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

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

Replication timing in the band 10A1-2.

<p>Control 10A-UAS; GAL4DBD chromosomes (A,B), 10A-UAS; CHRO^GAL4DBD (C,D), CHRO (green), PCNA (red). Arrow indicates the decondensation site.</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

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

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

Molecular maps of the genomic regions surrounding the insertions of UAS-bearing transgenes at 10A1-2 (A-C), 11A6-9 (D-F), and 59D1-4 (G-I).

<p>A, D, G—organization of 10A-UAS, EY01976, EY00353, and EY13417 transposons and adjacent genes. EcoRV and SacI are the restriction sites used for mapping DHSes in 10A1-2 and 11A6-9 regions, respectively. B, E, H—span of the bands 10A1-2, 11A6-9, 59D1-4 and positions of UAS insertions. C, F, I—schematic illustration of how the bands are expected to split in two fragments separated by an interband (thick and thin arrows) as a result of local CHRO or dCTCF tethering. Red block denotes a fragment of CG15208 gene used for FISH analysis (C).</p

10A1-2 band splits upon CHRO^GAL4DBD tethering.

<p>Phase contrast (left column). Overlay of phase contrast and immunostaining (A, C) or FISH (B, D) signals (right column). Black and white arrows point to the tethering region, red arrow indicates the position of <i>CG15208</i> on the edge of band in control chromosomes (B) or in the distal fragment that has split from 10A1-2 upon tethering (D). CHRO (green), MYC (red), FISH signal (red).</p