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. 2006 Aug 17;25(17):3955–3965. doi: 10.1038/sj.emboj.7601273

Roles of HIPK1 and HIPK2 in AML1- and p300-dependent transcription, hematopoiesis and blood vessel formation

Yukiko Aikawa 1, Lan Anh Nguyen 1, Kyoichi Isono 2, Nobuyuki Takakura 3, Yusuke Tagata 1, M Lienhard Schmitz 4, Haruhiko Koseki 2, Issay Kitabayashi 1,a
PMCID: PMC1560355  PMID: 16917507

Abstract

Histone acetyltransferases (HATs) p300 and CREB-binding protein (CBP) function as co-activators for a variety of sequence-specific transcription factors, including AML1. Here, we report that homeodomain-interacting protein kinase-2 (HIPK2) forms a complex with AML1 and p300, and phosphorylates both AML1 and p300 to stimulate transcription activation as well as HAT activities. Phosphorylation of p300 is triggered by phosphorylated AML1 as well as by PU.1, c-MYB, c-JUN and c-FOS, and is inhibited by dominant-negative HIPK2. Phosphorylation of p300 and AML1 is impaired in Hipk1/2 double-deficient mouse embryos. Double-deficient mice exhibit defects in primitive/definitive hematopoiesis, vasculogenesis, angiogenesis and neural tube closure. These phenotypes are in part similar to those observed in p300- and CBP-deficient mice. HIPK2 also phosphorylates another co-activator, MOZ, in an AML1-dependent manner. We discuss a possible mechanism by which transcription factors could regulate local histone acetylation and transcription of their target genes.

Keywords: histone acetyltransferase, protein phosphorylation, transcription regulation

Introduction

Covalent modification of histones (acetylation, phosphorylation, methylation, ubiquitination and sumoylation) is essential for regulating gene expression. These modifications appear to be inter-dependent and represent an evolutionarily conserved ‘histone code' that alters the interactions between histones and DNA, as well as marks binding sites on histones for effector proteins that regulate gene expression (Jenuwein and Allis, 2001). Histone acetylation is regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). HATs and HDACs, directly or through binding to transcription cofactors, interact with transcription factors, which recognize specific DNA sequences in their target genes, to regulate the acetylation of local histones (Marmorstein and Roth, 2001; Thiagalingam et al, 2003). The HAT p300 and the closely related CREB-binding protein (CBP) have been shown to function as co-activators for a wide variety of transcription factors and to play central roles in transcriptional control in response to a diverse range of physiological stimuli (Legube and Trouche, 2003).

The AML1 (RUNX1) gene is the most frequent target of leukemia-associated chromosome translocations, which are frequently detected in human leukemia (Look, 1997). AML1 protein forms a heterodimer with CBFβ and binds to a specific DNA sequence to regulate the expression of a number of hematopoietic genes (Meyers et al, 1993; Ogawa et al, 1993). Both AML1 and CBFβ are essential for the development of all definitive hematopoiesis lineages (Okuda et al, 1996; Sasaki et al, 1996; Wang et al, 1996a, 1996b; Niki et al, 1997; Okada et al, 1998). AML1 forms complexes with PML and HATs, such as p300/CBP and MOZ, to activate transcription (Kitabayashi et al, 1998b, 2001; Nguyen et al, 2005). AML1 can act synergistically with other transcription factors, such as AP-1 (Cockerill et al, 1996), C/EBPα (Zhang et al, 1996), c-Myb (Britos-Bray and Friedman, 1997), PU.1 (Petrovick et al, 1998) and Ets-1 (Kim et al, 1999). Inducible phosphorylation of AML1 is correlated with increased amounts of MOZ in the AML1 complex during differentiation of M1 myeloid cells (Kitabayashi et al, 2001), suggesting that phosphorylation of AML1 is important for regulation of AML1 complex formation and transcriptional activation.

Homeodomain-interacting protein kinases (HIPKs) are nuclear serine/threonine kinases, and three members of the HIPK family (HIPK1, HIPK2 and HIPK3) have been reported (Kim et al, 1998). The functional importance of HIPKs has chiefly been studied using HIPK2 and estimated by means of their interacting proteins and phosphorylation targets, including homeoproteins, p53, CtBP1 and Myb (Kim et al, 1998; Choi et al, 1999; D'Orazi et al, 2002; Hofmann et al, 2002; Zhang et al, 2003; Kanei-Ishii et al, 2004). All of these factors represent critical regulators of transcription, suggesting that HIPKs play critical roles in transcriptional regulation.

The activity of CBP or p300 HAT has been shown to be stimulated by a variety of sequence-specific transcription factors, such as HNF1α, HNF4, Sp1, Zta, NF-E2, C/EBPs and phosphorylated Elk1 (Chen et al, 2001; Soutoglou et al, 2001; Kovacs et al, 2003; Li et al, 2003; Schwartz et al, 2003). Through this stimulation, these sequence-specific transcription factors are thought to increase the acetylation of histones at their target promoters. However, the mechanisms regulating the activity of the transcription factor/HAT complexes on their target genes remain unclear. Here, we report that phosphorylation of p300 is induced by phosphorylated (active) AML1 but not by dephosphorylated (inactive) AML1. AML1 interacts with HIPK2 and mediates phosphorylation of p300 to stimulate HAT activity and induction of transcription. HIPK2-dependent phosphorylation of p300 is also induced by other transcription factors, such as PU.1, c-MYB, c-JUN and c-FOS, suggesting that the present novel mechanism of transcriptional regulation is of general relevance. The importance of HIPK1 and HIPK2 for inducible phosphorylation of p300 was revealed in double-deficient mice, which have defective p300 phosphorylation and show in part a similar phenotype to p300- and CBP-deficient mice.

Results

AML1 is phosphorylated at Ser249 and Ser276

Phosphorylation of AML1 is suggested to be important for its function in transcription and cell differentiation. To determine the phosphorylation sites of AML1, a full-length AML1 protein, AML1b, was purified from L-G myeloid cells, in which AML1 is highly phosphorylated. AML1b was then digested with trypsin and subjected to LC/MS/MS analysis. The predicted phosphorylation sites were Ser249 and Ser276. Comparison of these sites and their surrounding sequences among the different species indicated that the phosphorylation sites of AML1 have been evolutionarily conserved (Figure 1A). Mutants in which Ser249 and/or Ser276 were substituted by alanine, migrated faster than wild type (WT) when expressed in L-G cells (Figure 1B). Phosphatase-treated WT AML1b migrated to a similar position as the S249/276A mutant (Figure 1C). These results suggest that Ser249 and Ser276 of AML1 are phosphorylated in L-G cells. Alteration of Ser266, which is specifically phosphorylated by Erk (Tanaka et al, 1996), to alanine did not affect migration (Figure 1B).

Figure 1.

Figure 1

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AML1 is phosphorylated at Ser249 and Ser276 by HIPK2. (A) Phosphorylation sites are evolutionarily conserved. (B) Migration of WT and mutant AML1 in L-G cells. (C) Phosphatase treatment of AML1. Purified WT and S249/276A mutant AML1b were treated with (+) or without (−) calf intestine alkaline phosphatase (CIAP) and analyzed by immunobloting with anti-AML1 antibody. (D) AML1 complex contains HIPK2. Purified AML1 complexes were analyzed by immunobloting with anti-HIPK2 antibody. (E) Interaction of AML1 with HIPK2. 293T cells were transfected with HA-AML1b together with either FLAG-tagged WT or KD HIPK2. Immunoprecipitates with anti-FLAG were analyzed by immunoblotting with anti-HA antibody. (F) Phosphorylation of AML1 by HIPK2. Cell extracts from 293T cells expressing WT or mutants of HA-tagged AML1 and HIPK2 were subjected to immunoblot analysis with anti-HA antibody. (G) Phosphorylation of AML1 in vitro. Purified WT or mutant AML1b proteins were tested for in vitro kinase activity. (H) HIPK2 activates AML1-mediated transcription. SaOS2 cells were transfected with 50 ng of MPO-luc, 200 ng of LNCX-AML1b, 250 ng of MOZ or p300, 250 ng of WT or KD mutant of HIPK2 and 2 ng of phRL-cmv. Cell lysates were analyzed for luciferase activity at 24 h after transfection.

HIPK2 phosphorylates AML1 at Ser249 and Ser276

It has been reported that AML1b can be phosphorylated by Erk in 3T3 fibroblasts (Tanaka et al, 1996). However, treatment of L-G cells with inhibitors of Erk pathways did not affect the phosphorylation of AML1b (Supplementary Figure S1), suggesting the involvement of other kinases in this phosphorylation. We previously purified the AML1b complex from L-G cells (Kitabayashi et al, 2001) and identified the serine/threonine kinase HIPK2 as a component of the AML1 complexes (Supplementary Figure S2). Immunoblot analysis confirmed that HIPK2 is present in the purified complex (Figure 1D). To confirm this interaction, 293T cells were transfected with HA-tagged AML1b together with WT or kinase-dead (KD) point mutant FLAG-tagged HIPK2. HIPK2 proteins were immunoprecipitated with anti-FLAG antibodies. On immunoblot analysis, HA-AML1b was co-precipitated with the WT and mutant HIPK2 (Figure 1E).

To determine whether HIPK2 phosphorylates AML1, we tested the phosphorylation of AML1 by HIPK2 in vivo and in vitro. 293T cells were transfected with WT and mutant AML1 together with HIPK2. Immunoblot analysis demonstrated that co-transfection of HIPK2 leads to retardation of the protein band for WT AML1b (Figure 1F). Note that AML1b phosphorylation levels are low (i.e., its migration is fast) when AML1 is transiently transfected into 293T cells. Substitution of either Ser249 or Ser276 by alanine diminished the changes in band migration caused by HIPK2. In in vitro kinase assays, HIPK2 strongly phosphorylated WT AML1 as well as itself (Figure 1G). Mutations in Ser249 and Ser276 strongly inhibited HIPK2-mediated phosphorylation of AML1. These results indicate that HIPK2 is able to phosphorylate AML1 at Ser249 and Ser276.

HIPK2 stimulates cooperation between AML1, MOZ and p300 in transcription activation

In order to clarify the roles of phosphorylation in the function of AML1, we tested the effects of HIPK2 on AML1-mediated transcription. WT/mutant AML1 was co-transfected with p300/MOZ HAT, HIPK2 and MPO-luc or CCP1-luc reporter genes containing AML1-binding sites. As shown in Figure 1H, MOZ and p300 stimulated AML1-dependent transcription, as reported previously (Kitabayashi et al, 2001). MOZ-mediated and p300-mediated transcription activation was strongly enhanced by WT HIPK2 and was completely abolished by KD HIPK2, which functions in a dominant-negative manner. These results suggest that HIPK2 plays an important role in AML1-mediated transcription, particularly in its cooperation with MOZ and p300. HIPK2-, MOZ- and p300-induced transcription activation was almost completely abolished by substitution of Ser249 and Ser276 by alanine (S249/276A). In contrast, this activation activity was strongly enhanced when Ser249 and Ser276 were changed to aspartic acid, which mimics phosphorylated amino acids (S249/276D). These results suggest that phosphorylation of Ser249 and Ser276 on AML1 is essential for transcription activation.

Phosphorylation of p300 by activated AML1 and HIPK2

Because the above results suggest that HIPK2 stimulates AML1 cooperation with p300 and MOZ in transcription, we inferred that HIPK2-mediated AML1 phosphorylation stimulates interaction with p300 and/or MOZ. We then tested the effects of HIPK2 on these interactions. Co-immunoprecipitation analysis indicated that co-transfection with WT or KD HIPK2 had no effect on the interaction between AML1 and p300 (Figure 2A). Unexpectedly, however, we observed differences in mobility of the p300 band after transfection with AML1 and HIPK2. As shown in the upper panel of Figure 2A, co-transfection with AML1 resulted in mobility retardation of p300 (lane 2). This shift in mobility was due to phosphorylation, as treatment with phosphatase restored mobility (Figure 2B). A similar shift in mobility was also induced by co-transfection with WT HIPK2 (Figure 2A, lane 3). More importantly, the AML1-induced mobility shift of p300 was completely inhibited by co-transfection with KD HIPK2 (Figure 2A, lane 6). In vitro kinase assays revealed direct phosphorylation of p300 by HIPK2 (Figure 2C). These results suggest that AML1 is able to induce HIPK2-mediated phosphorylation of p300.

Figure 2.

Figure 2

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HIPK2 mediates AML1-dependent phosphorylation of p300. (A) AML1 induces phosphorylation of p300. 293T cells were transfected with FLAG-AML1b, HA-p300 and HIPK2. Cell lysates were immunoprecipitated with anti-FLAG antibodies. Cell lysates (1%) and immunoprecipitates were then analyzed by immunoblotting with anti-HA antibody. (B) CIAP-treatment of AML1–p300 complex. 293T cells were transfected with HA-p300 and FLAG-AML1b. After immunoprecipitation with anti-HA antibodies, immunoprecipitates were treated with CIAP and analyzed by immunoblotting with anti-HA antibody. (C) Phosphorylation of p300 in vitro. Purified WT p300 protein was incubated with or without HIPK2 in the presence of [γ32P]ATP and was separated by 7% SDS–PAGE. Gels were dried and subjected to autoradiography. (D) Phosphorylation of AML1 is required for AML1-induced phosphorylation of p300. 293T cells were transfected with HA-p300 and FLAG-tagged WT, S249/276A (2A) or S249/276A (2D) mutant AML1b. After immunoprecipitation with anti-FLAG antibodies, cell lysates (1%) and immunoprecipitates were analyzed by immunoblotting with anti-HA antibody. (E) HIPK2 and AML1 stimulates HAT activity of p300. 293T cells were transfected with FLAG-p300 together with either WT or KD HIPK2, or AML1b. After immunoprecipitation with anti-FLAG antibodies, immunoprecipitates were analyzed for HAT activity. (F) HIPK2 and AML1 stimulate transcription activation by a Gal4-p300 fusion protein. 293T cells were transfected with 500 ng of pFR-luc(Gal4-luc), 100 ng of Gal4-p300, 400 ng of WT or KD HIPK2, or AML1b and 2 ng of phRL-cmv. Cell lysates were analyzed for luciferase activity at 24 h after transfection.

The mobility shift of p300 was not induced when co-transfection was performed with an alanine mutant (2A=S249/276A), which lacks phosphorylation sites (upper panel in Figure 2D). In contrast, an aspartic acid mutant (2D=S249/276D), which mimics phosphorylated AML1, induced a greater mobility shift than WT AML1. These results suggest that activation of AML1 by phosphorylation is required for AML1-induced phosphorylation of p300. Co-immunoprecipitation experiments indicated that neither the alanine nor aspartic acid mutation affected the interaction between AML1 and p300 (Figure 2D, lower panel). In addition, neither of these mutations affected the interaction between AML1 and HIPK2 (Supplementary Figure S3).

HIPK2 activates p300

In order to investigate the role of phosphorylation by HIPK2 on p300 function, we tested the effects of HIPK2 on p300 HAT and transcription activation activities. To test HAT activity, FLAG-tagged p300 was co-transfected with WT HIPK2 and KD mutant, purified using anti-FLAG antibodies and tested for HAT activity. Intrinsic HAT activity of p300 was activated by WT HIPK2 and AML1b, and was inhibited by KD HIPK2 (Figure 2E). Reporter analysis indicated that WT HIPK2 and AML1 stimulated transcription activation by Gal4-p300 (Figure 2F). These results suggest that HIPK2 stimulates HAT activity and p300-mediated transcription activation.

The p300 sites phosphorylated by HIPK2 were investigated using various mutants of Gal4-p300, in which the putative phosphorylation sites (SP and TP) were substituted with AP, and these mutants were tested for both transcription activation and phosphorylation by HIPK2. Transcription activation and phosphorylation of p300 were markedly reduced by substitution of the 23 SP/TP sites (Figure 3).

Figure 3.

Figure 3

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Phosphorylation sites of p300 by HIPK2. (A) Schematic representation of p300 mutants used for determining HIPK2-induced phosphorylation sites. (B) Amino-acid sequence of putative HIPK2 phosphorylation sites of p300. Serines or threonines shown in red were substituted with alanines. (C) HIPK2-induced activation of transcription by Gal4-p300 mutants. 293T cells were transfected with 500 ng of pFR-luc(Gal4-luc), 100 ng of WT or mutant Gal4-p300, 400 ng of HIPK2 and 2 ng of phRL-cmv. Cell lysates were prepared at 24 h after transfection and were analyzed for luciferase activity. (D) Phosphorylation of p300 mutants by HIPK2. 293T cells were transfected with WT or mutants of Gal4-p300 together with HIPK2. Cell lysates were subjected to immunoblot analysis with anti-Gal4 antibody.

Interaction between HIPK2 and p300

Overexpressed HIPK2 was able to phosphorylate p300, even in the absence of AML1, thus suggesting a physical interaction between HIPK2 and p300. To test this interaction, FLAG-tagged HIPK2 or AML1 were co-transfected with HA-p300 or truncated versions thereof as schematically shown in Figure 4A. On IP-Western analysis, p300 was co-precipitated with WT and KD HIPK2 in addition to AML1 (Figure 4B). Reciprocal co-immunoprecipitation experiments also indicated that WT and KD HIPK2 were co-precipitated with p300 (Figure 4C). Deletion analysis of p300 allowed mapping of the HIPK2-binding domain (1304–1571) and AML1-binding domain (142–597) (Figure 4D). Interactions between AML1 and p300 were markedly reduced by deletion of the 142–957 region of p300, but the interaction could be still detected weakly after overexposure (Figure 4D), suggesting an indirect interaction through other factors or another AML1-interacting domain. GST pull-down analysis indicated that HIPK2, which was translated in vitro, bound to bacterially produced GST-AML1b and GST-p300(HAT) proteins (Figure 4E), suggesting that HIPK2 directly interacts with AML1 and p300.

Figure 4.

Figure 4

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Interaction of p300 and HIPK2. (A) Schematic representation of p300 deletion mutants used for determining HIPK2-binding domains. (B) Interaction of p300 with HIPK2. 293T cells were transfected with p300 together with FLAG-tagged WT or KD HIPK2, or AML1b. (B–D) Cell lysates (1%) and immunoprecipitates with anti-FLAG antibodies were analyzed by immunoblotting with anti-p300, anti-HA or anti-FLAG antibodies. (C) Reciprocal coimmunoprecipitation of HIPK2 and p300. (D) Interaction of p300 mutants with HIPK2 and AML1. 293T cells were transfected with p300 mutants as shown together with KD HIPK2 or AML1b. The lower panel represents a longer exposure of the same membrane shown in the middle panel. (E) HIPK2 translated in vitro and labelled with 35S was tested for binding to bacterially produced GST, GST-AML1b and GST-p300(HAT) proteins as shown. The input lane represents 10% of the material used for binding to the GST fusion protein.

Phosphorylation of p300 by various transcription factors

Various transcription factors are known to utilize p300 as a co-activator. It was recently reported that the C/EBP family of transcription factors induce phosphorylation of p300 and CBP (Kovacs et al, 2003; Schwartz et al, 2003). To test whether phosphorylation of p300 is induced by other transcription factors that bind to p300, several transcription factors were co-transfected with p300. Mobility shifts of p300 were induced by AML/runx family members, PU.1, C/EBPα, C/EBPɛ, c-MYB, c-JUN and c-FOS, but were not induced by IRF3, p53, RARα or ATF2 (Figures 5A and C). To determine whether these shifts were mediated by HIPK2, the dominant-negative HIPK2 was co-transfected with these factors. As shown in Figure 5B, the dominant-negative HIPK2 completely inhibited the phosphorylation of p300 induced by AML1/2/3, PU.1, c-MYB, c-JUN and c-FOS, which suggests that HIPK2 mediates p300 phosphorylation by these factors. On the other hand, phosphorylation induced by C/EBPα and C/EBPɛ was only partially inhibited by the dominant-negative HIPK2, which suggests that other kinase(s) are involved (Figure 5C).

Figure 5.

Figure 5

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HIPK2 mediates phosphorylation of p300 induced by a set of transcription factors. (A) Phosphorylation of p300 by various transcription factors. 293T cells were transfected with HA-p300 together with various transcription factors. Cell lysates were subjected to immunoblot analysis with anti-HA antibody. (B) Effects of WT and KD HIPK2 on transcription factor-induced phosphorylation of p300. 293T cells were transfected with HA-p300 and the transcription factors indicated together with WT and KD HIPK2. (C) Effects of KD HIPK2 on phosphorylation of p300. 293T cells were transfected with HA-p300 (0.2 μg) and either C/EBPɛ, C/EBPα or AML1b (0.4 μg) together with increasing amounts (0.2 and 0.4 μg) of KD HIPK2.

Roles of HIPK family members in phosphorylation and transcription

We generated Hipk1- and Hipk2-deficient mice by targeted disruption of the corresponding genes (Isono et al, 2006). Hipk1 and Hipk2 single-deficient mice are grossly normal (Kondo et al, 2003; Wiggins et al, 2004), but Hipk1/Hipk2 double homozygotes die between E9.5 and E12.5 (Isono et al, 2006). These findings suggest functional redundancy between HIPK1 and HIPK2. We thus addressed the physiological roles of HIPK family proteins by using Hipk1/Hipk2 double mutants. To investigate the phosphorylation of p300, proteins were extracted from whole embryos at E10.0 and were analyzed by immunoblottng with anti-p300 antibodies. Although significant amounts of highly phosphorylated forms of p300 were observed in Hipk1-deficient, Hipk2-deficient and WT embryos, highly phosphorylated forms were severely reduced in Hipk1/Hipk2 double-deficient embryos (Figure 6A). It has been reported that phosphorylation of p300 is stimulated during retinoic acid-induced differentiation of embryonic stem (ES) cells (Kitabayashi et al, 1995). To test whether HIPK1 and HIPK2 are required for p300 phosphorylation, we investigated changes in phosphorylation of p300 after retinoic acid-induced differentiation of WT and HIPK1/HIPK2 double knockout ES cells. Phosphorylation of p300 was stimulated and was associated with expression of HIPK2 during differentiation of WT ES cells but not in double knockout ES cells (Figure 6B). Induction of p300 phosphorylation and HIPK2 expression was also observed in G-CSF-induced differentiation of 32Dcl3 cells (Figure 6C).

Figure 6.

Figure 6

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HIPK1 and HIPK2 are essential for phosphorylation of p300 and AML1. (A) Phosphorylation of p300 in HIPK1- and/or HIPK2-deficient embryos. Proteins were extracted from WT and mutant E10.0 embryos and were subjected to immunoblot analysis with anti-p300 antibody. (B) Expression of HIPK2 and phosphorylation of p300 during differentiation of ES cells. WT and Hipk1/2 double-deficient ES cells were exposed to 1 μM retinoic acid for 2 and 4 days. The cell lysates were subjected to immunoblot analysis with anti-HIPK2 and anti-p300 antibodies. (C) Expression of HIPK2 and phosphorylation of p300 during differentiation of myeloid cells. 32Dcl3 cells were exposed to 10 ng G-CSF for indicated number of days. (D) Phosphorylation of AML1 in HIPK1- and/or HIPK2-deficient yolk sac. Yolk sac cells from E10.0 embryos were cultured on OP9 cells for 5 days. Cell lysates were subjected to immunoblot analysis with anti-AML1 and phospho-S249-specific AML1 antibodies. (E) Phosphorylation of p300 and AML1 by HIPK1 and HIPK2. 293T cells were transfected with HA-p300 or AML1 (0.2 μg) together with increasing amounts (0.05, 0.1, 0.2 μg) of HIPK1 or HIPK2. Cell lysates were subjected to immunoblot analysis with anti-HA antibody. (F) 293T cells were transfected with WT or mutant Gal4-p300 or HA-AML1 together with HIPK1 or HIPK2. Cell lysates were subjected to immunoblot analysis with anti-Gal4 or anti-HA antibody.

To confirm the phosphorylation of AML1, which is predominantly expressed in hematopoietic cells, nonadherent cells were prepared from the yolk sac of mutant embryos and were expanded by culture in vitro for 5 days. Immunoblot analysis using anti-AML1 antibody indicated that hypophosphorylated forms of AML1 were increased in Hipk1/Hipk2 double-deficient cells when compared with those in single-deficient cells or WT cells. Immunoblot analysis using phospho-specific antibody (Supplementary Figure S4), which detects AML1 phosphorylated at Ser249, indicated that phosphorylated AML1 was markedly decreased in Hipk1/Hipk2 double-deficient cells (Figure 6D).

In order to test whether HIPK1 can induce phosphorylation of p300 and AML1, 293T cells were transfected with p300 and AML1 together with HIPK1. Phosphorylation of p300 and AML1 was strongly induced by HIPK1 as well as by HIPK2 (Figure 6E). These phosphorylations were largely inhibited by substitution with alanine at the phosphorylation sites (Figure 6F). Co-immunoprecipitation analysis indicated that HIPK1 as well as HIPK2 interact with AML1 (Supplementary Figure S5).

Reporter analysis indicated that HIPK1 also stimulates transcription activation by Gal4-p300 and by AML1 (Figure 7A and B). Reporter analysis using WT and HIPK1/2 double-deficient mouse embryonic fibroblasts showed an impaired AML1-dependent transcription in HIPK1/HIPK2 double-deficient cells (Figure 7C). Thus, we conclude that both HIPK1 and HIPK2 mediate the phosphorylation of p300 and AML1, and enhance AML1- and p300-dependent transcription.

Figure 7.

Figure 7

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HIPK1 and HIPK2 are involved in p300- and AML1-mediated transcription. (A) 293T cells were transfected with 500 ng of pFR-luc(Gal4-luc), 100 ng of Gal4-p300, 400 ng of HIPK1 or HIPK2, and 2 ng of phRL-cmv. (B) SaOS2 cells were transfected with 50 ng of MPO-luc, 200 ng of LNCX-AML1b, 250 ng of MOZ or p300, 250 ng of HIPK1 or HIPK2, and 2 ng of phRL-cmv. (C) WT and HIPK1/2 double-deficient MEFs were transfected with 50 ng of MPO-luc, 200 ng of LNCX-AML1b, 500 ng of MOZ or p300, and 2 ng of phRL-cmv. Cell lysates were analyzed for luciferase activity at 24 h after transfection.

Hematopoiesis defects in HIPK1/2-deficient mice

Because AML1, p300 and the closely related CBP are essential for hematopoiesis (Okuda et al, 1996; Wang et al, 1996a; Oike et al, 1999; Tanaka et al, 2000; Kasper et al, 2002; Rebel et al, 2002), we analyzed hematopoietic cells in the yolk sac and the paraaortic splanchnopleural (P-Sp) region, in which definitive hematopoiesis is first detected during development. A marked decrease in the total number of cells was observed in Hipk1−/−Hipk2−/− yolk sacs (Figure 8A). To investigate definitive hematopoiesis, Hipk1−/−Hipk2+/− mice were mated, and the P-Sp region of embryos at 9.5-dpc were dissected and cultured on OP9 cells. After 7 days of culture, colony-forming cells (CFCs) from nonadherent cells were examined on methylcellulose medium. Yolk sac cells were directly tested for CFCs. As shown in Figure 8B and C, the total number of CFCs in the yolk sac and cultured P-Sp cells from double-deficient (Hipk1−/−Hipk2−/−) mutants was markedly reduced when compared with those from control littermates (Hipk1−/−Hipk2+/− or Hipk1−/−Hipk2+/+). These results suggest that primitive as well as definitive hematopoiesis is defective in Hipk1/Hipk2 double-deficient embryos.

Figure 8.

Figure 8

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Hematopoiesis in HIPK1/2 mutant embryos. (A) Yolk sac cells from embryos at E9.5 were dispersed into single-cell suspensions and the numbers of nucleated erythroid cells were counted. (B) Colony-forming cells in P-Sp cultures. P-Sp explants derived from E9.5 embryos obtained by intercrossing HIPK1−/−,HIPK2+/− mice were cultured on OP9 stromal cells for 7 days and were subjected to colony-formation assay. Dot-filled and oblique-line-filled boxes represent the number of erythroid and myeloid colonies, respectively. (C) Colony-forming cells in yolk sac. Yolk sac cells were directly subjected to colony-formation assay.

Defects in vasculogenesis/angiogenesis in HIPK1/2-deficient mice

Morphologic evaluation of embryos revealed severe defects in vessel formation in Hipk1/Hipk2 double-homozygous mutant embryos and yolk sacs at E10.0 (Figure 9A and B). Similar defects in vessel formation are also observed in p300- and cbp-deficient mice (Yao et al, 1998; Oike et al, 1999; Tanaka et al, 2000). To further study the vascular system, whole embryos were stained with anti-PECAM-1 monoclonal antibody, which detects differentiated endothelial cells. Hipk1/Hipk2 double-deficient embryos failed to form an organized vascular network (Figure 9C). Gross defects in vascular branching were observed, particularly in the head and trunk (Figure 9D and E). To further investigate the defects in the vascular networks in Hipk1/Hipk2 double mutant embryos, P-Sp regions were dissected from E8.75 embryos obtained by intercrossing Hipk1−/−Hipk2+/− mutant mice, cultured on OP9 stromal cells, and stained with anti-PECAM-1 antibody (Figure 9F). Both sheet (Figure 9G) and network (Figure 9H) formations, which are indicative of vasculogenesis and angiogenesis, respectively, were severely affected in P-Sp cultures from Hipk1/Hipk2 double-homozygous mutant embryos, while cultures from littermate embryos developed normally. The double-homozygous mutant embryos at E8.75 were comparable in size to their littermates (data not shown). Thus, we concluded that vascular formation in Hipk1/Hipk2 double-homozygotes is impaired rather than delayed.

Figure 9.

Figure 9

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HIPK1/2 mutant embryos showed defective vasculogenesis and angiogenesis. (A, B) Phenotypic comparison of E10.0 Hipk1−/−, Hipk2+/+ and Hipk1−/−,Hipk2−/− embryos with (A) and without (B) yolk sac. (C) Whole-mount immunohistostaining with anti-CD31 (PECAM-1) mAb of E9.5 Hipk1−/−,Hipk2+/+ and Hipk1−/−,Hipk2−/− embryos. (D) Primary vascular plexus observed in mutant heads. (E) Migration of endothelial cells forming intersomitic vessels. (F–H) Vasculo-angiogenesis in P-Sp culture. P-Sp explants derived from E8.75 embryos obtained by intercrossing Hipk1−/−,Hipk2+/− mice were cultured on OP9 stromal cells and were stained with anti-CD31 mAb. Representative results are shown in (F). The average values of areas of vascular sheet formation and vascular network formation are shown in (G) and (H), respectively.

Discussion

AML1 stimulates HIPK2-mediated phosphorylation of p300 and activates transcription

In the present study, we found that co-expression of AML1 induced phosphorylation of p300. This effect was inhibited by the dominant-negative form of HIPK2 and was enhanced by WT HIPK2. These results suggest that HIPK2 mediates the AML1-induced phosphorylation of p300. HIPK2-mediated phosphorylation of p300 stimulates its HAT activity and activates p300-dependent transcription. AML1 interacts with both p300 and HIPK2, and thus AML1 induces phosphorylation of p300 by stimulating the physical interaction between the enzyme HIPK2 and the substrate p300. Interestingly, the alanine mutant AML1-S249/276A, which lacks phosphorylation sites, did not induce p300 phosphorylation, while the aspartic acid mutant AML1-S246/276D, which mimicks the phosphorylated form, induced phosphorylation of p300 more efficiently than WT AML1. Moreover, AML1/p300-mediated transcriptional activation was inhibited by alanine mutations and enhanced by aspartic acid mutations on the phosphorylation sites of AML1. These findings suggest that phosphorylation of AML1 is required for the HIPK2-mediated phosphorylation of p300 and subsequent transcription activation. Thus, AML1 not only recruits HIPK2 to the substrate p300 but also functions as a regulatory subunit for HIPK2 to control p300 phosphorylation, local histone acetylation and transcription of target genes.

HIPK-induced phosphorylation of p300 is common to regulation by various transcription factors

Phosphorylation of p300 was also induced by transcription factors such as the AML1/runx family members, PU.1, c-MYB, c-JUN and c-FOS. These effects were inhibited by co-expression of dominant-negative HIPK2, suggesting that HIPK family members are involved in the phosphorylation of p300 by these transcription factors. It is likely that these transcription factors also regulate transcription by recruiting HIPKs and inducing p300 phosphorylation on their target genes in the same way as AML1. Thus, HIPK-mediated phosphorylation of p300 is a common event in transcriptional regulation by a range of transcription factors. Phosphorylation of p300 was not induced by transcription factors such as IRF3, p53, RARα and ATF2 under the conditions tested, although these transcription factors were able to interact with p300. As these factors are activated in response to a range of physiological stimuli, it is possible that additional modifications or factors induced by these stimuli are required for p300 phosphorylation.

Highly phosphorylated forms of p300 were scarcely detected in Hipk1/2 double-deficient embryos, suggesting that phosphorylation of p300 is largely regulated by HIPK1 and HIPK2 during embryonic development. A variety of transcription factors, including jun, fos, runx and ets family members, are probably involved in the regulation of p300 phosphorylation during embryogenesis and hematopoiesis. It has been reported that phosphorylation of p300 is stimulated and is associated with expression of c-JUN during retinoic acid-induced differentiation of F9 embryonic carcinoma cells and ES cells (Kitabayashi et al, 1995). We found here that the induction of p300 phosphorylation was inhibited in Hipk1/2 double-deficient ES cells. Thus, the phosphorylation of p300 in this process is likely to be mediated by HIPK1 and/or HIPK2 in combination with transcription factors, including c-JUN.

It has been reported that AML1 activates transcription synergistically with other transcription factors, such as AP-1 (jun/fos), C/EBPα, c-Myb, PU.1 and Ets-1, to activate transcription (Cockerill et al, 1996; Zhang et al, 1996; Britos-Bray and Friedman, 1997; Petrovick et al, 1998; Kim et al, 1999). We found here that phosphorylation of p300 is a common effect induced by these transcription factors, thus suggesting that phosphorylated forms of p300 and HIPK2 are involved in the above-mentioned synergistic effects.

Role of phosphorylation in p300/CBP function

HIPK1 and HIPK2 double-deficient embryos died between E9.5 and E12.5 and exhibited defects in vasculogenesis, angiogenesis, and primitive and definitive hematopoiesis. The double homozygous mutants exhibited developmental retardation and failed to close the anterior neuropore (Isono et al, 2006). These phenotypes were in part similar to those observed in p300- and CBP-deficient mice. Both p300- and CBP-deficient mice show embryonic lethality and die between E9.5 and E12.5 (Yao et al, 1998; Oike et al, 1999; Tanaka et al, 2000). Moreover, double heterozygosity for p300 and cbp is invariably associated with embryonic death, suggesting the overlapping function and overall gene dosage requirement of p300 and CBP. p300- and cbp-deficient embryos exhibited defective blood vessel formation, defective neural tube closure and apparent developmental retardation, as well as defects in both primitive and definitive hematopoiesis. Phosphorylation of p300 was inhibited in Hipk1/Hipk2-double deficient embryos. Taken together these findings suggest that phosphorylation of p300/CBP by HIPK1/HIPK2 is essential for p300 and CBP function in embryonic development. However, we cannot exclude a possibility that HIPK1/HIPK2 may have other substrates whose phosphorylation is required for hematopoiesis and development. p300 null embryos show other defects, in addition to those found in Hipk1/Hipk2-double deficient embryos, including anomalous heart formation, turning of the embryo and edema formation (Yao et al, 1998). That these defects are not observed in Hipk1/Hipk2-double deficient embryos suggests that some of the function of p300 is independent of HIPK1/2-mediated phosphorylation.

HIPK2 regulates transcription via multiple factors

We also found that co-expression of AML1 and HIPK2 induced phosphorylation of MOZ (Supplementary Figure S6). The phosphorylation of MOZ was not efficiently induced by either AML1 or HIPK2 alone, and thus interaction between these three molecules is likely to be required for efficient MOZ phosphorylation. AML1 probably stimulates MOZ phosphorylation by accelerating the physical interaction between HIPK2 and MOZ. Moreover, a highly phosphorylated form of MOZ was preferentially co-precipitated with AML1, suggesting that AML1 interacts with the highly phosphorylated form of MOZ more strongly than with the hypophosphorylated form. Thus, HIPK2 stabilizes the AML1/MOZ complex by phosphorylating MOZ. We generated MOZ mutant mice and found that MOZ homozygous mutant mice die around E15.5 and exhibit severe defects in definitive hematopoiesis (Katsumoto et al, 2006). Hipk1/Hipk2-deficient mice also show defective definitive hematopoiesis. These defects appear to be more severe than those observed in cbp- or p300-deficient mice. This is probably due to reduced MOZ activity, in addition to inactive AML1 and p300/CBP, in Hipk1/Hipk2-deficient mice.

It was recently reported that the Groucho corepressor is phosphorylated by Drosophila HIPK2 and subsequently dissociates from the corepressor complex (Choi et al, 2005). TLE, the human homolog of Groucho, also reportedly interacts with AML1 and suppresses AML1-induced transactivation (Imai et al, 1998; Levanon et al, 1998). Taken together with these findings, the present results suggest that HIPKs play a key role in regulating the transcription of target genes by relieving Groucho-mediated transcriptional repression as well as by activating transcription factor/HAT complexes.

Materials and methods

Cells and retroviruses

Culture of L-G, BOSC23 and SaOS2 cells were performed as described previously (Kitabayashi et al, 2001). Retrovirus infection of cells was performed as described previously (Kitabayashi et al, 1998a).

Plasmids

The AML1 expression vectors pLNCX-FLAG-AML1a, pLNCX-FLAG-AML1b, pLNCX-HA-p300, pLNCX-HA-MOZ, mCCP1-luc and MPO-luc were as described previously (Kitabayashi et al, 2001). LNCX-FLAG-HIPK2 and LNCX-HA-HIPK2 were generated by subcloning WT and KD HIPK2 (Hofmann et al, 2002) into a retrovirus-derived LNCX vector. CMV-HIPK1 was as described by Isono et al (2006).

Mass spectrometry

AML1 proteins were purified from L-G cells, digested with trypsin and subjected to LC/MS/MS analysis as described previously (Kitabayashi et al, 2001). Phosphopeptides were identified using TurboSEQUEST software.

Immunoprecipitation, immunoblotting and antibodies

Immunoprecipitation and immunoblotting analyses were performed as described previously (Kitabayashi et al, 2001). The following primary antibodies were used in the present study: anti-FLAG (M2) antibody (Sigma), anti-HA (3F10) antibody (Roche), anti-AML1 antibody (Kitabayashi et al, 1998a), anti-HIPK2 antibody (Hofmann et al, 2002) and anti-p300 antibody (N15) (Santa Cruz). The anti-phospho-S249 AML1 antibody was generated by immunizing rabbits with phosphopeptides RQIQPpSPPWSY corresponding to residues 244-254 of human AML1b.

HAT activity and kinase activity in vitro

For HAT activity, 293 cells were transfected with FLAG-tagged p300 together with WT or KD HIPK2. FLAG-p300 was purified by immunoprecipitation as described above and incubated in a 10-μl volume containing 50 mM Tris (pH 8.0), 10% glycerol, 1 mM DTT, 1 mM PMSF, 10 mM sodium butyrate, 0.5 μl of [1-14C]-acetyl-CoA (50 μCi/ml, Amersham), and 0.5 μg each of histones H2A, H2B, H3 and H4 at 37°C for 1 h. Reaction mixtures were analyzed by 18% SDS–PAGE, and gels were subjected to autoradiography. Histone acetylation was measured using a BAS2000 imaging analyzer (Fuji).

For kinase activity, FLAG-tagged WT and mutant AML1b, WT HIPK2 and p300 were purified by immunoprecipitation, as described above, and were incubated in 20 mM Tris–Cl (pH 7.5), 10 mM MgCl2, 1 mM DTT, and 50 μM ATP, 5 μCi [γ-32P]-ATP at 37°C for 30 min. Reaction mixtures were analyzed by SDS–PAGE, and gels were subjected to autoradiography.

Luciferase assay

Reporter analysis was performed as described previously (Kitabayashi et al, 2001).

GST pull-down assay

The GST pull-down assay was performed as described previously (Hofmann et al, 2002). GST-AML1b has been described previously (Kitabayashi et al, 1998b). GST-p300(HAT) was generated by subcloning of an EcoRI–StuI fragment into pGEX.

In vitro culture of P-Sp

P-Sp culture conditions were as described (Takakura et al, 2000).

In vitro hematopoietic colony assays

P-Sp cultures from mouse embryos were dispersed into single-cell suspensions and cultured on 1% methylcellulose in Iscove's modified Dulbecco's medium (IMDM) containing 15% fetal bovine serum, 1% bovine serum albumin, 10 μg/ml rh insulin, 200 μg/ml human transferrin, 100 μM 2-mercaptoethanol, 2 mM L-glutamine, 50 ng/ml rm SCF, 10 ng/ml rmIL-3, 10 ng/ml rh IL-6 and 3 U/ml erythropoietin (MethoCult GF M3434) (Stem Cell Technology Inc., Vancouver, Canada). Cultures were maintained at 37°C under humidified conditions with 5% CO2. Colonies containing more than 50 cells were counted on day 12, and myeloid CFUs, erythroid burst forming units (BFU-E) and CFU-Mix were defined based on morphology.

Isolation and differentiation of ES cells

Hipk1−/−Hipk2+/− mice were mated and blastocysts were harvested on day E3.5 then individually cultured in ES cell medium (containing 20% fetal calf serum and leukemia inhibitory factor (LIF)) on a feeder layer of mitomycin C-treated embryonic fibroblasts, as previously described (Auerbach et al, 2000). ES cells were cultured in ES cell medium containing 10% fetal calf serum plus LIF. Cells were then plated on gelatinized 100-mm plates at a density of 1 × 106 cells/plate in the absence of LIF for 24 h, and then they were treated with 1 μM RA for various periods of time.

Supplementary Material

Supplementary Figure S1

7601273s1.tiff (115.9KB, tiff)

Supplementary Figure S2

7601273s2.tiff (211.3KB, tiff)

Supplementary Figure S3

7601273s3.tiff (154.1KB, tiff)

Supplementary Figure S4

7601273s4.tiff (131.7KB, tiff)

Supplementary Figure S5

7601273s5.tiff (106.6KB, tiff)

Supplementary Figure S6

7601273s6.tiff (283.3KB, tiff)

Acknowledgments

We would like to thank Dr M Kurokawa and Dr M Nakagawa for their advice on preparation of hematopoietic cells in the P-Sp culture, Dr T Nakano for supplying the OP9 cells, and Ms N Aikawa and Ms M Shino for technical assistance. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Health, Labor and Welfare and from the Ministry of Education, Culture, Sports, Science and Technology, and by the Program for Promotion of Fundamental Studies from the National Institute of Biomedical Innovation of Japan.

References

  1. Auerbach W, Dunmore JH, Fairchild-Huntress V, Fang Q, Auerbach AB, Huszar D, Joyner AL (2000) Establishment and chimera analysis of 129/SvEv- and C57BL/6-derived mouse embryonic stem cell lines. Biotechniques 29: 1024–1028, 1030, 1032 [DOI] [PubMed] [Google Scholar]
  2. Britos-Bray M, Friedman AD (1997) Core binding factor cannot synergistically activate the myeloperoxidase proximal enhancer in immature myeloid cells without c-Myb. Mol Cell Biol 17: 5127–5135 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chen CJ, Deng Z, Kim AY, Blobel GA, Lieberman PM (2001) Stimulation of CREB binding protein nucleosomal histone acetyltransferase activity by a class of transcriptional activators. Mol Cell Biol 21: 476–487 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Choi CY, Kim YH, Kim YO, Park SJ, Kim EA, Riemenschneider W, Gajewski K, Schulz RA, Kim Y (2005) Phosphorylation by the DHIPK2 protein kinase modulates the corepressor activity of Groucho. J Biol Chem 280: 21427–21436 [DOI] [PubMed] [Google Scholar]
  5. Choi CY, Kim YH, Kwon HJ, Kim Y (1999) The homeodomain protein NK-3 recruits Groucho and a histone deacetylase complex to repress transcription. J Biol Chem 274: 33194–33197 [DOI] [PubMed] [Google Scholar]
  6. Cockerill PN, Osborne CS, Bert AG, Grotto RJ (1996) Regulation of GM-CSF gene transcription by core-binding factor. Cell Growth Differ 7: 917–922 [PubMed] [Google Scholar]
  7. D'Orazi G, Cecchinelli B, Bruno T, Manni I, Higashimoto Y, Saito S, Gostissa M, Coen S, Marchetti A, Del Sal G, Piaggio G, Fanciulli M, Appella E, Soddu S (2002) Homeodomain-interacting protein kinase-2 phosphorylates p53 at Ser 46 and mediates apoptosis. Nat Cell Biol 4: 11–19 [DOI] [PubMed] [Google Scholar]
  8. Hofmann TG, Moller A, Sirma H, Zentgraf H, Taya Y, Droge W, Will H, Schmitz ML (2002) Regulation of p53 activity by its interaction with homeodomain-interacting protein kinase-2. Nat Cell Biol 4: 1–10 [DOI] [PubMed] [Google Scholar]
  9. Imai Y, Kurokawa M, Tanaka K, Friedman AD, Ogawa S, Mitani K, Yazaki Y, Hirai H (1998) TLE, the human homolog of groucho, interacts with AML1 and acts as a repressor of AML1-induced transactivation. Biochem Biophys Res Commun 252: 582–589 [DOI] [PubMed] [Google Scholar]
  10. Isono K, Nemoto K, Li Y, Takada Y, Suzuki R, Katsuki M, Nakagawara A, Koseki H (2006) Overlapping roles for homeodomain-interacting protein kinases hipk1 and hipk2 in the mediation of cell growth in response to morphogenetic and genotoxic signals. Mol Cell Biol 26: 2758–2771 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jenuwein T, Allis CD (2001) Translating the histone code. Science 293: 1074–1080 [DOI] [PubMed] [Google Scholar]
  12. Kanei-Ishii C, Ninomiya-Tsuji J, Tanikawa J, Nomura T, Ishitani T, Kishida S, Kokura K, Kurahashi T, Ichikawa-Iwata E, Kim Y, Matsumoto K, Ishii S (2004) Wnt-1 signal induces phosphorylation and degradation of c-Myb protein via TAK1, HIPK2, and NLK. Genes Dev 18: 816–829 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kasper LH, Boussouar F, Ney PA, Jackson CW, Rehg J, van Deursen JM, Brindle PK (2002) A transcription-factor-binding surface of coactivator p300 is required for haematopoiesis. Nature 419: 738–743 [DOI] [PubMed] [Google Scholar]
  14. Katsumoto T, Aikawa Y, Iwama A, Ueda S, Ichikawa H, Ochiya T, Kitabayashi I (2006) MOZ is essential for maintenance of hematopoietic stem cells. Genes Dev 20: 1321–1330 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kim WY, Sieweke M, Ogawa E, Wee HJ, Englmeier U, Graf T, Ito Y (1999) Mutual activation of Ets-1 and AML1 DNA binding by direct interaction of their autoinhibitory domains. EMBO J 18: 1609–1620 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kim YH, Choi CY, Lee SJ, Conti MA, Kim Y (1998) Homeodomain-interacting protein kinases, a novel family of co-repressors for homeodomain transcription factors. J Biol Chem 273: 25875–25879 [DOI] [PubMed] [Google Scholar]
  17. Kitabayashi I, Aikawa Y, Nguyen LA, Yokoyama A, Ohki M (2001) Activation of AML1-mediated transcription by MOZ and inhibition by the MOZ-CBP fusion protein. EMBO J 20: 7184–7196 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kitabayashi I, Eckner R, Arany Z, Chiu R, Gachelin G, Livingston DM, Yokoyama KK (1995) Phosphorylation of the adenovirus E1A-associated 300 kDa protein in response to retinoic acid and E1A during the differentiation of F9 cells. EMBO J 14: 3496–3509 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kitabayashi I, Ida K, Morohoshi F, Yokoyama A, Mitsuhashi N, Shimizu K, Nomura N, Hayashi Y, Ohki M (1998a) The AML1-MTG8 leukemic fusion protein forms a complex with a novel member of the MTG8(ETO/CDR) family, MTGR1. Mol Cell Biol 18: 846–858 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kitabayashi I, Yokoyama A, Shimizu K, Ohki M (1998b) Interaction and functional cooperation of the leukemia-associated factors AML1 and p300 in myeloid cell differentiation. EMBO J 17: 2994–3004 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kondo S, Lu Y, Debbas M, Lin AW, Sarosi I, Itie A, Wakeham A, Tuan J, Saris C, Elliott G, Ma W, Benchimol S, Lowe SW, Mak TW, Thukral SK (2003) Characterization of cells and gene-targeted mice deficient for the p53-binding kinase homeodomain-interacting protein kinase 1 (HIPK1). Proc Natl Acad Sci USA 100: 5431–5436 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kovacs KA, Steinmann M, Magistretti PJ, Halfon O, Cardinaux JR (2003) CCAAT/enhancer-binding protein family members recruit the coactivator CREB-binding protein and trigger its phosphorylation. J Biol Chem 278: 36959–36965 [DOI] [PubMed] [Google Scholar]
  23. Legube G, Trouche D (2003) Regulating histone acetyltransferases and deacetylases. EMBO Rep 4: 944–947 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Levanon D, Goldstein RE, Bernstein Y, Tang H, Goldenberg D, Stifani S, Paroush Z, Groner Y (1998) Transcriptional repression by AML1 and LEF-1 is mediated by the TLE/Groucho corepressors. Proc Natl Acad Sci USA 95: 11590–11595 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Li QJ, Yang SH, Maeda Y, Sladek FM, Sharrocks AD, Martins-Green M (2003) MAP kinase phosphorylation-dependent activation of Elk-1 leads to activation of the co-activator p300. EMBO J 22: 281–291 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Look AT (1997) Oncogenic transcription factors in the human acute leukemias. Science 278: 1059–1064 [DOI] [PubMed] [Google Scholar]
  27. Marmorstein R, Roth SY (2001) Histone acetyltransferases: function, structure, and catalysis. Curr Opin Genet Dev 11: 155–161 [DOI] [PubMed] [Google Scholar]
  28. Meyers S, Downing JR, Hiebert SW (1993) Identification of AML-1 and the (8;21) translocation protein (AML-1/ETO) as sequence-specific DNA-binding proteins: the runt homology domain is required for DNA binding and protein-protein interactions. Mol Cell Biol 13: 6336–6345 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nguyen LA, Pandolfi PP, Aikawa Y, Tagata Y, Ohki M, Kitabayashi I (2005) Physical and functional link of the leukemia-associated factors AML1 and PML. Blood 105: 292–300 [DOI] [PubMed] [Google Scholar]
  30. Niki M, Okada H, Takano H, Kuno J, Tani K, Hibino H, Asano S, Ito Y, Satake M, Noda T (1997) Hematopoiesis in the fetal liver is impaired by targeted mutagenesis of a gene encoding a non-DNA binding subunit of the transcription factor, polyomavirus enhancer binding protein 2/core binding factor. Proc Natl Acad Sci USA 94: 5697–5702 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ogawa E, Inuzuka M, Maruyama M, Satake M, Naito-Fujimoto M, Ito Y, Shigesada K (1993) Molecular cloning and characterization of PEBP2 beta, the heterodimeric partner of a novel Drosophila runt-related DNA binding protein PEBP2 alpha. Virology 194: 314–331 [DOI] [PubMed] [Google Scholar]
  32. Oike Y, Takakura N, Hata A, Kaname T, Akizuki M, Yamaguchi Y, Yasue H, Araki K, Yamamura K, Suda T (1999) Mice homozygous for a truncated form of CREB-binding protein exhibit defects in hematopoiesis and vasculo-angiogenesis. Blood 93: 2771–2779 [PubMed] [Google Scholar]
  33. Okada H, Watanabe T, Niki M, Takano H, Chiba N, Yanai N, Tani K, Hibino H, Asano S, Mucenski ML, Ito Y, Noda T, Satake M (1998) AML1(−/−) embryos do not express certain hematopoiesis-related gene transcripts including those of the PU.1 gene. Oncogene 17: 2287–2293 [DOI] [PubMed] [Google Scholar]
  34. Okuda T, van Deursen J, Hiebert SW, Grosveld G, Downing JR (1996) AML1, the target of multiple chromosomal translocations in human leukemia, is essential for normal fetal liver hematopoiesis. Cell 84: 321–330 [DOI] [PubMed] [Google Scholar]
  35. Petrovick MS, Hiebert SW, Friedman AD, Hetherington CJ, Tenen DG, Zhang DE (1998) Multiple functional domains of AML1: PU.1 and C/EBPalpha synergize with different regions of AML1. Mol Cell Biol 18: 3915–3925 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rebel VI, Kung AL, Tanner EA, Yang H, Bronson RT, Livingston DM (2002) Distinct roles for CREB-binding protein and p300 in hematopoietic stem cell self-renewal. Proc Natl Acad Sci USA 99: 14789–14794 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sasaki K, Yagi H, Bronson RT, Tominaga K, Matsunashi T, Deguchi K, Tani Y, Kishimoto T, Komori T (1996) Absence of fetal liver hematopoiesis in mice deficient in transcriptional coactivator core binding factor beta. Proc Natl Acad Sci USA 93: 12359–12363 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Schwartz C, Beck K, Mink S, Schmolke M, Budde B, Wenning D, Klempnauer KH (2003) Recruitment of p300 by C/EBPbeta triggers phosphorylation of p300 and modulates coactivator activity. EMBO J 22: 882–892 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Soutoglou E, Viollet B, Vaxillaire M, Yaniv M, Pontoglio M, Talianidis I (2001) Transcription factor-dependent regulation of CBP and P/CAF histone acetyltransferase activity. EMBO J 20: 1984–1992 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Takakura N, Watanabe T, Suenobu S, Yamada Y, Noda T, Ito Y, Satake M, Suda T (2000) A role for hematopoietic stem cells in promoting angiogenesis. Cell 102: 199–209 [DOI] [PubMed] [Google Scholar]
  41. Tanaka T, Kurokawa M, Ueki K, Tanaka K, Imai Y, Mitani K, Okazaki K, Sagata N, Yazaki Y, Shibata Y, Kadowaki T, Hirai H (1996) The extracellular signal-regulated kinase pathway phosphorylates AML1, an acute myeloid leukemia gene product, and potentially regulates its transactivation ability. Mol Cell Biol 16: 3967–3979 [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Tanaka Y, Naruse I, Hongo T, Xu M, Nakahata T, Maekawa T, Ishii S (2000) Extensive brain hemorrhage and embryonic lethality in a mouse null mutant of CREB-binding protein. Mech Dev 95: 133–145 [DOI] [PubMed] [Google Scholar]
  43. Thiagalingam S, Cheng KH, Lee HJ, Mineva N, Thiagalingam A, Ponte JF (2003) Histone deacetylases: unique players in shaping the epigenetic histone code. Ann NY Acad Sci 983: 84–100 [DOI] [PubMed] [Google Scholar]
  44. Wang Q, Stacy T, Binder M, Marin-Padilla M, Sharpe AH, Speck NA (1996a) Disruption of the Cbfa2 gene causes necrosis and hemorrhaging in the central nervous system and blocks definitive hematopoiesis. Proc Natl Acad Sci USA 93: 3444–3449 [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wang Q, Stacy T, Miller JD, Lewis AF, Gu TL, Huang X, Bushweller JH, Bories JC, Alt FW, Ryan G, Liu PP, Wynshaw-Boris A, Binder M, Marin-Padilla M, Sharpe AH, Speck NA (1996b) The CBFbeta subunit is essential for CBFalpha2 (AML1) function in vivo. Cell 87: 697–708 [DOI] [PubMed] [Google Scholar]
  46. Wiggins AK, Wei G, Doxakis E, Wong C, Tang AA, Zang K, Luo EJ, Neve RL, Reichardt LF, Huang EJ (2004) Interaction of Brn3a and HIPK2 mediates transcriptional repression of sensory neuron survival. J Cell Biol 167: 257–267 [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yao TP, Oh SP, Fuchs M, Zhou ND, Ch'ng LE, Newsome D, Bronson RT, Li E, Livingston DM, Eckner R (1998) Gene dosage-dependent embryonic development and proliferation defects in mice lacking the transcriptional integrator p300. Cell 93: 361–372 [DOI] [PubMed] [Google Scholar]
  48. Zhang DE, Hetherington CJ, Meyers S, Rhoades KL, Larson CJ, Chen HM, Hiebert SW, Tenen DG (1996) CCAAT enhancer-binding protein (C/EBP) and AML1 (CBF alpha2) synergistically activate the macrophage colony-stimulating factor receptor promoter. Mol Cell Biol 16: 1231–1240 [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Zhang Q, Yoshimatsu Y, Hildebrand J, Frisch SM, Goodman RH (2003) Homeodomain interacting protein kinase 2 promotes apoptosis by downregulating the transcriptional corepressor CtBP. Cell 115: 177–186 [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Figure S1

7601273s1.tiff (115.9KB, tiff)

Supplementary Figure S2

7601273s2.tiff (211.3KB, tiff)

Supplementary Figure S3

7601273s3.tiff (154.1KB, tiff)

Supplementary Figure S4

7601273s4.tiff (131.7KB, tiff)

Supplementary Figure S5

7601273s5.tiff (106.6KB, tiff)

Supplementary Figure S6

7601273s6.tiff (283.3KB, tiff)

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