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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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WDHD1 associates with the centromere-encoded RNA transcripts. (A and B) RNA pull-down assay was carried out using biotinylated RNA transcripts. (A) In vitro transcribed RNAs corresponding to approximately one repeat of the centromeric major (lane 3) and minor (lane 5) satellite sequences were used as the baits. Precipitated proteins were visualized by western blotting using anti-WDHD1 antibody. Input represents direct loading that equals to 1/25 or 1/50 of the lysate protein used in the pull-down (lanes 1 and 2). Transcripts corresponding to 300 nt (lane 4) and 162 nt (lane 6) of the 18S rRNA sequence serve as controls for the major (lane 3) and minor (lane 5) satellite transcripts, respectively. (B) Dicer association with centromeric RNA sequences attenuated upon WDHD1 depletion. Cell lysates were subjected to immunodepletion using control IgG or WDHD1-specific antibodies, followed by RNA pull-down assay as in (A). (C) RNA-immunoprecipitation assay was performed using antibodies against the indicated proteins (WDHD1 and Dicer). RNA was extracted from the immunoprecipitates and subjected to dot blot analysis using probes corresponding to the sequences of the minor (upper) or major (lower) satellite repeats. (D–F) Association of WDHD1 with Dicer. (D) Anti-Dicer immunoprecipitates (IP) using extracts (input) prepared from NIH-3T3 (left) or 293T (right) cells were probed with antibodies against WDHD1 (top) and Dicer (bottom). (E) 293T cells were transfected with constructs encoding FLAG or FLAG-tagged WDHD1. Immunoprecipitation (IP) of the cell lysates was done using FLAG antibodies, and subsequently analyzed by immunoblotting with antibodies against FLAG (top) and Dicer (bottom). Extent of overexpression and subcellular localization of the FLAG-WDHD1 protein are shown in the Supplementary Information (Supplementary Figure S3). (F) 293T cell lysates were treated with (+) or without (−) RNase A prior to anti-Dicer immunoprecipiation as above. (G) Dicer occupancy of centromeric repeat regions. ChIP assays were performed on crosslinked chromatin from NIH-3T3 cells using antibodies specific for Dicer or control rabbit antibodies (IgG) (left panel). Sequential ChIP analysis was done to assess co-occupancy of the centromere by both WDHD1 and Dicer (right panel). Products of final PCR analysis using primers specific to major satellite repeat DNA sequence are resolved in agarose gel.
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Figure 4: WDHD1 associates with the centromere-encoded RNA transcripts. (A and B) RNA pull-down assay was carried out using biotinylated RNA transcripts. (A) In vitro transcribed RNAs corresponding to approximately one repeat of the centromeric major (lane 3) and minor (lane 5) satellite sequences were used as the baits. Precipitated proteins were visualized by western blotting using anti-WDHD1 antibody. Input represents direct loading that equals to 1/25 or 1/50 of the lysate protein used in the pull-down (lanes 1 and 2). Transcripts corresponding to 300 nt (lane 4) and 162 nt (lane 6) of the 18S rRNA sequence serve as controls for the major (lane 3) and minor (lane 5) satellite transcripts, respectively. (B) Dicer association with centromeric RNA sequences attenuated upon WDHD1 depletion. Cell lysates were subjected to immunodepletion using control IgG or WDHD1-specific antibodies, followed by RNA pull-down assay as in (A). (C) RNA-immunoprecipitation assay was performed using antibodies against the indicated proteins (WDHD1 and Dicer). RNA was extracted from the immunoprecipitates and subjected to dot blot analysis using probes corresponding to the sequences of the minor (upper) or major (lower) satellite repeats. (D–F) Association of WDHD1 with Dicer. (D) Anti-Dicer immunoprecipitates (IP) using extracts (input) prepared from NIH-3T3 (left) or 293T (right) cells were probed with antibodies against WDHD1 (top) and Dicer (bottom). (E) 293T cells were transfected with constructs encoding FLAG or FLAG-tagged WDHD1. Immunoprecipitation (IP) of the cell lysates was done using FLAG antibodies, and subsequently analyzed by immunoblotting with antibodies against FLAG (top) and Dicer (bottom). Extent of overexpression and subcellular localization of the FLAG-WDHD1 protein are shown in the Supplementary Information (Supplementary Figure S3). (F) 293T cell lysates were treated with (+) or without (−) RNase A prior to anti-Dicer immunoprecipiation as above. (G) Dicer occupancy of centromeric repeat regions. ChIP assays were performed on crosslinked chromatin from NIH-3T3 cells using antibodies specific for Dicer or control rabbit antibodies (IgG) (left panel). Sequential ChIP analysis was done to assess co-occupancy of the centromere by both WDHD1 and Dicer (right panel). Products of final PCR analysis using primers specific to major satellite repeat DNA sequence are resolved in agarose gel.

Mentions: Next, to unequivocally elucidate whether WDHD1 is directly involved in the processing of centromeric transcripts, we sought to determine if WDHD1-centromeric RNAs complexes exist. To demonstrate such association, we first performed RNA pull-down assays using streptavidin-coated beads and in vitro transcribed, biotinylated minor and major satellite RNAs, and probed for the presence of endogenous WDHD1 in the precipitated material. The immunoblotting results showed that WDHD1 in nuclear extracts was efficiently retained on the major and minor satellite RNA (respectively, lanes 3 and 5 of Figure 4A). As a control, no association was observed between WDHD1 and in vitro 18S rRNA transcripts (lanes 4 and 6). Furthermore, we also detected a specific pull-down of Dicer by the major satellite RNA (Figure 4B), consistent with its previously reported role. Next, to characterize the functional relevance of the observed WDHD1-RNA association, we assessed RNA binding of Dicer in the absence of WDHD1. To this end, cell extracts for the pull-down assay were first subjected to immunodepletion by the control or a WDHD1-specific antibody. After pull-down reactions, precipitates were then probed by immunoblotting. Interestingly, our results revealed that the amount of recovered Dicer was diminished after WDHD1 depletion (Figure 4B), implying that a stable association of Dicer with centromeric RNA may require WDHD1.Figure 4.


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)

WDHD1 associates with the centromere-encoded RNA transcripts. (A and B) RNA pull-down assay was carried out using biotinylated RNA transcripts. (A) In vitro transcribed RNAs corresponding to approximately one repeat of the centromeric major (lane 3) and minor (lane 5) satellite sequences were used as the baits. Precipitated proteins were visualized by western blotting using anti-WDHD1 antibody. Input represents direct loading that equals to 1/25 or 1/50 of the lysate protein used in the pull-down (lanes 1 and 2). Transcripts corresponding to 300 nt (lane 4) and 162 nt (lane 6) of the 18S rRNA sequence serve as controls for the major (lane 3) and minor (lane 5) satellite transcripts, respectively. (B) Dicer association with centromeric RNA sequences attenuated upon WDHD1 depletion. Cell lysates were subjected to immunodepletion using control IgG or WDHD1-specific antibodies, followed by RNA pull-down assay as in (A). (C) RNA-immunoprecipitation assay was performed using antibodies against the indicated proteins (WDHD1 and Dicer). RNA was extracted from the immunoprecipitates and subjected to dot blot analysis using probes corresponding to the sequences of the minor (upper) or major (lower) satellite repeats. (D–F) Association of WDHD1 with Dicer. (D) Anti-Dicer immunoprecipitates (IP) using extracts (input) prepared from NIH-3T3 (left) or 293T (right) cells were probed with antibodies against WDHD1 (top) and Dicer (bottom). (E) 293T cells were transfected with constructs encoding FLAG or FLAG-tagged WDHD1. Immunoprecipitation (IP) of the cell lysates was done using FLAG antibodies, and subsequently analyzed by immunoblotting with antibodies against FLAG (top) and Dicer (bottom). Extent of overexpression and subcellular localization of the FLAG-WDHD1 protein are shown in the Supplementary Information (Supplementary Figure S3). (F) 293T cell lysates were treated with (+) or without (−) RNase A prior to anti-Dicer immunoprecipiation as above. (G) Dicer occupancy of centromeric repeat regions. ChIP assays were performed on crosslinked chromatin from NIH-3T3 cells using antibodies specific for Dicer or control rabbit antibodies (IgG) (left panel). Sequential ChIP analysis was done to assess co-occupancy of the centromere by both WDHD1 and Dicer (right panel). Products of final PCR analysis using primers specific to major satellite repeat DNA sequence are resolved in agarose gel.
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Figure 4: WDHD1 associates with the centromere-encoded RNA transcripts. (A and B) RNA pull-down assay was carried out using biotinylated RNA transcripts. (A) In vitro transcribed RNAs corresponding to approximately one repeat of the centromeric major (lane 3) and minor (lane 5) satellite sequences were used as the baits. Precipitated proteins were visualized by western blotting using anti-WDHD1 antibody. Input represents direct loading that equals to 1/25 or 1/50 of the lysate protein used in the pull-down (lanes 1 and 2). Transcripts corresponding to 300 nt (lane 4) and 162 nt (lane 6) of the 18S rRNA sequence serve as controls for the major (lane 3) and minor (lane 5) satellite transcripts, respectively. (B) Dicer association with centromeric RNA sequences attenuated upon WDHD1 depletion. Cell lysates were subjected to immunodepletion using control IgG or WDHD1-specific antibodies, followed by RNA pull-down assay as in (A). (C) RNA-immunoprecipitation assay was performed using antibodies against the indicated proteins (WDHD1 and Dicer). RNA was extracted from the immunoprecipitates and subjected to dot blot analysis using probes corresponding to the sequences of the minor (upper) or major (lower) satellite repeats. (D–F) Association of WDHD1 with Dicer. (D) Anti-Dicer immunoprecipitates (IP) using extracts (input) prepared from NIH-3T3 (left) or 293T (right) cells were probed with antibodies against WDHD1 (top) and Dicer (bottom). (E) 293T cells were transfected with constructs encoding FLAG or FLAG-tagged WDHD1. Immunoprecipitation (IP) of the cell lysates was done using FLAG antibodies, and subsequently analyzed by immunoblotting with antibodies against FLAG (top) and Dicer (bottom). Extent of overexpression and subcellular localization of the FLAG-WDHD1 protein are shown in the Supplementary Information (Supplementary Figure S3). (F) 293T cell lysates were treated with (+) or without (−) RNase A prior to anti-Dicer immunoprecipiation as above. (G) Dicer occupancy of centromeric repeat regions. ChIP assays were performed on crosslinked chromatin from NIH-3T3 cells using antibodies specific for Dicer or control rabbit antibodies (IgG) (left panel). Sequential ChIP analysis was done to assess co-occupancy of the centromere by both WDHD1 and Dicer (right panel). Products of final PCR analysis using primers specific to major satellite repeat DNA sequence are resolved in agarose gel.
Mentions: Next, to unequivocally elucidate whether WDHD1 is directly involved in the processing of centromeric transcripts, we sought to determine if WDHD1-centromeric RNAs complexes exist. To demonstrate such association, we first performed RNA pull-down assays using streptavidin-coated beads and in vitro transcribed, biotinylated minor and major satellite RNAs, and probed for the presence of endogenous WDHD1 in the precipitated material. The immunoblotting results showed that WDHD1 in nuclear extracts was efficiently retained on the major and minor satellite RNA (respectively, lanes 3 and 5 of Figure 4A). As a control, no association was observed between WDHD1 and in vitro 18S rRNA transcripts (lanes 4 and 6). Furthermore, we also detected a specific pull-down of Dicer by the major satellite RNA (Figure 4B), consistent with its previously reported role. Next, to characterize the functional relevance of the observed WDHD1-RNA association, we assessed RNA binding of Dicer in the absence of WDHD1. To this end, cell extracts for the pull-down assay were first subjected to immunodepletion by the control or a WDHD1-specific antibody. After pull-down reactions, precipitates were then probed by immunoblotting. Interestingly, our results revealed that the amount of recovered Dicer was diminished after WDHD1 depletion (Figure 4B), implying that a stable association of Dicer with centromeric RNA may require WDHD1.Figure 4.

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