Spatial Distribution and Function of Sterol Regulatory Element-Binding Protein 1a and 2 Homo- and Heterodimers by In Vivo Two-Photon Imaging and Spectroscopy Fluorescence Resonance Energy Transfer

Abstract

Sterol regulatory element-binding proteins (SREBPs) are a subfamily of basic helix-loop-helix-leucine zipper proteins that regulate lipid metabolism. We show novel evidence of the in vivo occurrence and subnuclear spatial localization of both exogenously expressed SREBP-1a and -2 homodimers and heterodimers obtained by two-photon imaging and spectroscopy fluorescence resonance energy transfer. SREBP-1a homodimers localize diffusely in the nucleus, whereas SREBP-2 homodimers and the SREBP-1a/SREBP-2 heterodimer localize predominantly to nuclear speckles or foci, with some cells showing a diffuse pattern. We also used tethered SREBP dimers to demonstrate that both homo- and heterodimeric SREBPs activate transcription in vivo. Ultrastructural analysis revealed that the punctate foci containing SREBP-2 are electron-dense nuclear bodies, similar or identical to structures containing the promyelocyte (PML) protein. Immunofluorescence studies suggest that a dynamic interplay exists between PML, as well as another component of the PML-containing nuclear body, SUMO-1, and SREBP-2 within these nuclear structures. These findings provide new insight into the overall process of transcriptional activation mediated by the SREBP family.

ACKNOWLEDGMENTS

We acknowledge Bruce J. Tromberg, director of the Laser Microbeam and Medical Program; Michael W. Berns, Medical Free Electron Laser Program; and Kathryn E. Osann for statistical analysis and expert advice and Tatiana B. Krasieva, Linda Li, Katie Lee, and Marisa Magana for technical expertise.

This work was supported by the National American Heart Association (993005N to V.L.); the National Institutes of Health (HL48044 to T.O.); the National Institutes of Health Research Resource, Laser Microbeam, and Medical Program (P41R01192); and the Air Force of Scientific Research, Medical Free Electron Laser Program (FA9550-04-1-01-01).