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Mediator Subunit MED1 Is Required for E2A-PBX1–Mediated Oncogenic Transcription and Leukemic Cell Growth

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Mediator subunit MED1 is required for E2A-PBX1–mediated oncogenic transcription 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 transcription factor E2A-PBX1, containing the N-terminal coactivator 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 chromatin 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 gene 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 genes 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 proteins that serve as PBX1 interactions as potential therapeutic targets. oncogenic drivers. One such fusion protein 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 immunoprecipitation 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 promoter/enhancer-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.
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  • Structures of KIX Domain of CBP in Complex with Two Foxo3a Transactivation Domains Reveal Promiscuity and Plasticity in Coactivator Recruitment

    Structures of KIX Domain of CBP in Complex with Two Foxo3a Transactivation Domains Reveal Promiscuity and Plasticity in Coactivator Recruitment

    Structures of KIX domain of CBP in complex with two FOXO3a transactivation domains reveal promiscuity and plasticity in coactivator recruitment Feng Wanga,b, Christopher B. Marshalla,b, Kazuo Yamamotob,c, Guang-Yao Lia,b, Geneviève M. C. Gasmi-Seabrooka,b, Hitoshi Okadab,c, Tak W. Makb,c, and Mitsuhiko Ikuraa,b,1 aOntario Cancer Institute, University Health Network (UHN), Toronto, ON, Canada M5G 1L7; bDepartment of Medical Biophysics, University of Toronto, Toronto, ON, Canada M5G 2M9; and cCampbell Family Institute for Breast Cancer Research, Ontario Cancer Institute, UHN, Toronto, ON, Canada M5G 2C1 Edited by Robert G. Roeder, The Rockefeller University, New York, NY, and approved March 5, 2012 (received for review November 20, 2011) Forkhead box class O 3a (FOXO3a) is a transcription factor and FOXOs recruit CBP/p300 to the FRE, which, in turn, recruits tumor suppressor linked to longevity that determines cell fate general transcriptional machinery and also remodels chromatin through activating transcription of cell differentiation, survival, and through histone acetyltransferase activity (10). However, CBP/ apoptotic genes. Recruitment of the coactivator CBP/p300 is a p300 also acetylates the FH domain, which impairs DNA binding crucial step in transcription, and we revealed that in addition to and attenuates transcription (11). The CR2, which consists of conserved region 3 (CR3) of FOXO3a, the C-terminal segment of CR2 three separate regions (A, B, and C) (Fig. 1A), may also play (CR2C) binds CBP/p300 and contributes to transcriptional activity. a role in the transactivation activity of FOXOs. Chromosomal CR2C and CR3 of FOXO3a interact with the KIX domain of CBP/p300 translocations create MLL-FOXO fusion proteins composed of at both “MLL” and “c-Myb” binding sites simultaneously.