Nuclear repositioning of the VSG promoter during developmental silencing in Trypanosoma brucei

Abstract

Interphase nuclear repositioning of chromosomes has been implicated in the epigenetic regulation of RNA polymerase (pol) II transcription. However, little is known about the nuclear position-dependent regulation of RNA pol I-transcribed loci. Trypanosoma brucei is an excellent model system to address this question because its two main surface protein genes, procyclin and variant surface glycoprotein (VSG), are transcribed by pol I and undergo distinct transcriptional activation or downregulation events during developmental differentiation. Although the monoallelically expressed VSG locus is exclusively localized to an extranucleolar body in the bloodstream form, in this study, we report that nonmutually exclusive procyclin genes are located at the nucleolar periphery. Interestingly, ribosomal DNA loci and pol I transcription activity are restricted to similar perinucleolar positions. Upon developmental transcriptional downregulation, however, the active VSG promoter selectively undergoes a rapid and dramatic repositioning to the nuclear envelope. Subsequently, the VSG promoter region was subjected to chromatin condensation. We propose a model whereby the VSG expression site pol I promoter is selectively targeted by temporal nuclear repositioning during developmental silencing.

Figure 1.

GFP-LacI tagging of the procyclin locus in the nucleus of the T. brucei procyclic form. (a) In vivo fluorescence detection of the GFP-LacI bound to lac operator sequences (green) inserted into the procyclin locus. DNA was stained with DAPI (blue), and the cell was visualized by phase contrast (gray). A single optical section from a deconvolved two-channel 3D dataset is shown using a 0.5-μm z step (slice animation available as Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200607174/DC1). (a′) Higher magnification of the nucleus showing GFP-LacI and DAPI fluorescence signals. (b) 3D IF analysis of a procyclic-form interphase nucleus revealing localization of the GFP-LacI–tagged procyclin chromosomal locus using an anti-GFP monoclonal antibody (green) and localization of pol I using a polyclonal antibody against pol I (TbRPA1; red). DAPI staining (blue) reveals the nucleus as well as the mitochondrial DNA and indirectly indicates the position of the nucleolus because of its lack of DAPI staining. Pol I is exclusively localized in the nucleolus and displays a characteristic U-shape structure. The procyclin locus is detected in the nucleolar periphery in living and fixed cells. Maximum intensity projection of a three-channel 3D stack (b) or maximum anti-GFP intensity slice (b′) are shown. Dotted line indicates the nuclear outline. Bars, 1 μm.

Figure 2.

BrUTP labeling of nascent RNA in permeabilized procyclic-form nuclei. Double IF using anti–pol I antiserum (red) and a monoclonal antibody against BrdUTP (green) together with DNA staining using DAPI (blue) in PFA-fixed cells. (a) Total nuclear transcription in situ as revealed by the incorporation of BrUTP into nascent RNA in the absence of α-amanitin. (b) BrUTP-labeled nascent RNA transcribed by pol I in the presence of 100 μg/ml α-amanitin. Pol I–mediated transcription is confined to several perinucleolar foci (green), indicating that pol I transcription occurs largely along the periphery of the nucleolus. Single deconvolved slices are shown. Bars, 1 μm.

Figure 3.

Localization of a GFP-LacI–tagged rDNA chromosomal site in the procyclic and bloodstream forms of T. brucei. (a) Localization of the rDNA locus tagged with lac operators in the procyclic form visualized by an anti-GFP monoclonal antibody (green) and anti–pol I antiserum (red) in the nucleolus (arrow). (b) As in panel a, but using bloodstream-form parasites. Anti-pol antibody (red) stained the nucleolus (arrow) and the extranucleolar pol I–containing body, the ESB (arrowhead). In both developmental stages, the rDNA chromosomal sites are located in the nucleolar periphery. (c) The ESB (arrow) is associated with the GFP-tagged active VSG ES promoter, which was detected by double labeling using anti–pol I (red) and anti-GFP (green) antibodies in the PFA-fixed bloodstream form. Note the U-shape distribution of pol I in the nucleolus (arrowhead). Maximum intensity projections of 3D stacks (a–c) or maximum anti-GFP intensity slices (a′–c′) are shown. Dotted lines indicate the nuclear outline. Bars, 1 μm.

Figure 4.

Changes in nuclear localization and chromatin accessibility of the active ES promoter upon in vitro differentiation. (a) The GFP-tagged active ES promoter (green) localized to the nuclear periphery upon differentiation, as indicated by double labeling with an anti-GFP monoclonal antibody (green), anti–pol I antiserum (red), and DAPI staining (blue). 5 h after the induction of differentiation, the extranucleolar ESB is not detectable, and the GFP-tagged active VSG ES is now located at the nuclear envelope (as determined by the edge of DAPI staining). (b) In contrast, the GFP-LacI–tagged rDNA chromosomal site does not show nuclear repositioning. Maximum intensity projections of three-channel 3D datasets (a and b) or maximum anti-GFP intensity slices (a′ and b′) are shown. Dotted lines indicate the nuclear outline. Bars, 1 μm. (c) Statistical analysis on the number of GFP dot–positive cells where the GFP-LacI dot is in contact with the nuclear periphery in cells tagged in the rDNA, 121VSG BC, inactive ES, or active ES promoter loci. BSF, bloodstream form. (d) Statistical analysis of the number of GFP-expressing cells in which a clear GFP-LacI dot is visible in cells tagged in the rDNA, 121VSG BC, inactive ES, or active ES promoter regions. The profound lack of detection of the GFP-LacI bound to chromatin in the active ES promoter suggests a reduced chromosomal accessibility of this locus upon differentiation.