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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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Related in: MedlinePlus

Roles of WDHD1 in the expression of centromeric repeat non-coding RNA. (A) Expression levels of centromeric non-coding RNA spanning major and minor satellite repeats was examined by RT–PCR analysis of RNA isolated from control (ctrl) or WDHD1 knockdown NIH-3T3 cells. Total RNA samples were treated with DNase I prior to reverse transcription. ‘−’ denotes RT-minus reactions in which no reverse transcriptase was added. Expression levels of the housekeeping gene GAPDH, and WDHD1 are also shown. (B) Effect of WDHD1 knockdown on the transcription rates of minor and major satellite repeat region, as indicated. Nuclear run-on assays were performed to monitor newly transcribed centromeric RNA from nuclei of control (ctrl) and WDHD1 knockdown NIH-3T3 cells. U5 snRNA, which remained unchanged in both cell types, was used to demonstrate uniformity of input RNA. ‘–’ denotes RT-minus reactions in which no reverse transcriptase was added. Quantitative results are shown by bar graph below, and represent the mean ± SD of three independent experiments. For statistical significance of quantitative comparisons, calculations were done by (*P < 0.01; NS, P > 0.05). (C) NIH-3T3 cells were treated with siRNA targeting the indicated genes or control siRNA (ctrl). Degree of knockdown was monitored by western blot analysis as shown on the right. Equal amounts of total RNA isolated from these cells were resolved in a denaturing 8% acrylamide gel. The gel was subjected to northern blot analysis using probes specific for major satellite repeats. Blot was re-hybridized with a U6 snRNA-complementary probe to demonstrate equal loading (bottom on the left). (D) Expression of the homologous small RNA fragments. Total RNA from control (ctrl) or WDHD1 knockdown NIH-3T3 cells was size fractionated, and equivalents amounts of small RNA fraction (<200 nt) were resolved in a denaturing 12% acrylamide gel (EtBr staining shown in the middle). The gel was subjected to northern blot analysis using probes specific for major or minor satellite repeats, as indicated on the bottom. Blot was re-hybridized with a U6 snRNA-complementary probe to demonstrate equal loading (bottom).
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Figure 3: Roles of WDHD1 in the expression of centromeric repeat non-coding RNA. (A) Expression levels of centromeric non-coding RNA spanning major and minor satellite repeats was examined by RT–PCR analysis of RNA isolated from control (ctrl) or WDHD1 knockdown NIH-3T3 cells. Total RNA samples were treated with DNase I prior to reverse transcription. ‘−’ denotes RT-minus reactions in which no reverse transcriptase was added. Expression levels of the housekeeping gene GAPDH, and WDHD1 are also shown. (B) Effect of WDHD1 knockdown on the transcription rates of minor and major satellite repeat region, as indicated. Nuclear run-on assays were performed to monitor newly transcribed centromeric RNA from nuclei of control (ctrl) and WDHD1 knockdown NIH-3T3 cells. U5 snRNA, which remained unchanged in both cell types, was used to demonstrate uniformity of input RNA. ‘–’ denotes RT-minus reactions in which no reverse transcriptase was added. Quantitative results are shown by bar graph below, and represent the mean ± SD of three independent experiments. For statistical significance of quantitative comparisons, calculations were done by (*P < 0.01; NS, P > 0.05). (C) NIH-3T3 cells were treated with siRNA targeting the indicated genes or control siRNA (ctrl). Degree of knockdown was monitored by western blot analysis as shown on the right. Equal amounts of total RNA isolated from these cells were resolved in a denaturing 8% acrylamide gel. The gel was subjected to northern blot analysis using probes specific for major satellite repeats. Blot was re-hybridized with a U6 snRNA-complementary probe to demonstrate equal loading (bottom on the left). (D) Expression of the homologous small RNA fragments. Total RNA from control (ctrl) or WDHD1 knockdown NIH-3T3 cells was size fractionated, and equivalents amounts of small RNA fraction (<200 nt) were resolved in a denaturing 12% acrylamide gel (EtBr staining shown in the middle). The gel was subjected to northern blot analysis using probes specific for major or minor satellite repeats, as indicated on the bottom. Blot was re-hybridized with a U6 snRNA-complementary probe to demonstrate equal loading (bottom).

Mentions: To enrich for the small RNA fraction with sizes of ≤200 nt, a mirVana RNA isolation kit (Ambion) was used according to the manufacturer’s instructions. For northern blot analysis, 50 μg of total RNA (Figure 3C) or 25 μg of the small RNA fraction (Figure 3D) was separated, respectively, on 8 or 12% polyacrylamide/8 M urea denaturing gels and transferred to GeneScreen Plus membranes (PerkinElmer). The oligonucleotides used to probe for satellite repeats (major: mixture of Maj23 and Maj12; minor: mixture of Min1, Min2 and Min3) and U6 snRNA were designed based on a previous report (16) and are listed in the Supplementary Table. Oligonucleotide probes were 5′-end-labeled with [γ-32P] ATP using T4 polynucleotide kinase (New England Biolabs; Ipswich, MA, USA). Prehybridization of the filters was carried out in 6× SSC, 0.2% SDS, 5× Denhardt’s solution and 20 mg/ml sheared, denatured salmon sperm DNA. Hybridizations were performed in the same solution at 42°C, with addition of the denatured, labeled probes. After hybridization, the blots were subjected to stringent washes with 6× SSC, 0.2% SDS at 42°C twice for 10 min, and subsequently autoradiography.


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)

Roles of WDHD1 in the expression of centromeric repeat non-coding RNA. (A) Expression levels of centromeric non-coding RNA spanning major and minor satellite repeats was examined by RT–PCR analysis of RNA isolated from control (ctrl) or WDHD1 knockdown NIH-3T3 cells. Total RNA samples were treated with DNase I prior to reverse transcription. ‘−’ denotes RT-minus reactions in which no reverse transcriptase was added. Expression levels of the housekeeping gene GAPDH, and WDHD1 are also shown. (B) Effect of WDHD1 knockdown on the transcription rates of minor and major satellite repeat region, as indicated. Nuclear run-on assays were performed to monitor newly transcribed centromeric RNA from nuclei of control (ctrl) and WDHD1 knockdown NIH-3T3 cells. U5 snRNA, which remained unchanged in both cell types, was used to demonstrate uniformity of input RNA. ‘–’ denotes RT-minus reactions in which no reverse transcriptase was added. Quantitative results are shown by bar graph below, and represent the mean ± SD of three independent experiments. For statistical significance of quantitative comparisons, calculations were done by (*P < 0.01; NS, P > 0.05). (C) NIH-3T3 cells were treated with siRNA targeting the indicated genes or control siRNA (ctrl). Degree of knockdown was monitored by western blot analysis as shown on the right. Equal amounts of total RNA isolated from these cells were resolved in a denaturing 8% acrylamide gel. The gel was subjected to northern blot analysis using probes specific for major satellite repeats. Blot was re-hybridized with a U6 snRNA-complementary probe to demonstrate equal loading (bottom on the left). (D) Expression of the homologous small RNA fragments. Total RNA from control (ctrl) or WDHD1 knockdown NIH-3T3 cells was size fractionated, and equivalents amounts of small RNA fraction (<200 nt) were resolved in a denaturing 12% acrylamide gel (EtBr staining shown in the middle). The gel was subjected to northern blot analysis using probes specific for major or minor satellite repeats, as indicated on the bottom. Blot was re-hybridized with a U6 snRNA-complementary probe to demonstrate equal loading (bottom).
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Figure 3: Roles of WDHD1 in the expression of centromeric repeat non-coding RNA. (A) Expression levels of centromeric non-coding RNA spanning major and minor satellite repeats was examined by RT–PCR analysis of RNA isolated from control (ctrl) or WDHD1 knockdown NIH-3T3 cells. Total RNA samples were treated with DNase I prior to reverse transcription. ‘−’ denotes RT-minus reactions in which no reverse transcriptase was added. Expression levels of the housekeeping gene GAPDH, and WDHD1 are also shown. (B) Effect of WDHD1 knockdown on the transcription rates of minor and major satellite repeat region, as indicated. Nuclear run-on assays were performed to monitor newly transcribed centromeric RNA from nuclei of control (ctrl) and WDHD1 knockdown NIH-3T3 cells. U5 snRNA, which remained unchanged in both cell types, was used to demonstrate uniformity of input RNA. ‘–’ denotes RT-minus reactions in which no reverse transcriptase was added. Quantitative results are shown by bar graph below, and represent the mean ± SD of three independent experiments. For statistical significance of quantitative comparisons, calculations were done by (*P < 0.01; NS, P > 0.05). (C) NIH-3T3 cells were treated with siRNA targeting the indicated genes or control siRNA (ctrl). Degree of knockdown was monitored by western blot analysis as shown on the right. Equal amounts of total RNA isolated from these cells were resolved in a denaturing 8% acrylamide gel. The gel was subjected to northern blot analysis using probes specific for major satellite repeats. Blot was re-hybridized with a U6 snRNA-complementary probe to demonstrate equal loading (bottom on the left). (D) Expression of the homologous small RNA fragments. Total RNA from control (ctrl) or WDHD1 knockdown NIH-3T3 cells was size fractionated, and equivalents amounts of small RNA fraction (<200 nt) were resolved in a denaturing 12% acrylamide gel (EtBr staining shown in the middle). The gel was subjected to northern blot analysis using probes specific for major or minor satellite repeats, as indicated on the bottom. Blot was re-hybridized with a U6 snRNA-complementary probe to demonstrate equal loading (bottom).
Mentions: To enrich for the small RNA fraction with sizes of ≤200 nt, a mirVana RNA isolation kit (Ambion) was used according to the manufacturer’s instructions. For northern blot analysis, 50 μg of total RNA (Figure 3C) or 25 μg of the small RNA fraction (Figure 3D) was separated, respectively, on 8 or 12% polyacrylamide/8 M urea denaturing gels and transferred to GeneScreen Plus membranes (PerkinElmer). The oligonucleotides used to probe for satellite repeats (major: mixture of Maj23 and Maj12; minor: mixture of Min1, Min2 and Min3) and U6 snRNA were designed based on a previous report (16) and are listed in the Supplementary Table. Oligonucleotide probes were 5′-end-labeled with [γ-32P] ATP using T4 polynucleotide kinase (New England Biolabs; Ipswich, MA, USA). Prehybridization of the filters was carried out in 6× SSC, 0.2% SDS, 5× Denhardt’s solution and 20 mg/ml sheared, denatured salmon sperm DNA. Hybridizations were performed in the same solution at 42°C, with addition of the denatured, labeled probes. After hybridization, the blots were subjected to stringent washes with 6× SSC, 0.2% SDS at 42°C twice for 10 min, and subsequently autoradiography.

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