Application of electrophoretic mobility shift assay and chromatin immunoprecipitation in the study of transcription in adipose cells.
Chromatin, long thought to be no more than a scaffold supporting DNA compaction inside the cell nucleus, has emerged in the last few years as a major regulatory element involved in the control of gene expression both acutely during interphase and programmatically throughout complex processes of development and differentiation. Adipogenesis is the result of an intertwined network of transcription factors and coregulators with chromatin-modifying activities and offers an excellent model for the study of transcriptional regulation. In this regard, electrophoretic mobility shift assay and immunoprecipitation of chromatin are complementary methods that can be used to study the binding of nuclear proteins to DNA and to characterize how these proteins interact with and modify chromatin to regulate gene expression and, more globally, cell differentiation. This chapter provides some strategies to perform these two assays using 3T3-L1 cells and rodent primary preadipocytes and adipocytes.
Musri
Melina M
MM
Endocrinology and Nutrition Unit, Institut d'Investigacions Biomèdiques August Pi i Sunyer Hospital Clínic, Universitat de Barcelona, Barcelona, Spain.
Gomis
Ramon
R
Parrizas
Marcelina
M
D041721:3T3-L1 Cells; D017667:Adipocytes / Q000502:physiology*; D000818:Animals; D047369:Chromatin Immunoprecipitation / Q000295:instrumentation / Q000379:methods; D024202:Electrophoretic Mobility Shift Assay / Q000295:instrumentation / Q000379:methods; D051379:Mice; D014158:Transcription, Genetic