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

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Summary

Introduction

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

Antibody selection and validation
Tissue or developmental-stage selection
ChIP controls
ChIP quantification
Embryo collection
Larva collection
Embryo extract preparation
Larva extract preparation
Quantifying extract protein
Input DNA purification
Chromatin immunoprecipitation
ChIP reagents
Introduction to DamID
Experimental planning for DamID
Design and expression of DamID fusion proteins
Validation of DamID fusions
Biological material for DamID
DamID controls
DamID evaluation
Cloning
Transgenesis
Validation of DamID fusion proteins
Immunofluorescence
Western blot
Nematode culture
Purification and amplification of Dam-methylated DNA
For embryos
Go to step 5 3x
Methods for ChIP and DamID quantification
DNA microarrays
High-throughput sequencing
Data analysis
Experimental planning
Cis tagging—simple extrachromosomal arrays
Trans tagging—simple and complex extrachromosomal arrays
Bombardment-mediated insertion of lacO arrays next to sequences of interest
Preparing agarose pads for microscopy
Preparing embryos for live imaging
Imaging live embryos and worms
Compaction state
Radial nuclear localization
Findings
36. Abstract
Full Text
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