The trithorax group proteins Kismet and ASH1 promote H3K36 dimethylation to counteract Polycomb group repression in Drosophila

Abstract

Members of the Polycomb group of repressors and trithorax group of activators maintain heritable states of transcription by modifying nucleosomal histones or remodeling chromatin. Although tremendous progress has been made toward defining the biochemical activities of Polycomb and trithorax group proteins, much remains to be learned about how they interact with each other and the general transcription machinery to maintain on or off states of gene expression. The trithorax group protein Kismet (KIS) is related to the SWI/SNF and CHD families of chromatin remodeling factors. KIS promotes transcription elongation, facilitates the binding of the trithorax group histone methyltransferases ASH1 and TRX to active genes, and counteracts repressive methylation of histone H3 on lysine 27 (H3K27) by Polycomb group proteins. Here, we sought to clarify the mechanism of action of KIS and how it interacts with ASH1 to antagonize H3K27 methylation in Drosophila. We present evidence that KIS promotes transcription elongation and counteracts Polycomb group repression via distinct mechanisms. A chemical inhibitor of transcription elongation, DRB, had no effect on ASH1 recruitment or H3K27 methylation. Conversely, loss of ASH1 function had no effect on transcription elongation. Mutations in kis cause a global reduction in the di- and tri-methylation of histone H3 on lysine 36 (H3K36) - modifications that antagonize H3K27 methylation in vitro. Furthermore, loss of ASH1 significantly decreases H3K36 dimethylation, providing further evidence that ASH1 is an H3K36 dimethylase in vivo. These and other findings suggest that KIS antagonizes Polycomb group repression by facilitating ASH1-dependent H3K36 dimethylation.

Keywords: Chromatin; Histone methylation; Polycomb group; Transcription; Trithorax group.

Fig. 1.

ASH1 is not required for transcription elongation. (A-F) Polytene chromosomes from wild-type (A,C,E) and ash1 mutant (B,D,F) Drosophila larvae were stained with antibodies against ASH1 (A,B), RNA Pol IIo^ser2 (C,D) and SPT6 (E,F). (G) The relative fluorescence intensity is significantly reduced for ASH1 (P=0.0003), but is not significantly reduced for SPT6 (P=0.11) or Pol IIo^ser2 (P=0.52). Error bars in all figures indicate s.d.

Fig. 2.

DRB inhibits phosphorylation of RNA Pol II on serine 2. (A-L) Polytene chromosomes were treated with DMSO as a control (A,B,E,F,I,J) or the drug DRB (65 μM) (C,D,G,H,K,L) and stained with antibodies against RNA Pol IIa (A,C), RNA Pol IIo^ser5 (E,G), RNA Pol IIo^ser2 (I,K) and KIS-L (B,D,F,H,J,L). (M) The average fluorescence intensity normalized to the DMSO control shows an 85% reduction in RNA Pol IIo^ser2 fluorescence (P=5×10^-5) on the DRB-treated chromosomes, but no change in KIS-L or RNA Pol IIa and only a 60% reduction in RNA Pol IIo^ser5 (P=0.0005).

Fig. 3.

ASH1 does not require elongating RNA Pol II to localize to chromatin. (A-D,F-I) Polytene chromosomes were treated with DMSO (A,B,F,G) or DRB (C,D,H,I) and stained with antibodies against RNA Pol IIo^ser2 (green, B,D,G,I) and either SPT6 (red, A,C) or ASH1 (red, F,H). (E,J) The relative fluorescence intensity of SPT6 was reduced 70% (P=0.001) on the DRB-treated chromosomes (E). By contrast, the fluorescence intensity of ASH1 was not significantly reduced (P=0.77) on the DRB-treated chromosomes (J).

Fig. 4.

SPT6 but not ASH1 colocalizes with elongating RNA Pol II on polytene chromosomes. (A-C) Merged image (A) of polytene chromosomes stained with antibodies against SPT6 (red) and RNA Pol IIo^ser2 (green). The chromosome arm in A (boxed) is magnified in B, showing the banding patterns of SPT6 and Pol IIo^ser2, alone and split. A comparison of the band size and intensity for SPT6 and Pol IIo^ser2 (C) confirms the highly coincident pattern. (D-F) Merged image (D) of polytene chromosomes stained with antibodies against ASH1 (red) and RNA Pol IIo^ser2 (green). The chromosome arm in D (boxed) is magnified in E, showing the banding pattern of ASH1 and Pol IIo^ser2, alone and split. The band intensity distribution of ASH1 and Pol IIo^ser2 (F) shows that although Pol IIo^ser2 is present at many sites of ASH1, the intensities of each are not well correlated.

Fig. 5.

ASH1 is not recruited to heat-shock puffs by elongating RNA Pol II. (A) Merged image of polytene chromosomes from heat shocked third instar larvae stained with antibodies against ASH1 (red) and Pol IIo^ser2 (green). (B) Magnification of the region encompassing the 87A/C heat-shock puffs (arrowheads) from A. (C,D) ASH1 staining alone (C) and higher magnification (D) show that elongating RNA Pol II does not recruit ASH1 to heat-shock puffs (arrowheads).

Fig. 6.

KIS does not directly recruit ASH1 to chromatin. (A-F) Polytene chromosomes from larvae expressing lacZ (A-C) or KIS-L (D-F) in the salivary gland were stained with antibodies against KIS-L (red, A,D), ASH1 (green, B,E) or Pol IIo^ser2 (blue, C,F). (G) The average fluorescence intensity normalized to the lacZ control shows a ∼3-fold increase in KIS-L staining (P=2×10^-5) on polytene chromosomes from KIS-L-expressing larvae. By contrast, the fluorescence intensity of ASH1 is not significantly increased (P=0.57) and Pol IIo^ser2 staining is only slightly increased (P=0.01) on polytene chromosomes from KIS-L-expressing larvae.

Fig. 7.

DRB treatment does not affect the trimethylation of histone H3 on lysine 27. (A-C) Colocalization of the histone modification H3K27me3 (red) and Pol IIo^ser2 (green) is shown on polytene chromosomes (A). The chromosome arm in A (boxed) is magnified in B. Bands of H3K27me3 and Pol IIo^ser2 are often adjacent and occasionally partially overlapping, as shown in the split image and in the distribution of band intensities (C). (D-G) Polytene chromosomes treated with DMSO (D,E) or DRB (F,G) and stained with antibodies against H3K27me3 (D,F) and elongating Pol II (E,G). (H) No significant difference (P=0.08) in the relative fluorescence intensity of H3K27me3 was observed on polytene chromosomes from salivary glands treated with DRB.

Fig. 8.

Tri- and di-methylation of histone H3 on lysine 36 are reduced in kis mutants. (A-H) Polytene chromosomes from wild-type (A,B,E,F) and kis mutant (C,D,G,H) larvae were stained with antibodies against total RNA Pol II (A,C), H3K36me3 (B,D), H3K36me2 (E,G) and H3K27me3 (F,H). (I) The relative fluorescence intensity is significantly reduced for total Pol II (P=1.2×10^-6), H3K36me3 (P=9×10^-7) and H3K36me2 (P=0.0007) and significantly increased for H3K27me3 (P=0.0006).

Fig. 9.

ASH1 promotes H3K36 dimethylation in vivo. (A,B) Colocalization of ASH1 (green) and H3K36me2 (red) on the distal arm of a polytene chromosome (A), together with a comparison of the band intensities along the arm (B). (C-H) Polytene chromosomes from wild-type (C-E) and ash1 mutant (F-H) larvae were stained with antibodies against ASH1 (C,F), H3K36me2 (D,G) and H3K36me3 (E,H). (I) A 68% reduction in the average fluorescence intensity of H3K36me2 (P=7.5×10^-7) and no significant change in H3K36me3 (P=0.088) were observed in ash1 mutants.

Fig. 10.

Decrease in H3K36me2 is correlated with increase in H3K27me3 in ash1 mutants. (A,B) The banding patterns of H3K27me3 (green) and H3K36me2 (red) from a polytene chromosome arm (A), together with the distribution of pixel intensities along an arm (B). (C-F) Polytene chromosomes from wild-type (C,D) and ash1 mutant (E,F) larvae stained with antibodies against H3K27me3 (C,E) and H3K36me2 (D,F). (G) Relative fluorescence intensities show a significant increase in H3K27me3 (P=4.8×10^-6) and decrease in H3K36me2 (P=1.2×10^-6).

Fig. 11.

KIS promotes histone modifications that antagonize H3K27 methylation. KIS promotes H3K36 dimethylation in the vicinity of active genes by promoting the association of ASH1 with chromatin. KIS also promotes TRX binding, which acetylates H3K27 through its association with CBP in the TAC1 complex (Tie et al., 2009). KIS independently promotes transcription elongation, resulting in H3K36 trimethylation by SET2 at the 3′ end of genes. Each of these histone modifications antagonizes repressive H3K27 catalyzed by the E(Z) subunit of PRC2. GTFs, general transcription factors.