Abstract
In recent years, Caenorhabditis elegans has emerged as a new model to investigate the relationships between nuclear architecture, cellular differentiation, and organismal development. On one hand, C. elegans with its fixed lineage and transparent body is a great model organism to observe gene functions in vivo in specific cell types using microscopy. On the other hand, two different techniques have been applied in nematodes to identify binding sites for chromatin-associated proteins genome-wide: chromatin immunoprecipitation (ChIP), and Dam-mediated identification (DamID). We summarize here all three techniques together as they are complementary. We also highlight strengths and differences of the individual approaches.
Highlights
Introduction to chromatin immunoprecipitation (ChIP) Chromatin immunoprecipitation is a technique that is used to study protein-DNA interactions
A ChIP experiment is composed of the following steps (Figure 1): (1) worms are incubated with formaldehyde that crosslinks chromatin-associated proteins to each other and to DNA; (2) chromatin is sheared to shorter fragments and the protein of interest is immunoprecipitated along with bound DNA; and (3) bound DNA is identified and quantified. 2.2
The principle of DNA adenine methyltransferase (Dam)-mediated identification (DamID) is simple—genomic DNA is extracted from animals expressing Dam fused to the protein of interest and animals expressing a fusion of Dam to GFP
Summary
The modENCODE consortium as well as the vast majority of published ChIP and DamID studies use either entire worms or developing embryos. In C. elegans, the only system used up to now is the lacO/lacI couple (Carmi et al, 1998; Kaltenbach et al, 2000; Gonzalez-Serricchio and Sternberg, 2006; Yuzyuk et al, 2009; Meister et al, 2010b; Yuen et al, 2011; Towbin et al, 2012; Cochella and Hobert, 2012; Rohner et al, 2013). - analyze in vivo the interaction of a given protein (e.g., a transcription factor) with its cognate binding site, in a cell-type and developmental stage specific manner (Carmi et al, 1998; Kaltenbach et al, 2000; Updike and Mango, 2006; Kiefer et al, 2007; Yuzyuk et al, 2009). We discuss image acquisition and analysis techniques
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