The sub-nucleolar localization of PHF6 defines its role in rDNA transcription and early processing events

Abstract

Ribosomal RNA synthesis occurs in the nucleolus and is a tightly regulated process that is targeted in some developmental diseases and hyperactivated in multiple cancers. Subcellular localization and immunoprecipitation coupled mass spectrometry demonstrated that a proportion of plant homeodomain (PHD) finger protein 6 (PHF6) protein is localized within the nucleolus and interacts with proteins involved in ribosomal processing. PHF6 sequence variants cause Börjeson-Forssman-Lehmann syndrome (BFLS, MIM#301900) and are also associated with a female-specific phenotype overlapping with Coffin-Siris syndrome (MIM#135900), T-cell acute lymphoblastic leukemia (MIM#613065), and acute myeloid leukemia (MIM#601626); however, very little is known about its cellular function, including its nucleolar role. HEK 293T cells were treated with RNase A, DNase I, actinomycin D, or 5,6-dichloro-β-D-ribofuranosylbenzimadole, followed by immunocytochemistry to determine PHF6 sub-nucleolar localization. We observed RNA-dependent localization of PHF6 to the sub-nucleolar fibrillar center (FC) and dense fibrillar component (DFC), at whose interface rRNA transcription occurs. Subsequent ChIP-qPCR analysis revealed strong enrichment of PHF6 across the entire rDNA-coding sequence but not along the intergenic spacer (IGS) region. When rRNA levels were quantified in a PHF6 gain-of-function model, we observed an overall decrease in rRNA transcription, accompanied by a modest increase in repressive promoter-associated RNA (pRNA) and a significant increase in the expression levels of the non-coding IGS36RNA and IGS39RNA transcripts. Collectively, our results demonstrate a role for PHF6 in carefully mediating the overall levels of ribosome biogenesis within a cell.

Figure 1

A subset of nuclear PHF6 localizes to the nucleolus. (a) Schematic representation of the three sub-nucleolar compartments: the fibrillar center (FC), the dense fibrillar component (DFC), and the granular component (GC). The image depicts the cell nucleus counterstained with DAPI (blue) and the nucleoli visualized by NCL staining (red). (b) Representative images of immunofluorescently labeled of HeLa cells with antibodies to PHF6 (green) and specific markers (red) for the FC (UBF), DFC (FBRL), and GC (NCL).

Figure 2

PHF6 co-localizes with the FC and partially co-localizes with the DFC. (a) Nucleoli can be partially or completely disassembled in response to chemical treatment with Act D or DRB. During nucleolar reorganization, proteins that are found in the same nucleolar compartments commonly remain associated in the same nascent foci. HEK 293T cells were treated with 25 μg/ml DRB or 0.5 μg/ml Act D for 2 h and then fixed and labeled with antibodies to PHF6 (green) and (b) UBF, (c) FBRL, or (d) NCL (red). Representative cell images are shown. See Supplementary Figure S1 for additional controls.

Figure 3

PHF6 localizes to the nucleolus in an RNA-dependent manner, where it binds to rDNA-coding sequences. (a, b) HEK 293T cells were treated with DNase I (10 μg/ml) or RNase A (100 μg/ml) and then immunofluorescently stained with antibodies to PHF6 (green) and UBF (A; red) or NCL (B; red). Representative cell images are shown. (c) Schematic diagram of the rDNA gene repeat (GenBank: U13369.1), showing the intergenic spacer (IGS) region (red), the rDNA promoter (yellow), and the rRNA-coding region (green). Primer pairs were designed to amplify IGS sequences (A: −15634/−15523; B: −6942/−6839; C: −3712/−3610), the promoter sequence (D: −1017/−924), and the coding sequence (E: −47/+32; F: +307/+445; G: +8204/+8300; H: +12855/+12970). The locations of sequences corresponding to non-coding IGS and unprocessed pRNA (pre-pRNA) transcripts are indicated in blue. PHF6 enrichment throughout the rDNA repeat was determined by ChIP-qPCR (n=18) analysis performed from approximately 10⁶ HEK 293T cells (per experiment). CTCF (n=9) and UBF (n=13) ChIP-qPCR products were quantitated as controls for binding to the coding rDNA promoter and coding sequence, respectively. For each antibody, the individual bars on the graph represent the binding (relative to 1% input) for individual primer sets in sequential order: IGS (primer sets A–C, red), promoter (primer set D, yellow), and coding sequence (primer sets E–H, green). See Supplementary Figure S2 for additional controls.

Figure 4

PHF6 mediates the expression of rDNA coding and non-coding transcripts. (a) qRT-PCR analysis (n=8) of RNA isolated from HEK 293T cell lines expressing empty vector or one of two PHF6 overexpression constructs (PHF6-NTAP, PHF6-CTAP). All Ct values were normalized to GAPDH amplification and fold changes were calculated relative to the empty vector dCt value for all treatments. (b) qRT-PCR analysis (n=6) of RNA isolated from wild-type HEK 293T cells that were treated with 0.5 μg/ml Act D (2 h), 25 μg/ml DRB (2 h), 10 ng/ml trichostatin A (24 h), or 50 μM 5-azacytidine (24 h). The y axis is logarithmic (base 10). Bars represent SE (*P<0.05, **P<0.01, two-tailed Student's t-test). (c) Proposed model to explain how PHF6 overexpression could lead to the arrest of rRNA transcription and RNA Pol I-mediated expression of non-coding IGS transcripts. See Supplementary Figure S4 for additional controls.