Proteomic discovery of Max as a novel interacting partner of C/EBPalpha: a Myc/Max/Mad link

Authors:
Zada AA, Pulikkan JA, Bararia D, Geletu M, Trivedi AK, Balkhi MY, Hiddemann WD, Tenen DG, Behre HM, Behre G
In:
Source: Leukemia
Publication Date: (2006)
Issue: 20(12): 2137-2146
Research Area:
Cancer Research/Cell Biology
Immunotherapy / Hematology
Cells used in publication:
U-937
Species: human
Tissue Origin: blood
CD34+ cell, human
Species: human
Tissue Origin: blood
Platform:
Nucleofectorâ„¢ I/II/2b
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.