Haematopoietic malignancies caused by dysregulation of a chromatin-binding PHD finger

Abstract

Histone H3 lysine 4 methylation (H3K4me) has been proposed as a critical component in regulating gene expression, epigenetic states, and cellular identities1. The biological meaning of H3K4me is interpreted by conserved modules including plant homeodomain (PHD) fingers that recognize varied H3K4me states. The dysregulation of PHD fingers has been implicated in several human diseases, including cancers and immune or neurological disorders. Here we report that fusing an H3K4-trimethylation (H3K4me3)-binding PHD finger, such as the carboxy-terminal PHD finger of PHF23 or JARID1A (also known as KDM5A or RBBP2), to a common fusion partner nucleoporin-98 (NUP98) as identified in human leukaemias, generated potent oncoproteins that arrested haematopoietic differentiation and induced acute myeloid leukaemia in murine models. In these processes, a PHD finger that specifically recognizes H3K4me3/2 marks was essential for leukaemogenesis. Mutations in PHD fingers that abrogated H3K4me3 binding also abolished leukaemic transformation. NUP98-PHD fusion prevented the differentiation-associated removal of H3K4me3 at many loci encoding lineage-specific transcription factors (Hox(s), Gata3, Meis1, Eya1 and Pbx1), and enforced their active gene transcription in murine haematopoietic stem/progenitor cells. Mechanistically, NUP98-PHD fusions act as 'chromatin boundary factors', dominating over polycomb-mediated gene silencing to 'lock' developmentally critical loci into an active chromatin state (H3K4me3 with induced histone acetylation), a state that defined leukaemia stem cells. Collectively, our studies represent, to our knowledge, the first report that deregulation of the PHD finger, an 'effector' of specific histone modification, perturbs the epigenetic dynamics on developmentally critical loci, catastrophizes cellular fate decision-making, and even causes oncogenesis during mammalian development.

Figure 1. The PHD finger-containing NUP98-JARID1A fusion isoform (NJL), but not that lacking the PHD finger (NJS), confers leukomogenic potentials to hematopoietic stem/progenitor cells

a, NUP98-JARID1A and NUP98-PHF23 structure (see Supplementary Fig.1 for details). b, Immunoblot of hematopoietic cells transduced with empty vector (lanes 1-2) or that encoding FLAG-tagged NJS (lanes 3-4) or NJL (lanes 5-6). c, Proliferation kinetics of lineage-negative hematopoietic cells after transduction of empty vector, NJL or NJS. Data are presented as mean ±s.d. of 6 experiments. d, Wright-Giemsa staining (insert, microscopy image) and e, FACS of NJL-transformed cells. f, Leukemia kinetics in mice (12 each group) after transplantation of bone marrow transduced with vector, NJL or NLS. g, Hematoxylin-Eosin staining of spleen section and h, Wright-Giemsa staining of bone marrow from NJL-induced AML mice. Scale bar, 20μM.

Figure 2. JARID1A_PHD3, an essential motif for NJL-mediated leukemia, specifically recognizes H3K4me3/2 marks

a, Capability of JARID1A_PHD3, PHF23_PHD and JARID1A_PHD1 (the first PHD finger of JARID1A, Supplementary Fig.1) to interact with H3 peptides harboring different Kme in peptide pull-down assay. JARID1A_PHD1 interacted with H3K4me0 as BHC80_PHD. b, Crystal structure of JARID1A_PHD3 (cyan) complexed with H3K4me3 peptide (yellow) and a close-up view of the H3K4me3-binding channel (inset) formed by two orthogonally aligned Trp residues. The residue of JARID1A_PHD3 and H3 is shown in red and black, respectively. c, Capability of wildtype or mutant JARID1A_PHD3 to bind to H3K4me3/2. d, CoIP showing that NJL containing the wildtype, but not mutant (W1625A) PHD finger, associated with H3K4me3 or H3 in transiently trasfected 293 cells.

Figure 3. NUP98-JARID1A enforced high H3K4me3 and active transcription associated with developmentally critical loci such as Hox

a, ChIP for NJL- or Ezh2-binding to A-cluster Hox promoters in committed myeloid progenitor line (cell 1) or in hematopoietic stem/progenitor cells three weeks after transduction of control vector (cell 2) or 3xFlag-tagged NJL (cell 3-4). b, ChIP of H3K4me3, H3K27me3 and general H3 among Hox-A gene cluster in hematopoietic progenitors three weeks after transduction of vector or NJL. c, Hoxa9/a10 expression in hematopoietic stem/progenitor cells after days of in vitro cultivation (day 0, 4, 8, 12), macrophages (mϕ), NIH-3T3 fibroblasts or NJL-transformed progenitors. d, α-Hoxa9 blot in marrow progenitors 10 days after transduction of MLL-ENL, empty vector, NJS or NJL. e, ChIP for Hoxa9/a10 promoter-associated H3K4me3 in hematopoietic stem/progenitor cells after days of in vitro culture, mϕ and marrow progenitors 20 days post transduction of vector or NJL. n=3, error bar indicates s.d; *, P<0.01; **, P<0.001; ***, P <10^-4.

Figure 4. The H3K4me3/2 engagement by NUP98-JARID1A perturbs the epigenetic state of developmentally critical loci during hematopoiesis

a, Impact of mutations on the Flag-NJL binding to HOXA9 in 293 cells. b, Immunoblot of hematopoietic progenitors ten days post transduction of vector, wildtype or mutant NJL. Phospho-c-Kit, marker of mast cells. c, ChIP for Hoxa9 promoter-associated NUP98-fusion proteins (3xFlag-tagged) and H3K4me3 in marrow progenitors 10 days after transduction. d, Transforming capacities after introducing mutation to NJL or e, those by NUP98-PHF23 or after replacing JARID1A_PHD3 with another PHD finger that engages either H3K4me3/2 or H3K4me0. Total progenitor number was counted at day 1, 10, 25 and 40. f, ChIP for SUZ12 and g, MLL2 binding to Hoxa9/a11 and h, Hoxa9-associated H3 acetylation in marrow progenitors 15 days after transduction of vector or NJL. Error bar indicates s.d (n=3); *, P<0.05; **, P<0.005; ***, P<10^-4; *****, P<10^-6. i, A scheme that NUP98-PHD fusion acts as “boundary factor” and prevents the spreading of polycomb factors from Hoxa13/a11 to Hoxa9, thus inhibiting H3K4me3 removal and H3K27me3 addition during hematopoiesis.