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

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 α-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. 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 α-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. Transcription factors encoded by the gene E2A are crucial for normal development and implicated in acute leukemia. E2A-null mice manifest a complete block of B-lymphoid development at the pro-B cell stage, prior to immunoglobulin gene rearrangement, and a propensity to develop T-lymphoblastic lymphoma (1Bain G. Robanus Maandag E.C. Izon D.J. Amsen D. Kruisbeek A.M. Weintraub B.C. Krop I. Schlissel M.S. Feeney A.J. van Roon M. van der Valk M. te Riele H.P.J. Berns A. Murre C. Cell. 1994; 79: 885-892Abstract Full Text PDF PubMed Scopus (647) Google Scholar, 2Zhuang Y. Soriano P. Weintraub H. Cell. 1994; 79: 875-884Abstract Full Text PDF PubMed Scopus (578) Google Scholar, 3Bain G. Engel I. Robanus Maandag E.C. te Riele H.P. Voland J.R. Sharp L.L. Chun J. Huey B. Pinkel D. Murre C. Mol. Cell Biol. 1997; 17: 4782-4791Crossref PubMed Google Scholar). Despite these particular defects in the lymphoid compartment, the E2A gene products, called E12 and E47 (or “E2A proteins” for simplicity) are expressed widely in tissues and appear to function more generally in cell type specification and maturation (4Rutherford M.N. LeBrun D.P. Am. J. Pathol. 1998; 153: 165-173Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 5Aronheim A. Shiran R. Rosen A. Walker M.D. Nucleic Acids Res. 1993; 21: 1601-1606Crossref PubMed Scopus (18) Google Scholar). The E2A proteins belong to the basic helix-loop-helix (bHLH) 1The abbreviations used are: bHLH, basic helix-loop-helix; AD1, -2, activation domains 1 and 2; PCAF, p300/CREB-binding protein-associated factor; ALL, acute lymphoblastic leukemia; GST, glutathione S-transferase; PBS, phosphate-buffered saline; FBS, fetal bovine serum; IMDM, Iscove's modified Dulbecco's medium; IL-3, interleukin-3.1The abbreviations used are: bHLH, basic helix-loop-helix; AD1, -2, activation domains 1 and 2; PCAF, p300/CREB-binding protein-associated factor; ALL, acute lymphoblastic leukemia; GST, glutathione S-transferase; PBS, phosphate-buffered saline; FBS, fetal bovine serum; IMDM, Iscove's modified Dulbecco's medium; IL-3, interleukin-3. class of transcription factors (6Massari M.E. Murre C. Mol. Cell Biol. 2000; 20: 429-440Crossref PubMed Scopus (1373) Google Scholar). The bHLH domain, located toward the C terminus in E2A proteins, mediates dimerization and DNA binding (see Fig. 1). Dimerization is required for DNA binding by bHLH proteins. E2A proteins bind DNA at sites, called “E-boxes,” which contain the simple consensus sequence CANNTG. The widely expressed E2A proteins can heterodimerize with bHLH proteins whose expression is restricted to particular cell types to regulate target gene transcription in a tissue-specific manner. E2A proteins are transcriptional activators. Two activation domains have been mapped, largely using transient co-transfection of reporter plasmids with portions of E2A fused to heterologous DNA-binding domains (7Henthorn P. Kiledjian M. Kadesch T. Science. 1990; 247: 467-470Crossref PubMed Scopus (336) Google Scholar, 8Quong M.W. Massari M.E. Zwart R. Murre C. Mol. Cell Biol. 1993; 13: 792-800Crossref PubMed Scopus (130) Google Scholar, 9Aronheim A. Shiran R. Rosen A. Walker M.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8063-8067Crossref PubMed Scopus (105) Google Scholar). Activation domain 1 (AD1) encompasses 99 amino acids at the extreme N terminus of the proteins, whereas activation domain 2 (AD2) is located more centrally (see Fig. 1). These domains are considered to function independently through distinct mechanisms. For example, whereas AD1 can trans-activate reporter genes in various cell types, AD2 is differentially active in cells of pancreatic β-cell origin (5Aronheim A. Shiran R. Rosen A. Walker M.D. Nucleic Acids Res. 1993; 21: 1601-1606Crossref PubMed Scopus (18) Google Scholar). Transcriptional activation domains mediate contacts between DNA-bound transcription factors and other proteins involved in gene transcription (10Naar A.M. Lemon B.D. Tjian R. Annu. Rev. Biochem. 2001; 70: 475-501Crossref PubMed Scopus (434) Google Scholar). They can recruit large, multiprotein assemblies called transcriptional co-activator complexes. These often include proteins with intrinsic acetyltransferase activity. Acetyltransferases catalyze the acetylation of histones and other chromatin-associated proteins, including transcription factors. The mechanism by which acetylation of these proteins facilitates gene transcription is poorly understood and likely complex (11Felsenfeld G. Groudine M. Nature. 2003; 421: 448-453Crossref PubMed Scopus (807) Google Scholar). Among the best characterized transcriptional co-activator proteins are CREB binding protein (CBP) and its close paralog p300. CBP and p300 are large, ubiquitously expressed nuclear proteins (12Goodman R.H. Smolik S. Genes Dev. 2000; 14: 1553-1577PubMed Google Scholar). They were originally identified separately and are encoded on separate genes, but their polypeptide sequences are highly conserved; they are often referred to collectively as CBP/p300. Although lacking a DNA-binding domain, CBP/p300 can be recruited through protein-protein interactions to the promoters of target genes regulated by DNA-bound transcription factors. These include both oncogenic proteins, including c-Myb and c-Jun, and tumor suppressors such as p53 and pRb. The manner of involvement of CBP/p300 in oncogenesis appears complex and context-specific. PCAF, another well characterized and potent acetyltransferase, is an ortholog of the prototypic yeast acetyltransferase protein GCN5 (13Schiltz R.L. Nakatani Y. Biochim. Biophys. Acta. 2000; 1470: M37-M53Crossref PubMed Scopus (95) Google Scholar). PCAF exists as a stable complex with other proteins (the “PCAF complex”) and, like CBP/p300, can function as a transcriptional co-activator through transcription factor-dependent recruitment to target gene promoters. Furthermore, PCAF can associate directly with CBP/p300 and this probably contributes to its co-recruitment with CBP/p300 to some promoters. Functional and biochemical evidence indicates that E2A proteins can interact with CBP/p300. For example, p300, CBP, and PCAF can be co-immunoprecipitated with E2A proteins from crude nuclear extracts and co-elute with E2A proteins on fractionation of nuclear extracts by size exclusion chromatography (14Bradney C. Hjelmeland M. Komatsu Y. Yoshida M. Yao T.P. Zhuang Y. J. Biol. Chem. 2003; 278: 2370-2376Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The E2A locus on chromosome 19 is involved in at two chromosomal with acute lymphoblastic D.P. 2003; PubMed Scopus Google Scholar). the E2A-PBX1 which oncogenic proteins called and in of cases of D.P. 2003; PubMed Scopus Google Scholar). In these the of E2A proteins, which AD1 and AD2, is fused to of including its 1). this been that E2A-PBX1 oncogenic cell through the transcriptional of target genes that are regulated by transcriptional complexes. with this reporter plasmids consensus binding are by but proteins Murre C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: PubMed Scopus Google Scholar, Mol. Cell Biol. 1994; 14: PubMed Google Scholar, D.P. 1994; Google Scholar). Furthermore, that by such as that include AD1, also LeBrun D.P. Mol. Cell Biol. 1994; 14: PubMed Scopus Google Scholar, Google Scholar, R. LeBrun D.P. Mol. Cell Biol. 2000; 20: PubMed Scopus Google Scholar). appears likely as with transcriptional by E2A proteins, by E2A-PBX1 on interactions of polypeptide elements in AD1 AD2 with more transcriptional In the work described here, we elucidate functional interactions between the portion of E2A in E2A-PBX1 amino acids and transcriptional co-activators. Our findings that E2A can interact directly with the KIX domain of CBP. interaction involves conserved helical domains within AD1 and AD2 and is with cell proliferation in retrovirally transduced primary expression of glutathione proteins were using the and the CBP were used as for the The were with and (the were in the and the expression with the using were to the of expression of proteins were by the various from and, using the and sites, the expression with a portion of the amino acids of the DNA-binding two activation domains in The by from the by The reporter a from and of expression plasmids on were by protein in of cells by the of to and the cells were by in of to and the at for by the to a and of were The at with and to a chromatography The were with of in and with of proteins were in of protein were and at The with an of and in at The and of the proteins by of with with of bovine and extracts were from cells using the and Nucleic Acids Res. PubMed Scopus Google Scholar). For of protein with of in to a of and at for 2 The were in and and protein binding by in 1 bovine and The were in of nuclear protein in and at with The were and in They were and the proteins were in The with in for and proteins, of protein in of and bovine of to block protein-protein interactions by the portion of the recombinant proteins. for 2 at with of protein were and the for a 2 1 of and the The were in and and in of For proteins were on and to a The by for at 1 in and and with the primary in at The and with a to for 2 the by to the The were used in this and by and and cells were in with fetal bovine For transient cells in of complete of DNA of expression on of reporter and of a expression of were in an and to an using a the cells were of complete in and were using to the at The were to the and the and were For the cells were well of a in Dulbecco's modified The the cells were with expression and reporter plasmids of of 2 of and of by using a described previously B.D. M. R. S. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The cells were in and a portion of the using and an and marrow on 1 of the from the and of mice at of and in fetal bovine IL-3, and cell factor; recombinant were from cells were with and plasmids by the in On the bone marrow cells were in fetal and in The cells were to of and The the bone marrow cells were and the the cells in On the bone marrow cells were and on the cells in with The the bone marrow cells were and to in On the cells were in FBS, and and for is as in the The as described previously C. R. J. 1997; PubMed Scopus Google Scholar). retrovirally transduced cells were on 1 in with FBS, IL-3, and Cell at cells were The cells were in IMDM, and cells were in for the in the The in the Cell by as described previously M.N. T. H. LeBrun D.P. 2001; PubMed Scopus Google Scholar). The of E2A with by the that interaction with transcriptional a in we to the interaction between E2A-PBX1 and CBP/p300. map we using portions of expressed and as proteins with glutathione These were in to and with nuclear from the cell These cells were from a of and are with and expression of E2A-PBX1. on the were by by with an The that the oncogenic portion of E2A E2A which is between E12 and E47, but is of with CBP the of this proteins were to map elements within the E2A portion of E2A-PBX1. E2A which encompasses AD1 but AD2, also CBP, E2A of the from the E2A that E2A of the CBP by the polypeptide of the amino acids from E2A the CBP of E2A the CBP interaction to E2A but the amino acids of AD1 are required for the CBP interaction in the of E2A E2A AD2 but CBP but at E2A more CBP is by the of the CBP by E2A and E2A a interaction between elements within these two protein with CBP and PCAF within AD1 and AD2 of E2A acids of E2A are conserved and to a α-helical M.E. Murre C. Mol. Cell Biol. PubMed Scopus Google Scholar). helical domain with the transcriptional co-activator complex in yeast and is required in the transcriptional function of Our that the α-helical domain of AD1 also be important for CBP recruitment in the of E2A of of this domain in the of E2A but CBP recruitment in the we that elements within AD2 be to some for the of the AD1 helical domain from E2A Although sequence exists between the two E2A activation AD2, like AD1, a conserved domain that is to a and required in the transcriptional function of AD2 M.W. Massari M.E. Zwart R. Murre C. Mol. Cell Biol. 1993; 13: 792-800Crossref PubMed Scopus (130) Google Scholar). of the AD2 helical domain CBP recruitment to E2A whereas of both helical domains the interaction of E2A with CBP the with a PCAF recruitment of this protein by E2A and in that of CBP. Although the of this is the in at in these helical domains within both AD1 and AD2 to the recruitment of CBP and PCAF from lymphoid nuclear the AD1 helical domain a E2A with the KIX of is a protein with protein-protein interaction domains. map elements on CBP, proteins were with of CBP that collectively the These were used in with nuclear proteins on the were with an of CBP, from to E2A proteins the KIX domain also called the a well characterized protein-protein interaction domain to mediate interactions with transcriptional activation including the prototypic interaction with CREB M. R.H. Nature. 1993; PubMed Scopus Google Scholar, D. T. R. C. Mol. Cell Biol. PubMed Scopus Google Scholar). We to confirm interaction of E2A with the KIX domain of CBP. of the crucial within the KIX domain in the of the of CBP of with E2A the interaction Furthermore, a of CBP acids the KIX domain with amino acids of with E2A proteins. In some CBP an for with E2A proteins to the proteins the of the in the and of Fig. the of these the KIX domain of CBP is required and to mediate the interaction with E2A proteins. CBP can interact with proteins PCAF and E2A, the as to the interaction with E2A is direct. we to the interaction between E2A and CBP using purified, recombinant proteins. in with that the KIX and using a the N terminus of CBP. of the proteins with an of the of these the α-helical domains within AD1 and AD2 of E2A for the CBP of of CBP. of both of these the CBP of the E2A protein with which the KIX domain, with an the which as a of a of the various proteins These that E2A can interact directly with CBP the of proteins. Furthermore, they suggest that both of the α-helical domains within AD1 and AD2 direct with the KIX domain elements in its within amino of These using proteins from from crude nuclear In the the AD2 helical domain the AD1 domain in CBP whereas in the of proteins the two helical domains appear and be to our of of proteins in the the in this of proteins that have the interaction in the of from crude nuclear of CBP with Transcriptional of E2A of the of these findings to transcriptional by E2A proteins and we to our with transcriptional function in we plasmids that expression in cells of portions of E2A fused to a heterologous DNA-binding domain from These were cells with a reporter that expression regulated by binding of with which AD1, the reporter of that with E2A E2A which AD2 but is active E2A more active E2A In to these findings suggest that AD2 can be active in lymphoid cells and in this to be more active AD1 A. Shiran R. Rosen A. Walker M.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8063-8067Crossref PubMed Scopus (105) Google Scholar). Furthermore, in that the of polypeptide elements within and of E2A a on both co-activator recruitment and transcriptional activation the suggest functional cooperativity between elements within these portions of the of the helical domain from AD1 in the of E2A to of that with E2A whereas of the AD2 helical domain a more to of both domains in an of transcriptional to of that with E2A of the various proteins were as by the cell used in the reporter gene with an Furthermore, using the nuclear of of the proteins We a to the interaction in the of of cells with E2A fused to transcriptional activation and in potent activation of the reporter in a manner that on elements within both E2A and KIX of the AD1 helical domain to of that by E2A of the AD2 helical domain a to is to the of the on by E2A Fig. These findings that the interaction between E2A and the KIX domain of CBP can within the of cells and are with involvement of the helical domains of AD1 and between and CBP with in of primary bone marrow with E2A-PBX1 the in of highly cells with of 1994; Google Scholar). We used this to involvement of the interaction in oncogenesis by E2A-PBX1. The to the AD1 AD2 helical domains were and the of cell from retrovirally cells expression and nuclear of the recombinant proteins transduced bone marrow cells were in and the were The cells were in and in for the in the the of in the of a of by cells with the cell with of the these cells a in ability as of the of with the of cells transduced with In these the and at a through as demonstrated retrovirally transduced E2A-PBX1 is of the in of primary M. D. van Mol. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). of the AD1 helical domain in with the AD2 helical domain in in to the this to a for CBP in the of with expression of E2A-PBX1. we that the AD1 to with of by retrovirally transduced of proliferation cells and their of the retrovirally transduced cells in in with the demonstrated a of cell with of the AD1 but the AD2 helical domain of the AD2 helical domain in the of an AD1 helical domain, whereas both helical domains cell proliferation to of the AD1 domain Cell in the a the The and were with (see cell proliferation by cell on of the In cells transduced with the and of were in In the to in with the AD1 and in with in both AD1 and were in the with of the cell transduced with the by expression of These in primary cells suggest a particular for the AD1 helical domain and, by the CBP/p300 interaction in the induction of proliferation by E2A-PBX1. The interaction between E2A proteins and CBP/p300 in p300 by elements in reporter plasmids in R. Yao T.P. Genes Dev. PubMed Scopus Google Scholar). In an using nuclear from an a complex by E47 on an DNA p300 by the E2A activation domains expressed as heterologous proteins with the DNA-binding domain in cells Y. A. R. Mol. Cell Biol. 1998; PubMed Scopus Google Scholar). expressed interact with in p300 in a J. Y. Mol. Cell Biol. 1997; 17: PubMed Scopus Google Scholar). In p300, CBP, and PCAF were co-immunoprecipitated with E2A proteins from lymphoid nuclear the with E2A proteins on fractionation of nuclear extracts by size exclusion chromatography (14Bradney C. Hjelmeland M. Komatsu Y. Yoshida M. Yao T.P. Zhuang Y. J. Biol. Chem. 2003; 278: 2370-2376Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Our work these by that the E2A-CBP interaction is elements at and in the of involvement of the KIX domain of CBP, the functional of the AD1 and AD2 helical domains in the of portions of E2A and a between the CBP/p300 interaction and induction of proliferation by E2A-PBX1 in primary In of the of of AD1 in cell we were to transcriptional to AD2 in cells A. Shiran R. Rosen A. Walker M.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8063-8067Crossref PubMed Scopus (105) Google Scholar). we in in which A. Shiran R. Rosen A. Walker M.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8063-8067Crossref PubMed Scopus (105) Google of AD1 to AD2 be to in the E2A used proteins amino acids (AD1) (AD2) of E2A, whereas we used the of by AD2 by and have been to exclusion of polypeptide elements from their The KIX domain amino from to of CBP. in a complex with a from the CREB activation domain been by I. D. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). KIX is a domain from amino acids that reside in the a on the of the KIX domain that the KIX domain mediates interactions with transcriptional activation domains in transcription factors, including c-Jun, and D. M. T. A. I. A. M. Mol. Cell Biol. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, T. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, I. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, P. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The a amino is often in 2000; PubMed Scopus Google Scholar). interactions between the KIX domain and the CREB and c-Myb activation domains are well characterized D. M. T. A. I. A. M. Mol. Cell Biol. PubMed Scopus Google Scholar, T. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the of c-Myb with the α-helical domain of E2A and its of the and amino acids 1). the c-Myb activation domain directly with the KIX domain of CBP, the sequence with E2A our of a direct interaction between E2A and The is within the CBP/p300-interactive domain of the of transcriptional that function in by nuclear S. A. J. J. Genes Dev. 1998; PubMed Scopus Google Scholar). The is also conserved in bHLH proteins, as a direct of the domain that contributes to the interaction with the complex in yeast M.E. Murre C. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). the domain is conserved in and the appears to be restricted to the is yeast ortholog of CBP/p300, the between the of CBP/p300 and the in bHLH proteins is with a functional Our suggest like AD1, AD2 also direct with the KIX domain of CBP. The helical domain of AD2 sequence with that of domain is in AD2, a sequence to the is are required to binding of AD1 and AD2 to the KIX domain and map the and J. van Nature. PubMed Scopus Google demonstrated a for the KIX domain in Using were the of mice as to amino acids to mediate interactions between KIX and CREB mice of the CBP KIX domain were in p300 in several of these including and were also in (1Bain G. Robanus Maandag E.C. Izon D.J. Amsen D. Kruisbeek A.M. Weintraub B.C. Krop I. Schlissel M.S. Feeney A.J. van Roon M. van der Valk M. te Riele H.P.J. Berns A. Murre C. Cell. 1994; 79: 885-892Abstract Full Text PDF PubMed Scopus (647) Google Scholar, 2Zhuang Y. Soriano P. Weintraub H. Cell. 1994; 79: 875-884Abstract Full Text PDF PubMed Scopus (578) Google Scholar). CBP and p300 are highly conserved within the KIX domain, the we for CBP are probably to p300 I. D. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). In of our involvement of the KIX domain in the the between the and for a for the interaction in normal The by AD1 and AD2 to transcriptional induction with their in CBP recruitment and the AD2 helical domain appears important for transcriptional induction and CBP recruitment by E2A in the of appears at in the of AD1, in the induction of cell proliferation by Our for this which by proteins domains in transcriptional cell by E2A proteins. Our primary in this work the of protein-protein interactions that to by E2A-PBX1. this we have demonstrated that of the conserved helical domains within AD1 and AD2 both the direct interaction with CBP and the induction of proliferation in primary bone marrow cells these appear to the of by E2A-PBX1 that interactions with factors are that the within AD1 and AD2 the CBP/p300 interaction in the of this our that the of the E2A portion to oncogenesis by E2A-PBX1 be understood at in in of its ability to mediate a direct interaction with the KIX domain of CBP/p300. with

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