1. ¹Bone Marrow Transplantation Unit, State Center for Cell and Gene Therapy, Clinic Internal Medicine IV, Martin-Luther-University, Halle, Germany
2. ²Department of Internal Medicine III, University Hospital Grosshadern, Munich, Germany
3. ³Division of Hematology/Oncology, Harvard Institutes of Medicine, Boston, MA, USA
4. ⁴Andrology Section, Clinic Krollwitz, Martin-Luther-University, Halle, Germany

Correspondence: Professor Dr G Behre, Bone Marrow Transplantation Unit, State Center for Cell and Gene Therapy, Clinic Internal Medicine IV, Martin-Luther-University, Ernst-Grube-Str. 40, 06097 Halle, Germany. E-mail: gerhard.behre@medizin.uni-halle.de

Received 26 April 2006; Revised 10 September 2006; Accepted 14 September 2006; Published online 2 November 2006.

Abstract

The transcription factor CCAAT/enhancer binding protein a (C/EBP) 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/EBP 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/EBP in our screen. We confirmed the in vivo interaction of C/EBP with Max and showed that this interaction involves the basic region of C/EBP. Endogenous C/EBP 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/EBP on a minimal promoter. A chromatin immunoprecipitation assay revealed occupancy of the human C/EBP promoter in vivo by Max and Myc under cellular settings and by C/EBP and Max under retinoic acid induced granulocytic differentiation. Interestingly, enforced expression of Max and C/EBP 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/EBP, indicating the importance of C/EBP–Max in myeloid progenitor differentiation. Taken together, our data reveal Max as a novel co-activator of C/EBP functions, thereby suggesting a possible link between C/EBP and Myc–Max–Mad network.