The INTACT method for cell type-specific gene expression and chromatin profiling in Arabidopsis thaliana

Abstract

Genomic studies of cell differentiation and function within a whole organism depend on the ability to isolate specific cell types from a tissue, but this is technically difficult. We developed a method called INTACT (isolation of nuclei tagged in specific cell types) that allows affinity-based isolation of nuclei from individual cell types of a tissue, thereby circumventing the problems associated with mechanical purification techniques. In this method nuclei are affinity-labeled through transgenic expression of a biotinylated nuclear envelope protein in the cell type of interest. Total nuclei are isolated from transgenic plants and biotin-labeled nuclei are then purified using streptavidin-coated magnetic beads, without the need for specialized equipment. INTACT gives high yield and purity of nuclei from the desired cell types, which can be used for genome-wide analysis of gene expression and chromatin features. The entire procedure, from nuclei purification through cDNA preparation or chromatin immunoprecipitation (ChIP), can be completed within 2 d. The protocol we present assumes that transgenic lines are already available, and includes procedural details for amplification of cDNA or ChIP DNA prior to microarray or deep sequencing analysis.

Figure 1.. Nuclear targeting fusion (NTF) protein and transgenic lines for the INTACT system

(a) The three-part structure of the NTF is shown. The chimeric protein consists of the WPP domain of RanGAP1 for nuclear envelope targeting, green fluorescent protein (GFP) to allow visualization, and the biotin ligase recognition peptide (BLRP), which is biotinylated by BirA. (b) Confocal projection image of an Arabidopsis root expressing the NTF in the epidermal non-hair cells. GFP is shown in green and cell walls are shown in red. Scale bar is approximately 20 μm. (c) Confocal section of a root expressing the NTF in non-hair cells, showing localization to the nuclear envelope. Scale bar is approximately 5 μm. (d) Streptavidin western blots of protein extracts from plants expressing BirA only or both the NTF and BirA. Asterisk indicates the position of the 42 kDa NTF. Bands found in both BirA only and NTF/BirA protein extracts are endogenous biotinylated proteins. Molecular weights (in kDa) are indicated on the right.

Figure 2.. Purification of tagged nuclei with the INTACT system

(a) Diagram of the apparatus used for purification of bead-bound nuclei. The core of the device consists of a 1 ml micropipette tip inserted into a groove in the surface of a miniMACS magnet. After binding of magnetic streptavidin-coated beads to biotinylated nuclei in solution, this mixture of beads and nuclei is diluted and drawn into a 10 ml serological pipette, and the pipette is then attached to the 1 ml tip in the magnet. The mixture of beads and nuclei in the 10 ml pipette is allowed to flow slowly past the magnet, allowing capture of beads and nuclei on the wall of the 1 ml tip. Once the liquid is drained, the 1 ml tip is attached to a pipette and the purified nuclei and beads are released into solution by drawing liquid into and out of the pipette tip. (b) Fluorescence microscopy images of NTF-labeled nuclei that have been bound by streptavidin-coated magnetic beads prior to capture on a magnet. DNA is shown in blue and GFP fluorescence of the NTF, as well as fluorescence from the beads themselves, are shown in green. Scale bar is approximately 3 μm in each panel. The right panel is at a higher magnification than the left and center panels. (c) Low magnification brightfield images of bead-bound nuclei and free beads after magnetic purification. The left panel shows the purified material from a transgenic line expressing NTF and BirA in root epidermal non-hair cells, while the right panel shows that from a line expressing only BirA. Labeled nuclei become fully coated with beads during capture on the magnet and therefore appear as conspicuous clusters of beads after purification. One such bead cluster is highlighted by a red dotted box in the NTF/BirA panel. No beads clusters are seen in the BirA only panel, indicating that the purification specifically recovers biotinylated nuclei from the cell type of interest. Scale bar is approximately 100 μm in each panel. (d) Higher magnification fluorescence image of purified material from the NTF/BirA line, as shown in the left panel of (c). The fluorescence of DNA is shown in blue and the fluorescence of beads is shown in green. Purity of the nuclei can be assessed by comparing the number of bead-bound nuclei that show green fluorescence from the beads (red box) to the number of free nuclei that show only DAPI fluorescence and are not bound by beads (white box). Scale bar is approximately 10 μm.