Plant homeodomain finger protein 6 (PHF6) is a chromatin adaptor protein structurally defined by its two zinc-knuckle-atypical PHD (ZaP) domains. This structural configuration mediates its interaction with dsDNA, miRNA, the nucleosome remodeling and deacetylase (NuRD) complex and regulators of rDNA transcription (Upstream binding factor (UBF) and RNA polymerase-associated factor 1 complex (Paf1C)), ultimately facilitating its role as a chromatin adaptor protein and regulator of gene expression. Mutations in the gene are implicated in Börjeson–Forssman–Lehmann syndrome (BFLS), a rare X-linked intellectual disability disorder characterized by large ears, truncal obesity, and long tapering fingers. BFLS is primarily caused by missense and nonsense mutations while deletions, frameshifts and mutations disrupting the structural integrity of the ZaP domains have been described in T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML) patients. To provide more insight into PHF6 and BFLS, we have generated a Phf6 transgenic mouse line with a patient-related nonsense mutation (R342X). We show that the mutation drastically reduced Phf6 transcript levels and produced a truncated protein at very low levels in the developing brain. Mice were born at normal Mendelian ratios but mutant mice were significantly smaller than control littermates. Volumetric analysis of the brain via high resolution MRI revealed increased sizes of the amygdala, periaqueductal gray, and hypothalamus, and decreased volumes within the striatum, hippocampus and cerebellum. Studies of the pituitary gland revealed a postnatal defect in the growth of the anterior pituitary but not the posterior or intermediate regions. This change was reflected in altered expression levels of several hormones in the hypothalamic-pituitary-adrenal axis. Preliminary behavioral tests highlighted deficits in the anxiety and depression response of the mutant mice. Additional studies to fully characterize these mice are ongoing.

Defective chromatin remodeling proteins are associated with both germline and acquired human disease. PHF6 is encoded by an X-linked gene that is predominantly expressed in the brain and thymus. Structurally, PHF6 contains nuclear and nucleolar localization sequences as well as two ZaP domains, which bind dsDNA. Germline mutations in PHF6 are the cause of BFLS, an XLID, while somatic PHF6 mutations have been identified in T-ALL, AML, and CML. Indeed, screening of a pediatric cohort of nine T-ALL patients revealed a novel H329Q mutation. In a further clinical analysis, T-ALL onset occurred in a 9-year old male BFLS patient with an R342X mutation, suggesting that BFLS might be a cancer predisposition syndrome. To better understand its protein function, recombinant PHF6 was co-immunoprecipitated for a mass spectrometry based proteomic screen. Notably, PHF6 co-purified with multiple constituents of the NuRD complex, an important transcriptional regulator during embryogenesis and lineage commitment with particularly well characterized responsibilities during lymphogenesis. PHF6-NuRD localization was restricted to the nucleoplasm, however PHF6 also co-purified with several ribosomal and splicing proteins. When examined further, PHF6 was found to be recruited to the nucleolus by an RNA-mediated interaction and co-localized within the subnucleolar FC and DFC compartments. ChIP-qPCR revealed that PHF6 binds to transcribed regions of rDNA, resulting in the repression of rRNA. These data thus present a model of PHF6 acting as a tumour suppressor by mediating both nucleoplasmic and nucleolar transcriptional events.