E2A-PBX1 Interacts Directly with the KIX Domain of CBP/p300 in the Induction of Proliferation in Primary Hematopoietic Cells

Richard Bayly,Luan Chuen,Richard A Currie,Brandy D Hyndman,Richard Casselman,Gerd A Blobel,David P Lebrun

doi:10.1074/jbc.m408654200

Abstract

The E2A gene encodes DNA-binding transcription factors, called E12 and E47, involved in cell specification and maturation. E2A is also involved in a chromosomal translocation that leads to the expression of an oncogenic transcription factor called E2A-PBX1 in cases of acute leukemia. In the work described here, we elucidate the interaction between E2A-PBX1 and transcriptional co-activators. We confirm that the E2A portion can interact with CBP and PCAF and map required elements on E2A and CBP. On CBP, the interaction involves the KIX domain, a well characterized domain that mediates interactions with several other oncogenic transcription factors. On E2A, the interaction with CBP requires conserved alpha-helical domains that reside within activation domains 1 and 2 (AD1 and AD2, respectively). Using purified, recombinant proteins, we show that the E2A-CBP interaction is direct. Notwithstanding the previously demonstrated ability of AD1 and AD2 to function independently, some of our findings suggest functional cooperativity between these two domains. Finally, we show that the CBP/p300-interactive helical domains of E2A are important in the induction of proliferation in cultured primary bone marrow cells retrovirally transduced with E2A-PBX1. Our findings suggest that some aspects of E2A-PBX1 oncogenesis involve a direct interaction with the KIX domain of CBP/p300.

Highlights

The E2A gene encodes DNA-binding transcription factors, called E12 and E47, involved in cell specification and maturation
The Oncogenic Portion of E2A Proteins Interacts with CREB binding protein (CBP)— Motivated primarily by the consideration that interaction with transcriptional co-activators could play a role in leukemia induction, we set out to characterize the interaction between E2A-PBX1 and CBP/p300
The results indicate that the oncogenic portion of E2A (i.e. E2A 1– 483), which is invariant between E12 and E47, but not full-length PBX1a, is capable of interacting with CBP under the conditions of this experiment (Fig. 2B)

Summary

EXPERIMENTAL PROCEDURES

Plasmids—Plasmids conferring bacterial expression of glutathione S-transferase (GST) fusion proteins were constructed using PCR amplification. For pull-down experiments, 10 ␮g of GST fusion protein was combined with 20 ␮l of pre-swollen glutathione-Sepharose beads in PBS containing 1% Triton X-100 to a final volume of 300 ␮l and rocked at 4 °C for 2 h. The day, the cells were transfected with mammalian expression and reporter plasmids (3.5 ␮g of pCMV-GAL4-CBP 586 – 673, 0.5 ␮g of pCMV2xVP16-E2A, 2 ␮g of p5xGAL4 LUC, and 0.3 ␮g of PRL-CVM (a Renilla luciferase-expressing plasmid kindly provided by Dr Chris Mueller)) using a liposome-based technique described previously [23]. Retroviral Transductions and Immortalization Assays—Bone marrow was harvested on day 1 of the experiment from the femurs and tibia of five CD-1 mice at 12–14 weeks of age and placed in prestimulation mix (IMDM medium containing 15% fetal bovine serum (FBS), 10 ng/ml IL-3, 10 ng/ml IL-6, and 75 ng/ml murine stem cell factor; all recombinant cytokines were purchased from Peprotech Inc., Rocky Hill, NJ). Cell cycle analysis by flow cytometry was carried out as described previously [25]

RESULTS

CBP can interact with numerous proteins besides PCAF and

DISCUSSION