Mutation in Mouse Hei10, an E3 Ubiquitin Ligase, Disrupts Meiotic Crossing Over

Crossing over during meiotic prophase I is required for sexual reproduction in mice and contributes to genome-wide genetic diversity. Here we report on the characterization of an N-ethyl-N-nitrosourea-induced, recessive allele called mei4, which causes sterility in both sexes owing to meiotic defects. In mutant spermatocytes, chromosomes fail to congress properly at the metaphase plate, leading to arrest and apoptosis before the first meiotic division. Mutant oocytes have a similar chromosomal phenotype but in vitro can undergo meiotic divisions and fertilization before arresting. During late meiotic prophase in mei4 mutant males, absence of cyclin dependent kinase 2 and mismatch repair protein association from chromosome cores is correlated with the premature separation of bivalents at diplonema owing to lack of chiasmata. We have identified the causative mutation, a transversion in the 5′ splice donor site of exon 1 in the mouse ortholog of Human Enhancer of Invasion 10 (Hei10; also known as Gm288 in mouse and CCNB1IP1 in human), a putative B-type cyclin E3 ubiquitin ligase. Importantly, orthologs of Hei10 are found exclusively in deuterostomes and not in more ancestral protostomes such as yeast, worms, or flies. The cloning and characterization of the mei4 allele of Hei10 demonstrates a novel link between cell cycle regulation and mismatch repair during prophase I

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

Whole mount embryo in situ hybridization reveals that ncRNAs are not detectable at the known <i>en</i> and <i>inv</i> PREs.

<p>Grey Line indicates genomic DNA, with the coordinates listed at both ends (genome version R5.1). DIG-labeled RNA probes were generated to cover the entire region shown, on both strands. (A) Positive controls showing robust signal from <i>en</i> and <i>inv</i> probes, and from a probe against <i>miR-iab-8</i>, a miRNA in the BX-C <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048765#pone.0048765-Bender2" target="_blank">[30]</a>. (B) Selected in situ results from <i>inv-en</i> region. Panels 1–4 and 7, 8 show non-specific background staining using probes to detect RNAs transcribed in the regions of the <i>inv</i> and <i>en</i> PREs. Several probes yielded specific signals. Panels 5 and 9 show an <i>en</i>-like pattern at stage 9, panels 6 and 10 show a pair-rule pattern at stage 5, and panels 11–13 show late CNS staining at stage 16. Embryos located above the genomic DNA line were hybridized with antisense probes (with respect to <i>inv</i>), embryos located below the line were hybridized with sense probes (with respect to <i>inv</i>). Filled red boxes are the locations of PREs (as evidence by PcG binding and by PRE activity in transgenes). PcG protein binding sites, depicted with open red box, are where Pho was reported to bind in ChIP/chip studies in larvae and embryos <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048765#pone.0048765-Oktaba2" target="_blank">[39]</a>. Green boxes indicate the locations of regions reported to be transcribed <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048765#pone.0048765-Hild1" target="_blank">[31]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048765#pone.0048765-Kopczynski1" target="_blank">[32]</a>.</p

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

Stable expression of Pho-FLAG in cells that express En or Ci.

<p>UAS-Pho-FLAG expression by the <i>en</i>-GAL4 or <i>ci</i> -GAL4 driver. Anti-FLAG staining is red, anti-En staining is green. (A–C) 3^rd instar wing imaginal disc collected from a UAS-Pho-FLAG, <i>en</i>-GAL4 cross. Panel C shows nearly complete overlap of anti-FLAG and anti-En staining. (D–F) 3^rd instar wing imaginal disc collected from a UAS-Pho-FLAG, <i>ci</i>-GAL4 cross. Panel F shows complementary staining of anti-FLAG and anti-En. Note that the size of the anterior compartment, where Ci is expressed is about twice the size of the posterior compartment, where En is expressed <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048765#pone.0048765-Neufeld1" target="_blank">[35]</a>. (G) qRT-PCR showing that there is about twice as much Pho-FLAG transcript when it is driven by <i>ci</i>-Gal4 than by <i>en</i>-Gal4 (*** P≤0.001).</p

FLAG-tagged PcG proteins co-localize with endogenous PcG proteins on polytene chromosomes.

<p>FLAG-tagged proteins were driven by <i>arm</i>-Gal4.</p

Pho-FLAG and Sce-FLAG binding peaks at PRE2.

<p>(A) A map of the <i>en</i> gene showing the location of the PREs and the probes used for the qPCR (#1–8). (B,C) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048765#s2" target="_blank">Results</a> of X-ChIP experiment with Sce-FLAG (B) and Pho-FLAG (C) driven by <i>en</i>-Gal4 (open bars) or <i>ci</i>-Gal4 (closed bars). Pho binding was also done on all chromatin preparations. The results of a representative experiment are shown. These experiments were done with a different batch of FLAG antibody and different ChIP reagents than those done in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048765#pone-0048765-g005" target="_blank">Fig. 5</a>. Further, 20 larvae were used for each sample instead of 10. Under these conditions, we did not see a difference in the level of binding to the PREs between the “ON” and the “OFF” states; however, the qualitative result, PcG proteins binding to PRE2 in both the “ON” and “OFF” states was the same in these experiments and those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048765#pone-0048765-g005" target="_blank">Fig. 5</a>.</p

Enhancer-promoter communication at the Drosophila engrailed locus

Enhancers are often located many tens of kilobases away from the promoter they regulate, sometimes residing closer to the promoter of a neighboring gene. How do they know which gene to activate? We have used homing P[en] constructs to study the enhancer-promoter communication at the engrailed locus. Here we show that engrailed enhancers can act over large distances, even skipping over other transcription units, choosing the engrailed promoter over those of neighboring genes. This specificity is achieved in at least three ways. First, early acting engrailed stripe enhancers exhibit promoter specificity. Second, a proximal promoter-tethering element is required for the action of the imaginal disc enhancer(s). Our data suggest that there are two partially redundant promoter-tethering elements. Third, the long-distance action of engrailed enhancers requires a combination of the engrailed promoter and sequences within or closely linked to the promoter proximal Polycomb-group response elements. These data show that multiple mechanisms ensure proper enhancer-promoter communication at the Drosophila engrailed locus