Understanding the Logic of TCF Target Location in Drosophila Wnt Signaling.

The TCF/LEF family of transcription factors are major mediators of Wnt/beta-catenin signaling in metazoans. How TCFs mediate the pleiotropic effects of Wnt signaling is poorly understood. All TCFs contain a High Mobility Group (HMG) sequence specific DNA binding domain. Many TCFs also contain a second domain, the C-clamp, which binds DNA motifs called Helper sites. Both sequences are indispensable for activation of multiple W-CRMs (Wnt-responsive cis-Regulatory Elements). However, the rules concerning what constitutes a functional HMG-Helper pair were unknown. I used in vitro binding, reporter gene analysis and bioinformatics to address this question, using Drosophila TCF/Pangolin as a model. Surprisingly, I found that TCF displays remarkable functional flexibility. A Helper site near an HMG site in all four orientations can increase in vitro binding and transcriptional response; however, spacing preferences are orientation specific. In many tissues, such as the larval imaginal discs, there is a positive correlation between in vitro binding affinity and transcriptional activation levels; and altering an HMG-Helper pair from a low to high affinity configuration dramatically increases W-CRM responsiveness. However, in other tissues, this correlation is not seen, and low affinity motifs drive robust expression. Analysis of the distribution of bipartite motif configurations in TCF bound genomic regions indicates that bipartite motifs in all four orientations are enriched at a wide range of intersite spacing, but this enrichment is most pronounced for high affinity configurations. These data suggest motif configuration plays an instructive role in setting the threshold, magnitude, and tissue-specificity of W-CRM driven transcriptional responses. Computational searches for high affinity motifs identified several novel W-CRMs. All tested elements drove reporter gene expression in patterns overlapping Wg (a fly Wnt) protein expression domains in transgenic Drosophila. Novel elements on Chromosome 3R require intact Wnt signal transduction for activation, indicating they are bona-fide W-CRMs. One of these elements drives expression in the larval prothoracic gland, a tissue not previously linked to Wnt signaling. This finding highlights the importance of W-CRM discovery in understanding the many facets of Wnt/beta-catenin signaling.PHDCellular & Molecular BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/108923/1/hilaryar_1.pd

DDX3X and specific initiation factors modulate FMR1 repeat‐associated non‐AUG‐initiated translation

A CGG trinucleotide repeat expansion in the 5′ UTR of FMR1 causes the neurodegenerative disorder Fragile X‐associated tremor/ataxia syndrome (FXTAS). This repeat supports a non‐canonical mode of protein synthesis known as repeat‐associated, non‐AUG (RAN) translation. The mechanism underlying RAN translation at CGG repeats remains unclear. To identify modifiers of RAN translation and potential therapeutic targets, we performed a candidate‐based screen of eukaryotic initiation factors and RNA helicases in cell‐based assays and a Drosophila melanogaster model of FXTAS. We identified multiple modifiers of toxicity and RAN translation from an expanded CGG repeat in the context of the FMR1 5′UTR. These include the DEAD‐box RNA helicase belle/DDX3X, the helicase accessory factors EIF4B/4H, and the start codon selectivity factors EIF1 and EIF5. Disrupting belle/DDX3X selectively inhibited FMR1 RAN translation in Drosophila in vivo and cultured human cells, and mitigated repeat‐induced toxicity in Drosophila and primary rodent neurons. These findings implicate RNA secondary structure and start codon fidelity as critical elements mediating FMR1 RAN translation and identify potential targets for treating repeat‐associated neurodegeneration.SynopsisFragile X‐associated tremor/ataxia syndrome is caused by CGG repeat‐associated non‐AUG (RAN) translation that initiates within the 5′UTR of FMR1. A candidate‐based screen identified several initiation factors—DDX3X/Belle, eIF4B, eIF4H, eIF1, and eIF5—critical for FMR1 RAN translation.Knockdown of the RNA helicase DDX3X selectively suppresses FMR1 RAN translation in Drosophila melanogaster, cultured HeLa cells, and in vitro translation assays.DDX3X knockdown reduces CGG repeat‐associated toxicity in Drosophila and mammalian neurons.Eukaryotic initiation factors that modulate RNA‐RNA secondary structure (DDX3X, EIF4B, EIF4H) or start codon fidelity (EIF1, EIF5) impact FMR1 RAN translation.FXTAS is caused by CGG repeat‐associated non‐AUG (RAN) translation that initiates within the 5′UTR of FMR1. A candidate‐based screen identified several initiation factors—DDX3X/Belle, eIF4B, eIF4H, eIF1, and eIF5—critical for FMR1 RAN translation.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/151325/1/embr201847498.reviewer_comments.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151325/2/embr201847498-sup-0001-Appendix.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151325/3/embr201847498_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151325/4/embr201847498.pd

Abnormal RNA Stability in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) share key features, including accumulation of the RNA-binding protein TDP-43. TDP-43 regulates RNA homeostasis, but it remains unclear whether RNA stability is affected in these disorders. We use Bru-seq and BruChase-seq to assess genome-wide RNA stability in ALS patient-derived cells, demonstrating profound destabilization of ribosomal and mitochondrial transcripts. This pattern is recapitulated by TDP-43 overexpression, suggesting a primary role for TDP-43 in RNA destabilization, and in postmortem samples from ALS and FTD patients. Proteomics and functional studies illustrate corresponding reductions in mitochondrial components and compensatory increases in protein synthesis. Collectively, these observations suggest that TDP-43 deposition leads to targeted RNA instability in ALS and FTD, and may ultimately cause cell death by disrupting energy production and protein synthesis pathways

Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans

Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same regio

Bipartite Recognition of DNA by TCF/Pangolin Is Remarkably Flexible and Contributes to Transcriptional Responsiveness and Tissue Specificity of Wingless Signaling

<div><p>The T-cell factor (TCF) family of transcription factors are major mediators of Wnt/β-catenin signaling in metazoans. All TCFs contain a High Mobility Group (HMG) domain that possesses specific DNA binding activity. In addition, many TCFs contain a second DNA binding domain, the C-clamp, which binds to DNA motifs referred to as Helper sites. While HMG and Helper sites are both important for the activation of several Wnt dependent cis-regulatory modules (W-CRMs), the rules of what constitutes a functional HMG-Helper site pair are unknown. In this report, we employed a combination of in vitro binding, reporter gene analysis and bioinformatics to address this question, using the <i>Drosophila</i> family member TCF/Pangolin (TCF/Pan) as a model. We found that while there were constraints for the orientation and spacing of HMG-Helper pairs, the presence of a Helper site near a HMG site in any orientation increased binding and transcriptional response, with some orientations displaying tissue-specific patterns. We found that altering an HMG-Helper site pair from a sub-optimal to optimal orientation/spacing dramatically increased the responsiveness of a W-CRM in several fly tissues. In addition, we used the knowledge gained to bioinformatically identify two novel W-CRMs, one that was activated by Wnt/β-catenin signaling in the prothoracic gland, a tissue not previously connected to this pathway. In sum, this work extends the importance of Helper sites in fly W-CRMs and suggests that the type of HMG-Helper pair is a major factor in setting the threshold for Wnt activation and tissue-responsiveness.</p></div

HMG-Helper pair configuration preferences in imaginal discs.

<p>Brightfield images of imaginal discs from 3^rd instar larva containing the indicated lacZ reporter constructs stained for lacZ activity. (A) Wing imaginal discs. (B) Eye/antennal discs. The FF0 and AK6 reporters display the highest expression in these tissues. Neither the promoter alone (HSP70) nor the HMG site only (HMG1 shown) constructs have detectable expression. At least 20 discs for each reporter were analyzed, with representative images shown. The same base pair sequences used in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004591#pgen-1004591-g003" target="_blank">Figure 3</a> were utilized for the transgenic reporters (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004591#pgen.1004591.s011" target="_blank">Table S1</a> for sequences).</p

Binding preferences of TCF/Pan for various HMG-Helper site configurations <i>in vitro</i>.

<p>(A) Competition electromobility shift assay (EMSA) experiments performed with a recombinant TCF fragment containing the HMG and C-Clamp domains, an AK6 IR-labeled probe, and competitor oligonucleotides containing one of the four orientations at 0 or 6 spaces. Images were taken on the Licor Odyssey, and binding intensity quantified with Image Studio 2.0. AK6 and FF0 were the strongest competitors, but Helper sites in all positions improve binding affinity when compared to binding of the HMG sites alone (HMG only 1 and 2). (B) The IC₅₀ value (the measure of DNA concentration required to reduce binding of the labeled probe to 50% of uncompeted levels) for each competitor was calculated using Prism 6.1 (Graphpad). (C) Semilog graphs depicting competition results from three independent experiments. Error bars indicate SD. Sequences of the HMG and Helper motifs the same as shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004591#pgen-1004591-g001" target="_blank">Figure 1D</a> (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004591#pgen.1004591.s011" target="_blank">Table S1</a> for full sequence of each competitor).</p

Optimizing motif architecture increases transcriptional output.

<p>(A) Cartoon of the <i>nkd</i>UPE2 W-CRM. A RW4 motif (green box) was altered to an AK6, point mutations indicated in lowercase. (B–D) Brightfield images of 3^rd instar imaginal discs stained with X-gal. The “optimized” AK6 containing (OPT) W-CRM (B′, C′, D′) drives higher expression levels of the reporter transgene than the wildtype (WT) W-CRM (B,C,D), and expands the region which responds to Wg signaling (red arrows). Representative wing (B), haltere (C), and eye/antennal (D) imaginal discs shown. (E,F) Confocal images of lacZ immunostained <i>nkd</i>UPE2 W-CRM reporters at embryonic stage 12 (E, E′) and stage 13 (F, F′). The “optimized” reporter has a dramatic increase in expression.</p

TCF/Pan-bound chromatin is enriched for HMG-Helper site pairs.

<p>(A) Distribution of HMG-Helper pairs in genomic sequences bound by TCF/Pan (blue bars) versus random DNA (yellow bars). TCF/Pan-bound sequences were obtained from a ChIP-seq data set from germband extended <i>Drosophila</i> embryos <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004591#pgen.1004591-Junion1" target="_blank">[28]</a>. Equivalent amounts of random DNA from intergenic, intronic and 5′/3′UTR regions were analyzed, with the average of ten such runs displayed. Nearly all the HMG-Helper site configurations were enriched in TCF/Pan bound regions, with the highest degree of enrichment in FF0-2 and AK0-6. Due to the semi-palindromic nature of the Helper site, many sites with nucleotide mismatches were called as both AK/RW or FF/KK. The overlap is indicated in the Venn diagram to the right of each pair of graphs. (B) AK and FF motifs are highly enriched in peaks which are uniquely bound by TCF/Pan (see text for further explanation). Enrichment is less dramatic in TCF/Pan peaks that are shared by Pannier or pMAD.</p

HMG and Helper site configurations in W-CRMs.

<p>(A) Previously characterized W-CRMs <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004591#pgen.1004591-Chang1" target="_blank">[44]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004591#pgen.1004591-Chang2" target="_blank">[57]</a> with location of predicted HMG (red arrows) and Helper (blue arrows) indicated (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004591#pgen.1004591.s003" target="_blank">Figure S3</a> for PWMs of these motifs). Cutoffs for HMG and Helper sites were 5.35 and 6.5, respectively. Direction of arrow indicates orientation of motif (see inset for consensus sequences in both orientations). Number of nucleotides between each motif is indicated. Black asterisks indicate sites that contributed to W-CRM activation by Wnt signaling in cell culture when mutated individually <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004591#pgen.1004591-Chang1" target="_blank">[44]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004591#pgen.1004591-Chang2" target="_blank">[57]</a>. Red and blue asterisks denote function when all indicated HMG or Helper sites were mutated simultaneously. (B) Systematic mutagenesis of second Helper site in the <i>nkd</i>IntE W-CRM reporter indicates all seven positions contribute to W-CRM activation. Letters indicate mutated nucleotides, with dashes denoting the wild-type sequence. Reporter constructs were transfected into <i>Drosophila</i> Kc cells with or without a plasmid expressing Arm*. Fold activation represents the ratio of Arm*/control, SD equals standard deviation of three biological replicates. (C) HMG and Helper sites work in closely spaced pairs. The <i>nkd</i>IntE reporter contains three functional HMG and two functional Helper sites <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004591#pgen.1004591-Chang1" target="_blank">[44]</a>, which were mutated (striped arrows) in different combinations, and tested for Arm* activation in Kc cells. Values represent the mean of three biological replicates ± SD. A Student's T-test was employed to determine significance for data in panels B and C. (D) Nomenclature and symbology for the four possible HMG-Helper site pair orientations. A right pointing arrow indicates the consensus sequence is read 5′ to 3′ on the “top” strand, a left arrow indicates the consensus is read 5′ to 3′ on the “bottom” strand. The sequences shown for HMG and Helper sites are identical to the ones used for DNA binding experiments in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004591#pgen-1004591-g002" target="_blank">Figure 2</a> and synthetic Wnt reporter constructs (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004591#pgen-1004591-g003" target="_blank">Figure 3A</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004591#pgen-1004591-g004" target="_blank">4</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004591#pgen-1004591-g005" target="_blank">5</a>).</p