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

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

A model explaining differences in the Su(Hw) complex recruitment to the consensus sites and to sites in the <i>gypsy</i> insulator.

<p>(A) The Su(Hw) complex is recruited to consensus site(s) consisting of three modules. Most of ZFs are involved in stable interaction with the consensus site. The interactions of C190 and Mod(mdg4) with neighboring proteins (dotted arrows) are not critical for recruiting the insulator complex. (B) The insulator complex carrying Su(Hw) with mutated ZF10 is recruited to the sites in the <i>gypsy</i> insulator. The binding of the Su(Hw) complex to DNA is unstable and strongly depends on CP190 and Mod(mdg4)-67.2 (thick arrows) that may stabilize interaction with surrounding proteins. (C) The insulator complex with the Su(Hw)⁺ protein is recruited to the <i>gypsy</i> sites. ZFs 1–4 are not involved in binding to these sites and may recruit unknown proteins/complex (un-p) that improve the enhancer-blocking activity of the Su(Hw) complex in the absence of the Mod(mdg4)-67.2 protein.</p

Role of ZF10 in Su(Hw) for interaction between the insulator proteins.

<p>A structural schematic of Su(Hw). The borders between the Su(Hw) domains (NTAD, N-terminal acidic domain; CTAD, C-terminal acidic domain; ZF, zinc-finger domain) are indicated by numbers. Gray pentagon indicates H-to-Y substitution in the Su(Hw)^f mutant; dotted line, deletion of ZF10 in the Su(Hw)^Δ10 mutant. The number of plus signs indicates the relative strength of interaction in the Y2H assay (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193497#pone.0193497.s003" target="_blank">S1 Fig</a>); the minus sign denoted an absence of interactions; an asterisk, a reduction in the Y2H signal due to the repressive effect of Su(Hw) C-terminal domain on the transcription in yeast [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193497#pone.0193497.ref072" target="_blank">72</a>]. All the results were reproduced in three independent experiments. Numbers in brackets refer to the amino acid residues that flank protein regions included in the analysis. (B) Co-immunoprecipitation between different Su(Hw) variants fused to the FLAG epitope and the insulator proteins under normal conditions. The FLAG-Su(Hw)⁺, FLAG-Su(Hw)^f, and FLAG-Su(Hw)^Δ10 were expressed in the S2 cells. The immunoprecipitated complexes were washed with buffers containing 150 mM NaCl before loading onto the SDS-PAGE for Western blot analysis. The PVDF membrane was consecutively probed with antibodies against the indicated proteins (CP190 or Mod-67.2) or FLAG epitope. Each column represents a single FLAG-immunoprecipitation experiment with the particular mutant variant. Each lane shows the result of a subsequent hybridization of each immunoprecipitate with different antibodies on the same membrane. "Input" is the input fraction (10% of the lysate used for the immunoprecipitation); "Output IP," the supernatant after the immunoprecipitation; "IP," the immunoprecipitate. The results were obtained in three independent experiments.</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

Effects of the su(Hw) mutations on the activity of <i>gypsy</i> insulator in the <i>mod(mdg4)</i>^<i>u1</i> background.

<p>(A) Effects of the <i>mod(mdg4)</i>^<i>u1</i> mutation on <i>yellow</i> expression in transgenic lines. A schematic of the <i>y</i>^<i>2</i> allele (drawn not to scale): the <i>yellow</i> wing (Ew) and body (Eb) enhancers are shown as partially overlapping gray ovals; the bristle enhancer (Ebr), as a gray oval in the <i>yellow</i> intron; the transcription start site is indicated by an arrowhead. The <i>gypsy</i> insertion is shown as a triangle with the black circle (Gy) marking Su(Hw) binding sites. Analysis of the transgenic lines was performed in the <i>y</i>^<i>2</i><i>sc</i>^<i>D1</i><i>ct</i>^<i>6</i>; <i>mod(mdg4)</i>⁺<i>/mod(mdg4)</i>⁺ background (mod⁺) or in the <i>y</i>^<i>2</i><i>sc</i>^<i>D1</i><i>ct</i>^<i>6</i>; <i>mod(mdg4)</i>^<i>u1</i><i>/mod(mdg4)</i>^<i>u1</i> background (mod⁻). Numbers in the "mod⁺" and "mod^Ȣ" lines indicate a relative level of <i>yellow</i> expression in the body cuticle/wing blades/bristles, which ranged from 2 (pigmentation as in the <i>y</i>^<i>2</i> allele) to 5 (pigmentation as in the wild-type flies). (B) ChIP-qPCR analysis of Su(Hw), Mod-67.2, and CP190 binding at the mid-pupal stage in the transgenic lines expressing different variants of Su(Hw) 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>mod(mdg4)</i>^<i>u1</i><i>/ su(Hw)</i>^{<i>e04061</i>} <i>mod(mdg4)</i>^<i>u1</i> lines (mod⁻ in designations of the lines). The abbreviations of transgenes P{Su(Hw)} are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193497#pone.0193497.g003" target="_blank">Fig 3</a>. Quantitative PCR (qPCR) was performed on the intergenic regions bound by Su(Hw). PCR products were amplified from two separate immunoprecipitates of three different chromatin preparations. The <i>ras64B</i> coding region (Ras) was used as a control devoid of Su(Hw) binding sites. The recovery percentage 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

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

Supplementary materials: Figs S1 - S6, methods and supplementary table from Multiple interactions are involved in a highly specific association of the Mod(mdg4)-67.2 isoform with the Su(Hw) sites in <i>Drosophila</i>

This file contains Supplementary Figures, material and methods, and primer sequences used in PCR for ChIP analysis

Testing (A) <i>aurora</i> or (B) <i>CG3281</i> promoter in the scs' insulator.

<p>Asterisks indicate that the transgene was codirectionally inserted (*) in the first intron of the <i>effet</i> gene (3R:10565091) transcribed in the same direction as the <i>mini-white</i> gene or (**) in the first intron of the <i>Dek</i> gene (2R:12744143). Other designations are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062690#pone-0062690-g001" target="_blank">Figures 1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062690#pone-0062690-g002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062690#pone-0062690-g005" target="_blank">5</a>.</p