Limits...
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.

Show MeSH

Related in: MedlinePlus

The H3K4me3/2 engagement by NUP98-JARID1A perturbs the epigenetic state of developmentally critical loci during hematopoiesisa, 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 JARID1APHD3 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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2697266&req=5

Figure 4: The H3K4me3/2 engagement by NUP98-JARID1A perturbs the epigenetic state of developmentally critical loci during hematopoiesisa, 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 JARID1APHD3 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.

Mentions: It has been reported that the A-cluster Hox gene expression is high in hematopoietic stem cells (HSC) and early progenitors, followed by down-regulation and shut-off during terminal differentiation24. Our ex vivo hematopoietic stem/progenitor cell system recapitulated such dynamics— coincident to the silencing of HSC marker and activation of differentiation marker (Supplementary Fig.9f), Hoxa9/a10 were down regulated >10- or 60-fold respectively in 8 days of culture (Fig.3c); Concurrent loss of Hoxa9/a10-associated H3K4me3 was observed in these cells (Fig.3e). Strikingly, NJL persistently enforced high levels of Hoxa9/a10 expression and Hoxa9/a10-associated H3K4me3 in marrow cells, whereas Hoxa9/a10 was silenced ten days after transduction of vector or NJS in similarly maintained cells (Fig.3c-e). To rigorously test the role of H3K4me3 recognition during leukemogenesis, we mutated the H3K4me3-engaging residues. NJL harboring mutation on the residueW1625 or W1635 failed to bind to H3K4me3 or H3 (Fig. 2d), failed to bind to the Hoxa9 promoter that exhibited high H3K4me3 in 293 cells (Fig.4a; Supplementary Fig.9i), failed to enforce the Hoxa9 expression (Fig.4b) or Hoxa9-associated H3K4me3 in hematopoietic progenitors (Fig.4c), and failed to transform the hematopoietic cells (Fig.4d), whereas the irrelevant mutation (V1609G) did not affect these activities (Supplementary Fig.10e). To assess whether NJL-induced phenotype was unique to JARID1APHD3, we investigated another similar de novo translocation, NUP98-PHF23 (Fig.1a)5, and also swapped JARID1APHD3 with other PHD fingers reported before. PHF23PHD specifically engaged H3K4me3/2 as predicted1 (Fig.2a); NUP98-PHF23 robustly enforced Hoxa9-associated H3K4me3 and transformed hematopoietic progenitors (Fig.4c,e; Supplementary Fig.10). Strikingly, swapping JARID1APHD3 with another H3K4me3/2-binding PHD finger from ING28 or even S. cerevisiae Yng119 also succeeded in the transformation, whereas replacing it with an H3K4me0-binding PHD finger, either BHC80PHD11 or JARID1APHD1 (Fig.2a), abolished the transformation (Fig.4c,e). Therefore, engaging H3K4me3/2 by NUP98-PHD fusion causes leukemia by enforcing an active state on developmentally critical loci.


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)

The H3K4me3/2 engagement by NUP98-JARID1A perturbs the epigenetic state of developmentally critical loci during hematopoiesisa, 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 JARID1APHD3 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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2697266&req=5

Figure 4: The H3K4me3/2 engagement by NUP98-JARID1A perturbs the epigenetic state of developmentally critical loci during hematopoiesisa, 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 JARID1APHD3 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.
Mentions: It has been reported that the A-cluster Hox gene expression is high in hematopoietic stem cells (HSC) and early progenitors, followed by down-regulation and shut-off during terminal differentiation24. Our ex vivo hematopoietic stem/progenitor cell system recapitulated such dynamics— coincident to the silencing of HSC marker and activation of differentiation marker (Supplementary Fig.9f), Hoxa9/a10 were down regulated >10- or 60-fold respectively in 8 days of culture (Fig.3c); Concurrent loss of Hoxa9/a10-associated H3K4me3 was observed in these cells (Fig.3e). Strikingly, NJL persistently enforced high levels of Hoxa9/a10 expression and Hoxa9/a10-associated H3K4me3 in marrow cells, whereas Hoxa9/a10 was silenced ten days after transduction of vector or NJS in similarly maintained cells (Fig.3c-e). To rigorously test the role of H3K4me3 recognition during leukemogenesis, we mutated the H3K4me3-engaging residues. NJL harboring mutation on the residueW1625 or W1635 failed to bind to H3K4me3 or H3 (Fig. 2d), failed to bind to the Hoxa9 promoter that exhibited high H3K4me3 in 293 cells (Fig.4a; Supplementary Fig.9i), failed to enforce the Hoxa9 expression (Fig.4b) or Hoxa9-associated H3K4me3 in hematopoietic progenitors (Fig.4c), and failed to transform the hematopoietic cells (Fig.4d), whereas the irrelevant mutation (V1609G) did not affect these activities (Supplementary Fig.10e). To assess whether NJL-induced phenotype was unique to JARID1APHD3, we investigated another similar de novo translocation, NUP98-PHF23 (Fig.1a)5, and also swapped JARID1APHD3 with other PHD fingers reported before. PHF23PHD specifically engaged H3K4me3/2 as predicted1 (Fig.2a); NUP98-PHF23 robustly enforced Hoxa9-associated H3K4me3 and transformed hematopoietic progenitors (Fig.4c,e; Supplementary Fig.10). Strikingly, swapping JARID1APHD3 with another H3K4me3/2-binding PHD finger from ING28 or even S. cerevisiae Yng119 also succeeded in the transformation, whereas replacing it with an H3K4me0-binding PHD finger, either BHC80PHD11 or JARID1APHD1 (Fig.2a), abolished the transformation (Fig.4c,e). Therefore, engaging H3K4me3/2 by NUP98-PHD fusion causes leukemia by enforcing an active state on developmentally critical loci.

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