Tethering of CHROMATOR and dCTCF proteins results in decompaction of condensed bands in the <i>Drosophila melanogaster</i> polytene chromosomes but does not affect their transcription and replication timing

<div><p>Instulator proteins are central to domain organization and gene regulation in the genome. We used ectopic tethering of CHROMATOR (CHRIZ/CHRO) and dCTCF to pre-defined regions of the genome to dissect the influence of these proteins on local chromatin organization, to analyze their interaction with other key chromatin proteins and to evaluate the effects on transcription and replication. Specifically, using UAS-GAL4DBD system, CHRO and dCTCF were artificially recruited into highly compacted polytene chromosome bands that share the features of silent chromatin type known as intercalary heterochromatin (IH). This led to local chromatin decondensation, formation of novel DHSes and recruitment of several “open chromatin” proteins. CHRO tethering resulted in the recruitment of CP190 and Z4 (PZG), whereas dCTCF tethering attracted CHRO, CP190, and Z4. Importantly, formation of a local stretch of open chromatin did not result in the reactivation of silent marker genes <i>yellow</i> and <i>mini</i>-<i>white</i> immediately adjacent to the targeting region (UAS), nor did RNA polII become recruited into this chromatin. The decompacted region retained late replicated, similarly to the wild-type untargeted region.</p></div

Summary of protein immunodetection data in the chromosomes from CHRO^GAL4DBD- and dCTCF^GAL4DBD-expressing animals.

<p>Summary of protein immunodetection data in the chromosomes from CHRO^GAL4DBD- and dCTCF^GAL4DBD-expressing animals.</p

11A6-9 band splits upon tethering of CHRO^GAL4DBD (A-D) and dCTCF^GAL4DBD (E,F) into the regions of EY01976 (A,B) and EY00353 (C-F) insertions.

<p>Phase contrast (left column). Overlay of phase contrast and immunostaining (right column). Thin arrow indicates EY01976 insertion (A—control, B—CHRO^GAL4DBD expression and splitting of the band 11A6-9 in its distal part), thick arrow indicates EY00353 insertion in the middle of the band (C,E—control; D,F—tethering of CHRO^GAL4DBD and dCTCF^GAL4DBD, respectively).</p

11A6-9 band splits upon tethering of CHRO^GAL4DBD (A-D) and dCTCF^GAL4DBD (E,F) into the regions of EY01976 (A,B) and EY00353 (C-F) insertions.

<p>Phase contrast (left column). Overlay of phase contrast and immunostaining (right column). Thin arrow indicates EY01976 insertion (A—control, B—CHRO^GAL4DBD expression and splitting of the band 11A6-9 in its distal part), thick arrow indicates EY00353 insertion in the middle of the band (C,E—control; D,F—tethering of CHRO^GAL4DBD and dCTCF^GAL4DBD, respectively).</p

Overlap between 4HMM fragments and chromatin states and types.

<p>5 principal chromatin colors reported in Filion et al., [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157147#pone.0157147.ref004" target="_blank">4</a>] (A); 9 chromatin states by Kharchenko et al., [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157147#pone.0157147.ref008" target="_blank">8</a>], S2 cells (B), BG3 cells (C); and 3 chromatin compactization classes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157147#pone.0157147.ref010" target="_blank">10</a>] (D).</p

Enhancers and protein distributions across different 4HMM chromatin types.

<p>(A) Ratio of the observed fraction of overlapping fragments to the expected one. Observed fraction means the ratio of the total length of genomic regions associated with a protein of interest to the total length of the chromatin type in IH. Expected fraction is the fraction of overlap expected by chance (under random distribution model). Only the values above the “expected” threshold are shown. Asterisks denote probabilities of occurrence by chance *–p<0.05; **–p<1E^-3; ***–p<1E^-25. (B) Probability values that the observed overlap happened by chance. Bar height (-log₁₀[P]) shows the significance levels for the enrichment of a chromatin type with regulatory elements or proteins indicated on the X axis. The probabilities were computed by the permutation Monte-Carlo test, as described in Materials and Methods section. Enhancers (1) and (2) are taken from RedFly [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157147#pone.0157147.ref036" target="_blank">36</a>] and Kvon et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157147#pone.0157147.ref029" target="_blank">29</a>], respectively.</p

Ectopic tethering of CHRO^GAL4DBD does not lead to transcription of the decompacted chromatin.

<p>(A)–anti-MYC signal (UAS) and anti-RNA PolII Ser5 signal do not co-localize in the decompacted interband-like regions formed in the bands 10A1-2 and 11A6-9. Upper row: phase contrast of the split-band morphology, bottom row: immunostaining signals for RNA PolII Ser5 (green) and MYC (red). Positions of UASes are denoted by arrows; (B)–wing bristle pigmentation in <i>Oregon</i> R (dark), EY00353; DBDGAL4 (brown) and EY00353; CHRO^GAL4DBD (brown) flies indicates that the reporter <i>yellow</i>⁺ gene present in EY elements is not induced upon CHRO^GAL4DBD tethering.</p

59D1-4 band splits in the heterozygotes for EY13417 insertion upon CHRO^GAL4DBD tethering.

<p>Control EY13417/+; GAL4DBD chromosomes (A). Tethering of CHRO^GAL4DBD to one UAS-bearing homolog manifests as a partial splitting of the 59D1-4 band in its central part (B).</p

Immunodetection of insulator proteins in the decompacted regions formed within the bands 10A1-2 (A, B) and 11A6-9 (C–F).

<p>Left column: 10A1-2 and 11A6-9 region of the X chromosome (phase contrast), right column: overlay of phase contrast and immunostaining data. Arrows indicate the position of decompaction zone within 10A1-2 (thin arrows) and 11A6-9 (thick arrows) bands.</p

Replication timing gradient in the IH region 47A1-2.

<p>Comparison of positions of 4HMM-derived chromatin types with replication timing. Data on the distribution of ORC2 protein and replication timing were taken from the figures published in Belyaeva et al., [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157147#pone.0157147.ref023" target="_blank">23</a>].</p