Czapiewski Rafal, de Las Heras Jose I, Schirmer Eric C
Institute of Cell Biology, University of Edinburgh, Edinburgh, UK.
MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh, UK.
Methods Mol Biol. 2025;2958:287-325. doi: 10.1007/978-1-0716-4714-1_19.
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.
每个组织都有独特的空间基因组组织模式，这些模式有助于组织特异性的基因调控，而这些模式在衰老、癌症以及诸如肌肉萎缩症和脂肪营养不良等发育性疾病中常常被破坏。核膜蛋白与特定基因集之间的相互作用推动了许多这种组织特异性的基因组组织；然而，由于这些蛋白的不溶性以及它们与核纤层聚合物的相互作用，确定这些蛋白的特定基因靶点可能具有挑战性。DamID方法是克服这些限制的一种强大方法，因为与染色质免疫沉淀（ChIP）方法不同，它不需要分离核膜蛋白；然而，与连接特定染色质的核膜蛋白融合的dam甲基化酶可能会干扰它们与基因组相互作用的特异性。因此，将dam甲基化酶与核纤层蛋白B1融合，以确定在核膜蛋白存在或不存在时核膜上的所有基因，通过比较应该能够识别受核膜蛋白位置控制的基因。然而，DamID需要针对每个组织进行优化；所以，在新组织中建立DamID时，将每个步骤编目并能够比较该步骤的不同修改是很有帮助的。我们在此展示了一种针对前脂肪细胞和分化脂肪细胞的核纤层蛋白B1-DamID优化方案以及两种分析方法。我们还展示了一种在脂肪细胞中进行三维免疫荧光原位杂交（3D Immuno-FISH）以确认DamID结果的方法。这种方法需要专门的方法来使脂肪细胞通透，否则脂肪细胞会有显著的自发荧光。
