Limits...
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.

Show MeSH

Related in: MedlinePlus

H3K4 methylation is not sufficient for the recruitment of KIS-L to chromatin.A) CD2 of KIS-L is aligned with CD2 of other CHD proteins and the chromodomains of Drosophila HP1 and PC. Identical and conserved amino acids are highlighted in black and grey, respectively. Aromatic amino acids that are important for binding of methylated histone tails by the CD2 of yeast CHD1 are marked by stars. B) The in vitro binding of HIS-tagged CD2 of KIS-L, CD1 and 2 of human CHD1 and the Drosophila HP1 chromodomain to histone H3K4me2, H3K4me3, H3K9me2 and histone H3 peptides were examined. Input (I), unbound protein (S) and the bound proteins (P) were detected by western blotting using anti-HIS tag antibody. Note that the chromodomains of HP1 and human CHD1, but not KIS-L, specifically bound methylated H3K9 and H3K4 peptides, respectively. C–D) The distributions of H3K4me2 (C, red) and H3K4me3 (D, red) were compared to that of KIS-L (C and D, green) on a representative region of wild-type polytene chromosomes. The arrowheads represent H3K4me2 and H3K4me3 bands that do not overlap with KIS-L, respectively, while the arrows represent bands of KIS-L that do not overlap with H3K4me2 and H3K4me3 bands, respectively. E–F) The distributions of KIS-L and H3K4me3 over the fkh gene were 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. For KIS-L, the percentages of DNA immunoprecipitated for regions E, P, B and C2 were normalized to the percentage of DNA immunoprecipitated for region C1 (E). The ratio of DNA immunoprecipitated with antibodies against H3K4me3 and histone H3 are shown for each region (F). Note that KIS-L is enriched over the transcription start site of fkh while H3K4me3 is enriched over both the transcription start site and the body of fkh gene. The bars represent the average of independent biological experiments (n = 4 for H3K4me3 and n = 5 for KIS-L) with the corresponding standard deviations.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2563034&req=5

pgen-1000217-g003: H3K4 methylation is not sufficient for the recruitment of KIS-L to chromatin.A) CD2 of KIS-L is aligned with CD2 of other CHD proteins and the chromodomains of Drosophila HP1 and PC. Identical and conserved amino acids are highlighted in black and grey, respectively. Aromatic amino acids that are important for binding of methylated histone tails by the CD2 of yeast CHD1 are marked by stars. B) The in vitro binding of HIS-tagged CD2 of KIS-L, CD1 and 2 of human CHD1 and the Drosophila HP1 chromodomain to histone H3K4me2, H3K4me3, H3K9me2 and histone H3 peptides were examined. Input (I), unbound protein (S) and the bound proteins (P) were detected by western blotting using anti-HIS tag antibody. Note that the chromodomains of HP1 and human CHD1, but not KIS-L, specifically bound methylated H3K9 and H3K4 peptides, respectively. C–D) The distributions of H3K4me2 (C, red) and H3K4me3 (D, red) were compared to that of KIS-L (C and D, green) on a representative region of wild-type polytene chromosomes. The arrowheads represent H3K4me2 and H3K4me3 bands that do not overlap with KIS-L, respectively, while the arrows represent bands of KIS-L that do not overlap with H3K4me2 and H3K4me3 bands, respectively. E–F) The distributions of KIS-L and H3K4me3 over the fkh gene were 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. For KIS-L, the percentages of DNA immunoprecipitated for regions E, P, B and C2 were normalized to the percentage of DNA immunoprecipitated for region C1 (E). The ratio of DNA immunoprecipitated with antibodies against H3K4me3 and histone H3 are shown for each region (F). Note that KIS-L is enriched over the transcription start site of fkh while H3K4me3 is enriched over both the transcription start site and the body of fkh gene. The bars represent the average of independent biological experiments (n = 4 for H3K4me3 and n = 5 for KIS-L) with the corresponding standard deviations.

Mentions: CD2 of KIS-L is highly related to chromodomains that directly bind methylated histone tails (Figure 3A), including CD2 of yeast CHD1, which binds both di- and tri-methylated H3K4 [33]. This similarity suggested that KIS-L might directly bind methylated H3K4. To investigate this possibility, we examined the ability of recombinant KIS-L proteins to bind immobilized synthetic peptides corresponding to N-terminal histone tails. A recombinant protein corresponding to KIS-L CD2 (residues 1937 to 1997) did not bind unmodified histone H3 tails or a variety of methylated H3 tails (including H3K4me2, H3K4me3, and H3K9me2), even at relatively low (150 mM) salt concentrations (Figure 3B). By contrast, we were able to detect the binding of the Drosophila HP1 chromodomain to H3K9me2 using this assay (Figure 3B), as previously observed by others [45],[46]. Recent studies of the human CHD1 protein have shown that both CD1 and CD2 are required for binding of methylated H3K4 in vitro [32],[34]. While we were able to reproduce this result (Figure 3B), a comparable recombinant protein spanning CD1 and CD2 of KIS-L – as well as full-length KIS-L proteins from embryo extracts – bound both unmodified and methylated H3 and H4 tails (data not shown), presumably due to non-specific ionic interactions with the positively charged tails. We were therefore unable to determine if regions outside of CD2 enable KIS-L to bind methylated histone tails. Thus, although KIS-L CD2 failed to interact with methylated histone tails in vitro, it remains possible that the full-length KIS-L protein recognizes one or more histone modifications in vivo.


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

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

H3K4 methylation is not sufficient for the recruitment of KIS-L to chromatin.A) CD2 of KIS-L is aligned with CD2 of other CHD proteins and the chromodomains of Drosophila HP1 and PC. Identical and conserved amino acids are highlighted in black and grey, respectively. Aromatic amino acids that are important for binding of methylated histone tails by the CD2 of yeast CHD1 are marked by stars. B) The in vitro binding of HIS-tagged CD2 of KIS-L, CD1 and 2 of human CHD1 and the Drosophila HP1 chromodomain to histone H3K4me2, H3K4me3, H3K9me2 and histone H3 peptides were examined. Input (I), unbound protein (S) and the bound proteins (P) were detected by western blotting using anti-HIS tag antibody. Note that the chromodomains of HP1 and human CHD1, but not KIS-L, specifically bound methylated H3K9 and H3K4 peptides, respectively. C–D) The distributions of H3K4me2 (C, red) and H3K4me3 (D, red) were compared to that of KIS-L (C and D, green) on a representative region of wild-type polytene chromosomes. The arrowheads represent H3K4me2 and H3K4me3 bands that do not overlap with KIS-L, respectively, while the arrows represent bands of KIS-L that do not overlap with H3K4me2 and H3K4me3 bands, respectively. E–F) The distributions of KIS-L and H3K4me3 over the fkh gene were 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. For KIS-L, the percentages of DNA immunoprecipitated for regions E, P, B and C2 were normalized to the percentage of DNA immunoprecipitated for region C1 (E). The ratio of DNA immunoprecipitated with antibodies against H3K4me3 and histone H3 are shown for each region (F). Note that KIS-L is enriched over the transcription start site of fkh while H3K4me3 is enriched over both the transcription start site and the body of fkh gene. The bars represent the average of independent biological experiments (n = 4 for H3K4me3 and n = 5 for KIS-L) with the corresponding standard deviations.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2563034&req=5

pgen-1000217-g003: H3K4 methylation is not sufficient for the recruitment of KIS-L to chromatin.A) CD2 of KIS-L is aligned with CD2 of other CHD proteins and the chromodomains of Drosophila HP1 and PC. Identical and conserved amino acids are highlighted in black and grey, respectively. Aromatic amino acids that are important for binding of methylated histone tails by the CD2 of yeast CHD1 are marked by stars. B) The in vitro binding of HIS-tagged CD2 of KIS-L, CD1 and 2 of human CHD1 and the Drosophila HP1 chromodomain to histone H3K4me2, H3K4me3, H3K9me2 and histone H3 peptides were examined. Input (I), unbound protein (S) and the bound proteins (P) were detected by western blotting using anti-HIS tag antibody. Note that the chromodomains of HP1 and human CHD1, but not KIS-L, specifically bound methylated H3K9 and H3K4 peptides, respectively. C–D) The distributions of H3K4me2 (C, red) and H3K4me3 (D, red) were compared to that of KIS-L (C and D, green) on a representative region of wild-type polytene chromosomes. The arrowheads represent H3K4me2 and H3K4me3 bands that do not overlap with KIS-L, respectively, while the arrows represent bands of KIS-L that do not overlap with H3K4me2 and H3K4me3 bands, respectively. E–F) The distributions of KIS-L and H3K4me3 over the fkh gene were 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. For KIS-L, the percentages of DNA immunoprecipitated for regions E, P, B and C2 were normalized to the percentage of DNA immunoprecipitated for region C1 (E). The ratio of DNA immunoprecipitated with antibodies against H3K4me3 and histone H3 are shown for each region (F). Note that KIS-L is enriched over the transcription start site of fkh while H3K4me3 is enriched over both the transcription start site and the body of fkh gene. The bars represent the average of independent biological experiments (n = 4 for H3K4me3 and n = 5 for KIS-L) with the corresponding standard deviations.
Mentions: CD2 of KIS-L is highly related to chromodomains that directly bind methylated histone tails (Figure 3A), including CD2 of yeast CHD1, which binds both di- and tri-methylated H3K4 [33]. This similarity suggested that KIS-L might directly bind methylated H3K4. To investigate this possibility, we examined the ability of recombinant KIS-L proteins to bind immobilized synthetic peptides corresponding to N-terminal histone tails. A recombinant protein corresponding to KIS-L CD2 (residues 1937 to 1997) did not bind unmodified histone H3 tails or a variety of methylated H3 tails (including H3K4me2, H3K4me3, and H3K9me2), even at relatively low (150 mM) salt concentrations (Figure 3B). By contrast, we were able to detect the binding of the Drosophila HP1 chromodomain to H3K9me2 using this assay (Figure 3B), as previously observed by others [45],[46]. Recent studies of the human CHD1 protein have shown that both CD1 and CD2 are required for binding of methylated H3K4 in vitro [32],[34]. While we were able to reproduce this result (Figure 3B), a comparable recombinant protein spanning CD1 and CD2 of KIS-L – as well as full-length KIS-L proteins from embryo extracts – bound both unmodified and methylated H3 and H4 tails (data not shown), presumably due to non-specific ionic interactions with the positively charged tails. We were therefore unable to determine if regions outside of CD2 enable KIS-L to bind methylated histone tails. Thus, although KIS-L CD2 failed to interact with methylated histone tails in vitro, it remains possible that the full-length KIS-L protein recognizes one or more histone modifications in vivo.

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