Specific residues in the Pbx homeodomain differentially modulate the DNA-binding activity of Hox and Engrailed proteins

Two classes of homeodomain proteins, Hox and Engrailed, have been shown to act in concert with the atypical homeodomain proteins Pbx and extradenticle. We now show that specific residues located within the Pbx homeodomain are essential for cooperative DNA binding with Hox and Engrailed gene products. Within the N-terminal region of the Pbx homeodomain, we have identified a residue that is required for cooperative DNA binding with three Hox gene products but not for cooperativity with Engrailed-2 (En-2). Furthermore, there are similarities between heterodimeric interactions involving the yeast mating type proteins MATa1 and MATalpha2 and those that allow the formation of Pbx/Hox and Pbx/En-2 heterodimers. Specifically, residues located in the a1 homeodomain that were previously shown to form a hydrophobic pocket allowing the alpha2 C-terminal tail to bind, are also required for Pbx/Hox and Pbx/En-2 cooperativity. Furthermore, we show that three residues located in the turn between helix 1 and helix 2, characteristic of many atypical homeodomain proteins, are required for cooperative DNA binding involving both Hox and En-2. Replacement of the three residues located in the turn between helix 1 and helix 2 of the Pbx homeodomain with those of the atypical homeodomain proteins controlling cell fate in the basidiomycete Ustilago maydis, bE5 and bE6, allows cooperative DNA binding with three Hox members but abolishes interactions with En-2. The data suggest that the molecular mechanism of homeodomain protein interactions that control cell fate in Saccharomyces cerevisiae and in the basidiomycetes may well be conserved in part in multicellular organisms.

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The Drosophila extramacrochaetae protein antagonizes sequence-specific DNA binding by daughterless/achaete-scute protein complexes

Development ◽

10.1242/dev.113.1.245 ◽

1991 ◽

Vol 113 (1) ◽

pp. 245-255 ◽

Cited By ~ 32

Author(s):

M. Van Doren ◽

H.M. Ellis ◽

J.W. Posakony

Keyword(s):

Dna Binding ◽

Cell Fate ◽

Protein Complexes ◽

Competitive Inhibitor ◽

Binding Activity ◽

Regulatory Function ◽

Biochemical Basis ◽

Dna Binding Activity

In Drosophila, a group of regulatory proteins of the helix-loop-helix (HLH) class play an essential role in conferring upon cells in the developing adult epidermis the competence to give rise to sensory organs. Proteins encoded by the daughterless (da) gene and three genes of the achaete-scute complex (AS-C) act positively in the determination of the sensory organ precursor cell fate, while the extramacrochaetae (emc) and hairy (h) gene products act as negative regulators. In the region upstream of the achaete gene of the AS-C, we have identified three ‘E box’ consensus sequences that are bound specifically in vitro by hetero-oligomeric complexes consisting of the da protein and an AS-C protein. We have used this DNA-binding activity to investigate the biochemical basis of the negative regulatory function of emc. Under the conditions of our experiments, the emc protein, but not the h protein, is able to antagonize specifically the in vitro DNA-binding activity of da/AS-C and putative da/da protein complexes. We interpret these results as follows: the heterodimerization capacity of the emc protein (conferred by its HLH domain) allows it to act in vivo as a competitive inhibitor of the formation of functional DNA-binding protein complexes by the da and AS-C proteins, thereby reducing the effective level of their transcriptional regulatory activity within the cell.

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DNA Damage Induced Hyperphosphorylation of Replication Protein A. 2. Characterization of DNA Binding Activity, Protein Interactions, and Activity in DNA Replication and Repair†

Biochemistry ◽

10.1021/bi048057b ◽

2005 ◽

Vol 44 (23) ◽

pp. 8438-8448 ◽

Cited By ~ 34

Author(s):

Steve M. Patrick ◽

Greg G. Oakley ◽

Kathleen Dixon ◽

John J. Turchi

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Dna Binding ◽

Protein Interactions ◽

Protein A ◽

Replication Protein A ◽

Binding Activity ◽

Dna Binding Activity ◽

Dna Replication And Repair

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Transcriptional and DNA Binding Activity of the Foxp1/2/4 Family Is Modulated by Heterotypic and Homotypic Protein Interactions

Molecular and Cellular Biology ◽

10.1128/mcb.24.2.809-822.2004 ◽

2004 ◽

Vol 24 (2) ◽

pp. 809-822 ◽

Cited By ~ 191

Author(s):

Shanru Li ◽

Joel Weidenfeld ◽

Edward E. Morrisey

Keyword(s):

Dna Binding ◽

Protein Interactions ◽

Leucine Zipper ◽

Binding Activity ◽

Transcriptional Repressors ◽

Transcription Analysis ◽

Dna Binding Activity ◽

The Family ◽

Two Hybrid ◽

Repression Domain

ABSTRACT Foxp1, Foxp2, and Foxp4 are large multidomain transcriptional regulators belonging to the family of winged-helix DNA binding proteins known as the Fox family. Foxp1 and Foxp2 have been shown to act as transcriptional repressors, while regulatory activity of the recently identified Foxp4 has not been determined. Given the importance of this Fox gene subfamily in neural and lung development, we sought to elucidate the mechanisms by which Foxp1, Foxp2, and Foxp4 repress gene transcription. We show that like Foxp1 and Foxp2, Foxp4 represses transcription. Analysis of the N-terminal repression domain in Foxp1, Foxp2, and Foxp4 shows that this region contains two separate and distinct repression subdomains that are highly homologous termed subdomain 1 and subdomain 2. However, subdomain 2 is not functional in Foxp4. Screening for proteins that interact with subdomains 1 and 2 of Foxp2 using yeast two-hybrid analysis revealed that subdomain 2 binds to C-terminal binding protein 1, which can synergistically repress transcription with Foxp1 and Foxp2, but not Foxp4. Subdomain 1 contains a highly conserved leucine zipper similar to that found in N-myc and confers homo- and heterodimerization to the Foxp1/2/4 family members. These interactions are dependent on the conserved leucine zipper motif. Finally, we show that the integrity of this subdomain is essential for DNA binding, making Foxp1, Foxp2, and Foxp4 the first Fox proteins that require dimerization for DNA binding. These data reveal a complex regulatory mechanism underlying Foxp1, Foxp2, and Foxp4 activity, demonstrating that Foxp1, Foxp2, and Foxp4 are the first Fox proteins reported whose activity is regulated by homo- and heterodimerization.

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Multifunctional Role of the Pitx2 Homeodomain Protein C-Terminal Tail

Molecular and Cellular Biology ◽

10.1128/mcb.19.10.7001 ◽

1999 ◽

Vol 19 (10) ◽

pp. 7001-7010 ◽

Cited By ~ 79

Author(s):

Brad A. Amendt ◽

Lillian B. Sutherland ◽

Andrew F. Russo

Keyword(s):

Amino Acid ◽

Dna Binding ◽

Inhibitory Effect ◽

Binding Activity ◽

Homeodomain Protein ◽

Rieger Syndrome ◽

Amino Acid Peptide ◽

Dna Binding Activity ◽

C Terminus

ABSTRACT Pitx2 is a newly described bicoid-like homeodomain transcription factor that is defective in Rieger syndrome and shows a striking leftward developmental asymmetry. We have previously shown that Pitx2 (also called Ptx2 and RIEG) transactivates a reporter gene containing abicoid enhancer and synergistically transactivates the prolactin promoter in the presence of the POU homeodomain protein Pit-1. In this report, we focused on the C-terminal region which is mutated in some Rieger patients and contains a highly conserved 14-amino-acid element. Deletion analysis of Pitx2 revealed that the C-terminal 39-amino-acid tail represses DNA binding activity and is required for Pitx2-Pit-1 interaction and Pit-1 synergism. Pit-1 interaction with the Pitx2 C terminus masks the inhibitory effect and promotes increased DNA binding activity. Interestingly, cotransfection of an expression vector encoding the C-terminal 39 amino acids of Pitx2 specifically inhibits Pitx2 transactivation activity. In contrast, the C-terminal 39-amino-acid peptide interacts with Pitx2 to increase its DNA binding activity. These data suggest that the C-terminal tail intrinsically inhibits the Pitx2 protein and that this inhibition can be overcome by interaction with other transcription factors to allow activation during development.

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Synergistic activation of transcription is mediated by the N-terminal domain of Drosophila fushi tarazu homeoprotein and can occur without DNA binding by the protein

Molecular and Cellular Biology ◽

10.1128/mcb.13.3.1599-1609.1993 ◽

1993 ◽

Vol 13 (3) ◽

pp. 1599-1609

Author(s):

J Ananthan ◽

R Baler ◽

D Morrissey ◽

J Zuo ◽

Y Lan ◽

...

Keyword(s):

Dna Binding ◽

Protein Interactions ◽

Binding Activity ◽

Protein Protein Interactions ◽

Fushi Tarazu ◽

Combinatorial Regulation ◽

Dna Binding Activity ◽

Culture Cells ◽

Synergistic Activation ◽

Activation Of Transcription

Synergistic activation of transcription by Drosophila segmentation genes in tissue culture cells provides a model with which to study combinatorial regulation. We examined the synergistic activation of an engrailed-derived promoter by the pair-rule proteins paired (PRD) and fushi tarazu (FTZ). Synergistic activation by PRD requires regions of the homeodomain or adjacent sequences, and that by FTZ requires the first 171 residues. Surprisingly, deletion of the FTZ homeodomain does not reduce the capacity of the protein for synergistic activation, although this mutation abolishes any detectable DNA-binding activity. This finding suggests that FTZ can function through protein-protein interactions with PRD or other components of the homeoprotein transcription complex, adding a new layer of mechanisms that could underlie the functional specificities and combinatorial regulation of homeoproteins.

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Inefficient homooligomerization contributes to the dependence of myogenin on E2A products for efficient DNA binding.

Molecular and Cellular Biology ◽

10.1128/mcb.11.7.3633 ◽

1991 ◽

Vol 11 (7) ◽

pp. 3633-3641 ◽

Cited By ~ 39

Author(s):

T Chakraborty ◽

T J Brennan ◽

L Li ◽

D Edmondson ◽

E N Olson

Keyword(s):

Dna Binding ◽

Gene Activation ◽

Binding Activity ◽

Specific Gene ◽

Gene Products ◽

Basic Domain ◽

High Affinity ◽

Dna Binding Activity

Myogenin is a muscle-specific transcription factor that can activate myogenesis; it belongs to a family of transcription factors that share homology within a basic region and an adjacent helix-loop-helix (HLH) motif. Although myogenin alone binds DNA inefficiently, in the presence of the widely expressed HLH proteins E12 and E47 (encoded by the E2A gene), it forms heterooligomers that bind with high affinity to a DNA sequence known as a kappa E-2 site. In contrast, E47 and to a lesser extent E12 are both able to bind the kappa E-2 site relatively efficiently as homooligomers. To define the relative contributions of the basic regions of myogenin and E12 to DNA binding and muscle-specific gene activation, we created chimeras of the two proteins by swapping their basic regions. We showed that myogenin's weak affinity for the kappa E-2 site is attributable to inefficient homooligomerization and that the myogenin basic domain alone can mediate high-affinity DNA binding when placed in E12. Within a heterooligomeric complex, two basic regions were required to form a high-affinity DNA-binding domain. Basic-domain mutants of myogenin or E2A gene products that cannot bind DNA retained the ability to oligomerize and could abolish DNA binding of the wild-type proteins in vitro. These myogenin and E2A mutants also acted as trans-dominant inhibitors of muscle-specific gene activation in vivo. These findings support the notion that muscle-specific gene activation requires oligomerization between myogenin and E2A gene products and that E2A gene products play an important role in myogenesis by enhancing the DNA-binding activity of myogenin, as well as other myogenic HLH proteins.

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