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WDHD1 modulates the post-transcriptional step of the centromeric silencing pathway.

Hsieh CL, Lin CL, Liu H, Chang YJ, Shih CJ, Zhong CZ, Lee SC, Tan BC - Nucleic Acids Res. (2011)

Bottom Line: As a consequence, such reduced epigenetic silencing is manifested in disrupted heterochromatic state of the centromere and a defective mitosis.This role is mediated at the post-transcriptional level and likely through stabilizing Dicer association with centromeric RNA.Collectively, these findings suggest that WDHD1 may be a critical component of the RNA-dependent epigenetic control mechanism that sustains centromere integrity and genomic stability.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan.

ABSTRACT
The centromere is a highly specialized chromosomal element that is essential for chromosome segregation during mitosis. Centromere integrity must therefore be properly preserved and is strictly dependent upon the establishment and maintenance of surrounding chromatin structure. Here we identify WDHD1, a WD40-domain and HMG-domain containing protein, as a key regulator of centromere function. We show that WDHD1 associates with centromeres in a cell cycle-dependent manner, coinciding with mid-to-late S phase. WDHD1 down-regulation compromises HP1α localization to pericentric heterochromatin and leads to altered expression of epigenetic markers associated with this chromatin region. As a consequence, such reduced epigenetic silencing is manifested in disrupted heterochromatic state of the centromere and a defective mitosis. Moreover, we demonstrate that a possible underlying mechanism of WDHD1's involvement lies in the proper generation of the small non-coding RNAs encoded by the centromeric satellite repeats. This role is mediated at the post-transcriptional level and likely through stabilizing Dicer association with centromeric RNA. Collectively, these findings suggest that WDHD1 may be a critical component of the RNA-dependent epigenetic control mechanism that sustains centromere integrity and genomic stability.

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Loss of heterochromatic features at the centromere in the absence of WDHD1. (A) Representative indirect immunofluorescence of HP1α in NIH-3T3 cells transfected with control (ctrl) or WDHD1 siRNA. Loss of HP1α was also measured by calculating the percentage of cells stained positively for HP1α foci in each cell type (n = 500). Percentages given are averages of three independent experiments. (B and C) Chromatin fragments were prepared from control (ctrl) or WDHD1 knockdown cells. ChIP was carried out with control (IgG), H3K9me3, H4K20me3, or H3K27me antibody, as denoted. Semi-quantitative determination of the bound minor satellite DNA in the anti-H3K9me3 ChIP is depicted by the gel figure in (B). Input chromatin was also loaded and equals to 1/10 of IP. Quantitative determination of the bound major satellite DNA, as assessed by quantitative real-time PCR, is shown by bar graphs in (C). Results are expressed as percentages of input chromatin, normalized to control IgG samples, and represent the mean ± SD of at least three independent experiments. (D) DNA methylation analysis of genomic DNA derived from control (−) and knockdown (+) cells. Equivalent amounts of DNA were digested with MspI and HpaII, and subsequently purified and gel separated (EtBr staining on the left). Southern blot analysis was next carried out with hybridizing probes corresponding to minor (middle) or major (right) satellite sequences. Blot was re-hybridized with a mitochondria DNA probe to demonstrate equal loading (bottom). A shorter-exposed version of the blot is shown in the Supplementary Figure S6A. (E) Chromatin structure on the centromeric region was examined in the control (−) and WDHD1 knockdown (+) cells based on the Micrococcal nuclease (MNase)-Southern blot analysis. Isolated intact nuclei were treated with 4.8, 2.4, 1.2, 0.6 and 0 U of MNase. DNA was subsequently purified and separated on an agarose gel (EtBr staining shown in the left panel). In the right panel, the gel was blotted and probed with a radiolabeled probe specific for major satellite repeats.
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Figure 5: Loss of heterochromatic features at the centromere in the absence of WDHD1. (A) Representative indirect immunofluorescence of HP1α in NIH-3T3 cells transfected with control (ctrl) or WDHD1 siRNA. Loss of HP1α was also measured by calculating the percentage of cells stained positively for HP1α foci in each cell type (n = 500). Percentages given are averages of three independent experiments. (B and C) Chromatin fragments were prepared from control (ctrl) or WDHD1 knockdown cells. ChIP was carried out with control (IgG), H3K9me3, H4K20me3, or H3K27me antibody, as denoted. Semi-quantitative determination of the bound minor satellite DNA in the anti-H3K9me3 ChIP is depicted by the gel figure in (B). Input chromatin was also loaded and equals to 1/10 of IP. Quantitative determination of the bound major satellite DNA, as assessed by quantitative real-time PCR, is shown by bar graphs in (C). Results are expressed as percentages of input chromatin, normalized to control IgG samples, and represent the mean ± SD of at least three independent experiments. (D) DNA methylation analysis of genomic DNA derived from control (−) and knockdown (+) cells. Equivalent amounts of DNA were digested with MspI and HpaII, and subsequently purified and gel separated (EtBr staining on the left). Southern blot analysis was next carried out with hybridizing probes corresponding to minor (middle) or major (right) satellite sequences. Blot was re-hybridized with a mitochondria DNA probe to demonstrate equal loading (bottom). A shorter-exposed version of the blot is shown in the Supplementary Figure S6A. (E) Chromatin structure on the centromeric region was examined in the control (−) and WDHD1 knockdown (+) cells based on the Micrococcal nuclease (MNase)-Southern blot analysis. Isolated intact nuclei were treated with 4.8, 2.4, 1.2, 0.6 and 0 U of MNase. DNA was subsequently purified and separated on an agarose gel (EtBr staining shown in the left panel). In the right panel, the gel was blotted and probed with a radiolabeled probe specific for major satellite repeats.

Mentions: Because small RNA molecules transcribed from the CT/PCT regions are important for the initiation and maintenance of repressive chromatin modifications and structure, we next aimed to dissect the role of WDHD1 in regulating these physical attributes. We first examined the status of HP1α on heterochromatin following WDHD1 knockdown in mouse NIH-3T3 cells. Indirect immunofluorescence analysis revealed a significant loss of HP1α nuclear foci in the knockdown versus control cells (Figure 5A), suggesting a delocalization of this heterochromatin structural protein. Next, ChIP assays followed by PCR using primers spanning the minor (Figure 5B) or major (Figure 5C) satellite regions were performed to compare the levels of several resident histone modifications between control and WDHD1 RNAi cells. As shown in Figure 5B and C, these regions underwent a decline in the repression-associated marks (H3K9me3, H4K20me3 and H3K27me1) in response to WDHD1 knockdown. Significant reduction in the association of H3K9me3 with pericentric heterochromatin was also illustrated by the immunostaining analysis (Supplementary Figure S5A). On the contrary, there was a coincident increase in the level of acetylated H4, an activating mark. We further confirmed that such altered centromeric heterochromatin association was not due to changes in the overall abundance of these epigenetic marks (Supplementary Figure S5B).Figure 5.


WDHD1 modulates the post-transcriptional step of the centromeric silencing pathway.

Hsieh CL, Lin CL, Liu H, Chang YJ, Shih CJ, Zhong CZ, Lee SC, Tan BC - Nucleic Acids Res. (2011)

Loss of heterochromatic features at the centromere in the absence of WDHD1. (A) Representative indirect immunofluorescence of HP1α in NIH-3T3 cells transfected with control (ctrl) or WDHD1 siRNA. Loss of HP1α was also measured by calculating the percentage of cells stained positively for HP1α foci in each cell type (n = 500). Percentages given are averages of three independent experiments. (B and C) Chromatin fragments were prepared from control (ctrl) or WDHD1 knockdown cells. ChIP was carried out with control (IgG), H3K9me3, H4K20me3, or H3K27me antibody, as denoted. Semi-quantitative determination of the bound minor satellite DNA in the anti-H3K9me3 ChIP is depicted by the gel figure in (B). Input chromatin was also loaded and equals to 1/10 of IP. Quantitative determination of the bound major satellite DNA, as assessed by quantitative real-time PCR, is shown by bar graphs in (C). Results are expressed as percentages of input chromatin, normalized to control IgG samples, and represent the mean ± SD of at least three independent experiments. (D) DNA methylation analysis of genomic DNA derived from control (−) and knockdown (+) cells. Equivalent amounts of DNA were digested with MspI and HpaII, and subsequently purified and gel separated (EtBr staining on the left). Southern blot analysis was next carried out with hybridizing probes corresponding to minor (middle) or major (right) satellite sequences. Blot was re-hybridized with a mitochondria DNA probe to demonstrate equal loading (bottom). A shorter-exposed version of the blot is shown in the Supplementary Figure S6A. (E) Chromatin structure on the centromeric region was examined in the control (−) and WDHD1 knockdown (+) cells based on the Micrococcal nuclease (MNase)-Southern blot analysis. Isolated intact nuclei were treated with 4.8, 2.4, 1.2, 0.6 and 0 U of MNase. DNA was subsequently purified and separated on an agarose gel (EtBr staining shown in the left panel). In the right panel, the gel was blotted and probed with a radiolabeled probe specific for major satellite repeats.
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Figure 5: Loss of heterochromatic features at the centromere in the absence of WDHD1. (A) Representative indirect immunofluorescence of HP1α in NIH-3T3 cells transfected with control (ctrl) or WDHD1 siRNA. Loss of HP1α was also measured by calculating the percentage of cells stained positively for HP1α foci in each cell type (n = 500). Percentages given are averages of three independent experiments. (B and C) Chromatin fragments were prepared from control (ctrl) or WDHD1 knockdown cells. ChIP was carried out with control (IgG), H3K9me3, H4K20me3, or H3K27me antibody, as denoted. Semi-quantitative determination of the bound minor satellite DNA in the anti-H3K9me3 ChIP is depicted by the gel figure in (B). Input chromatin was also loaded and equals to 1/10 of IP. Quantitative determination of the bound major satellite DNA, as assessed by quantitative real-time PCR, is shown by bar graphs in (C). Results are expressed as percentages of input chromatin, normalized to control IgG samples, and represent the mean ± SD of at least three independent experiments. (D) DNA methylation analysis of genomic DNA derived from control (−) and knockdown (+) cells. Equivalent amounts of DNA were digested with MspI and HpaII, and subsequently purified and gel separated (EtBr staining on the left). Southern blot analysis was next carried out with hybridizing probes corresponding to minor (middle) or major (right) satellite sequences. Blot was re-hybridized with a mitochondria DNA probe to demonstrate equal loading (bottom). A shorter-exposed version of the blot is shown in the Supplementary Figure S6A. (E) Chromatin structure on the centromeric region was examined in the control (−) and WDHD1 knockdown (+) cells based on the Micrococcal nuclease (MNase)-Southern blot analysis. Isolated intact nuclei were treated with 4.8, 2.4, 1.2, 0.6 and 0 U of MNase. DNA was subsequently purified and separated on an agarose gel (EtBr staining shown in the left panel). In the right panel, the gel was blotted and probed with a radiolabeled probe specific for major satellite repeats.
Mentions: Because small RNA molecules transcribed from the CT/PCT regions are important for the initiation and maintenance of repressive chromatin modifications and structure, we next aimed to dissect the role of WDHD1 in regulating these physical attributes. We first examined the status of HP1α on heterochromatin following WDHD1 knockdown in mouse NIH-3T3 cells. Indirect immunofluorescence analysis revealed a significant loss of HP1α nuclear foci in the knockdown versus control cells (Figure 5A), suggesting a delocalization of this heterochromatin structural protein. Next, ChIP assays followed by PCR using primers spanning the minor (Figure 5B) or major (Figure 5C) satellite regions were performed to compare the levels of several resident histone modifications between control and WDHD1 RNAi cells. As shown in Figure 5B and C, these regions underwent a decline in the repression-associated marks (H3K9me3, H4K20me3 and H3K27me1) in response to WDHD1 knockdown. Significant reduction in the association of H3K9me3 with pericentric heterochromatin was also illustrated by the immunostaining analysis (Supplementary Figure S5A). On the contrary, there was a coincident increase in the level of acetylated H4, an activating mark. We further confirmed that such altered centromeric heterochromatin association was not due to changes in the overall abundance of these epigenetic marks (Supplementary Figure S5B).Figure 5.

Bottom Line: As a consequence, such reduced epigenetic silencing is manifested in disrupted heterochromatic state of the centromere and a defective mitosis.This role is mediated at the post-transcriptional level and likely through stabilizing Dicer association with centromeric RNA.Collectively, these findings suggest that WDHD1 may be a critical component of the RNA-dependent epigenetic control mechanism that sustains centromere integrity and genomic stability.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan.

ABSTRACT
The centromere is a highly specialized chromosomal element that is essential for chromosome segregation during mitosis. Centromere integrity must therefore be properly preserved and is strictly dependent upon the establishment and maintenance of surrounding chromatin structure. Here we identify WDHD1, a WD40-domain and HMG-domain containing protein, as a key regulator of centromere function. We show that WDHD1 associates with centromeres in a cell cycle-dependent manner, coinciding with mid-to-late S phase. WDHD1 down-regulation compromises HP1α localization to pericentric heterochromatin and leads to altered expression of epigenetic markers associated with this chromatin region. As a consequence, such reduced epigenetic silencing is manifested in disrupted heterochromatic state of the centromere and a defective mitosis. Moreover, we demonstrate that a possible underlying mechanism of WDHD1's involvement lies in the proper generation of the small non-coding RNAs encoded by the centromeric satellite repeats. This role is mediated at the post-transcriptional level and likely through stabilizing Dicer association with centromeric RNA. Collectively, these findings suggest that WDHD1 may be a critical component of the RNA-dependent epigenetic control mechanism that sustains centromere integrity and genomic stability.

Show MeSH
Related in: MedlinePlus