title: 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.
authors.affiliation: Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada.|Department of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada.
authors.forename: Matthew A M
authors.initials: MA
authors.lastname: Todd
authors.affiliation: Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Huh
authors.affiliation: Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada.|Department of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada.|Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada.
authors.forename: David J
authors.initials: DJ
authors.lastname: Picketts
mesh_terms: D021581:Active Transport, Cell Nucleus; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012335:RNA, Ribosomal / Q000235:genetics*; D012097:Repressor Proteins
chemical_list: D002352:Carrier Proteins; C502542:PHF6 protein, human; D012335:RNA, Ribosomal; D012097:Repressor Proteins