Limits...
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.

Show MeSH

Related in: MedlinePlus

Characterization of the molecular determinants underlying WDHD1′s centromeric localization. (A) WDHD1 was isolated from NIH-3T3 cell lysates by immunoprecipitation (IP) using α-WDHD1 antibody. Western blot analysis was subsequently performed with antibodies against selected components of the DNA replication and RNA transcription complexes, as indicated. IgG refers to control rabbit or mouse Ab IP using whole cell lysates derived from the same cells. Input equals 1/10 of IP. (B and C) Indirect immunofluorescence analysis was performed to observe localization of endogenous WDHD1 (red) and HP1α (green) in the inhibitor-treated NIH-3T3 cells. (B) Before immunostaining, cells arrested at the G1/S junction were released in the presence of 100 µM DRB for 2 h to inhibit Pol II activity (+DRB) or in its absence (−DRB) and subsequently pulse-labeled with BrdU. Nuclear DNA was stained by DAPI (blue). Images were captured by laser scanning confocal microscopy and single sections are shown (scale bar is 10 µm). (C) NIH-3T3 cells grown on coverslips were first permeablized, and subsequently mock- (−) or RNase A- (+) treated before fixation. Immunostaining were performed with antibodies specific for WDHD1 (red) and HP1α (green). Nuclear DNA was stained by DAPI (blue). Confocal microscopy was done as above (scale bar is 10 µm). Removal of RNA was monitored by gel electrophoresis of total RNA extracted from the cells, as shown by the EtBr staining image (right).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3105424&req=5

Figure 2: Characterization of the molecular determinants underlying WDHD1′s centromeric localization. (A) WDHD1 was isolated from NIH-3T3 cell lysates by immunoprecipitation (IP) using α-WDHD1 antibody. Western blot analysis was subsequently performed with antibodies against selected components of the DNA replication and RNA transcription complexes, as indicated. IgG refers to control rabbit or mouse Ab IP using whole cell lysates derived from the same cells. Input equals 1/10 of IP. (B and C) Indirect immunofluorescence analysis was performed to observe localization of endogenous WDHD1 (red) and HP1α (green) in the inhibitor-treated NIH-3T3 cells. (B) Before immunostaining, cells arrested at the G1/S junction were released in the presence of 100 µM DRB for 2 h to inhibit Pol II activity (+DRB) or in its absence (−DRB) and subsequently pulse-labeled with BrdU. Nuclear DNA was stained by DAPI (blue). Images were captured by laser scanning confocal microscopy and single sections are shown (scale bar is 10 µm). (C) NIH-3T3 cells grown on coverslips were first permeablized, and subsequently mock- (−) or RNase A- (+) treated before fixation. Immunostaining were performed with antibodies specific for WDHD1 (red) and HP1α (green). Nuclear DNA was stained by DAPI (blue). Confocal microscopy was done as above (scale bar is 10 µm). Removal of RNA was monitored by gel electrophoresis of total RNA extracted from the cells, as shown by the EtBr staining image (right).

Mentions: Having established the spatiotemporal patterns of WDHD1 nuclear localization, we next aimed to further explore the molecular basis of such attribute. Towards this end, we first performed selective profiling of WDHD1’s associated proteins in the NIH-3T3 cell nucleus (Figure 2A). Immunoprecipitation assays revealed that, in contrast to the previous findings, our WDHD1 specific antibodies did not co-precipitate DNA polymerase δ. Architectural components of pericentric replication foci, PCNA, CAF-1 and HP1α, also were not present in the WDHD1 immunoprecipitates. Conversely, we were able to detect association of RNA Pol II as well as known post-transcriptional regulators of the CT/PCT, such as ADAR and Vigilin, with WDHD1. These findings collectively raised the possibility that the distinct overlap of WDHD1 with PCT replication foci may actually be coupled with transcriptional and/or post-transcriptional events taking place in the vicinity. In line with such notion, we observed that inhibition of Pol II transcription by DRB treatment in a mid-late S phase population of cells resulted in the disappearance of WDHD1 staining at pericentric heterochromatin domains in the vast majority of cells (Figure 2B), thus suggesting that such localization requires ongoing RNA transcription. However, this change in staining did not result from a loss of RNA, as RNase-treated cells retained significant WDHD1 focal signals (Figure 2C). Incidentally, colocalization was evident between the RNase-resistant WDHD1 foci and signals corresponding to the RNase-insensitive, replication-specific HP1α pool (42) (Figure 2C), again affirming the spatiotemporal mode of WDHD1 pericentromere recruitment. Given that transcription of the satellite repeats underlies the establishment of replicated CT/PCT heterochromatin, our results may support a link between WDHD1 and the regulation of satellite repeats-encoded RNA expression in cells.Figure 2.


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)

Characterization of the molecular determinants underlying WDHD1′s centromeric localization. (A) WDHD1 was isolated from NIH-3T3 cell lysates by immunoprecipitation (IP) using α-WDHD1 antibody. Western blot analysis was subsequently performed with antibodies against selected components of the DNA replication and RNA transcription complexes, as indicated. IgG refers to control rabbit or mouse Ab IP using whole cell lysates derived from the same cells. Input equals 1/10 of IP. (B and C) Indirect immunofluorescence analysis was performed to observe localization of endogenous WDHD1 (red) and HP1α (green) in the inhibitor-treated NIH-3T3 cells. (B) Before immunostaining, cells arrested at the G1/S junction were released in the presence of 100 µM DRB for 2 h to inhibit Pol II activity (+DRB) or in its absence (−DRB) and subsequently pulse-labeled with BrdU. Nuclear DNA was stained by DAPI (blue). Images were captured by laser scanning confocal microscopy and single sections are shown (scale bar is 10 µm). (C) NIH-3T3 cells grown on coverslips were first permeablized, and subsequently mock- (−) or RNase A- (+) treated before fixation. Immunostaining were performed with antibodies specific for WDHD1 (red) and HP1α (green). Nuclear DNA was stained by DAPI (blue). Confocal microscopy was done as above (scale bar is 10 µm). Removal of RNA was monitored by gel electrophoresis of total RNA extracted from the cells, as shown by the EtBr staining image (right).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105424&req=5

Figure 2: Characterization of the molecular determinants underlying WDHD1′s centromeric localization. (A) WDHD1 was isolated from NIH-3T3 cell lysates by immunoprecipitation (IP) using α-WDHD1 antibody. Western blot analysis was subsequently performed with antibodies against selected components of the DNA replication and RNA transcription complexes, as indicated. IgG refers to control rabbit or mouse Ab IP using whole cell lysates derived from the same cells. Input equals 1/10 of IP. (B and C) Indirect immunofluorescence analysis was performed to observe localization of endogenous WDHD1 (red) and HP1α (green) in the inhibitor-treated NIH-3T3 cells. (B) Before immunostaining, cells arrested at the G1/S junction were released in the presence of 100 µM DRB for 2 h to inhibit Pol II activity (+DRB) or in its absence (−DRB) and subsequently pulse-labeled with BrdU. Nuclear DNA was stained by DAPI (blue). Images were captured by laser scanning confocal microscopy and single sections are shown (scale bar is 10 µm). (C) NIH-3T3 cells grown on coverslips were first permeablized, and subsequently mock- (−) or RNase A- (+) treated before fixation. Immunostaining were performed with antibodies specific for WDHD1 (red) and HP1α (green). Nuclear DNA was stained by DAPI (blue). Confocal microscopy was done as above (scale bar is 10 µm). Removal of RNA was monitored by gel electrophoresis of total RNA extracted from the cells, as shown by the EtBr staining image (right).
Mentions: Having established the spatiotemporal patterns of WDHD1 nuclear localization, we next aimed to further explore the molecular basis of such attribute. Towards this end, we first performed selective profiling of WDHD1’s associated proteins in the NIH-3T3 cell nucleus (Figure 2A). Immunoprecipitation assays revealed that, in contrast to the previous findings, our WDHD1 specific antibodies did not co-precipitate DNA polymerase δ. Architectural components of pericentric replication foci, PCNA, CAF-1 and HP1α, also were not present in the WDHD1 immunoprecipitates. Conversely, we were able to detect association of RNA Pol II as well as known post-transcriptional regulators of the CT/PCT, such as ADAR and Vigilin, with WDHD1. These findings collectively raised the possibility that the distinct overlap of WDHD1 with PCT replication foci may actually be coupled with transcriptional and/or post-transcriptional events taking place in the vicinity. In line with such notion, we observed that inhibition of Pol II transcription by DRB treatment in a mid-late S phase population of cells resulted in the disappearance of WDHD1 staining at pericentric heterochromatin domains in the vast majority of cells (Figure 2B), thus suggesting that such localization requires ongoing RNA transcription. However, this change in staining did not result from a loss of RNA, as RNase-treated cells retained significant WDHD1 focal signals (Figure 2C). Incidentally, colocalization was evident between the RNase-resistant WDHD1 foci and signals corresponding to the RNase-insensitive, replication-specific HP1α pool (42) (Figure 2C), again affirming the spatiotemporal mode of WDHD1 pericentromere recruitment. Given that transcription of the satellite repeats underlies the establishment of replicated CT/PCT heterochromatin, our results may support a link between WDHD1 and the regulation of satellite repeats-encoded RNA expression in cells.Figure 2.

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