subunit MED1 is required for E2A-PBX1–mediated oncogenic and leukemic cell growth

Yu-Ling Leea, Keiichi Itoa,1, Wen-Chieh Pib,1, I-Hsuan Linc, Chi-Shuen Chua, Sohail Malika, I-Hsin Chengb, Wei-Yi Chena,b,d,2, and Robert G. Roedera,2

aLaboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, NY 10065; bInstitute of Biochemistry and Molecular Biology, National Yang-Ming University, 112 Taipei, Taiwan; cResearch Center of Cancer Translational Medicine, Taipei Medical University, 110 Taipei, Taiwan; and dCancer Progression Research Center, National Yang-Ming University, 112 Taipei, Taiwan

Contributed by Robert G. Roeder, December 18, 2020 (sent for review January 3, 2020; reviewed by Ananda Roy and Harinder Singh) The chimeric E2A-PBX1, containing the N-terminal complex specifically in B cells (7). Since many different activation domains of E2A fused to the C-terminal DNA-binding domain transcriptional activators have been shown to interact physically and of PBX1, results in 5% of pediatric acute lymphoblastic leukemias (ALL). functionally with Mediator, which is generally required for Pol II- We recently have reported a mechanism for RUNX1-dependent recruit- mediated transcription (8, 9), this latter result is consistent with the ment of E2A-PBX1 to in pre-B leukemic cells; but the subse- possibility of Mediator recruitment by E2A activation domains, quent E2A-PBX1 functions through various coactivators and the general which may further contribute to activation by E2A in a transcriptional machinery remain unclear. The Mediator complex plays context-dependent manner. a critical role in cell-specific gene activation by serving as a key coac- Given the previously characterized functional domains present tivator for gene-specific transcription factors that facilitates their func- in E2A-PBX1, it had been generally assumed that the PBX1 DNA- tion through the RNA polymerase II transcriptional machinery, but whether Mediator contributes to aberrant expression of E2A-PBX1 tar- binding domain mediates the binding to oncogenic target and get genes remains largely unexplored. Here we show that Mediator that the E2A activation domains facilitate their transcription. Con- interacts directly with E2A-PBX1 through an interaction of the MED1 sistent with this idea, E2A activation domains AD1 and AD2 were CELL BIOLOGY subunit with an E2A activation domain. Results of MED1 depletion by reported to mediate p300 interactions (4) and to be essential for CRISPR/Cas9 further indicate that MED1 is specifically required for E2A- E2A-PBX1–mediated gene activation and cell transformation (10, PBX1–dependent gene activation and leukemic cell growth. Integrated 11). However, our recent study also uncovered a mechanism for transcriptome and cistrome analyses identify pre-B cell receptor and cell RUNX1-mediated recruitment of E2A-PBX1 to RUNX1 target cycle regulatory genes as direct cotargets of MED1 and E2A-PBX1. No- genes in pre-B leukemic cells through a direct interaction between tably, complementary biochemical analyses also demonstrate that re- RUNX1 and the PBX1 homeodomain (12). Beyond these studies, cruitment of E2A-PBX1 to a target DNA template involves a direct there have been no comprehensive analyses of the general interaction with DNA-bound RUNX1 that can be further stabilized by EBF1. These findings suggest that E2A-PBX1 interactions with RUNX1 Significance and MED1/Mediator are of functional importance for both gene-specific transcriptional activation and maintenance of E2A-PBX1–driven leuke- mia. The MED1 dependency for E2A-PBX1–mediated gene activation Current challenges in B cell acute lymphoblastic leukemia and leukemogenesis may provide a potential therapeutic opportunity (B-ALL) include a comprehensive understanding of mechanistic by targeting MED1 in E2A-PBX1+ pre-B leukemia. effects of associated oncogenic factors, the development of efficacious therapeutic regimens, and the identification of B-ALL subgroups with characteristic molecular features that Mediator | E2A-PBX1 leukemia | MED1 | RUNX1 | EBF1 can be targeted in cancer treatment. Here we show that MED1 is required for a identified interaction between the Mediator cell acute lymphoblastic leukemia (B-ALL) accounts for the coactivator complex and oncogenic E2A-PBX1, for the prolif- Bhighest incidence of cancer in children. Standard treatment eration, specifically, of E2A-PBX1–driven leukemic cells and for for B-ALL involves chemotherapy, with a cure rate up to 80%. the activation of E2A-PBX1 target genes. RUNX1 directs the Nevertheless, about 20% of remissions suffer a relapse with a recruitment of E2A-PBX1 to target genes, including cell cycle very poor prognosis (1). B-ALLs are subcategorized based on regulatory E2F5 and survival signaling pre-B cell receptor their karyotypes, cell types, and immunophenotypes, and many genes. These results provide insights into mechanisms under- are associated with the development of chromosomal aberra- lying E2A-PBX1–mediated leukemogenesis and MED1–E2A- tions and expression of the resulting fusion that serve as PBX1 interactions as potential therapeutic targets. oncogenic drivers. One such fusion is E2A-PBX1, which results from the t(1;19)(q23;p13) chromosomal translocation and Author contributions: Y.-L.L. and R.G.R. designed research; Y.-L.L., K.I., W.-C.P., C.-S.C., is present in 5 to 7% of pediatric ALLs. This genomic rear- S.M., and W.-Y.C. performed research; Y.-L.L., I.-H.L., I.-H.C., W.-Y.C., and R.G.R. analyzed rangement fuses the N-terminal transcriptional activation do- data; and Y.-L.L., W.-Y.C., and R.G.R. wrote the paper. mains (AD1, AD2, and AD3) of E2A (exons 1 to 16, E2AN)to Reviewers: A.R., NIH; and H.S., University of Pittsburgh School of Medicine. the C-terminal HOX cooperative motif and homeodomain of the The authors declare no competing interest. C protein encoded by PBX1 (exons 4 to 9, PBX1 ), a proto- Published under the PNAS license. oncogene with a critical role in hematopoiesis and lymphopoi- See online for related content such as Commentaries. esis (2, 3). Activation of E2A target genes is mediated, in part, 1K.I. and W.-C.P. contributed equally to this work. through AD-mediated interactions of E2A with transcriptional 2To whom correspondence may be addressed. Email: [email protected] or roeder@ coactivators p300 (via AD1 and AD2) (4, 5) and TAF4 (via rockefeller.edu. AD3) (6). In addition, a chromatin fol- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ lowed by next-generation sequencing (ChIP-seq) analysis showed doi:10.1073/pnas.1922864118/-/DCSupplemental. that E2A colocalizes with the MED1 subunit of the Mediator Published February 4, 2021.

PNAS 2021 Vol. 118 No. 6 e1922864118 https://doi.org/10.1073/pnas.1922864118 | 1of11 Downloaded by guest on September 27, 2021 coactivators, such as the Mediator complex, in mediating the onco- specifically for the oncogenic proliferation of E2A-PBX1–driven genic functions of E2A-PBX1. leukemic cells and for expression of selected direct target genes. In this study, we sought: 1) To understand whether the Mediator Finally, our biochemical analyses further establish an E2A-PBX1 complex, possibly through specific subunits, plays an important role recruitment mechanism involving direct E2A-PBX1 interactions in regulating expression of E2A-PBX1 target genes; 2) to identify with /-bound RUNX1. key direct target genes; and 3) to establish biochemical mechanisms for E2A-PBX1 recruitment and subsequent function on target Results genes. Toward these objectives, we show a direct interaction of MED1 Subunit of the Mediator Complex Is Targeted by E2A-PBX1. To E2A-PBX1 with the Mediator through the MED1 subunit both investigate whether the Mediator complex plays a role in activating in vitro and in vivo. Further studies with MED1-depleted E2A- E2A-PBX1 target genes, we first tested for an interaction between – PBX1+ and E2A-PBX1 pre-B cells show that MED1 is required purified Mediator and E2A-PBX1 proteins (SI Appendix,Fig.

Fig. 1. The MED1 subunit in the Mediator complex is directly targeted by E2A-PBX1. (A) Co-IP assay, using highly purified proteins, showing a direct in- teraction of Mediator complex and E2A-PBX1. IPs were performed with anti–E2A-PBX1 antibody or mouse IgG control. Mediator complex was detected by immu- noblotting with indicated antibodies. (B and C) Coupled IP-DSP cross-linking analyses showing that the MED1 subunit in the Mediator is directly targeted by E2A-PBX1 (B)andE2A(C). The immunocomplexes of purified E2A-PBX1 or E2A and purified Mediator were treated without or with DSP followed by washing with a buffer containing 8 M urea. Bound (cross-linked) proteins were then released by cross-link reversal and visualized by immunoblotting with indicated antibodies. (D) Immobilized DNA template assay demonstrating an E2A-mediated recruitment of Mediator to the IGLL1 promoter DNA template. The NCAPD2 promoter DNA template served as a control. (E) Cell-free co-IP assay showing an AD2-dependent interaction of E2A and MED1. (Upper) Coomassie-stained gel showing the purified full-length E2As (wild-type, mutated [m] AD1 and/or AD2, deleted [Δ] AD3 mutants). Bovine serum albumin (BSA) standards were used to quantify the amounts of E2A proteins. (Lower) immunoblots of precipitated MED1 and E2As using, respectively, anti-GST and anti-Flag antibodies. The level of MED1-GST in the wild type E2A lane was set to 1. (F) Purified (Coomassie blue-stained, Left) and diagrammed (Right) GST-fused MED1 full-length (FL), and derived N-terminal (N), middle (M), and C-terminal (C) fragments. (G) Cell-free co-IP assays showing E2A-PBX1 interactions with the N- and C- terminal fragments of MED1. (H) Cell-free co-IP assays, using purified E2A-PBX1 and cell extracts of wild-type (WT) or MED1 KO 697 lines, demonstrating a MED1-dependent interaction of Mediator and E2A-PBX1.

2of11 | PNAS Lee et al. https://doi.org/10.1073/pnas.1922864118 Mediator subunit MED1 is required for E2A-PBX1–mediated oncogenic transcription and leukemic cell growth Downloaded by guest on September 27, 2021 S1 A–C). A direct physical E2A-PBX1–Mediator interaction was MED1 Is Required for the Cell Growth and Cell Cycle Progression of observed in a cell-free coimmunoprecipitation (co-IP) assay E2A-PBX1+ Leukemia. Interestingly, while generating the MED1 (Fig. 1A, lane 3). To identify the Mediator subunits that directly KO 697 cells (Fig. 2A), we observed low survival rates and sig- interact with E2A-PBX1, a coupled IP-diithiobis succinimidyl pro- nificantly slow proliferation of the MED1-inactivated clones. pionate (DSP) cross-linking analysis (13) was performed using pu- Therefore, to further establish the functional significance of the rified E2A-PBX1 or E2A and purified Mediator. The immobilized MED1-dependent E2A-PBX1–Mediator interaction, we also immunoprecipitated complex was cross-linked with increasing doses genetically inactivated MED1 loci in E2A-PBX1+ RCH-ACV – of DSP and then treated with urea/SDS to dissociate the uncross- and E2A-PBX1 NALM6 pre–B-ALL lines for comparison. linked Mediator subunits. Bound (cross-linked) subunits were re- Complete MED1 inactivation was confirmed by immunoblots of leased by cross-link reversal and analyzed by immunoblot. In MED1 levels in RCH-ACV (Fig. 2B) and NALM6 (Fig. 2C) agreement with the co-IP assays, representative Mediator subunits clonal cells. To assess the consequences of MED1 inactivation, were detected in the IP of E2A-PBX1 prior to DSP and urea/SDS cell cycle profiles and proliferation of these lines were analyzed. treatment (Fig. 1B, lane 4), whereas only MED1 of the tested Notably, a striking MED1-dependent cell cycle progression subunits remained associated with E2A-PBX1 after the cross- (Fig. 2 D–F) and proliferation (Fig. 2 G–I) were specifically – linking, denaturation, and washing procedures (Fig. 1B,lane8). observed in E2A-PBX1+, but not E2A-PBX1 , leukemic cells. In These results indicate a direct interaction between the MED1 addition, further functional studies using a colony formation subunit and E2A-PBX1. To determine which part of E2A-PBX1 assay (CFU) also showed that MED1 is essential for the survival interacts with MED1/Mediator, a cell-free co-IP assay was used to of E2A-PBX1+ leukemic cells (Fig. 2J). To confirm the MED1 assess Mediator interaction with purified full-length E2A or PBX1 dependency in E2A-PBX1 leukemic cells, ectopic MED1 ex- (SI Appendix,Fig.S1C). The results show a direct Mediator inter- pression vectors were lentivirally transduced into a MED1 KO action with E2A (SI Appendix,Fig.S1D, lane 3), but not with PBX1 697 clone (Fig. 2K, last lane). As expected, the reintroduced (SI Appendix,Fig.S1E, lane 3). In a further cross-linking analysis, MED1 restored normal cell cycle progression and proliferation the direct interaction of E2A with Mediator (Fig. 1C, lane 3) was of MED1 KO cells (Fig. 2 L and M), confirming that MED1 is + mapped, at least in part, to the MED1 subunit (Fig. 1C,lane6). required for the proliferation of E2A-PBX1 leukemia cells. Given that E2A-PBX1 and E2A share the same N-terminal AD, Altogether, these results indicate that MED1 has an indispens- these results suggest that the direct E2A-PBX1–Mediator interac- able role for the proliferation, cell cycle progression, and main- + tion is primarily, but not necessarily exclusively, through the E2A tenance of the leukemogenic properties of E2A-PBX1 , but not – AD and the Mediator MED1 subunit. E2A-PBX1 , leukemic cells. To investigate whether an E2A AD–Mediator interaction re- CELL BIOLOGY sults in Mediator recruitment to DNA templates, and since E2A- MED1 and E2A-PBX1 Cooccupy and Coregulate Pre-B Cell Survival and PBX1 does not bind directly to its target genes (12), we carried Growth Genes. To identify common targets of E2A-PBX1 and out a surrogate immobilized template assay with purified E2A MED1/Mediator, we used an anti-MED1 antibody to perform proteins and the IGLL1 promoter that contains four E2A ChIP-seq in 697 cells. We then interrogated these data with binding sites (E-boxes). Notably, whereas E2A binds indepen- previous genome-wide profiling (ChIP-seq) data both for MED1 dently to the IGLL1 promoter DNA template (Fig. 1D, lane 4), and for E2A-PBX1 (12), which was monitored with anti-HA Mediator is recruited only in the presence, and not in the ab- antibody in 697 pre–B-ALL cells stably expressing a sub- sence, of E2A (Fig. 1D, lane 3 vs. lane 5). As a control, and even endogenous level of C-terminal HA-Flag–tagged E2A-PBX1 in the presence of E2A, Mediator is not recruited to the DNA (E2A-PBX1-hf) and with antibodies recognizing either the N template of the NCAPD2 promoter that contains no E-boxes terminus (E2AN) or the C terminus (PBX1C) of endogenous (Fig. 1D, lane 9). Coupled with demonstrations of direct Medi- E2A-PBX1. Analyses of two MED1 ChIP-seq datasets defined a ator interactions with E2A and E2A-PBX1, these results estab- total of 9,500 MED1 ChIP-seq peaks (Dataset S1), in which lish an E2A AD-mediated recruitment of Mediator, through a ∼35% (3,386) of the peaks overlap with previously identified direct interaction, to the DNA template in vitro. To further E2A-PBX1 peaks. Reciprocally, the analysis also showed that identify which activation domain (AD1, AD2, or AD3) of E2A ∼39% (3,519) of the E2A-PBX1 peaks coincide with the MED1 interacts with MED1 in the E2A–Mediator interaction, full- peaks (Fig. 3A and Dataset S1). (GO) analyses length E2A proteins with AD1 (L16A) or AD2 (D398A/ indicated that 3,350 genomic regions cobound by E2A-PBX1 and E399A) point that perturb the E2A interaction with MED1 are significantly involved in hematopoietic processes, p300 (4, 5) or an AD3 deletion (211 to 300 deleted) that disrupts including the B lymphocyte differentiation and activation path- the TAF4 interaction (6), were tested for MED1 binding in cell- ways (Fig. 3B), whereas regions bound by E2A-PBX1 only are free co-IP assays. Notably, the results revealed a reduction of weakly involved in the T lympocyte differentiation and activation MED1 binding by AD2-mutated E2A proteins, whereas the wild- pathways (SI Appendix, Fig. S2). type, AD1-mutated, and AD3-deleted E2A forms displayed These results suggested that recruitment of Mediator (through similar levels of MED1 binding (Fig. 1E), thus indicating a direct MED1) may participate in the activation of a subset of E2A- MED1–AD2 interaction. Using purified MED1 fragments PBX1–driven genes that contribute to B cell lymphopoiesis (Fig. 1F), this direct E2A–MED1 interaction was further and leukemogenesis. To further study the function of MED1 in mapped to the N- and C-terminal regions of MED1 (Fig. 1G, regulating the E2A-PBX1 targets, we identified 993 MED1 and lanes 1 and 4). To document a MED1-dependent Mediator in- E2A-PBX1 cobound genes that contain overlapping MED1 and teraction with the oncogenic E2A-PBX1, cell-free co-IP assays E2A-PBX1 ChIP-seq peaks that lie within an area extending were performed with cell lysates from wild-type or MED1 from −10 Kb upstream of the transcription start site to +1 Kb kockout (KO) E2A-PBX1+ 697 pre–B-ALL cells (Fig. 2A). The downstream of the transcription end site (Fig. 3C). As a com- results show that purified E2A-PBX1 interacts with the MED1- plement to the genomic profiles, we also performed RNA- containing Mediator complex in the extract from wild-type cells sequencing (RNA-seq) analyses of parental and MED1 KO (Fig. 1H, lane 3), but not with the MED1-deficient Mediator 697 lines (Dataset S2). Integrating the cistrome and tran- complex in the extract from MED1 KO cells (Fig. 1H, lane 4). It scriptome results, we identified 105 MED1 and E2A-PBX1 may be noted that the Mediator complex remains intact even in cobound genes that were dysregulated (51 down-regulated and the absence of MED1 (14, 15). Collectively, these data indicate 55 up-regulated transcripts) in response to MED1 inactivation that the E2A-PBX1–Mediator interaction is dependent on the (Fig. 3C). Notably, these dysregulated transcripts could be Mediator MED1 subunit. rescued by ectopic MED1 in MED1 KO 697 lines (Fig. 3D and

Lee et al. PNAS | 3of11 Mediator subunit MED1 is required for E2A-PBX1–mediated oncogenic transcription https://doi.org/10.1073/pnas.1922864118 and leukemic cell growth Downloaded by guest on September 27, 2021 Fig. 2. Selective induction of cell cycle arrest by MED1 deficiency in E2A-PBX1+ leukemic cells. (A–C) Generation of MED1 KO E2A-PBX1+ 697 (A), E2A-PBX1+ RCH-ACV (B), and E2A-PBX1− NALM6 (C)pre–B-ALL cells by a CRISPR/Cas9-mediated approach. Immunoblots of MED1 showing partial (clones 5-14 and 5-16) and complete (clones 13-4, 13-3, and 5-1) inactivation of MED1 loci in 697 lines. (D–F) Cell cycle profiling by propidium iodide staining in wild-type and MED1 KO clones of 697 (D), RCH-ACV (E), and NALM6 (F) lines. (G–I) Proliferation assays with ALAMAR blue staining in wild-type and MED1 KO clones of 697 (G), RCH-ACV (H), and NALM6 (I) lines. (J) Colony formation assay (CFU) for wild-type (WT) and MED1 KO (clone 13-4) 697 lines. (K) Immunoblots of MED1 in MED1 KO 697 cells and cells infected with control (13-4-Ctrl) or MED1-expressing (13-4-MED1) lentivirus. (L and M) Cell cycle profiling (L) and cell proliferation (M)of MED1 KO 697 cells (clone 13-4) infected with control or MED1-expressing lentivirus. Means ± SD from three biological experiments are shown. Student’s t test, ***P < 0.001.

4of11 | PNAS Lee et al. https://doi.org/10.1073/pnas.1922864118 Mediator subunit MED1 is required for E2A-PBX1–mediated oncogenic transcription and leukemic cell growth Downloaded by guest on September 27, 2021 CELL BIOLOGY

Fig. 3. MED1 plays an essential role in the activation of E2A-PBX1–bound pre-BCR and cell cycle genes. (A) Venn diagram showing the overlap between E2A- PBX1-hf and MED1 ChIP-seq peaks. (B) GO biological processes of MED1 and E2A-PBX1–associated ChIP-peaks using GREAT analysis. (C) Venn diagram showing the overlap between MED1– and E2A-PBX1–bound genes and down- or up-regulated transcripts in MED1 KO 697 line. (D) Heatmaps of RNA-seq results revealing the identified 51 MED1- and E2A-PBX1–bound genes that were down-regulated in MED1 KO 697 lines (Left, four biological repeats) and activated by the reexpressed MED1 (Right, three biological repeats). EV, MED1 KO with empty vector; KO, MED1 knockout; reMED1, MED1 KO with re- expressed MED1. (E) ChIP-seq profiles of E2A-PBX1-hf in stable 697 line and the N-terminal region of E2A (E2AN), C-terminal region of PBX1 (PBX1C), p300, and MED1 (two repeats), and RUNX1 (two repeats) antibodies in 697 lines at IGLL1 (Left), E2F5 (Center), and VPREB3 (Right) loci. E2F5 is an identified cotarget of E2A-PBX1 and MED1. Black arrows indicate the direction of transcription.

Lee et al. PNAS | 5of11 Mediator subunit MED1 is required for E2A-PBX1–mediated oncogenic transcription https://doi.org/10.1073/pnas.1922864118 and leukemic cell growth Downloaded by guest on September 27, 2021 SI Appendix,Fig.S3), thereby confirming the direct function of and MED1 ChIP-seq signals at IGLL1, E2F5, and VPREB3 MED1 in the expression of these E2A-PBX1–bound genes. In- genes (Fig. 3E). These results suggest a direct function of MED1 terestingly, in the list of down-regulated transcripts in MED1 KO in regulating the E2A-PBX1–bound growth-related genes and cells, we identified: 1) Several genes involved in B cell survival/ leukemic cell growth. development, including pre-B cell receptor IGLL1, VPREB3, POU2AF1(OCA-B) (16), and MEF2C (17); and 2) several genes E2F5 and IGLL1 Are Direct Cotargets of E2A-PBX1 and MED1 and involved in general cell growth and survival pathways, including Highly Expressed in E2A-PBX1+ Pre–B-ALL Cells. Since both Media- E2F5, CCND3 (18), CREB5 (19), MTOR (20, 21), MND1 (22), tor and E2A-PBX1 predominantly activate (or maintain) ex- EPHA3 (23), ERMP1 (24), and JAKMIP1 (25) (Fig. 3D). How- pression of direct target genes, and since MED1-inactivated + ever, we did not identify growth-related genes in the list of up- E2A-PBX1 leukemic cells show significant growth retardation regulated transcripts (SI Appendix,Fig.S3). Consistent with our compared to control cells (Fig. 2 G and H), further analyses were previous demonstration of RUNX1-mediated recruitment of focused on down-regulated genes that are involved in cell sur- E2A-PBX1 (13), gene tracks displayed significant overlap of vival and growth, in particular, the IGLL1 gene that encodes an RUNX1, p300, E2A-PBX1 (E2A-PBX1-hf, E2AN, and PBX1C), essential factor for pre-B cell receptor (pre-BCR)–mediated

Fig. 4. Depletion of E2A-PBX1 down-regulates the expression of IGLL1 and E2F5 genes. (A and B) Immunoblots of E2A-PBX1 and MED1 in 697 (A) and RCH- ACV (B) cells that were treated with scramble (Ctrl) or indicated PBX1 shRNAs. (C and D) qRT-PCR showing the expression levels of indicated genes in 697 (C)or RCH-ACV (D) cells treated with a scramble shRNA or two different PBX1 shRNAs. (E and F) qRT-PCR showing the expression levels of indicated genes in 697 (E) or RCH-ACV (F) cells treated with a scramble shRNA or two different E2A/E2A-PBX1 shRNAs that target the E2A portion of E2A-PBX1. Means ± SD from three biological experiments are shown. Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001.

6of11 | PNAS Lee et al. https://doi.org/10.1073/pnas.1922864118 Mediator subunit MED1 is required for E2A-PBX1–mediated oncogenic transcription and leukemic cell growth Downloaded by guest on September 27, 2021 survival signaling (26) and the E2F5 gene that encodes a cell suggest that both IGLL1 and E2F5 are direct cotargets of E2A- cycle regulator (27). The joint function of cobound MED1 and PBX1 and MED1 and indispensable for the survival and growth E2A-PBX1 in expression of these genes was analyzed by qRT- of leukemic cells. PCR assays in both E2A-PBX1+ (697 and RCH-ACV) and E2A- − PBX1 (NALM6) pre–B-ALL lines following E2A-PBX1 de- RUNX1-Depedent Recruitment and EBF1-Dependent Stabilization of pletion by short-hairpin RNA (shRNA) or MED1 inactivation by E2A-PBX1 DNA Binding. To further dissect the molecular mecha- CRISPR/Cas9. The efficiencies of E2A-PBX1 depletion were nisms underlying E2A-PBX1 recruitment to MED1-occupied analyzed by immunoblot (Fig. 4 A and B) and qRT-PCR targets, motif analysis was performed with genomic regions (Fig. 4 C and D) and the efficiencies of MED1 inactivation, as cobound by E2A-PBX1 and MED1 (Fig. 3A and Dataset S1). well as MED1 restoration by lentivirus transduction (rescue ex- Consistent with results of our previous analysis focused on E2A- periment), were also analyzed by immunoblot (Fig. 2 A–C and K) PBX1 genomic localization (12), motif analysis showed signifi- and qRT-PCR (Fig. 5 A–C). cant and equivalent enrichment of RUNX1 and EBF1 binding The complementary qRT-PCR assays in the E2A-PBX1–depleted motifs with E2A-PBX1 and MED1 sites (SI Appendix, Fig. S6). (Fig. 4 C–F)andMED1-inactivated (Fig. 5 A and B) 697 and RCH- To further detail the binding of RUNX1 and EBF1 to the MED1 ACV cells confirmed a joint E2A-PBX1 and MED1 requirement for and E2A-PBX1 cobound regions, we carried out genome-wide optimal expression of the representative IGLL1 and E2F5 genes. As profiling of EBF1 and integrated the data with previous ChIP- controls, the qRT-PCR assays in Fig. 5D demonstrate the ability of seq data for RUNX1 in 697 leukemic cells (12). Notably, over ectopically expressed MED1 to rescue the expression of these genes 76% of MED1 and E2A-PBX1 cobound regions are also cooc- in MED1-KO 697 cells, whereas the assays in Fig. 5C demonstrate cupied by RUNX1 and EBF1, while nearly 92% and 81% of the lack of a MED1 requirement for expression of these genes in the these regions are specifically targeted by RUNX1 and EBF1, – E2A-PBX1 NALM6 leukemic cells. Consistent with the expression respectively (SI Appendix, Fig. S6B). Interestingly, an expression analyses, ChIP-qPCR assays demonstrated the binding of MED1 and analysis using the Cancer Cell Line Encyclopedia dataset (17) + E2A-PBX1 to IGLL1 and E2F5 genes only in E2A-PBX1 697 and also indicated that RUNX1 and EBF1 are selectively up- – RCH-ACV cells, but not in E2A-PBX1 NALM6 cells (Fig. 5 E regulated in B-ALL (SI Appendix, Fig. S7). Furthermore, and F). among the RUNX family RUNX1 is also specifically up-regulated We have previously reported that the gene encoding the pre- in E2A-PBX1+ leukemia cells, whereas EBFs are not differen- BCR component IGLL1 is directly targeted by E2A-PBX1 (12) tially expressed in these B-ALL leukemia cells (SI Appendix, Fig. and highly up-regulated in E2A-PBX1+ pre-B ALL cases (28). S8). Together, these observations raised the possibility that both However, little is known about the function of E2F5 in RUNX1 and EBF1 may play important roles in E2A-PBX1 CELL BIOLOGY pre–B-ALL. E2F5 was found to be highly associated with the binding to identified genes involved in pre-BCR signaling and progression of various cancers, including prostate, pancreatic, cell growth. Therefore, we sought to determine the function of glioblastoma, ovarian, and colorectal cancers (29–32), although RUNX1 and EBF1 on these identified genes. While we were it may be dispensable for the growth of normal cells (27, 33). unable to identify shRNAs that efficiently knock down EBF1, Interestingly, E2F5 is the only gene to be highly expressed in our earlier transcriptomic analyses of RUNX1-depleted 697 + E2A-PBX1 pre–B-ALL relative to other members of the E2F leukemic cells revealed that RUNX1 also plays an essential role gene family (SI Appendix, Fig. S4). Notably, as mentioned above in expression of these genes (Fig. 6A) in the context of the broader group of E2A-PBX1 target genes: Toward a mechanistic analysis, we next analyzed whether 1) MED1, RUNX1, p300, and E2A-PBX1 peaks coincide at the E2A-PBX1 interacts directly with RUNX1 and/or EBF1 in co-IP E2F5 locus in ChIP-seq analyses (Fig. 3E); 2) E2F5 transcript assays with purified recombinant proteins (SI Appendix, Fig. levels are substantially down-regulated by knockdown of E2A- S1C). Notably, in this assay, RUNX1 and EBF1 were found to PBX1 (Fig. 4 C–F); 3) E2F5 transcription is also down-regulated interact independently with E2A-PBX1 (Fig. 6B, lanes 2 and 3), + in MED1 KO E2A-PBX1 cells (Figs. 3D and 5 A and B), but but showed no apparent cooperative binding (Fig. 6B, lane 4). To − not in E2A-PBX1 leukemic cells (Fig. 5C), and is restored by further elucidate the mechanism of action, we used an immobi- reexpressed MED1 (Fig. 5D)–confirming the specificity of lized DNA template assay with an IGLL1 promoter fragment MED1 dependency; and 4) consistent with the functional assays, that is adjacent to the native core promoter and transcription MED1 (Fig. 5F) and E2A-PBX1 (Fig. 5G) bind at the E2F5 start site and contains two EBF1 and two RUNX1 binding sites + locus in E2A-PBX1 697 and RCH-ACV cells, but not in E2A- and, as a control, an NCAPD2 promoter fragment that contains − PBX1 NALM6 cells. These results establish E2F5 as a direct no EBF1 or RUNX1 sites (Fig. 6C). Notably, E2A-PBX1 re- cotarget of MED1 and E2A-PBX1, as well as a MED1 dependency cruitment to the IGLL1 promoter was critically dependent upon in regulating E2F5 expression and cell cycle progression specifically RUNX1 in the absence of EBF1 (Fig. 6D, lane 6 vs. lane 4) and in the context of E2A-PBX1–driven leukemia. Although the basis further stabilized by EBF1 (Fig. 6D, lane 7 vs. lane 6). Curiously, forthispre–B-ALL subtype specificity for the MED1 dependency whereas RUNX1 alone showed independent binding to the of E2F5 expression is as yet unknown, but potentially related, we have template as expected (Fig. 6D, lane 10), demonstrable EBF1 found that whereas ectopic overexpression of E2A-PBX1 markedly binding was observed only in the presence of E2A-PBX1, with a enhances E2F5 expression in E2A-PBX1+ 697 pre–B-ALL cells, it coincident low level of EBF1-dependent E2A-PBX1 binding − does not do so in the E2A-PBX1 NALM6 pre–B-ALL cells, again (Fig. 6D, lane 5 vs. lanes 4 and 9). This result further indicates a demonstrating pre–B-ALL subtype specific regulation of E2F5 (SI direct E2A-PBX1–EBF1 interaction that somehow stabilizes Appendix,Fig.S5). EBF1 binding to the template. Direct in vitro binding of RUNX1 To investigate the role of E2F5 in MED1-mediated cell to promoter DNA elements was further confirmed by an alter- growth of E2A-PBX1+ leukemic cells, cell viability was assessed native colorimetric DNA-binding assay (Fig. 6E, see legend). To in 697 cells treated with four individual shRNAs targeting E2F5. functionally validate the requirement for RUNX1 and EBF1 Notably, decreased cell viability was observed in E2F5-depleted binding sites, a cell-based reporter assay was carried out with an leukemic cells (Fig. 5H). Furthermore, in agreement with the IGLL1 promoter-driven luciferase construct in E2A-PBX1+ 697 RUNX1-depedent recruitment of E2A-PBX1 and the direct cells. Notably, whereas of the EBF1 sites significantly E2A-PBX1–MED1 interaction (discussed above), RUNX1 deceased the promoter activity, mutation of the RUNX1 sites knockdown reduced the binding of both E2A-PBX1 and MED1 completely impaired IGLL1 promoter function (SI Appendix, to the E2F5 locus (Fig. 5I), as well as the mRNA levels of E2F5 Fig. S9B). Moreover, EMSAs with recombinant proteins and a and IGLL1, in 697 cells (Fig. 5J). Collectively, these results DNA probe spanning the proximal RUNX1 site of the IGLL1

Lee et al. PNAS | 7of11 Mediator subunit MED1 is required for E2A-PBX1–mediated oncogenic transcription https://doi.org/10.1073/pnas.1922864118 and leukemic cell growth Downloaded by guest on September 27, 2021 Fig. 5. MED1 binds to and activates the expression of IGLL1 and E2F5 genes in an E2A-PBX1–dependent manner. (A–D) qRT-PCR showing the relative ex- pression of indicated genes in MED1 KO 697 (A), RCH-ACV (B), and NALM6 (C) cell lines and in the MED1 KO 697 clone 13-4 infected with control (13-4 Ctrl Exp) or MED1-expressing (13-4 MED1 Exp) lentivirus (D). (E) ChIP-qPCR showing the binding of MED1 at the IGLL1 promoter in WT and MED1 KO (KO) 697, RCH-ACV (RCH), and NALM6 (NAL) cell lines. IgG as a negative control. (F and G) ChIP-qPCR showing the binding of MED1 (F) and E2A-PBX1 (G)attheE2F5 locus in WT and MED1 KO 697, RCH-ACV (RCH), and NALM6 (NAL) cell lines. (H) Viability of 697 cells treated with scramble or indicated E2F5 shRNAs. Data presented as viability relative to the control. (I) ChIP-qPCR showing the binding of RUNX1, E2A-PBX1, and MED1 at the E2F5 locus in 697 cells treated with scramble (Ctrl) or RUNX1 shRNAs. Means ± SD from three biological experiments are shown. (J) Quantitative RT-PCR assays showing the relative expression of indicated genes in 697 cells treated with scramble (Ctrl) or RUNX1 shRNAs. Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001.

8of11 | PNAS Lee et al. https://doi.org/10.1073/pnas.1922864118 Mediator subunit MED1 is required for E2A-PBX1–mediated oncogenic transcription and leukemic cell growth Downloaded by guest on September 27, 2021 CELL BIOLOGY

Fig. 6. Recruitment and stabilization of E2A-PBX1 DNA binding by interactions with RUNX1 and EBF1. (A) Heatmap of RNA-seq results revealing the down- regulation of pre-BCR component and B cell transcription (co)factor genes (blue colored) and cell growth-related (red colored) genes in 697 line treated with two differentially designed shRUNX1s. Occupancies of RUNX1 and EBF1 were identified by ChIP-seq assays and are denoted on the right. (B) Cell-free co-IP assays showing direct E2A-PBX1 interactions with RUNX1 and EBF1. IPs were carried out with full-length recombinant proteins and anti–E2A-PBX1 antibody. Bound proteins were visualized by immunoblotting with anti-Flag antibody. (C) Diagram of the IGLL1 promoter. The binding sites for E2A (E boxes, blue), RUNX1 (red), and EBF1 (purple) are shown. The yellow arrow indicates the region used in the immobilized DNA template assays. (D) Immobilized template assays showing a RUNX1-dependent recruitment of E2A-PBX1 with stabilization by EBF1 on the IGLL1 promoter. “N” represents an NCAPD2 promoter fragment that lacks E2A-PBX1, RUNX1, or EBF1 binding sites. Bound proteins were visualized by immunoblotting with anti-Flag antibody. (E) Colorimetric DNA–protein binding assays indicating RUNX1 binding to both RUNX1 sites of the IGLL1 promoter. Means ± SD from three biological experiments are shown. Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001. (F) Schematic model for E2A-PBX1 recruitment and subsequent transcriptional function through direct protein-protein interactions. E2A-PBX1 is primarily recruited to the DNA template by interaction with RUNX1 (12) and further stabilized by DNA-bound EBF1. The Mediator coactivator complex is recruited to the target locus through an MED1-E2A (AD2) interaction (reported here), while TFIID and p300 are recruited through TAF4 TAFH-E2A AD3 (6) and p300 KIX-E2A AD1/AD2 (4, 5) interactions, respectively.

Lee et al. PNAS | 9of11 Mediator subunit MED1 is required for E2A-PBX1–mediated oncogenic transcription https://doi.org/10.1073/pnas.1922864118 and leukemic cell growth Downloaded by guest on September 27, 2021 promoter further demonstrated an E2A-PBX1–enhanced and have demonstrated direct interactions of E2A-PBX1 with the RUNX1 site-dependent DNA binding of RUNX1 (SI Appendix, 30-subunit Mediator complex that serves as the main means of Fig. S9C). Collectively, our results suggest a mechanism for communication between enhancer/promoter-bound transcriptional RUNX1-dependent recruitment and EBF1-dependent stabili- activators and the general transcription machinery, with RNA po- zation of E2A-PBX1 DNA binding to the IGLL1 promoter lymerase II being a critical target of the Mediator (8, 9). We have (Fig. 6F). further demonstrated that 1) the E2A-PBX1–Mediator interaction involves E2A AD2, 2) E2A-PBX1 interacts directly with the MED1 Discussion subunitofMediator,3)MED1is necessary for E2A-PBX1–Mediator Current challenges in B-ALL related to the t(1;19) translocation interactions and, importantly, 4) MED1 is important both for acti- are to identify druggable oncogenic factors that are regulated by vation of representative E2A-PBX1 target genes and for maintenance E2A-PBX1, associated E2A-PBX1 recruitment mechanisms, and of leukemic cell growth. It is noteworthy that previous studies have additional transcriptional cofactors that serve as direct effectors established critical functions for MED1, through direct interactions, for the promoter-bound E2A-PBX1 functions in leukemic cell in the function of nuclear hormone receptors (37) and in the function growth. Here, we have uncovered an essential and previously of GATA factors in hematopoiesis (38). The latter result and our unrecognized role for the MED1 subunit of the Mediator current results raise the possibility of additional functions of MED1 in coactivator complex in the transcriptional regulation and pro- hematopoiesis and hematopoietic malignancies. In this regard, we + liferation of E2A-PBX1 leukemic cells. We show that MED1 is recently have demonstrated a critical role for MED1 in mediating the required for interaction of the Mediator with E2A-PBX1, for function of the B cell coactivator OCA-B, essential for germinal activation of E2A-PBX1 target genes, and for oncogenic prolif- center formation, in activation of the BCL6 enhancer in a germinal – eration, specifically, of E2A-PBX1 driven leukemic cells. Toward center B cell-derived lymphoma (39). Notably, our present studies an understanding of underlying mechanisms, we established direct showed an important role for MED1 in the proliferation of E2A- – MED1 E2A-PBX1 targets that include genes involved in pre-BCR PBX1+ pre–B-ALL cells (697 and RCH-ACV), but not of E2A- – survival signaling, lymphocyte activation/differentiation, and cell PBX1 NALM6 pre–B-ALL cells. This observation suggests that cycle control and established an E2A-PBX1 recruitment mech- MED1 is not generally required for cell viability, consistent with our anism that involves direct interactions with enhancer-bound IGLL1 earlier demonstration of very early embryonic development in the RUNX1 on the native promoter template of , a cotarget of absence of MED1 and viability of MED1 null fibroblasts (40). MED1 and E2A-PBX1. These findings suggest that, at least on It is notable that the previously described E2A activation some genes, E2A-PBX1 acts as an intermediary coactivator with domains (AD1, AD2, and AD3) are retained in the E2A-PBX1 additional downstream interactions with other factors, such as fusion, such that gene activation mechanisms previously identi- the Mediator. fied for E2A through these domains might reasonably be expected to be functional for E2A-PBX1—as indicated in the E2A-PBX1 Acts as a Transcriptional Coactivator, Rather than a Primary model in Fig. 6F. These effector functions include AD1 and AD2 DNA Binding Factor, and Requires Enhancer-Bound RUNX1 for Target interactions with p300 that effect histone acetylation (4, 5) and Gene Binding (Recruitment) and Activation. Given the structure of – E2A-PBX1, in which the DNA-binding domain of E2A is the AD3 TAF4 interaction that enhances TFIID binding and replaced by the DNA-binding homeodomain of PBX1, it has facilitates preinitiation complex formation (6). It thus will be generally been assumed that direct E2A-PBX1 target genes important to determine whether these interactions, like the would contain PBX1 sites, especially since E2A-PBX1 can bind AD2-MED1/Mediator interaction, are important for E2A-PBX1 to natural PBX1 sites in vitro (11). In this regard, a recent study functions and whether the requirements vary with the nature of suggested that E2A-PBX1 self-association through the PBX1 the E2A-PBX1 target genes. Of note, these interactions could PBC-B domain facilitates binding to target genes, although the represent potential therapeutic targets, especially by targeting ability of E2A-PBX1 to associate with HOX proteins is not es- the MED1 subunit that is not generally required for cell viability. sential (34). Our recent ChIP-seq and RNA-seq analyses iden- tified direct E2A-PBX1 target genes (12). Surprisingly, however, Role of Mediator in Cancer through Gene-Specific Transcriptional motif analyses showed no apparent PBX1 sites but, instead, Activation. Consistent with our findings, a potential role of revealed binding sites for major transcription factors (most notably MED1 in cancers has been reported in prostate cancers by RUNX1 and EBF1) with critical roles in B cell development and regulating androgen-dependent expression of endogenous an- hematologic malignancies. Importantly, in the representative drogen receptor target genes. Knockdown of MED1 has been E2A-PBX1 target genes (IGLL1 and E2F5) analyzed here, cell- shown to inhibit cell proliferation and induce in based assays established that RUNX1 is required both for the prostate cancer cells, thus suggesting that Mediator plays an direct recruitment of E2A-PBX1 and for optimal transcription of important coregulatory role for in prostate these genes. These results, as well as our earlier study emphasizing cancer cell proliferation and survival (41). Functions for MED1, other E2A-PBX1 target genes that included RUNX1 itself (12), through its phosphorylation, have also been reported in hormone are consistent with reported RUNX1 requirements for leukemo- resistance in both prostate cancer (42) and breast cancer (43). genesis, proliferation, and survival of leukemic cells (35, 36). Similarly, knockdown of MED19 has also been shown to inhibit Mechanistically, the results of our biochemical and cell-based cell growth and proliferation of breast cancer cells (44). In leu- studies suggest that E2A-PBX1 acts more like a transcriptional kemogenesis, interaction of BRD4 and Mediator is required for coactivator in the activation of E2A-PBX1 target genes. In this gene-specific transcriptional activation and for acute myeloid regard, our biochemical analyses with purified proteins unequiv- leukemia maintenance (45). Hence, targeting of Mediator sub- ocally established direct interactions of E2A-PBX1 with RUNX1 units, as shown in previous studies (41, 46), may provide a po- and EBF1 and, importantly, a clear RUNX1-dependent binding of tential therapeutic strategy for antagonizing the oncogenic E2A-PBX1 to the natural IGLL1 promoter that is enhanced transcription programs driven by E2A-PBX1. Mediator isolated by EBF1. from MED1 KO cell lines is structurally stable and transcrip- tionally active, suggesting that MED1 is not essential for the Role of Mediator Subunit MED1 in E2A-PBX1 Function and Oncogenicity. integrity and general function of the Mediator complex, as also Given the recruitment of E2A-PBX1 to target genes, a key question evidenced by the viability of MED1-deficient fibroblasts (15). concerns the nature of its effector functions in stimulating tran- Therefore, the specific oncogenic function of MED1 in E2A- scription by the general transcription machinery. In this regard, we PBX1–driven leukemogenesis may provide a potential therapeutic

10 of 11 | PNAS Lee et al. https://doi.org/10.1073/pnas.1922864118 Mediator subunit MED1 is required for E2A-PBX1–mediated oncogenic transcription and leukemic cell growth Downloaded by guest on September 27, 2021 target for E2A-PBX1 t(1;19) leukemia without affecting normal expression and purification, immobilized template protein recruitment assay, tissue homeostasis. in vitro co-IP, in vitro DNA-protein binding colorimetric assay, EMSA, shRNA- mediated gene silencing, ChIP-qPCR, ChIP-seq, RNA-seq, bioinformatic analyses, E2F5, a Cell-Cycle Regulator That Is Highly Expressed in Various and data availability, as well as related materials (plasmids, primers, and anti- Cancers, Is Coregulated by E2A-PBX1 and MED1. The E2F family bodies), can be found in SI Appendix. of transcription factors plays a crucial role in the control of the cell + cycle. Here we show that E2F5 is highly expressed in E2A-PBX1 Data Availability. ChIP-seq and RNA-seq data data have been deposited in the pre–B-ALL cells, that the transcriptional activation of E2F5 is cor- European Bioinformatics Institute (EMBL-EBI), https://www.ebi.ac.uk/ (ac- egulated by E2A-PBX1, RUNX1 and MED1, and that E2F5 cession nos. E-MTAB-8177, E-MTAB-8178, and E-MTAB-8179). knockdown impairs the leukemic cell growth. Consistent with our findings, E2A-PBX1 has been implicated in cell-cycle dysregulation ACKNOWLEDGMENTS. We thank Tomoyoshi Nakadai, Miho Shimada, CDKN2C Xiangdong Lu, Shu-Ping Wang, Alan Gerber, Zhanyun Tang, and Takashi by suppressing the expression of through targeting Onikubo for helpful advice. R.G.R. was supported by grants from the SETDB2 (47). In addition, the oncogenic function of E2F5 in pre-B National Cancer Institute (CA178765) and the Leukemia and Lymphoma cell leukemia may be similar to its contribution in a various type of Society (SCOR 7021-20). W.-Y.C. was supported by grants from the Ministry cancers, including prostate, pancreatic, glioblastoma, ovarian, and of Science and Technology in Taiwan (MOST 107-2320-B-010-024-MY3) and colorectal cancers (29–32). Overexpression and dependence of E2F5 the Cancer Progression Research Center, National Yang-Ming University, provide a rationale for its consideration as a biomarker and thera- from the Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education in Taiwan. peutic target for broad subtypes of cancers, including leukemias. Y.-L.L. was supported by a Fellowship for Overseas Project from the Ministry of Science and Technology in Taiwan (MOST 104-2917-I-564-005) and in part Materials and Methods by Grant UL1TR001866 from the National Center for Advancing Transla- Methods detailing cell culture, CRISPR/Cas9-mediated gene editing, recombinant tional Sciences (National Institutes of Health Clinical and Translational DNA construction, flow cytometry, cell proliferation assays, recombinant protein Science Award).

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Lee et al. PNAS | 11 of 11 Mediator subunit MED1 is required for E2A-PBX1–mediated oncogenic transcription https://doi.org/10.1073/pnas.1922864118 and leukemic cell growth Downloaded by guest on September 27, 2021