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Highly condensed chromatins are formed adjacent to subtelomeric and decondensed silent chromatin in fission yeast.

Matsuda A, Chikashige Y, Ding DQ, Ohtsuki C, Mori C, Asakawa H, Kimura H, Haraguchi T, Hiraoka Y - Nat Commun (2015)

Bottom Line: Knob regions span ∼50 kb of sequence devoid of methylated histones.Disruption of methylation at lysine 36 of histone H3 (H3K36) eliminates knob formation and gene repression at the subtelomeric and adjacent knob regions.Thus, epigenetic marks at H3K36 play crucial roles in the formation of a unique chromatin structure and in gene regulation at those regions in S. pombe.

View Article: PubMed Central - PubMed

Affiliation: 1] Advanced ICT Research Institute Kobe, National Institute of Information and Communications Technology, 588-2, Iwaoka, Iwaoka-cho, Kobe 651-2492, Japan [2] Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita 565-0871, Japan.

ABSTRACT
It is generally believed that silent chromatin is condensed and transcriptionally active chromatin is decondensed. However, little is known about the relationship between the condensation levels and gene expression. Here we report the condensation levels of interphase chromatin in the fission yeast Schizosaccharomyces pombe examined by super-resolution fluorescence microscopy. Unexpectedly, silent chromatin is less condensed than the euchromatin. Furthermore, the telomeric silent regions are flanked by highly condensed chromatin bodies, or 'knobs'. Knob regions span ∼50 kb of sequence devoid of methylated histones. Knob condensation is independent of HP1 homologue Swi6 and other gene silencing factors. Disruption of methylation at lysine 36 of histone H3 (H3K36) eliminates knob formation and gene repression at the subtelomeric and adjacent knob regions. Thus, epigenetic marks at H3K36 play crucial roles in the formation of a unique chromatin structure and in gene regulation at those regions in S. pombe.

No MeSH data available.


Related in: MedlinePlus

Knob condensation is dynamically regulated in the cell cycle.(a) Live imaging of three independent cells expressing H2B-GFP. Pictures were taken every 7.5 min but here images are shown in 15-min steps. Deconvolved optical sections with the brightest dots at each time frame are shown. Scale bar, 1 μm. (b) Frequency of knob-containing nuclei. The number of knobs in a nucleus is indicated by different colours. Standard growth conditions (wild type in EMM2 5S at 26 °C) are indicated as ‘−' and any differences from these conditions are indicated. ‘H3S10ph' means standard cells positively stained with anti-H3S10ph antibody, which is specific for the M phase. Cell cycle was arrested with hydroxyurea (‘HU'), temperature-sensitive mutations or nitrogen starvation (‘-N') at S, G1 or G2. Numbers in parenthesis indicate the number of nuclei examined. (c) An optical section of a typical anaphase nucleus (DAPI staining; blue) without a knob near the subtelomeric markers (Taz1-GFP in green and H3K9me3 in red). The lower panel shows DAPI staining in white marked with Taz1-GFP (green contours) and H3K9me3 foci (red contours) coexisting at a single chromosome end. The chromosomes are pulled in the direction of the arrows. The bar indicates 500 nm for c,d. (d) An optical section of a typical DAPI-stained nucleus containing two knobs, commonly found in HU-treated cells.
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f5: Knob condensation is dynamically regulated in the cell cycle.(a) Live imaging of three independent cells expressing H2B-GFP. Pictures were taken every 7.5 min but here images are shown in 15-min steps. Deconvolved optical sections with the brightest dots at each time frame are shown. Scale bar, 1 μm. (b) Frequency of knob-containing nuclei. The number of knobs in a nucleus is indicated by different colours. Standard growth conditions (wild type in EMM2 5S at 26 °C) are indicated as ‘−' and any differences from these conditions are indicated. ‘H3S10ph' means standard cells positively stained with anti-H3S10ph antibody, which is specific for the M phase. Cell cycle was arrested with hydroxyurea (‘HU'), temperature-sensitive mutations or nitrogen starvation (‘-N') at S, G1 or G2. Numbers in parenthesis indicate the number of nuclei examined. (c) An optical section of a typical anaphase nucleus (DAPI staining; blue) without a knob near the subtelomeric markers (Taz1-GFP in green and H3K9me3 in red). The lower panel shows DAPI staining in white marked with Taz1-GFP (green contours) and H3K9me3 foci (red contours) coexisting at a single chromosome end. The chromosomes are pulled in the direction of the arrows. The bar indicates 500 nm for c,d. (d) An optical section of a typical DAPI-stained nucleus containing two knobs, commonly found in HU-treated cells.

Mentions: The genome of S. pombe has well-studied, discrete silent heterochromatic regions marked by histone H3 methylated at K9 (H3K9me). The subtelomeric and pericentromeric regions are the two largest heterochromatic regions (∼50 kb each) and are suitable for cytological analysis using 3DSIM because of their large size in S. pombe. Unexpectedly, known silent regions indicated by H3K9me3, as well as regions indicated by markers for the centromere (Cnp1-GFP) and the telomere (Taz1-GFP) had the lowest DNA concentration of the regions examined in this study (Fig. 2b,c). In particular, the Cnp1-GFP region is the innermost kinetochore region of the centromere (cnt) where foreign genes are known to be silenced11, but exhibited the lowest condensation level (Fig. 2b,c). Taz1 is the orthologue of TRF in mammals and recognizes telomere-associated sequences12. Although the length of the Taz1-binding sites could be too short to image with 3DSIM, the results showed that telomere-associated sequences were separate from any nearby condensed regions (Fig. 2c). H3K9me3 foci were found close to Cnp1- or Taz1-GFP foci, and often found in between the chromatin mass and the outside regions (Fig. 2c; see also Figs 3a, 4c and 5c). This was also the case when we used histone H3, instead of DAPI, as the chromatin marker (Fig. 2d, Supplementary Fig. 2a). The results for H3K9me2 were essentially identical to those for H3K9me3, although the antibody against H3K9me3 produced a more specific and intense signal than that against H3K9me2 (Supplementary Fig. 2b,c). In addition, the overlap with DAPI staining was similar for pericentromeric and subtelomeric H3K9me3 loci (Supplementary Fig. 2d,e). Silent regions marked by H3K9me3 (about 50 kb each) should be large enough to recognize with 3DSIM as we were able to visualize other chromatin regions of a similar size, that is, ‘knob' (see below). Decondensed silent chromatin was already predicted from reported micrococcal nuclease I (MNaseI) protection assays, where subtelomeric and pericentromeric heterochromatin regions had lower MNaseI protection than the rest of the genome1314 (summarized in Supplementary Fig. 5; also see Fig. 7a). Also, origins of replication in pericentromeric regions and the silent mating-type locus are replicated early in S phase1516. Furthermore, the cnt region of centromere is known to be modified by H3K4me3 (ref. 17). Collectively, the evidence indicates that silent regions in S. pombe are in fact less condensed than active regions.


Highly condensed chromatins are formed adjacent to subtelomeric and decondensed silent chromatin in fission yeast.

Matsuda A, Chikashige Y, Ding DQ, Ohtsuki C, Mori C, Asakawa H, Kimura H, Haraguchi T, Hiraoka Y - Nat Commun (2015)

Knob condensation is dynamically regulated in the cell cycle.(a) Live imaging of three independent cells expressing H2B-GFP. Pictures were taken every 7.5 min but here images are shown in 15-min steps. Deconvolved optical sections with the brightest dots at each time frame are shown. Scale bar, 1 μm. (b) Frequency of knob-containing nuclei. The number of knobs in a nucleus is indicated by different colours. Standard growth conditions (wild type in EMM2 5S at 26 °C) are indicated as ‘−' and any differences from these conditions are indicated. ‘H3S10ph' means standard cells positively stained with anti-H3S10ph antibody, which is specific for the M phase. Cell cycle was arrested with hydroxyurea (‘HU'), temperature-sensitive mutations or nitrogen starvation (‘-N') at S, G1 or G2. Numbers in parenthesis indicate the number of nuclei examined. (c) An optical section of a typical anaphase nucleus (DAPI staining; blue) without a knob near the subtelomeric markers (Taz1-GFP in green and H3K9me3 in red). The lower panel shows DAPI staining in white marked with Taz1-GFP (green contours) and H3K9me3 foci (red contours) coexisting at a single chromosome end. The chromosomes are pulled in the direction of the arrows. The bar indicates 500 nm for c,d. (d) An optical section of a typical DAPI-stained nucleus containing two knobs, commonly found in HU-treated cells.
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Related In: Results  -  Collection

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Show All Figures
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f5: Knob condensation is dynamically regulated in the cell cycle.(a) Live imaging of three independent cells expressing H2B-GFP. Pictures were taken every 7.5 min but here images are shown in 15-min steps. Deconvolved optical sections with the brightest dots at each time frame are shown. Scale bar, 1 μm. (b) Frequency of knob-containing nuclei. The number of knobs in a nucleus is indicated by different colours. Standard growth conditions (wild type in EMM2 5S at 26 °C) are indicated as ‘−' and any differences from these conditions are indicated. ‘H3S10ph' means standard cells positively stained with anti-H3S10ph antibody, which is specific for the M phase. Cell cycle was arrested with hydroxyurea (‘HU'), temperature-sensitive mutations or nitrogen starvation (‘-N') at S, G1 or G2. Numbers in parenthesis indicate the number of nuclei examined. (c) An optical section of a typical anaphase nucleus (DAPI staining; blue) without a knob near the subtelomeric markers (Taz1-GFP in green and H3K9me3 in red). The lower panel shows DAPI staining in white marked with Taz1-GFP (green contours) and H3K9me3 foci (red contours) coexisting at a single chromosome end. The chromosomes are pulled in the direction of the arrows. The bar indicates 500 nm for c,d. (d) An optical section of a typical DAPI-stained nucleus containing two knobs, commonly found in HU-treated cells.
Mentions: The genome of S. pombe has well-studied, discrete silent heterochromatic regions marked by histone H3 methylated at K9 (H3K9me). The subtelomeric and pericentromeric regions are the two largest heterochromatic regions (∼50 kb each) and are suitable for cytological analysis using 3DSIM because of their large size in S. pombe. Unexpectedly, known silent regions indicated by H3K9me3, as well as regions indicated by markers for the centromere (Cnp1-GFP) and the telomere (Taz1-GFP) had the lowest DNA concentration of the regions examined in this study (Fig. 2b,c). In particular, the Cnp1-GFP region is the innermost kinetochore region of the centromere (cnt) where foreign genes are known to be silenced11, but exhibited the lowest condensation level (Fig. 2b,c). Taz1 is the orthologue of TRF in mammals and recognizes telomere-associated sequences12. Although the length of the Taz1-binding sites could be too short to image with 3DSIM, the results showed that telomere-associated sequences were separate from any nearby condensed regions (Fig. 2c). H3K9me3 foci were found close to Cnp1- or Taz1-GFP foci, and often found in between the chromatin mass and the outside regions (Fig. 2c; see also Figs 3a, 4c and 5c). This was also the case when we used histone H3, instead of DAPI, as the chromatin marker (Fig. 2d, Supplementary Fig. 2a). The results for H3K9me2 were essentially identical to those for H3K9me3, although the antibody against H3K9me3 produced a more specific and intense signal than that against H3K9me2 (Supplementary Fig. 2b,c). In addition, the overlap with DAPI staining was similar for pericentromeric and subtelomeric H3K9me3 loci (Supplementary Fig. 2d,e). Silent regions marked by H3K9me3 (about 50 kb each) should be large enough to recognize with 3DSIM as we were able to visualize other chromatin regions of a similar size, that is, ‘knob' (see below). Decondensed silent chromatin was already predicted from reported micrococcal nuclease I (MNaseI) protection assays, where subtelomeric and pericentromeric heterochromatin regions had lower MNaseI protection than the rest of the genome1314 (summarized in Supplementary Fig. 5; also see Fig. 7a). Also, origins of replication in pericentromeric regions and the silent mating-type locus are replicated early in S phase1516. Furthermore, the cnt region of centromere is known to be modified by H3K4me3 (ref. 17). Collectively, the evidence indicates that silent regions in S. pombe are in fact less condensed than active regions.

Bottom Line: Knob regions span ∼50 kb of sequence devoid of methylated histones.Disruption of methylation at lysine 36 of histone H3 (H3K36) eliminates knob formation and gene repression at the subtelomeric and adjacent knob regions.Thus, epigenetic marks at H3K36 play crucial roles in the formation of a unique chromatin structure and in gene regulation at those regions in S. pombe.

View Article: PubMed Central - PubMed

Affiliation: 1] Advanced ICT Research Institute Kobe, National Institute of Information and Communications Technology, 588-2, Iwaoka, Iwaoka-cho, Kobe 651-2492, Japan [2] Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita 565-0871, Japan.

ABSTRACT
It is generally believed that silent chromatin is condensed and transcriptionally active chromatin is decondensed. However, little is known about the relationship between the condensation levels and gene expression. Here we report the condensation levels of interphase chromatin in the fission yeast Schizosaccharomyces pombe examined by super-resolution fluorescence microscopy. Unexpectedly, silent chromatin is less condensed than the euchromatin. Furthermore, the telomeric silent regions are flanked by highly condensed chromatin bodies, or 'knobs'. Knob regions span ∼50 kb of sequence devoid of methylated histones. Knob condensation is independent of HP1 homologue Swi6 and other gene silencing factors. Disruption of methylation at lysine 36 of histone H3 (H3K36) eliminates knob formation and gene repression at the subtelomeric and adjacent knob regions. Thus, epigenetic marks at H3K36 play crucial roles in the formation of a unique chromatin structure and in gene regulation at those regions in S. pombe.

No MeSH data available.


Related in: MedlinePlus