Genome-Wide Ultrabithorax Binding Analysis Reveals Highly Targeted Genomic Loci at Developmental Regulators and a Potential Connection to Polycomb-Mediated Regulation

Hox homeodomain transcription factors are key regulators of animal development. They specify the identity of segments along the anterior-posterior body axis in metazoans by controlling the expression of diverse downstream targets, including transcription factors and signaling pathway components. The Drosophila melanogaster Hox factor Ultrabithorax (Ubx) directs the development of thoracic and abdominal segments and appendages, and loss of Ubx function can lead for example to the transformation of third thoracic segment appendages (e.g. halters) into second thoracic segment appendages (e.g. wings), resulting in a characteristic four-wing phenotype. Here we present a Drosophila melanogaster strain with a V5-epitope tagged Ubx allele, which we employed to obtain a high quality genome-wide map of Ubx binding sites using ChIP-seq. We confirm the sensitivity of the V5 ChIP-seq by recovering 7/8 of well-studied Ubx-dependent cis-regulatory regions. Moreover, we show that Ubx binding is predictive of enhancer activity as suggested by comparison with a genome-scale resource of in vivo tested enhancer candidates. We observed densely clustered Ubx binding sites at 12 extended genomic loci that included ANTP-C, BX-C, Polycomb complex genes, and other regulators and the clustered binding sites were frequently active enhancers. Furthermore, Ubx binding was detected at known Polycomb response elements (PREs) and was associated with significant enrichments of Pc and Pho ChIP signals in contrast to binding sites of other developmental TFs. Together, our results show that Ubx targets developmental regulators via strongly clustered binding sites and allow us to hypothesize that regulation by Ubx might involve Polycomb group proteins to maintain specific regulatory states in cooperative or mutually exclusive fashion, an attractive model that combines two groups of proteins with prominent gene regulatory roles during animal development

Probing the canonicity of the Wnt/Wingless signaling pathway

The hallmark of canonical Wnt signaling is the transcriptional induction of Wnt target genes by the beta-catenin/TCF complex. Several studies have proposed alternative interaction partners for beta-catenin or TCF, but the relevance of potential bifurcations in the distal Wnt pathway remains unclear. Here we study on a genome-wide scale the requirement for Armadillo (Arm, Drosophila beta-catenin) and Pangolin (Pan, Drosophila TCF) in the Wnt/Wingless(Wg)-induced transcriptional response of Drosophila Kc cells. Using somatic genetics, we demonstrate that both Arm and Pan are absolutely required for mediating activation and repression of target genes. Furthermore, by means of STARR-sequencing we identified Wnt/Wg-responsive enhancer elements and found that all responsive enhancers depend on Pan. Together, our results confirm the dogma of canonical Wnt/Wg signaling and argue against the existence of distal pathway branches in this system

Optimizing sgRNA position markedly improves the efficiency of CRISPR/dCas9-mediated transcriptional repression

CRISPR interference (CRISPRi) represents a newly developed tool for targeted gene repression. It has great application potential for studying gene function and mapping gene regulatory elements. However, the optimal parameters for efficient single guide RNA (sgRNA) design for CRISPRi are not fully defined. In this study, we systematically assessed how sgRNA position affects the efficiency of CRISPRi in human cells. We analyzed 155 sgRNAs targeting 41 genes and found that CRISPRi efficiency relies heavily on the precise recruitment of the effector complex to the target gene transcription start site (TSS). Importantly, we demonstrate that the FANTOM5/CAGE promoter atlas represents the most reliable source of TSS annotations for this purpose. We also show that the proximity to the FANTOM5/CAGE-defined TSS predicts sgRNA functionality on a genome-wide scale. Moreover, we found that once the correct TSS is identified, CRISPRi efficiency can be further improved by considering sgRNA sequence preferences. Lastly, we demonstrate that CRISPRi sgRNA functionality largely depends on the chromatin accessibility of a target site, with high efficiency focused in the regions of open chromatin. In summary, our work provides a framework for efficient CRISPRi assay design based on functionally defined TSSs and features of the target site chromatin

Probing the canonicity of the Wnt/Wingless signaling pathway

<div><p>The hallmark of canonical Wnt signaling is the transcriptional induction of Wnt target genes by the beta-catenin/TCF complex. Several studies have proposed alternative interaction partners for beta-catenin or TCF, but the relevance of potential bifurcations in the distal Wnt pathway remains unclear. Here we study on a genome-wide scale the requirement for Armadillo (Arm, <i>Drosophila</i> beta-catenin) and Pangolin (Pan, <i>Drosophila</i> TCF) in the Wnt/Wingless(Wg)-induced transcriptional response of <i>Drosophila</i> Kc cells. Using somatic genetics, we demonstrate that both Arm and Pan are absolutely required for mediating activation and repression of target genes. Furthermore, by means of STARR-sequencing we identified Wnt/Wg-responsive enhancer elements and found that all responsive enhancers depend on Pan. Together, our results confirm the dogma of canonical Wnt/Wg signaling and argue against the existence of distal pathway branches in this system.</p></div

Ubx binds to active enhancers.

<p>(A) The bar plot shows the percentage of Vienna tiles (VTs) without or with Ubx binding sites (black and red bars, respectively) that is active at any stage of embryogenesis (left) or at the indicated embryonic stages. Hypergeometric p-value: **P<10⁻¹⁰. (B) The left panel shows the fraction of all VTs (top) and the fraction of Ubx-bound VTs (bottom) that overlaps HOT regions (dark shading). The bar plot on the right shows the percentage of active tiles for the four subsets of VTs defined on the left panel. NS–not significant. Hypergeometric p-value: **P<10⁻¹⁹.</p

Mutagenesis of the <i>pan</i> gene.

<p>(A) Schematic of the <i>pan</i> gene-locus. Untranslated regions (UTR) are indicated in grey boxes, translated exons in black. HMG box in green. (B) CRISPR targeting strategy: Target sites of both sgRNAs are represented by black triangles. The PAM site is highlighted in blue, the HMG box in green. Sequences as they are present in the pan^-/--AF1AD26 (pan^-/-) cells are depicted below. (C) Wild-type (WT) and pan^-/--AF1AD26 (pan^-/-) cells were transfected with the <i>wingful</i> luciferase reporter expression vector and Renilla expression vector 24 h prior stimulation with WCM (as control CM was used). After 24h stimulation, reporter activity was analyzed. (D) Wild-type (WT) and pan^-/--AF1AD26 (pan^-/-) cells were transfected with Pangolin overexpression vector under the control of the Actin promoter together with <i>wingful</i> luciferase reporter expression vector and Renilla expression vector 24 h prior stimulation with WCM (as control CM was used). After 24h stimulation, reporter activity was analyzed.</p

Gene expression analysis of Wg/Wnt target genes in arm^-/--AFII7/8 cells.

<p>(A) Heat map of Wnt/Wg target genes showing their log2 fold change (FC) of expression after to before WCM treatment in wild-type (WT) and arm^-/--AFII7/8 (arm^-/-) cells. The genes are listed according to the strength of the induction of their expression in WT cells. Strongest up-regulated genes are on top. Up-regulated genes are shown in red, down-regulated genes in blue, no expression in white. (B) Boxplots showing the difference in gene activity for up- and down-regulated genes after WCM stimulation in wild-type (WT) and arm^-/--AFII7/8 (arm^-/-) cells. Paired t-test: * ≤ 0.05, *** ≤ 0.0001.</p

Genomic location of Ubx binding sites and recovery of known Ubx-dependent enhancers.

<p>(A) Genomic distribution of Ubx peaks (right) in comparison to the genome (left). (B-H) UCSC Genome Browser screenshots [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161997#pone.0161997.ref083" target="_blank">83</a>] of Ubx (blue), mock (green) ChIP-seq fragment density tracks and the Ubx peak calls at known Ubx-dependent enhancers (red bars) (see the main text for references). Panel (B) also contains the fragment density tracks for the two input samples (grey). (I) UCSC Genome Browser view of the <i>hth</i> locus and examples of Ubx-bound embryonic enhancers and their activity patterns [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161997#pone.0161997.ref022" target="_blank">22</a>]. (J) UCSC Genome Browser view of the <i>Con</i> locus and Ubx-bound embryonic enhancer [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161997#pone.0161997.ref022" target="_blank">22</a>]. Vienna tiles (VT) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161997#pone.0161997.ref022" target="_blank">22</a>] are marked by slate blue boxes and their ID numbers are indicated.</p

Creation of an epitope-tagged Ubx allele by homologous recombination.

<p>(A) Design of the targeting construct (top), the integration to the endogenous Ubx locus and the location of the probe for Southern blotting (middle) and the locus after the cassette removal (bottom). (B) The eye color and haltere morphology for candidate flies before (left) and after cassette removal (white-eyed fly) (right). (C) Southern blot confirming the correct integration for two independently recombined <i>Drosophila</i> lines #11 and #12 (heterozygous for the insert). w¹¹¹⁸ flies were used as a control.</p