Identification of a Co-repressor That Inhibits the Transcriptional and Growth-Arrest Activities of CCAAT/Enhancer-binding Protein α

Abstract

We used a yeast two-hybrid screening approach to identify novel interactors of CCAAT/enhancer-binding protein ␣ (C/EBP␣) that may offer insight into its mechanism of action and regulation. One clone obtained was that for CA150, a nuclear protein previously characterized as a transcriptional elongation factor. In this report, we show that CA150 is a widely expressed co-repressor of C/EBP proteins. Two-hybrid and co-immunoprecipitation analyses indicated that CA150 interacts with C/EBP␣. Overexpression of CA150 inhibited the transactivation produced by C/EBP␣ and was also able to reverse the enhancing effect of the co-activator p300 on C/EBP␤-mediated transactivation. Analysis of C/EBP␣ mutants indicated that CA150 interacts with C/EBP␣ primarily through a domain spanning amino acids 135-150. Chromatin immunoprecipitation assays showed that CA150 was present on a promoter that is repressed by C/EBP␣ but not present on a promoter that is activated by C/EBP␣. Finally, we showed that in cells in which growth arrest had been induced by ectopic expression of C/EBP␣, CA150 was able to release them from growth arrest. Interestingly, CA150 could not reverse the growth arrest produced by the minimal growth-arrest domain of C/EBP␣ (amino acids 175-217), suggesting that the effect of CA150 was directed at a region of C/EBP␣ outside of this minimal domain, consistent with our two-hybrid analysis. Taken together, these data indicate that CA150 is a co-repressor of C/EBP proteins and provides a possible mechanism for how C/EBP␣ can repress transcription of specific genes. CCAAT/enhancer binding proteins (C/EBPs)2 are eukaryotic transcription factors that regulate a large number of genes. There are eight members in the C/EBP protein family, and the first two members that were identified, C/EBP␣ and C/EBP␤, are the most extensively examined and characterized (1). Although they are expressed in a variety of cell types, both family members are enriched in liver and have been demonstrated to regulate the expression of a number of genes that are associated with energy metabolism (2).C/EBP␣ and -␤ have been generally observed to be activators of transcription. The transactivation domains of these proteins reside in the N terminus of each protein, and can function independently of the basic region-leucine zipper DNA-binding domain if linked to a heterologous DNA-binding domain (1). There appear to be several sub-domains within the transactivation domains that mediate transactivation (3)(4)(5)(6)(7)(8), although the precise boundaries of these regions are difficult to determine from the available studies because they are assigned based on the arbitrary design of the mutants used. For C/EBP␣, studies indicate that the majority of the transactivation potential lies within amino acid residues 1-150 (5), whereas for C/EBP␤, the transactivation domain appears to lie within amino acid residues 1-108 (9).In addition to its transcriptional role, C/EBP␣ is also a strong inhibitor of cell proliferation. It inhibits proliferation of cultured cells...

Growth Hormone Corrects Proliferation and Transcription of Phosphoenolpyruvate Carboxykinase in Livers of Old Mice via Elimination of CCAAT/Enhancer-binding Protein α-Brm Complex

“…This observation is consistent with previous publications showing that C/EBP␣ negatively regulates the HNF6 promoter through a direct binding (9,20). Recent work from Roesler's group (27) suggested that this C/EBP␣-mediated negative regulation involves a recruitment of CA150 protein to the HNF6 promoter. Taken together, the studies of effects of GH on the expression of liver-specific genes showed that GH restores transcriptional activities of C/EBP␣.…”

C/EBPα and DEK coordinately regulate myeloid differentiation

“…C/EBP␣ functions as a primary molecular determinant of granulopoiesis and mediates the differentiation of granulocyte-monocyte progenitors through interactions with a diverse set of chromatin modifiers and transcription factors that collectively activate (CBP/p300, 10 c-Jun, 11 histone acetyltransferase TIP60, 12 and Max 13 ) or repress (CDP/cut 45 and CA150 46 ) specific target genes. Posttranslational modifications represent an additional regulatory mechanism for C/EBP␣ function.…”

Complexity of CEBPA Dysregulation in Human Acute Myeloid Leukemia

“…Also, the impact of repressors of CEBPA activity, such as CA150, has to be tested in AML as well as the role of CEBPA acetylation or changes in subcellular localization during malignant transformation (49)(50)(51).…”