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

Wang GG, Song J, Wang Z, Dormann HL, Casadio F, Li H, Luo JL, Patel DJ, Allis CD - Nature (2009)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Chromatin Biology & Epigenetics, The Rockefeller University, New York, New York 10065, USA.

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.

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JARID1APHD3, an essential motif for NJL-mediated leukemia, specifically recognizes H3K4me3/2 marksa, Capability of JARID1APHD3, PHF23PHD and JARID1APHD1 (the first PHD finger of JARID1A, Supplementary Fig.1) to interact with H3 peptides harboring different Kme in peptide pull-down assay. JARID1APHD1 interacted with H3K4me0 as BHC80PHD11. b, Crystal structure of JARID1APHD3 (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 JARID1APHD3 and H3 is shown in red and black, respectively. c, Capability of wildtype or mutant JARID1APHD3 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.
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Figure 2: JARID1APHD3, an essential motif for NJL-mediated leukemia, specifically recognizes H3K4me3/2 marksa, Capability of JARID1APHD3, PHF23PHD and JARID1APHD1 (the first PHD finger of JARID1A, Supplementary Fig.1) to interact with H3 peptides harboring different Kme in peptide pull-down assay. JARID1APHD1 interacted with H3K4me0 as BHC80PHD11. b, Crystal structure of JARID1APHD3 (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 JARID1APHD3 and H3 is shown in red and black, respectively. c, Capability of wildtype or mutant JARID1APHD3 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.

Mentions: The fact that NJS failed to induce leukemia indicated that the PHD finger is required for leukemogenesis. Indeed, deletion of JARID1APHD3, but not JARID1A sequences prior to or following it, abolished NJL-mediated transformation of hematopoietic cells (Supplementary Fig.2f-h). We next asked whether JARID1APHD3 recognizes histone methylation. First, only histone H3 associated with recombinant JARID1APHD3 using total histone extracts (Supplementary Fig.3a). When a mini-library of H3 peptides harboring either unmodified, mono-, di- or tri-methylated K4, K9, K27, K36 or K79 were screened in biotinylated peptide pull-down, JARID1APHD3 only interacted with those containing H3K4me3/2 (Fig.2a; Supplementary Fig.3b). Such specificity was further confirmed by immunostaining and co-immunoprecipitation using Flag-NJL stable expression cells— NJL exhibited a speckled nuclear staining pattern and significantly co-localized with H3K4me3, but not H3K9me3 (Supplementary Fig.4); The vast majority of NJL were bound to mononuclesomes containing H3K4me3, but not H3K27me3 (Supplementary Fig.3c). Calorimetry-based measurements revealed a dissociation constant (Kd) of ∼0.75 μM for JARID1APHD3 binding to H3K4me3, with reduced affinities to H3K4me2/1/0 (Supplementary Fig.3d).


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

Wang GG, Song J, Wang Z, Dormann HL, Casadio F, Li H, Luo JL, Patel DJ, Allis CD - Nature (2009)

JARID1APHD3, an essential motif for NJL-mediated leukemia, specifically recognizes H3K4me3/2 marksa, Capability of JARID1APHD3, PHF23PHD and JARID1APHD1 (the first PHD finger of JARID1A, Supplementary Fig.1) to interact with H3 peptides harboring different Kme in peptide pull-down assay. JARID1APHD1 interacted with H3K4me0 as BHC80PHD11. b, Crystal structure of JARID1APHD3 (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 JARID1APHD3 and H3 is shown in red and black, respectively. c, Capability of wildtype or mutant JARID1APHD3 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.
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Related In: Results  -  Collection

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Figure 2: JARID1APHD3, an essential motif for NJL-mediated leukemia, specifically recognizes H3K4me3/2 marksa, Capability of JARID1APHD3, PHF23PHD and JARID1APHD1 (the first PHD finger of JARID1A, Supplementary Fig.1) to interact with H3 peptides harboring different Kme in peptide pull-down assay. JARID1APHD1 interacted with H3K4me0 as BHC80PHD11. b, Crystal structure of JARID1APHD3 (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 JARID1APHD3 and H3 is shown in red and black, respectively. c, Capability of wildtype or mutant JARID1APHD3 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.
Mentions: The fact that NJS failed to induce leukemia indicated that the PHD finger is required for leukemogenesis. Indeed, deletion of JARID1APHD3, but not JARID1A sequences prior to or following it, abolished NJL-mediated transformation of hematopoietic cells (Supplementary Fig.2f-h). We next asked whether JARID1APHD3 recognizes histone methylation. First, only histone H3 associated with recombinant JARID1APHD3 using total histone extracts (Supplementary Fig.3a). When a mini-library of H3 peptides harboring either unmodified, mono-, di- or tri-methylated K4, K9, K27, K36 or K79 were screened in biotinylated peptide pull-down, JARID1APHD3 only interacted with those containing H3K4me3/2 (Fig.2a; Supplementary Fig.3b). Such specificity was further confirmed by immunostaining and co-immunoprecipitation using Flag-NJL stable expression cells— NJL exhibited a speckled nuclear staining pattern and significantly co-localized with H3K4me3, but not H3K9me3 (Supplementary Fig.4); The vast majority of NJL were bound to mononuclesomes containing H3K4me3, but not H3K27me3 (Supplementary Fig.3c). Calorimetry-based measurements revealed a dissociation constant (Kd) of ∼0.75 μM for JARID1APHD3 binding to H3K4me3, with reduced affinities to H3K4me2/1/0 (Supplementary Fig.3d).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Chromatin Biology & Epigenetics, The Rockefeller University, New York, New York 10065, USA.

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.

Show MeSH
Related in: MedlinePlus