CHD7 functions in the nucleolus as a positive regulator of ribosomal RNA biogenesis - PubMed (original) (raw)

. 2010 Sep 15;19(18):3491-501.

doi: 10.1093/hmg/ddq265. Epub 2010 Jun 29.

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CHD7 functions in the nucleolus as a positive regulator of ribosomal RNA biogenesis

Gabriel E Zentner et al. Hum Mol Genet. 2010.

Abstract

De novo mutation of the gene encoding chromodomain helicase DNA-binding protein 7 (CHD7) is the primary cause of CHARGE syndrome, a complex developmental disorder characterized by the co-occurrence of a specific set of birth defects. Recent studies indicate that CHD7 functions as a transcriptional regulator in the nucleoplasm. Here, we report based on immunofluorescence and western blotting of subcellular fractions that CHD7 is also constitutively localized to the nucleolus, the site of rRNA transcription. Standard chromatin immunoprecipitation (ChIP) assays indicate that CHD7 physically associates with rDNA, a result that is also observable upon alignment of whole-genome CHD7 ChIP coupled with massively parallel DNA sequencing data to the rDNA reference sequence. ChIP-chop analyses demonstrate that CHD7 specifically associates with hypomethylated, active rDNA, suggesting a role as a positive regulator of rRNA synthesis. Consistent with this hypothesis, siRNA-mediated depletion of CHD7 results in hypermethylation of the rDNA promoter and a concomitant reduction of 45S pre-rRNA levels. Accordingly, cells overexpressing CHD7 show increased levels of 45S pre-rRNA compared with control cells. Depletion of CHD7 also reduced cell proliferation and protein synthesis. Lastly, compared with wild-type ES cells, the levels of 45S pre-rRNA are reduced in both Chd7(+/-) and Chd7(-/-) mouse ES cells, as well as in Chd7(-/-) whole mouse embryos and multiple tissues dissected from Chd7(+/-) embryos. Together with previously published studies, these results indicate that CHD7 dually functions as a regulator of both nucleoplasmic and nucleolar genes and provide a novel avenue for investigation into the pathogenesis of CHARGE syndrome.

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Figures

Figure 1.

Figure 1.

CHD7 localizes to the nucleoplasm and nucleolus. (A) Immunofluorescent staining of DLD1-A2 cells with FLAG and nucleolin antibodies. The arrow indicates nucleolar localization of CHD7, whereas the asterisk marks nucleoplasmic CHD7. Scale bar = 4 µm. (B) Western blot analysis of subcellular fractions from DLD1-A2 and DLD1-WT cells. The purity of cytoplasmic (CP), nucleoplasmic (NP) and nucleolar (No) fractions was assessed by blotting for tubulin, NUP62 and UBF, respectively. Densitometric quantification of CHD7 in the nucleoplasmic and nucleolar fractions is also shown and expressed as a percentage of total CHD7 signal.

Figure 2.

Figure 2.

CHD7 binds to rDNA. (A) Schematic representation of a mammalian rDNA repeat. Approximate locations of qPCR primers for human ChIP are denoted by black boxes and mouse ChIP by open boxes. (B) ChIP–PCR analysis of FLAG-CHD7 binding to the human rDNA locus. (C) ChIP–PCR analysis of CHD7 binding to the mouse rDNA locus. For ChIP–PCR, non-target regions are included as controls for specificity of CHD7 enrichment. Error bars represent the mean + SD for triplicates. ChIP-seq of FLAG-CHD7 at human rDNA in DLD1-A2 cells (D) and mouse rDNA in mES cells (E) recapitulates the binding patterns of CHD7 seen by ChIP–PCR. *P < 0.05; **P < 0.01; ***P < 0.001 by _t_-test versus non-target regions.

Figure 3.

Figure 3.

CHD7 positively regulates rRNA transcription. (A) qRT–PCR and western blot analysis of CHD7 mRNA and protein levels following the treatment of DLD1-A2 cells with non-target siRNA pools (siNT-1/2), a CHD7 siRNA pool (siCHD7-1) and a CHD7 siRNA not present in the pool (siCHD7-2). (B) qRT–PCR for the 45S pre-rRNA in cells treated with control or CHD7 siRNA (n = 2–5). (C) qRT–PCR and western blot analysis of CHD7 mRNA and protein levels in mES cells derived from Whirligig embryos. (D) qRT–PCR for the 45S pre-rRNA in mES cells (n = 4–7). (E) qRT–PCR analysis of 45S pre-rRNA levels in DLD1-A2 cells transfected with empty vector or plasmid encoding untagged human CHD7 protein (n = 2). The FLAG-tagged CHD7 expressed in DLD1-A2 cells is not detectable with the Abcam CHD7 antibody and thus, blotting with this antibody only reveals the untagged CHD7 that was transfected (see

Supplementary Material, Fig. S2

). Error bars represent mean + SEM for biological replicates. *P < 0.05; **P < 0.01; ***P < 0.001 by _t_-test.

Figure 4.

Figure 4.

Loss of CHD7 impairs cell proliferation and protein synthesis. (A) Cell counting assay performed in DLD1-A2 cells treated with the indicated siRNA. Error bars represent mean + SD (n = 3). (B) Quantification of BrdU labeling 5 days post-transfection in DLD1-A2 cells treated with control or CHD7 siRNA. Error bars represent mean + SEM (n = 2–3). (C) Representative image of BrdU labeling in control and BrdU labeling in DLD1-A2 cells showing a qualitative reduction in labeling in CHD7 siRNA-treated cells. Fields are approximately matched for cell number (160–180 cells/field). Scale bar = 32 µm. (D) Measurement of global protein synthesis in DLD1-A2 cells after CHD7 knockdown by [35S]methionine radiolabeling at the indicated time points after siRNA transfection. Scintillation counts were normalized to total protein. (E) Western blots of FLAG-CHD7 and p53 in DLD1-A2 cells. These blots indicate that the CHD7 siRNA is effective up to 5 days post-transfection and that p53 protein levels are not altered. (F) qRT–PCR analysis of CHD7, p53 and p21 transcript levels in DLD1-A2 cells 4 days post-transfection with siRNA (n = 4). *P < 0.05; **P < 0.01; ***P < 0.001 by _t_-test.

Figure 5.

Figure 5.

CHD7 is associated with active rDNA repeats and counteracts rDNA promoter methylation. (A) Schematic representation of active and inactive rDNA loci showing their respective epigenetic features. Open circles represent unmethylated CpG dinucleotides and methylated CpG dinucleotides are represented as filled circles. (B) ChIP-chop analysis of FLAG-CHD7, H3K4me2 and H3K9me2 binding to the human rDNA locus in DLD1-A2 cells (n = 2). (C) Analysis of rDNA promoter methylation in DLD1-A2 cells by _Hpa_II/_Msp_I digest and qPCR (n = 3). (D) Analysis of rDNA promoter methylation in mES cells by _Hpa_II/_Msp_I digest and qPCR (n = 3). Error bars represent the mean + SEM for biological replicates. *P < 0.05 by _t_-test.

Figure 6.

Figure 6.

Pre-rRNA levels are reduced in embryonic Chd7 mutant CHARGE tissues. (A) qRT–PCR for Chd7 and pre-rRNA in whole E10.5 Chd7 gene-trap embryos (n = 2–3). (B) qRT–PCR for the 45S pre-rRNA in Chd7 mutant tissues. (C) qRT–PCR for the 45S pre-rRNA in Chd7 mutant tissues. Error bars represent the mean + SEM for biological replicates (n = 3–4). *P < 0.05, **P < 0.01 by _t_-test.

Figure 7.

Figure 7.

CHD7 promotes association of treacle with rDNA. (A) ChIP analysis of treacle binding to rDNA in Chd7 +/+ and Chd7 −/− mES cells (n = 2). Two nucleoplasmic non-target regions are included as controls for ChIP specificity. (B) qRT–PCR and western blot analysis of Tcof1 mRNA and treacle protein levels, respectively, in Chd7 +/+ and Chd7 −/− mES cells (n = 3). Error bars represent the mean + SEM for biological replicates. *P < 0.05 by _t_-test.

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