Repression and Coactivation of CCAAT/Enhancer-binding Protein ϵ by Sumoylation and Protein Inhibitor of Activated STATx Proteins

Abstract

CCAAT/enhancer-binding protein ⑀ (C/EBP⑀) is a neutrophil-specific transcription factor whose activity is controlled by juxtaposed activating and regulatory domains. We previously determined that the function of the major regulatory domain (RD1) in C/EBP⑀ was dependent on the integrity of a five-amino acid motif that was identical to the recognition site for members of the small ubiquitin-like modifier (SUMO) family of ubiquitin-related proteins. We show here that the SUMO attachment site (the regulatory domain motif) is necessary and sufficient both for the intrinsic inhibitory function of RD1 and for coactivation by PIASx␣ and PIASx␤, two members of the protein inhibitor of activated STAT (PIAS) family of SUMO E3 ligases. PIASx␤ was a more potent coactivator than PIASx␣ of both fulllength C/EBP⑀ and fusion proteins containing the Nterminal portion of C/EBP⑀, whereas PIASx␣ was more active on fusion proteins containing a heterologous activation domain. Two modes of coactivation were observed, one that was dependent on the integrity of the RING finger (RF) domain and was shared by both PIASx␣ and PIASx␤ and a second mode that was independent of the RF and was only observed with PIASx␤. Sumoylation of C/EBP⑀ was enhanced by coexpression of PIASx␣, suggesting that this modification is associated with the enhanced activity of the target protein. These results suggest that a complex interplay of accessory factors, including SUMO and PIAS proteins, modulates the activity of C/EBP⑀.

Acetylation of C/EBPε is a prerequisite for terminal neutrophil differentiation

“…K121 is located in the repression domain I of C/EBPe, which has previously been identified as a key element of the conserved repression motif (RDM) of various C/EBP family members, including C/EBPb. 21,31 The regulation of C/EBPe transcriptional activity has previously been associated with levels of K121 sumoylation, suggesting a delicate balance between repression and the recruitment of coactivators, resulting in increased C/EBPe transcriptional activity. 30,31 According to our data, acetylation of K121 is important for DNA binding and transcriptional activity.…”

“…21,31 The regulation of C/EBPe transcriptional activity has previously been associated with levels of K121 sumoylation, suggesting a delicate balance between repression and the recruitment of coactivators, resulting in increased C/EBPe transcriptional activity. 30,31 According to our data, acetylation of K121 is important for DNA binding and transcriptional activity. This was confirmed by increased C/EBPe activity in luciferase reporter assays and the rescue of DNA binding using a C/EBPe mutant in which lysine 121 was replaced by a glutamine residue (K121Q), mimicking acetylation (supplemental Figures 4C and 6).…”

“…28,29 Sumoylation of lysine residues within the repression domain has been linked to a conserved repression motif of C/EBPe, similar to other C/EBP family members. 30,31 Recently, Kyme et al have demonstrated that C/EBPe can be acetylated in mature peripheral blood neutrophils. 32 Here we demonstrate that C/EBPe acetylation is required for neutrophil development.…”

Human C/EBP-ϵ activator and repressor isoforms differentially reprogram myeloid lineage commitment and differentiation

“…The RDI domain contains a highly conserved "VKEEP" sumoylation consensus sequence (boxed in panel B), through which sumoylation increases the transcriptional activity of the murine activator isoforms. 23,49,50 C/EBP-␣ and C/EBP-␤ in a dose-dependent manner 2 and inhibits endogenous MBP1 gene transcription in AML14.3D10 eosinophils transduced with an HIV Tat-C/EBP-⑀ 14 fusion protein 22 support a repressor role for this isoform.…”

“…Functional domains and sequence alignments of the C/EBP-⑀ isoforms. Functional domains and predicted transcriptional activities of the 4 C/EBP-⑀ isoforms (32,30,27, and 14 kDa), based on mutagenesis and transactivation studies of the human and murine full-length isoforms 16,23,49,50 and shorter human isoforms, 2,22 are shown schematically in panel A. Alignments of their amino acid sequences and locations of the various transactivation, repression, and bZIP domains are shown in panel B. All 4 isoforms are identical at their carboxyl terminus, which encodes the RDII repressor, basic DNA binding, and bZIP dimerization domains.…”