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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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KIS-L is required for the association of ASH1 and TRX with chromatin.The distribution of ASH1 (A, C, red) and TRX (E, G, red) on salivary gland polytene chromosomes isolated from wild-type and kisk13416 larvae were detected by indirect immunofluorescence microscopy. The chromosomes were also stained with an antibody against Pol IIa (B, D, F, H, green) as an internal control. The loss of KIS-L function dramatically reduces the levels of ASH1 and TRX, but not Pol IIa, associated with polytene chromosomes.
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pgen-1000217-g006: KIS-L is required for the association of ASH1 and TRX with chromatin.The distribution of ASH1 (A, C, red) and TRX (E, G, red) on salivary gland polytene chromosomes isolated from wild-type and kisk13416 larvae were detected by indirect immunofluorescence microscopy. The chromosomes were also stained with an antibody against Pol IIa (B, D, F, H, green) as an internal control. The loss of KIS-L function dramatically reduces the levels of ASH1 and TRX, but not Pol IIa, associated with polytene chromosomes.

Mentions: In some cases, chromatin-remodeling factors stimulate transcription by recruiting histone-modifying enzymes to promoters [52],[53]. We therefore examined if KIS-L is required for the association of ASH1 and TRX with chromatin. The loss of kis function resulted in a significant reduction in the levels of both ASH1 and TRX associated with polytene chromosomes (Figure 6A–G). This is unlikely to result from the decreased expression of ASH1 or TRX, as western blotting indicated that both proteins, though slightly reduced, were still present in kis mutants (Figure S2). A few residual bands of relatively strong ASH1 and TRX staining were observed in the mutants (Figure 6C and G), suggesting that the recruitment of the two trithorax group proteins to a small number of chromosomal sites may be independent of KIS-L. These results demonstrate that KIS-L is required for the recruitment of ASH1 and TRX to the majority of their target genes 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)

KIS-L is required for the association of ASH1 and TRX with chromatin.The distribution of ASH1 (A, C, red) and TRX (E, G, red) on salivary gland polytene chromosomes isolated from wild-type and kisk13416 larvae were detected by indirect immunofluorescence microscopy. The chromosomes were also stained with an antibody against Pol IIa (B, D, F, H, green) as an internal control. The loss of KIS-L function dramatically reduces the levels of ASH1 and TRX, but not Pol IIa, associated with polytene chromosomes.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1000217-g006: KIS-L is required for the association of ASH1 and TRX with chromatin.The distribution of ASH1 (A, C, red) and TRX (E, G, red) on salivary gland polytene chromosomes isolated from wild-type and kisk13416 larvae were detected by indirect immunofluorescence microscopy. The chromosomes were also stained with an antibody against Pol IIa (B, D, F, H, green) as an internal control. The loss of KIS-L function dramatically reduces the levels of ASH1 and TRX, but not Pol IIa, associated with polytene chromosomes.
Mentions: In some cases, chromatin-remodeling factors stimulate transcription by recruiting histone-modifying enzymes to promoters [52],[53]. We therefore examined if KIS-L is required for the association of ASH1 and TRX with chromatin. The loss of kis function resulted in a significant reduction in the levels of both ASH1 and TRX associated with polytene chromosomes (Figure 6A–G). This is unlikely to result from the decreased expression of ASH1 or TRX, as western blotting indicated that both proteins, though slightly reduced, were still present in kis mutants (Figure S2). A few residual bands of relatively strong ASH1 and TRX staining were observed in the mutants (Figure 6C and G), suggesting that the recruitment of the two trithorax group proteins to a small number of chromosomal sites may be independent of KIS-L. These results demonstrate that KIS-L is required for the recruitment of ASH1 and TRX to the majority of their target genes 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