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Molecular landscape of modified histones in Drosophila heterochromatic genes and euchromatin-heterochromatin transition zones.

Yasuhara JC, Wakimoto BT - PLoS Genet. (2007)

Bottom Line: We found that H3-di-methylated-at-lysine 9 (H3K9me2) was depleted at the 5' ends but enriched throughout transcribed regions of heterochromatic genes.Moreover, the profile was only subtly affected by a Su(var)3-9 mutation, implicating a histone methyltransferase other than SU(VAR)3-9 as responsible for most H3K9me2 associated with heterochromatic genes in embryos.The results are also relevant for understanding the effects of chromosome aberrations and the megabase scale over which epigenetic position effects can operate in multicellular organisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
Constitutive heterochromatin is enriched in repetitive sequences and histone H3-methylated-at-lysine 9. Both components contribute to heterochromatin's ability to silence euchromatic genes. However, heterochromatin also harbors hundreds of expressed genes in organisms such as Drosophila. Recent studies have provided a detailed picture of sequence organization of D. melanogaster heterochromatin, but how histone modifications are associated with heterochromatic sequences at high resolution has not been described. Here, distributions of modified histones in the vicinity of heterochromatic genes of normal embryos and embryos homozygous for a chromosome rearrangement were characterized using chromatin immunoprecipitation and genome tiling arrays. We found that H3-di-methylated-at-lysine 9 (H3K9me2) was depleted at the 5' ends but enriched throughout transcribed regions of heterochromatic genes. The profile was distinct from that of euchromatic genes and suggests that heterochromatic genes are integrated into, rather than insulated from, the H3K9me2-enriched domain. Moreover, the profile was only subtly affected by a Su(var)3-9 mutation, implicating a histone methyltransferase other than SU(VAR)3-9 as responsible for most H3K9me2 associated with heterochromatic genes in embryos. On a chromosomal scale, we observed a sharp transition to the H3K9me2 domain, which coincided with increased retrotransposon density in the euchromatin-heterochromatin (eu-het) transition zones on the long chromosome arms. Thus, a certain density of retrotransposons, rather than specific boundary elements, may demarcate Drosophila pericentric heterochromatin. We also demonstrate that a chromosome rearrangement that created a new eu-het junction altered H3K9me2 distribution and induced new euchromatic sites of enrichment as far as several megabases away from the breakpoint. Taken together, the findings argue against simple classification of H3K9me as the definitive signature of silenced genes, and clarify roles of histone modifications and repetitive DNAs in heterochromatin. The results are also relevant for understanding the effects of chromosome aberrations and the megabase scale over which epigenetic position effects can operate in multicellular organisms.

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Distribution of Modified Histones in Representative Heterochromatic and Euchromatic Genes(A) ChIP results for the light gene. The top diagram summarizes gene structure, using the key shown in the figure. Gray blocks on the top line indicate whether the region is repetitive (high bar) or unique (no bar). For this diagram, non-overlapping 100 bp windows were individually BLASTed against the whole genome sequence and each hit with E < 10−3 was counted as one copy. The colored arrows show orientation and types of transposable elements located in the repetitive regions (INE-1 is a highly abundant TE that constitutes its own category). The black arrow depicts the exons (black blocks) and introns (black lines). Pairs of opposing black triangles with identification numbers show the locations of ten PCR primer pairs used in the ChIP assay. Left panels show the gel assay of quantitative duplex PCR of input or ChIP samples as indicated, with the numbers identifying the corresponding PCR primer pair used for each lane. The common bottom band in each lane is the normalization control Pdi (Materials and Methods). The bar graphs summarize ChIP results across the length of the gene (x-axis), with bar heights showing average enrichment factors (y-axis) and standard errors for three trials for H3K9me2 (top graph), and two trials each for H3K4me2 (middle) and H3K9/14acet (bottom). ChIP results for (B) concertina and (C) Chitinase with the same number of trials as described for lt. ChIP results for two euchromatic control genes, (D) Moca-cyp and (E) CG1646 with two trials each.
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pgen-0040016-g001: Distribution of Modified Histones in Representative Heterochromatic and Euchromatic Genes(A) ChIP results for the light gene. The top diagram summarizes gene structure, using the key shown in the figure. Gray blocks on the top line indicate whether the region is repetitive (high bar) or unique (no bar). For this diagram, non-overlapping 100 bp windows were individually BLASTed against the whole genome sequence and each hit with E < 10−3 was counted as one copy. The colored arrows show orientation and types of transposable elements located in the repetitive regions (INE-1 is a highly abundant TE that constitutes its own category). The black arrow depicts the exons (black blocks) and introns (black lines). Pairs of opposing black triangles with identification numbers show the locations of ten PCR primer pairs used in the ChIP assay. Left panels show the gel assay of quantitative duplex PCR of input or ChIP samples as indicated, with the numbers identifying the corresponding PCR primer pair used for each lane. The common bottom band in each lane is the normalization control Pdi (Materials and Methods). The bar graphs summarize ChIP results across the length of the gene (x-axis), with bar heights showing average enrichment factors (y-axis) and standard errors for three trials for H3K9me2 (top graph), and two trials each for H3K4me2 (middle) and H3K9/14acet (bottom). ChIP results for (B) concertina and (C) Chitinase with the same number of trials as described for lt. ChIP results for two euchromatic control genes, (D) Moca-cyp and (E) CG1646 with two trials each.

Mentions: The exons of the 2Lh genes are unique sequence, but the bulk of introns and flanking sequences consist of repetitive TE-like sequences (Figure 1). This structure required careful design of PCR primer pairs for the ChIP assay since detection of recovered fragments is hybridization-based. PCR primer pairs were designed so: (i) at least one primer in the pair was unique sequence, or (ii) both primers in the pair were different repetitive sequences but the juxtaposition of the two sequences was a unique occurrence in the genome. There were three instances in the case of the lt gene in which both primers in a pair were anchored in repetitive sequences. We verified specificity of these primer sets by showing that PCR fragments were amplified from DNA of normal embryos, but not from embryos deleted for the lt gene region (Figure S2). This scheme allowed specific detection of 2Lh gene sequences, despite the fact that most regions are repeated many times in the genome.


Molecular landscape of modified histones in Drosophila heterochromatic genes and euchromatin-heterochromatin transition zones.

Yasuhara JC, Wakimoto BT - PLoS Genet. (2007)

Distribution of Modified Histones in Representative Heterochromatic and Euchromatic Genes(A) ChIP results for the light gene. The top diagram summarizes gene structure, using the key shown in the figure. Gray blocks on the top line indicate whether the region is repetitive (high bar) or unique (no bar). For this diagram, non-overlapping 100 bp windows were individually BLASTed against the whole genome sequence and each hit with E < 10−3 was counted as one copy. The colored arrows show orientation and types of transposable elements located in the repetitive regions (INE-1 is a highly abundant TE that constitutes its own category). The black arrow depicts the exons (black blocks) and introns (black lines). Pairs of opposing black triangles with identification numbers show the locations of ten PCR primer pairs used in the ChIP assay. Left panels show the gel assay of quantitative duplex PCR of input or ChIP samples as indicated, with the numbers identifying the corresponding PCR primer pair used for each lane. The common bottom band in each lane is the normalization control Pdi (Materials and Methods). The bar graphs summarize ChIP results across the length of the gene (x-axis), with bar heights showing average enrichment factors (y-axis) and standard errors for three trials for H3K9me2 (top graph), and two trials each for H3K4me2 (middle) and H3K9/14acet (bottom). ChIP results for (B) concertina and (C) Chitinase with the same number of trials as described for lt. ChIP results for two euchromatic control genes, (D) Moca-cyp and (E) CG1646 with two trials each.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211541&req=5

pgen-0040016-g001: Distribution of Modified Histones in Representative Heterochromatic and Euchromatic Genes(A) ChIP results for the light gene. The top diagram summarizes gene structure, using the key shown in the figure. Gray blocks on the top line indicate whether the region is repetitive (high bar) or unique (no bar). For this diagram, non-overlapping 100 bp windows were individually BLASTed against the whole genome sequence and each hit with E < 10−3 was counted as one copy. The colored arrows show orientation and types of transposable elements located in the repetitive regions (INE-1 is a highly abundant TE that constitutes its own category). The black arrow depicts the exons (black blocks) and introns (black lines). Pairs of opposing black triangles with identification numbers show the locations of ten PCR primer pairs used in the ChIP assay. Left panels show the gel assay of quantitative duplex PCR of input or ChIP samples as indicated, with the numbers identifying the corresponding PCR primer pair used for each lane. The common bottom band in each lane is the normalization control Pdi (Materials and Methods). The bar graphs summarize ChIP results across the length of the gene (x-axis), with bar heights showing average enrichment factors (y-axis) and standard errors for three trials for H3K9me2 (top graph), and two trials each for H3K4me2 (middle) and H3K9/14acet (bottom). ChIP results for (B) concertina and (C) Chitinase with the same number of trials as described for lt. ChIP results for two euchromatic control genes, (D) Moca-cyp and (E) CG1646 with two trials each.
Mentions: The exons of the 2Lh genes are unique sequence, but the bulk of introns and flanking sequences consist of repetitive TE-like sequences (Figure 1). This structure required careful design of PCR primer pairs for the ChIP assay since detection of recovered fragments is hybridization-based. PCR primer pairs were designed so: (i) at least one primer in the pair was unique sequence, or (ii) both primers in the pair were different repetitive sequences but the juxtaposition of the two sequences was a unique occurrence in the genome. There were three instances in the case of the lt gene in which both primers in a pair were anchored in repetitive sequences. We verified specificity of these primer sets by showing that PCR fragments were amplified from DNA of normal embryos, but not from embryos deleted for the lt gene region (Figure S2). This scheme allowed specific detection of 2Lh gene sequences, despite the fact that most regions are repeated many times in the genome.

Bottom Line: We found that H3-di-methylated-at-lysine 9 (H3K9me2) was depleted at the 5' ends but enriched throughout transcribed regions of heterochromatic genes.Moreover, the profile was only subtly affected by a Su(var)3-9 mutation, implicating a histone methyltransferase other than SU(VAR)3-9 as responsible for most H3K9me2 associated with heterochromatic genes in embryos.The results are also relevant for understanding the effects of chromosome aberrations and the megabase scale over which epigenetic position effects can operate in multicellular organisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
Constitutive heterochromatin is enriched in repetitive sequences and histone H3-methylated-at-lysine 9. Both components contribute to heterochromatin's ability to silence euchromatic genes. However, heterochromatin also harbors hundreds of expressed genes in organisms such as Drosophila. Recent studies have provided a detailed picture of sequence organization of D. melanogaster heterochromatin, but how histone modifications are associated with heterochromatic sequences at high resolution has not been described. Here, distributions of modified histones in the vicinity of heterochromatic genes of normal embryos and embryos homozygous for a chromosome rearrangement were characterized using chromatin immunoprecipitation and genome tiling arrays. We found that H3-di-methylated-at-lysine 9 (H3K9me2) was depleted at the 5' ends but enriched throughout transcribed regions of heterochromatic genes. The profile was distinct from that of euchromatic genes and suggests that heterochromatic genes are integrated into, rather than insulated from, the H3K9me2-enriched domain. Moreover, the profile was only subtly affected by a Su(var)3-9 mutation, implicating a histone methyltransferase other than SU(VAR)3-9 as responsible for most H3K9me2 associated with heterochromatic genes in embryos. On a chromosomal scale, we observed a sharp transition to the H3K9me2 domain, which coincided with increased retrotransposon density in the euchromatin-heterochromatin (eu-het) transition zones on the long chromosome arms. Thus, a certain density of retrotransposons, rather than specific boundary elements, may demarcate Drosophila pericentric heterochromatin. We also demonstrate that a chromosome rearrangement that created a new eu-het junction altered H3K9me2 distribution and induced new euchromatic sites of enrichment as far as several megabases away from the breakpoint. Taken together, the findings argue against simple classification of H3K9me as the definitive signature of silenced genes, and clarify roles of histone modifications and repetitive DNAs in heterochromatin. The results are also relevant for understanding the effects of chromosome aberrations and the megabase scale over which epigenetic position effects can operate in multicellular organisms.

Show MeSH
Related in: MedlinePlus