Critical Role for a Single Leucine Residue in Leukemia Induction by E2A-PBX1

Abstract

In roughly 5% of cases of acute lymphoblastic leukemia, a chromosomal translocation leads to expression of the oncogenic protein E2A-PBX1. The N-terminal portion of E2A-PBX1, encoded by the E2A gene, is identical in sequence to the corresponding portion of the E proteins E12/E47 and includes transcriptional activation domains. The C terminus consists of most of the HOX interacting transcription factor PBX1, including its DNA-binding homeodomain. Structure-function correlative experiments have suggested that oncogenesis by E2A-PBX1 requires an activation domain, called AD1, at the extreme N terminus. We recently demonstrated that a potentially helical portion of AD1 interacts directly with the transcriptional coactivator protein cyclic AMP response element-binding protein (CBP) and that this interaction is essential in the immortalization of primary bone marrow cells in tissue culture. Here we show that a conserved LXXLL motif within AD1 is required in the interaction between E2A-PBX1 and the KIX domain of CBP. We show by circular dichroism spectroscopy that the LXXLL-containing portion of AD1 undergoes a helical transition upon interacting with the KIX domain and that amino acid substitutions that prevent helix formation prevent both the KIX interaction and cell immortalization by E2A-PBX1. Perhaps most strikingly, substitution of a single, conserved leucine residue (L20) within the LXXLL motif impairs leukemia induction in mice after transplantation with E2A-PBX1-expressing bone marrow. The KIX domain of CBP mediates well-characterized interactions with several transcription factors of relevance to leukemia induction. Circumstantial evidence suggests that the side chain of L20 might interact with a deep hydrophobic pocket in the KIX domain. Therefore, our results serve to identify a potential new drug target.The neoplastic cells in acute lymphoblastic leukemia (ALL) frequently contain recurrent somatic chromosomal abnormalities that contribute to their abnormal accumulation and function. A reciprocal translocation between chromosome bands 1q23 and 19p13.3 is detectable by conventional cytogenetic techniques in roughly 5% of cases of ALL (10). Of that 5% of cases, in 90 to 95% the presence of t(1;19) leads to the fusion of portions of the E2A gene on chromosome 19 with portions of the PBX1 gene on chromosome 1 and to expression of chimeric transcription factors called E2A-PBX1a and E2A-PBX1b (these isoforms are generated by alternative splicing of the PBX1 portion of the premRNA) (20,30). Beyond the consistent association with leukemia, the idea of the oncogenic potential of E2A-PBX1 is supported by abundant experimental evidence (reviewed in reference 24). Therefore, it seems likely that elucidating the molecular mechanisms by which E2A-PBX1 contributes to abnormal cellular behavior may uncover novel avenues in the development of better therapies for cases of ALL.E2A-PBX1 incorporates a PBX1-derived homeodomain near its C terminus and, like wild-type PBX1 proteins, can bind to PBX1 recognition sequences in coop...

Differential Roles for the E2A Activation Domains in B Lymphocytes and Macrophages

“…The AD1 domain has been shown to interact with the coactivator proteins p300/CBP and the SAGA histone acetyltransferase complex through an LDFS motif (18,33,34). A recent study demonstrated that the AD2 domain of E2-2, which is highly conserved with the E2A AD2 domain, also interacts with p300/CBP (35).…”

Structural basis of CBP/p300 recruitment in leukemia induction by E2A-PBX1

“…The assay was performed in 293T cells as previously described, 17 except for the quantities of plasmid DNA used (0.01 g/well pCMV-GAL4 construct, 0.01 g/well pCMV-2xVP16 construct, 0.7 g/ well p5xGAL luciferase reporter, and 0.1 g/well pCMV-Renilla internal control). Statistical significance was measured using a one-way ANOVA with Tukey posthoc test.…”

“…12 Mutation of the common leucine residue of the overlapping LXXLL and LDFS sequences in the PCET motif of AD1 markedly impaired both KIX binding and leukemogenesis by E2A-PBX1 in a murine bone marrow transplantation model. 17 A detailed structural characterization of the PCET/KIX interaction will provide molecular insights into both the critical role of E-proteins to lymphopoiesis and that of E2A-PBX1 in leukemia induction.…”

“…10,[12][13][14][15] The LDFS sequence of yeast bHLH transcription factors was shown to be important for recruiting the SAGA histone acetyltransferase complex, 16 and more recently its importance in E2A recruitment of human histone acetyltransferase complexes, such as CBP/p300, has been proposed. 1,17,18 Among the E-proteins, E2A is critical for the differentiation of B-lymphoid progenitors through direct activation of multiple target genes, primarily EBF, 19,20 with E2A Ϫ/Ϫ mice displaying a blockage at the earliest stages of B-lymphoid specification. 21 Given the importance of E2A in lymphoid differentiation, it is perhaps not surprising that in 4%-12% of pediatric acute lymphoblastic leukemia cases, regions of the E2A and PBX1 genes are fused by a chromosomal translocation between chromosomes 1 and 19 [t(1; 19)(q23;p13.3)].…”

Association of Transcription Factor 4 (TCF4) variants with schizophrenia and intellectual disability

“…By contrast, the class V HLH proteins (eg., inhibition of DNA binding 2 (ID2)) also associate with Eproteins, but since they lack a DNA-binding domain they sequester E-proteins into transcriptionally inactive heterodimers in a dominant-negative manner. In addition to the HLH family of proteins, TCF4 may also be regulated by interactions with the transcriptional co-repressor runt-related transcription factor 1 (RUNX1T1) and the E1A binding protein p300 (EP300) histone deacetylase capable of binding to TCF3 [43,44]. TCF3 has been found to co-purify with numerous proteins in human embryonic kidney 293 T (HEK293T) and pro-B cells suggesting that E-proteins may function as part of an oligomeric protein complex [45•].…”