Structure/function analysis of C/EBPα and C/EBPβ

Abstract

CCAAT/enhancer binding proteins (C/EBPs) are modular transcription factors that play important roles in energy metabolism. For example, C/EBP transcription factors are important regulators of the expression of phosphoenolpyruvate carboxykinase (PEPCK), a key regulatory enzyme in gluconeogenesis in the liver. In support of this, C/EBP knockout analysis and antisense methodologies have uncovered a role for C/EBPα in mediating the cAMP responsiveness of the PEPCK gene promoter. The work in this thesis is dedicated to defining the regions within C/EBPα and C/EBPα that mediate the constitutive and PKA-inducible activity towards the PEPCK promoter.

Examination of the regions of C/EBPα and C/EBPβ previously characterized to mediate a robust PKA-inducible response identified three conserved regions (CRs). Deletion analysis, in conjunction with GAL4 methodology and dominant negative transfection analysis, showed that CR2 (amino acids (aa) 61-70) was critical for mediating the PKA-inducible activity when C/EBPα occupied the distal sites on the PEPCK promoter. However, CR3(aa96-106) and amino acids 6-50 of C/EBPα were found to be important for mediating the PKA-inducible response when it bound to the cAMP Response Element site (CRE). In contrast, the CRs within C/EBPβ did not appear to contribute to the PKA-inducible activity regardless of where it bound to the PEPCK promoter.

GST-pull down analysis indicated that aa 6-50 and CR3, but not CR2, were required for C/EBPα to physically interact with TATA-binding protein (TBP) and Transcription Factor II B (TFIIB). This protein-protein analysis, in conjunction with transient transfection analysis, suggested that the domains within C/EBPα that mediate the constitutive and PKA inducible activities vary depending on which cis-element it occupies on the PEPCK promoter. For example, when C/EBPα binds to the CRE site, amino acids 6-50 and CR3 may mediate the constitutive and PKA-inducible activity through a mechanism that may involve physical interaction with TBP and TFIIB. However, when C/EBPα binds to the distal promoter region, CR2 may mediate the PKA-inducible response by interacting with different proteins, possibly a co-activator. The data suggest that the precise location of a transcription factor on a promoter may influence which domains within the protein are employed to mediate transactivation.