Mapping of protein- and chromatin-interactions at the nuclear lamina

Abstract

The nuclear envelope and lamina define the nuclear periphery and are implicated in many nuclear processes, including chromatin organization, transcription and DNA replication. Mutations in lamin A proteins, major components of the lamina, interfere with these functions and cause a set of phenotypically diverse diseases referred to as laminopathies. The phenotypic diversity of laminopathies is thought to be the result of alterations in specific protein- and chromatin interactions due to lamin A mutations. Systematic identification of lamin A-protein and -chromatin interactions will be critical to uncover the molecular etiology of laminopathies. Here we summarize and critically discuss recent technology to analyze lamina protein- and chromatin interactions.

Figures and Tables

Figure 1 A schematic view of lamina and lamina-interacting protein fractions purified by various techniques. The nuclear periphery consists of an inner nuclear membrane (INM), outer nuclear membrane (ONM) and is connected to the endoplasmic reticulum (ER). (Left) Salt solubilizes weakly attached lamina proteins, but not the nuclear lamina. Detergents preferentially dissolve membrane proteins that are not anchored in the detergent resistant lamina.28,65 Chaotropes and alkaline extraction generate an insoluble fraction mainly consisting of integral membrane proteins.65 Immunoprecipitations (right) with an antibody directed against lamin A/C, using mild lysis conditions (for example 0.1% NP-40, 250 mM NaCl12), preferentially dissolve and precipitate nucleosoluble A-type lamins and protein interactors.35 For a lamin A OST pull-down assay,46 cross-linking, indicated by crosses, captures protein-protein and protein-chromatin interactions and allows solubilization of the total lamin A/C pool while preserving interactions.

Figure 2 Schematic overview of techniques to identify chromatin interactions, which are categorized in enzymatic- and affinity-based approaches. For DamID58 a DNA adenine methyltransferase (Dam) tag (ball on stick) is fused to the protein of interest and adenylates (star) bound chromatin in vivo, enabling in vitro selective DpnI (scissor) restriction and subsequent amplification of restricted chromatin by ligation mediated PCR (LMPCR). For in vivo chromatin endogenous cleavage (ChEC)63 a protein of interest is fused to a micrococcal nuclease (MNase) tag, which introduces DNA double strand breaks (scissors) upon introduction of calcium chloride to weakly permeabilized cells. Due to the mild permeabilization of cells prior to addition of calcium chloride for activation, the MNase digestion step is indicated as being partially in vitro and in vivo. Restricted DNA is amplified by LMPCR. For chromatin immunocleavage (ChIC)63 cells are cross-linked (crosses). In vitro, MNase-conjugated antibody interacts with the epitope of interest and induces DNA breaks enabling LMPCR amplification of cleaved chromatin. For chromatin immunoprecipitation (ChIP) chromatin-protein interactions are cross-linked and chromatin is randomly sheared, typically by ultrasonication, (lightning arrow and stripes). Antibodies are used to precipitate the endogenous protein of interest with the help of antibody binding beads (big ball). In a OneSTrEP (OST) pull-down a OST-tagged protein is expressed.58 Cells are cross-linked and ultrasonicated. The OST-protein is highly efficiently precipitated by a streptactin matrix (big square).

Table 1 Techniques to identify protein interactions at the nuclear lamina

Table 2 Solubilization conditions to identify protein interactions at the nuclear lamina.

Table 3 Techniques to identify chromatin interactions at the nuclear lamina