title: Proteomic discovery of Max as a novel interacting partner of C/EBPalpha: a Myc/Max/Mad link.
abstract: The transcription factor CCAAT/enhancer binding protein a (C/EBPalpha) is important in the regulation of granulopoiesis and is disrupted in human acute myeloid leukemia. In the present study, we sought to identify novel C/EBPalpha interacting proteins in vivo through immunoprecipitation using mass spectrometry-based proteomic techniques. We identified Max, a heterodimeric partner of Myc, as one of the interacting proteins of C/EBPalpha in our screen. We confirmed the in vivo interaction of C/EBPalpha with Max and showed that this interaction involves the basic region of C/EBPalpha. Endogenous C/EBPalpha and Max, but not Myc and Max, colocalize in intranuclear structures during granulocytic differentiation of myeloid U937 cells. Max enhanced the transactivation capacity of C/EBPalpha on a minimal promoter. A chromatin immunoprecipitation assay revealed occupancy of the human C/EBPalpha promoter in vivo by Max and Myc under cellular settings and by C/EBPalpha and Max under retinoic acid induced granulocytic differentiation. Interestingly, enforced expression of Max and C/EBPalpha results in granulocytic differentiation of the human hematopoietic CD34(+) cells, as evidenced by CD11b, CD15 and granulocyte colony-stimulating factor receptor expression. Silencing of Max by short hairpin RNA in CD34(+) and U937 cells strongly reduced the differentiation-inducing potential of C/EBPalpha, indicating the importance of C/EBPalpha-Max in myeloid progenitor differentiation. Taken together, our data reveal Max as a novel co-activator of C/EBPalpha functions, thereby suggesting a possible link between C/EBPalpha and Myc-Max-Mad network.
authors.affiliation: Bone Marrow Transplantation Unit, State Center for Cell and Gene Therapy, Clinic Internal Medicine IV, Martin-Luther-University, Halle, Germany.
authors.forename: A A
authors.initials: AA
authors.lastname: Zada
authors.forename: J A
authors.initials: JA
authors.lastname: Pulikkan
authors.forename: D
authors.initials: D
authors.lastname: Bararia
authors.forename: M
authors.initials: M
authors.lastname: Geletu
authors.forename: A K
authors.initials: AK
authors.lastname: Trivedi
authors.forename: M Y
authors.initials: MY
authors.lastname: Balkhi
authors.forename: W D
authors.initials: WD
authors.lastname: Hiddemann
authors.forename: D G
authors.initials: DG
authors.lastname: Tenen
authors.forename: H M
authors.initials: HM
authors.lastname: Behre
authors.forename: G
authors.initials: G
authors.lastname: Behre
mesh_terms: D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / Q000032:analysis / Q000502:physiology*; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000032:analysis / Q000737:chemistry / Q000502:physiology*; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D019281:Dimerization; D006412:Hematopoietic Stem Cells / Q000166:cytology; D006801:Humans; D019891:Leukopoiesis; D011401:Promoter Regions, Genetic; D040901:Proteomics; D016271:Proto-Oncogene Proteins c-myc / Q000032:analysis / Q000502:physiology*; D034741:RNA, Small Interfering / Q000494:pharmacology; D012097:Repressor Proteins / Q000502:physiology*; D013937:Thymidine Kinase / Q000235:genetics
chemical_list: D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D022763:CCAAT-Enhancer-Binding Protein-alpha; C499330:MAX protein, human; C079439:MXD1 protein, human; C489427:MYC protein, human; D016271:Proto-Oncogene Proteins c-myc; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D013937:Thymidine Kinase