Florian A. Steiner

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

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 biotin-labeling 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.

Structural features of small RNA precursors determine Argonaute loading in Caenorhabditis elegans

In C. elegans, DCR-1 is required for the maturation of both short interfering RNAs (siRNAs) and microRNAs (miRNAs), which are subsequently loaded into different Argonaute proteins to mediate silencing via distinct mechanisms. We used in vivo analyses to show that precursors of small RNAs contain structural features that direct the small RNAs into the RNA interference (RNAi) pathway or the miRNA-processing pathway. Nucleotide changes in the pre-let-7 miRNA precursor that make its stem fully complementary cause the resulting small RNA to be recognized as siRNA and induce binding to RDE-1, which leads to RNAi. Mismatches of 1 to 3 nucleotides at various positions in the stem of the precursor restore direction into the miRNA pathway, as the largest portion of such small RNA variants is associated with ALG-1. The Argonaute proteins to which the small RNAs are bound determine the silencing mode, and no functional overlap between RDE-1 and ALG-1 was detected.

RDE-1 slicer activity is required only for passenger-strand cleavage during RNAi in Caenorhabditis elegans

RNA interference (RNAi) is a process in which double-stranded RNA is cleaved into small interfering RNAs (siRNAs) that induce the destruction of homologous single-stranded mRNAs. Argonaute proteins are essential components of this silencing process; they bind siRNAs directly and can cleave RNA targets using a conserved RNase H motif. In Caenorhabditis elegans, the Argonaute protein RDE-1 has a central role in RNAi. In animals lacking RDE-1, the introduction of double-stranded RNA does not trigger any detectable level of RNAi. Here we show that RNase H activity of RDE-1 is required only for efficient removal of the passenger strand of the siRNA duplex and not for triggering the silencing response at the target-mRNA level. These results uncouple the role of the RDE-1 RNase H activity in small RNA maturation from its role in target-mRNA silencing in vivo.

Holocentromeres are dispersed point centromeres localized at transcription factor hotspots

Centromeres vary greatly in size and sequence composition, ranging from ‘point’ centromeres with a single cenH3-containing nucleosome to ‘regional’ centromeres embedded in tandemly repeated sequences to holocentromeres that extend along the length of entire chromosomes. Point centromeres are defined by sequence, whereas regional and holocentromeres are epigenetically defined by the location of cenH3-containing nucleosomes. In this study, we show that Caenorhabditis elegans holocentromeres are organized as dispersed but discretely localized point centromeres, each forming a single cenH3-containing nucleosome. These centromeric sites co-localize with kinetochore components, and their occupancy is dependent on the cenH3 loading machinery. These sites coincide with non-specific binding sites for multiple transcription factors (‘HOT’ sites), which become occupied when cenH3 is lost. Our results show that the point centromere is the basic unit of holocentric organization in support of the classical polycentric model for holocentromeres, and provide a mechanistic basis for understanding how centromeric chromatin might be maintained. DOI: http://dx.doi.org/10.7554/eLife.02025.001

MicroRNA–Directed siRNA Biogenesis in Caenorhabditis elegans

RNA interference (RNAi) is a post-transcriptional silencing process, triggered by double-stranded RNA (dsRNA), leading to the destabilization of homologous mRNAs. A distinction has been made between endogenous RNAi–related pathways and the exogenous RNAi pathway, the latter being essential for the experimental use of RNAi. Previous studies have shown that, in Caenorhabditis elegans, a complex containing the enzymes Dicer and the Argonaute RDE-1 process dsRNA. Dicer is responsible for cleaving dsRNA into short interfering RNAs (siRNAs) while RDE-1 acts as the siRNA acceptor. RDE-1 then guides a multi-protein complex to homologous targets to trigger mRNA destabilization. However, endogenous role(s) for RDE-1, if any, have remained unexplored. We here show that RDE-1 functions as a scavenger protein, taking up small RNA molecules from many different sources, including the microRNA (miRNA) pathway. This is in striking contrast to Argonaute proteins functioning directly in the miRNA pathway, ALG-1 and ALG-2: these proteins exclusively bind miRNAs. While playing no significant role in the biogenesis of the main pool of miRNAs, RDE-1 binds endogenous miRNAs and triggers RdRP activity on at least one perfectly matching, endogenous miRNA target. The resulting secondary siRNAs are taken up by a set of Argonaute proteins known to act as siRNA acceptors in exogenous RNAi, resulting in strong mRNA destabilization. Our results show that RDE-1 in an endogenous setting is actively screening the transcriptome using many different small RNAs, including miRNAs, as a guide, with implications for the evolution of transcripts with a potential to be recognized by Dicer.

Knocking out the Argonautes.

Argonaute proteins are key players in gene silencing involving small RNAs. In this issue, Yigit et al. (2006) report a comprehensive study of Argonautes in the worm that places many of the 27 family members into a complex gene-silencing network.

Cell type-specific affinity purification of nuclei for chromatin profiling in whole animals.

Analyzing cell differentiation during development in a complex organism requires the analysis of expression and chromatin profiles in individual cell types. Our laboratory has developed a simple and generally applicable strategy to purify specific cell types from whole organisms for simultaneous analysis of chromatin and expression. The method, termed INTACT for Isolation of Nuclei TAgged in specific Cell Types, depends on the expression of an affinity-tagged nuclear envelope protein in the cell type of interest. These nuclei can be affinity-purified from the total pool of nuclei and used as a source for RNA and chromatin. The method serves as a simple and scalable alternative to FACS sorting or laser capture microscopy to circumvent the need for expensive equipment and specialized skills. This chapter provides detailed protocols for the cell-type specific purification of nuclei from Caenorhabditis elegans.

Recombinant Cell Lines Stably Expressing Functional Ion Channels

Ion channels are membrane proteins that gate the flow of ions into and out of a cell. They are present in the membranes of human, animal, plant, and bacterial cells. They are profoundly involved in diverse tasks ranging from neuronal functions to hormonal secretion and cell division. Biophysical characterization and modulation of ion channel targets are important approaches in modern drug discovery. With the heterologous expression of the nicotinic acetylcholine receptor alpha7 (nAChRalpha7) in a host cell, we show a way to construct and use such a stable cell-based expression system for electrophysiological assays.