Proteomic Discovery of Max as a Novel Interacting Partner of C/EBPa: A Myc/Max/Mad Link.

In the present study, we sought to identify novel C/EBPa 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/EBPa in our screen. We confirmed the in vivo interaction of C/EBPa with Max and showed that this interaction involves the basic region of C/EBPa. Endogenous C/EBPa and Max, but not Myc and Max co-localize in intranuclear structures during granulocytic differentiation of myeloid U937 cells. Max enhanced the transactivation capacity of C/EBPa on a minimal promoter. A chromatin-immunoprecipitation assay revealed occupancy of the human C/EBPa promoter in vivo by Max under cellular settings and by C/EBPa and Max under retinoic acid induced granulocytic differentiation. Interestingly, enforced expression of Max and C/EBPa, results in granulocytic differentiation of the human hematopoietic CD34+ cells as evidenced by CD11b and CD15 expression and by real-time PCR for various genes. Silencing of Max by short hairpin RNA in CD34+ and U937 cells strongly reduced the differentiation-inducing potential of C/EBPa, indicating the importance of C/EBPa-Max in myeloid progenitor differentiation. Taken together, our data reveal Max as a novel co-activator of C/EBPa functions thereby suggesting a possible link between CEBPa and Myc-Max-Mad network.