Pairing Lamin B1-DamID and Immuno-3D-FISH to Resolve and Verify Peripheral Genome Organization of Adipogenesis.

Each tissue has unique patterns of spatial genome organization that contribute to tissue-specific gene regulation, and these patterns are often disrupted in aging, cancer, and developmental diseases such as muscular dystrophy and lipodystrophy. Interactions between nuclear membrane proteins and specific sets of genes drive much of this tissue-specific genome organization; however, identifying specific gene targets of these proteins can be challenging because of their insolubility and interactions with the lamin polymer. The DamID method is a powerful approach to overcome these limitations because, unlike chromatin immunoprecipitation (ChIP) approaches, it does not require isolation of the nuclear membrane protein; however, dam methylase fusions to the nuclear membrane proteins that tether specific chromatin may interfere with the specificity of their genome interactions. Thus, dam methylase is fused to lamin B1 to determine all genes at the nuclear envelope when the nuclear membrane protein is present or absent, and the comparison should identify genes under nuclear membrane protein positional control. However, DamID needs to be optimized for each tissue; so, it is helpful to have each procedure catalogued and to be able to compare different modifications of the procedure when establishing DamID in a new tissue. We present here an optimized protocol for lamin B1-DamID on pre-adipocytes and differentiated adipocytes along with two approaches for analysis. We also present a method for 3D Immuno-FISH in adipocytes to confirm DamID results. This method requires specialized approaches to permeabilize fat cells which otherwise have significant autofluorescence.