The H3.3K27M oncohistone antagonizes reprogramming in Drosophila

Abstract

Development proceeds by the activation of genes by transcription factors and the inactivation of others by chromatin-mediated gene silencing. In certain cases development can be reversed or redirected by mis-expression of master regulator transcription factors. This must involve the activation of previously silenced genes, and such developmental aberrations are thought to underlie a variety of cancers. Here, we express the wing-specific Vestigial master regulator to reprogram the developing eye, and test the role of silencing in reprogramming using an H3.3K27M oncohistone mutation that dominantly inhibits histone H3K27 trimethylation. We find that production of the oncohistone blocks eye-to-wing reprogramming. CUT&Tag chromatin profiling of mutant tissues shows that H3K27me3 of domains is generally reduced upon oncohistone production, suggesting that a previous developmental program must be silenced for effective transformation. Strikingly, Vg and H3.3K27M synergize to stimulate overgrowth of eye tissue, a phenotype that resembles that of mutations in Polycomb silencing components. Transcriptome profiling of elongating RNA Polymerase II implicates the mis-regulation of signaling factors in overgrowth. Our results demonstrate that growth dysregulation can result from the simple combination of crippled silencing and transcription factor mis-expression, an effect that may explain the origins of oncohistone-bearing cancers.

Fig 1. Vg expression transforms the eye into wing tissue.

(A-C) Side and dorsal views of the head and eyes of wildtype eyGAL/CyO adults (A) and eyGAL >Vg adults (B,C). About half of all adults with >Vg production display pigmented wing-like projections, while the rest lack eyes entirely. (D,E) Eye-antennal imaginal discs from wildtype eyGAL >RFP larvae (D) and from eyGAL >RFP >Vg larvae (E). Discs were immunostained for the Vg protein and for ELAV, a marker of differentiating photoreceptors. The antennal (a) and eye (e) portions of the disc are indicated, and the morphogenetic furrow that separates mitotically active undifferentiated cells in the anterior of the eye from differentiating photoreceptors in the posterior is marked with an orange arrowhead. The eye portions of discs with Vg production are distorted, with detectable Vg, low RFP signal, and very few differentiating photoreceptors at the posterior edge.

Fig 2. H3.3K27M oncohistones block direct reprogramming.

(A-C) Side views of eyes of wildtype un-induced UAS-H3.3K27M adults (A), adults with heterozygous for eyGAL and H3.3K27M transgenes (B), or homozygous for two eyGAL and H3.3K27M transgenes (C). Production of the oncohistone reduces the size of the eye. (D-F) Eye-antennal imaginal discs from larvae with eyGAL-induced production of RFP, H3.3K27M, and Vg. Discs were immunostained for the K27M epitope and for the photoreceptor ELAV marker. The oncohistone is produced throughout the developing eye portion of the disc. (G-J) Eye-antennal imaginal discs with eyGAL-induced production of RFP, H3.3K27M, and Vg, and immunostained for the H3K27me3 histone modification and for ELAV. Histone methylation is reduced in the eye portion of the disc relative to staining in the antennal portion in discs producing the oncohistone. (K, L) Examples of adults with co-production of Vg and H3.3K27M in the eye. Eyes are overgrown and convoluted (K, K’) or overgrown with extreme projections (L, L’).

Fig 3. H3.3K27M and Vg stimulate cell death and proliferation.

(A-D) Eye-antennal discs immunostained for the H3S10-phosphorylation marker of mitosis (A-D) with higher magnification imaging around the morphogenetic furrow (arrowheads, A’-D’). (E-H) Discs immunostained for the cleaved DCP-1 marker of apoptosis (E-H). The wildtype ‘+’ strain was w¹¹¹⁸, and all discs were immunostained for ELAV.

Fig 4. Tissue-specific gene expression changes during direct reprogramming.

(A) Side view of a vg^CL7A mutant adult with eyGAL-induced Vg production in the eye. Eye size is reduced, but not transformed into a wing. (B) Side view of a vg^U/+ head with eyGAL-induced H3.3K27M and Vg co-production in the eye. The eye is convoluted but reduced in size instead of overgrown. (C-F) Eye-antennal discs with eyGAL-induced production of RFP, H3.3K27M and Vg and immunostained for the eye-specific Dac protein and for ELAV. Neither Vg nor H3.3K27M production prevent Dac expression, although these discs are disorganized. The wildtype ‘+’ strain was eyGAL4>UASRFP/CyO. (G-K) Wildtype (‘+’, eyGAL4>UASRFP/CyO) wing imaginal discs (G) and eye-antennal discs with eyGAL-induced production of H3.3K27M and Vg (H-K) immunostained for the wing-specific Nub protein and for ELAV. The wing pouch is heavily labeled with Nub, and eye discs with Vg production have patches with low level Nub staining. No Nub staining is visible in eye discs with H3.3K27M production or in discs with H3.3K27M and Vg co-produced.

Fig 5. Chromatin profiling of H3K27me3 domains in reprogrammed eye imaginal discs.

(A) Chromatin profiling at the ANTP-C H3K27me3 domain in wing and eye imaginal discs. Arrowheads mark the positions of major Polycomb-bound PREs. (B) Changes in average fragment density (CPKM) of H3K27me3 in 166 annotated Polycomb domains between wing and eye imaginal discs. Domains are ranked by decreasing average H3K27me3 CPKM in wing and eye imaginal discs. Selected domains containing eye-specific genes (pink), wing-specific genes (blue) or common repressed genes (red) are marked. (C) Selected domains including eye-specific genes (pink), wing-specific genes (blue) or common domains (grey) are shown for wing and eye controls and production of Vg and H3.3K27M. (D-F) Changes in average fragment density (CPKM) of H3K27me3 in Polycomb domains between w¹¹¹⁸ eye imaginal discs and discs with H3.3K27M and Vg production. Selected domains with the largest changes in chromatin methylation are marked. (G) Average H3K27me3 coverage around 700 Polycomb-bound PREs [24] within H3K27me3 domains (solid lines), and around promoters (dashed line).

Fig 6. Chromatin profiling of elongating RNAPII and H3K4me2 in reprogrammed eye imaginal discs.

(A,B) RNAPII-S2p and H3K4me2 signal across the ANTP-C (A) and the ey gene (B). Arrowheads indicate alternative promoters of the Antp and the ey genes, and the green arrow indicates the direction of transcription. (C-F) Volcano plots of differentially-expressed genes between wildtype (w¹¹¹⁸) eye imaginal discs, wing imaginal discs, and eye discs with H3.3K27M and Vg production. Selected genes that are significantly differentially-expressed are marked.

Fig 7. Effects of reduced PRC1 and PRC2 on direct reprogramming and cell proliferation.

(A-B) Eye-antennal discs with eyGAL-induced expression of RFP and RNAi constructs directed against E(z) (A) or Pc (B) transcripts. Both discs are immunostained for the wing-specific Nub protein and for ELAV. The eye and antennal portions of discs are greatly reduced with E(z) knockdown, while Pc knockdown results in overgrowth of the eye portion and expression of Nub at the dorsal edge of the disc. (C-D) Heads of dissected dying pharate pupae with E(z) knockdown (C, C’) or E(z) knockdown and Vg production (D, D’). Loss of E(z) ablates all tissue derived from the eye-antennal disc including the head capsule and eyes, without affecting mouthparts. (E-F) Heads of dissected dying pharate pupae with Pc knockdown (E, E’) or Pc knockdown and Vg production (F, F’). Loss of Pc induces ectopic wing-like structures at the dorsal edges of eyes (arrowhead) and transforms antennae into legs (arrow). Vg production with Pc knockdown results greatly reduced eyes and expanded and flattened wing-like structures (bracket). (G) Volcano plot of differentially-expressed genes between wildtype (w¹¹¹⁸) eye imaginal discs and eye discs with Pc knockdown. Antp and Ubx are the most significantly differentially-expressed.