DamID Analysis of Nuclear Organization in Caenorhabditis elegans

Abstract

The development of genomics and next generation sequencing platforms has dramatically improved our insight into chromatin structure and organization and its fine interplay with gene expression. The nuclear envelope has emerged as a key component in nuclear organization via extensive contacts between the genome and numerous proteins at the nuclear periphery. These contacts may have profound effects on gene expression as well as cell proliferation and differentiation. Indeed, their perturbations are associated with several human pathologies known as laminopathies or nuclear envelopathies. However, due to their dynamic behavior the contacts between nuclear envelope proteins and chromatin are challenging to identify, in particular in intact tissues. Here, we propose the DamID technique as an attractive method to globally characterize chromatin organization in the popular model organism Caenorhabditis elegans. DamID is based on the in vivo expression of a chromatin-associated protein of interest fused to the Escherichia coli DNA adenine methyltransferase, which produces unique identification tags at binding site in the genome. This marking is simple, highly specific and can be mapped by sensitive enzymatic and next generation sequencing approaches.

Loss of an H3K9me anchor rescues laminopathy-linked changes in nuclear organization and muscle function in an Emery-Dreifuss muscular dystrophy model

“…While the LMN-Y59C mutant gain-of-function sequestration of the myo-3p reporter was intriguing, it was important to determine how LMN-Y59C expression affects the spatial distribution of endogenous chromatin. To test this we performed muscle-specific genome-wide emerin-DamID, a method in which the muscle-specific expression of an INM protein (emerin or EMR-1) fused to the E. coli DNA adenine methyltransferase (Dam) enables mapping of chromatin at the INM of muscle nuclei (Gómez-Saldivar et al 2016;Muñoz-Jiménez et al 2017). We expressed Dam∷EMR-1 in a muscle-specific manner thanks to a FLP-FRT system driven by muscle-specific FLP expression ( Fig.…”

“…DamID was performed and analyzed largely as described in Gómez-Saldivar et al (2016) and Cabianca et al (2019). In this study, three biological replicas were prepared and all strains were grown in parallel in each replica and for all strains the analysis was performed in biological triplicates.…”

Evolution of theD. melanogasterchromatin landscape and its associated proteins

“…For instance, we do not know how chromatin states differ in Drosophila over development and between tissues. Tackling other organisms, such as human cell lines, mouse (M. musculus), worm (C. elegans), and plant (A. thaliana), is in principle possible, as the experimental technique that underpins the data set in Drosophila, DamID, has been successfully adapted to each of these species (Vogel et al 2006;Zhang et al 2007;Gómez-Saldivar et al 2016;Tosti et al 2018). Comparing different species is crucial to determine if the evolutionary scenarios that we propose indeed hold true and how they may need to be refined or reconsidered.…”

DamID identifies targets of CEH-60/PBX that are associated with neuron development and muscle structure in Caenorhabditis elegans

“…DamID sampling and library preparation were carried out essentially as described in [ 33 ]. Strains carrying either the gfp :: dam (LSC1722) or dam :: ceh-60 (LSC1724) transgene were grown under standard conditions.…”