Repression of Transcriptional Activity of C/EBPα by E2F-Dimerization Partner Complexes

Abstract

The transcription factor CCAAT/enhancer-binding protein ␣ (C/EBP␣) coordinates proliferation arrest and the differentiation of myeloid progenitors, adipocytes, hepatocytes, keratinocytes, and cells of the lung and placenta. C/EBP␣ transactivates lineage-specific differentiation genes and inhibits proliferation by repressing E2F-regulated genes. The myeloproliferative C/EBP␣ BRM2 mutant serves as a paradigm for recurrent human C-terminal bZIP C/EBP␣ mutations that are involved in acute myeloid leukemogenesis. BRM2 fails to repress E2F and to induce adipogenesis and granulopoiesis. The data presented here show that, independently of pocket proteins, C/EBP␣ interacts with the dimerization partner (DP) of E2F and that C/EBP␣-E2F/DP interaction prevents both binding of C/EBP␣ to its cognate sites on DNA and transactivation of C/EBP target genes. The BRM2 mutant, in addition, exhibits enhanced interaction with E2F-DP and reduced affinity toward DNA and yet retains transactivation potential and differentiation competence that becomes exposed when E2F/DP levels are low. Our data suggest a tripartite balance between C/EBP␣, E2F/DP, and pocket proteins in the control of proliferation, differentiation, and tumorigenesis.

Functional interaction of CCAAT/enhancer-binding-protein-α basic region mutants with E2F transcription factors and DNA

“…Individual C/EBPα mutations (I294A, R297A, R297Q and R297K) were introduced by Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer's protocol and confirmed by sequencing. The C/EBP responsive -82 cMGF-luciferase reporter as well as HA-tagged DP1, HA-tagged E2F1, and pE2Fx6-TATA-luciferase reporter were previously described [37]. …”

“…The pBabePuro-based retroviral C/EBPα basic region point mutants (BRM2: I294A and R297A) were obtained from Claus Nerlov [31] and cloned into pcDNA3 plasmid as described [37]. WT C/EBPα fused to a C-terminal FLAG-tag was cloned into pcDNA3.…”

“…ChIP and qPCR from differentiated C/EBPα-/-MEFs were performed as described [37]. pPCR primers for the ACSL1 promotor (appr.…”

“…Expression profiling in combination with chromatin immunoprecipitation demonstrated extensive crossregulation between C/EBP and E2F target genes in cell cycle entry during liver regeneration [34]. Disparate interpretations, however, exist about the importance of C/EBPα DNA binding versus binding to E2F-DP complexes, respectively [35][36][37], leaving the mechanism unresolved, bZip C/EBPα mutations and E2F…”

“…Colonies were stained with crystal violet (Sigma) after 6 days. Adipogenesis and shRNA experiments were performed as described [37].…”