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Visualization by BiFC of different C/EBP? dimers and their interaction with HP1? 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/EBP? not only associates with pericentromeric heterochromatin but also interacts with the nucleoskeleton upon induction of adipocyte differentiation of 3T3-L1 cells. Different C/EBP? 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 HP1?. HP1? inhibits LAP transcriptional capacity and occupies the promoter of the C/EBP?-dependent gene c/ebp? in 3T3-L1 preadipocytes. When adipogenesis is induced, HP1? binding decreases from c/ebp? promoter, allowing transcription. Thus, the equilibrium among different pools of C/EBP? associated with chromatin or nucleoskeleton, and dynamic changes in their interaction with HP1?, play key roles in the regulation of C/EBP target genes during adipogenesis.

SUBMITTER: Susperreguy S

PROVIDER: S-EPMC3138133 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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

Susperreguy Sebastián S Prendes Luciana P LP Desbats María A MA Charó Nancy L NL Brown Karen K MacDougald Ormond A OA Kerppola Tom T Schwartz Jessica J Piwien-Pilipuk Graciela G

Experimental cell research 20101128 6

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 inducti ...[more]

PMID: 21122806

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Project description:Bimolecular Fluorescence Complementation (BiFC) assay is a method used to directly visualize protein-protein interaction in vivo using live-cell imaging or fixed cells. This protocol described here is based on our recent paper describing the functional association of human chromatin adaptor and transcription cofactor Brd4 with p53 tumor suppressor protein (Wu et al., 2013). BiFC was first described by Hu et al. (2002) using two non-fluorescent protein fragments of enhanced yellow fluorescent protein (EYFP), which is an Aequorea victoria GFP variant protein, fused respectively to a Rel family protein and a bZIP family transcription factor to investigate interactions between these two family members in living cells. The YFP was later improved by introducing mutations to reduce its sensitivity to pH and chloride ions, thus generating a super-enhanced YFP, named Venus fluorescent protein, without showing diminished fluorescence at 37 °C as typically observed with EYFP (Nagai et al., 2006). The fluorescence signal is regenerated by complementation of two non-fluorescent fragments (e.g., the Venus N-terminal 1-158 amino acid residues, called Venus-N, and its C-terminal 159-239 amino acid residues, named Venus-C; see Figure 1A and Gully et al., 2012; Ding et al., 2006; Kerppola, 2006) that are brought together by interaction between their respective fusion partners (e.g., Venus-N to p53, and Venus-C to the PDID domain of human Brd4; see Figure 1B and 1C). The intensity and cellular location of the regenerated fluorescence signals can be detected by fluorescence microscope. The advantages of the proximity-based BiFC assay are: first, it allows a direct visualization of spatial and temporal interaction between two partner proteins in vivo; second, the fluorescence signal provides a sensitive readout for detecting protein-protein interaction even at a low expression level comparable to that of the endogenous proteins; third, the intensity of the fluorescence signal is proportional to the strength of protein-protein interaction (Morell et al., 2008); and fourth, the BiFC signals are derived from intrinsic protein-protein interaction, rather than from extrinsic fluorophores that may not reflect true protein-protein interaction due to their nonspecific association with cellular macromolecules or subcellular compartments. However, some limitations of BiFC include slow maturation (T1/2 ~ 1 h) of an eventually stable BiFC complex (Hu et al., 2002), making it unsuitable for real-time observation of transient interaction that disappears prior to BiFC detection, and enhanced BiFC background at high expression levels due to fusion-independent association between two non-fluorescent fragments association. BiFC signals generated by in vivo protein-protein interaction can be validated by amino acid mutation introduced at the protein-protein contact surfaces. This imaging technique has been widely used in different cell types and organisms (Kerppola, 2006).

Dataset Information

Visualization by BiFC of different C/EBP? dimers and their interaction with HP1? reveals a differential subnuclear distribution of complexes in living cells.

Publications

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

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