Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2012 Nov 15.
Published in final edited form as: Cancer Cell. 2011 Nov 15;20(5):563–575. doi: 10.1016/j.ccr.2011.09.008

CBX8, a Polycomb Group Protein, is Essential for MLL-AF9-Induced Leukemogenesis

Jiaying Tan 1, Morgan Jones 2, Haruhiko Koseki 3, Manabu Nakayama 4, Andrew Muntean 1, Ivan Maillard 5, Jay L Hess 1,6
PMCID: PMC3220883  NIHMSID: NIHMS334999  PMID: 22094252

SUMMARY

Chromosomal translocations involving the mixed lineage leukemia (MLL) gene lead to the development of acute leukemias. Constitutive HOX gene activation by MLL fusion proteins is required for MLL-mediated leukemogenesis; however, the underlying mechanisms remain elusive. Here, we show that chromobox homolog 8 (CBX8), a Polycomb Group protein that interacts with MLL-AF9 and TIP60, is required for MLL-AF9-induced transcriptional activation and leukemogenesis. Conversely, both CBX8 ablation and specific disruption of the CBX8 interaction by point mutations in MLL-AF9 abrogate HOX gene upregulation and abolish MLL-AF9 leukemic transformation. Surprisingly, Cbx8 deficient mice are viable and display no apparent hematopoietic defects. Together, our findings demonstrate that CBX8 plays an essential role in MLL-AF9 transcriptional regulation and leukemogenesis.

INTRODUCTION

Mixed lineage leukemia (MLL), a human homolog of the Drosophila trithorax group (TrxG) protein, is a histone H3 lysine 4 specific methyltransferase commonly associated with transcriptional activation (Krivtsov and Armstrong, 2007; Nakamura et al., 2002). MLL is essential for both embryonic development and normal hematopoiesis, mainly through transcriptional regulation of the homeobox (HOX) gene family and their cofactors (Dou and Hess, 2008). Chromosome translocations at the MLL locus that generate oncogenic MLL fusion proteins are one of the major genetic lesions leading to acute leukemias. In total, MLL translocations account for up to 80% of infant leukemias and approximately 10% of adult acute leukemias with generally poor prognosis (Aplan, 2006; Muntean et al., 2010). To date, more than 50 different translocation partners have been identified, of which the most common ones are the transcriptional activators AF9, ENL and AF4 (Krivtsov and Armstrong, 2007; Monroe et al., 2010; Yokoyama et al., 2010).

It is well-established that constitutive activation of HOX genes, particularly HOXA9, is a key feature of MLL leukemia pathogenesis; however, the molecular mechanisms governing the aberrant HOX gene activation have not been completely deciphered (Sitwala et al., 2008; Yokoyama and Cleary, 2008). Extensive studies have been conducted to explore the functional significance of both the retained MLL portion and the translocation partners of MLL fusion proteins in transcriptional regulation. On the one hand, the amino-terminal portion of MLL has been shown to be required for the localization of MLL fusion proteins, due to its DNA-binding ability (Ayton et al., 2004; Slany et al., 1998) and the Menin-LEDGF association (Yokoyama and Cleary, 2008). Moreover, we and others have shown that the polymerase associated factor complex (PAFc), an important component of the basal transcriptional machinery, interacts with this region to facilitate transcriptional activation and leukemic transformation (Milne et al., 2010; Muntean et al., 2010; Tan et al., 2010). On the other hand, the mechanisms, by which the major fusion partners contribute to MLL-rearranged leukemogenesis, are beginning to be defined (Monroe et al., 2010). It has been reported that a complex of proteins termed ENL-associated proteins (EAPs), or a closely related complex named AEP for AF4 family/ENL family/P-TEFb complex, interacts with the major MLL fusion partners AF9, ENL and AF4 (Lin et al., 2010; Muntean et al., 2010; Yokoyama et al., 2010). The EAP complex includes not only the common MLL fusion partners but also the histone methyltransferase DOT1L and the P-TEFb complex (consisting of CDK9 and cyclin T1), positively regulating transcription elongation (Krivtsov et al., 2008; Mueller et al., 2007). Meanwhile, other investigators have described an H3K79 methyltransferase complex, DotCom, containing several frequent MLL fusion partners, including AF9, ENL and AF10, that plays a positive role in leukemogenesis (Mohan et al., 2010a). The components of these complexes partially overlap, suggesting the presence of separate complexes that contribute to MLL-rearranged leukemogenesis (Mohan et al., 2010b; Mueller et al., 2007). Interestingly, chromobox homolog 8 (CBX8), a Polycomb Group (PcG) protein generally associated with transcription repression, is also present in complexes recruited by MLL fusion proteins (Monroe et al., 2010; Mueller et al., 2007). However, the significance of this association has not been defined.

CBX8, also known as HPC3 (Human Polycomb 3), belongs to the CBX protein family (including CBX2, 4, 6, 7 and 8) that are homologs of the Drosophila Polycomb (Pc) protein (Kerppola, 2009). CBX8 was originally characterized as a transcriptional repressor, interacting with RING1a/b and associating with BMI1 in the polycomb repressive complex 1 (PRC1) (Bardos et al., 2000). A previous study has reported that as a PRC1 component, CBX8 represses the INK4a/ARF expression in fibroblasts (Dietrich et al., 2007). Further studies showed that several distinct PRC1 complexes colocalize and regulate the INK4a/ARF expression, suggesting that the INK4a/ARF locus is a general target for PRC1 complexes, rather than a CBX8-specific downstream target (Maertens et al., 2009). Therefore, the exact role of CBX8 in transcriptional regulation remains largely undefined. It has been reported that certain CBX proteins, such as CBX4, can associate with protein complexes other than PRC1, thereby playing a PRC1-independent role in transcriptional regulation (Kerppola, 2009). However, it remains unknown whether CBX8 has a PRC1-independent function and what its biological significance may be.

In the present study, we investigated the role of CBX8 in MLL-AF9-induced leukemogenesis and explored the underlying mechanisms in relation to its involvement in PRC1.

RESULTS

CBX8 Specifically Interacts with MLL-AF9 at the C-Terminal Domain (CTD)

Previous studies have reported that the MLL fusion partner AF9 directly interacts with CBX8 through the evolutionarily conserved CTD (Figure 1A) (Garcia-Cuellar et al., 2001; Hemenway et al., 2001; Monroe et al., 2010). However, whether this interaction is retained in MLL-AF9 fusion protein has not been defined. To address this question, we transiently co-expressed epitope-tagged MLL-AF9 and CBX8 in human embryonic kidney 293 cells, using a FLAG-tagged “empty” vector as a negative control. Specific interaction between CBX8 and MLL-AF9 was detected by immunoprecipitation (IP) experiments. When using AF9-conjugated agarose beads to pull down the full-length fusion protein, we consistently observed that CBX8 coprecipitated with MLL-AF9 (Figure 1B). To further characterize this interaction, we performed IP experiments in the presence of Benzonase. Using anti-FLAG antibody to pull down FLAG-tagged MLL-AF9, we detected endogenous CBX8 coprecipitating with the fusion protein, indicating that CBX8 interacts with MLL-AF9 in a DNA-independent manner (Figures 1C and S1A). Next, we characterized the critical CBX8 interaction sites on MLL-AF9, by generating 15 point mutants within the CTD through single amino acid substitution. By Co-IP experiments, we identified two point mutants (T542A and T554A) that specifically disrupt the CBX8 interaction (Figures 1A and 1D). This observation was further supported by reciprocal Co-IP experiments, using anti-FLAG or anti-Myc antibodies to pull down CBX8 or CxxC-AF9, respectively (Figures 1E and S1B), in which case CxxC-AF9, a previous characterized MLL-AF9 fragment, was used as a surrogate for the full-length fusion protein (Muntean et al., 2010).

Figure 1. CBX8 Specifically Interacts with MLL-AF9 at the C-terminal Domain (CTD).

Figure 1

Open in a new tab

(A) Schematic of full-length MLL-AF9. The amino acid sequence of the evolutionarily conserved CTD of AF9 is aligned with Drosophila, Rattus norvegicus and Mus musculus AF9 homologs. Red arrows indicate the evolutionarily-conserved threonine residues converted to alanine used below. (B) Co-immunoprecipitation of FLAG-tagged CBX8 with fMLL-AF9 (f-MA9). (C) Co-immunoprecipitation of endogenous CBX8 with f-MA9, after Benzonase treatment. (D) Co-immunoprecipitation of endogenous CBX8 with WT FLAG-MA9, but not with the mutants (T542A and T554A). (E) Co-immunoprecipitation of FLAG-CBX8 with WT Myc-CxxC-AF9, but not the mutants. (F) Co-immunoprecipitation of endogenous CDK9, CYCLIN T1 and AF5q31 with both WT Myc-CxxC-AF9 and the mutants. (G) Co-immunoprecipitation of both WT Myc-CxxC-AF9 and the mutants with HA-DOT1L. All of the Co-IP experiments included an epitope-tagged empty vector (FLAG-V or Myc-V) as a control and were performed in 293 cells. A fraction (3%) of cell lysate was used for input control. See also Figure S1.

Apart from CBX8, AF9 also associates either directly or indirectly with DOT1L, the P-TEFb complex (CDK9 and CYCLINT1) and AF5q31 (Monroe et al., 2010). Therefore, we asked whether the CBX8 interaction is required for interaction with any of these cofactors. To this end, we transiently transfected Myc-tagged CxxC-AF9 (WT or the mutants) in 293 cells and found that the P-TEFb complex (CDK9 and CYCLINT1) and AF5q31 coprecipitated with both the WT CxxC-AF9 fragment and the mutants (Figure 1F). Moreover, the interaction between DOT1L and CxxC-AF9 was also retained in the T542A and T554A mutants, as shown by reciprocal IP experiments using anti-FLAG or anti-HA antibodies to pull down CBX8 or DOT1L, respectively (Figures 1G and S1C). This observation was further confirmed by IP experiments in the context of full-length MLL-AF9 (Figure S1D). Together, our results showed that CBX8 specifically interacts with MLL-AF9 at the CTD, and that disrupting the CBX8 interaction does not affect the interaction with either P-TEFb or DOT1L, both of which are required for MLL-AF9-induced leukemogenesis.

CBX8 Is Essential for Both Initiation and Maintenance of MLL-AF9 Leukemic Transformation

To assess the importance of the CBX8 interaction in MLL-AF9-induced transformation, we first used bone marrow transformation (BMT) assays to examine the transformation ability of the MLL-AF9 mutants (T542A and T554A), which lack the CBX8 interaction. Briefly, Lin- hematopoietic cells derived from primary murine bone marrow (BM) were retrovirally transduced with either WT MLL-AF9 or the mutants, followed by three consecutive rounds of plating (Figure 2A). Despite the comparable expression of the fusion transcripts, as confirmed by real-time quantitative polymerase chain reaction (RT-PCR), the T542A and T554A mutations completely abolished myeloid transformation very early on, whereas the WT control potently transformed primary hematopoietic cells, forming a large number of colonies (Figures 2B and 2C). The tertiary colonies formed by WT MLL-AF9-transduced cells displayed a dense, compact morphology, indicative of immortalization. Wright Giemsa staining shows that these colonies are composed of myeloblasts (Figure 2D). In contrast, the MLL-AF9 mutant-transduced cells failed to form colonies in the second round of selection, and they were composed primarily of monocytes and macrophages (Figure 2D). To further confirm that Cbx8 is required for MLL-AF9-induced transformation, we transduced the MLL-AF9-transformed BM cells with either the control shRNA or a shRNA directed against Cbx8 after the third round plating, followed by puromycin selection (Figure S2A). Cbx8 expression, as measured by RT-qPCR, was effectively downregulated (Figure S2B), whereas the MLL-AF9 expression level was not significantly affected (p>0.05, Figure S2C). As expected, knockdown of Cbx8 significantly reduced the colony formation ability of MLL-AF9-transduced cells, compared to the control (p<0.01, Figures S2D and S2E). Together, these results suggest that the CBX8/MLL-AF9 interaction is required for MLL-AF9-mediated immortalization.

Figure 2. CBX8/MLL-AF9 Interaction Is Essential for MLL-AF9 Leukemic Transformation.

Figure 2

Open in a new tab

(A) Experimental scheme of the BMT assays evaluating the leukemic transformation ability of WT MLL-AF9 and MLL-AF9 mutants (T542A and T554A). (B) RT-qPCR analysis of the expression levels of WT MLL-AF9 and the mutants in Lin- BM after retroviral transduction. (C) Colony-forming units (CFU) per 20,000 plated cells in each round of plating in methylcellulose. Error bars represent ± standard deviation (SD) from three independent experiments. (D) Morphology of representative colonies from primary BM cells transduced with indicated constructs. The first row shows the representative colony morphology in methylcellulose. Scale bar, 500 μm. The second row shows the p-iodonitro tetrazolium violet (INT)-stained colonies after two rounds of plating. Dense red colonies are visible from WT MLL-AF9. The third row shows the Wright-Giemsa-stained cells isolated after two rounds of plating. Scale bar, 50 μm. See also Figure S2.

We then used a conditional Cbx8 knockout mouse model (generated by Dr. Koseki) to further assess the role of Cbx8 in initiation and maintenance of transformation by MLL-fusion proteins in vitro and in vivo (Figure 3A). Cbx8f/f mice were bred with Rosa26-Cre-ERT2 mice to generate Cbx8 conditional knockout mice. Treatment with 4-hydroxyltamoxifen (4-OHT) induced efficient Cbx8 excision in primary BM cells from Cbx8f/f; Cre+ mice (Figure 3B). To assess the role of Cbx8 in initiation and maintenance of MLL-AF9 leukemic transformation, we induced Cbx8 excision by 4-OHT treatment, simultaneously with MLL-AF9 transduction or after selecting MLL-AF9-transformed cells by three consecutive rounds of plating, respectively, with BM from Cbx8f/f; Cre− mice serving as a control (Figure 3C). The expression level of MLL-AF9 was not significantly altered by 4-OHT treatment in either of these experimental settings (Figures S3B and S3C). Strikingly, loss of Cbx8 completely abolished colony formation by MLL-AF9-transduced cells under both conditions (Figures 3D-3F and S3A). In contrast to the colonies formed by Cbx8f/f; Cre+ cells with the control treatment and the Cbx8f/f; Cre− control cells with or without 4-OHT treatment, which showed dense morphology and were composed predominantly of myeloblasts (Figure 3G; data not shown), Cbx8-depleted cells failed to form colonies and were composed of monocytes and macrophages (Figure 3G). Together, our results strongly indicate that Cbx8 is essential for both initiation and maintenance of MLL-AF9 leukemic transformation.

Figure 3. Cbx8 Is Required for Both Initiation and Maintenance of MLL-AF9 Leukemic Transformation.

Figure 3

Open in a new tab

(A) Schematic showing the floxed Cbx8 and the primers used for detecting the floxed and the excised Cbx8. (B) Genotype analysis showing the efficiency of Cbx8 excision induced by 4-OHT treatment in primary BM from Cbx8f/f; Cre+ mice, with ethanol treatment as a control (EtOH). (C) Experimental scheme for the BMT assays with Cbx8 excision in primary BM from Cbx8f/f; Cre+ and Cbx8f/f; Cre− mice. The first experimental procedure was performed as described in Figure 2A, except 4-OHT or ethanol was added during MLL-AF9 retroviral transduction. The second experiment was performed as described in Figure S2A, except using 4-OHT treatment instead of shCbx8 transduction. (D and E) Relative CFU of MLL-AF9-transduced cells in the two experimental settings. Error bars represent ± SD from two independent experiments. (F) Representative INT-stained colonies in methylcellulose. (G) Wright-Giemsa-stained cells isolated after transformation selection. Scale bar, 50 μm. (H) Kaplan-Meier survival analysis of mice transplanted with Cbx8Δ/Δ (n=5) or Cbx8f/f (n=5) donor BM (I) GFP expression of BM from the transplanted mice, assessed by flow cytometry. (J) Wright-Giemsa staining of peripheral blood smear and BM and histology of liver and spleen from the transplanted mice. See also Figure S3.

Given our findings with the BMT assay, an in vitro surrogate for assessing myeloid transformation ability (Cheung et al., 2007; Lavau et al., 1997; Smith et al., 2011), we then tested the role of Cbx8 in MLL-AF9 leukemogenesis in vivo. The MigR1-MLL-AF9 construct, which expresses both MLL-AF9 and GFP, was used to retrovirally transduce Lin- BM cells derived from the Cbx8f/f; Cre+ mice, in the presence or absence of 4-OHT. These cells were then transplanted into syngeneic mice for accessing their leukemogenic potential. Complete Cbx8 excision in the donor cells was achieved by 4-OHT treatment, as confirmed by genotyping the peripheral blood of the recipient mice three weeks post transplant (Figure S3M). Consistent with our in vitro findings, mice receiving Cbx8-deficient, MLL-AF9-transduced cells failed to develop leukemia, whereas mice receiving WT MLL-AF9-transduced BM all died from leukemia, as evidenced by marked splenomegaly and extensive infiltration of peripheral blood, spleen and liver (Figures 3H, S3N and 3J). As expected, flow cytometry analysis showed that BM from the leukemic mice was replaced by GFP-positive, MLL-AF9-transformed cells (>99%). In contrast, BM from the mice receiving Cbx8-depleted donor cells was negative for GFP expression (Figure 3I). These results strongly demonstrate that CBX8 is required for MLL-AF9-induced leukemogenesis.

Notable, a previous study has shown that CBX8 also interacts with another MLL fusion partner, ENL, which is also a component of the EAP (or the related AEP) complex (Garcia-Cuellar et al., 2001). Therefore, it is likely that CBX8 is not only required for MLL-AF9 leukemogenesis but also involved in leukemic transformation by other MLL fusion proteins that interact with the EAP (or the related AEP and the Dotcom) complex, such as MLL-ENL. Indeed, similar to MLL-AF9, Cbx8 is essential for initiation and maintenance of leukemic transformation induced by MLL-ENL, as shown by BMT assays (Figures S3D–S3G). This finding suggests that the dependence on CBX8 of leukemic transformation is not restricted to MLL-AF9 but may apply to other MLL fusion proteins as well.

CBX8 Is Crucial for Proliferation and Survival of MLL-AF9-transformed Leukemic Cells and for MLL-AF9-Induced Transcriptional Activation

To explore the underlying mechanisms of Cbx8-dependent oncogenic transformation, we first investigated whether the Cbx8 dependence is specific for certain MLL-rearranged transformation or for leukemic transformation in general. Using the conditional Cbx8 knockout mice, we assessed the impact of Cbx8 deletion on leukemic transformation by E2A-HLF, a leukemogenic fusion protein that transforms through Hox-independent pathways (Ayton and Cleary, 2003). Despite the complete depletion of the Cbx8 protein achieved by 4-OHT treatment, neither the initiation nor the maintenance of E2A-HLF-induced leukemic transformation was affected, suggesting the specificity of Cbx8-dependent transformation (Figures S3H–S3L). Similar results were observed with Hoxa9/Meis1-transformed cells (data not shown). Together, these findings suggest that Cbx8 plays a specific role in leukemic transformation by certain MLL fusion proteins, such as MLL-AF9.

We then examined whether Cbx8 is important in regulating the proliferation of MLL-AF9 leukemic cells and found that the Cbx8 shRNA, but not the scrambled control, decreased the growth rate of MLL-AF9 leukemic cells (Figure 4A). The phenotype was even more dramatic in primary murine BM cells, where we observed a complete growth arrest in liquid cultured primary BM cells (Cbx8f/f; Cre+) with Cbx8 excision by 4-OHT treatment, whereas no such effect was observed in control cells (Cbx8f/f; Cre−) (Figures 4B and 4C). In agreement with these observations, the apoptotic population of MLL-AF9 leukemic cells increased upon Cbx8 depletion by 4-OHT treatment, but not in the control cells (Figure S4A). Additionally, we consistently observed a slight decrease of the S-phase cell population upon Cbx8 depletion in MLL-AF9 leukemic cells (Figure S4B). However, the effect was rather minor, suggesting that the dramatic proliferation defect of MLL-AF9 cells upon Cbx8 depletion is not mainly due to cell cycle arrest.

Figure 4. CBX8 Is Crucial for Proliferation and Survival of MLL-AF9-transformed Leukemic Cells and for MLL-AF9-Induced Transcriptional Activation.

Figure 4

Open in a new tab

(A) Growth curve of MLL-AF9 leukemic cells transduced with shCbx8 or control (shScram). Error bars represent ± SD from duplicate experiments. Results from one of three independent experiments are shown. (B and C) Growth curves of MLL-AF9-transformed primary BM from Cbx8f/f; Cre+ and Cbx8f/f; Cre− mice, with 4-OHT treatment compared to the control. Error bars represent ± SD from a duplicate experiment. Results from one of two independent experiments are shown. (D) RT-PCR analysis of the expression of Cbx8 and Hoxa9 in MLL-AF9-transformed primary BM, with shCbx8 transduction compared to the control (shScram). (E) RT-qPCR analysis of the expression of Cbx8 and Hoxa9 in MLL-AF9-transformed primary BM from Cbx8f/f; Cre+ mice, with 4-OHT treatment compared to the control (EtOH). (F) RT-PCR analysis of the Hoxa9 expression in primary BM transduced by WT MLL-AF9 or MLL-AF9 mutants, compared to the vector control. (G) Luciferase assay with a Hoxa9 promoter-driven reporter activated by WT or mutant MLL-AF9 (T542A and T554A) in 293 cells. Error bars represent ± SD from three independent experiments. (H) Luciferase assay with the Hoxa9 promoter-driven reporter activated by MLL-AF9, with CBX8 knockdown (siCBX8) or control treatment (siScram) in HeLa cells. Error bars represent ± SD from three independent experiments. (I) Western blot showing CBX8 expression with siCBX8 treatment compared to the control (siScram). (J–L) Relative binding of Cbx8, RNAP II and MLL-AF9 together with WT AF9 to the Hoxa9 promoter in MLL-AF9-transformed cells from Cbx8f/f; Cre+ mice, with 4-OHT treatment compared to the control (EtOH). See also Figure S4.

A well-established oncogenic mechanism of MLL-AF9 transformation is the constitutive activation of the HOX genes, particularly HOXA9 along with the HOX cofactor MEIS1 (Armstrong et al., 2002; Ayton and Cleary, 2003; Kumar et al., 2004), whereas CBX8 was previously shown to be involved in transcriptional repression (Dietrich et al., 2007; Maertens et al., 2009). The seemingly opposite effects of CBX8 and MLL-AF9 on transcriptional regulation raise an intriguing question: what role does CBX8 play in MLL-AF9-induced transcriptional activation? To address this question, we examined Hoxa9 expression in MLL-AF9-transformed primary BM transduced with the Cbx8 shRNA. Compared to the control, Cbx8 downregulation led to a marked suppression of Hoxa9 expression (Figure 4D). A similar effect was observed in MLL-AF9-transformed Cbx8f/f; Cre+ BM, following Cbx8 excision by 4-OHT treatment, but not in the control cells (Figures 4E and S4C). To further confirm that the impact of Cbx8 on Hoxa9 expression is dependent on the interaction between Cbx8 and MLL-AF9, we compared the Hoxa9 expression in primary BM cells transduced by WT MLL-AF9 or by the mutants lacking the Cbx8 interaction (T542A and T554A). Notably, MLL-AF9 mutant-transduced cells show significantly reduced Hoxa9 expression, compared to the cells transduced by WT MLL-AF9 (Figure 4F). It is noteworthy that the cells examined in this experiment were harvested after the second round of selection because very few mutant-transformed cells survived the third round of selection. Therefore, few residual non-transformed progenitors may account for the detected Hoxa9 expression in the mutant-transformed cells, suggesting that the reduction of Hoxa9 expression in the mutant-transduced cells could be even greater. Nevertheless, these data strongly indicate that Cbx8 serves as a co-activator of MLL-AF9, promoting Hoxa9 upregulation in MLL-AF9-transformed cells. To further assess the specificity of the role of Cbx8 in Hoxa9 transcriptional regulation, we examined the effect of Cbx8 knockdown on Hoxa9 expression in several human and murine leukemic cell lines. CBX8 inducible knockdown stable cell lines were generated by lentiviral transduction of a TRIPZ-RFP-shCBX8 construct in three human leukemic cell lines. The THP-1 and Mono Mac 6 (MM 6) cells are transformed by MLL-AF9, whereas K562 is a BCR-ABL-transformed cell line that serves as a control. As expected, knocking down of CBX8 induced by doxycycline treatment significantly decreased HOXA9 expression in both MLL-AF9-transformed cell lines (MM 6 and THP-1), but not in the control cell line (Figure S4D). Consistent with this observation, Cbx8 knockdown by shRNA led to a marked decrease of Hoxa9 expression in a murine MLL-AF9 cell line, but not in the Hoxa9-independent E2A-HLF cell line (Figure S4E). These findings suggest that Cbx8 specifically contributes to MLL-AF9-induced Hoxa9 transcriptional activation.

In order to mechanistically understand how Cbx8 facilitates MLL-AF9-induced Hoxa9 upregulation, we investigated the effect of Cbx8 on Hoxa9 promoter activity in the presence of MLL-AF9. We first performed dual luciferase assays in 293 cells transfected with a MLL-AF9 responsive luciferase construct, under the control of the murine Hoxa9 promoter (Hoxa9-LUC). Our data show that disrupting the CBX8 interaction by the point mutation of T542A or T554A significantly decreased the activation of the Hoxa9 promoter by MLL-AF9 (T542A: p<0.01, T554A: p<0.01; Figures 4G). Consistent with this result, knocking down the CBX8 level by siRNAs reduced the MLL-AF9 induced transcriptional activation by around 50% (p<0.01, Figures 4H and 4I). Notably, neither the point mutations nor CBX8 knockdown significantly affected MLL-AF9 expression, indicating that the reduction in Hoxa9 promoter activity was not due to a general decrease in the MLL-AF9 level (Figures S4F and S4G). A similar response was observed using another MLL-AF9 responsive luciferase reporter containing the thymidine kinase promoter and multimerized Myc E box, further supporting the importance of Cbx8 in MLL-AF9-induced transcriptional activation (Figures S4H and S4I). We then carried out chromatin immunoprecipitation (ChIP) in MLL-AF9-transformed murine hematopoietic cells, to examine changes at the Hoxa9 promoter in response to Cbx8 depletion. In agreement with the suppression of Hoxa9 activation, a significant decrease of RNA polymerase II (RNAP II) binding to the Hoxa9 promoter was detected following Cbx8 depletion by 4-OHT treatment, while as expected, Cbx8 binding was essentially ablated (Figures 4J and 4K). Moreover, the collective binding of MLL-AF9 fusion protein and WT AF9 was not affected by Cbx8 depletion, as shown by ChIP using an anti-AF9 antibody (Figure 4L). Because WT AF9 is also a component of the MLL-AF9 complex, and our previous results already showed that the Cbx8 interaction is not required for the assembly between the EAP complex and the MLL-AF9 fusion protein (Figures 1F, 1G, S1C and S1D), this observation suggest that the recruitment of the MLL-AF9 complex to the Hoxa9 promoter is not significantly affected by the loss of Cbx8, which is also consistent with previous reports regarding the importance of the retaining MLL portion in MLL fusion complex localization, rather than the fusion partner portion (Ayton et al., 2004; Milne et al., 2010; Muntean et al., 2010; Slany et al., 1998; Yokoyama and Cleary, 2008). Similar findings were observed using CBX8 inducible knockdown MLL-AF9-transformed cell lines (Figures S4J–S4M), further supporting that Cbx8 regulates MLL-AF9 target promoter activity, thereby contributing to MLL-AF9-induced transcriptional activation, without affecting the collective localization of MLL-AF9 and WT AF9.

Role of CBX8 in MLL-AF9 Leukemic Transformation and Transcriptional Activation Is Independent of PRC1

To date, the only reported functional characterization of Cbx8 is its role as a transcriptional repressor in PRC1, whereas our data indicate that Cbx8 serves as a transcriptional coactivator in the presence of MLL-AF9. These opposing transcriptional regulatory roles suggest that Cbx8 functions in a PRC1-independent manner in MLL-AF9 leukemic transformation. It has been shown that Ring1b, another PRC1 component, is required for the stability of PRC1 complexes (Leeb and Wutz, 2007; van der Stoop et al., 2008). In addition, previous studies have indicated that Ring1b also interacts with AF9 (Monroe et al., 2010), which we confirmed by IP experiments showing that endogenous RING1b consistently coprecipitates with the MLL-AF9 fragment, CxxC-AF9 (Figure 5A). Therefore, to test our hypothesis of the potential PRC1-independent function of Cbx8, we first assessed the impact of Ring1b on MLL-AF9 leukemic transformation by BMT assays. Two individual shRNA molecules specifically targeting Ring1b were used to effectively knock down Ring1b expression in MLL-AF9-transformed leukemic cells. Reduction in Ring1b expression in these experiments did not impair the transformation ability of MLL-AF9 (Figures 5B, 5C and 5F). Knocking down Ring1b did not significantly affect the growth rate or Hoxa9 expression in MLL-AF9 cells either (Figures 5D and 5E). We also performed dual luciferase assays to examine the impact of knocking down RING1b by siRNA on the MLL-AF9 target promoter activity. Despite the marked reduction of RING1b expression shown by western-blot analysis, MLL-AF9-induced transactivation of the target promoters was not suppressed by RING1b knockdown (Figures 5G–5I). Similar to our observations with Ring1b, knockdown of Bmi1, another core PRC1 component, did not affect the transformation ability, growth rate or transcriptional activation in MLL-AF9 leukemic cells (Figures S5B–S5I). Consistent with these observations, Cbx8 depletion in MLL-AF9-transformed BM cells did not affect the global levels of Ring1b and Bmi1, as shown by western-blot analysis (Figure S5A). Taken together, these findings suggest that Cbx8 functions as an MLL-AF9 cofactor to promote leukemogenesis in a PRC1-independent manner.

Figure 5. Ring1b Knockdown Does not Recapitulate the Effects of Cbx8 Knockdown in MLL-AF9 Leukemic Transformation.

Figure 5

Open in a new tab

(A) Co-immunoprecipitation of endogenous RING1b with Myc-CxxC-AF9 in 293 cells. A fraction (3%) of cell lysate was used for input control. (B) Relative CFU of MLL-AF9 leukemic cells with Ring1b knockdown by two individual shRing1b molecules, compared to the control (shScram). Error bars represent ± SD from three independent experiments. (C) Representative INT-stained colonies in methylcellulose. (D) Growth curve of MLL-AF9 leukemic cells with Ring1b knockdown, compared to the control. Error bars represent ± SD from a duplicate experiment. Results from one of three independent experiments are shown. (E) RT-PCR analysis of Hoxa9 expression in MLL-AF9 leukemic cells with Ring1b knockdown, compared to the control. (F) RT-PCR analysis of Ring1b expression in MLL-AF9 leukemic cells, confirming the knockdown efficiency. (G and H) Experiments were performed as described in Figures 4H and S4I, except using siRNAs specifically targeting RING1b (siRING1b) in place of siCBX8. Error bars represent ± SD from three independent experiments. (I) Western blot showing RING1b expression with siRING1b treatment, compared to the control (siScram). See also Figure S5.

Notably, given the finding that CBX8 is involved in the INK4a/ARF transcriptional repression by PRC1 in fibroblasts (Dietrich et al., 2007), it is important to determine whether the observed effect of CBX8 on MLL-AF9 transcriptional activation is related to its role in INK4a/ARF regulation. Therefore, we examined the impact of Cbx8 on Ink4a/Arf expression by RT-PCR in MLL-AF9 leukemic cells. Neither downregulation nor depletion of Cbx8 led to Ink4a/Arf activation (Figures S8A and S8B), confirming that the Cbx8-dependent MLL-AF9 transformation is not due to Ink4a/Arf repression.

CBX8 Regulation of TIP60 Localization Contributes to MLL-AF9 Leukemic Transformation

The characterization of the PRC1 independence of Cbx8 functions in MLL-AF9 leukemic transformation prompted us to explore the possible involvement of other Cbx8 interacting proteins that may explain the role of Cbx8 in transcriptional activation. A previous study has reported that CBX8 directly interacts with the histone acetyltransferase HIV Tat-interacting protein of 60 kDa (TIP60) by high-throughput yeast two-hybrid screens and mass spectroscopy analysis (Stelzl et al., 2005). However, this observation has not yet been verified in any mammalian cell system; therefore, the functional implication of this interaction remains an open question. To first confirm this interaction, we transiently expressed FLAG-tagged CBX8 in 293 cells. Specific interaction between CBX8 and TIP60 was detected by IP experiments: using an anti-FLAG antibody to pull down CBX8, we observed that CBX8 consistently coprecipitated with endogenous TIP60 in the presence of Benzonase, indicating CBX8 interacts with TIP60 in a DNA-independent manner (Figures 6A and S6A). This finding implied an intriguing possibility that CBX8 promotes MLL-AF9 leukemic transformation, at least partially through its interaction with the transcriptional coactivator TIP60. To test this hypothesis, we first assessed the impact of Tip60 on MLL-AF9 leukemic transformation by BMT assays. Using shRNA molecules specifically targeting Tip60, we observed a reduction in the colony formation ability of MLL-AF9 leukemic cells (Figures 6B, 6C and 6F). Moreover, Tip60 downregulation by shRNA led to a decrease in the growth rate and Hoxa9 expression of MLL-AF9-transformed cells (Figures 6D and 6E). Similar results were obtained using a different shRNA pool, further supporting that Tip60 positively contributes to MLL-AF9 leukemic transformation (Figures S6B–S6F).

Figure 6. CBX8 Regulates the Localization of TIP60, Whose Downregulation Phenocopies the Effects of Cbx8 Knockdown in MLL-AF9 Leukemic Transformation.

Figure 6

Open in a new tab

(A) Co-immunoprecipitation of endogenous TIP60 with FLAG-CBX8 in 293 cells, after Benzonase treatment. A fraction (3%) of cell lysate was used for input control. (B) Relative CFU of MLL-AF9 leukemic cells with Tip60 knockdown by shRNA, compared to the control (shScram). Error bars represent ± SD from three independent experiments. (C) Representative INT-stained colonies in methylcellulose. (D) Growth curve of MLL-AF9 leukemic cells with Tip60 knockdown, compared to the control. Error bars represent ± SD from a duplicate experiment. Results from one of three independent experiments are shown. (E) RT-PCR analysis of Hoxa9 expression in MLL-AF9 leukemic cells with Tip60 knockdown, compared to the control. (F) RT-PCR analysis of Tip60 expression in MLL-AF9 leukemic cells, showing the knockdown efficiency. (G and H) Experiments were performed as described in Figures 4H and S4I, except using siRNAs specifically targeting TIP60 (siTIP60) instead of siCBX8. Error bars represent ± SD from three independent experiments. (I) Relative binding of Tip60 to the Hoxa9 promoter in MLL-AF9-transformed cells from Cbx8f/f; Cre+ mice, with Cbx8 excision induced by 4-OHT treatment compared to the control (EtOH). See also Figure S6.

To further characterize the role of Tip60 in MLL-AF9-induced transcriptional activation, we performed dual luciferase assays to examine the impact of TIP60 downregulation by siRNA on the MLL-AF9 target promoter activity. A significant reduction of TIP60 expression was confirmed by western-blot analysis (Figures S6G). As expected, the MLL-AF9-induced transcriptional activation of the target promoters was significantly suppressed by TIP60 knockdown (Figures 6G and 6H). Collectively, these data demonstrate that knocking down Tip60 phenocopies the effect of Cbx8 knockdown in MLL-AF9 leukemic cells, suggesting a functional significance of the CBX8/TIP60 interaction in MLL-AF9 leukemic transformation. To confirm the observed role of Tip60 is indeed associated to the CBX8/TIP60 interaction, we performed ChIP assays in MLL-AF9-transformed leukemic cells, following Cbx8 depletion by 4-OHT treatment. As expected, Cbx8 depletion resulted in decreased Tip60 binding at the Hoxa9 promoter (Figure 6I). Similar findings were seen using MLL-AF9-transformed cell lines, MM6 and THP-1, engineered for inducible knockdown of CBX8. In these cells, TIP60 binding at the Hoxa9 promoter was reduced upon CBX8 downregulation induced by doxycycline treatment (Figure S6I, data not shown). Together, our results suggest that CBX8 regulates the localization of TIP60, which plays a positive role in MLL-AF9 leukemic transformation. Of note, Tip60 downregulation in MLL-AF9 leukemic cells did not lead to Ink4a/Arf activation (Figures S8C–S8D), consistent with previous observations from Cbx8 knockdown, further supporting that Ink4a/Arf repression does not account for the role of Cbx8 in MLL-AF9 leukemogenesis.

Cbx8 Is not Required for Normal Hematopoiesis

The profound impact of Cbx8 on MLL-AF9 leukemogenesis prompted us to examine the role of Cbx8 in normal hematopoiesis. We first examined the effect of Cbx8 depletion on hematopoietic steady-state conditions in vivo. Constitutive depletion of Cbx8 showed no aberrant phenotype, and deletion of Cbx8 by 4-OHT treatment in adult animals had no detectable effect on any measured peripheral blood population as measured by complete blood count (CBC) analysis (Figures 7A-7D and S7A). Moreover, both the cellularity of major hematopoietic organs (BM, spleen and thymus) and the cell numbers of mature hematopoietic populations as defined by flow cytometry were similar between Cbx8-deficient mice and controls (Figures 7E-7H and S7C–S7E). To address potential effects of Cbx8 deletion on primitive long-term hematopoietic stem cells (LT-HSCs), we combined flow cytometry for characterization of progenitor populations and competitive BM transplantation assays. These analyses revealed no detectable differences in LT-HSC numbers (Figure 7I) or hematopoietic reconstitution ability of Cbx8 WT or deficient BM in lethally irradiated recipients (Figure 7K). In addition, the total progenitor output from the BM of Cbx8-deficient animals was similar to controls, as measure by colony forming assays (Figures 7J and S7B). Together, these findings indicate that Cbx8 is not required for steady state hematopoiesis, LT-HSC maintenance, or stem and progenitor cell function.

Figure 7. Cbx8-Depleted Mice Shows Normal Hematopoiesis.

Figure 7

Open in a new tab

(A–D) Peripheral blood CBC analysis of (A) platelets, (B) white blood cells, (C) red blood cells, and (D) hemoglobin content of Cbx8 floxed (f/f) and deleted (Δ/Δ) mice. The experiment was performed four weeks after complete Cbx8 excision in vivo. (E–H) Absolute quantification of total bone marrow cellularity (E), erythroid cells (Ter119+; F), developing B lymphocytes (AA4.1+CD19+B220+; G), and myeloid cells (CD11b+Gr1+; H) from Cbx8f/f and Cbx&Delta;/Δ, analyzed by flow cytometry (bars represent mean + SD; n=5 mice/genotype). (I) Flow cytometric analysis of LT-HSCs (CD150+CD48-LSK; I) from Cbx8f/f and Cbx8Delta;/Δ mice (bars represent mean + SD; n=5 mice/genotype). (J) Colony forming assays using BM from Cbx8f/f and Cbx8 Delta;/Δ mice. The data included 4 independent experiments per genotype (bars represent mean + SD; n=3 plate/experiment). (K) Competitive BM transplantation at competitor: tester ratios of 1:1 (n=10/group), 1:3 (n=5 mice per group), and 1:9 (n=5 mice per group). No difference between Cbx8f/f and Cbx8 Delta;/Δ mice was observed in tester contribution to myeloid reconstitution at any ratio (data represent mean ± SD). (E–K) were performed 5–7 months after complete Cbx8 excision in vivo. See also Figure S7.

DISCUSSION

Our study establishes CBX8 as an essential cofactor required for MLL-AF9-induced transcriptional activation and leukemic transformation (Figure 8). CBX8 is one of the five human homologs of the Drosophila Pc protein. Although all CBX proteins share highly conserved chromodomains and Pc boxes, their different sizes and the presence of other motifs suggest potentially different functions (Whitcomb et al., 2007). Indeed, previous studies have reported that mice deficient for different PRC1 components show only a partial overlap in phenotype, raising the possibility of PRC1-independent functions of these components that may be context dependent and involve other protein complexes (de Napoles et al., 2004; Katoh-Fukui et al., 1998; Leeb and Wutz, 2007; Suzuki et al., 2002; Voncken et al., 2003). The present study has uncovered such a PRC1-independent function of CBX8 in MLL-AF9-induced transcriptional regulation. Interestingly, contrary to its role as a transcriptional repressor in PRC1, CBX8 serves as a transcriptional coactivator in the MLL-AF9 complex. Furthermore, CBX8 is present in the EAP (or the related AEP and the Dotcom) transcriptional activation complex (Mohan et al., 2010b; Monroe et al., 2010; Yokoyama et al., 2010) and is also required for the leukemic transformation induced by MLL-ENL, another EAP-interacting MLL fusion protein, implying a broader role of CBX8 in MLL-rearranged leukemogenesis, which warrants further exploration. Importantly, consistent with our observations, a recent study showed that Bmi1 is not required for MLL-AF9-induced leukemogenesis (Smith et al., 2011). Moreover, our findings that neither Cbx8 downregulation or depletion nor Tip60 knockdown induced Ink4a/Arf expression further support that the role of CBX8 in MLL-AF9 leukemogenesis is independent of PRC1.

Figure 8. Schematic Model Illustrating Role of CBX8 in Promoting MLL-AF9-Induced Leukemogenesis.

Figure 8

Open in a new tab

Top: recruitment of WT MLL is required for transcriptional regulation of Hox gene expression in HSCs and early progenitor cells. Left: During normal hematopoiesis, Hox gene expression decreases due to the transcriptional repression of Polycomb Group proteins. Right: In MLL-AF9 leukemic cells, CBX8 interacts with MLL-AF9 at the target gene loci to facilitate transcriptional activation, possibly by recruiting transcriptional cofactors such as TIP60, thereby promoting leukemogenesis. See also Figure S8.

In addition to the MLL fusion partners, CBX8 has previously been shown to directly interact with the HAT TIP60 (Stelzl et al., 2005). Tip60 is a member of the MYST (Moz, Ybf2/Sas3, Sas2, Tip60) protein family, the largest family of HATs that are present in all eukaryotes (Voss and Thomas, 2009). Histone acetylation near promoters is associated with transcriptional activation; therefore, HATs generally promote transcriptional activation (Mills, 2010). Several TrxG complexes are known to recruit HATs during normal development. For example, the HAT MYST1 has been purified in WT MLL complex, and the HAT CREB-binding protein (CBP) is known to interacts with both MLL and another TrxG protein ASH1 (Bantignies et al., 2000; Dou et al., 2005; Ernst et al., 2001; Petruk et al., 2001). Under normal physiological conditions, HATs function as a coactivator to facilitate TrxG-induced transcriptional activation, antagonizing the transcriptional repressive effect of the PcG complex (Mills, 2010; Pasini et al., 2010). This mechanism contributes to the active HOXA9 expression in hematopoietic stem cells and early progenitors (Figure 8). Moreover, MLL is fused to CBP or P300 in a subset of acute leukemias (Wang et al., 2005). However, previous studies have not reported HATs as a component of the EAP complex, which is recruited by the most common MLL fusion partners. Therefore, whether HATs contribute to transcriptional activation induced by common MLL-rearranged oncoproteins remains unknown. Our finding of the CBX8-dependent TIP60 localization at the HOXA9 promoter indicates that this transcriptional activation mechanism is likely to be adopted by MLL fusion proteins to activate target gene expression, such as HOXA9. Interestingly, a previous RNAi screening study in mouse embryonic stem cells (ESCs) showed that Tip60 is required for pluripotency, while MLL myeloid leukemia stem cells have been shown to share the transcriptional program with ESCs, rather than adult stem cells (Fazzio et al., 2008; Somervaille et al., 2009). Together, these observations suggest a possible functional association between the TIP60-regulated signaling network and the transcriptional program in MLL-rearranged leukemic cells, raising the possibility that TIP60 may be involved in establishing the transcriptional program required for MLL-AF9-induced leukemogenesis (Figure 8). Given the broad involvement of TIP60 in multiple biological processes (Sapountzi et al., 2006), it is unclear whether the role of TIP60 in MLL-AF9 leukemogenesis is as specific as that of CBX8. This mechanism warrants further investigation. Finally, our identification of the critical role of the CBX8/MLL-AF9 interaction in leukemogenesis but not viability or normal hematopoiesis suggest that developing small molecule inhibitors targeting CBX8 represents a promising therapeutic strategy for MLL-rearranged leukemias.

EXPERIMENTAL PROCEDURES

Cell Culture and Animal Use

HeLa and 293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and non-essential amino acids. MLL-AF9, MLL-ENL and E2A-HLF cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 15% fetal calf serum (FCS, Stem Cell Technologies). THP-1, Mono Mac 6 (MM6), and K562 cells were cultured in RPMI-1640 medium supplemented with 10% FBS. The Tripz-RFP-shCBX8 expression was induced by 0.5 μg/ml doxycycline. Full details of conditional gene targeting of Cbx8 and analysis of Cbx8Delta;/Δ embryos will be provided in a subsequent manuscript (H.K., unpublished). All animal experiments in this study were approved by the University of Michigan Committee on Use and Care of Animals and Unit for Laboratory Animal Medicine (ULAM).

In Vivo Leukemogenesis Assays

Lin- BM was isolated from 6 to 8-week-old mice (Cbx8f/f; Cre+) injected with 5-fluorouacil (see Supplemental Information). The harvested Lin- BM cells were retrovirally transduced with MigR1-MLL-AF9 by two rounds of spinoculation in the presence of either 4-OHT (100 nM) or ethanol as a control. The cells were then counted and injected intravenously through the tail vein to cohorts of lethally irradiated (900 rads) C57BL/6 mice (3.5 × 104 cells per injection). Recipient mice were maintained on antibiotics for 2 weeks after transplantation.

Complete Blood Count Analysis

For in vivo Cbx8 excision, Cbx8f/f; Cre+ mice were treated with corn oil or 50 mg/kg 4-OHT by i.p. injection for five continuous days. Four weeks after injection, peripheral blood was used harvested in EDTA (ethylenediaminetetraacetic acid)-containing Microtainer tubes (BD Biosciences) and subjected for analysis performed by the ULAM laboratory. Meanwhile, genotyping was performed using genomic DNA extracted from peripheral blood.

Flow Cytometry and Cell Sorting

After blocking non-specific binding with unlabeled rat plus mouse IgG (Sigma-Aldrich), cells were stained on ice in PBS plus 4% fetal calf serum (FCS) and sorted on FACSAria (BD Biosciences). Analysis was performed on LSR II, FACSCanto, or FACSAria (BD Biosciences). Files were analyzed in FlowJo (TreeStar).

Statistical Analysis

Statistical significance was determined by Student’s t test using the Excel software (Microsoft 2007); p< 0.05 was considered statistically significant.

Supplementary Material

01

Highlights.

  • CBX8 is essential for both initiation and maintenance of MLL-AF9 transformation

  • CBX8 is crucial for MLL-AF9-induced transcriptional activation

  • Role of CBX8 in MLL-AF9 leukemogenesis is Independent of PRC1

  • CBX8 regulation of TIP60 localization contributes to MLL-AF9 transformation

SIGNIFICANCE.

MLL translocations that generate MLL-rearranged oncoproteins are a common cause of human acute leukemias. Although aberrant target gene activation is known as the primary driver of MLL-rearranged leukemogenesis, the underlying mechanisms remain poorly understood. Here, we demonstrate that chromobox homolog 8 (CBX8), a previously characterized transcription repressor, is a crucial cofactor required for MLL-AF9-induced leukemogenesis. Contrary to its role in the polycomb repressive complex 1, CBX8 facilitates the transcriptional activation of MLL-AF9 target genes, possibly through regulating the recruitment of the histone acetyltransferase TIP60. Strikingly, despite its essential role in MLL-AF9 leukemic transformation, CBX8 appears dispensable for normal hematopoiesis. Our findings suggest that disrupting the interaction between CBX8 and MLL-AF9 may be an effective therapeutic strategy in MLL-rearranged leukemias.

Acknowledgments

We thank Ann Friedman and Julien Schira for technical assistance. J.L.H. is supported by the National Institutes of Health (R01-CA92251) and by a SCOR grant from the Leukemia and Lymphoma Society (Jonathan Licht: PI).

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Aplan PD. Chromosomal translocations involving the MLL gene: molecular mechanisms. DNA Repair (Amst) 2006;5:1265–1272. doi: 10.1016/j.dnarep.2006.05.034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Armstrong SA, Staunton JE, Silverman LB, Pieters R, den Boer ML, Minden MD, Sallan SE, Lander ES, Golub TR, Korsmeyer SJ. MLL translocations specify a distinct gene expression profile that distinguishes a unique leukemia. Nat Genet. 2002;30:41–47. doi: 10.1038/ng765. [DOI] [PubMed] [Google Scholar]
  3. Ayton PM, Chen EH, Cleary ML. Binding to nonmethylated CpG DNA is essential for target recognition, transactivation, and myeloid transformation by an MLL oncoprotein. Mol Cell Biol. 2004;24:10470–10478. doi: 10.1128/MCB.24.23.10470-10478.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ayton PM, Cleary ML. Transformation of myeloid progenitors by MLL oncoproteins is dependent on Hoxa7 and Hoxa9. Genes Dev. 2003;17:2298–2307. doi: 10.1101/gad.1111603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bantignies F, Goodman RH, Smolik SM. Functional interaction between the coactivator Drosophila CREB-binding protein and ASH1, a member of the trithorax group of chromatin modifiers. Mol Cell Biol. 2000;20:9317–9330. doi: 10.1128/mcb.20.24.9317-9330.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bardos JI, Saurin AJ, Tissot C, Duprez E, Freemont PS. HPC3 is a new human polycomb orthologue that interacts and associates with RING1 and Bmi1 and has transcriptional repression properties. J Biol Chem. 2000;275:28785–28792. doi: 10.1074/jbc.M001835200. [DOI] [PubMed] [Google Scholar]
  7. Cheung N, Chan LC, Thompson A, Cleary ML, So CW. Protein arginine-methyltransferase-dependent oncogenesis. Nat Cell Biol. 2007;9:1208–1215. doi: 10.1038/ncb1642. [DOI] [PubMed] [Google Scholar]
  8. de Napoles M, Mermoud JE, Wakao R, Tang YA, Endoh M, Appanah R, Nesterova TB, Silva J, Otte AP, Vidal M, et al. Polycomb group proteins Ring1A/B link ubiquitylation of histone H2A to heritable gene silencing and X inactivation. Dev Cell. 2004;7:663–676. doi: 10.1016/j.devcel.2004.10.005. [DOI] [PubMed] [Google Scholar]
  9. Dietrich N, Bracken AP, Trinh E, Schjerling CK, Koseki H, Rappsilber J, Helin K, Hansen KH. Bypass of senescence by the polycomb group protein CBX8 through direct binding to the INK4A-ARF locus. EMBO J. 2007;26:1637–1648. doi: 10.1038/sj.emboj.7601632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dou Y, Hess JL. Mechanisms of transcriptional regulation by MLL and its disruption in acute leukemia. Int J Hematol. 2008;87:10–18. doi: 10.1007/s12185-007-0009-8. [DOI] [PubMed] [Google Scholar]
  11. Dou Y, Milne TA, Tackett AJ, Smith ER, Fukuda A, Wysocka J, Allis CD, Chait BT, Hess JL, Roeder RG. Physical association and coordinate function of the H3 K4 methyltransferase MLL1 and the H4 K16 acetyltransferase MOF. Cell. 2005;121:873–885. doi: 10.1016/j.cell.2005.04.031. [DOI] [PubMed] [Google Scholar]
  12. Ernst P, Wang J, Huang M, Goodman RH, Korsmeyer SJ. MLL and CREB bind cooperatively to the nuclear coactivator CREB-binding protein. Mol Cell Biol. 2001;21:2249–2258. doi: 10.1128/MCB.21.7.2249-2258.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fazzio TG, Huff JT, Panning B. An RNAi screen of chromatin proteins identifies Tip60-p400 as a regulator of embryonic stem cell identity. Cell. 2008;134:162–174. doi: 10.1016/j.cell.2008.05.031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Garcia-Cuellar MP, Zilles O, Schreiner SA, Birke M, Winkler TH, Slany RK. The ENL moiety of the childhood leukemia-associated MLL-ENL oncoprotein recruits human Polycomb 3. Oncogene. 2001;20:411–419. doi: 10.1038/sj.onc.1204108. [DOI] [PubMed] [Google Scholar]
  15. Hemenway CS, de Erkenez AC, Gould GC. The polycomb protein MPc3 interacts with AF9, an MLL fusion partner in t(9;11)(p22;q23) acute leukemias. Oncogene. 2001;20:3798–3805. doi: 10.1038/sj.onc.1204478. [DOI] [PubMed] [Google Scholar]
  16. Katoh-Fukui Y, Tsuchiya R, Shiroishi T, Nakahara Y, Hashimoto N, Noguchi K, Higashinakagawa T. Male-to-female sex reversal in M33 mutant mice. Nature. 1998;393:688–692. doi: 10.1038/31482. [DOI] [PubMed] [Google Scholar]
  17. Kerppola TK. Polycomb group complexes--many combinations, many functions. Trends Cell Biol. 2009;19:692–704. doi: 10.1016/j.tcb.2009.10.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Krivtsov AV, Armstrong SA. MLL translocations, histone modifications and leukaemia stem-cell development. Nat Rev Cancer. 2007;7:823–833. doi: 10.1038/nrc2253. [DOI] [PubMed] [Google Scholar]
  19. Krivtsov AV, Feng Z, Lemieux ME, Faber J, Vempati S, Sinha AU, Xia X, Jesneck J, Bracken AP, Silverman LB, et al. H3K79 methylation profiles define murine and human MLL-AF4 leukemias. Cancer Cell. 2008;14:355–368. doi: 10.1016/j.ccr.2008.10.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kumar AR, Hudson WA, Chen W, Nishiuchi R, Yao Q, Kersey JH. Hoxa9 influences the phenotype but not the incidence of Mll-AF9 fusion gene leukemia. Blood. 2004;103:1823–1828. doi: 10.1182/blood-2003-07-2582. [DOI] [PubMed] [Google Scholar]
  21. Lavau C, Szilvassy SJ, Slany R, Cleary ML. Immortalization and leukemic transformation of a myelomonocytic precursor by retrovirally transduced HRX-ENL. EMBO J. 1997;16:4226–4237. doi: 10.1093/emboj/16.14.4226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Leeb M, Wutz A. Ring1B is crucial for the regulation of developmental control genes and PRC1 proteins but not X inactivation in embryonic cells. J Cell Biol. 2007;178:219–229. doi: 10.1083/jcb.200612127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lin C, Smith ER, Takahashi H, Lai KC, Martin-Brown S, Florens L, Washburn MP, Conaway JW, Conaway RC, Shilatifard A. AFF4, a component of the ELL/P-TEFb elongation complex and a shared subunit of MLL chimeras, can link transcription elongation to leukemia. Mol Cell. 2010;37:429–437. doi: 10.1016/j.molcel.2010.01.026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Maertens GN, El Messaoudi-Aubert S, Racek T, Stock JK, Nicholls J, Rodriguez-Niedenfuhr M, Gil J, Peters G. Several distinct polycomb complexes regulate and co-localize on the INK4a tumor suppressor locus. PLoS One. 2009;4:e6380. doi: 10.1371/journal.pone.0006380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mills AA. Throwing the cancer switch: reciprocal roles of polycomb and trithorax proteins. Nat Rev Cancer. 2010;10:669–682. doi: 10.1038/nrc2931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Milne TA, Kim J, Wang GG, Stadler SC, Basrur V, Whitcomb SJ, Wang Z, Ruthenburg AJ, Elenitoba-Johnson KS, Roeder RG, Allis CD. Multiple interactions recruit MLL1 and MLL1 fusion proteins to the HOXA9 locus in leukemogenesis. Mol Cell. 2010;38:853–863. doi: 10.1016/j.molcel.2010.05.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mohan M, Herz HM, Takahashi YH, Lin C, Lai KC, Zhang Y, Washburn MP, Florens L, Shilatifard A. Linking H3K79 trimethylation to Wnt signaling through a novel Dot1-containing complex (DotCom) Genes Dev. 2010a;24:574–589. doi: 10.1101/gad.1898410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mohan M, Lin C, Guest E, Shilatifard A. Licensed to elongate: a molecular mechanism for MLL-based leukaemogenesis. Nat Rev Cancer. 2010b;10:721–728. doi: 10.1038/nrc2915. [DOI] [PubMed] [Google Scholar]
  29. Monroe SC, Jo SY, Sanders DS, Basrur V, Elenitoba-Johnson KS, Slany RK, Hess JL. MLL-AF9 and MLL-ENL alter the dynamic association of transcriptional regulators with genes critical for leukemia. Exp Hematol. 2010 doi: 10.1016/j.exphem.2010.09.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mueller D, Bach C, Zeisig D, Garcia-Cuellar MP, Monroe S, Sreekumar A, Zhou R, Nesvizhskii A, Chinnaiyan A, Hess JL, Slany RK. A role for the MLL fusion partner ENL in transcriptional elongation and chromatin modification. Blood. 2007;110:4445–4454. doi: 10.1182/blood-2007-05-090514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Muntean AG, Tan J, Sitwala K, Huang Y, Bronstein J, Connelly JA, Basrur V, Elenitoba-Johnson KS, Hess JL. The PAF complex synergizes with MLL fusion proteins at HOX loci to promote leukemogenesis. Cancer Cell. 2010;17:609–621. doi: 10.1016/j.ccr.2010.04.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Nakamura T, Mori T, Tada S, Krajewski W, Rozovskaia T, Wassell R, Dubois G, Mazo A, Croce CM, Canaani E. ALL-1 is a histone methyltransferase that assembles a supercomplex of proteins involved in transcriptional regulation. Mol Cell. 2002;10:1119–1128. doi: 10.1016/s1097-2765(02)00740-2. [DOI] [PubMed] [Google Scholar]
  33. Pasini D, Malatesta M, Jung HR, Walfridsson J, Willer A, Olsson L, Skotte J, Wutz A, Porse B, Jensen ON, Helin K. Characterization of an antagonistic switch between histone H3 lysine 27 methylation and acetylation in the transcriptional regulation of Polycomb group target genes. Nucleic Acids Res. 2010;38:4958–4969. doi: 10.1093/nar/gkq244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Petruk S, Sedkov Y, Smith S, Tillib S, Kraevski V, Nakamura T, Canaani E, Croce CM, Mazo A. Trithorax and dCBP acting in a complex to maintain expression of a homeotic gene. Science. 2001;294:1331–1334. doi: 10.1126/science.1065683. [DOI] [PubMed] [Google Scholar]
  35. Sapountzi V, Logan IR, Robson CN. Cellular functions of TIP60. Int J Biochem Cell Biol. 2006;38:1496–1509. doi: 10.1016/j.biocel.2006.03.003. [DOI] [PubMed] [Google Scholar]
  36. Sitwala KV, Dandekar MN, Hess JL. HOX proteins and leukemia. Int J Clin Exp Pathol. 2008;1:461–474. [PMC free article] [PubMed] [Google Scholar]
  37. Slany RK, Lavau C, Cleary ML. The oncogenic capacity of HRX-ENL requires the transcriptional transactivation activity of ENL and the DNA binding motifs of HRX. Mol Cell Biol. 1998;18:122–129. doi: 10.1128/mcb.18.1.122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Smith LL, Yeung J, Zeisig BB, Popov N, Huijbers I, Barnes J, Wilson AJ, Taskesen E, Delwel R, Gil J, et al. Functional Crosstalk between Bmi1 and MLL/Hoxa9 Axis in Establishment of Normal Hematopoietic and Leukemic Stem Cells. Cell Stem Cell. 2011;8:649–662. doi: 10.1016/j.stem.2011.05.004. [DOI] [PubMed] [Google Scholar]
  39. Somervaille TC, Matheny CJ, Spencer GJ, Iwasaki M, Rinn JL, Witten DM, Chang HY, Shurtleff SA, Downing JR, Cleary ML. Hierarchical maintenance of MLL myeloid leukemia stem cells employs a transcriptional program shared with embryonic rather than adult stem cells. Cell Stem Cell. 2009;4:129–140. doi: 10.1016/j.stem.2008.11.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Stelzl U, Worm U, Lalowski M, Haenig C, Brembeck FH, Goehler H, Stroedicke M, Zenkner M, Schoenherr A, Koeppen S, et al. A human protein-protein interaction network: a resource for annotating the proteome. Cell. 2005;122:957–968. doi: 10.1016/j.cell.2005.08.029. [DOI] [PubMed] [Google Scholar]
  41. Suzuki M, Mizutani-Koseki Y, Fujimura Y, Miyagishima H, Kaneko T, Takada Y, Akasaka T, Tanzawa H, Takihara Y, Nakano M, et al. Involvement of the Polycomb-group gene Ring1B in the specification of the anterior-posterior axis in mice. Development. 2002;129:4171–4183. doi: 10.1242/dev.129.18.4171. [DOI] [PubMed] [Google Scholar]
  42. Tan J, Muntean AG, Hess JL. PAFc, a Key Player in MLL-rearranged Leukemogenesis. Oncotarget. 2010;1:461–465. doi: 10.18632/oncotarget.181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. van der Stoop P, Boutsma EA, Hulsman D, Noback S, Heimerikx M, Kerkhoven RM, Voncken JW, Wessels LF, van Lohuizen M. Ubiquitin E3 ligase Ring1b/Rnf2 of polycomb repressive complex 1 contributes to stable maintenance of mouse embryonic stem cells. PLoS One. 2008;3:e2235. doi: 10.1371/journal.pone.0002235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Voncken JW, Roelen BA, Roefs M, de Vries S, Verhoeven E, Marino S, Deschamps J, van Lohuizen M. Rnf2 (Ring1b) deficiency causes gastrulation arrest and cell cycle inhibition. Proc Natl Acad Sci U S A. 2003;100:2468–2473. doi: 10.1073/pnas.0434312100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Voss AK, Thomas T. MYST family histone acetyltransferases take center stage in stem cells and development. Bioessays. 2009;31:1050–1061. doi: 10.1002/bies.200900051. [DOI] [PubMed] [Google Scholar]
  46. Wang J, Iwasaki H, Krivtsov A, Febbo PG, Thorner AR, Ernst P, Anastasiadou E, Kutok JL, Kogan SC, Zinkel SS, et al. Conditional MLL-CBP targets GMP and models therapy-related myeloproliferative disease. EMBO J. 2005;24:368–381. doi: 10.1038/sj.emboj.7600521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Whitcomb SJ, Basu A, Allis CD, Bernstein E. Polycomb Group proteins: an evolutionary perspective. Trends Genet. 2007;23:494–502. doi: 10.1016/j.tig.2007.08.006. [DOI] [PubMed] [Google Scholar]
  48. Yokoyama A, Cleary ML. Menin critically links MLL proteins with LEDGF on cancer-associated target genes. Cancer Cell. 2008;14:36–46. doi: 10.1016/j.ccr.2008.05.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Yokoyama A, Lin M, Naresh A, Kitabayashi I, Cleary ML. A higher-order complex containing AF4 and ENL family proteins with P-TEFb facilitates oncogenic and physiologic MLL-dependent transcription. Cancer Cell. 2010;17:198–212. doi: 10.1016/j.ccr.2009.12.040. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

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

Supplementary Materials

01
NIHMS334999-supplement-01.doc (2.7MB, doc)

RESOURCES