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. 2015 May 7:8:18.
doi: 10.1186/s13072-015-0008-6. eCollection 2015.

Drosophila Cyclin G and epigenetic maintenance of gene expression during development

Affiliations

Drosophila Cyclin G and epigenetic maintenance of gene expression during development

Camille A Dupont et al. Epigenetics Chromatin. .

Abstract

Background: Cyclins and cyclin-dependent kinases (CDKs) are essential for cell cycle regulation and are functionally associated with proteins involved in epigenetic maintenance of transcriptional patterns in various developmental or cellular contexts. Epigenetic maintenance of transcription patterns, notably of Hox genes, requires the conserved Polycomb-group (PcG), Trithorax-group (TrxG), and Enhancer of Trithorax and Polycomb (ETP) proteins, particularly well studied in Drosophila. These proteins form large multimeric complexes that bind chromatin and appose or recognize histone post-translational modifications. PcG genes act as repressors, counteracted by trxG genes that maintain gene activation, while ETPs interact with both, behaving alternatively as repressors or activators. Drosophila Cyclin G negatively regulates cell growth and cell cycle progression, binds and co-localizes with the ETP Corto on chromatin, and participates with Corto in Abdominal-B Hox gene regulation. Here, we address further implications of Cyclin G in epigenetic maintenance of gene expression.

Results: We show that Cyclin G physically interacts and extensively co-localizes on chromatin with the conserved ETP Additional sex combs (ASX), belonging to the repressive PR-DUB complex that participates in H2A deubiquitination and Hox gene silencing. Furthermore, Cyclin G mainly co-localizes with RNA polymerase II phosphorylated on serine 2 that is specific to productive transcription. CycG interacts with Asx, PcG, and trxG genes in Hox gene maintenance, and behaves as a PcG gene. These interactions correlate with modified ectopic Hox protein domains in imaginal discs, consistent with a role for Cyclin G in PcG-mediated Hox gene repression.

Conclusions: We show here that Drosophila CycG is a Polycomb-group gene enhancer, acting in epigenetic maintenance of the Hox genes Sex combs reduced (Scr) and Ultrabithorax (Ubx). However, our data suggest that Cyclin G acts alternatively as a transcriptional activator or repressor depending on the developmental stage, the tissue or the target gene. Interestingly, since Cyclin G interacts with several CDKs, Cyclin G binding to the ETPs ASX or Corto suggests that their activity could depend on Cyclin G-mediated phosphorylation. We discuss whether Cyclin G fine-tunes transcription by controlling H2A ubiquitination and transcriptional elongation via interaction with the ASX subunit of PR-DUB.

Keywords: Cyclin G; Homeotic; Polycomb; Trithorax.

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Figures

Figure 1
Figure 1
Cyclin G physically interacts with ASX in vivo. (A) Schematic representation of Cyclin G full-length (CycG) and truncated forms (CycG 1 to 130 and CycG 130 to 566 corresponding to amino-acids 1 to 130 and 130 to 566, respectively). Grey box: Cyclin domain; red box: PEST sequence. (B-D) Cyclin G interacts with ASX-C (amino-acids 1139 to 1668) in S2 cells; black arrowheads indicate co-immunoprecipitation. FLAG-CycG (B) and FLAG-CycG 130–566 (D) co-immunoprecipitate with Myc-ASX-C. Note that FLAG-CycG 130 to 566 co-migrates with IgG heavy chains. (C) Myc-ASX-C co-immunoprecipitates with FLAG-CycG 1 to 130. Immunoprecipitations were performed with anti-Myc, anti-FLAG, or anti-HA (Mock) antibodies. Immunoprecipitated proteins were revealed by Western blot, using anti-Myc (top panel) or anti-FLAG antibodies (bottom panel). In (B) and (D), asterisks indicate IgG heavy chains. S: supernatant after immunoprecipitation; IP: protein G-agarose beads. Five percent of the input or supernatant and 50% of the immunoprecipitate were loaded onto the gels. (E) Myc-Cyclin G co-immunoprecipitates with endogenous ASX in da > Myc-CycG ΔP third instar larvae. Immunoprecipitated proteins were revealed by Western blot, using anti-Myc antibody.
Figure 2
Figure 2
Cyclin G co-localizes with ASX and binds mainly active chromatin. Immunostaining of polytene chromosomes from w 1118 third instar larvae. DNA was stained with DAPI (blue). (A) Cyclin G (green) and ASX (red) co-localize at many sites. Bottom: close-up view of the region framed by a white rectangle. (B) Cyclin G (green) binds DAPI interbands. Right: close-up view of the region framed by a white rectangle. (C) Cyclin G (green) and H3K27me3 (red) are almost completely exclusive. Rare co-localizations are shown with white arrows. (D) Cyclin G (green) co-localizes at many sites with RNA Pol II phosphorylated on serine 2 (red). Bottom: close-up view of the region framed by a white rectangle.
Figure 3
Figure 3
CycG genetically interacts with Asx. (A, B) Effect of CycG misregulation on the penetrance of ectopic sex combs on L2 induced by (A) Asx XF23 and (B) Asx 3. (C, D) Penetrance of A4 to A5 (C) and A5 to A4 (D) abdominal segment transformations in Asx 3/+, Asx 3/+;da > CycG RNAi and Asx 3/+;da > CycG ΔP/+ males. (E) Posterior abdomens of adult males showing wild-type male pigmentation in A4 to A6, A4 to A5 transformation in Asx 3/+ male (black arrowhead), and A5 to A4 transformation in Asx 3/+;da > CycG ΔP/+ male (white arrowhead). Fisher’s exact test, *P < 0.01 **P < 0.0001 (n ≥ 30). L2: mesothoracic leg. A4, A5, A6: abdominal segments 4, 5, and 6.
Figure 4
Figure 4
CycG misregulation alters ectopic sex comb phenotypes of an mxc mutant. (A) Expressivity of mesothoracic leg (L2) and metathoracic leg (L3) transformations into prothoracic leg (L1). The wild-type sex comb is marked by a white arrowhead. mxc G46/Y males present ectopic sex comb teeth on distal L1, on L2 and L3 (black arrowheads). Only phenotypes affecting L2 and L3 were rated. These phenotypes are enhanced by CycG loss of function. (B) Penetrance of the ectopic sex combs phenotype on male legs. CycG loss of function enhances mxc G46 induced ectopic sex combs on L2 and L3, whereas CycG gain of function suppresses ectopic sex combs on both legs. Fisher’s exact test, *P < 0.05 **P < 0.0001 (n ≥ 50).
Figure 5
Figure 5
CycG misregulation alters homeotic transformations of a Pc mutant. (A) Penetrance of L2 and L3 ectopic sex combs in Pc 3/+, Pc 3/+;da > CycG RNAi and Pc 3/da > CycG ΔP males. Fisher’s exact test, **P < 0.0001 (n ≥ 58). (B) Penetrance of wing to haltere transformations in Pc 3/+, Pc 3/+;da > CycG RNAi and Pc 3/da > CycG ΔP females and males. Fisher’s exact test, **P < 0.0001 (n ≥ 31). (C) Representative Pc 3/+ adult female wing with posterior deformation corresponding to a partial transformation into haltere tissue (arrowhead). Representative Pc 3/da > CycG ΔP female wing with suppressed haltere to wing transformation. (D) Penetrance of A4 to A5 abdominal segment transformations in Pc 3/+, Pc 3/+;da > CycG RNAi and Pc 3/da > CycG ΔP males. Fisher’s exact test, *P < 0.05, **P < 0.0001 (n ≥ 29). (E) Abdomens of Pc 3 and Pc 3/+;da > CycG RNAi males with representative A4 to A5 transformations (arrowheads). A4, A5, A6: abdominal segments 4, 5, and 6.
Figure 6
Figure 6
CycG gain of function enhances abdominal transformations of trxG mutants. (A, D) Penetrance of A5 to A4 transformations in adult male cuticles. Effect of CycG misregulation on brm 2-induced (A) or trx E2-induced (D) A5 to A4 transformations. (B, C) Male abdominal cuticles. Representative trx E2/+ (B) and trx E2/da > CycG ΔP (C) males present, respectively, small and large light-pigmented cuticle patches on A5, denoting a partial transformation of abdominal segment A5 into A4 (white and black arrowheads). Fisher’s exact test, **P < 0.0001 (n ≥ 30). A4, A5, A6: abdominal segments 4, 5, and 6.
Figure 7
Figure 7
CycG misregulation modulates Hox protein profiles of PcG mutants. (A) Anti-SCR immunostainings of third instar larval leg imaginal discs from wild-type (wt), mxc G46/Y, mxc G46/Y;da > CycG RNAi or mxc G46/Y;da > CycG ΔP males. (B) Anti-UBX immunostaining of third instar haltere (H), wing (W), and prothoracic leg (L3) imaginal discs from a wild-type female larva, and anti-UBX staining of wing discs from Pc 3/+, Pc 3/+;da > CycG RNAi or Pc 3/da > CycG ΔP females. Note the reduced ectopic staining in the Pc 3/da > CycG ΔP female wing disc (arrowhead).

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