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

Department of Molecular & Integrative Physiology (T.X.C., G.P.-P., J.K., J.S.), Program in Cellular and Molecular Biology (J.S.H., J.S.), Departments of Biological Chemistry and Obstetrics & Gynecology (R.K.), University of Michigan, Ann Arbor, Michigan 48109

Address all correspondence and requests for reprints to: Dr. Jessica Schwartz, Department of Molecular & Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0622. E-mail: jeschwar{at}umich.edu.

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

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