Homolog Pairing at the Push of a Button

Homologous chromosomes pair in somatic cells in Drosophila, but how this occurs is poorly understood. In this issue of Developmental Cell, Viets et al. (2019) show that proteins and chromatin structure mediate pairing and argue against a DNA sequence-based mechanism

An Sp1/KLF binding site is important for the activity of a Polycomb group response element from the Drosophila engrailed gene

Polycomb-group response elements (PREs) are DNA elements through which the Polycomb-group (PcG) of transcriptional repressors act. Many of the PcG proteins are associated with two protein complexes that repress gene expression by modifying chromatin. Both of these protein complexes specifically associate with PREs in vivo, however, it is not known how they are recruited or held at the PRE. PREs are complex elements, made up of binding sites for many proteins. Our laboratory has been working to define all the sequences and DNA binding proteins required for the activity of a 181 bp PRE from the Drosophila engrailed gene. Here we show that one of the sites necessary for PRE activity, Site 2, can be bound by members of the Sp1/KLF family of zinc finger proteins. There are 10 Sp1/KLF family members in Drosophila, and nine of them bind to Site 2. We derive a consensus binding site for the Sp1/KLF Drosophila family members and show that this consensus sequence is present in most of the molecularly characterized PREs. These data suggest that one or more Sp1/KLF family members play a role in PRE function in Drosophila

Hierarchical Recruitment of Polycomb Group Silencing Complexes

AbstractPolycomb group (PcG) proteins maintain the transcriptional silence of target genes through many cycles of cell division. Here, we provide evidence for the sequential binding of PcG proteins at a Polycomb response element (PRE) in proliferating cells in which the sequence-specific DNA binding Pho and Phol proteins directly recruit E(z)-containing complexes, which in turn methylate histone H3 at lysine 27 (H3mK27). This provides a tag that facilitates binding by a Pc-containing complex. In wing imaginal discs, these PcG proteins also are present at discrete locations at or downstream of the promoter of a silenced target gene, Ubx. E(z)-dependent H3mK27 is also present near the Ubx promoter and is needed for Pc binding. The location of E(z)- and Pc-containing complexes downstream of the Ubx transcription start site suggests that they may inhibit transcription by interfering with assembly of the preinitiation complex or by blocking transcription initiation or elongation

P-Element Homing Is Facilitated by engrailed Polycomb-Group Response Elements in Drosophila melanogaster

P-element vectors are commonly used to make transgenic Drosophila and generally insert in the genome in a nonselective manner. However, when specific fragments of regulatory DNA from a few Drosophila genes are incorporated into P-transposons, they cause the vectors to be inserted near the gene from which the DNA fragment was derived. This is called P-element homing. We mapped the minimal DNA fragment that could mediate homing to the engrailed/invected region of the genome. A 1.6 kb fragment of engrailed regulatory DNA that contains two Polycomb-group response elements (PREs) was sufficient for homing. We made flies that contain a 1.5kb deletion of engrailed DNA (enΔ1.5) in situ, including the PREs and the majority of the fragment that mediates homing. Remarkably, homing still occurs onto the enΔ1. 5 chromosome. In addition to homing to en, P[en] inserts near Polycomb group target genes at an increased frequency compared to P[EPgy2], a vector used to generate 18,214 insertions for the Drosophila gene disruption project. We suggest that homing is mediated by interactions between multiple proteins bound to the homing fragment and proteins bound to multiple areas of the engrailed/invected chromatin domain. Chromatin structure may also play a role in homing

An ancient yet flexible cis-regulatory architecture allows localized Hedgehog tuning by patched/Ptch1

The Hedgehog signaling pathway is part of the ancient developmental-evolutionary animal toolkit. Frequently co-opted to pattern new structures, the pathway is conserved among eumetazoans yet flexible and pleiotropic in its effects. The Hedgehog receptor, Patched, is transcriptionally activated by Hedgehog, providing essential negative feedback in all tissues. Our locus-wide dissections of the cis-regulatory landscapes of fly patched and mouse Ptch1 reveal abundant, diverse enhancers with stage- and tissue-specific expression patterns. The seemingly simple, constitutive Hedgehog response of patched/Ptch1 is driven by a complex regulatory architecture, with batteries of context-specific enhancers engaged in promoter-specific interactions to tune signaling individually in each tissue, without disturbing patterning elsewhere. This structure—one of the oldest cis-regulatory features discovered in animal genomes—explains how patched/Ptch1 can drive dramatic adaptations in animal morphology while maintaining its essential core function. It may also suggest a general model for the evolutionary flexibility of conserved regulators and pathways. DOI: http://dx.doi.org/10.7554/eLife.13550.00

Polycomb Group Proteins Bind an <em>engrailed</em> PRE in Both the “ON” and “OFF” Transcriptional States of <em>engrailed</em>

<div><p>Polycomb group (PcG) and trithorax Group (trxG) proteins maintain the “OFF” and “ON” transcriptional states of HOX genes and other targets by modulation of chromatin structure. In Drosophila, PcG proteins are bound to DNA fragments called Polycomb group response elements (PREs). The prevalent model holds that PcG proteins bind PREs only in cells where the target gene is “OFF”. Another model posits that transcription through PREs disrupts associated PcG complexes, contributing to the establishment of the “ON” transcriptional state. We tested these two models at the PcG target gene <em>engrailed</em>. <em>engrailed</em> exists in a gene complex with <em>invected</em>, which together have 4 well-characterized PREs. Our data show that these PREs are not transcribed in embryos or larvae. We also examined whether PcG proteins are bound to an <em>engrailed</em> PRE in cells where <em>engrailed</em> is transcribed. By FLAG-tagging PcG proteins and expressing them specifically where <em>engrailed</em> is “ON” or “OFF”, we determined that components of three major PcG protein complexes are present at an <em>engrailed</em> PRE in both the “ON” and “OFF” transcriptional states in larval tissues. These results show that PcG binding per se does not determine the transcriptional state of <em>engrailed</em>.</p> </div

FLAG-tagged PcG proteins are bound to the <i>en</i> PRE in both the “ON” and “OFF” transcriptional states.

<p>(A–D) X-ChIP (with α-FLAG) was performed on third instar imaginal discs and CNS, with <i>en</i>-GAL4 or <i>ci</i>-GAL4 driven Pho-FLAG (A), Sce-FLAG (B), Esc-FLAG (C), FLAG-Scm (D). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048765#s2" target="_blank">Results</a> are shown as a percentage of the input DNA, collected prior to ChIP. ns = not significant, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 (un-paired, two-tailed t-tests). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048765#s2" target="_blank">Results</a> shown are from three independent biological samples with 2 replicates each. (E) Fold increase (PRE/control) using the means from the experiments shown in A–D. The UAS-lines are shown on the left, with the drivers <i>en</i>-Gal4 (<i>en</i>) and ci-Gal4 (<i>ci</i>) on top.</p