13 research outputs found

    Targets of Heterochromatin Assembly in Drosophila melanogaster

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    Heterochromatin is classically defined as densely staining regions of the genome; these domains are typically late replicating and show little recombination. Correct assembly of heterochromatin is critical for chromosome stability. Assembly begins with histone deacetylation and H3 lysine 9 di- and trimethylation: H3K9me2/3); the methylated H3 is typically bound by Heterochromatin Protein 1a: HP1a). Heterochromatin predominates at pericentric and telomeric domains --regions abundant in transposable elements: TEs) and satellite repeats. Transcription of these TEs has been found to generate a platform for assembly of heterochromatin through RNAi in S. pombe and A. thaliana, and may play a critical role in Drosophila melanogaster. However, the precise role of RNAi in heterochromatin assembly for a metazoan system such as flies remains unclear. However, 1360, a DNA transposable element in D. melanogaster, has been found to be sufficient to promote heterochromatin assembly in a repeat-rich region, as shown by a variegating phenotype of a hsp70-white reporter. RNAi components and heterochromatin factors such as HP1a were both implicated in this 1360-sensitive variegation, a form of position effect variegation: PEV). Here, I sought to determine the extent and mechanism of TE-sensitive PEV. A collection of 1360-sensitive landing pad insertion lines containing the hsp70-w reporter was generated. This tool allows for the repeated sampling of altered 1360 constructs in a variety of chromatin contexts, a useful a platform to study the attributes of 1360-sensitive variegation as well as PEV generally. We found 1360-sensitive PEV to extend to sites outside of annotated heterochromatin, although most sensitive sites lie within or proximal to heterochromatic masses. I used biochemical approaches to show that 1360-sensitive PEV corresponds to HP1a accumulation over the hsp70-w promoter region, confirming that the silencing is due to heterochromatin assembly. The deletion of sites within the 1360 element with homology to the PIWI-interacting RNAs: piRNAs) in 1360 suppressed PEV, as did dominant mutations in PIWI domain proteins. Similar results were obtained using Invader4, a retrotransposon, in the same landing pad site. The results support a mechanism that uses piRNAs for transposon-sensitive HP1a-silencing, likely early in development, with persistent effects observed in the adult somatic tissue of the eye. To determine if the sequence determinants required for 1360-sensitive silencing in a euchromatic region: as seen above) also operate in a repetitious sequence environment, where interspersed signals may operate cooperatively, I investigated a 1360-sensitive site in the piRNA generating locus 42AB. We find that mutations in piwi, along with many prototypical Su(var) mutations, result in weak suppression of variegation at this site, while an ago2 mutation enhances variegation. Tests of various fragments of the TEs do not reveal a strong dependency on piRNA matching sequences, contrary to the euchromatic site driven to a heterochromatic form by the added TE. These findings indicate that suppression of PEV by mutations in the genes for RNAi components occurs in a limited number of heterochromatic domains, predominantly those near gene clusters - sites typically found at the border between euchromatin and heterochromatin. Thus chromosomal context appears to be an important determinant for RNAi-dependent 1360-sensitive PEV. This finding helps to reconcile reports of inconsistent PEV effects from mutations in RNAi components that have been carried out using reporters in different domains. Collectively, these results indicate the TEs can act as sequence determinants of heterochromatin assembly at a subset of genomic sites using an RNAi-mediated targeting mechanism

    Ectopic assembly of heterochromatin in Drosophila melanogaster triggered by transposable elements

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    A persistent question in biology is how cis-acting sequence elements influence trans-acting factors and the local chromatin environment to modulate gene expression. We reported previously that the DNA transposon 1360 can enhance silencing of a reporter in a heterochromatic domain of Drosophila melanogaster. We have now generated a collection of variegating phiC31 landing-pad insertion lines containing 1360 and a heat-shock protein 70 (hsp70)-driven white reporter to explore the mechanism of 1360-sensitive silencing. Many 1360-sensitive sites were identified, some in apparently euchromatic domains, although all are close to heterochromatic masses. One such site (line 1198; insertion near the base of chromosome arm 2L) has been investigated in detail. ChIP analysis shows 1360-dependent Heterochromatin Protein 1a (HP1a) accumulation at this otherwise euchromatic site. The phiC31 landing pad system allows different 1360 constructs to be swapped with the full-length element at the same genomic site to identify the sequences that mediate 1360-sensitive silencing. Short deletions over sites with homology to PIWI-interacting RNAs (piRNAs) are sufficient to compromise 1360-sensitive silencing. Similar results were obtained on replacing 1360 with Invader4 (a retrotransposon), suggesting that this phenomenon likely applies to a broader set of transposable elements. Our results suggest a model in which piRNA sequence elements behave as cis-acting targets for heterochromatin assembly, likely in the early embryo, where piRNA pathway components are abundant, with the heterochromatic state subsequently propagated by chromatin modifiers present in somatic tissue

    Targeting heterochromatin formation to transposable elements in Drosophila: potential roles of the piRNA system

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    Successful heterochromatin formation is critical for genome stability in eukaryotes, both to maintain structures needed for mitosis and meiosis and to silence potentially harmful transposable elements. Conversely, inappropriate heterochromatin assembly can lead to inappropriate silencing and other deleterious effects. Hence targeting heterochromatin assembly to appropriate regions of the genome is of utmost importance. Here we focus on heterochromatin assembly in Drosophila melanogaster, the model organism in which variegation, or cell-to-cell variable gene expression resulting from heterochromatin formation, was first described. In particular, we review the potential role of transposable elements as genetic determinants of the chromatin state and examine how small RNA pathways may participate in the process of targeted heterochromatin formation

    A distinct type of heterochromatin at the telomeric region of the Drosophila melanogaster Y chromosome

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    Heterochromatin assembly and its associated phenotype, position effect variegation (PEV), provide an informative system to study chromatin structure and genome packaging. In the fruit fly Drosophila melanogaster, the Y chromosome is entirely heterochromatic in all cell types except the male germline; as such, Y chromosome dosage is a potent modifier of PEV. However, neither Y heterochromatin composition, nor its assembly, has been carefully studied. Here, we report the mapping and characterization of eight reporter lines that show male-specific PEV. In all eight cases, the reporter insertion sites lie in the telomeric transposon array (HeT-A and TART-B2 homologous repeats) of the Y chromosome short arm (Ys). Investigations of the impact on the PEV phenotype of mutations in known heterochromatin proteins (i.e., modifiers of PEV) show that this Ys telomeric region is a unique heterochromatin domain: it displays sensitivity to mutations in HP1a, EGG and SU(VAR)3-9, but no sensitivity to Su(z)2 mutations. It appears that the endo-siRNA pathway plays a major targeting role for this domain. Interestingly, an ectopic copy of 1360 is sufficient to induce a piRNA targeting mechanism to further enhance silencing of a reporter cytologically localized to the Ys telomere. These results demonstrate the diversity of heterochromatin domains, and the corresponding variation in potential targeting mechanisms

    The histone deacetylase complex MiDAC regulates a neurodevelopmental gene expression program to control neurite outgrowth

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    The mitotic deacetylase complex (MiDAC) is a recently identified histone deacetylase (HDAC) complex. While other HDAC complexes have been implicated in neurogenesis, the physiological role of MiDAC remains unknown. Here, we show that MiDAC constitutes an important regulator of neural differentiation. We demonstrate that MiDAC functions as a modulator of a neurodevelopmental gene expression program and binds to important regulators of neurite outgrowth. MiDAC upregulates gene expression of pro-neural genes such as those encoding the secreted ligands SLIT3 and NETRIN1 (NTN1) by a mechanism suggestive of H4K20ac removal on promoters and enhancers. Conversely, MiDAC inhibits gene expression by reducing H3K27ac on promoter-proximal and -distal elements of negative regulators of neurogenesis. Furthermore, loss of MiDAC results in neurite outgrowth defects that can be rescued by supplementation with SLIT3 and/or NTN1. These findings indicate a crucial role for MiDAC in regulating the ligands of the SLIT3 and NTN1 signaling axes to ensure the proper integrity of neurite development

    A Survey of Validation Strategies for CRISPR-Cas9 Editing

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    Abstract The T7 endonuclease 1 (T7E1) mismatch detection assay is a widely used method for evaluating the activity of site-specific nucleases, such as the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system. To determine the accuracy and sensitivity of this assay, we compared the editing estimates derived by the T7E1 assay with that of targeted next-generation sequencing (NGS) in pools of edited mammalian cells. Here, we report that estimates of nuclease activity determined by T7E1 most often do not accurately reflect the activity observed in edited cells. Editing efficiencies of CRISPR-Cas9 complexes with similar activity by T7E1 can prove dramatically different by NGS. Additionally, we compared editing efficiencies predicted by the Tracking of Indels by Decomposition (TIDE) assay and the Indel Detection by Amplicon Analysis (IDAA) assay to that observed by targeted NGS for both cellular pools and single-cell derived clones. We show that targeted NGS, TIDE, and IDAA assays predict similar editing efficiencies for pools of cells but that TIDE and IDAA can miscall alleles in edited clones

    A Distinct Type of Heterochromatin at the Telomeric Region of the <i>Drosophila melanogaster</i> Y Chromosome

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    <div><p>Heterochromatin assembly and its associated phenotype, position effect variegation (PEV), provide an informative system to study chromatin structure and genome packaging. In the fruit fly <i>Drosophila melanogaster</i>, the Y chromosome is entirely heterochromatic in all cell types except the male germline; as such, Y chromosome dosage is a potent modifier of PEV. However, neither Y heterochromatin composition, nor its assembly, has been carefully studied. Here, we report the mapping and characterization of eight reporter lines that show male-specific PEV. In all eight cases, the reporter insertion sites lie in the telomeric transposon array (<i>HeT-A</i> and <i>TART-B2</i> homologous repeats) of the Y chromosome short arm (Ys). Investigations of the impact on the PEV phenotype of mutations in known heterochromatin proteins (i.e., modifiers of PEV) show that this Ys telomeric region is a unique heterochromatin domain: it displays sensitivity to mutations in HP1a, EGG and SU(VAR)3-9, but no sensitivity to <i>Su(z)2</i> mutations. It appears that the endo-siRNA pathway plays a major targeting role for this domain. Interestingly, an ectopic copy of <i>1360</i> is sufficient to induce a piRNA targeting mechanism to further enhance silencing of a reporter cytologically localized to the Ys telomere. These results demonstrate the diversity of heterochromatin domains, and the corresponding variation in potential targeting mechanisms.</p></div

    Impacts of mutations in components of the small RNA pathways on the level of variegation of the Y-linked reporters.

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    <p>(a) Pigment level quantification indicating the extent of the suppression of PEV. (Bars represent the average pigment level ± standard error. Asterisks are used to indicate mutant alleles that show statistical significant modifier activities, single, p<0.05; double, p<0.005.) Pathways requiring the genes tested are indicated below the allele names. (b) Representative pictures showing the dominant impacts on PEV in the fly eye from mutations disrupting small RNA pathways.</p

    Impact of mutations in HMTs and of dosage of the 4<sup>th</sup> chromosome on the level of variegation of the Y-linked reporters.

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    <p>(a) Pigment level quantification showing the level of expression. (Bars represent the average pigment level ± standard error. Asterisks are used to indicate mutant alleles that show statistical significant modifier activities, single, p<0.05; double, p<0.005.) 1X 4<sup>th</sup> and 3X 4<sup>th</sup> represent the copy number of the 4<sup>th</sup> chromosome in the assayed flies. Note that the control line <i>yw<sup>67C23</sup></i> has 2X 4<sup>th</sup> (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086451#pone.0086451.s001" target="_blank">Table S1</a> for information on the fly lines used.), the t test on 1X 4<sup>th</sup> examines E(var) activities of the allele (see materials and methods for details). (b) Representative pictures showing the dominant impacts on PEV in the fly eye from mutations disrupting HMT activities.</p

    Sequencing and <i>in situ</i> hybridization mapping locate the Y-linked PEV reporters in the telomeric transposon arrays of Ys.

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    <p>(a) Alignment of reporter insertion flanking sequences to a consensus sequence of telomeric retro-transposon <i>TART-B2</i>. The 1 Kb region in the 3′-UTR harboring the insertion sites is magnified. The effective sequence read length for each reporter line is represented by the red line aligned to the region. The red dot at the end of each red line indicates the 5′ end of the reporter. The reporter flanking sequence of line 5m298 starts in the 3′ end of a <i>TART-B2</i> element and extends upstream to a neighboring <i>HeT-A</i> element, suggesting insertion into a partial fragment of <i>TART</i>. (b) <i>In situ</i> hybridization images of the metaphase chromosomes from third instar larval brain squashes. Only the Y chromosome of the representative metaphase spread is shown. DAPI staining is pseudo-colored in blue and the hybridization signal is in red. (C: centromere, Y<sup>L</sup>: long arm, Y<sup>S</sup>: short arm).</p
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