In order to purify endogenous Jarid2 complexes, we used recombineering technology to construct a fly line that contains a FLAG-HA sequence at the N terminus of the Jarid2 ORF (see Materials and Methods). The resulting FLAG-HA-Jarid2 line was able to rescue lethality of the two strongest available Jarid2 mutants, Jarid2MB00996 and Jarid2e03131 (data not shown; see also Fig. S1A to C in the supplemental material). Also, colocalization studies on polytene chromosomes confirm that FLAG-HA-Jarid2 shows a distribution pattern similar to that of untagged endogenous Jarid2 (A), demonstrating that in the tagged line Jarid2 is not mislocalized. FLAG immunopurification was performed on extracts from 6- to 18-h-old Drosophila embryos to isolate Jarid2 and any associated proteins (B to D). However, full-length FLAG-HA-Jarid2 appears to be highly unstable as only a truncated form of FLAG-HA-Jarid2 could reproducibly be detected on a silver-stained SDS-PAGE gel (B, lane 2, asterisk) or in the purified fraction after FLAG peptide elution on a Western blot (C, lane 4, upper panel). In contrast, nuclear extracts from Drosophila embryos contain the full-length form of FLAG-HA-Jarid2 (~250 kDa) as well as the truncated version that runs around 130 kDa (C, lane 2, upper panel).
Fig 1 Purification of an endogenous Jarid2 complex in Drosophila. (A) Colocalization of Jarid2 and FLAG-HA-Jarid2 on polytene chromosomes from third-instar larvae showing overlap of endogenous Jarid2 with FLAG-HA-Jarid2. (A) Merged image of Jarid2 and HA antibody (more ...)
Multidimensional protein identification technology (MudPIT) analysis readily detects the PRC2 core components Enhancer of zeste [E(z)], Suppressor of zeste 12 [Su(z)12], Extra sex combs (Esc), and chromatin assembly factor 1 (Caf1, also known as p55 or Nurf55) as factors that copurify with Jarid2 (D). The relative quantities of PRC2 core components appear to be slightly higher than those of FLAG-HA-Jarid2 because the truncated version of FLAG-HA-Jarid2 might possibly result in fewer detectable peptides, and the relative abundance calculation assumes full-length Jarid2. The interaction of FLAG-HA-Jarid2 and E(z) was further confirmed by Western blotting (C, lane 4, lower panel). However, Jarid2 colocalizes only to some degree with E(z) on polytene chromosomes (E), and their banding patterns differ significantly (compare E′ with E″). In addition to these core components of PRC2, another protein, Jing, which is the Drosophila
homolog of mammalian AEBP2, was copurified with FLAG-HA-Jarid2 (D) (3
). In summary, these studies demonstrate that Jarid2 is a conserved member of PRC2 complexes in Drosophila
but differs significantly in its distribution pattern from canonical PRC2 core components such as E(z) on polytene chromosomes.
To determine the role of Jarid2 in PRC2 function in an in vivo system, we studied the effect of Jarid2 overexpression (A to D) and of Jarid2 mutations (E) on H3K27 methylation, the mark implemented by PRC2. Overexpression of Jarid2 was confirmed by a Jarid2 antibody (A). The white arrow points out the posterior compartment where Jarid2 is overexpressed (A′). Jarid2 overexpression results in reduction of H3K27me3 levels (compare B′, arrow, with C′, arrow), but no changes for H3K27me1 could be observed (D′, arrow). To assess H3K27me3 in Jarid2 mutant tissue, we created Jarid2 eye-antenna imaginal disc mosaics consisting of patches of Jarid2 mutant tissue (E, no GFP expression and therefore negatively marked) and wild-type tissue (E, expresses GFP and therefore marked in green) within the same disc. This allowed us to directly compare H3K27me3 levels between Jarid2 mutant tissue and wild-type tissue side by side under exactly the same experimental conditions. Our studies show that H3K27me3 is increased in Jarid2 mutant clones in the posterior region of eye-antenna imaginal discs where photoreceptor differentiation is in progress (E′; two large Jarid2 mutant clones are outlined with dashed lines and highlighted by arrows). No modifications in Jarid2 mutant clones were observed for other methyl marks tested (see Table S1 in the supplemental material). Quantitative RT-PCR experiments confirmed a very strong decrease of Jarid2 mRNA in Jarid2 mutant animals that were used to create mutant clones in eye-antenna imaginal discs (see Fig. S1A and B in the supplemental material) compared to wild-type controls (see Fig. S1A and B). This is also evident on the protein level as assessed by Western blotting (see Fig. S1C). Even though a global increase and reduction in H3K27me3 could be reproducibly detected under both Jarid2 mutant (E′, arrows) and Jarid2-overexpressing conditions, respectively (B′, white arrow), they are relatively weak and do not compare to the effects observed by RNA interference (RNAi) knockdown of E(z) in the posterior half of wing imaginal discs (F), which essentially eliminates all H3K27me3 (F′, arrow; see also Fig. S1D and E). However, these studies support the biochemical data in that Jarid2 only alters the levels of H3K27 methylation, as expected of a modulator of the PRC2 complex.
Fig 2 Jarid2 modulates H3K27 methylation in vivo. (A to D) GAL4-driven overexpression of Jarid2 in the posterior part of the wing imaginal disc under the control of the engrailed (en) promoter. GFP expression in green marks the posterior part (highlighted by (more ...)
In order to assess a possible role of Jarid2 in recruiting PRC2 complexes to chromatin, we compared Jarid2 (see Fig. S2A to D in the supplemental material), Su(z)12 (A to D) and H3K27me3 (E to H) and Polycomb (Pc) (I to L) localization patterns on polytene chromosomes in the wild type (A, E, and I; see also Fig. S2A), Jarid2 mutant (B, F, and J; see also Fig. S2B), and under Jarid2-overexpressing (C, G, and K; see also Fig. S2C) and E(z) mutant (D, H, and L; see also Fig. S2D) conditions. Jarid2 is widely distributed in wild-type animals (see Fig. S2A′) and is completely lost from chromatin in Jarid2 mutants (see Fig. S2B′). Surprisingly, Su(z)12 binding to chromatin (compare A′ and B′) and H3K27me3 levels (compare E′ and F′) are not detectably affected in Jarid2 mutants. Similarly, the pattern of Pc (compare I′ and J′), a core component of Polycomb repressive complex 1 (PRC1), is not considerably altered in Jarid2 mutants. This seems to suggest that, at least in Drosophila, Jarid2 is not globally required for recruitment of the PRC2 or PRC1 complex to Polycomb response elements (PREs).
Fig 3 The role of Jarid2 in recruitment of Polycomb repressive complexes 2 and 1 and modulation of H3K27me3 on chromatin. (A to L) Squashes of polytene chromosomes from salivary glands of wild-type, Jarid2 mutant, Jarid2-overexpressing (UAS-Jarid2), and E( (more ...)
Upon overexpression, Jarid2 binding to chromatin is strongly enhanced (see Fig. S2C′ in the supplemental material), and in some cases, the polytene chromosomes appear shortened, bloated, and poorly banded, indicating defects in higher-order chromosome compaction (C; see also Fig. S2C). In other cases this bloating phenotype is weaker (K) or not distinctly observable (G). Similar bloating phenotypes have been described for chromatin remodelers such as Imitation SWI (Iswi) (5
), and it has been shown that mutants of another chromatin remodeling factor, the trithorax group protein Kismet (Kis), globally affect H3K27 methylation (47
). This phenotype is interesting in the light of a recent report which suggests a genetic interaction between yet another chromatin remodeler, brahma
), and Jarid2
), providing further evidence for a possible role of Jarid2 in regulating higher-order chromatin structure. Interestingly, in Jarid2
-overexpressing larvae, both Su(z)12 recruitment (compare A′ and C′) and H3K27me3 levels (compare E′ and G′) are negatively affected. Pc localization on polytene chromosomes was marginally reduced when Jarid2
was overexpressed (compare I′ and K′).
The concept that PRC2 is generally not required for recruitment of PRC1 was further confirmed when E
mutants were analyzed (4
). Upon a temperature shift to 29°C for 24 h, E
mutant third-instar larvae show almost a complete loss of Su(z)12 from polytene chromosomes (compare A′ and D′) and consequently H3K27me3 is lost (H′). Nevertheless, Pc recruitment is not appreciably altered (compare I′ and L′). This strongly supports a PRC2-independent recruitment of PRC1 to chromatin (see also references 29
) and furthermore suggests that at least in vivo
the H3K27me3 mark is not the primary determinant for Pc binding, in contrast to previous proposals based largely on in vitro
). The absence of E(z) does also not impair Jarid2 localization on polytene chromosomes (compare Fig. S2A′ and D′ in the supplemental material), suggesting that Jarid2 can be recruited to chromatin independently of PRC2.
To address whether Jarid2 affects recruitment of PRC2 at a molecular level, ChIP-seq experiments for Jarid2, Su(z)12, and H3K27me3 in the wild type and Jarid2
mutants were performed. Most Jarid2 and Su(z)12 peaks are found at transcription start sites (TSSs) (A; see also Fig. S3A in the supplemental material). Of the peaks that were detected upstream, downstream, or inside genes, some could define TSSs of novel transcription units, as recently proposed for Polycomb repressive complex 1 (PRC1) (8
). In wild-type larvae the binding patterns of Jarid2 and Su(z)12 correlate very well with each other (Pearson coefficient, 0.76) (B) except for a small group of genes, which display high Su(z)12 occupancy and low Jarid2 occupancy (B, highlighted in red). Interestingly, in this class, 7 out of 30 genes are Hox
genes (see Table S2 in the supplemental material). The binding patterns of Jarid2 and Su(z)12 on the Bithorax
complex illustrate this finding (C). Occupancy of canonical PRC2 components such as Su(z)12 is usually very high on this locus and was distributed in several peaks (C, red tracks). At many of these highly occupied Su(z)12 sites, Jarid2 occupancy is relatively low (C, compare red with green tracks). This indicates a fundamentally different Jarid2 binding behavior than that of canonical PRC2 members for some of the major PRC2 target genes such as Hox
Fig 4 Genome-wide localization patterns by ChIP-seq of H3K27me3, Su(z)12, and Jarid2 in wild-type and Jarid2 mutant tissue. (A) Pie charts showing the genome-wide distribution of Jarid2 and Su(z)12 peaks in wild-type larvae. Gene localizations are represented (more ...)
Despite substantial colocalization of Jarid2 and Su(z)12 genome-wide, only comparatively few genes show significant changes in Su(z)12 binding (C, red tracks) in Jarid2 mutants, confirming our observations on polytene chromosomes. No general effects on Su(z)12 binding were apparent when wild-type Jarid2 mutant tissues in eye imaginal discs were compared (data not shown). Nonetheless, in a few cases, a significant increase or reduction (≥2 or ≤0.5) of Su(z)12 occupancy in Jarid2 mutants can be observed (C, red tracks, black arrows). In contrast to the mammalian findings, Su(z)12 occupancy can also be increased on certain genes in Jarid2 mutants (C, red tracks, left arrow). Neither genes with reduced Su(z)12 occupancy (C, red tracks, right arrow; see also Fig. S3B in the supplemental material) nor genes with increased Su(z)12 occupancy (C, red tracks, left arrow; see also Fig. S3C) displayed any significant changes (≥2 or ≤0.5) in H3K27me3 enrichment in Jarid2 mutants (C, blue tracks) or represented genes with no or low H3K27me3 enrichment (see Fig. S3B and C, upper two tracks). Although genes with changes in Su(z)12 occupancy and the majority of other genes [Su(z)12/Jarid2 bound and unbound] do not generally display significant changes (≥2 or ≤0.5) in H3K27me3 enrichment, a relatively big group of genes shows a very weak but statistically observable increase in H3K27me3 in Jarid2 mutant versus wild-type larvae (see Fig. S3D, dashed box). Likewise, the sum of these small increases in H3K27me3 enrichment of a large group of genes might be the reason why H3K27me3 is globally increased in Jarid2 mutant eye-antenna imaginal disc clones (E).
To gain further insight as to how Jarid2 regulates the expression of its target genes, we performed gene expression analyses from wild-type, Jarid2 mutant, and E(z)-RNAi larvae (; see Fig. S4 and S5 in the supplemental material), A total of 204 Jarid2/Su(z)12 cobound genes showed at least a 1.5-fold gain (A, highlighted in blue), and 136 Jarid2/Su(z)12 cobound genes had a more than 1.5-fold loss (A, highlighted in red) in expression in Jarid2 mutant larvae. Genes with a significant increase/reduction in Su(z)12 localization and/or increased/reduced H3K27me3 enrichment in Jarid2 mutants (≥2 or ≤0.5) did not generally correlate with gene expression changes (data not shown). Not all Jarid2/Su(z)12 cobound genes that are transcriptionally changed in Jarid2 mutants are also modified in E(z)-RNAi larvae (B) and vice versa see Fig. S4A and B). We find evidence for coregulation of PRC2 target genes by Jarid2 and E(z) (C and D show individual examples), confirming a role for Jarid2 in PRC2-mediated regulation of transcription. Jarid2 and E(z) only not appear to function in maintenance of transcriptional repression (C) but are also possibly involved in activation of common target genes (D). However, the possibility exists that a gene might be cobound by Jarid2/Su(z)12 and yet would not directly be transcriptionally controlled by these factors. Despite the binding of Jarid2/Su(z)12, a change in transcription in the corresponding mutant/RNAi animal could be a secondary consequence of other genes that are under direct control of Jarid2/Su(z)12. Generally, Jarid2/Su(z)12 cobound genes that show transcriptional changes in Jarid2 mutants and/or E(z)-RNAi larvae do not correlate with changes (≥2 or ≤0.5) in Su(z)12 occupancy (E) and/or changes in H3K27me3 enrichment (F) in Jarid2 mutants. We also identified Jarid2/Su(z)12 cobound genes that are regulated by Jarid2 independently of canonical PRC2 members [E(z)] (see Fig. S4C and D) and target genes solely controlled by canonical PRC2 members [E(z)] independently of Jarid2 (see Fig. S4E and F).
Fig 5 PRC2-dependent function of Jarid2 in active transcription and repression. (A) MA plot of the gene expression analysis in Jarid2MB00996 mutant larvae compared to the wild type. The y axis shows the fold change of expression levels of the Jarid2MB00996 (more ...)
It needs to be stressed that both ChIP-seq experiments and gene expression analyses have been carried out in larvae which represent a mixed population of cells. This could prevent the identification of genes that are regulated by Jarid2 in a tissue-specific manner. Alterations in Su(z)12 occupancy and H3K27me3 enrichment might be masked because only a subpopulation of cells is controlled by Jarid2. Therefore, we also performed ChIP-seq studies for Su(z)12 occupancy and gene expression analyses in eye-antenna imaginal discs (see Fig. S5A and B in the supplemental material). Similar to the results obtained in larvae, gene expression changes in Jarid2 mutant eye-antenna imaginal disc clones usually do not correlate with altered Su(z)12 occupancy (see Fig. S5B). Taken together, these results imply a role for Jarid2 in PRC2-dependent and -independent regulation of transcription. Our data support a role for Jarid2 and PRC2 in maintenance of transcriptional repression (C) and suggest the possibility for both Jarid2 and PRC2 to function in transcriptional activation or elongation (D). For most of its target genes, Jarid2 seems to carry out its role in transcriptional regulation independently of changes in H3K27me3 or PRC2 recruitment. However, the general tendency implies that most Jarid2/PRC2-coregulated genes have relatively low levels of H3K27me3 (F).