Altering the DNA-binding Specificity of the Yeast Matα2 Homeodomain Protein

Mathias, Jonathan R.; Zhong, Hualin; Jin, Yisheng; Vershon, Andrew K.

doi:10.1074/jbc.m103097200

Cited by 14 publications

(10 citation statements)

References 37 publications

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“…The amino acid sequences of the homeodomains are highly conserved among Dlx family members, sharing 80-88% positional identity. Critically, all 4 of the residues in helices three to four responsible for basepairspecific contacts between homeodomain proteins and the DNA major groove are identical (Dave et al, 2000;Laughon, 1991;Mathias et al, 2001). Therefore, these constructs are predicted to modulate target genes of all known Dlx family members, which is advantageous given their overlapping expression.…”

Section: Resultsmentioning

confidence: 99%

Dlx proteins position the neural plate border and determine adjacent cell fates

et al. 2003

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The lateral border of the neural plate is a major source of signals that induce primary neurons, neural crest cells and cranial placodes as well as provide patterning cues to mesodermal structures such as somites and heart. Whereas secreted BMP, FGF and Wnt proteins influence the differentiation of neural and non-neural ectoderm, we show here that members of the Dlx family of transcription factors position the border between neural and non-neural ectoderm and are required for the specification of adjacent cell fates. Inhibition of endogenous Dlx activity in Xenopus embryos with an EnR-Dlx homeodomain fusion protein expands the neural plate into non-neural ectoderm tissue whereas ectopic activation of Dlx target genes inhibits neural plate differentiation. Importantly, the stereotypic pattern of border cell fates in the adjacent ectoderm is reestablished only under conditions where the expanded neural plate abuts Dlx-positive non-neural ectoderm. Experiments in which presumptive neural plate was grafted to ventral ectoderm reiterate induction of neural crest and placodal lineages and also demonstrate that Dlx activity is required in non-neural ectoderm for the production of signals needed for induction of these cells. We propose that Dlx proteins regulate intercellular signaling across the interface between neural and non-neural ectoderm that is critical for inducing and patterning adjacent cell fates.

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Section: Resultsmentioning

confidence: 99%

Dlx proteins position the neural plate border and determine adjacent cell fates

et al. 2003

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“…Position 50 of the homeodomain appears to be involved in dimerization of PITX2a in at least two other homeodomain proteins in addition to its well-studied important role in DNA binding (1,12,22,32). The Drosophila Paired homeodomains dimerize with resultant cooperative binding to DNA, and mutation of a serine to a glutamine at position 50 results in fourfold greater cooperativity than WT (35,36).…”

Section: Discussionmentioning

confidence: 99%

Dominant Negative Dimerization of a Mutant Homeodomain Protein in Axenfeld-Rieger Syndrome

Saadi¹,

Kuburas

Engle

et al. 2003

Molecular and Cellular Biology

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Axenfeld-Rieger syndrome is an autosomal-dominant disorder caused by mutations in the PITX2 homeodomain protein. We have studied the mechanism underlying the dominant negative K88E mutation, which occurs at position 50 of the homeodomain. By using yeast two-hybrid and in vitro pulldown assays, we have documented that PITX2a can form homodimers in the absence of DNA. Moreover, the K88E mutant had even stronger dimerization ability, primarily due to interactions involving the C-terminal region. Dimerization allowed cooperative binding of wild-type (WT) PITX2a to DNA containing tandem bicoid sites in a head-to-tail orientation (Hill coefficient, 1.73). In contrast, the WT-K88E heterodimer bound the tandem sites with greatly reduced cooperativity and decreased transactivation activity. To further explore the role of position 50 in PITX2a dimerization, we introduced a charge-conservative mutation of lysine to arginine (K88R). The K88R protein had greatly reduced binding to a TAATCC element and did not specifically bind any other TAATNN motif. Like K88E, K88R formed relatively stronger dimers with WT. As predicted by our model, the K88R protein acted in a dominant negative manner to suppress WT PITX2a activity. These results suggest that the position 50 residue in the PITX2 homeodomain plays an important role in both DNA binding and dimerization activities.The PITX2 gene was cloned based on its linkage in Axenfeld-Rieger syndrome (ARS) (30). ARS is an autosomal-dominant human disorder characterized by ocular anterior chamber anomalies causing glaucoma in more than 50% of affected individuals as well as dental hypoplasia, mild craniofacial dysmorphism, and umbilical stump abnormalities. Other features associated with this syndrome include abnormal cardiac, limb, and pituitary development (28,30). Three isoforms of PITX2 (designated a, b, and c) that differ only in their amino termini have been identified (9, 15). PITX2a and the related PITX1 protein have been shown to interact with Pit-1, a transcription factor that regulates pituitary cell differentiation, including the expression of the thyroid-stimulating hormone, growth hormone, and prolactin genes (4, 16, 31). We have used the prolactin promoter as a model target for studying PITX2a activities. Transient transfection assays with the prolactin promoter, which contains both Pit-1 and PITX2a binding sites, have shown a strong synergistic effect of PITX2a and Pit-1 on transactivation (4). Consistent with these data, knockout mice lacking Pitx2 show arrested pituitary gland development (10).PITX2a is a 33-kDa homeodomain protein that is a functional member of the bicoid homeodomain subfamily, as defined by a lysine at residue 50 of the homeodomain (4). It has been demonstrated that this residue in the bicoid protein interacts with base pairs 5 and 6 of the hexanucleotide consensus 5Ј-TAATCC-3Ј (12, 32). PITX2a has been shown to specifically bind to this consensus sequence and transactivate corresponding reporter genes (4). We have previously reported an ARS mutation th...

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“…However, these analyses have utilized data for only a handful of DBD classes and species, and they contrast with numerous demonstrations that mutation of one or a few critical DBD AAs can alter the sequence preferences of a TF (e.g. (Aggarwal et al, 2010; Cook et al, 1994; De Masi et al, 2011; Mathias et al, 2001; Noyes et al, 2008)), which suggest that prediction of DNA binding preferences by homology should be highly error-prone. To our knowledge, rigorous and exhaustive analyses of the accuracy and limitations of inference approaches to predicting TF DNA-binding motifs using DBD sequences has not been done.…”

Section: Introductionmentioning

confidence: 99%

Determination and Inference of Eukaryotic Transcription Factor Sequence Specificity

Weirauch

Yang

Albu

et al. 2014

Cell

1,621

1,912

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SUMMARY Transcription factor (TF) DNA sequence preferences direct their regulatory activity, but are currently known for only ~1% of all eukaryotic TFs. Broadly sampling DNA-binding domain (DBD) types from multiple eukaryotic clades, we determined DNA sequence preferences for >1,000 TFs encompassing 54 different DBD classes from 131 diverse eukaryotes. We find that closely related DBDs almost always have very similar DNA sequence preferences, enabling inference of motifs for ~34% of the ~170,000 known or predicted eukaryotic TFs. Sequences matching both measured and inferred motifs are enriched in ChIP-seq peaks and upstream of transcription start sites in diverse eukaryotic lineages. SNPs defining expression quantitative trait loci in Arabidopsis promoters are also enriched for predicted TF binding sites. Importantly, our motif “library” (http://cisbp.ccbr.utoronto.ca) can be used to identify specific TFs whose binding may be altered by human disease risk alleles. These data present a powerful resource for mapping transcriptional networks across eukaryotes.

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Altering the DNA-binding Specificity of the Yeast Matα2 Homeodomain Protein

Cited by 14 publications

References 37 publications

Dlx proteins position the neural plate border and determine adjacent cell fates

Dlx proteins position the neural plate border and determine adjacent cell fates

Dominant Negative Dimerization of a Mutant Homeodomain Protein in Axenfeld-Rieger Syndrome

Determination and Inference of Eukaryotic Transcription Factor Sequence Specificity

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