Cell-type-specific nuclei purification from whole animals for genome-wide expression and chromatin profiling

Abstract

An understanding of developmental processes requires knowledge of transcriptional and epigenetic landscapes at the level of tissues and ultimately individual cells. However, obtaining tissue-or cell-type-specific expression and chromatin profiles for animals has been challenging. Here we describe a method for purifying nuclei from specific cell types of animal models that allows simultaneous determination of both expression and chromatin profiles. The method is based on in vivo biotinlabeling of the nuclear envelope and subsequent affinity purification of nuclei. We describe the use of the method to isolate nuclei from muscle of adult Caenorhabditis elegans and from mesoderm of Drosophila melanogaster embryos. As a case study, we determined expression and nucleosome occupancy profiles for affinity-purified nuclei from C. elegans muscle. We identified hundreds of genes that are specifically expressed in muscle tissues and found that these genes are depleted of nucleosomes at promoters and gene bodies in muscle relative to other tissues. This method should be universally applicable to all model systems that allow transgenesis and will make it possible to determine epigenetic and expression profiles of different tissues and cell types.

Cell-type-specific profiling of protein–DNA interactions without cell isolation using targeted DamID with next-generation sequencing

“…The first approach focuses on specifically labelling and isolating the subset of cells of interest before undertaking conventional ChIP profiling. Examples include BITS-ChIP, where ells 14 or fixed nuclei 15,16 are isolated by fluorescent activated cell sorting (FACS), and the INTACT method, were nuclei are isolate by biotin labelling and pulldown [17][18][19] . These methods have the advantage that they allow a large range of factors to be profiled via a single transgenic animal.…”

“…The digested DNA is column purified to exclude un-cut, genomic DNA from uninduced cells (steps [12][13][14], and adaptors for PCR-amplification are ligated to the DpnI cut fragments (steps [15][16][17][18]. To prevent unmethylated regions of DNA from being aberrantly amplified, the material is digested with DpnII, which only cuts non-methylated GATC sites (steps [19][20][21]). The resulting DNA is then used as template for a PCR reaction to amplify the methylated fragments (steps [22][23][24][25].…”

RNA Polymerase II transcription elongation and Pol II CTD Ser2 phosphorylation

“…Isolation of tissue-specific material, either through flow cytometry, cell-type specific tagging, 170 or the recently developed INTACT (isolation of nuclei tagged in specific cell types) method 171,172 is likely to have a major impact on the ability to understand tissue-specific phenomenon. This method has already been used to isolate C. elegans muscle nuclei 172 and it seems likely that this could be easily adapted to additional somatic tissues or the germline. As we move toward the development of methods that can dissect these events in multicellular organisms, it is likely that we will find unique exceptions to well-established models of transcription.…”

Affinity-based Isolation of Tagged Nuclei from <em>Drosophila</em> Tissues for Gene Expression Analysis

“…Biotin-labeled nuclei can be subsequently purified from mixed populations using streptavidin-based affinity isolation. Using this approach, nuclei were successfully labeled and isolated from the mesoderm of Drosophila embryos in which a nuclear envelope fusion protein was expressed under control of a mesoderm-specific enhancer 8 . The authors also generated nuclear envelope fusion proteins that can be expressed in any cell type under control of the Gal4 regulatory sequence, UAS .…”

“…To overcome these limitations, several groups have utilized affinity-based isolation techniques to purify nuclei that are labeled with a specific epitope in a particular cell type. The isolation of nuclei tagged in specific cell types (INTACT) method developed for use in Arabidopsis thaliana 6,7 has recently been adapted for use in Drosophila 8 . In this method, a nuclear envelope fusion protein that is a substrate for in vivo biotinylation is coexpressed with the Escherichia coli biotin ligase BirA in specific cell types.…”