Evolution of the activation domain in a Hox transcription factor

Linking changes in amino acid sequences to the evolution of transcription regulatory domains is often complicated by the low sequence complexity and high mutation rates of intrinsically disordered protein regions. For the Hox transcription factor Ultrabithorax (Ubx), conserved motifs distributed throughout the protein sequence enable direct comparison of specific protein regions, despite variations in the length and composition of the intervening sequences. In cell culture, the strength of transcription activation by Drosophila melanogaster Ubx correlates with the presence of a predicted helix within its activation domain. Curiously, this helix is not preserved in species more divergent than flies, suggesting the nature of transcription activation may have evolved. To determine whether this helix contributes to Drosophila Ubx function in vivo, wild-type and mutant proteins were ectopically expressed in the developing wing and the phenotypes evaluated. Helix mutations alter Drosophila Ubx activity in the developing wing, demonstrating its functional importance in vivo. The locations of activation domains in Ubx orthologues were identified by testing the ability of truncation mutants to activate transcription in yeast one-hybrid assays. In Ubx orthologues representing 540 million years of evolution, the ability to activate transcription varies substantially. The sequence and the location of the activation domains also differ. Consequently, analogous regions of Ubx orthologues change function over time, and may activate transcription in one species, but have no activity, or even inhibit transcription activation in another species. Unlike homeodomain-DNA binding, the nature of transcription activation by Ubx has substantially evolved.

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An activation domain of the helix-loop-helix transcription factor E2A shows cell type preference in vivo in microinjected zebra fish embryos.

Molecular and Cellular Biology ◽

10.1128/mcb.16.4.1714 ◽

1996 ◽

Vol 16 (4) ◽

pp. 1714-1721 ◽

Cited By ~ 19

Author(s):

F Argenton ◽

Y Arava ◽

A Aronheim ◽

M D Walker

Keyword(s):

Gene Expression ◽

Nervous System ◽

Transcription Factor ◽

Transcription Activation ◽

Cell Types ◽

Zebra Fish ◽

Reporter Plasmid ◽

Activation Domains ◽

Helix Loop Helix

The E2A protein is a mammalian transcription factor of the helix-loop-helix family which is implicated in cell-specific gene expression in several cell lineages. Mouse E2A contains two independent transcription activation domains, ADI and ADII; whereas ADI functions effectively in a variety of cultured cell lines, ADII shows preferential activity in pancreatic beta cells. To analyze this preferential activity in an in vivo setting, we adapted a system involving transient gene expression in microinjected zebra fish embryos. Fertilized one- to four-cell embryos were coinjected with an expression plasmid and a reporter plasmid. The expression plasmids used encode the yeast Gal4 DNA-binding domain (DBD) alone, or Gal4 DBD fused to ADI, ADII, or VP16. The reporter plasmid includes the luciferase gene linked to a promoter containing repeats of UASg, the Gal4-binding site. Embryo extracts prepared 24 h after injection showed significant luciferase activity in response to each of the three activation domains. To determine the cell types in which the activation domains were functioning, a reporter plasmid encoding beta-galactosidase and then in situ staining of whole embryos were used. Expression of ADI led to activation in all major groups of cell types of the embryo (skin, sclerotome, myotome, notochord, and nervous system). On the other hand, ADII led to negligible expression in the sclerotome, notochord, and nervous system and much more frequent expression in the myotome. Parallel experiments conducted with transfected mammalian cells have confirmed that ADII shows significant activity in myoblast cells but little or no activity in neuronal precursor cells, consistent with our observations in zebra fish. This transient-expression approach permits rapid in vivo analysis of the properties of transcription activation domains: the data show that ADII functions preferentially in cells of muscle lineage, consistent with the notion that certain activation domains contribute to selective gene activation in vivo.

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A high-throughput screen for transcription activation domains reveals their sequence characteristics and permits reliable prediction by deep learning

10.1101/2019.12.11.872986 ◽

2019 ◽

Author(s):

Ariel Erijman ◽

Lukasz Kozlowski ◽

Salma Sohrabi-Jahromi ◽

James Fishburn ◽

Linda Warfield ◽

...

Keyword(s):

Amino Acid ◽

Transcription Activation ◽

Amino Acid Sequences ◽

Large Set ◽

Hydrophobic Residues ◽

Activation Domains ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Protein Interfaces ◽

Wide Range

AbstractTranscription activation domains (ADs) are encoded by a wide range of seemingly unrelated amino acid sequences, making it difficult to recognize features that permit their dynamic behavior, fuzzy interactions and target specificity. We screened a large set of random 30-mer peptides for AD function and trained a deep neural network (ADpred) on the AD-positive and negative sequences. ADpred correctly identifies known ADs within protein sequences and accurately predicts the consequences of mutations. We show that functional ADs are (1) located within intrinsically disordered regions with biased amino acid composition, (2) contain clusters of hydrophobic residues near acidic side chains, (3) are enriched or depleted for particular dipeptide sequences, and (4) have higher helical propensity than surrounding regions. Taken together, our findings fit the model of “fuzzy” binding through hydrophobic protein-protein interfaces, where activator-coactivator binding takes place in a dynamic hydrophobic environment rather than through combinations of sequence-specific interactions.

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Faculty Opinions recommendation of Intrinsically disordered regions direct transcription factor in vivo binding specificity.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.738147787.793576666 ◽

2020 ◽

Author(s):

Andrew D Sharrocks

Keyword(s):

Transcription Factor ◽

Binding Specificity ◽

Direct Transcription ◽

In Vivo Binding ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Disordered Regions

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Faculty Opinions recommendation of Intrinsically disordered regions direct transcription factor in vivo binding specificity.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.738147787.793575950 ◽

2020 ◽

Author(s):

Vladimir Uversky

Keyword(s):

Transcription Factor ◽

Binding Specificity ◽

Direct Transcription ◽

In Vivo Binding ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Disordered Regions

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Transcriptional Activation in Yeast Cells Lacking Transcription Factor IIA

Genetics ◽

10.1093/genetics/153.4.1573 ◽

1999 ◽

Vol 153 (4) ◽

pp. 1573-1581 ◽

Cited By ~ 2

Author(s):

Susanna Chou ◽

Sukalyan Chatterjee ◽

Mark Lee ◽

Kevin Struhl

Keyword(s):

Transcription Factor ◽

Transcriptional Activation ◽

Large Subunit ◽

Yeast Cells ◽

Specific Cell ◽

Wild Type ◽

Activation Domains ◽

Cycle Arrest ◽

General Transcription Factor

Abstract The general transcription factor IIA (TFIIA) forms a complex with TFIID at the TATA promoter element, and it inhibits the function of several negative regulators of the TATA-binding protein (TBP) subunit of TFIID. Biochemical experiments suggest that TFIIA is important in the response to transcriptional activators because activation domains can interact with TFIIA, increase recruitment of TFIID and TFIIA to the promoter, and promote isomerization of the TFIID-TFIIA-TATA complex. Here, we describe a double-shut-off approach to deplete yeast cells of Toa1, the large subunit of TFIIA, to <1% of the wild-type level. Interestingly, such TFIIA-depleted cells are essentially unaffected for activation by heat shock factor, Ace1, and Gal4-VP16. However, depletion of TFIIA causes a general two- to threefold decrease of transcription from most yeast promoters and a specific cell-cycle arrest at the G2-M boundary. These results indicate that transcriptional activation in vivo can occur in the absence of TFIIA.

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Transcription Activation Domains of the Yeast Factors Met4 and Ino2: Tandem Activation Domains with Properties Similar to the Yeast Gcn4 Activator

Molecular and Cellular Biology ◽

10.1128/mcb.00038-18 ◽

2018 ◽

Vol 38 (10) ◽

Cited By ~ 12

Author(s):

Derek Pacheco ◽

Linda Warfield ◽

Michelle Brajcich ◽

Hannah Robbins ◽

Jie Luo ◽

...

Keyword(s):

Transcription Initiation ◽

Transcription Activation ◽

Binding Studies ◽

Eukaryotic Transcription ◽

Activation Domains ◽

Conserved Sequence ◽

Functional Studies ◽

Intrinsically Disordered ◽

Binding Domains ◽

Optimal Function

ABSTRACTEukaryotic transcription activation domains (ADs) are intrinsically disordered polypeptides that typically interact with coactivator complexes, leading to stimulation of transcription initiation, elongation, and chromatin modifications. Here we examined the properties of two strong and conserved yeast ADs: Met4 and Ino2. Both factors have tandem ADs that were identified by conserved sequence and functional studies. While the AD function of both factors depended on hydrophobic residues, Ino2 further required key conserved acidic and polar residues for optimal function. Binding studies showed that the ADs bound multiple Med15 activator-binding domains (ABDs) with similar orders of micromolar affinity and similar but distinct thermodynamic properties. Protein cross-linking data show that no unique complex was formed upon Met4-Med15 binding. Rather, we observed heterogeneous AD-ABD contacts with nearly every possible AD-ABD combination. Many of these properties are similar to those observed with yeast activator Gcn4, which forms a large heterogeneous, dynamic, and fuzzy complex with Med15. We suggest that this molecular behavior is common among eukaryotic activators.

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Reconstitution of transcriptional activation domains by reiteration of short peptide segments reveals the modular organization of a glutamine-rich activation domain

Molecular and Cellular Biology ◽

10.1128/mcb.14.9.6056-6067.1994 ◽

1994 ◽

Vol 14 (9) ◽

pp. 6056-6067

Author(s):

M Tanaka ◽

W Herr

Keyword(s):

Dna Binding ◽

Transcriptional Activation ◽

Binding Domain ◽

Dna Binding Domain ◽

Activation Domain ◽

Activation Domains ◽

Transcriptional Activation Domain ◽

Pou Domain

The POU domain activator Oct-2 contains an N-terminal glutamine-rich transcriptional activation domain. An 18-amino-acid segment (Q18III) from this region reconstituted a fully functional activation domain when tandemly reiterated and fused to either the Oct-2 or GAL4 DNA-binding domain. A minimal transcriptional activation domain likely requires three tandem Q18III segments, because one or two tandem Q18III segments displayed little activity, whereas three to five tandem segments were active and displayed increasing activity with increasing copy number. As with natural Oct-2 activation domains, in our assay a reiterated activation domain required a second homologous or heterologous activation domain to stimulate transcription effectively when fused to the Oct-2 POU domain. These results suggest that there are different levels of synergy within and among activation domains. Analysis of reiterated activation domains containing mutated Q18III segments revealed that leucines and glutamines, but not serines or threonines, are critical for activity in vivo. Curiously, several reiterated activation domains that were inactive in vivo were active in vitro, suggesting that there are significant functional differences in our in vivo and in vitro assays. Reiteration of a second 18-amino-acid segment from the Oct-2 glutamine-rich activation domain (Q18II) was also active, but its activity was DNA-binding domain specific, because it was active when fused to the GAL4 than to the Oct-2 DNA-binding domain. The ability of separate short peptide segments derived from a single transcriptional activation domain to activate transcription after tandem reiteration emphasizes the flexible and modular nature of a transcriptional activation domain.

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Direct photoresponsive inhibition of a p53-like transcription activation domain in PIF3 by Arabidopsis phytochrome B

10.1101/2021.05.11.443493 ◽

2021 ◽

Author(s):

Chan Yul Yoo ◽

Qing Sang ◽

Jiangman He ◽

Yongjian Qiu ◽

Lingyun Long ◽

...

Keyword(s):

Transcription Factors ◽

Target Genes ◽

Transcription Activation ◽

Phytochrome B ◽

Activation Domain ◽

Light Responses ◽

Dark Light ◽

Transactivation Activity ◽

Transcription Activation Domain

Phytochrome B (PHYB) triggers diverse light responses in Arabidopsis by binding to a group of antagonistically acting PHYTOCHROME-INTERACTING transcription FACTORs (PIFs) to promote PIF degradation, consequently downregulating PIF target genes. However, whether PHYB directly controls the transactivation activity of PIFs remains ambiguous. Here we show that the prototypic PIF, PIF3, possesses a p53-like transcription activation domain (TAD) consisting of a sequence-specific, hydrophobic activator motif surrounded by acidic residues. A PIF3mTAD mutant in which the activator motif is replaced with alanines fails to activate PIF3 target genes in Arabidopsis in dark, light, and shade conditions, validating the in vivo functions of the PIF3 TAD. Intriguingly, binding of the N-terminal photosensory module of PHYB to the PHYB-binding site adjacent to the TAD inhibits its transactivation activity. These results unveil a photoresponsive transcriptional switching mechanism in which photoactivated PHYB directly masks the transactivation activity of PIF3. Our study also suggests the unexpected conservation of sequence-specific TADs between the animal and plant kingdoms.

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Inhibition of Xbra transcription activation causes defects in mesodermal patterning and reveals autoregulation of Xbra in dorsal mesoderm

Development ◽

10.1242/dev.122.8.2427 ◽

1996 ◽

Vol 122 (8) ◽

pp. 2427-2435 ◽

Cited By ~ 34

Author(s):

F.L. Conlon ◽

S.G. Sedgwick ◽

K.M. Weston ◽

J.C. Smith

Keyword(s):

Transcription Activation ◽

Zebrafish Embryos ◽

Transcription Activator ◽

Activation Domain ◽

Activation Domains ◽

Dorsal Mesoderm ◽

Transcription Activators ◽

Carboxy Terminal ◽

Genetic Mutants ◽

Axial Development

The Brachyury (T) gene is required for formation of posterior mesoderm and for axial development in both mouse and zebrafish embryos. In this paper, we first show that the Xenopus homologue of Brachyury, Xbra, and the zebrafish homologue, no tail (ntl), both function as transcription activators. The activation domains of both proteins map to their carboxy terminal regions, and we note that the activation domain is absent in two zebrafish Brachyury mutations, suggesting that it is required for gene function. A dominant-interfering Xbra construct was generated by replacing the activation domain of Xbra with the repressor domain of the Drosophila engrailed protein. Microinjection of RNA encoding this fusion protein allowed us to generate Xenopus and zebrafish embryos which show striking similarities to genetic mutants in mouse and fish. These results indicate that the function of Brachyury during vertebrate gastrulation is to activate transcription of mesoderm-specific genes. Additional experiments show that Xbra transcription activation is required for regulation of Xbra itself in dorsal, but not ventral, mesoderm. The approach described in this paper, in which the DNA-binding domain of a transcription activator is fused to the engrailed repressor domain, should assist in the analysis of other Xenopus and zebrafish transcription factors.

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