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

Jinyong Kim,Savitha Sharma,Yamin Li,Everardo Cobos,Jorma J Palvimo,Simon C Williams

doi:10.1074/jbc.m413771200

Abstract

CCAAT/enhancer-binding protein epsilon (C/EBPepsilon) 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/EBPepsilon 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 PIASxalpha and PIASxbeta, two members of the protein inhibitor of activated STAT (PIAS) family of SUMO E3 ligases. PIASxbeta was a more potent coactivator than PIASxalpha of both full-length C/EBPepsilon and fusion proteins containing the N-terminal portion of C/EBPepsilon, whereas PIASxalpha 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 PIASxalpha and PIASxbeta and a second mode that was independent of the RF and was only observed with PIASxbeta. Sumoylation of C/EBPepsilon was enhanced by coexpression of PIASxalpha, 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/EBPepsilon.

Highlights

The CCAAT/enhancer-binding proteins form a subgroup within the basic region/leucine zipper superfamily of transcriptional regulatory proteins [1, 2]
To test whether the regulatory domain motif (RDM) was sufficient for transcription repression in the context of Gal4 fusion proteins, we constructed expression vectors encoding Gal4 fusion proteins consisting of the C/EBP⑀ AD1 region joined to a short peptide containing either the wild type RDM sequence (VKEEP) or a variant sequence in which the lysine that serves as the attachment site for small ubiquitin-like modifier (SUMO) proteins was changed to arginine (VREEP)
We report that two members of the protein inhibitor of activated STAT (PIAS) family of RING finger domain proteins function as differential coactivators of the neutrophil-specific transcription factor C/EBP⑀

Summary

The abbreviations used are

C/EBP, CCAAT/enhancer-binding protein; RD1 and -2, regulatory domain 1 and 2, respectively; SUMO, small ubiquitin-like modifier; RDM, regulatory domain motif; PIAS, protein inhibitor of activated STAT; RF, RING finger; AD, activation domain; STAT, signal transducers and activators of transcription; NTA, nitrilotriacetic acid; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase. Sequence comparisons of RD1 domains from each C/EBP revealed a conserved five-amino acid motif with the consensus sequence (I/V/L)KXEP We named this sequence the regulatory domain motif (RDM) and noted its similarity both to the synergy control element initially found in members of the nuclear hormone receptor superfamily [23, 24] and to the consensus sequence for attachment of SUMO proteins [25]. We showed previously that the RDM of C/EBP⑀ was an attachment site for SUMO-1 and that the three other C/EBPs that contain RDM-like sequences (C/EBP␣, C/EBP␤, and C/EBP␦) were sumoylation targets [22]. We have extended our earlier studies on the regulation of C/EBP⑀ by sumoylation to attempt to define the mechanism by which C/EBP⑀ activity is controlled

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION