Endogenous CCAAT/Enhancer Binding Protein β and p300 Are Both Regulated by Growth Hormone to Mediate Transcriptional Activation

Abstract

The regulation of c-fos transcription by GH involves multiple factors, including CCAAT/enhancer binding protein (C/EBP) beta. Knockdown of C/EBPbeta by RNA interference prevents stimulation of endogenous c-fos mRNA by GH, indicating a key role for C/EBPbeta in GH-stimulated c-fos transcription. GH rapidly increases the occupancy of both endogenous C/EBPbeta and p300 on the c-fos promoter in 3T3-F442A preadipocytes as indicated by chromatin immunoprecipitation. The transient occupancy of p300 on c-fos and the presence of p300 in the anti-C/EBPbeta immunoprecipitate coincide with the transient increase in c-fos transcription with GH, suggesting that a nuclear complex containing both p300 and C/EBPbeta occupies the c-fos promoter in response to GH. Expression of p300 with C/EBPbeta markedly increases c-fos promoter activity when neither alone is effective, indicating that p300 coactivates C/EBPbeta-mediated c-fos promoter activation. Such coactivation can determine a baseline for c-fos activation by GH. Furthermore, the occupancy of phosphorylated murine C/EBPbeta (T188) on c-fos upon GH treatment is simultaneous with increased occupancy by p300, suggesting that phospho-C/EBPbeta recruits p300 in response to GH. Thus, endogenous C/EBPbeta and p300 on c-fos are dynamically regulated by GH to determine transcriptional activation. Phosphorylated C/EBPbeta and p300 appear to function as part of a regulated complex that mediates GH-stimulated transcription.

Stress Activation of Mammary Epithelial Cell Xanthine Oxidoreductase Is Mediated by p38 MAPK and CCAAT/Enhancer-binding Protein-β

“…Chromatin Immunoprecipitation (ChIP), PCR, and Quantitation-ChIP analysis was performed essentially as described (23)(24)(25) using the EZ-ChIP kit from Upstate (catalog no. 17-371) and the modifications indicated here.…”

“…C/EBP␤-specific ChIP (23)(24)(25) was performed on HC11 cells that were untreated (control), exposed to 10 g/ml cycloheximide for 5 h, or treated with 20 M SB202190 for 45 min and then exposed to cycloheximide for 5 h. PCR products obtained from C/EBP␤-specific immunoprecipitation (antibody C-19) showed magnesium and DNA concentration dependence, and PCR band intensities were increased by 2.2-8-fold by cycloheximide (Fig. 7D).…”

CCAAT/Enhancer-binding Protein (C/EBP) β Is Acetylated at Multiple Lysines

“…SDS protein dye (50 mM Tris, 1% SDS, 0.001% bromphenol blue, 10% glycerol, 10% ␤-mercaptoethanol) was then added to the beads, and samples were boiled, separated by SDS-PAGE, and immunoblotted as described previously (55). Bands on immunoblots were visualized using IRDye 700-coupled antimouse IgG (1:10,000) or IRDye 800-coupled anti-rabbit IgG (1:10,000) on the Odyssey infrared scanning system (LI-COR, Inc., Lincoln, NE) as described previously (47). Molecular weight was estimated using Kaleidoscope protein molecular weight standard (Bio-Rad).…”

“…Cells were lysed in Lysis Buffer; samples were immunoprecipitated with anti-HA as described and analyzed by immunoblotting with anti-Ac-K and anti-C/ EBP␤. Membranes were scanned using the Odyssey infrared scanning system (47), and bands were quantified using Odyssey software. Acetylation of C/EBP␤ was calculated using values for acetylation (anti-Ac-K) divided by total C/EBP␤ (anti-C/EBP␤ or anti-HA).…”

“…Using an antiacetyl-lysine antibody that recognizes acetylated Lys-39, endogenous C/EBP␤ was found to be acetylated in preadipocytes, in which C/EBP␤ is an important factor during adipogenesis. C/EBP␤ is a critical mediator of GH-regulated transcription of c-fos (46,47). A nonacetylatable mutation at Lys-39 of C/EBP␤ impaired GH-stimulated c-fos promoter activation, and GH was found to increase acetylation of C/EBP␤.…”