CCAAT/enhancer-binding proteins (C/EBPs) are basic region/leucine zipper transcription factors that function as regulators of cell growth and differentiation in numerous cell types. We previously localized transcriptional activation and inhibitory regions in one family member, C/EBP⑀. Here we describe the further characterization of a C/EBP⑀ inhibitory domain termed regulatory domain I. We show that functionally related domains are present in C/EBP␣, C/EBP␤, and C/EBP␦. These domains contain an evolutionarily conserved five-amino acid motif (the regulatory domain motif (RDM)) that conforms to the consensus sequence (I/V/ L)KXEP. Mutagenesis studies revealed that the residues at positions 1, 2, and 4 of the RDM are critical for inhibitory domain function. Data base searches identified RDM-like sequences in a number of nuclear proteins. We found that small regions from c-Jun, JunB, and JunD containing this sequence also function as transcriptional inhibitory domains. Importantly, the RDM is similar to the recognition sequence for attachment of the ubiquitin-like protein, small ubiquitin-like modifier-1 (SUMO-1), and the conserved lysine residue of each C/EBP RDM served as an attachment site for SUMO-1. SUMO-1 attachment decreased the inhibitory effect of the C/EBP⑀ regulatory domain, suggesting that sumoylation may play an important role in modulating C/EBP⑀ activity as well as that of the other C/EBP family members.

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⑀.