SU(VAR)3–9 and JIL-1 interact <i>in vivo</i>.

<p>(<b>A</b>) Coexpression of JIL-1 and SU(VAR)3–9 in SF9 cells using a baculoviral expression system. <i>left</i>: Western Blot of whole cell Sf9 extract. Proteins were detected using the indicated antibodies. <i>right:</i> Histone methyltransferase assay after co-infection of SF9 cells with flag-JIL-1 and his-SU(VAR)3–9 followed by affinity purification on a Talon™ metal affinity resin. (<b>B</b>) Precipitation of JIL-1 from an SF9 cell extract co-purifies active SU(VAR)3–9. <i>left</i>: Western Blot analysis of flag-coimmunoprecipitations using SF9 whole cell extract after co-infection with the indicated viruses. After the Co-IP flag M2-beads were washed and eluted with the flag peptide. 10% of the eluates were separated by SDS PAGE and analyzed by Western blotting using specific antibodies. <i>right:</i> Histone methyltransferase assay after co-infection of SF9 cells with flag-JIL-1, a flag-JIL^D392A and his-SU(VAR)3–9 followed by affinity purification on a flag M2 affinity resin.</p

SU(VAR)3–9 can repress transcription when tethered to a promoter.

<p>(<b>A</b>) SU(VAR)3–9 represses transcription in a TSA-dependent manner. (<b>B</b>) An N-terminal truncation of SU(VAR)3–9 lacking the domain that interacts with JIL-1 and RPD3 can no longer repress transcription. (<b>C</b>) SU(VAR)3–9's transcriptional repression capacity is 2 fold reduced in a S191A mutated SU(VAR)3–9. <i>Drosophila</i> SL2 cells were co-transfected with expression constructs for dorsal and twist and the reporter construct pG₅DE₅-tkluc together with the indicated plasmids coding for: GAL4-DBD or GAL4 fusion proteins of SU(VAR)3–9 (and deletions/mutations thereof) and RPD3 in the presence or absence of the histone deacetylase inhibitor trichostatin A (TSA). <i>left:</i> luciferase assay of the activated reporter gene after transfection with the indicated plasmids. The repression capacity of GAL4-SU(VAR)3–9 was set to 100% and all other values were normalized accordingly. <i>right</i>: Western Blot analysis of the extracts used for the luciferase assay using an antibody specific against the GAL4-DBD [G: GAL4 DBD; R: GAL4-Rpd3; S: GAL4- SU(VAR)3–9].</p

Transcript levels in the <i>Digitor</i> null mutant relative to wild type.

<p>Relative transcript levels of <i>Digitor/dASCIZ</i>, <i>Cut up</i>, <i>CG32280</i>, and <i>Skeletor</i> in <i>Digitor</i> mutant (<i>EP(3)3709/EP(3)3709</i>) third instar larvae as compared to wild type. The transcript levels of all four genes were determined by qRT-PCR and fold change was calculated according to the 2-ΔΔCt method. Each determination was performed in triplicate and the average is shown with S.D.</p

Confocal time-lapse analysis of 3xHA-Digitor-mCitrine during mitosis in a syncytial <i>Drosophila</i> embryo.

<p>The figure shows the relative dynamics of the 3xHA-Digitor-mCitrine construct (in green) and tubulin-mCherry (in red) at interphase and metaphase, respectively. Arrows indicate the localization of the 3xHA-Digitor-mCitrine construct in the spindle region after NEB and microtubule spindle formation at metaphase.</p

Transgenic expression of 3xHA-Digitor-mCitrine in salivary gland cells.

<p>(A) Diagram of 3xHA-Digitor-mCitrine. (B) Polytene squash preparation labeled with anti-HA antibody to detect the 3xHA-Digitor-mCitrine construct (in green) and with Hoechst to label DNA (in blue/grey). In the composite image (comp) examples of developmental puff regions with higher expression of 3xHA-Digitor-mCitrine are indicated by arrowheads. (C) Polytene squash preparation labeled with anti-HA antibody to detect the 3xHA-Digitor-mCitrine construct (in green) and with anti-Skeletor antibody (in red). In the composite image (comp) co-localization between 3xHA-Digitor-mCitrine and Skeletor is indicated by yellow/orange colors. Examples of developmental puff regions positive for 3xHA-Digitor-mCitrine but not for Skeletor are indicated by arrowheads. The relatively low exposure of anti-HA antibody-labeling of 3xHA-Digitor-mCitrine in (B) shows the prominent labeling of the developmental puffs whereas the longer exposure in (C) highlights the labeling of the weaker labeled interband regions.</p

<i>Digitor</i> mutant third instar larvae have a severely reduced brain size.

<p>(A) Images of dissected brains from wild type (WT) and <i>Digitor</i> mutant (<i>EP(3)3709/EP(3)3709</i>) third instar larvae at the same scale. (B) Comparison of the average area of the two optic lobes from 20 brains of wild type and <i>Digitor</i> mutant larvae with S.D., respectively. The difference in size was statistically significant (P value < 0.001, Student's two-tailed t-test). (C) Images of wild type and <i>Digitor</i> mutant third instar larvae side by side. (D) Comparison of the average from four determinations of the body weight of 22 wild type and <i>Digitor</i> mutant larvae with S.D., respectively. The difference in weight was not statistically significant (P value ≥ 0.8, Student's two-tailed t-test). (E) Acridine Orange labeling of brains from wild type and <i>Digitor</i> mutant larvae. (F) TUNEL labeling of brains from wild type and <i>Digitor</i> mutant larvae. (G) BrdU incorporation into brains from wild type and <i>Digitor</i> mutant larvae. (H) Anti-H3S10ph antibody labeling of brains from wild type and <i>Digitor</i> mutant larvae. (I) Comparison of the average fluorescence intensity of BrdU incorporation from the two optic lobes and thoracic segments from 10 brains of wild type and <i>Digitor</i> mutant larvae with S.D., respectively. The difference was statistically significant (P value < 0.01, Student's two-tailed t-test). (J) Comparison of the average fluorescence intensity of anti-H3S10ph antibody labeling from the two optic lobes and thoracic segments from 8 brains of wild type and <i>Digitor</i> mutant larvae with S.D., respectively. The difference was statistically significant (P value < 0.001, Student's two-tailed t-test).</p

Localization of 3xHA-Digitor-mCitrine before and after heat shock treatment.

<p>The figure shows images of polytene squash preparations before heat shock as well as 5 min, 1.5 hr, and 5 hr after heat shock. The preparations were labeled with anti-HA antibody to detect the 3xHA-Digitor-mCitrine construct (in green), with anti-Pol IIo^ser5 antibody (in red), and with Hoechst for DNA (in blue/grey). Arrows indicate Pol IIo^ser5-positive heat shock-induced puffs 5 min after treatment.</p

Histone H3S10ph and H3S28ph antibody labelings of male salivary gland nuclei smush preparations.

<p>(A) Double labeling with H3S10ph antibody (in red) and JIL-1 antibody (in green) demonstrating co-localization and the characteristic upregulation of JIL-1 and H3S10ph labeling on the male X chromosome (X). (B) Double labeling with H3S28ph (CS) antibody (in red) and JIL-1 antibody (in green). (C) Double labeling with H3S28ph (UP) antibody (in red) and JIL-1 antibody (in green). In contrast to the labeling of the H3S10ph antibody there was no discernable labeling above background of the autosomes or the male X chromosome by either of the two H3S28ph antibodies.</p

14-3-3 antibody labeling of salivary gland chromosomes and nuclei.

<p>(A–B) 14-3-3 antibody labeling of male polytene squash preparations before and after heat shock treatment. (A) Wild-type squash preparations labeled with 14-3-3 (SZ) or 14-3-3 (CS) antibody (in red), JIL-1 antibody (in green), and Hoechst (DNA, in blue/gray). (B) Wild-type squash preparations after heat shock treatment labeled with 14-3-3 (SZ) or 14-3-3 (CS) antibody (in red), Pol IIo^ser5 antibody (in green), and Hoechst (DNA, in blue/gray). No or little specific labeling above background of either 14-3-3 antibody was discernable. (C–E) 14-3-3 localizes to the nuclear matrix surrounding the chromosomes. (C–D) Confocal sections of whole-mount salivary gland nuclei labeled with 14-3-3 (SZ) or 14-3-3 (CS) antibody (in red) and Hoechst (DNA in blue). (E) Confocal section of a live salivary gland nuclei from a 14-3-3ε-GFP (in green) enhancer trap line co-expressing histone H2Av-RFP (in red).</p

Live imaging of transiently transfected S2 cells with Digitor-GFP and Cut up-mCherry.

<p>(A) Co-transfection of Cut up-mCherry together with Digitor-GFP. Cut up-mCherry in singly transfected cells was localized both to the nucleus and the cytoplasm (arrows); however, in co-transfected cells, Cut up-mCherry was found only in the nucleus together with Digitor-GFP (Arrowhead). (B) In cells co-transfected with Cut up-mCherry and an H2Av-GFP control construct, Cut up-mCherry retained its dual localization to both the nucleus and cytoplasm (arrows).</p