The results of tests for EAST interaction with insulator proteins in S2 cells.

<p>(A) Co-immunoprecipitation of V5 epitope-tagged insulator proteins Su(Hw), Mod(mdg4)-67.2 (Mod-67.2), and CP190 by antibodies against the FLAG epitope fused to either EAST or its truncated forms EAST^1-1995 and EAST^933-2362. Immunoprecipitated complexes were washed several times with 150 mM KCl-containing buffers and resolved by SDS-PAGE for Western blot analysis with the indicated antibodies. InPut, input fraction (10% of lysate using for immunoprecipitation); OutPut, supernatant after immunoprecipitation; IP, immunoprecipitate. (B) Co-immunoprecipitation of FLAG-tagged EAST variants by antibodies against the V5 epitope fused to either of the insulator proteins. (C) Co-immunoprecipitation between the insulator proteins and the EAST variants fused in frame with FLAG. The S2 cells were transfected with different EAST truncated proteins fused in frame with FLAG. Immunoprecipitation was performed with antibodies raised to insulator proteins Su(Hw), Mod(mdg4)-67.2 (Mod-67.2), or CP190. The bottom panel (Ab in IP) shows the result of immunoprecipitation of target insulator proteins, which was performed in each assay. (D) Co-immunoprecipitation of Su(Hw), Mod(mdg4)-67.2, and CP190 by antibodies against the FLAG epitope fused to each of the EAST variants.</p

Comparison of the enhancer-blocking activity of the <i>gypsy</i> (Gy) and S^x4 insulators.

<p>(A) Effects of the <i>su(Hw)</i> and <i>mod(mdg4)</i> mutations on <i>yellow</i> and <i>white</i> expression in transgenic lines. In the scheme of the construct (drawn not to scale), the <i>yellow</i> wing (Ew) and body (Eb) enhancers are shown as shaded ovals; the eye enhancer (Ee) inserted between them, as a white oval; the <i>yellow</i> (Y) and <i>white</i> (W) genes, as arrows indicating the direction of transcription; and the <i>gypsy</i> and S^×4 insulators, as shaded triangles. Downward arrows indicate <i>loxP</i> target sites for the Cre recombinase; the same sites in the construct names are denoted by parentheses. The “<i>yellow</i>” column shows the numbers of transgenic lines with the <i>yellow</i> pigmentation level in the abdominal cuticle (reflecting the activity of the body enhancer); in most of the lines, the pigmentation levels in wing blades (reflecting the activity of the wing enhancer) closely correlated with these scores. The level of pigmentation (i.e., of <i>y</i> expression) was estimated on an arbitrary five-grade scale, with wild-type expression and the absence of expression assigned scores of 5 and 1, respectively. Wild-type <i>white</i> expression determined the bright red eye color (R); in the absence of <i>white</i> expression, the eyes were white (W). Intermediate levels of pigmentation, with the eye color ranging from pale yellow to yellow (Y) from dark yellow to orange (Or), from dark orange to brownish red (Br) reflect the increasing levels of <i>white</i> expression. In the N/T ratio, N is the number of lines in which flies acquired a new <i>y</i> phenotype upon deletion (Δ) of the specified DNA fragment, or on the mutant background, and T is the total number of lines examined for each particular construct. (B) ChIP-qPCR analysis of insulator proteins binding to the <i>gypsy</i> or S^×4 insulator in transgenic lines. The Eye(S^×4)YW lines included in the analysis are designated # 1 and # 2. The <i>ras64B</i> coding region (Ras) was used as a control devoid of Su(Hw) binding sites. The percent recovery of immunoprecipitated DNA (Y axis) was calculated relative to the amount of input DNA. Error bars indicate standard deviation of three independent biological replicates. The abbreviations of mutant backgrounds: <i>su(Hw)</i>⁺—<i>su(Hw)</i>^<i>v</i>/<i>TM6,Tb</i>; <i>su(Hw)</i>^‒—<i>su(Hw)</i>^<i>v</i>/<i>su(Hw)</i>^{<i>e04061</i>}; <i>su(Hw)</i>^<i>f</i>–<i>su(Hw)</i>^<i>v</i>/<i>su(Hw)</i>^<i>f</i>; <i>mod</i>^-–<i>mod(mdg4)</i>^<i>u1</i>/<i>mod(mdg4)</i>^<i>u1</i>.</p

Role of EAST and CP190 in the expression of genes whose promoters are bound to by the insulator proteins.

<p>(A) Changes in the expression of individual genes after CP190-specific RNAi knockdown in <i>Drosophila</i> S2 cells relative to that after GFP RNAi treatment (taken to be 1). The <i>RpL32</i> and <i>eh</i> genes that showed no change in expression after the loss of CP190 were used as control. Anti-Tubulin staining (Tub) was used as loading control. The experiments were performed on two samples involving independent RNAi, RNA preparations, and RNA reverse transcription into cDNA. (B) Changes in the expression of individual genes after overexpression of EAST^1-2362, EAST^1-1995, or EAST^933-2362. The experiments were performed on three samples involving independent transfections with a full-length EAST or its fragments, RNA preparations, and RNA reverse transcription into cDNA. Error bars indicate standard deviation of two or three independent biological replicates *<i>P</i> ≤ 0.05 (Student’s <i>t</i>-test), in other cases <i>P</i> ≤ 0.01.</p

Evaluation of binding of the insulator protein in the <i>mod(mdg4)</i>⁺ transgenic lines.

<p>Variants of the Su(Hw) protein were expressed in the <i>y</i>^<i>2</i><i>sc</i>^<i>D1</i><i>ct</i>^<i>6</i>; <i>P{Su(Hw)}-38D/P{Su(Hw)}-38D</i>; <i>su(Hw)</i>^<i>v</i><i>/su(Hw)</i>^{<i>e04061</i>} lines, where P{Su(Hw)} are Su(Hw)⁺-Act5C –<i>P{w</i>⁺;<i>WAB-Su(Hw)1-945-FLAG}/ P{w</i>⁺;<i>WAB-Su(Hw)1-945-FLAG}</i>; Su(Hw)^Δ10-Act5C –<i>P{w</i>⁺;<i>WAB-Su(Hw)</i>^<i>Δ10</i><i>-FLAG}/ P{w</i>⁺;<i>WAB-Su(Hw)</i>^<i>Δ10</i><i>-FLAG}</i>; Su(Hw)^f-Act5C –<i>P{w</i>⁺;<i>WAB-Su(Hw)</i>^<i>f</i> <i>-FLAG}/ P{w</i>⁺;<i>WAB-Su(Hw)</i>^<i>f</i> <i>-FLAG}</i>; Su(Hw)^f-Ubi–<i>P{w</i>⁺;<i>UbqW-Su(Hw)</i>^<i>f</i> <i>-FLAG}/ P{w</i>⁺;<i>UbqW-Su(Hw)</i>^<i>f</i> <i>-FLAG}</i>. The <i>y</i>^<i>2</i><i>sc</i>^<i>D1</i><i>ct</i>^<i>6</i>; <i>su(Hw)</i>^<i>v</i><i>/su(Hw)</i>^{<i>e04061</i>} line is designated as Su(Hw)⁻. Quantitative PCR (qPCR) was performed at the selected Su(Hw) regions. The <i>ras64B</i> coding region (Ras) was used as a negative control. The percent recovery of immunoprecipitated DNA (Y axis) was calculated relative to the amount of input DNA. Error bars indicate standard deviation of three independent biological replicates.</p

EAST Organizes <i>Drosophila</i> Insulator Proteins in the Interchromosomal Nuclear Compartment and Modulates CP190 Binding to Chromatin

<div><p>Recent data suggest that insulators organize chromatin architecture in the nucleus. The best studied <i>Drosophila</i> insulator proteins, dCTCF (a homolog of the vertebrate insulator protein CTCF) and Su(Hw), are DNA-binding zinc finger proteins. Different isoforms of the BTB-containing protein Mod(mdg4) interact with Su(Hw) and dCTCF. The CP190 protein is a cofactor for the dCTCF and Su(Hw) insulators. CP190 is required for the functional activity of insulator proteins and is involved in the aggregation of the insulator proteins into specific structures named nuclear speckles. Here, we have shown that the nuclear distribution of CP190 is dependent on the level of EAST protein, an essential component of the interchromatin compartment. EAST interacts with CP190 and Mod(mdg4)-67.2 proteins in <i>vitro</i> and in <i>vivo</i>. Over-expression of EAST in S2 cells leads to an extrusion of the CP190 from the insulator bodies containing Su(Hw), Mod(mdg4)-67.2, and dCTCF. In consistent with the role of the insulator bodies in assembly of protein complexes, EAST over-expression led to a striking decrease of the CP190 binding with the dCTCF and Su(Hw) dependent insulators and promoters. These results suggest that EAST is involved in the regulation of CP190 nuclear localization.</p></div

Comparison of the antisilencing activity of <i>gypsy</i> and S^×4 insulators.

<p>The 660-bp PRE is shown as a black oval; the bristle enhancer (Ebr), as a gray oval in the intron of the <i>yellow</i> gene. The “<i>yellow</i>” column shows the numbers of transgenic lines with the <i>yellow</i> pigmentation level in bristles. The degree of variegation in bristles of the thorax and head: 1, loss of pigmentation in all bristles at thorax and head; e-v, extreme variegation (only 1–3 bristles on thorax and head are pigmented); m-v, moderate variegation (about half of bristles are yellow); w-v, weak variegation (only 1–3 bristles on thorax and head are yellow); 5, pigmentation of all bristles as in wild-type flies. Other designations are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193497#pone.0193497.g005" target="_blank">Fig 5</a>.</p

Analysis of insulator proteins binding to chromatin after RNAi knockdown of EAST in S2 cells.

<p>ChIP was performed with antibodies against Su(Hw), specific isoform Mod(mdg4)-67.2 (Mod-67.2), and CP190 in normal S2 cells (+) and S2 cells after knockdown of EAST (RNAi EAST). Quantitative qPCR was performed on five Su(Hw)-depended insulator sites and the promoter regions of five genes bound by the Su(Hw) insulator complex. Primers were positioned in the middle of the binding region identified in ModEncode by ChIP-seq. Error bars indicate standard deviation of four independent biological replicates. *<i>P</i> ≤ 0.05 (Student’s <i>t</i>-test), in other cases <i>P</i> ≤ 0.01. Other designations are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140991#pone.0140991.g006" target="_blank">Fig 6</a>.</p

Scheme of Su(Hw) binding with a full consensus binding site, showing which ZFs are involved in recognition of specific cores (from Baxley et al. 2017) [57].

<p>Scheme of Su(Hw) binding with a full consensus binding site, showing which ZFs are involved in recognition of specific cores (from Baxley et al. 2017) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193497#pone.0193497.ref057" target="_blank">57</a>].</p

Role of Su(Hw) zinc finger 10 and interaction with CP190 and Mod(mdg4) proteins in recruiting the Su(Hw) complex to chromatin sites in <i>Drosophila</i>

<div><p>Su(Hw) belongs to the class of proteins that organize chromosome architecture and boundaries/insulators between regulatory domains. This protein contains a cluster of 12 zinc finger domains most of which are responsible for binding to three different modules in the consensus site. Su(Hw) forms a complex with CP190 and Mod(mdg4)-67.2 proteins that binds to well-known <i>Drosophila</i> insulators. To understand how Su(Hw) performs its activities and binds to specific sites in chromatin, we have examined the previously described <i>su(Hw)</i>^<i>f</i> mutation that disrupts the 10th zinc finger (ZF10) responsible for Su(Hw) binding to the upstream module. The results have shown that Su(Hw)^f loses the ability to interact with CP190 in the absence of DNA. In contrast, complete deletion of ZF10 does not prevent the interaction between Su(Hw)^Δ10 and CP190. Having studied insulator complex formation in different mutant backgrounds, we conclude that both association with CP190 and Mod(mdg4)-67.2 partners and proper organization of DNA binding site are essential for the efficient recruitment of the Su(Hw) complex to chromatin insulators.</p></div

Speckles formed at high and low salt concentrations.

<p>Immunofluorescent localization of insulator proteins in the nuclei of nontransfected S2 cells (control) and S2 cells overexpressing EAST^933-2362 tagged with FLAG×3 (EAST). Immunostaining with antibodies to Su(Hw) (green), CP190 (red), common part of Mod(mdg4) (Mod-Com, blue), and FLAG×3 (blue). Cells were stained in standard SFX medium (isotonic conditions), the same medium diluted fourfold with deionized water (hypotonic conditions), or after treatment with 250 mM NaCl (hypertonic conditions). Scale bars, 5 μm.</p