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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

DNA concentration in the interphase nucleus of S. pombe.(a) Single optical sections obtained with 3DSIM of fixed, immunostained nuclei using antibodies recognizing the indicated epitopes (green) and counterstained with DAPI (magenta). ‘RNAPIIser5p' and ‘RNAPIIser2p' denote RNAPII phosphorylated at serine 5 and 2 of the C-terminal domain, respectively. Scale bar, 500 nm. (b) Quantitative representation of the overlap of each chromatin marker with DAPI staining. Bar shows s.d. of nuclei. The value 1.0 indicates perfect overlap of the immunofluorescence with the DAPI fluorescence. Numbers in parenthesis indicate the number of nuclei examined. (c) The merged images (left panels) show centromeric (Cnp1-GFP) and telomeric (Taz1-GFP) regions in green and silent loci marked by trimethylation at lysine 9 of histone H3 (H3K9me3) in red, superimposed on DAPI images in blue. The right two columns show the corresponding DAPI images (white) marked with regions of Cnp1-GFP or Taz1-GFP (the green contour) and H3K9me3 regions (the red contour). (d) The merged image (left panel) shows H3K9me3 (red) superimposed on the image of histone H3-GFP (green); the right panel shows the corresponding histone H3-GFP image (white) marked with the H3K9me3 region (the red contour).
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f2: DNA concentration in the interphase nucleus of S. pombe.(a) Single optical sections obtained with 3DSIM of fixed, immunostained nuclei using antibodies recognizing the indicated epitopes (green) and counterstained with DAPI (magenta). ‘RNAPIIser5p' and ‘RNAPIIser2p' denote RNAPII phosphorylated at serine 5 and 2 of the C-terminal domain, respectively. Scale bar, 500 nm. (b) Quantitative representation of the overlap of each chromatin marker with DAPI staining. Bar shows s.d. of nuclei. The value 1.0 indicates perfect overlap of the immunofluorescence with the DAPI fluorescence. Numbers in parenthesis indicate the number of nuclei examined. (c) The merged images (left panels) show centromeric (Cnp1-GFP) and telomeric (Taz1-GFP) regions in green and silent loci marked by trimethylation at lysine 9 of histone H3 (H3K9me3) in red, superimposed on DAPI images in blue. The right two columns show the corresponding DAPI images (white) marked with regions of Cnp1-GFP or Taz1-GFP (the green contour) and H3K9me3 regions (the red contour). (d) The merged image (left panel) shows H3K9me3 (red) superimposed on the image of histone H3-GFP (green); the right panel shows the corresponding histone H3-GFP image (white) marked with the H3K9me3 region (the red contour).

Mentions: To analyse which genomic regions were more condensed or decondensed in the interphase nucleus, immunofluorescence of histone modifications and chromatin factors were observed with 3DSIM (Fig. 2a). The chromatin condensation level of each domain was expressed as the DNA concentration measured by 4′,6-diamidino-2-phenylindole (DAPI) staining. Although DAPI has a higher affinity for AT-rich sequences, the GC-content of the genome in S. pombe is relatively constant at this resolution. Thus, the immunofluorescence signal overlapping with the DAPI staining should directly reflect the DNA concentration in the target region. The overlap was determined with our original algorithm that measures the fraction of signal that co-localizes with the DAPI signal (see ‘Algorithm for the co-localization analysis' in Methods). Our algorithm allows measurements with or without thresholding. Thresholding has the advantage that it limits the co-localization analysis only to the target signal, but has the disadvantage that it relies on human interpretation of the object. This potential disadvantage is overcome by taking measurements without thresholding, though the difference among data is then modest due to the weak background signal. Here we took measurements using both methods for comparison.


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)

DNA concentration in the interphase nucleus of S. pombe.(a) Single optical sections obtained with 3DSIM of fixed, immunostained nuclei using antibodies recognizing the indicated epitopes (green) and counterstained with DAPI (magenta). ‘RNAPIIser5p' and ‘RNAPIIser2p' denote RNAPII phosphorylated at serine 5 and 2 of the C-terminal domain, respectively. Scale bar, 500 nm. (b) Quantitative representation of the overlap of each chromatin marker with DAPI staining. Bar shows s.d. of nuclei. The value 1.0 indicates perfect overlap of the immunofluorescence with the DAPI fluorescence. Numbers in parenthesis indicate the number of nuclei examined. (c) The merged images (left panels) show centromeric (Cnp1-GFP) and telomeric (Taz1-GFP) regions in green and silent loci marked by trimethylation at lysine 9 of histone H3 (H3K9me3) in red, superimposed on DAPI images in blue. The right two columns show the corresponding DAPI images (white) marked with regions of Cnp1-GFP or Taz1-GFP (the green contour) and H3K9me3 regions (the red contour). (d) The merged image (left panel) shows H3K9me3 (red) superimposed on the image of histone H3-GFP (green); the right panel shows the corresponding histone H3-GFP image (white) marked with the H3K9me3 region (the red contour).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4525289&req=5

f2: DNA concentration in the interphase nucleus of S. pombe.(a) Single optical sections obtained with 3DSIM of fixed, immunostained nuclei using antibodies recognizing the indicated epitopes (green) and counterstained with DAPI (magenta). ‘RNAPIIser5p' and ‘RNAPIIser2p' denote RNAPII phosphorylated at serine 5 and 2 of the C-terminal domain, respectively. Scale bar, 500 nm. (b) Quantitative representation of the overlap of each chromatin marker with DAPI staining. Bar shows s.d. of nuclei. The value 1.0 indicates perfect overlap of the immunofluorescence with the DAPI fluorescence. Numbers in parenthesis indicate the number of nuclei examined. (c) The merged images (left panels) show centromeric (Cnp1-GFP) and telomeric (Taz1-GFP) regions in green and silent loci marked by trimethylation at lysine 9 of histone H3 (H3K9me3) in red, superimposed on DAPI images in blue. The right two columns show the corresponding DAPI images (white) marked with regions of Cnp1-GFP or Taz1-GFP (the green contour) and H3K9me3 regions (the red contour). (d) The merged image (left panel) shows H3K9me3 (red) superimposed on the image of histone H3-GFP (green); the right panel shows the corresponding histone H3-GFP image (white) marked with the H3K9me3 region (the red contour).
Mentions: To analyse which genomic regions were more condensed or decondensed in the interphase nucleus, immunofluorescence of histone modifications and chromatin factors were observed with 3DSIM (Fig. 2a). The chromatin condensation level of each domain was expressed as the DNA concentration measured by 4′,6-diamidino-2-phenylindole (DAPI) staining. Although DAPI has a higher affinity for AT-rich sequences, the GC-content of the genome in S. pombe is relatively constant at this resolution. Thus, the immunofluorescence signal overlapping with the DAPI staining should directly reflect the DNA concentration in the target region. The overlap was determined with our original algorithm that measures the fraction of signal that co-localizes with the DAPI signal (see ‘Algorithm for the co-localization analysis' in Methods). Our algorithm allows measurements with or without thresholding. Thresholding has the advantage that it limits the co-localization analysis only to the target signal, but has the disadvantage that it relies on human interpretation of the object. This potential disadvantage is overcome by taking measurements without thresholding, though the difference among data is then modest due to the weak background signal. Here we took measurements using both methods for comparison.

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