Degenerate Pax2 and Senseless binding motifs improve detection of low-affinity sites required for enhancer specificity

<div><p>Cells use thousands of regulatory sequences to recruit transcription factors (TFs) and produce specific transcriptional outcomes. Since TFs bind degenerate DNA sequences, discriminating functional TF binding sites (TFBSs) from background sequences represents a significant challenge. Here, we show that a <i>Drosophila</i> regulatory element that activates Epidermal Growth Factor signaling requires overlapping, low-affinity TFBSs for competing TFs (Pax2 and Senseless) to ensure cell- and segment-specific activity. Testing available TF binding models for Pax2 and Senseless, however, revealed variable accuracy in predicting such low-affinity TFBSs. To better define parameters that increase accuracy, we developed a method that systematically selects subsets of TFBSs based on predicted affinity to generate hundreds of position-weight matrices (PWMs). Counterintuitively, we found that degenerate PWMs produced from datasets depleted of high-affinity sequences were more accurate in identifying both low- and high-affinity TFBSs for the Pax2 and Senseless TFs. Taken together, these findings reveal how TFBS arrangement can be constrained by competition rather than cooperativity and that degenerate models of TF binding preferences can improve identification of biologically relevant low affinity TFBSs.</p></div

A high affinity Sens site results in repression of <i>RhoA</i> in abdominal C1-SOPs.

<p><b>(A)</b> The SELEX-seq [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007289#pgen.1007289.ref023" target="_blank">23</a>] Sens logo aligned with <i>RhoA</i> variants. Mis-matches are in red font, and sequence variants that improve the match are in green font. The Pax2, Exd, Hth, and Hox TFBSs are highlighted. <b>(B, C)</b> EMSAs using the indicated <i>RhoA</i> probes with either purified Sens (0, 23.5, 57, 114, and 228 ng) or Pax2 (0, 10.25, 20.5, 41, and 82 ng). Full gels are shown in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007289#pgen.1007289.s003" target="_blank">S3 Fig</a></b>. <b>(D-G)</b> Lateral view of stage 11 <i>RhoBAD-lacZ</i> (D), <i>RhoBAD-SS-lacZ</i> (E), <i>RhoBAD-PM-lacZ</i> (F), and <i>RhoBAD-PSSS-lacZ</i> (G) embryos immunostained for β-gal. β-gal intensity is represented by a heat-map at left. “A1” indicates the first abdominal segment. <b>(H)</b> Quantification of β-gal intensity in abdominal C1-SOPs in age-matched embryos. Each box represents measurements from a single embryo. <i>RhoBAD-SS-lacZ</i>, <i>RhoBAD-PM-lacZ</i>, and <i>RhoBAD-PSSS-lacZ</i> embryos were processed and imaged separately, each with <i>RhoBAD-lacZ</i> control embryos. Quantification for a representative set of <i>RhoBAD-lacZ</i> embryos are shown. β-gal intensities for each variant are reported as relative to the average β-gal intensity of control embryos. Two-tailed Welch’s T-test with Bonferroni correction was done to compare β-gal intensities to <i>RhobAD-SS</i> (* p < 0.05, ** p < 0.001, *** p < 0.0001), n = 12 (WT), 9 (SS), 13 (PM), and 19 (PSSS). (<b>I-K</b>) Lateral view of <i>RhoBAD-rho</i>^<i>cDNA</i> (I), <i>RhoBAD-SS-rho</i>^<i>cDNA</i> (J), and <i>RhoBAD-PSSS-rho</i>^<i>cDNA</i> embryos in a <i>rho</i>^7M background (stage 15) immunostained for an oenocyte marker (HNF4). Note, at least 10 embryos with transgenes containing high affinity Sens sites were analyzed and no oenocytes were observed.</p

Overlapping activator and repressor binding sites are required for abdomen-specific <i>RhoA</i> activity.

<p><b>(A)</b> Sequences of tested <i>RhoA</i> variants. <i>RhoA-RDM</i> contains random nucleotides downstream of the Hox site. <i>RhoA-SM</i> contains mutations that decrease Sens binding, and <i>RhoA-SM/SWT</i>, <i>RhoA-SM/SS</i> and <i>RhoA-SM/SM</i> add either a low affinity (WT), high affinity (SS), or mutant (SM) Sens site downstream of the Hox site. <b>(B-C)</b> Quantification of β-gal immunostaining intensities in C1-SOPs in <i>RhoBAD-LacZ versus RhoBAD-RDM-LacZ</i> (B) or <i>RhoBAD-SM-LacZ</i> (C). Each box summarizes measurements from a single embryo. Two-tailed Welch’s T-test was used to compare <i>RhoBAD-SM</i> and <i>RDM</i> mutants to wildtype, n = 12 (WT) and 18 (RDM) in (B) and n = 12 (WT) and 15 (SM) in (C). <b>(D)</b> EMSAs comparing binding of purified Sens to <i>RhoA</i> probes (0, 57, 114, and 228 ng of Sens). <b>(E)</b> EMSAs assessing competition between purified Sens (114 or 228 ng) against purified AbdA (189 ng) and Exd/Hth (59.2 ng) on <i>RhoA-SS</i> and <i>RhoA-SM-SS</i>. <b>(E’)</b> Close-up view of Exd/Hth/Hox and Exd/Hth/Hox/Sens complexes on DNA probes. Schematics denote the formation of each transcription factor complex. <b>(F-I</b>) Lateral view of stage 11 <i>RhoBAD-SM-lacZ</i> (F), <i>RhoBAD-SM/SWT-lacZ</i> (G), <i>RhoBAD-SM/SM-lacZ</i> (H), and <i>RhoBAD-SM/SS-lacZ</i> (I) embryos immunostained for β-gal. Intensity of β-gal stain is represented by heat-map at left. “A1” indicates first abdominal segment. Note, no <i>RhoBAD-SM/SS</i> activity is detected in the PNS and the activity that is observed is in cells of the gut. <b>(J)</b> Quantification of β-gal intensities in thoracic and abdominal C1-SOPs of noted <i>RhoBAD-lacZ</i> embryos. Each box represents measurements from a single embryo. Statistical analysis was done using Kruskal-Wallis test followed by post-hoc pairwise Mann-Whitney U test with Bonferroni correction, n = 25 (SM), 23 (SM/SM), 22 (SM/SWT), and 24 (SM/SS). For all statistical comparisons, n.s. p ≥ 0.05; * p < 0.05, ** p < 0.001, *** p < 0.0001.</p

High information content PWMs are less accurate at identifying TFBSs obtained from both <i>in vitro</i> and <i>in vivo</i> binding events.

<p><b>(A)</b> Schematic describing how PWMs were created by sub-sampling Sens and Pax2 B1H hits. Each B1H hit was placed into quartiles based on 8-mer sequence frequency within the pool of B1H hits. 100 PWMs were generated by iteratively sampling 50 B1H hits from each quartile. 100 PWMs were also generated by sampling 50 B1H hits from the entire pool (Control PWMs). The range of total information content (I.C.) for PWMs in each quartile are indicated below the motifs. <b>(B)</b> Relative log-likelihood (RLL) score of each PWM for the <i>RhoA</i> sequence. <b>(C)</b> AUROC of each PWM for discriminating low-stringency B1H hits from shuffled sequences. <b>(D)</b> AUROC of each PWM for discriminating bound PBM probes (binned by fluorescence, as indicated on x-axis) from non-specifically bound probes (matched number of control probes randomly selected from the 50% of probes with the lowest fluorescence). <b>(E)</b> AUROC of each Sens PWM for discriminating <i>M</i>. <i>musculus</i> Gfi1 and Gfi1b ChIP-seq peaks from random, non-repetitive genomic sequences. Gfi1b ChIP-seq was conducted using multipotent Hematopoietic Progenitor cells (HPC-7) and Gfi1 ChIP was conducted using innate Type-2 Lymphocytes (ILC2) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007289#pgen.1007289.ref032" target="_blank">32</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007289#pgen.1007289.ref033" target="_blank">33</a>]. Analysis was limited to the 1000 peaks with greatest fold enrichment per ChIP dataset, and ChIP peaks were binned by fold enrichment as indicated on x-axis. For panels <b>C</b>-<b>E</b>, AUROCs represent the median using 10 different sets of negative sequences. All violin plots are scaled to have the same width. Statistical analysis was performed using Kurskal-Wallis test followed by a post-hoc pairwise Mann-Whitney U test. P-values were Bonferroni-adjusted due to multiple comparisons arising from groups of PWMs (all panels) and binning of sequences (panels <b>D</b> and <b>E</b>) (n.s. p ≥ 0.05; * p < 0.05; ** p < 0.01, *** p < 0.001).</p

<i>RhoA</i> contains low affinity Pax2 and Sens binding sites.

<p><b>(A, D)</b> Alignment of Pax2 (A) and Sens (D) logos derived from SELEX-seq [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007289#pgen.1007289.ref023" target="_blank">23</a>] to <i>RhoA</i> and selected B1H sites [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007289#pgen.1007289.ref024" target="_blank">24</a>]. Mismatches to the logos are highlighted in red. <b>(B, E)</b> Pax2 (B) and Sens (E) binding to <i>RhoA</i> and selected B1H hits using EMSAs. Each probe was incubated with 0, 106, or 212 ng of Sens or 0, 48, or 96 ng of Pax2. Full gels are shown in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007289#pgen.1007289.s002" target="_blank">S2 Fig</a></b>. <b>(C, F)</b> Correlation between proportion of probe bound in EMSAs versus proportion predicted by PWM energy models. The Spearman-rank correlation (ρ) and coefficient-of-determination (r²) are indicated on the plots. Linear regression of this relationship is shown in blue.</p

The <i>RhoA</i> enhancer activates gene expression in <i>Drosophila</i> abdominal C1-SOPs.

<p><b>(A, B)</b> Lateral view of <i>Drosophila RhoBAD-LacZ</i> (A) or <i>RhoAAA-LacZ</i> (B) embryos (stage 11) immunostained for β-gal (green) and AbdA (purple). Both reporters are active in a specific cell type (C1-SOP) with higher levels observed in abdominal segments (stained by AbdA, first abdominal segment marked by “A1”) than thoracic segments. <b>(C)</b> The <i>RhoA</i> sequence has binding sites for Pax2, Sens, Exd, Hth, and AbdA that are critical for proper <i>RhoBAD-LacZ</i> and <i>RhoAAA-LacZ</i> activity in <i>Drosophila</i> embryos [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007289#pgen.1007289.ref017" target="_blank">17</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007289#pgen.1007289.ref022" target="_blank">22</a>]. <b>(D)</b> Schematic model of competition between activator (Pax2/Exd/Hth/AbdA) and repressor (Sens) TFs. Sens binds and represses <i>RhoA</i> activity in the thorax; whereas AbdA and the activators outcompete Sens to promote gene activation in C1-SOP cells of the abdomen.</p

A Novel Variant of ATP5MC3 Associated with Both Dystonia and Spastic Paraplegia

Background: In a large pedigree with an unusual phenotype of spastic paraplegia or dystonia and autosomal dominant inheritance, linkage analysis previously mapped the disease to chromosome 2q24-2q31. Objective: The aim of this study is to identify the genetic cause and molecular basis of an unusual autosomal dominant spastic paraplegia and dystonia. Methods: Whole exome sequencing following linkage analysis was used to identify the genetic cause in a large family. Cosegregation analysis was also performed. An additional 384 individuals with spastic paraplegia or dystonia were screened for pathogenic sequence variants in the adenosine triphosphate (ATP) synthase membrane subunit C locus 3 gene (ATP5MC3). The identified variant was submitted to the “GeneMatcher” program for recruitment of additional subjects. Mitochondrial functions were analyzed in patient-derived fibroblast cell lines. Transgenic Drosophila carrying mutants were studied for movement behavior and mitochondrial function. Results: Exome analysis revealed a variant (c.318C \u3e G; p.Asn106Lys) (NM_001689.4) in ATP5MC3 in a large family with autosomal dominant spastic paraplegia and dystonia that cosegregated with affected individuals. No variants were identified in an additional 384 individuals with spastic paraplegia or dystonia. GeneMatcher identified an individual with the same genetic change, acquired de novo, who manifested upper-limb dystonia. Patient fibroblast studies showed impaired complex V activity, ATP generation, and oxygen consumption. Drosophila carrying orthologous mutations also exhibited impaired mitochondrial function and displayed reduced mobility. Conclusion: A unique form of familial spastic paraplegia and dystonia is associated with a heterozygous ATP5MC3 variant that also reduces mitochondrial complex V activity

<i>rho</i> enhancer activity maps to four neural precursors in the embryonic head.

<p>(A) The <i>rho</i> locus highlighting the Rho654 enhancer’s four conserved elements (RhoA-RhoD). (B-D) Immunostaining of <i>Rho654>H2B-YFP</i> (B), <i>RhoBB>H2B-YFP</i> (C) and <i>RhoAAA>H2B-YFP</i> (D) reporter lines demonstrating that the RhoB region of Rho654 mediates activity in four neural precursors within the head (arrowheads in B and C) while the RhoA region acts in abdominal SOPs (A1 denotes first the first abdominal segment in B and D). Early (C) and slightly later (B,D) stage 11, <i>z</i>-projected lateral views. (E-M) Comparison of <i>Rho654>H2B-YFP</i> (E-L) with published expression data for CNS neuroblasts and SOPs (M, adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134915#pone.0134915.ref017" target="_blank">17</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134915#pone.0134915.ref090" target="_blank">90</a>]). Co-expression of YFP with Dpn (E) indicates neural precursor identity. Lack of YFP co-expression with Msh (F), Ind (G), Vnd (H), Otd (I), or Fas2 (J) restricts Rho654 cell (green borders) identities to the Dv1/3 SOPs and Dv4/7 neuroblasts. Co-expression of YFP with DPax2 (K) and Ato (L) (arrowheads) is consistent with expression data for the Dv1/3 SOPs (M). Stage 11, <i>z</i>-projected ventral views with anterior up. PC: protocerebrum, DC: deutocerebrum, TC: tritocerebrum, VNC: ventral nerve cord. Bold dashed lines represent approximate neuromere boundaries.</p

Temporal control of toxin expression separates defects in development and viability.

<p>(A-B) Temperature-sensitive Gal80 expression controls Gal4-dependent GFP reporter activity. Stage 17 embryos, <i>z</i>-projected dorsal views. (C-D) Quantification of pupae formed (C) and surviving to adulthood (D) for larvae subject to different toxin expression conditions. (E-G) Analysis of age at pupariation (E), length of pupation (F) and age at eclosion (G) for non-toxin-targeted and embryonically-targeted flies. Since developmental timing is temperature dependent, data are shown as experimentals (<i>UAS-DTI</i> positive) normalized to controls (<i>UAS-DTI</i> negative). *p<0.01 or **p<0.001. (H) Comparison of non-targeted and embryonically-targeted adults. Scale bar, 1 mm. (I) Quantification of wing area for non-targeted and embryonically-targeted flies. <i>p</i>>0.1 for inter-sex comparisons.</p