Subnuclear localization of C/EBP beta is regulated by growth hormone and dependent on MAPK.
Localization of transcription regulatory proteins in the nucleus is dynamically regulated, and may alter nucleoplasmic concentrations and/or assembly of multimolecular transcription regulatory complexes, which ultimately regulate gene expression. Since growth hormone (GH) regulates multiple transcription factors including C/EBP beta, the effect of GH on the subcellular localization of C/EBP beta was examined in 3T3-F442A preadipocytes. Indirect immunofluorescence shows that C/EBP beta is diffusely distributed in nuclei of quiescent cells. Within 5 min of GH treatment, the diffuse pattern dramatically becomes punctate. The relocalization of C/EBP beta coincides with DAPI staining of heterochromatin. Further, C/EBP beta and heterochromatin protein (HP)-1 alpha colocalize in the nucleus, consistent with localization of C/EBP beta to pericentromeric heterochromatin. In contrast, C/EBP delta exhibits a diffuse distribution in the nucleus that is not modified by GH treatment. C/EBP beta is rapidly and transiently phosphorylated on a conserved MAPK consensus site in response to GH (Piwien-Pilipuk, G., MacDougald, O. A., and Schwartz, J. (2002) J. Biol. Chem. 277, 44557-44565). Indirect immunofluorescence using antibodies specific for C/EBP beta phosphorylated on the conserved MAPK site shows that GH also rapidly induces a punctate pattern of staining for the phosphorylated C/EBP beta. In addition, phosphorylated C/EBP beta colocalizes to pericentromeric heterochromatin. The satellite DNA present in heterochromatin contains multiple C/EBP binding sites. DNA binding analysis shows that C/EBP beta, C/EBP delta, and C/EBP alpha (p42 and p30 forms) can bind to satellite DNA as homo- or heterocomplexes in vitro. Importantly, GH rapidly and transiently increases binding of endogenous C/EBP beta from 3T3-F442A cells to satellite DNA. Further, the GH-promoted nuclear relocalization of C/EBP beta to pericentromeric heterochromatin was prevented by the MEK inhibitor U0126. This observation suggests that GH-dependent MAPK activation plays a role in the regulation of nuclear relocalization of C/EBP beta. Nuclear redistribution introduces a new level of transcriptional regulation in GH action, since GH-mediated phosphorylation and nuclear redistribution of C/EBP beta may be coordinated to achieve spatial-temporal control of gene expression.
Piwien Pilipuk
Graciela
G
Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan 48109, USA.
Galigniana
Mario D
MD
Schwartz
Jessica
J
D016475:3T3 Cells; D017667:Adipocytes / Q000166:cytology / Q000502:physiology; D000818:Animals; D001665:Binding Sites; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000378:metabolism*; D002453:Cell Cycle / Q000502:physiology*; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000502:physiology*; D004276:DNA, Satellite / Q000378:metabolism; D006570:Heterochromatin / Q000502:physiology*; D019382:Human Growth Hormone / Q000502:physiology*; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010766:Phosphorylation
D022782:CCAAT-Enhancer-Binding Protein-beta; D004276:DNA, Satellite; D006570:Heterochromatin; D019382:Human Growth Hormone; D020928:Mitogen-Activated Protein Kinases