Visualization by BiFC of different C/EBPβ dimers and their interaction with HP1α reveals a differential subnuclear distribution of complexes in living cells

Abstract

How the co-ordinated events of gene activation and silencing during cellular differentiation are influenced by spatial organization of the cell nucleus is still poorly understood. Little is known about the molecular mechanisms controlling subnuclear distribution of transcription factors, and their interplay with nuclear proteins that shape chromatin structure. Here we show that C/EBPβ not only associates with pericentromeric heterochromatin but also interacts with the nucleoskeleton upon induction of adipocyte differentiation of 3T3-L1 cells. Different C/EBPβ dimers localize in different nuclear domains. Using BiFC in living cells, we show that LAP (Liver Activating Protein) homodimers localize in euchromatin and heterochromatin. In contrast, LIP (Liver Inhibitory Protein) homodimers localize exclusively in heterochromatin. Importantly, their differential subnuclear distribution mirrors the site for interaction with HP1α. HP1α inhibits LAP transcriptional capacity and occupies the promoter of the C/EBPβ-dependent gene c/ebpα in 3T3-L1 preadipocytes. When adipogenesis is induced, HP1α binding decreases from c/ebpα promoter, allowing transcription. Thus, the equilibrium among different pools of C/EBPβ associated with chromatin or nucleoskeleton, as well as dynamic changes in their interaction with HP1α, play key roles in the regulation of C/EBP target genes during adipogenesis.

Unraveling transcription factor interactions with heterochromatin protein 1 using fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy

“…38 While C/EBPα and β share overlapping roles in the regulation of genes involved in energy metabolism, these isoforms have markedly different roles in the control of cellular differentiation and proliferation. The different roles of these BZip family proteins are reflected in the lack of sequence homology in their amino-terminal transactivation domains.…”

“…11 The earlier bimolecular fluorescence complementation study showed that the CD and the hinge region of HP1α could interact with the basic region of C/EBPβ. 38 This prompted us to focus our studies on the BZIP domain of C/EBPα.…”

“…37 More recently, others showed that C/EBPβ, which shares a high degree of homology with C/EBPα in the basic and leucine zipper regions, but not in the transactivation domain, interacts with HP1α. 38 That study demonstrated that the basic region of C/EBPβ interacted with HP1α. Therefore, we next determined whether the BZip domain of C/EBPα was sufficient for the interactions of HP1α.…”

NF-κB Transcriptional Activity Is Modulated by FK506-binding Proteins FKBP51 and FKBP52

“…Chromatin Immunoprecipitation (ChIP)-ChIP was performed following a modification of a previously described method (35). Briefly, proteins and DNA were cross-linked after HEK 293-T cell treatment with 1% formaldehyde for 10 min at 37°C and neutralized with glycine added at a final concentration of 0.125 M. Cells were rinsed twice with cold PBS, scraped, and centrifuged at 800 ϫ g for 5 min at 4°C.…”

C/EBPβ and C/EBPδ transcription factors: Basic biology and roles in the CNS

“…This nuclear relocalization is blocked by MAPK inhibitors (Piwien Pilipuk et al 2003) and by carnosine (Gaya et al 2013). Using bimolecular fluorescence complementation in living cells, it was observed that LAP homodimers localize in euchromatin and heterochromatin whereas LIP homodimers localize exclusively in heterochromatin (Susperreguy et al 2011). Subnuclear relocalization of C/EBPβ has also been reported in macrophages ) and endothelial cells ) but it has been much less characterized.…”