Causal role for inheritance of H3K27me3 in maintaining the OFF state of a Drosophila HOX gene

Abstract

Many eukaryotic cells can respond to transient environmental or developmental stimuli with heritable changes in gene expression that are associated with nucleosome modifications. However, it remains uncertain whether modified nucleosomes play a causal role in transmitting such epigenetic memories, as opposed to controlling or merely reflecting transcriptional states inherited by other means. Here, we provide in vivo evidence that H3K27 trimethylated nucleosomes, once established at a repressed Drosophila HOX gene, remain heritably associated with that gene and can carry the memory of the silenced state through multiple rounds of replication, even when the capacity to copy the H3K27me3 mark to newly incorporated nucleosomes is diminished or abolished. Hence, in this context, the inheritance of H3K27 trimethylation conveys epigenetic memory.

Figure 1. Role of the PRE in establishing ON and OFF states of the >PRE>UZ transgene

(A) Structure of >PRE>UZ and >UZ^∆PRE transgenes. The Ubx promoter drives expression of the lacZ coding sequence (red; arrow points in the direction of transcription) and is regulated by the PRE, an early enhancer (EE) and a disc enhancer (DE) (Methods). The PRE is embedded in a Flp-out cassette flanked by Flp recombinase target (FRT) sequences (encircled arrows) and contains a Tub.CD2 marker gene transcribed in the opposite direction (green). The transgene is marked by a yellow⁺ mini-gene. (B) Ubx.lacZ (UZ, red) and Tub.CD2 (CD2 green) expression from the >PRE>UZ transgene. UZ is first apparent in a central domain of blastoderm stage embryos delimited by the transcription factor Hunchback (Hb; turquoise), which represses EE enhancer activity, after which it is heritably ON in parasegments 6–12 and OFF in the remaining parasegments (gastrula and late stage germ band embryos). The gastrula image shows the three left-right pairs of the thoracic disc primordia, as well as three gnathal primordia on the lower side (marked by Distalless, Dll, turquoise). The wing and haltere primordia on one side are boxed and shown at higher magnification (here and elsewhere, anterior is to the left, parasegment 6 is indicated by a yellow asterisk and Hoechst (DNA; blue) provides a counterstain). The anterior boundary of UZ coincides with the anterior boundary of parasegment 6, which subdivides the haltere primordium into anterior (A) and posterior (P) compartments. CD2 is also transiently up-regulated in parasegments 6–12 at this stage (only parasegment 6 and 7 are apparent in this image). UZ expression in late germ band embryos closely resembles that of native Ubx (turquoise). (C) UZ expression in embryos carrying the >UZ^∆PRE transgene (stained as in B; absence of the >PRE> cassette is confirmed by the absence of CD2). UZ is not expressed at the blastoderm stage but comes on ubiquitously after gastrulation. (D) UZ expression in third instar wing and haltere discs carrying the >PRE>UZ transgene; the insert shows a haltere disc independently stained for UZ and Ubx. UZ is ON in the P compartment of the haltere but OFF in the A compartment as well as in the entire wing disc; CD2 expression is uniform. Native Ubx is ON in the entire haltere disc but does not over-ride the OFF state of the >PRE>UZ transgene in the A compartment. (E) UZ expression in wing and haltere discs carrying the >UZ^∆PRE transgene. UZ is expressed in all cells, albeit with stereotyped, position-dependent differences in level that correlate with the kinetics of release from silencing following PRE excision (Fig. 2).

Figure 2. Release from silencing following >PRE> excision depends on cell position and number of cell divisions

(A) Schedule of induction of >UZ^∆PRE clones (E = embryo; L1, 2 and 3 = first, second and third larval instars; AEL, time after egg laying (in hours). For all time points (arrow heads), clones were assayed in mature wing discs at the end of larval life (~120 AEL; inset shows the prospective notum (body wall), hinge, and wing blade domains of the columnar epithelium, as well as the peripodial epithelium). (B) Wing discs carrying the >PRE>UZ transgene stained for UZ (red top, white bottom) and CD2 (green, top) following >PRE> excision at the time points indicated. The columnar epithelium is shown for all discs, and the peripodial epithelium is also shown for the 108 AEL disc. >UZ^∆PRE clones are marked “black” by the absence of CD2 expression. Most clones induced in mid-stage embryos (9 AEL) cell-autonomously derepress UZ (e.g., arrow). However, clones induced later derepress UZ in a manner that depends on position within the disc and the time of clone induction (e.g., 18 and 48 AEL clones fail to derepress UZ, respectively, in most or all of the notum (arrowheads); 72 and 96 AEL clones fail to show release from silencing in most of the prospective notum, wing hinge, and some regions of the wing blade; and 108 AEL clones show release only in peripodial cells). (C) Schedule of induction of >UZ^∆PRE clones in “stalled” wing discs (Methods). In this background, cell division ceases when the imaginal discs reach full size at around 144 AEL, while larvae continue to feed and grow for up to three weeks. Arrowheads indicate the timing of >PRE> excision in the presence (E,F) or absence (D) of co-induced Tub>hh clones. (D) Stalled wing disc two weeks after >PRE> excision at 120AEL (stained as in B): no UZ expression is detected despite >PRE> excision having occurred two weeks previously (clones are not apparent at this magnification, having undergone only one or two divisions before the stall). (E) Stalled wing disc heat shocked to co-induce >UZ^∆PRE and Tub>hh clones during the first larval instar (24–48 AEL), assayed ~one week later. Large clonal patches are apparent in both the A and P compartments (the patches in A are exceptionally large owing to the extra growth induced by ectopic Hh). UZ is expressed throughout the clones located within the prospective wing blade. (F) Stalled wing disc heat shocked to co-induce >UZ^∆PRE and Tub>hh clones ~ 24 hrs before the stall and assayed ~2 weeks later. Clones in the A compartment continue to proliferate as a function of distance from the A/P boundary: clones located far from the A/P boundary are large and de-repress UZ throughout the prospective wing, whereas clones closer to the A/P boundary are smaller and show release from silencing in the vicinity of the D/V boundary (as in 96 AEL clones in B). In contrast, clones in the remainder of the disc (posterior A and the entirety of P) stop dividing after only a few divisions and do not express UZ. Excision clones displayed in this figure were induced by a 1 hr heat shock at 36±1°C.

Figure 3. Cell division-dependent dilution of H3K27me3 following PRE excision

(A) Probe pairs used for ChIP-qPCR of H3K27me3 and total H3 associated with the >PRE>UZ and >UZ^∆PRE transgenes (all probe pairs detect only DNA in the transgene and not endogenous Ubx or Tub DNA, and are indicated as blue, green or red segments with the amplified regions shown as white segments; not to scale; Table S7). (B) ChIP-qPCR for H3K27me3 and total H3 for entirely >PRE>UZ (white) and >UZ^∆PRE (grey) wing discs (120 AEL). H3K27me3 is detected for all of the probed regions of >PRE>UZ except for the ubiquitously expressed, CD2 encoding region amplified by probe pair #1. In contrast, no significant H3K27me3 signal was observed for any of probed regions for the >UZ^∆PRE transgene (total H3 levels were similar for all probed segments for both transgenes; here and elsewhere, see the indicated Table (Table S1 in this case) for detailed quantitation and mock ChIP controls). (C) ChIP-qPCR analysis of H3K27me3 following PRE excision during normal development. The schedule is shown at the top (as in Fig. 2B). ChIP-qPCR data are shown for “IN” probes, which detect only intact >PRE>UZ DNA (white columns, green) and “EX” probes, which detect only >UZ^∆PRE DNA (grey columns, red). The experimental design does not allow a meaningful “zero” time point for H3K27me3 associated with >UZ^∆PRE DNA immediately following PRE excision. Comparing the change in H3K27me3 between the 24–48, 48–72 and 24–72 time points, the EX probe data indicate a decline in H3K27me3 levels of ~10–12%/cell cycle for both the 24–48 and 48–72 hr time points, normalized by direct assessment of cell division in parallel experiments (Fig. S4); total H3 remained constant. H3K27me3 after 72 hours is still significantly above background, as monitored in the >UZ^∆PRE control. No significant change is detected by the IN probes for H3K27me3 or total H3 (Table S2). (D) ChIP-qPCR analysis of H3K27me3 following PRE excision in “stalled discs” during extended larval development. >UZ^∆PRE clones were induced around the time of the stall and assayed by ChIP-qPCR at 24, 48 and 120 hours afterwards (top, as in Fig. 2D). No significant change was observed in H3K27me3 or total H3 using the same probes as in C, even after 120 hrs (Table S3). Here, and in Figs. 4A and 5B, each data point indicates the percent input value for a single biological replicate and bars represent the mean±SEM of 4 independent replicates. Values above plots represent P values calculated by unpaired t test. “n/a” signifies that the DNA is not present for detection in the >UZ^∆PRE genotype. >UZ^∆PRE clones in C and D were induced by 1 hr heat shock at 38°C.

Figure 4. Spiking the H3⁺ pool with H3^K27R reduces H3K27me3 and accelerates loss of the OFF state

(A) ChIP-qPCR analysis of the >PRE>UZ transgene in wing discs in which the native histone gene complex is replaced by transgenes expressing 12 copies of the H3⁺ coding sequence plus 8 copies of the H3^K27R coding sequence (12K:8R; grey; probes as in Fig. 3A), or alternatively, by transgenes containing 24 copies of the H3⁺ coding sequence (24K; white; Methods). For each of the five probed regions that are H3K27 trimethylated in wild type wing discs (Fig. 3B), H3K27me3 is reduced by ~30–40% comparing 12K:8R to 24K discs (no significant difference is seen total H3 ChIP; Table S4). (B) Mature 12K:8R or 24K wing discs carrying multiple >UZ^∆PRE clones induced during the first larval instar (marked by the absence of CD2). In 24K discs (right panel), as in wild type discs (Fig. 2B), >UZ^∆PRE clones remains silenced in the prospective notum (boxed in yellow; magnified to the right). In 12K:8R discs (left panel), >UZ^∆PRE clones show extensive release from silencing. (C) The prospective notum of a 12K:8R wing disc carrying the >PRE>UZ transgene as well as a single copy of the native Histone gene complex (HisC⁺), and in which three kinds of clones have been co-induced during the first larval instar: (i) >UZ^∆PRE clones in which the transgene PRE has been excised, but HisC⁺ remains present (blue in the cartoon; marked “black” in the upper left image by the absence of CD2, green, and blue in the lower left image by the presence of a Ubi.GFP transgene linked in cis to the single HisC⁺ allele); (ii) 12K:8R clones that retain the intact >PRE>UZ transgene but have lost HisC⁺ (green in the cartoon; marked green in the upper left image and “black” in the lower left image); and (iii) >UZ^∆PRE12K:8R clones that have lost both the PRE and HisC⁺ (red in the cartoon, marked black in the upper and lower left images); cell populations that retain both the PRE and HisC⁺ are shown as turquois in the cartoon. Only the >UZ^∆PRE 12K:8R clones express UZ in the central portion of the notum (red), whereas the remaining two kinds of clones do not. Clones in B and C were induced by 1 hr heat shock at 37.5°C.

Figure 5. Transient reduction in PRC2 activity accelerates loss of H3K27me3 and the OFF state

(A) Schedule of induction of >UZ^∆PRE clones (generated by a one hour heat shock at 37°C; dark red) followed by a transient loss of PRC2 activity (24 hours, 29°C light red) relative to larval instars at 17°C. Transient loss of PRC2 was achieved by RNAi knock-down of E(z), the catalytic subunit of PRC2, under Gal80^ts/Gal4 control, which allows normal PRC2 activity at 17°C, but reduces or abolishes it at 29°C. (B) H3K27me3 ChIP-qPCR was performed using EX and IN probes, which assay the PRE deleted (>UZ) and intact (>PRE>UZ) transgenes (as in Fig. 3A; Table S5). RNAi (experimental) and no RNAi (control) discs were analyzed in strict parallel. Transient RNAi knock-down of PRC2 following PRE excision causes a 2.5 fold further decrease in H3K27me3 relative to the control (EX probe), in contrast to a modest 20% reduction in H3K27me3 observed in cells that did not excise the PRE (IN probe). (C) UZ expression in the notum region of no RNAi and E(z) RNAi wing discs, stained for UZ (red/white) and CD2 (green); >UZ^∆PRE clones are marked by the absence of CD2. Extensive UZ expression is observed in >UZ^∆PRE clones in E(z) RNAi discs, in contrast to sporadic, weak expression in control discs. >UZ^∆PRE clones in B and C were induced by 1 hr heat shock at 38°C.

Figure 6. Epigenetic memory of the OFF state by inheritance of H3K27me3

(A) The model. Maintenance of the OFF state of silenced HOX loci depends on H3K27me3 catalyzed by the Polycomb Repressive Complex 2 (PRC2), recruited by cis-acting Polycomb Response Elements (PREs). Following replication, parental H3K27me3 nucleosomes are efficiently re-deposited at the HOX locus, but diluted by half by incorporation of newly synthesized, naive nucleosomes; PRC2 is induced by binding to H3K27me3 on parental nucleosomes to copy the K27me3 mark onto neighboring, naïve nucleosomes (depicted only on the bottom daughter strand). Targeting of PRC2 to the PRE (“w/PRE”) is required for efficient copying, and the process reiterates after each replication cycle, propagating the epigenetic memory of the OFF state without requiring additional inputs to perpetuate the state. Following PRE excision (“∆PRE”), residual “free” PRC2 can copy the mark, but inefficiently, resulting in the serial dilution of H3K27me3 nucleosomes following each replication cycle, and the release from silencing when the level of H3K27me3 falls beneath a critical threshold. (B) The evidence. In otherwise wildtype cells, the rate of division-coupled dilution of H3K27me3 nucleosomes following PRE excision is slow (~10–12%/cell cycle; ∆PRE; green), indicating that sufficient PRC2 activity remains in the absence of the PRE to copy most of the H3K27me3 marks from parental nucleosomes to naïve nucleosomes. However, after sufficient divisions occur to dilute H3K27me3 nucleosomes below distinct thresholds, the memory of the OFF state is lost in a manner that depends on cell position, e.g., after ~4–6 divisions and a decline of >50% for cells located in the prospective wing (yellow) and after >8–9 divisions and a decline of >75% for cells in the prospective notum (grey). Compromising the copying capacity of PRC2 further by introducing a sub-population of K27R mutant histone H3 molecules that cannot be trimethylated [either from fertilization onwards (∆PRE in 12K:8R; dark pink) or concomitant with PRE excision (∆PRE 12K:8R in wt; light pink)], or alternatively, by transiently knocking down free PRC2 activity (∆PRE + 24 hr PRC2 KD, turquoise), causes a corresponding acceleration in both the dilution of H3K27 as well as the loss of the memory of the OFF state. Finally, in the extreme case in which the capacity of PRC2 to copy the mark is abruptly terminated by replacing all newly synthesized nucleosomes available for incorporation with the H3K27R mutant form (H3⁺ to H3^K27R; black), silencing of the native Ubx gene is lost in a manner that correlates with the number of cell divisions and appears to obey the same region-specific thresholds for release from silencing as the >PRE>UZ transgene, assuming a 50% dilution of parental H3K27me3 nucleosomes following each round of replication. Collectively, these results establish a causal relationship between inheritance of H3K27me3 and the epigenetic memory of the OFF state.