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Drosophila Kismet regulates histone H3 lysine 27 methylation and early elongation by RNA polymerase II.

Srinivasan S, Dorighi KM, Tamkun JW - PLoS Genet. (2008)

Bottom Line: Although we observed significant overlap between the distributions of KIS-L, ASH1, and TRX on polytene chromosomes, KIS-L did not bind methylated histone tails in vitro, and loss of TRX or ASH1 function did not alter the association of KIS-L with chromatin.By contrast, loss of kis function led to a dramatic reduction in the levels of TRX and ASH1 associated with chromatin and was accompanied by increased histone H3 lysine 27 methylation-a modification required for Polycomb group repression.Our findings suggest that KIS-L promotes early elongation and counteracts Polycomb group repression by recruiting the ASH1 and TRX histone methyltransferases to chromatin.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cell, and Developmental Biology, University of California Santa Cruz, Santa Cruz, CA, USA.

ABSTRACT
Polycomb and trithorax group proteins regulate cellular pluripotency and differentiation by maintaining hereditable states of transcription. Many Polycomb and trithorax group proteins have been implicated in the covalent modification or remodeling of chromatin, but how they interact with each other and the general transcription machinery to regulate transcription is not well understood. The trithorax group protein Kismet-L (KIS-L) is a member of the CHD subfamily of chromatin-remodeling factors that plays a global role in transcription by RNA polymerase II (Pol II). Mutations in CHD7, the human counterpart of kis, are associated with CHARGE syndrome, a developmental disorder affecting multiple tissues and organs. To clarify how KIS-L activates gene expression and counteracts Polycomb group silencing, we characterized defects resulting from the loss of KIS-L function in Drosophila. These studies revealed that KIS-L acts downstream of P-TEFb recruitment to stimulate elongation by Pol II. The presence of two chromodomains in KIS-L suggested that its recruitment or function might be regulated by the methylation of histone H3 lysine 4 by the trithorax group proteins ASH1 and TRX. Although we observed significant overlap between the distributions of KIS-L, ASH1, and TRX on polytene chromosomes, KIS-L did not bind methylated histone tails in vitro, and loss of TRX or ASH1 function did not alter the association of KIS-L with chromatin. By contrast, loss of kis function led to a dramatic reduction in the levels of TRX and ASH1 associated with chromatin and was accompanied by increased histone H3 lysine 27 methylation-a modification required for Polycomb group repression. A similar increase in H3 lysine 27 methylation was observed in ash1 and trx mutant larvae. Our findings suggest that KIS-L promotes early elongation and counteracts Polycomb group repression by recruiting the ASH1 and TRX histone methyltransferases to chromatin.

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Mutations in kis lead to increased H3K27 methylation.A–D) The level and distribution of H3K27me3 (A, B, red) and PC (C, D, green) on salivary gland polytene chromosomes of wild-type (A, C) and kisk13416 (B, D) larvae was examined by double-label indirect immunofluorescence microscopy. Split images of the distributions of H3K27me3 (red) and PC (green) on wild-type (E) and kisk13416 (F) polytene chromosomes are shown. Loss of kis function results in an increase in the levels of H3K27me3 without altering the distribution of this modification on polytene chromosomes. For panels E and F, the levels of H3K27me3 were independently processed using Adobe Photoshop software to facilitate the comparison of the methyl mark and PC. G) The distribution of H3K27me3 over the fkh gene was determined by ChIP using chromatin isolated from the salivary glands of wild-type (red bars) or kisk13416 (green bars) larvae. A map of the fkh gene is shown below the X axis; black bars represent the primers used to amplify the following regions: C1: region upstream of fkh, E: fkh enhancer, P: fkh transcription start site, B: fkh body, C2: region downstream of fkh. The ratio of DNA immunoprecipitated with antibodies against H3K27me3 and histone H3 are shown for each region. The bars represent the average of three independent biological experiments with the corresponding standard deviations. The loss of KIS-L function leads to a significant increase in H3K27me3 over the entire fkh region.
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pgen-1000217-g009: Mutations in kis lead to increased H3K27 methylation.A–D) The level and distribution of H3K27me3 (A, B, red) and PC (C, D, green) on salivary gland polytene chromosomes of wild-type (A, C) and kisk13416 (B, D) larvae was examined by double-label indirect immunofluorescence microscopy. Split images of the distributions of H3K27me3 (red) and PC (green) on wild-type (E) and kisk13416 (F) polytene chromosomes are shown. Loss of kis function results in an increase in the levels of H3K27me3 without altering the distribution of this modification on polytene chromosomes. For panels E and F, the levels of H3K27me3 were independently processed using Adobe Photoshop software to facilitate the comparison of the methyl mark and PC. G) The distribution of H3K27me3 over the fkh gene was determined by ChIP using chromatin isolated from the salivary glands of wild-type (red bars) or kisk13416 (green bars) larvae. A map of the fkh gene is shown below the X axis; black bars represent the primers used to amplify the following regions: C1: region upstream of fkh, E: fkh enhancer, P: fkh transcription start site, B: fkh body, C2: region downstream of fkh. The ratio of DNA immunoprecipitated with antibodies against H3K27me3 and histone H3 are shown for each region. The bars represent the average of three independent biological experiments with the corresponding standard deviations. The loss of KIS-L function leads to a significant increase in H3K27me3 over the entire fkh region.

Mentions: We previously noted that the majority (>80%) of PC binding sites in salivary gland polytene chromosomes are adjacent to sites of KIS-L binding [30]. The majority of sites of H3K27 methylation are also flanked on one or both sides by KIS-L (Figure S6). These observations, together with the lack of obvious changes in the level or distribution of PRC1 and PRC2 in kis mutants, suggested that KIS-L might counteract Polycomb group repression by antagonizing H3K27 methylation. To investigate this possibility, we stained salivary gland polytene chromosomes of wild-type and kisk13416 mutant larvae with an antibody that specifically recognizes this histone modification. Loss of kis function results in a large (∼7 fold) increase in the level of H3K27me3 on polytene chromosomes (Figure 9A and B) without altering the level or distribution of PC (Figure 9C and D). A similar increase in H3K27 trimethylation was observed over the entire fkh gene of kisk13416 mutant larvae by ChIP (Figure 9G).


Drosophila Kismet regulates histone H3 lysine 27 methylation and early elongation by RNA polymerase II.

Srinivasan S, Dorighi KM, Tamkun JW - PLoS Genet. (2008)

Mutations in kis lead to increased H3K27 methylation.A–D) The level and distribution of H3K27me3 (A, B, red) and PC (C, D, green) on salivary gland polytene chromosomes of wild-type (A, C) and kisk13416 (B, D) larvae was examined by double-label indirect immunofluorescence microscopy. Split images of the distributions of H3K27me3 (red) and PC (green) on wild-type (E) and kisk13416 (F) polytene chromosomes are shown. Loss of kis function results in an increase in the levels of H3K27me3 without altering the distribution of this modification on polytene chromosomes. For panels E and F, the levels of H3K27me3 were independently processed using Adobe Photoshop software to facilitate the comparison of the methyl mark and PC. G) The distribution of H3K27me3 over the fkh gene was determined by ChIP using chromatin isolated from the salivary glands of wild-type (red bars) or kisk13416 (green bars) larvae. A map of the fkh gene is shown below the X axis; black bars represent the primers used to amplify the following regions: C1: region upstream of fkh, E: fkh enhancer, P: fkh transcription start site, B: fkh body, C2: region downstream of fkh. The ratio of DNA immunoprecipitated with antibodies against H3K27me3 and histone H3 are shown for each region. The bars represent the average of three independent biological experiments with the corresponding standard deviations. The loss of KIS-L function leads to a significant increase in H3K27me3 over the entire fkh region.
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pgen-1000217-g009: Mutations in kis lead to increased H3K27 methylation.A–D) The level and distribution of H3K27me3 (A, B, red) and PC (C, D, green) on salivary gland polytene chromosomes of wild-type (A, C) and kisk13416 (B, D) larvae was examined by double-label indirect immunofluorescence microscopy. Split images of the distributions of H3K27me3 (red) and PC (green) on wild-type (E) and kisk13416 (F) polytene chromosomes are shown. Loss of kis function results in an increase in the levels of H3K27me3 without altering the distribution of this modification on polytene chromosomes. For panels E and F, the levels of H3K27me3 were independently processed using Adobe Photoshop software to facilitate the comparison of the methyl mark and PC. G) The distribution of H3K27me3 over the fkh gene was determined by ChIP using chromatin isolated from the salivary glands of wild-type (red bars) or kisk13416 (green bars) larvae. A map of the fkh gene is shown below the X axis; black bars represent the primers used to amplify the following regions: C1: region upstream of fkh, E: fkh enhancer, P: fkh transcription start site, B: fkh body, C2: region downstream of fkh. The ratio of DNA immunoprecipitated with antibodies against H3K27me3 and histone H3 are shown for each region. The bars represent the average of three independent biological experiments with the corresponding standard deviations. The loss of KIS-L function leads to a significant increase in H3K27me3 over the entire fkh region.
Mentions: We previously noted that the majority (>80%) of PC binding sites in salivary gland polytene chromosomes are adjacent to sites of KIS-L binding [30]. The majority of sites of H3K27 methylation are also flanked on one or both sides by KIS-L (Figure S6). These observations, together with the lack of obvious changes in the level or distribution of PRC1 and PRC2 in kis mutants, suggested that KIS-L might counteract Polycomb group repression by antagonizing H3K27 methylation. To investigate this possibility, we stained salivary gland polytene chromosomes of wild-type and kisk13416 mutant larvae with an antibody that specifically recognizes this histone modification. Loss of kis function results in a large (∼7 fold) increase in the level of H3K27me3 on polytene chromosomes (Figure 9A and B) without altering the level or distribution of PC (Figure 9C and D). A similar increase in H3K27 trimethylation was observed over the entire fkh gene of kisk13416 mutant larvae by ChIP (Figure 9G).

Bottom Line: Although we observed significant overlap between the distributions of KIS-L, ASH1, and TRX on polytene chromosomes, KIS-L did not bind methylated histone tails in vitro, and loss of TRX or ASH1 function did not alter the association of KIS-L with chromatin.By contrast, loss of kis function led to a dramatic reduction in the levels of TRX and ASH1 associated with chromatin and was accompanied by increased histone H3 lysine 27 methylation-a modification required for Polycomb group repression.Our findings suggest that KIS-L promotes early elongation and counteracts Polycomb group repression by recruiting the ASH1 and TRX histone methyltransferases to chromatin.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cell, and Developmental Biology, University of California Santa Cruz, Santa Cruz, CA, USA.

ABSTRACT
Polycomb and trithorax group proteins regulate cellular pluripotency and differentiation by maintaining hereditable states of transcription. Many Polycomb and trithorax group proteins have been implicated in the covalent modification or remodeling of chromatin, but how they interact with each other and the general transcription machinery to regulate transcription is not well understood. The trithorax group protein Kismet-L (KIS-L) is a member of the CHD subfamily of chromatin-remodeling factors that plays a global role in transcription by RNA polymerase II (Pol II). Mutations in CHD7, the human counterpart of kis, are associated with CHARGE syndrome, a developmental disorder affecting multiple tissues and organs. To clarify how KIS-L activates gene expression and counteracts Polycomb group silencing, we characterized defects resulting from the loss of KIS-L function in Drosophila. These studies revealed that KIS-L acts downstream of P-TEFb recruitment to stimulate elongation by Pol II. The presence of two chromodomains in KIS-L suggested that its recruitment or function might be regulated by the methylation of histone H3 lysine 4 by the trithorax group proteins ASH1 and TRX. Although we observed significant overlap between the distributions of KIS-L, ASH1, and TRX on polytene chromosomes, KIS-L did not bind methylated histone tails in vitro, and loss of TRX or ASH1 function did not alter the association of KIS-L with chromatin. By contrast, loss of kis function led to a dramatic reduction in the levels of TRX and ASH1 associated with chromatin and was accompanied by increased histone H3 lysine 27 methylation-a modification required for Polycomb group repression. A similar increase in H3 lysine 27 methylation was observed in ash1 and trx mutant larvae. Our findings suggest that KIS-L promotes early elongation and counteracts Polycomb group repression by recruiting the ASH1 and TRX histone methyltransferases to chromatin.

Show MeSH
Related in: MedlinePlus