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

doi:10.1038/nature08036

Nature. Author manuscript; available in PMC 2009 December 11.

Published in final edited form as:

Nature. 2009 June 11; 459(7248): 847–851.

doi: 10.1038/nature08036

PMCID: PMC2697266

NIHMSID: NIHMS107550

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

Gang G. Wang,¹ Jikui Song,² Zhanxin Wang,² Holger L. Dormann,¹ Fabio Casadio,¹ Haitao Li,² Jun-Li Luo,³ Dinshaw J. Patel,² and C. David Allis¹

¹Laboratory of Chromatin Biology & Epigenetics, The Rockefeller University, New York, NY 10065, USA

²Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA

³Department of Cancer Biology, The Scripps Research Institute, Scripps Florida, Jupiter, FL 33458, USA

Correspondence and requests for materials should be addressed to C.D.A. (Email: alliscd/at/rockefeller.edu).

Author contributions G.G.W. and C.D.A. designed the study. G.G.W. performed most of cellular & molecular experiments, and wrote the paper; J.S. performed NMR analyses and obtained protein crystals; Z.W. performed crystallographic analyses; H.L.D. and G.G.W. did immunostaining; F.C. helped on plasmid/protein generation; H.L. measured the Kd; G.G.W. and J.L. did animal studies; D.J.P. and C.D.A. supervised the structural and functional aspects of the project, respectively, and helped with manuscript preparation.

Author Information Coordinates of X-ray and NMR structures of JARID1A_PHD3 in the H3-bound or free state have been deposited to the Protein Data Bank (accession #3GL6, 2KGG and 2KGI), and the chemical shift assignment of NMR structures to BioMagResBank (accession # 16209 and 16210). Reprints and permissions information is available at www.nature.com/reprints.

The publisher's final edited version of this article is available at Nature

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Abstract

Histone H3 Lys4 methylation (H3K4me) was proposed as a critical component in regulating the gene expression, epigenetic states, and cellular identities¹. The biological meaning of H3K4me is interpreted via conserved modules including plant homeodomain (PHD) fingers that recognize varied H3K4me states¹^,². The dysregulation of PHD finger has been implicated in a variety of human diseases including cancers and immune or neurological disorders³. Here we report that fusing an H3K4-trimethylation (H3K4me3)-binding PHD finger, such as the C-terminal PHD finger of JARID1A or PHF23 (JARID1A_PHD3, PHF23_PHD), to a common fusion partner nucleoporin-98 (NUP98) as identified in human leukemias⁴^,⁵, generated potent oncoproteins that arrested hematopoietic differentiation and induced acute myeloid leukemia (AML). In these processes, a PHD finger that specifically recognizes H3K4me3/2 marks was essential for leukemogenesis. Mutations in PHD fingers that abrogated H3K4me3-binding also abolished leukemic transformation. NUP98-PHD fusion prevented the differentiation-associated removal of H3K4me3 at many loci encoding lineage-specific transcription factors (Hox(s), Gata3, Meis1, Eya1, Pbx1), and enforced their active gene transcription. Mechanistically, NUP98-PHD fusions act as ‘chromatin boundary factors’, dominating over polycomb-mediated gene silencing to ‘lock’ developmentally crucial loci into an active chromatin state (H3K4me3 with induced histone acetylation), a state that defined leukemia stem cells. Collectively, our studies represent the first report wherein the deregulation of PHD finger, ‘effector’ of specific histone modification, perturbs the epigenetic dynamics on developmentally critical loci, catastrophizes cellular fate decision-making, and even causes oncogenesis during development.

Recent studies have showed that an H3K4me3-binding PHD finger in the NURF, ING2 or TFIID complex helps to recruit and/or stabilize these effectors and associated factors onto appropriate target promoters during transcriptional regulation¹^,⁶^-¹⁰; An unmodified H3K4 (H3K4me0)-engaging PHD finger in the DNMT3L or LSD1-complex connects activities of DNA methylation or H3K4 demethylation to repressive chromatin¹¹^,¹². Interestingly, germ-line mutation in the PHD finger of RAG2 abrogates its recognition of H3K4me3 and causes immunodeficiency¹³; Mutations in the PHD finger of ING1 have been implicated in cancers³^,⁸^,¹⁴. However, evidence supporting a causal role for PHD finger mutation and inappropriate interpretation of histone modification in oncogenesis is still elusive.

In clinically reported AML patients⁴^,⁵, chromosomal translocation fuses the C-terminal PHD finger of JARID1A (also known as RBP2/KDM5A) or PHF23, together with nuclear localization signals, to NUP98, a common leukemia fusion partner that harbors transactivation activities¹⁵^-¹⁷ (Supplementary Fig.1). Notably, the JARID1A_PHD3 motif is excluded from an alternatively spliced isoform of JARID1A and the corresponding NUP98-JARID1A fusion (hereafter referred to as NJS), while it is retained in the longer fusion isoform (hereafter referred to as NJL; Fig.1a). We asked whether JARID1A_PHD3 as a putative chromatin-‘reading’ module is involved in hematopoietic malignancies. To test this, we examined leukemogenic potential of both fusion isoforms using hematopoeitic progenitor transformation assay¹⁸ (Supplementary Fig.2a). While bone marrow-derived hematopoietic stem/progenitor cells transduced with empty retrovirus or retrovirus encoding NJS proliferated transiently and differentiated into mature cells, those transduced with NJL proliferated indefinitely as undifferentiated progenitors (Fig.1b-c). NJL-transduced marrow cells proliferated in a cell-autonomous manner, exhibited typical myeloblast morphology (Fig.1d) and expressed early myeloid progenitor antigens (c-Kit⁺/Cd11b⁺/Cd34⁺/Gr-1^-/Cd19^-/B220^-/low; Fig.1e and Supplementary Fig. 2b). The arres of myeloid differentiation by NJL indicated that it would induce leukemia in vivo. Indeed, all of 12 mice transplanted with bone marrow progenitors transduced with NJL died of AML in an average of 69 days, whereas those reconstituted with vector- or NJS-transduced progenitors remained healthy after one year (Fig.1f). NJL-induced leukemia exhibited a myeloid phenotype (Supplementary Fig.2c-d), and typically presented with an enlarged spleen, packed progenitors in bone marrow, and massive increase in peripheral white blood cells (Supplementary Table 1; Fig.1g-h). Taken together, NJL represents a potent leukemia oncogene in both cellular and animal models.

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

The fact that NJS failed to induce leukemia indicated that the PHD finger is required for leukemogenesis. Indeed, deletion of JARID1A_PHD3, but not JARID1A sequences prior to or following it, abolished NJL-mediated transformation of hematopoietic cells (Supplementary Fig.2f-h). We next asked whether JARID1A_PHD3 recognizes histone methylation. First, only histone H3 associated with recombinant JARID1A_PHD3 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, JARID1A_PHD3 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 JARID1A_PHD3 binding to H3K4me3, with reduced affinities to H3K4me2/1/0 (Supplementary Fig.3d).

Figure 2

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

We determined the structure of JARID1A_PHD3:H3K4me3 complexes using X-ray crystallographic and NMR spectroscopic techniques. Both analyses revealed that the JARID1A_PHD3-H3K4me3 interaction was established via (i) anti-parallel β-sheet pairing between the H3 backbone and a β-sheet of JARID1A_PHD3, (ii) a hydrophobic cleft formed by two Trp residues (W1625, W1635) that anchor the H3K4me3 side chain, and (iii) positioning of H3R2 in an acidic pocket (Q1627/D1629/D1633) (Fig.2b; Supplementary Fig. 5b,6c). H3K4me3 is stacked between the indole rings of two orthogonally aligned Trp residues with intermolecular contacts showed in Fig.2b and Supplementary Fig.5b,6d. The X-ray (a domain-swapped dimer of one molecule and a crystallographic symmetry-related molecule) and solution NMR (monomer) analyses are summarized in Supplementary Fig.5 (statistics in Supplementary Table 2) and Supplementary Fig.6-7 (statistics in Supplementary Table 3), respectively. Comparison between JARID1A_PHD3 structures in the free and H3K4me3-bound state (Supplementary Fig.6a-b) revealed no overall conformational changes. Residues W1625 and W1635 are evolutionarily conserved among JARID1 homologues (Supplementary Fig.8a). Mutations targeting these Trp residues disrupted the H3K4me3-binding in vitro (Fig.2c) and in cells (Fig.2d). Such a two-sided H3K4me3-binding tryptophan channel is a varied form of the H3K4me3-engaging pocket involving 3-4 hydrophobic residues found in the PHD finger of BPTF⁷, ING2⁸, Yng1¹⁹ or RAG2¹³ (Supplementary Fig.8b-d). Yet, it exhibited a stronger H3K4me3-binding affinity (Kd=0.75μM). Collectively, the PHD finger, an essential motif of NUP98-JARID1A, uniquely recognizes H3K4me3/2 using an aromatic engaging channel.

To gain insight into mechanisms of NJL-induced AML, we used microarray analyses to compare the transcriptome of NJL-transformed progenitors and control cells— committed myeloid progenitors generated as described before¹⁸. Strikingly, a significant portion of genes upregulated in NJL-transformed progenitors were those either targeted by polycomb proteins²⁰^,²¹ or exhibiting ‘bivalent domain pattern’²² in stem cells, many of which encode developmentally critical transcription factors (Hoxa5/a7/a9/a10, Gata3, Meis1, Eya1, Pbx1; Supplementary Table 4). Such upregulation was further confirmed by RT-PCR using vector- versus NJL-transduced marrow cells (Supplementary Fig. 9a-c). Other Hox-A genes (a1, a2, a11, a13) were not expressed in NJL-transformed progenitors. We detected a similar target specificity for Hox-A genes using chromatin immunoprecipitation (ChIP)— NJL directly bound to the promoters of Hoxa6-a10, but not distal Hoxa1-a3 or Hoxa11-a13 (Fig.3a and Supplementary Fig.9d; green); NJL-binding specificity among Hox clusters was correlated to H3K4me3— H3K4me3 was abundant in Hoxa6-a10, while low/absent in Hoxa1-a4 or Hoxa11-a13 (Fig.3b). Enforced expression of Hox and Meis1 has been shown sufficient to induce AML²³. This indicated that NJL blocks hematopoietic differentiation and induces AML by enforcing the transcription of these genes.

Figure 3

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

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 JARID1A_PHD3, we investigated another similar de novo translocation, NUP98-PHF23 (Fig.1a)⁵, and also swapped JARID1A_PHD3 with other PHD fingers reported before. PHF23_PHD 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 JARID1A_PHD3 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 BHC80_PHD¹¹ or JARID1A_PHD1 (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.

Figure 4

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

Because the H3K4me3 recognition cannot provide DNA sequence specificity and yet NJL-upregulated genes were enriched with polycomb-targeted ²⁰^,²¹ or ‘bivalent domain’ genes²² in stem cells (e.g., Hox(s), Gata3, Meis1; Supplementary Table 4), we asked whether such specificity is due to their dynamically regulated characteristics. Towards this end, we examined the effect of NJL on two distinct gene classes— developmentally critical genes, and housekeeping genes that exhibit constitutive H3K4me3 (Supplementary Fig.11a, top panel). Interestingly, although NJL bound to housekeeping genes, it had little affect on their expression during cell differentiation (Supplementary Fig.11a, middle and bottom panels). Thus, NJL tends to affect the developmentally critical loci specifically during hematopoeisis. We next pursued the possibility that NJL interferes with activities of polycomb proteins at these developmentally critical loci. Using ChIP, we found that, while Ezh2 or Suz12 was spread throughout Hox-A clusters in vehicle-infected marrow progenitors that underwent differentiation, these polycomb factors were restricted within Hoxa11-a13 in NJL-infected progenitors (Fig.(Fig.3a3a,,4f,4f, red). In the NJL-transduced cells, H3K27me3 was also only detected at Hoxa13-a11— the differentiation-associated spreading of H3K27me3 was inhibited at a region from Hoxa10 to Hoxa1 (Fig.3b). The spreading of polycomb factors from distal Hox loci (a13-a11) seemed to be blocked at Hoxa10-a9 by NJL that were bound there (Fig.3a; Supplementary Fig.9d). Similar result was also found at Meis1 (Supplementary Fig.9e). Consistent to previous reports¹⁵^,¹⁶, the recruitment of p300 and dramatic elevation of H3 acetylation (H3K27ac by >2,000 fold) were observed on Hoxa9 in NJL-transduced cells (Fig.4h; Supplementary Fig.11b). Collectively, NUP98-PHD fusion dominated over the spreading of polycomb and enforced an H3K4me3/acetylated histone state at developmentally critical loci, an epigenetic state that defines leukemia stem cells.

In summary, we have demonstrated for the first time that fusing an H3K4me3-engaging PHD finger (plus nuclear localization signal) to a common partner NUP98 is sufficient to induce leukemia. We showed that NUP98-PHD fusion prevented the silencing of critical loci encoding master transcription factors (Hox(s), Gata3, Mesi1, Pbx1) during hematopoietic differentiation. NUP98 fusion partners can be grouped into two major groups, DNA-binding homoedomain and chromatin-associated factors including PHD fingers (JARID1A, PHF23)¹⁷. Although the existence of additional unknown ligand is possible for PHD fingers in the latter group (as H3K4 site cannot be mutated in mammals), the most straightforward interpretation of our findings is that binding H3K4me3/2 marks is responsible for leukemia described here. In support, a genetic interaction was demonstrated in yeast between H3K4 and the Yng1 PHD finger²⁵, a module that imparted similar oncogenic properties when swapping into our assays (Fig.4e). Several PHD fingers exist in NSD1, another NUP98-fusion partner¹⁶, however, none contains critical H3K4me3-engaging residues¹. Thus, our report represents the first example wherein inappropriate interpretation of histone modification can actively induce a deregulation of developmentally critical loci, perturb cellular/epigenetic identities, and even induce oncogenesis. NUP98-PHD fusion coordinates acts of H3K4me3/2 and histone acetylation, mimicking mechanisms utilized by evolutionarily conserved ING(s)-complexes for robust gene activation¹⁹^,²⁶ (Supplementary Fig.12). H3K4me3 bound by NUP98-PHD may serve as ‘seed’ of propagation mediated by WDR5-MLL2/3 complexes¹^,²⁷ that is also coupled with UTX/Jmjd3-mediated H3K27 demethylation²⁸^,²⁹, as we detected high levels of WDR5, RBBP5, and MLL2 on Hoxa9 in NJL-transduced marrow cells (Fig.4g; Supplementary Fig.11c-d). We suggest that NUP98-PHD acts as ‘boundary factors’, using the PHD finger to protect H3K4me3 from JARID1(s)-mediated demethylation²⁹ and also inducing H3K27ac to block H3K27me addition (Fig.4i). In support, we observed a ‘bivalent domain’ feature²² at Hoxa11-a10, the junction region of two antagonizing mechanisms (Fig.3b). Loss-of-function mutation of RAG2_PHD in immunodeficiency and gain-of-function mutation involving PHD fingers in malignancies described here indicate a new type of diseases that arise from ‘misinterpreting’ the ‘histone code’³^,³⁰. With ~200 PHD fingers in human genome and some intimately associated to diseases³, we expect similar ‘mis-reading’ mechanisms responsible for some unstudied diseases. These pathologies together with those caused by ‘mis-writing’ or ‘mis-erasing’²⁹ histone modification, underscore the significance in investigating the biological readout of histone marks.

METHODS SUMMARY

Hematopoietic cell transformation assays

Protocols for the culture of primary hematopoietic stem/progenitor cells were previously described¹⁸. Briefly, 100,000 lineage-negative bone marrow stem/progenitor cells were subjected to retroviral infection, followed by kinetics analyses of proliferation versus differentiation in ex vivo culture system as described before¹⁸.

Peptide pull-down assay

Pull-down using biotinylated histone peptide and recombinant protein was performed as described⁶^,¹¹. After binding, peptide-Avidin beads were washed extensively in solution containing 50mM Tris pH 7.5, 150mM NaCl (250mM as stringent washing), 0.05% NP-40, 0.3mg/ml BSA and 1mM DTT.

Supplementary Material

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Acknowledgement

We thank L. Baker, P. Chi, A. Ruthenberg and A. Xiao for critical reading and help in manuscript preparation, C. Hughes for antibodies, and other Allis lab member for their advice. We are extremely grateful to Drs. M. Kamps and S. Rafii for shared plasmids and expert advice. Thanks to E. Coutavas, W. Chen, E. Ezhkova, and the Bob Roeder lab for help on cell and animal work. G.G.W. is supported by a Leukemia & Lymphoma Society Fellow award and a Choh-Hao Li Memorial Fund Scholar award; H.L.D. is supported by a predoctoral Boehringer Ingelheim Foundation fellowship; This research was supported by the NIH grant and funds of Rockefeller University to C.D.A, funds of Abby Rockefeller Mauze Trust and the Dewitt Wallace and Maloris Foundations to D.J.P, U.S. Department of Defense CDMRP grant to J.L., and a joint Starr Foundation Cancer Consortium grant.

Appendix

METHODS

Plasmid construction and retroviral expression system

The NUP98-JARID1A fusion cDNA⁴ was generated by ligating NUP98 sequences encoding amino acids 1-514 to those encoding amino acids 1489-1690 of JARID1A transcript variant 1 (NCBI accession No. NM_001042603) or amino acids 1489-1641 of JARID1A transcript variant 2 (NCBI accession No. NM_005056), producing two fusion isoforms (NJL or NJS) respectively. The same method was used to generate NUP98-PHF23⁵. The fusion cDNA with an N-terminal 3xFLAG was cloned into MSCV retroviral expression vector (Clontech). JARID1A (RBP2), PHF23 and BHC80 cDNAs were purchased from Open Biosystems. NUP98 plasmids were kindly provided by Dr. J.M. van Deursen, Hoxa9 by Dr. M.P. Kamps, MLL-ENL by Dr. R.K. Slany, Yng1 by Dr. S.D. Taverna, CBX7 by Dr. E. Bernstein, and ING2 by Dr. Z. Tang. Mutations were generated by site-directed mutagenesis, and all used plasmids were confirmed by sequencing.

Purification and culture of primary hematopoietic cells

Bone marrow cells harvested from femur and tibia of balb/C or b/6 mice were subject to lineage-negative (Lin^-) enrichment using Hematopoietic Progenitor Enrichment Kit (StemCell Technologies or Miltenyi Biotec) to remove cells expressing differentiation antigens as described before¹⁶. ~400,000 of Lin^- enriched hematopoietic progenitors were obtained per mouse with ~10% c-Kit⁺Lin^-Sca1⁺ HSCs. Before retroviral infection, Lin^--enriched hematopoietic progenitors were stimulated in OptiMEM base medium (Invitrogen, cat#31985) complemented with 10% of FBS (Invitrogen, cat#16000-044), 1% of antibiotics, 50μM of β-mercaptoethanol and a cytokine cocktail containing SCF (supernatant of SCF-producer cells), 5ng/mL FLT3 ligand (Sigma), 5ng/mL IL3 and IL6 (Miltenyi) for 2-3 days as described¹⁸^,³¹. After retroviral infection and selection (1μg/mL puromycin), marrow cells were plated in the same medium with SCF as the sole cytokine. Cell splitting and replating to fresh medium were performed every 3-4 days to keep cell number <2 million per well (6- or 12-well plate). Cell morphology was examined by Wright-Giemsa staining. Macrophages were obtained by culture of marrow cells in M-CSF (Miltenyi) for 1-2 weeks as described³²; Immortalized cell lines that mimic committed neutrophil-macrophage progenitors were generated as described before¹⁸^,³¹^,³³.

Murine bone marrow transplantation leukemogenic assay

Leukemogenic potentials of oncogenes were evaluated in sublethally irradiated syngeneic mice after tail vein injection with 100,000 of bone marrow-derived Lin- cells that were infected with retrovirus encoding the fusion gene as described¹⁸. Mice exhibiting leukemic phenotype were subjected to pathological analyses.

Recombinant protein production and GST pull-down

JARID1A_PHD3 (amino acids 1601-1660) GST-fusion proteins were produced using a previously described protocol¹⁹. GST pull-down using total histone extracts was performed as described with modification⁹. Briefly, ~2μg GST-fusion protein bound to glutathione beads (Amersham) were incubated with 10μg of calf thymus histone extracts (Worthington) in a binding buffer containing 50mM Tris-HCl pH 7.5, 0.5 M NaCl, 0.5% NP-40, 0.2mM EDTA, 1mM DTT and protease inhibitor cocktail (Roche) at 4°C for 4 h.

Native co-immunoprecipitation (CoIP)

Mononucleosomes-containing fractions were prepared as described before⁶. Briefly, intact nuclei were subject to limited micrococcal nuclease (MNase) digestion so that the major form of released chromatin is mononucleosome. After the removal of insoluble fraction by centrifugation, supernatant containing mononucleosomes was then incubated with FLAG or HA-agarose beads (Sigma), or with Dynal magnetic beads (Invitrogen) coupled with αH3K4me3 (Abcam) or control antibodies. After extensive washing, precipitated proteins were subject to immunoblot.

Isothermal titration calorimetry (ITC) measurements

Calorimetric experiments were conducted at 25.0°C with an MicroCal iTC200 instrument (Northampton, MA) as described⁷. Recombinant JARID1A_PHD3 protein and H3_1-15K4me peptides were dialyzed overnight against 25mM Tris-HCl pH7.5, 50mM KCl, and 2mM β-mercatoethanol. Protein concentration was determined by absorbance spectroscopy (Tyr ε₂₈₀=1,420 M^-1cm^-1; Trp ε₂₈₀=5,600 M^-1cm^-1; Cys ε₂₈₀=125 M^-1 cm^-1). H3_1-15K4me peptides were quantified by the absorbance of an added C-terminal Tyr with ε₂₈₀=1,280 M^-1cm^-1 for peptide. Acquired calorimetric titration data were analyzed using software Origin7.0 (MicroCal, LLC ITC 200) based on a 1:1 binding stoichiometry.

Antibodies and immunoblot

Antibodies used were α-FLAG (Sigma; M2), α-HA (Covance, MMS101), α-Hoxa9 (Upstate, 07-178), α-Pbx1 (Santa Cruz, sc889), α-phosph-c-Kit (Cell signaling) and α-Tubulin (Sigma).

ChIP analysis

ChIP analysis was performed using Upstate ChIP kit and a protocol described before³⁴. 1~2 million cells per ChIP were used for histones, and 2~3 million for others. Antibodies and amount used were α-Flag (Sigma M2, 1-3μg), α-HA (Covance MMS101, 1-3μg), α-H3K4me3 (Upstate 07-473, 1μL; Abcam 8580, 0.5μg), α-H3K27me3 (Upstate 07-449, 0.5μg), α-acetyl-H3 (Upstate 06-599, 1μg), α-general H3 (Abcam 1791, 0.5μg), α-acetyl-H3K9 (Upstate 06-942, 1μg), α-acetyl-H3K27 (Abcam 4729, 1μg), α-Ezh2 (Cell signaling 4905, 4-5ul), α-Suz12 (Upstate 07-379, 2ul), α-MLL2 (Bethyl A300-113A, 4μg), α-WDR5 (Upstate 07-706, 2μg), α-RBBP5 (Bethyl A300-109A, 3μg; a gift of Dr. Christina Hughes) and α-p300 (Santa Cruz, N15/C20, 10μg). The same amount of nonspecific IgG (Upstate) was used as antibody control, and a silenced intragenic locus, Chr8Int, as locus control for H3K4me3 or activator binding as described²¹. The promoter sequence was acquired from UCSC genomic browser (http://genome.ucsc.edu). ChIP primers were shown in Supplementary Table 5. ChIP signals were represented in the percentage (%) of signals from total chromatin used, and fold of enrichment calculated by normalizing against signals of nonspecific IgG.

Microarray analysis

Total RNA was extracted and the transcript expression quantified using Affymetrix GeneChip Mouse arrays as described¹⁸. RNA hybridization, scanning and signal quantification were performed by RU Gemonic Resources Center. Hybridization signals were retrieved and normalized, followed by differential expression analysis and statistical analysis using GeneSpring Analysis Platform GX 7.0 (Agilent Technologies).

RT-PCR analysis

Reverse transcription of RNA was performed using the random hexamer and Invitrogen Superscript III kit. Usually the PCR amplicon (size ~90-200bp) is designed to span over large intron regions. Exon-intron information was obtained from UCSC genomic browser. Quantitative PCR was performed in triplicate using SYBR green master mix reagent (Applied Biosystem) on a Stratagene Mx3005P QPCR system. Primer information is shown in Supplementary Table 5.

Flow cytometry (FACS)

Cells were blocked with BD FcBlock (2.4G2) and stained on ice with fluro-conjugated antibodies (1:1,000 dilution of Cd117^FITC, Sca-I^PE:CY7, Cd34^APC, Cd34^FITC, Cd11b^APC, Gr-1^PE, Cd19^PE or B220^PE, BD Biosciences) and analyzed on BD FACSCalibor cytometer. Data was collected and analyzed using CellQuestPro and FlowJo software.

Immunofluorescence microscopy

Suspension cultured hematopoietic cells were attached to cover slips treated with 0.01% (w/v) poly-lysine, followed by 15-minute fixation in 4% of parafomaldehyde and 10-min solubilization in PBS, 0.2% Triton-X100 and 0.2% NP-40. After a 30-min block in PBS, 2.5% BSA and 10% normal goat serum, cells were stained with primary antibodies (M2 α-FLAG [1:1,000~2,000 dilution of 1mg/mL], rabbit α-H3K4me3 [Upstate 07-473 or Abcam 8580, 1:2,000] or rabbit α-H3K9me3 antibodies [Upstate 07-442, 1:1,000]) followed by washing and staining with fluorescent-labeled secondary antibodies. After washing, fluorescent signal was visualized and analyzed with a DeltaVision Image Restoration Microscope and a Cofocal Microscope (Applied Precision/Olympus). Deconvolution microscopy image analysis was performed to reassign the out-of-focus blurred light to its origin³⁵, and subcellular co-localization analysis was carried out from stacks of deconvolved images using ImageJ (Rasband WS, et al. NIH, Bethesda, USA; http://rsb.info.nih.gov/ij/) and the plugin JACoP³⁶. Confocal microscopy analysis was performed as previously described³⁷. Co-immunostaining statistics was analyzed using Pearson’s Coefficient of Correlation method. Image acquisition, processing and analyses were performed with the expert help from RU Bio-Imaging Center, and detailed protocols are available upon request.

Statistics

All results are presented as the mean and standard deviation (s.d). Statistical analyses were performed using Student’s t-test.

Protein preparation for structural studies

The gene fragment encoding JARID1A_PHD3 was fused to the C-terminus of a His(6x)-SUMO tag in a modified pRSFDuet-1 vector (Novagen), with a ubiquitin-like-protease (ULP) cleavage site located at the linker region. The bacterial expressed protein was purified using a Ni-NTA affinity column, followed by ULP cleavage, separation of JARID1A_PHD3 from His(6x)-SUMO via a second Ni-NTA chromatography step, and gel filtration. The JARIPD1A_PHD3-H3K4me3 complex was obtained by mixing JARIPD1A_PHD3 protein with an equal molar amount of H3_1-9K4me3 peptides (H3 amino acids 1-9, with Lys4 tri-methylated), then purified by gel filtration, and concentrated by ultrafiltration.

Crystal growth

The crystals of JARIPD1A_PHD3-H3K4me3 complexes were obtained by equilibrating a reservoir consisting of 20% (w/v) poly(ethylene glycol) monomethyl ether 2000, 10mM nickel (II) chloride hexahydrate, and 0.1 M Tris (pH 8.5) with a hanging drop consisting of 1 μL of the reservoir solution and 1 μL of a 27 mg/mL protein solution in 10 mM Tris pH 8.0, 0.1 mM ZnCl₂, 5mM DTT and 50 mM NaCl (Crystal Screen 2 kit, Hampton Research). A mixture of the well solution with 10% (v/v) glycerol was used as cryoprotectant.

Data collection and structure determination

An anomalous diffraction data set for JARID1A_PHD3-H3_1-9K4me3 complex was collected at the zinc anomalous peak wavelength (1.28215 Å) at beamline NE-CAT 24ID-C, Advanced Photon Source, Chicago. The data set was indexed, integrated and merged to 2.2 Å using the program HKL2000. The crystal belongs to I4₁ space group and contains one molecule per asymmetric unit. Heavy-atom search, SAD phasing and model building were performed with the PHENIX³⁸ software package. Three zinc atoms were unambiguously identified for SAD phasing, and ~90% residues of the protein-peptide complex were successfully built into the initial model. The PHENIX-model was further manually rebuilt using COOT³⁹ and refined using REFMAC5⁴⁰ in successive cycles. The final refined structure has R_work and R_free values of 0.200 and 0.234, respectively (Supplementary Table 2). One molecule forms a domain-swapped dimer with a crystallographic symmetry-related molecule (Supplementary Fig. 5a). The swapped segment spans the first 14 residues from the N-terminus.

Using a crystal of the complex following pH optimization of crystallization conditions, we were able to collect one 1.9 Å data set at wavelength 0.97949 Å at the same beamline. The crystal belongs to the same crystal form as the previous one. We solved the high-resolution structure by molecular replacement using PHASER⁴¹ with the above 2.2 Å model after removing all the water molecules and some flexible residues. Structure refinement was done using CNSsolve⁴², cycled with manual model building in COOT. Prior to the refinement, the same R_free set of reflections were transferred from the low-resolution data using the program Freerflag in CCP4 suite⁴³ for effective cross validation. For both data sets, ~10% reflections were selected in a ‘random’ mode throughout the resolution range. After resetting the overall B-factor to 20 Å² and rigid body refinement, simulated annealing starting at 5,000 K was performed to reduce model bias before extensive B-factor and positional refinement. The final model contains full length JARID1A_PHD3 (1609-1659) with one additional serine at the N-terminus from the expression vector, histone H3_1-8K4me3, three zinc ions and thirty-two water molecules. The JARID1A_PHD3-H3K4me3 complex in the crystal shows that one molecule forms a domain-swapped dimmer with a crystallographically symmetry-related molecule (Supplementary Fig. 5a). Two zinc ions are integral to the folding of the PHD finger, while the third zinc ion locates at the interface between two domain-swapped dimers, thereby mediating crystal packing (Supplementary Fig. 5c). The R_work and R_free of the final structure are 0.208 and 0.225 respectively (Supplementary Table 2).

Isotopic labeling, NMR data collection and structure determination

Samples used for NMR chemical shift assignments, ¹⁵N relaxation measurements, and structure determination contained 0.2-0.5 mM uniformly-[¹⁵N]- or [¹³C, ¹⁵N]-labeled JARID1A_PHD3 in the free state and in complex with unlabeled H3_1-9K4me3 peptide dissolved in NMR buffer (20 mM Na-phosphate, 1 mM ZnCl₂, 5 mM DTT, 90% H₂O/10% D₂O) at pH 7.0. The sample used for measurements of ¹⁵N-¹H residual dipolar couplings (RDCs) contained 0.2 mM JARID1A_PHD3 aligned in 12 mg/ml of bacteriophage Pf1 (Alsa, Riga, Latvia), 10 mM MOPS, 200 mM NaCl, pH 7.0.

All NMR spectra were collected at the New York Structural Biology Center (NYSBC) using 800 MHz Bruker NMR spectrometers equipped with ¹H, ¹⁵N, ¹³C triple-resonance cryogenic probes. Unless indicated otherwise, the sample temperature was controlled at 20°C. A suite of 3D heteronuclear NMR experiments, including HNCACB, CBCA(CO)NH, HNCO, HBHA(CO)NH, and HCCH-TOCSY were acquired for sequential backbone and non-aromatic side chain assignments of JARIPD1A_PHD3 both in the free state and in complex with H3_1-9K4me3 peptide in solution. 2D NOESY (τ_mix = 100 ms), 3D ¹⁵N-edited NOESY-HSQC (τ_mix = 100 ms), 3D aromatic ¹³C-edited NOESY-HSQC (τ_mix = 100 ms) and 3D aliphatic ¹³C-edited NOESY-HSQC (τ_mix = 100 ms) data sets were acquired and used for additional assignments (side chain amide and aromatic groups) and distance constraints. To selectively observe the NOEs between JARIPD1A_PHD3 and H3_1-9K4me3 peptide, a [¹³C,¹⁵N]-filtered,¹³C-edited NOESY (τ_mix = 120 ms) spectrum⁴⁴ of uniformly [¹⁵N,¹³C]-labeled JARIPD1A_PHD3 bound to unlabeled H3_1-9K4me3 peptide was recorded. One-bond N-H RDCs were determined by using the IPAP ¹⁵N-HSQC sequence at 25°C⁴⁵. Standard pulse sequences⁴⁶ were used for measurements of the ¹⁵N relaxation rates (R₁, R₂) of JARIPD1A_PHD3 at 25°C. The spectra were processed and analyzed, respectively, with the NMRPipe⁴⁷ and Sparky (http://www.cgl.ucsf.edu/home/sparky) software. The solution structures of JARIPD1A_PHD3 both in the free state and in complex with H3_1-9K4me3 peptide were first calculated using the CYANA program⁴⁸. Interproton distance constraints were derived from 2D NOESY, 3D ¹⁵N-edited NOESY-HSQC and 3D ¹³C-edited NOESY-HSQC spectra. Backbone ϕ and ψ angles were derived from TALOS-based analysis of backbone chemical shifts⁴⁹. A number of hydrogen bonds derived from chemical shift analysis and from observed NOEs characteristic for α-helices and β-sheets, were added in the final rounds of structure refinement. Of the 100 final structures calculated by CYANA, 20 structures with the lowest target functions were chosen for further refinement using the Xplor-NIH program⁵⁰, in which ¹D_NH RDC restraints, physical force field terms and explicit solvent terms⁵¹ were added to the calculation. The final structures were validated by Procheck-NMR⁵², and the statistics for the 20 final structures are listed in Supplementary Table 3.

Monomeric state of JARID1A_PHD3 in free and H3_1-9K4me3-bound states in solution

The oligomeric states of JARID1A_PHD3 (mol. wt. 5.8 kD) and JARID1A_PHD3-H3_1-9K4me3 complex (mol. wt. 6.8 kD) were first evaluated by comparing their elution volumes on a Superdex G75 16/60 column, with the calibration line derived from a number of molecular standards. As shown in Supplementary Fig. 7a, the elution volumes of both free JARID1A_PHD3 and JARID1A_PHD3-H3_1-9K4me3 complex are comparable with those expected for their monomeric states, but considerably larger that those expected for their dimeric states. This suggests that JARID1A_PHD3 is monomeric in solution, for both the free and H3_1-9K4me3 states. Furthermore, the rotational correlation times of JARID1A_PHD3 in the free and H3_1-9K4me3-bound states were estimated as 3.5 ns and 4.5 ns, respectively, based on an analysis of ¹⁵N R2/R1 relaxation time ratios (Supplementary Fig. 7b) using the quadratic diffusion program⁵³. These values are consistent with isotropic tumbling values of a monomeric protein of their respective sizes, further supporting that both free and complexed JARID1A_PHD3 exist as monomers in solution.

Thus, although JARID1A_PHD3-H3K4me3 complex exhibits a domain-swapped dimer in the crystal, gel filtration and NMR relaxation measurements (Supplementary Figure 7) clearly showed such a complex to be monomeric in solution. Hence the domain-swapped dimerization observed in the crystal is likely to be a characteristic feature of the crystalline state, originating perhaps in packing interactions.

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Footnotes

Full Methods and any associated references in the online version of the paper at www.nature.com/nature.

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