RNA SILENCING AND HIGHER ORDER CHROMATIN ORGANIZATION IN DROSOPHILA

Higher order chromatin organization influences gene expression, but mechanisms by which this phenomenon occurs are not well understood. RNA silencing, a conserved mechanism that involves small RNAs bound to an Argonaute protein, mediates gene expression via transcriptional or post-transcriptional regulation. Recently, a role for RNA silencing in chromatin has been emerging. In fission yeast, a major role of RNA interference (RNAi) is to establish pericentromeric heterochromatin. However, whether this mechanism is conserved throughout evolution is unclear. In Drosophila, a powerful model organism, there are multiple functionally distinct RNA silencing pathways. Previous studies have suggested the involvement of the Piwi-interacting RNA (piRNA) and endogenous small interfering RNA (endo-siRNA) pathways in heterochromatin formation in order to silence transposable elements in germline and somatic tissues, respectively, but direct evidence is lacking. We addressed whether the genomic locations generating these small RNAs may act as AGO-dependent platforms for heterochromatin recruitment. Our genetic and biochemical analyses revealed that heterochromatin is nucleated independently of endo-siRNA and piRNA pathways suggesting that RNAi-dependent heterochromatin assembly may not be conserved in metazoans. Chromatin insulators are regulatory elements characterized by enhancer blocking and barrier activity. Insulators form large nuclear foci termed insulator bodies that are tethered to the nuclear matrix and have been proposed to organize the genome into distinct transcriptional domains by looping out intervening DNA. In Drosophila, RNA silencing has been reported to affect nuclear organization of gypsy insulator complexes and formation of Polycomb repression bodies. Our studies revealed that AGO2 is required for CTCF/CP190-dependent Fab-8 insulator function independent of its catalytic activity or Dicer-2. Moreover, AGO2 associates with euchromatin but not heterochromatin genome-wide. Also, AGO2 associates physically with CP190 and CTCF, and mutation of CTCF, CP190, or AGO2 decreases chromosomal looping interactions and alters gene expression. We propose a novel RNAi-independent role for AGO2 in the nucleus. We postulate that insulator proteins recruit AGO2 to chromatin to promote or stabilize chromosomal interactions crucial for proper gene expression. Overall, our findings demonstrate novel mechanisms by which RNA silencing affects gene expression on the level of higher order chromatin organization

HP1 Recruitment in the Absence of Argonaute Proteins in Drosophila

Highly repetitive and transposable element rich regions of the genome must be stabilized by the presence of heterochromatin. A direct role for RNA interference in the establishment of heterochromatin has been demonstrated in fission yeast. In metazoans, which possess multiple RNA–silencing pathways that are both functionally distinct and spatially restricted, whether RNA silencing contributes directly to heterochromatin formation is not clear. Previous studies in Drosophila melanogaster have suggested the involvement of both the AGO2-dependent endogenous small interfering RNA (endo-siRNA) as well as Piwi-interacting RNA (piRNA) silencing pathways. In order to determine if these Argonaute genes are required for heterochromatin formation, we utilized transcriptional reporters and chromatin immunoprecipitation of the critical factor Heterochromatin Protein 1 (HP1) to monitor the heterochromatic state of piRNA clusters, which generate both endo-siRNAs and the bulk of piRNAs. Surprisingly, we find that mutation of AGO2 or piwi increases silencing at piRNA clusters corresponding to an increase of HP1 association. Furthermore, loss of piRNA production from a single piRNA cluster results in genome-wide redistribution of HP1 and reduction of silencing at a distant heterochromatic site, suggesting indirect effects on HP1 recruitment. Taken together, these results indicate that heterochromatin forms independently of endo-siRNA and piRNA pathways

Tissue-Specific Regulation of Chromatin Insulator Function

<div><p>Chromatin insulators organize the genome into distinct transcriptional domains and contribute to cell type–specific chromatin organization. However, factors regulating tissue-specific insulator function have not yet been discovered. Here we identify the RNA recognition motif-containing protein Shep as a direct interactor of two individual components of the <em>gypsy</em> insulator complex in <em>Drosophila</em>. Mutation of <em>shep</em> improves <em>gypsy</em>-dependent enhancer blocking, indicating a role as a negative regulator of insulator activity. Unlike ubiquitously expressed core <em>gypsy</em> insulator proteins, Shep is highly expressed in the central nervous system (CNS) with lower expression in other tissues. We developed a novel, quantitative tissue-specific barrier assay to demonstrate that Shep functions as a negative regulator of insulator activity in the CNS but not in muscle tissue. Additionally, mutation of <em>shep</em> alters insulator complex nuclear localization in the CNS but has no effect in other tissues. Consistent with negative regulatory activity, ChIP–seq analysis of Shep in a CNS-derived cell line indicates substantial genome-wide colocalization with a single <em>gypsy</em> insulator component but limited overlap with intact insulator complexes. Taken together, these data reveal a novel, tissue-specific mode of regulation of a chromatin insulator.</p> </div

Coimmunoprecipitation of <i>gypsy</i> insulator proteins with Shep isoforms.

<p>(A) Identification of Shep isoforms <i>in vivo</i>. Western blotting for Shep from larval extracts that are wildtype (lane 1), expressing <i>Act5C</i>::Gal4 driving single copy UAS-<i>shep</i> dsRNA (lane 2), expressing <i>Act5C</i>::Gal4 driving single copy UAS-<i>shep</i> C and E (lane 3), or containing a P-element insertion that disrupts the coding region of isoform A (lane 4). Pep is shown as a loading control. (B) Coimmunoprecipitation of <i>gypsy</i> insulator proteins with Shep. Embryo nuclear extracts (lane 1) were immunoprecipitated (IP) with either Pre-Immune (Pre Im; lanes 2 and 4) or α-Shep (lanes 3 and 5) serum. Shep, Mod(mdg4)2.2, Su(Hw), and CP190 were detected in nuclear extracts (Nuc Ext), supernatants (Sup) (lanes 2–3) and IPs (lanes 4–5) by Western blotting. Approximately 0.02% CP190, 0.02% Su(Hw), and 0.1% Mod(mdg4)2.2 of total were recovered in the IP.</p

Summary of <i>shep</i> homozygous P element and heterozygous deficiency alleles.

1<p>In <i>mod(mdg4)^u1</i> background; percentage shown is % viable homozygous adults with respect to number of homozygous' pupae; NQ = not quantified; see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003069#pgen.1003069.s006" target="_blank">Table S2</a> for number of flies and pupae counted.</p>2<p>Expresses isoforms C and E.</p>3<p>No results due to lethality.</p>4<p>Escaper phenotype.</p>5<p>Scored as heterozygotes.</p

Shep negatively regulates <i>gypsy</i> activity in the CNS.

<p>(A) Confocal imaging of Shep distribution in stage 14 wildtype Oregon R embryo by indirect immunofluorescence using guinea pig α-Shep (green) and mouse α-Elav (red) antibodies detected by α-guinea pig Alexa-488 and α-mouse Alexa-594 secondary antibodies. DAPI staining (blue) is also shown in the merged image. A, anterior; P, posterior; D, dorsal; V, ventral. (B) Western blotting of anterior third instar larval extracts (lane 1), brains (lane 2), eye discs (lane 3), leg discs (lane 4), wing discs (lane 5), and salivary glands (lane 6) for Shep, Su(Hw), Mod(mdg4)2.2, Pep, and Lamin. (C) Epifluorescence imaging of insulator body localization by indirect immunofluorescence using rabbit α-CP190 and α-rabbit Alexa-594 in whole mount brain, leg imaginal disc, or eye imaginal disc tissues in wild type; <i>mod(mdg4)^u1</i>; or <i>shep^BG00836</i>, <i>mod(mdg4)^u1</i> larvae. White dotted lines outline one example nucleus in each image. (D) Western blotting of larval extracts for Shep, Su(Hw), CP190, Mod(mdg4)2.2 and Pep in wildtype (lane 1), non-insulated (lanes 2–5), and insulated (lanes 6–9) luciferase lines. <i>Act5c</i>::Gal4 was used to drive single copy UAS-<i>su(Hw)</i> dsRNA (lanes 3 and 7), UAS-<i>shep</i> dsRNA (lanes 4 and 8) or Shep overexpression (UAS-<i>shep</i>, lanes 5 and 9). (E–G) Relative luciferase units were quantified in individual larvae expressing <i>Act5C</i>::Gal4 (E), <i>l(3)31-1</i>::Gal4 (F) <i>Mef2</i>::Gal4 (G), dsRNA hairpin, and/or UAS-<i>shep</i> as indicated. Luciferase values across the population are plotted as box and whisker plots where boxes represent upper and lower quartiles proximal to the median, and whiskers represent the range excluding outliers. Populations were compared by 1-way ANOVA, and pairwise <i>p</i> values were calculated by Tukey HSD <i>post hoc</i> tests. Outliers falling outside a normal distribution are shown (dots) but were not used to calculate <i>p</i> values. For each genotype, <i>n</i>≥12 larvae. For (F), non-insulated control vs. non-insulated <i>shep</i> RNAi, <i>p</i> = 0.18; for (G), insulated control vs. insulated <i>shep</i> RNAi, <i>p</i> = 0.99.</p

Comparison of Su(Hw), Mod(mdg4)2.2, and Shep ChIP–seq profiles in BG3 cells.

<p>(A) Screenshot of Su(Hw), Mod(mdg4)2.2, and Shep ChIP-seq signals at the <i>dnt</i> neuronal-expressed locus. The large gap in ChIP signal corresponds to a highly repetitive region to which sequence reads could not be aligned with high confidence. (B) Screenshot of the <i>caps</i> neuronal-expressed locus. (C) Classification of Su(Hw), Mod(mdg4)2.2, and Shep ChIP-seq peaks in BG3 cells. Number of sites and percentage of total in parentheses corresponding to TSS, transcription start site; CDS, coding sequence; 5′ UTR, 5′ untranslated region; 3′ UTR, 3′ untranslated region. See methods for classification hierarchy of overlapping categories. (D) Heat map of log₂ enrichment scores for pairwise comparisons of binding sites for Su(Hw), Mod(mdg4)2.2, Shep, and additional data sets. Color scale corresponding to enrichment value is indicated (right). Positive values indicate significant enrichment while negative values indicate significant negative correlation of enrichment. Self-self comparisons are indicated in grey, and pairwise comparisons that are not statistically significant (p>0.001) are indicated in white. Numbers along top of each column indicate the total number of features in each data set, and the number of sites overlapping with Shep are indicated in parentheses. Data from Richter (2011) were derived from larval brains and imaginal discs; all other datasets are derived from BG3 cells. Data from modENCODE are indicated by an asterisk. Full heat map with hierarchical clustering is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003069#pgen.1003069.s004" target="_blank">Figure S4</a>. (E) Binary heat map of Su(Hw), Mod(mdg4)2.2, and Shep binding sites in BG3 cells ordered by supervised hierarchical clustering. Each row represents a single genomic location, and a mark in a column represents the presence of a particular factor.</p

Shep associates directly with <i>gypsy</i> insulator complexes.

<p>(A) Diagram of Shep protein isoforms. RRMs (blue) and alternative amino acid stretches (not to scale, orange) are shown. Regions of Shep utilized for antibody production or contained in the yeast two-hybrid clone, which corresponds to exons present in isoform E, are indicated. (B) Coomassie staining of recombinant GST fusion proteins used for binding reactions in (C). Protein marker is run in lane 1. (C) Interaction of purified, soluble His-Mod(mdg4)2.2 (lane 1, 4.5% input) with immobilized GST (lane 2), GST-Su(Hw) (lane 3) or GST-Shep isoforms (lanes 4–6). Binding of His-Mod(mdg4)2.2 to GST-fusion proteins was detected by Western blotting. (D) Coomassie staining of recombinant GST fusion proteins used for binding reactions in (E). (E) Interaction of purified, soluble His-Su(Hw) (lane 1, 6.3% input) with immobilized GST (lane 2), GST-Mod(mdg4)2.2 (lane 3) or GST-Shep isoforms (lanes 4–6). Binding of His-Su(Hw) to GST-fusion proteins was detected by Western blotting.</p

Loss-of-function <i>shep</i> alleles disrupt <i>gypsy</i> insulator activity at <i>ct⁶</i>.

<p>(A) Effects of <i>shep</i> mutations on the <i>ct⁶</i> phenotype. All flies are homozygous for <i>mod(mdg4)^u1</i>. At the <i>shep</i> locus, flies are wildtype (<i>shep⁺</i>), harbor a heterozygous deficiency, or contain a homozygous P-element insertion as indicated. Percent of population scored on a scale of 0–4 is indicated for each genotype. 0, no notching; 1, slight notching in one wing; 2, slight notching in both wings; 3, pronounced notching in hinge distal wing margin; 4, severe notching in both hinge proximal and distal margins. Asterisks denote P-element insertions showing extensive synthetic lethal interaction with <i>mod(mdg4)^u1</i> for which rare escapers were scored (49≤<i>n</i>≤180 for all genotypes). (B) Hemizygous alleles of <i>shep</i> affect <i>ct⁶</i>. Phenotypes of <i>ct⁶</i> of <i>shep^BG00836</i> and <i>shep^d05714</i> mutations transheterozygous with <i>Df(3L)Exel6104</i>. All flies are homozygous for <i>mod(mdg4)^u1</i>. Flies were scored in parallel with those in (A) (85≤<i>n</i>≤180). (C) Male abdominal pigmentation due to <i>y²</i> expression is unchanged in <i>mod(mdg4)^u1</i> compared to <i>shep^BG00836</i>, <i>mod(mdg4)^u1</i> flies.</p

RNAi-independent role for Argonaute2 in CTCF/CP190 chromatin insulator function

Lei and colleagues explore the role of AGO2 in chromatin organization in Drosophila. AGO2 is found to localize to euchromatin, where it is required for CTCF/CP190-dependent Fab-8 insulator function in a manner independent of its catalytic activity and of Dicer-2. The work points to an RNAi-independent role for AGO2 in defining transcriptional domains through chromosomal looping