PHF19 promotes multiple myeloma tumorigenicity through PRC2 activation and broad H3K27me3 domain formation.
ABSTRACT: Dysregulation of polycomb repressive complex 2 (PRC2) promotes oncogenesis partly through its enzymatic function for inducing trimethylation of histone H3 lysine 27 (H3K27me3). However, it remains to be determined how PRC2 activity is regulated in normal and diseased settings. We here report a PRC2-associated cofactor, PHD finger protein 19 (PHF19; also known as polycomb-like 3), as a crucial mediator of tumorigenicity in multiple myeloma (MM). Overexpression and/or genomic amplification of PHF19 is found associated with malignant progression of MM and plasma cell leukemia, correlating to worse treatment outcomes. Using various MM models, we demonstrated a critical requirement of PHF19 for tumor growth in vitro and in vivo. Mechanistically, PHF19-mediated oncogenic effect relies on its PRC2-interacting and chromatin-binding functions. Chromatin immunoprecipitation followed by sequencing profiling showed a critical role for PHF19 in maintaining the H3K27me3 landscape. PHF19 depletion led to loss of broad H3K27me3 domains, possibly due to impaired H3K27me3 spreading from cytosine guanine dinucleotide islands, which is reminiscent to the reported effect of an "onco"-histone mutation, H3K27 to methionine (H3K27M). RNA-sequencing-based transcriptome profiling in MM lines also demonstrated a requirement of PHF19 for optimal silencing of PRC2 targets, which include cell cycle inhibitors and interferon-JAK-STAT signaling genes critically involved in tumor suppression. Correlation studies using patient sample data sets further support a clinical relevance of the PHF19-regulated pathways. Lastly, we show that MM cells are generally sensitive to PRC2 inhibitors. Collectively, this study demonstrates that PHF19 promotes MM tumorigenesis through enhancing H3K27me3 deposition and PRC2's gene-regulatory functions, lending support for PRC2 blockade as a means for MM therapeutics.
Project description:Polycomb group proteins are transcriptional repressors that play essential roles in regulating genes required for differentiation and embryonic development. The Polycomb repressive complex 2 (PRC2) contains the methyltransferase activity for lysine 27 on histone 3 (H3K27me3), which is a docking site for the PRC1 complex and leads to gene repression. However, the role of other histone modifications in regulating PRC2 activity is just beginning to be understood. Here we show that direct recognition of histone H3 methylated at lysine 36 (H3K36me), an mark associated with activation, by the PRC2 subunit Phf19 is required for the full enzymatic activity of the PRC2 complex. We provide structural evidence for this interaction by nuclear magnetic resonance spectroscopy (NMR). Using genome-wide chromatin binding analyses and expression analyses, we show that Phf19 binds to a subset of PRC2 targets in embryonic stem (ES) cells, and that this is required for their repression and for H3K27me3 deposition. These findings reveal that the H3K36me2/3-Phf19 interaction is essential for PRC2 complex activity and for proper regulation of gene repression in ES cells. We determined the genome binding/occupancy profile of Phf19, H3K36me3, H3K36me2, H3K27me3 and Suz12 by high throughput sequencing in mouse embryonic stem cells. For Phf19 two independent biological replicas were performed and Phf19 binding sites were defined as those sites (ChIP-seq peaks) present in both replicas. H3K27me3 was evaluated in control ES cells and cells depleted of Phf19 (shRd and shPhf19 respectively).
Project description:Dysregulation of Polycomb Repressive Complex 2 (PRC2) promotes oncogenesis partly through its enzymatic function for inducing tri-methylation of histone H3 lysine 27 (H3K27me3). However, it remains to be determined how PRC2 activity is regulated in normal and diseased settings. We here report a PRC2-associated cofactor, PHD finger protein 19 (PHF19, also known as Polycomb-like 3), as a crucial mediator of tumorigenicity in multiple myeloma (MM). Overexpression and/or genomic amplification of PHF19 is found associated with malignant progression of MM and plasma cell leukemia, correlating to worse treatment outcomes. Using various MM models, we demonstrated a critical requirement of PHF19 for tumor growth in vitro and in vivo. Mechanistically, PHF19-mediated oncogenic effect relies on its PRC2-interacting and chromatin-binding functions. ChIP-Seq profiling showed a critical role for PHF19 in maintaining the H3K27me3 landscape. PHF19 depletion led to loss of broad H3K27me3 domains possibly due to impaired H3K27me3 spreading from CpG islands, which is reminiscent to the reported effect of an ‘onco’-histone mutation, H3K27-to-methionine (H3K27M). RNA-Seq-based transcriptome profiling in MM lines also demonstrated a requirement of PHF19 for optimal silencing of PRC2 targets, which include cell cycle inhibitors and interferon-JAK-STAT signaling genes critically involved in tumor suppression. Overall design: Examination of transcriptome profiles in the MM.1S cells with stable expression of either empty vector (shEV) or shPHF19 for knockdown of PHF19 (PHF19-KD), as well as PHF19-KD cells rescued with shRNA-resistant PHF19 (KD_Rescued)
Project description:Despite improvements in outcome, 15-25% of newly diagnosed multiple myeloma (MM) patients have treatment resistant high-risk (HR) disease with a poor survival. The lack of a genetic basis for HR has focused attention on the role played by epigenetic changes. Aberrant expression and somatic mutations affecting genes involved in the regulation of tri-methylation of the lysine (K) 27 on histone 3 H3 (H3K27me3) are common in cancer. H3K27me3 is catalyzed by EZH2, the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2). The deregulation of H3K27me3 has been shown to be involved in oncogenic transformation and tumor progression in a variety of hematological malignancies including MM. Recently we have shown that aberrant overexpression of the PRC2 subunit PHD Finger Protein 19 (PHF19) is the most significant overall contributor to HR status further focusing attention on the role played by epigenetic change in MM. By modulating both the PRC2/EZH2 catalytic activity and recruitment, PHF19 regulates the expression of key genes involved in cell growth and differentiation. Here we review the expression, regulation and function of PHF19 both in normal and the pathological contexts of solid cancers and MM. We present evidence that strongly implicates PHF19 in the regulation of genes important in cell cycle and the genetic stability of MM cells making it highly relevant to HR MM behavior. A detailed understanding of the normal and pathological functions of PHF19 will allow us to design therapeutic strategies able to target aggressive subsets of MM.
Project description:The Polycomb-like protein PHF19/PCL3 associates with PRC2 and mediates its recruitment to chromatin in embryonic stem cells. PHF19 is also overexpressed in many cancers. However, neither PHF19 targets nor misregulated pathways involving PHF19 are known. Here, we investigate the role of PHF19 in prostate cancer cells. We find that PHF19 interacts with PRC2 and binds to PRC2 targets on chromatin. PHF19 target genes are involved in proliferation, differentiation, angiogenesis, and extracellular matrix organization. Depletion of PHF19 triggers an increase in MTF2/PCL2 chromatin recruitment, with a genome-wide gain in PRC2 occupancy and H3K27me3 deposition. Transcriptome analysis shows that PHF19 loss promotes deregulation of key genes involved in growth, metastasis, invasion, and of factors that stimulate blood vessels formation. Consistent with this, PHF19 silencing reduces cell proliferation, while promotes invasive growth and angiogenesis. Our findings reveal a role for PHF19 in controlling the balance between cell proliferation and invasiveness in prostate cancer.
Project description:The Polycomb repressive complex 2 (PRC2) is a multicomponent histone H3K27 methyltransferase complex, best known for silencing the Hox genes during embryonic development. The Polycomb-like proteins PHF1, MTF2, and PHF19 are critical components of PRC2 by stimulating its catalytic activity in embryonic stem cells. The Tudor domains of PHF1/19 have been previously shown to be readers of H3K36me3 in vitro. However, some other studies suggest that PHF1 and PHF19 co-localize with the H3K27me3 mark but not H3K36me3 in cells. Here, we provide further evidence that PHF1 co-localizes with H3t in testis and its Tudor domain preferentially binds to H3tK27me3 over canonical H3K27me3 in vitro. Our complex structures of the Tudor domains of PHF1 and PHF19 with H3tK27me3 shed light on the molecular basis for preferential recognition of H3tK27me3 by PHF1 and PHF19 over canonical H3K27me3, implicating that H3tK27me3 might be a physiological ligand of PHF1/19.
Project description:Polycomb-group proteins are transcriptional repressors with essential roles in embryonic development. Polycomb repressive complex 2 (PRC2) contains the methyltransferase activity for Lys27. However, the role of other histone modifications in regulating PRC2 activity is just beginning to be understood. Here we show that direct recognition of methylated histone H3 Lys36 (H3K36me), a mark associated with activation, by the PRC2 subunit Phf19 is required for the full enzymatic activity of the PRC2 complex. Using NMR spectroscopy, we provide structural evidence for this interaction. Furthermore, we show that Phf19 binds to a subset of PRC2 targets in mouse embryonic stem cells and that this is required for their repression and for H3K27me3 deposition. These findings show that the interaction of Phf19 with H3K36me2 and H3K36me3 is essential for PRC2 complex activity and for proper regulation of gene repression in embryonic stem cells.
Project description:Polycomb repressive complex 2 (PRC2) regulates pluripotency, differentiation, and tumorigenesis through catalysis of histone H3 lysine 27 trimethylation (H3K27me3) on chromatin. However, the mechanisms that underlie PRC2 recruitment and spreading on chromatin remain unclear. Here we report that histone H3 lysine 36 trimethylation (H3K36me3) binding activity is harbored in the Tudor motifs of PRC2-associated polycomb-like (PCL) proteins PHF1/PCL1 and PHF19/PCL3. Ectopically expressed PHF1 induced Tudor-dependent stabilization of PRC2 complexes on bulk chromatin and mediated spreading of PRC2 and H3K27me3 into H3K36me3-containing chromatin regions. In murine pluripotent stem cells, we identified coexistence of H3K36me3, H3K27me3, and PHF19/PCL3 at a subset of poised developmental genes and demonstrated that PHF19/PCL3 Tudor function is required for optimal H3K27me3 and repression of these loci. Collectively, our data suggest that PCL recognition of H3K36me3 promotes intrusion of PRC2 complexes into active chromatin regions to promote gene silencing and modulate the chromatin landscape during development.
Project description:Epigenetic deregulation is increasingly recognized as a contributing pathological factor in multiple myeloma (MM). In particular tri-methylation of H3 lysine 27 (H3K27me3), which is catalyzed by PHD finger protein 19 (PHF19), a subunit of the Polycomb Repressive Complex 2 (PRC2), has recently shown to be a crucial mediator of MM tumorigenicity. Overexpression of PHF19 in MM has been associated with worse clinical outcome. Yet, while there is mounting evidence that PHF19 overexpression plays a crucial role in MM carcinogenesis downstream mechanisms remain to be elucidated. In the current study we use a functional knock down (KD) of PHF19 to investigate the biological role of PHF19 and show that PHF19KD leads to decreased tumor growth in vitro and in vivo. Expression of major cancer players such as bcl2, myc and EGR1 were decreased upon PHF19KD further underscoring the role of PHF19 in MM biology. Additionally, our results highlighted the prognostic impact of PHF19 overexpression, which was significantly associated with worse survival. Overall, our study underscores the premise that targeting the PHF19-PRC2 complex would open up avenues for novel MM therapies.
Project description:Adult hematopoietic stem cells (HSCs) are rare multipotent cells in bone marrow that are responsible for generating all blood cell types. HSCs are a heterogeneous group of cells with high plasticity, in part, conferred by epigenetic mechanisms. PHF19, a subunit of the Polycomb repressive complex 2 (PRC2), is preferentially expressed in mouse hematopoietic precursors. Here, we now show that, in stark contrast to results published for other PRC2 subunits, genetic depletion of Phf19 increases HSC identity and quiescence. While proliferation of HSCs is normally triggered by forced mobilization, defects in differentiation impede long-term correct blood production, eventually leading to aberrant hematopoiesis. At molecular level, PHF19 deletion triggers a redistribution of the histone repressive mark H3K27me3, which notably accumulates at blood lineage-specific genes. Our results provide novel insights into how epigenetic mechanisms determine HSC identity, control differentiation, and are key for proper hematopoiesis.
Project description:The Polycomb group proteins are repressive chromatin modifiers with essential roles in metazoan development, cellular differentiation and cell fate maintenance. How Polycomb proteins access active chromatin in order to confer transcriptional silencing during lineage transitions remains unclear. Here we show that the Polycomb Repressive Complex 2 (PRC2) component PHF19 binds the active chromatin mark H3K36me3 via its tudor domain. PHF19 associates with the H3K36me3 demethylase NO66, and is required to recruit the PRC2 complex and NO66 to stem cells genes during differentiation, leading to PRC2 mediated H3K27 tri-methylation, loss of H3K36me3 and transcriptional silencing. We propose a model whereby PHF19 functions during ES cell differentiation to transiently bind the H3K36me3 mark via its tudor domain, forming essential contact points that allow recruitment of PRC2 and H3K36me3 demethylase activity to active gene loci during their transition to a Polycomb-repressed state. Examination of PHF19 genome-wide binding in mouse embryonic stem cells