We have developed the INTACT method to allow analysis of the specific gene expression programs and underlying epigenetic factors that define a given cell type. This approach is based on the premise that affinity-tagged nuclei can be produced in vivo
in a specific cell type, and these can then be isolated by standard affinity purification techniques. In this study we have shown that this method is easy to perform, does not require sophisticated instrumentation or specialized skills, and can produce large quantities of the desired nuclei at very high purity, in contrast to FACS and LCM-based methods for cell isolation. For example, INTACT provided recovery of >105
nuclei at nearly 100% purity, whereas we recovered <10% of hair cell-specific protoplasts with only 50% purity using FACS based on GFP fluorescence (Figure S2
). INTACT is also clearly suitable for isolating nuclei from relatively rare cell types, given that hair and non-hair cells each represent only about 10% of cells in the primary root (Dolan et al., 1993
). Given the high specificity and avidity of the biotin-streptavidin interaction, nuclei from cells with even lower abundance should also be possible to purify in sufficient quantities simply by starting with a larger amount of whole tissue. In addition, this approach should be applicable to any organism that can be transformed, and is limited only by the need for a suitable nuclear envelope-targeting domain and a promoter that is expressed in the cell type of interest and not in nearby cells. The RanGAP1 WPP domain is likely to be useful for many other, if not all, plant cell types, although it is not likely to work for non-plant cells given differences in the targeting of RanGAP to the nuclear envelope between plants and other organisms (Meier et al., 2008
). For adaptation of the method to non-plant systems, the C-terminus of RanGAP, or perhaps certain nuclear pore complex proteins could be used in place of the WPP domain for nuclear targeting. Thus, INTACT represents a universal strategy for cell type-specific profiling.
Gene expression profiling using INTACT-purified hair and non-hair cell nuclei revealed a large number of genes that are preferentially expressed in each of these cell types. Among the genes we classified as hair cell-enriched, we identified most of the reporter-confirmed hair cell-specific genes and observed increased expression of many of our putative hair cell genes in the gl2-8 mutant roots as compared to wild-type roots. Analysis of overrepresentation of GO terms within our gene sets revealed genes that were previously characterized as being involved in the specification of each of these cell types. In the case of hair cells we also observed an overabundance of genes involved in structural and physiological processes known to be important for the function of this cell type, such as translation, energy generation, cell expansion, vacuole function, and cytoskeletal dynamics. Furthermore, because nuclear and total RNA pools have a very similar composition, and INTACT provides nuclei at nearly 100% purity, the expression profiles generated from INTACT-purified nuclei should accurately represent the transcriptome of the cell type from which they were purified.
Profiling of two histone modifications, H3K4me3 and H3K27me3, in hair and non-hair cell nuclei showed that it is possible to produce robust and highly reproducible ChIP data from the number of nuclei obtained using INTACT. We found that both of these histone modifications showed distributions similar to those recently described in Arabidopsis
(Oh et al., 2008
; Zhang et al., 2009
; Zhang et al., 2007
). In addition, we show that in each cell type the level of H3K4me3 within a gene decreases with decreasing expression level and the H3K27me3 modification increases decreasing expression ( and S4
), as expected. These correlations between expression levels and well-studied chromatin modifications serve as an independent confirmation of the accuracy of our gene expression profiles for each cell type.
Previous profiling of H3K4me3 and H3K27me3 in Arabidopsis
suggested that many plant genes have overlapping regions of H3K4me3 and H3K27me3, as observed in mammalian cells, but because whole plant tissues were used in these experiments it was not clear whether these overlaps were in individual cells or were an artifact of amalgamation of signals from multiple cell types (Oh et al., 2008
; Zhang et al., 2009
; Zhang et al., 2007
). By profiling chromatin landscapes at cell type-resolution we are able to show that these modifications do indeed coexist in the same cell type, as has been observed in mammalian cells (Bernstein et al., 2006
; Roh et al., 2006
A comparison of each histone modification profile by subtraction of the non-hair cell profile from that of the hair cell showed that the largest expression differences between cell types often corresponded to an increase in H3K4me3 and a decrease in H3K27me3 in the cell type showing preferential expression of a given gene. This suggests that a balance between the activities of Trithorax group protein-mediated H3K4 trimethylation and Polycomb group protein-mediated trimethylation of H3K27 is involved in establishing cell type-specific expression. However, many differentially expressed genes showed little difference in histone modification levels between cell types over cell type-enriched genes, indicating that there are mechanisms for generating cell type-specific expression that are unrelated to the H3K4me3/H3K27me3 balance.
In conclusion, we have demonstrated that the INTACT method is a robust and simple technique for gene expression and chromatin profiling of individual cell types within a complex tissue, and this approach has many advantages over currently available methods. The INTACT method is far gentler than FACS or LCM in that extensive tissue manipulation is not required and the procedure is much faster, therefore minimizing the possibility of artifacts arising from tissue manipulation and the time required to obtain sufficient material for epigenomic profiling. An additional advantage is that because nuclei can be isolated simply by freezing and grinding of tissue, the INTACT method will be easier to apply to cells embedded within deep internal tissues as well as cells that are recalcitrant to dissociation from a tissue for other reasons. INTACT, being an affinity-based method, also has the distinct advantage of being insensitive to autofluorescence and other optical disturbances that will interfere with FACS-based purification. In addition, the INTACT method requires no specialized skills beyond those common to any molecular biologist, obviates the need for sophisticated and expensive equipment, and should be widely applicable.