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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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Down-regulation of WDHD1 leads to cell cycle progression deficiencies attributable to defective centromere. (A) Cell cycle profiles of the control (ctrl) and WDHD1 knockdown cells. Mock or WDHD1-knockdown 293T cells were subjected to FACS for measurement of DNA content. (B) Mitotic fidelity was assessed by scoring mitotic cells with chromosome bridges. Condensed mitotic chromosomes were visualized by phospho-histone H3 immunostaining. Representative immunofluorescence images are depicted on the left. Both anaphase and telophase nuclei are shown, as indicated. Panel on the right shows the percentages of mitotic cells with chromosome bridges in control (ctrl) versus knockdown cells (n = 250). The results represent averages of three independent experiments. (C) Nuclear morphology was altered in WDHD1-deficient cells. Control (ctrl) and knockdown HeLa cells were immunostained for WDHD1, and counterstained for F-actin (for demarcating cell membrane) and DNA (DAPI). Confocal microscopy was done as described before (scale bar is 10 µm). Percentages of cells with more than one nuclei staining, examples of which are indicated by arrowheads, are shown on the bottom (n = 1000) and represent averages of three independent experiments.
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Figure 6: Down-regulation of WDHD1 leads to cell cycle progression deficiencies attributable to defective centromere. (A) Cell cycle profiles of the control (ctrl) and WDHD1 knockdown cells. Mock or WDHD1-knockdown 293T cells were subjected to FACS for measurement of DNA content. (B) Mitotic fidelity was assessed by scoring mitotic cells with chromosome bridges. Condensed mitotic chromosomes were visualized by phospho-histone H3 immunostaining. Representative immunofluorescence images are depicted on the left. Both anaphase and telophase nuclei are shown, as indicated. Panel on the right shows the percentages of mitotic cells with chromosome bridges in control (ctrl) versus knockdown cells (n = 250). The results represent averages of three independent experiments. (C) Nuclear morphology was altered in WDHD1-deficient cells. Control (ctrl) and knockdown HeLa cells were immunostained for WDHD1, and counterstained for F-actin (for demarcating cell membrane) and DNA (DAPI). Confocal microscopy was done as described before (scale bar is 10 µm). Percentages of cells with more than one nuclei staining, examples of which are indicated by arrowheads, are shown on the bottom (n = 1000) and represent averages of three independent experiments.

Mentions: Next, to address the physiological significance of WDHD1-mediated regulation of centromere integrity, we wanted to assess the effects of WDHD1 knockdown upon the progression and fidelity of the cell cycle, particularly in the mitotic phase. Flow cytometry analysis demonstrated an accumulation of cells with a DNA content of 4 N (representative of G2/M phase) in the WDHD1 knockdown culture (Figure 6A), implying possibly a delay in G2 and/or mitosis progression. Immunofluorescence staining with phospho-H3 antibody, which demarcates condensed mitotic chromosomes, further revealed that silencing of WDHD1 resulted in pronounced increase in the occurrence of mitotic chromosome bridges (11.5–29%, Figure 6B), indicative of defects in chromosome segregation. This phenotype was also accompanied by abnormal nuclear morphology (marked by arrowheads in Figure 6C), characterized by significantly higher incidence of cells with multiple nuclei (15.2% in the knockdown cells compared to 1.7% in the control). We conclude that WDHD1 down-regulation, giving rise to dramatic loss of the centromeric small RNA and disruption of the associated heterochromatin structure, likely impacts on the centromere integrity and kinetochore assembly, and consequently faithful chromosome segregation during mitosis.Figure 6.


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)

Down-regulation of WDHD1 leads to cell cycle progression deficiencies attributable to defective centromere. (A) Cell cycle profiles of the control (ctrl) and WDHD1 knockdown cells. Mock or WDHD1-knockdown 293T cells were subjected to FACS for measurement of DNA content. (B) Mitotic fidelity was assessed by scoring mitotic cells with chromosome bridges. Condensed mitotic chromosomes were visualized by phospho-histone H3 immunostaining. Representative immunofluorescence images are depicted on the left. Both anaphase and telophase nuclei are shown, as indicated. Panel on the right shows the percentages of mitotic cells with chromosome bridges in control (ctrl) versus knockdown cells (n = 250). The results represent averages of three independent experiments. (C) Nuclear morphology was altered in WDHD1-deficient cells. Control (ctrl) and knockdown HeLa cells were immunostained for WDHD1, and counterstained for F-actin (for demarcating cell membrane) and DNA (DAPI). Confocal microscopy was done as described before (scale bar is 10 µm). Percentages of cells with more than one nuclei staining, examples of which are indicated by arrowheads, are shown on the bottom (n = 1000) and represent averages of three independent experiments.
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Figure 6: Down-regulation of WDHD1 leads to cell cycle progression deficiencies attributable to defective centromere. (A) Cell cycle profiles of the control (ctrl) and WDHD1 knockdown cells. Mock or WDHD1-knockdown 293T cells were subjected to FACS for measurement of DNA content. (B) Mitotic fidelity was assessed by scoring mitotic cells with chromosome bridges. Condensed mitotic chromosomes were visualized by phospho-histone H3 immunostaining. Representative immunofluorescence images are depicted on the left. Both anaphase and telophase nuclei are shown, as indicated. Panel on the right shows the percentages of mitotic cells with chromosome bridges in control (ctrl) versus knockdown cells (n = 250). The results represent averages of three independent experiments. (C) Nuclear morphology was altered in WDHD1-deficient cells. Control (ctrl) and knockdown HeLa cells were immunostained for WDHD1, and counterstained for F-actin (for demarcating cell membrane) and DNA (DAPI). Confocal microscopy was done as described before (scale bar is 10 µm). Percentages of cells with more than one nuclei staining, examples of which are indicated by arrowheads, are shown on the bottom (n = 1000) and represent averages of three independent experiments.
Mentions: Next, to address the physiological significance of WDHD1-mediated regulation of centromere integrity, we wanted to assess the effects of WDHD1 knockdown upon the progression and fidelity of the cell cycle, particularly in the mitotic phase. Flow cytometry analysis demonstrated an accumulation of cells with a DNA content of 4 N (representative of G2/M phase) in the WDHD1 knockdown culture (Figure 6A), implying possibly a delay in G2 and/or mitosis progression. Immunofluorescence staining with phospho-H3 antibody, which demarcates condensed mitotic chromosomes, further revealed that silencing of WDHD1 resulted in pronounced increase in the occurrence of mitotic chromosome bridges (11.5–29%, Figure 6B), indicative of defects in chromosome segregation. This phenotype was also accompanied by abnormal nuclear morphology (marked by arrowheads in Figure 6C), characterized by significantly higher incidence of cells with multiple nuclei (15.2% in the knockdown cells compared to 1.7% in the control). We conclude that WDHD1 down-regulation, giving rise to dramatic loss of the centromeric small RNA and disruption of the associated heterochromatin structure, likely impacts on the centromere integrity and kinetochore assembly, and consequently faithful chromosome segregation during mitosis.Figure 6.

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