The ISWI Chromatin Remodeler Organizes the hsrω ncRNA–Containing Omega Speckle Nuclear Compartments

The complexity in composition and function of the eukaryotic nucleus is achieved through its organization in specialized nuclear compartments. The Drosophila chromatin remodeling ATPase ISWI plays evolutionarily conserved roles in chromatin organization. Interestingly, ISWI genetically interacts with the hsrω gene, encoding multiple non-coding RNAs (ncRNA) essential, among other functions, for the assembly and organization of the omega speckles. The nucleoplasmic omega speckles play important functions in RNA metabolism, in normal and stressed cells, by regulating availability of hnRNPs and some other RNA processing proteins. Chromatin remodelers, as well as nuclear speckles and their associated ncRNAs, are emerging as important components of gene regulatory networks, although their functional connections have remained poorly defined. Here we provide multiple lines of evidence showing that the hsrω ncRNA interacts in vivo and in vitro with ISWI, regulating its ATPase activity. Remarkably, we found that the organization of nucleoplasmic omega speckles depends on ISWI function. Our findings highlight a novel role for chromatin remodelers in organization of nucleoplasmic compartments, providing the first example of interaction between an ATP-dependent chromatin remodeler and a large ncRNA

The Nucleosome-Remodeling ATPase ISWI Is Regulated by Poly-ADP-Ribosylation

ATP-dependent nucleosome-remodeling enzymes and covalent modifiers of chromatin set the functional state of chromatin. However, how these enzymatic activities are coordinated in the nucleus is largely unknown. We found that the evolutionary conserved nucleosome-remodeling ATPase ISWI and the poly-ADP-ribose polymerase PARP genetically interact. We present evidence showing that ISWI is target of poly-ADP-ribosylation. Poly-ADP-ribosylation counteracts ISWI function in vitro and in vivo. Our work suggests that ISWI is a physiological target of PARP and that poly-ADP-ribosylation can be a new, important post-translational modification regulating the activity of ATP-dependent nucleosome remodelers

The Nucleosome Remodeling Factor ISWI Functionally Interacts With an Evolutionarily Conserved Network of Cellular Factors

ISWI is an evolutionarily conserved ATP-dependent chromatin remodeling factor playing central roles in DNA replication, RNA transcription, and chromosome organization. The variety of biological functions dependent on ISWI suggests that its activity could be highly regulated. Our group has previously isolated and characterized new cellular activities that positively regulate ISWI in Drosophila melanogaster. To identify factors that antagonize ISWI activity we developed a novel in vivo eye-based assay to screen for genetic suppressors of ISWI. Our screen revealed that ISWI interacts with an evolutionarily conserved network of cellular and nuclear factors that escaped previous genetic and biochemical analyses

Trans-Reactivation: A New Epigenetic Phenomenon Underlying Transcriptional Reactivation of Silenced Genes

<div><p>In order to study the role played by cellular RNA pools produced by homologous genomic loci in defining the transcriptional state of a silenced gene, we tested the effect of non-functional alleles of the <i>white</i> gene in the presence of a functional copy of <i>white</i>, silenced by heterochromatin. We found that non-functional alleles of <i>white</i>, unable to produce a coding transcript, could reactivate <i>in trans</i> the expression of a wild type copy of the same gene silenced by heterochromatin. This new epigenetic phenomenon of transcriptional <i>trans</i>-reactivation is heritable, relies on the presence of homologous RNA’s and is affected by mutations in genes involved in post-transcriptional gene silencing. Our data suggest a general new unexpected level of gene expression control mediated by homologous RNA molecules in the context of heterochromatic genes.</p></div

Mutations in genes encoding for factors involved in PTGS and RNA methylation influence <i>trans</i>-reactivation.

<p><b>(A)</b> Loss of function mutations in <i>armitage</i> (<i>armi</i>^Δ99), <i>piwi</i> (<i>piwi</i>^<i>06843</i>), <i>aubergine</i> (<i>aub</i>^<i>QC42</i>), <i>hsp83</i> (<i>hsp83</i>^{<i>scratch</i>}), <i>spn-E</i> (<i>spn-E</i>^<i>1</i>), <i>argonaute2</i> (<i>ago2</i>), <i>argonaute1</i> (<i>ago1</i>^<i>04845</i>), <i>dicer1</i> (<i>dcr1</i>), <i>dicer2</i> (<i>dcr2</i>), <i>r2d2</i> (<i>r2d2</i>^<i>1</i>), and <i>dnmt2</i> (<i>dnmt2</i>), were tested in heterozygosis for their ability to modify the levels of eye pigmentation we scored in <i>w</i>^<i>m4h</i>/<i>w* trans</i>-reactivation. The Log₂ ratio of <i>w</i>^<i>m4h</i>/<i>w* trans-reactivation</i> in the mutant background over the normal levels of <i>w</i>^<i>m4h</i>/<i>w* trans-reactivation</i> was calculated and plotted (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005444#pgen.1005444.s007" target="_blank">S7D Fig</a>). A positive value indicates that the mutation enhances <i>trans-reactivation</i> (dark orange bars), while a negative value is indicative of a suppression of <i>trans-reactivation</i> (pale orange bars). The strong <i>trans-reactivating w</i>^<i>sey</i> allele was used in this assay. <b>(B)</b> A schematic representation of the biological significance of the data obtained shows that mutations in genes involved in the biogenesis of siRNA and piRNA counteract <i>trans</i>-reactivation (red bars). On the other hand, mutations in genes involved in the processing of miRNA and in RNA methylation (RdMT; <u>R</u>NA-<u>d</u>ependent <u>M</u>ethyl <u>T</u>ransferase) promote <i>trans</i>-reactivation (green arrows). <b>(C)</b> High throughput RNA-Seq reads mapped on the <i>Drosophila</i> genome at the <i>white</i> gene locus for long and short RNA species purified from Malpighian tubules of <i>Δw</i>, <i>w</i>^<i>sey</i>, <i>w</i>^<i>m4h</i> and <i>w</i>^<i>sey</i><i>/w</i>^<i>m4h</i> lines. <b>(D)</b> FPKM values plot obtained for the overall amount of long RNA species mapped at the <i>w</i> gene in the <i>Δw</i>, <i>w</i>^<i>sey</i>, <i>w</i>^<i>m4h</i> and <i>w</i>^<i>sey</i><i>/w</i>^<i>m4h</i> lines. <b>(E)</b> Eye pigment quantification of <i>w</i>^<i>m4h</i> females resulting from the injection of <i>w</i>^<i>m4h</i> eggs with buffer (-) or total RNA extracted from testis of either Δw or <i>w</i>^<i>sey</i><i>Drosophila</i> males. The asterisks indicate the statistical significance (< 0.05) of differences in eye pigment using Student's <i>t</i>-test.</p

Hypomorphic and loss-of-function alleles of the <i>white</i> gene increase <i>w</i>^<i>m4h</i> eye color pigmentation.

<p><b>(A)</b> Upper panel; schematic representation of the <i>In(1)w</i>^<i>m4h</i> inversion juxtaposing the <i>w</i> wildtype gene in the proximity of X chromosome pericentric heterochromatin, resulting in the generation of the <i>w</i>^<i>m4h</i> allele with silenced <i>w</i> expression in most eye cells and leading to a variegated eye color. Lower panel; parental (P) variegating <i>w</i>^<i>m4h</i> females were crossed with males carrying a hypomorphic or loss-of-function allele of the <i>white</i> gene (<i>w*</i>). The recovered progeny (F1) was scored for increased, decreased or unchanged eye color pigmentation. Number of alleles recovered (n) and corresponding percentage (%) of total alleles screened are reported. Eye pigment quantification of parental stocks (P) and of the resulting trans-heterozygous female (<i>w</i>^<i>m4h</i><i>/w*</i>) and control male (<i>w</i>^<i>m4h</i><i>/Y</i>) progeny (F1) for the <i>w</i>^<i>sey</i><b>(B)</b>, <i>w</i>^<i>1118</i><b>(C)</b> alleles and <i>Δw</i><b>(D)</b>, are shown together with representative eye pictures for each genotype tested. P and F1 eye pigment quantification graphs have different scale.</p

Effect of <i>w*</i> alleles on <i>E(var)</i>, <i>Sb</i>^<i>v</i> and <i>w</i> variegating autosomal insertions.

<p><b>(A)</b> Upper panel; eye pigment quantification of females <i>w</i>^<i>sey</i>, <i>w</i>^<i>1118</i> and <i>Δw</i> trans-heterozygous with <i>w</i>^<i>m4h</i>, carrying one copy of the dominant <i>E(var)3−1</i>^<i>01</i> mutation. Lower panel; schematic representation of the <i>E(var)3−1</i>^<i>01</i>; <i>w</i>^<i>m4h</i> line used in the eye variegation assay. <b>(B)</b> Upper panel; quantification of the effect of <i>Δw</i>, <i>w</i>^<i>1118</i>, <i>w</i>^<i>sey</i> and <i>Su(var)</i>^{<i>4–20</i>} on the number of short (<i>Sb</i>) vs normal (<i>wt</i>) bristle length in the <i>Sb</i>^<i>v</i> variegating line. Lower panel; representation of <i>T(2</i>:<i>3)Sb</i>^<i>v</i> translocation. Activation of the dominant <i>Sb</i> allele results in short bristles (white arrows). <b>(C-E)</b> Upper panels; eye pigment quantification of <i>w</i>^<i>sey</i>, <i>w</i>^<i>1118</i> and <i>Δw</i> males carrying one copy of the IV and II chromosome pericentric heterochromatin autosomal <i>w</i> variegating insertion 39C-12, 118E-10 and P-819. Lower panel; schematic representation of autosomal insertions <b>(F)</b> Upper panel; eye pigment quantification of <i>w</i>^<i>sey</i>, <i>w</i>^<i>1118</i> and <i>Δw</i> males carrying one copy of the III chromosome telomeric heterochromatin <i>w</i> variegating insertion <i>A4-4</i>. Lower panel; schematic representation of autosomal insertions. The asterisks indicate the statistical significance (< 0.05) of differences in eye pigment using Student's <i>t</i>-test. For cross details see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005444#pgen.1005444.s004" target="_blank">S4 Fig</a>.</p

<i>trans</i>-reactivation is mediated by a diffusible factor.

<p><b>(A)</b> Upper panel; schematic representation of normal fertilization leading to the generation of an adult fly. Lower panel; description of the process of gynogenesis in which a viable adult fly that does not carry any genetic contribution from the father is generated through the fertilization of a gynogenetic <i>gyn</i>^<i>2</i>, <i>gyn</i>^<i>3</i> egg (where diploidy is restored by the fusion of two non-sister nuclei out of the four egg pronuclei which result from the second meiotic division) by a <i>ms(3)</i>^<i>K81</i> sperm (one of a rare class of fly male sterile mutations in which sterility is caused by the elimination of male pronucleus after sperm entry into the egg). Despite, the male pronucleus (blue) does not contribute to the zygote, the male nucleoplasm (light blue) contributes together with the two female pronuclei (pink) to the development of a gynogenetic adult fly. <b>(B)</b> Cross scheme used to test if nucleoplasmic factors devoid of any male genomic contribution coming from the <i>w* trans</i>-reactivating allele can <i>trans</i>-reactivate <i>w</i>^<i>m4h</i> in female gynogenetic adult flies. Possible F1 eye phenotypes coming from the cross are boxed. Eye pigment measurements of parental and gynogenetic progeny were conducted to score the presence of a <i>trans</i>-reactivated <i>w</i>^<i>m4h</i> in <b>(C)</b><i>w</i>^<i>m4h</i><i>; gyn</i>^<i>2</i>, <i>gyn</i>^<i>3</i> female, <i>w</i>^<i>sey</i><i>; ms(3)</i>^<i>K81</i> male parental stocks, and F1 gynogenetic <i>w</i>^<i>m4h</i>; <i>gyn</i>^<i>2</i>, <i>gyn</i>^<i>3</i> progeny, or in control <b>(D)</b><i>w</i>^<i>m4h</i><i>; gyn</i>^<i>2</i>, <i>gyn</i>^<i>3</i> female, <i>Δw; ms(3)</i>^<i>K81</i> male parental stocks, and F1 gynogenetic <i>w</i>^<i>m4h</i>; <i>gyn</i>^<i>2</i>, <i>gyn</i>^<i>3</i> progeny. Eye pigment quantification graphs have a different scale.</p

<i>trans</i>-reactivation of <i>w*</i> over <i>w</i>^<i>m4h</i> is heritable.

<p><b>(A)</b> Cross scheme used to test trans-generational inheritance of <i>w</i>^<i>m4h</i><i>trans</i>-reactivation. Parental (P) variegating <i>w</i>^<i>m4h</i><i>/w</i>^<i>m4h</i> homozygous females were crossed with males carrying the <i>trans</i>-reactivating <i>w*</i> allele. The (F1) <i>trans</i>-reactivated <i>w</i>^<i>m4h</i><i>/w</i>^<i>m4h</i> female was back crossed with <i>w*</i>/Y males carrying the <i>trans</i>-reactivating <i>w*</i> allele. Finally, the F2 progeny was scored for eye color pigmentation in control <i>w</i>^<i>m4h</i>/<i>w*</i> females, <i>w*/Y</i> males and the experimental <i>w</i>^<i>m4h</i>/<i>Y</i> males progeny, where the <i>w* trans</i>-reactivating allele had segregated. The possible F2 <i>w</i>^<i>m4h</i>/<i>Y</i> males eye phenotypes coming from the cross are boxed. Eye pigment quantification of the P and F1 stocks, together with the resulting F2 control trans-heterozygous female (<i>w</i>^<i>m4h</i><i>/w*</i>), control male (<i>w</i>^<i>m4h</i><i>/Y</i>) and experimental segregating <i>w</i>^<i>m4h</i><i>/Y</i> male progeny for the <i>w</i>^<i>1118</i><b>(B)</b> and <i>w</i>^<i>sey</i><b>(C)</b> alleles, together with representative eye pictures for each genotype tested are shown. <b>(D)</b> Cross scheme used to test if the <i>Δw/Y</i> males stock, carrying a deletion of the entire <i>w</i> locus, was able to induce the inheritance of <i>w</i>^<i>m4h</i><i>trans</i>-reactivation in F2 <i>w</i>^<i>m4h</i><i>/Δw</i> females and <i>w</i>^<i>m4h</i><i>/Y</i> male progeny derived from segregating <i>w</i>^<i>m4h</i><i>/w</i>^<i>1118</i><b>(E)</b> and <i>w</i>^<i>m4h</i><i>/w</i>^<i>sey</i><b>(F)</b> F1 trans-heterozygous. <b>(G)</b> Cross scheme showing the possible F1 progeny arising from an homozygous <i>trans</i>-reactivated <i>w</i>^<i>m4h</i><i>/w</i>^<i>m4h</i> female crossed with a <i>trans</i>-reactivated <i>w</i>^<i>m4h</i>/<i>Y</i> male, <b>(H)</b> and the corresponding eye pigment quantification of the P and F1 progeny obtained with crosses with stably <i>trans</i>-reactivated parental <i>w</i>^<i>m4h</i><i>/w</i>^<i>m4h</i> females and <i>w</i>^<i>m4h</i>/<i>Y</i> males (produced with the <i>trans</i>-reactivating <i>w</i>^<i>sey</i> allele).</p

Increased levels of wildtype <i>white</i> coding transcripts and <i>w</i>^<i>m4h</i> chromatin locus opening by <i>w*</i> alleles.

<p><b>(A)</b> Structure of the <i>white</i> gene shows the 3’ region used to design the primers (arrows) for semi-quantitative RT-PCR conducted on total RNA, derived from adult heads dissected from the different genomic combinations shown. The graph shows the levels of amplified <i>w</i> transcripts. Primers designed against the <i>Act5C</i> transcript have been used as internal control. The two amplified bands represent unspliced and spliced forms of the <i>white</i> transcript. Polytene chromosome FISH using genomic probes covering the entire <i>w</i> gene (with the exception of the first intron) and coding sequences for the <i>hsp70</i> gene (mapping chromosome 3R) on homozygous <i>w</i>^<i>m4h</i><i>/w</i>^<i>m4h</i><b>(B)</b>, <i>w</i>^<i>1118</i><i>/w</i>^<i>1118</i><b>(C)</b>, <i>w</i>^<i>sey</i><i>/w</i>^<i>sey</i><b>(D)</b> and trans-heterozygous <i>w</i>^<i>m4h</i><i>/w</i>^<i>1118</i><b>(E)</b> and <i>w</i>^<i>m4h</i><i>/w</i>^<i>sey</i><b>(F)</b> allelic combinations. FISH signals for <i>white</i> (red) and <i>hsp70</i> (green) genomic sequences are indicated by arrowheads. The asterisks indicate the region of pericentric heterochromatin. The X indicates the chromosome where the <i>w</i> gene maps. FISH signals for the <i>w</i> locus are detected in the <i>w</i>^<i>1118</i><i>/w</i>^<i>1118</i> and <i>w</i>^<i>sey</i><i>/w</i>^<i>sey</i> chromosomes because in these genotypes the <i>w</i> genomic sequences reside on euchromatic accessible region.</p