1p but not Dig2p in yeast extracts. In contrast, recombinant glutathione S-transferase–Dig2p binds to the Ste12p DNA-binding domain (DBD). Expression of DIG2, but notDIG1, from a GAL promoter inhibits transcriptional activation by an Ste12p DBD-VP16 fusion. Furthermore, disruption of dig1, but not dig2, causes elevated transcriptional activation by a LexA–Ste12p(216–688) fusion. Ste12p has multiple regions within the C terminus (flanking residue 474) that can promote multimerization in vitro, and we demonstrate that these interactions can contribute to the activation of endogenous Ste12p by overproduced C-terminal fragments. These results demonstrate that Dig1p and Dig2p do not function by redundant mechanisms but rather inhibit pheromone-responsive transcription through interactions with separate regions of Ste12p.

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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 ◽

1994 ◽

Vol 14 (12) ◽

pp. 7899-7908 ◽

Cited By ~ 18

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.

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Biochemical and genetic characterization of a yeast TFIID mutant that alters transcription in vivo and DNA binding in vitro

Molecular and Cellular Biology ◽

10.1128/mcb.12.5.2372-2382.1992 ◽

1992 ◽

Vol 12 (5) ◽

pp. 2372-2382

Author(s):

K M Arndt ◽

S L Ricupero ◽

D M Eisenmann ◽

F Winston

Keyword(s):

Amino Acid ◽

Dna Binding ◽

Direct Repeat ◽

Single Amino Acid ◽

Wild Type ◽

Dna Binding Specificity ◽

Selection For

A mutation in the gene that encodes Saccharomyces cerevisiae TFIID (SPT15), which was isolated in a selection for mutations that alter transcription in vivo, changes a single amino acid in a highly conserved region of the second direct repeat in TFIID. Among eight independent spt15 mutations, seven cause this same amino acid change, Leu-205 to Phe. The mutant TFIID protein (L205F) binds with greater affinity than that of wild-type TFIID to at least two nonconsensus TATA sites in vitro, showing that the mutant protein has altered DNA binding specificity. Site-directed mutations that change Leu-205 to five different amino acids cause five different phenotypes, demonstrating the importance of this amino acid in vivo. Virtually identical phenotypes were observed when the same amino acid changes were made at the analogous position, Leu-114, in the first repeat of TFIID. Analysis of these mutations and additional mutations in the most conserved regions of the repeats, in conjunction with our DNA binding results, suggests that these regions of the repeats play equivalent roles in TFIID function, possibly in TATA box recognition.

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Two monomers of yeast transcription factor ADR1 bind a palindromic sequence symmetrically to activate ADH2 expression.

Molecular and Cellular Biology ◽

10.1128/mcb.11.3.1566 ◽

1991 ◽

Vol 11 (3) ◽

pp. 1566-1577 ◽

Cited By ~ 58

Author(s):

S K Thukral ◽

A Eisen ◽

E T Young

Keyword(s):

Transcription Factor ◽

Amino Acid ◽

Dna Binding ◽

Dimethyl Sulfate ◽

Binding Activity ◽

Single Amino Acid ◽

Minor Effect ◽

Palindromic Sequence ◽

Amino Terminal ◽

Dna Binding Activity

ADR1 is a transcription factor from Saccharomyces cerevisiae that regulates ADH2 expression through a 22-bp palindromic sequence (UAS1). Size fractionation studies revealed that full-length ADR1 and a truncated ADR1 protein containing the first 229 amino acids, which has the complete DNA-binding domain, ADR1:17-229, exist as monomers in solution. However, two complexes were formed with target DNA-binding sites. UV-cross-linking studies suggested that these two complexes represent one and two molecules of ADR1 bound to DNA. Studies of ADR1 complexes formed with wild-type UAS1, asymmetrically altered UAS1, and one half of UAS1 showed that ADR1 can bind to one half of UAS1 and gives rise to a complex containing one molecule of ADR1. Dimethyl sulfate interference studies were consistent with this interpretation and in addition indicated that purine contact sites in each half of UAS1 were identical. Increasing the distance between the two halves of UAS1 had at most a minor effect of the thermodynamics of formation of the two complexes. These data are more consistent with ADR1 binding as two independent monomers, one to each half of UAS1. However, binding of two ADR1 monomers at UAS1 is apparently essential for transactivation in vivo. Further, we have identified a stretch of 18 amino acid residues amino terminal to the zinc two-finger domains of ADR1 which is essential for DNA-binding activity. Single amino acid substitutions of residues in this region resulted in severely reduced DNA-binding activity.

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A single amino acid change in CUP2 alters its mode of DNA binding.

Molecular and Cellular Biology ◽

10.1128/mcb.10.9.4778 ◽

1990 ◽

Vol 10 (9) ◽

pp. 4778-4787 ◽

Cited By ~ 28

Author(s):

C Buchman ◽

P Skroch ◽

W Dixon ◽

T D Tullius ◽

M Karin

Keyword(s):

Amino Acid ◽

Dna Binding ◽

Binding Activity ◽

Binding Domain ◽

Single Amino Acid ◽

Dna Binding Domain ◽

Sequence Motifs ◽

Wild Type ◽

Mobility Shift ◽

Single Amino Acid Change

CUP2 is a copper-dependent transcriptional activator of the yeast CUP1 metallothionein gene. In the presence of Cu+ and Ag+) ions its DNA-binding domain is thought to fold as a cysteine-coordinated Cu cluster which recognizes the palindromic CUP1 upstream activation sequence (UASc). Using mobility shift, methylation interference, and DNase I and hydroxyl radical footprinting assays, we examined the interaction of wild-type and variant CUP2 proteins produced in Escherichia coli with the UASc. Our results suggest that CUP2 has a complex Cu-coordinated DNA-binding domain containing different parts that function as DNA-binding elements recognizing distinct sequence motifs embedded within the UASc. A single-amino-acid substitution of cysteine 11 with a tyrosine results in decreased Cu binding, apparent inactivation of one of the DNA-binding elements and a dramatic change in the recognition properties of CUP2. This variant protein interacts with only one part of the wild-type site and prefers to bind to a different half-site from the wild-type protein. Although the variant has about 10% of wild-type DNA-binding activity, it appears to be completely incapable of activating transcription.

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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.

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Properties of the DNA-binding domain of the Saccharomyces cerevisiae STE12 protein.

Molecular and Cellular Biology ◽

10.1128/mcb.11.12.5910 ◽

1991 ◽

Vol 11 (12) ◽

pp. 5910-5918 ◽

Cited By ~ 50

Author(s):

Y L Yuan ◽

S Fields

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Dna Binding ◽

Binding Affinity ◽

Binding Domain ◽

Upstream Region ◽

Dna Binding Domain ◽

Yeast Saccharomyces Cerevisiae ◽

Dnase I Footprinting

The STE12 protein of the yeast Saccharomyces cerevisiae binds to the pheromone response element (PRE) present in the upstream region of genes whose transcription is induced by pheromone. Using DNase I footprinting assays with bacterially made STE12 fragments, we localized the DNA-binding domain to 164 amino acids near the amino terminus. Footprinting of oligonucleotide-derived sequences containing one PRE, or two PREs in head-to-tail or tail-to-tail orientation, showed that the N-terminal 215 amino acids of STE12 has similar binding affinity to either of the dimer sites and a binding affinity 5- to 10-fold lower for the monomer site. This binding cooperativity was also evident on a fragment from the MFA2 gene, which encodes the a-factor pheromone. On this fragment, the 215-amino-acid STE12 fragment protected both a consensus PRE as well as a degenerate PRE containing an additional residue. Mutation of the degenerate site led to a 5- to 10-fold decrease in binding; mutation of the consensus site led to a 25-fold decrease in binding. The ability of PREs to function as pheromone-inducible upstream activation sequences in yeast correlated with their ability to bind the STE12 domain in vitro. The sequence of the STE12 DNA-binding domain contains similarities to the homeodomain, although it is highly diverged from other known examples of this motif. Moreover, the alignment between STE12 and the homeodomain postulates loops after both the putative helix 1 and helix 2 of the STE12 sequence.

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The TATA-Binding Protein (TBP) from the Human Fungal PathogenCandida albicans Can Complement Defects in Human and Yeast TBPs

Journal of Bacteriology ◽

10.1128/jb.180.7.1771-1776.1998 ◽

1998 ◽

Vol 180 (7) ◽

pp. 1771-1776 ◽

Cited By ~ 2

Author(s):

Ping Leng ◽

Philip E. Carter ◽

Alistair J. P. Brown

Keyword(s):

Amino Acid ◽

Dna Binding ◽

Transcription Initiation ◽

Binding Protein ◽

Tata Binding Protein ◽

Binding Domain ◽

Amino Acid Sequences ◽

Initiation Factor ◽

Dna Binding Domain

ABSTRACT Candida albicans is the major fungal pathogen in humans, yet little is known about transcriptional regulation in this organism. Therefore, we have isolated, characterized, and expressed theC. albicans TATA-binding protein (TBP) gene (TBP1), because this general transcription initiation factor plays a key role in the activation and regulation of eukaryotic promoters. Southern and Northern blot analyses suggest that a single C. albicans TBP1 locus is expressed at similar levels in the yeast and hyphal forms of this fungus. The TBP1 open reading frame is 716 bp long and encodes a functional TBP of 27 kDa. C. albicans TBP is capable of binding specifically to a TATA box in vitro, substituting for the human TBP to activate basal transcription in vitro, and suppressing the lethal Δspt15 mutation inSaccharomyces cerevisiae. The predicted amino acid sequences of TBPs from C. albicans and other organisms reveal a striking pattern of C-terminal conservation and N-terminal variability: the C-terminal DNA-binding domain displays at least 80% amino acid sequence identity to TBPs from fungi, flies, nematodes, slime molds, plants, and humans. Sequence differences between human and fungal TPBs in the DNA-binding domain may represent potential targets for antifungal therapy.

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