Precise temporal control of the eye regulatory gene Pax6 via enhancer-binding site affinity

Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Genes & development (Impact Factor: 10.8). 04/2010; 24(10):980-5. DOI: 10.1101/gad.1890410
Source: PubMed


How transcription factors interpret the cis-regulatory logic encoded within enhancers to mediate quantitative changes in spatiotemporally restricted expression patterns during animal development is not well understood. Pax6 is a dosage-sensitive gene essential for eye development. Here, we identify the Prep1 (pKnox1) transcription factor as a critical dose-dependent upstream regulator of Pax6 expression during lens formation. We show that Prep1 activates the Pax6 lens enhancer by binding to two phylogenetically conserved lower-affinity DNA-binding sites. Finally, we describe a mechanism whereby Pax6 levels are determined by transcriptional synergy of Prep1 bound to the two sites, while timing of enhancer activation is determined by binding site affinity.

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Available from: Salil Lachke, Mar 18, 2014
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    • "Recent work, however, demonstrates that DNA-binding properties also have a capacity to be modular as they can have secondary or alternative binding preferences in addition to their primary or most preferred binding site (Badis et al. 2009; Gordân et al. 2011; Busser et al. 2012; Nakagawa et al. 2013). Other work reveals that transcription factors need multiple binding sites that differ in affinity because they are crucial for executing unique developmental functions (Rowan et al. 2010; Peterson et al. 2012). In the Drosophila mesoderm, many homeodomain transcription factors are coexpressed and share a primary binding motif. "
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    ABSTRACT: Gene regulatory networks (GRNs) describe the progression of transcriptional states that take a single-celled zygote to a multicellular organism. It is well documented that GRNs can evolve extensively through mutations to cis-regulatory modules. Transcription factor proteins that bind these cis-regulatory modules may also evolve to produce novelty. Coding changes are considered to be rarer, however, because transcription factors are multifunctional and hence are more constrained to evolve in ways that will not produce widespread detrimental effects. Recent technological advances have unearthed a surprising variation in DNA binding abilities, such that individual transcription factors may recognize both a preferred primary motif and an additional secondary motif. This provides a source of modularity in function. Here, we demonstrate that orthologous transcription factors can also evolve a changed preference for a secondary binding motif, thereby offering an unexplored mechanism for GRN evolution. Using Protein Binding Microarray, Surface Plasmon Resonance, and in vivo reporter assays, we demonstrate an important difference in DNA binding preference between Tbrain protein orthologs in two species of echinoderms, the sea star, Patiria miniata, and the sea urchin, Strongylocentrotus purpuratus. While both orthologs recognize the same primary motif, only the sea star Tbr also has a secondary binding motif. Our in vivo assays demonstrate that this difference may allow for greater evolutionary change in timing of regulatory control. This uncovers a layer of transcription factor binding divergence that could exist for many pairs of orthologs. We hypothesize that this divergence provides modularity that allows orthologous transcription factors to evolve novel roles in gene regulatory networks through modification of binding to secondary sites.
    Full-text · Article · Jul 2014 · Molecular Biology and Evolution
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    • "Altered binding affinity has been shown to have substantial impacts on the contributions of TFBS [35]. In order to assess the potential impact of SNPs on TF binding, we next scored the predicted differential TF binding affinity between alleles of each SNP using PWMs. "
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    ABSTRACT: Background Genome wide association studies (GWAS) are a population-scale approach to the identification of segments of the genome in which genetic variations may contribute to disease risk. Current methods focus on the discovery of single nucleotide polymorphisms (SNPs) associated with disease traits. As there are many SNPs within identified risk loci, and the majority of these are situated within non-coding regions, a key challenge is to identify and prioritize variants affecting regulatory sequences that are likely to contribute to the phenotype assessed. Methods We focused investigation on SNPs within lung and breast cancer GWAS loci that reached genome-wide significance for potential roles in gene regulation with a specific focus on SNPs likely to disrupt transcription factor binding sites. Within risk loci, the regulatory potential of sub-regions was classified using relevant open chromatin and epigenetic high throughput sequencing data sets from the ENCODE project in available cancer and normal cell lines. Furthermore, transcription factor affinity altering variants were predicted by comparison of position weight matrix scores between disease and reference alleles. Lastly, ChIP-seq data of transcription associated factors and topological domains were included as binding evidence and potential gene target inference. Results The sets of SNPs, including both the disease-associated markers and those in high linkage disequilibrium with them, were significantly over-represented in regulatory sequences of cancer and/or normal cells; however, over-representation was generally not restricted to disease-relevant tissue specific regions. The calculated regulatory potential, allelic binding affinity scores and ChIP-seq binding evidence were the three criteria used to prioritize candidates. Fitting all three criteria, we highlighted breast cancer susceptibility SNPs and a borderline lung cancer relevant SNP located in cancer-specific enhancers overlapping multiple distinct transcription associated factor ChIP-seq binding sites. Conclusion Incorporating high throughput sequencing epigenetic and transcription factor data sets from both cancer and normal cells into cancer genetic studies reveals potential functional SNPs and informs subsequent characterization efforts.
    Full-text · Article · Jun 2014 · BMC Medical Genomics
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    • "Interestingly, comparison of the phenotypes of the Prep-null (Prep1 2/ 2 , embryonic lethal before E7.5), hypomorphic (Prep1 i/i , E17.5) and trans-heterozygous (Prep1 2/i , E12.5) mutants (Rowan et al., 2010), indicates that Prep1 has multiple critical and essential functions during embryogenesis. In addition, the few adult Prep1 i/i mice (25%) that escape embryonic lethality, live a normallength life but display a variety of phenotypes. "
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    ABSTRACT: TALE (three amino acids loop extension) homeodomain transcription factors are required in various steps of embryo development, in many adult physiological functions and are involved in important pathologies. This review focuses on the PREP, MEIS and PBX sub-families of TALE factors and aims at giving information on their biochemical properties, i.e. structure, interactors and interaction surfaces. Members of the three sets of protein form dimers in which the common partner is PBX but they can also directly interact with other proteins forming higher-order complexes, in particular HOX. Finally, recent advances in determining the genome-wide DNA-binding sites of PREP1, MEIS1 and PBX1, and their partial correspondence with the binding sites of some HOX proteins, are reviewed. These studies have generated a few general rules that can be applied to all members of the three gene families. PREP and MEIS recognize slightly different consensus sequences: PREP prefers to bind to promoters and to have PBX as a DNA-binding partner; MEIS prefers HOX as partner, and both PREP and MEIS drive PBX to their own binding sites. This outlines the clear individuality of the PREP and MEISs proteins, the former mostly devoted to basic cellular functions, the latter more to developmental functions. Developmental Dynamics, 2013. © 2013 Wiley Periodicals, Inc.
    Full-text · Article · Jan 2014 · Developmental Dynamics
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