WDHD1 modulates the post-transcriptional step of the centromeric silencing pathway.
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.
Affiliation: Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan.
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.
- DNA-Binding Proteins/analysis/antagonists & inhibitors/physiology*
- Gene Silencing*
- Cell Cycle
- Cell Line
- Epigenesis, Genetic
- RNA Processing, Post-Transcriptional
- S Phase
- Transcription, Genetic
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Figure 1: Dynamic patterns of WDHD1 subnuclear localization during S phase. (A) HeLa cells were grown on coverslips, pulse-labeled with BrdU (for marking cells in S phase) before being fixed, and subjected to immunostaining. Left, WDHD1; middle, BrdU; right, merged image (WDHD1, green; BrdU, red). Individual and merged images were captured by laser scanning confocal microscope and single sections are shown (scale bar is 10 µm). Arrowheads mark cells with punctate staining patterns of WDHD1. (B) Distinct focal localization of WDHD1 (green) and its partial colocalization (see merge) with BrdU (red) in S-phase cells. Immunostaining and laser confocal microscopy were performed as in (A). Cells in the indicated sub-stages of S phase (early, mid, or late) were distinguished on the basis of the BrdU incorporation patterns (a–f). (C) Cell-cycle localization of WDHD1 during S phase by immunofluorescence microscopy. NIH-3T3 cells were synchronized at G1/S junction and released into S (a–f). Before being fixed at the indicated post-release time points, cells were pulse-labeled with BrdU (15 min) for visualizing DNA replication site. S phase progression was also analyzed based on DNA content by flow cytometry (shown on the bottom). Images of early S-phase cells subjected to a shorter pulse of BrdU labeling (8 min) are shown for a more defined representation of early replication site (g and h, ‘shorter BrdU pulse’). (D) NIH-3T3 cells were briefly treated to remove the soluble pool of proteins, fixed, and subsequently stained to reveal WDHD1 and pericentric heterochromatin (DAPI). Confocal microscopy was done as above (scale bar is 10 µm). (E) Distinct patterns of WDHD1 localization at centromeric foci. NIH-3T3 cells in S phase were collected as in (C), and subjected to indirect immunofluorescence analysis. Three types of WDHD1 subnuclear localization pattern can be readily distinguished, based on staining signals relative to the DAPI foci: non-chromocenter, horse-shoe and sphere. Representative immunostaining images for the latter two types are shown. The merged images (‘merge’) and the schematic cartoon figure illustrate the relative positions of WDHD1 (‘WDHD1’, green) and the centromeric heterochromatin in nucleus (‘DAPI’, blue, but pseudo-color red in the merged images). The insets represent enlarged images denoted by arrows. The histograms on the right represents the local intensity distribution (diagonal white lines through the images) of WDHD1 in green and DAPI in red. Graph on the bottom shows quantification of cells with the indicated distinct focal patterns of WDHD1 staining at centromeres at 2, 3 and 4 h after release into S phase. One hundred percent represents total number of cells (n = 200) and percentages given are averages of three independent experiments. (F) WDHD1 occupancy of centromeric repeat regions. ChIP assays were performed on crosslinked chromatin from NIH-3T3 cells using antibodies specific for WDHD1 or control rabbit antibodies (IgG). Products of final PCR analysis using primers specific to major satellite repeat DNA sequence are resolved in agarose gel.
To begin investigating the biological function of WDHD1, we first performed indirect immunofluorescence analysis to examine its cellular localization in cycling HeLa cells. Consistent with previous observations, WDHD1 localized to the nucleus (Figure 1A, left panel). Interestingly, in a sub-population of cells, WDHD1 displayed a unique, punctate staining (indicated by arrowheads). These cells corresponded to those actively undergoing DNA replication, as indicated by BrdU incorporation (Figure 1A, middle panel). Furthermore, confocal images revealed a partial overlap between WDHD1 and BrdU staining signals (Figure 1A, right panel, arrowheads). A detailed breakdown of the S-phase cells (Figure 1B), based on the distribution of DNA replication sites, further pinpointed the distinct punctate appearance temporally at the mid-to-late stage of S phase (Figure 1B, c–e).Figure 1.