Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

Summary

Here, we describe the use of the single-molecule imaging method, DNA Curtains, to study the biophysical mechanism of EWS-FLI1 condensates assembling on DNA.

Abstract

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 (FET) family of fusion oncoproteins, is one of the most frequently involved fusion genes in Ewing sarcoma. These FET family fusion proteins typically harbor a low-complexity domain (LCD) of FET protein at their N-terminus and a DNA-binding domain (DBD) at their C-terminus. EWS-FLI1 has been confirmed to form biomolecular condensates at its target binding loci due to LCD-LCD and LCD-DBD interactions, and these condensates can recruit RNA polymerase II to enhance gene transcription. However, how these condensates are assembled at their binding sites remains unclear. Recently, a single-molecule biophysics method-DNA Curtains-was applied to visualize these assembling processes of EWS-FLI1 condensates. Here, the detailed experimental protocol and data analysis approaches are discussed for the application of DNA Curtains in studying the biomolecular condensates assembling on target DNA.

Introduction

Transcriptional regulation is a crucial step for precise gene expression in living cells. Many factors, such as chromosomal modification, transcription factors (TFs), and non-coding RNAs, participate in this complicated process^1,2,3. Among these factors, TFs contribute to the specificity of transcriptional regulation by recognizing and binding to specific DNA sequences known as promoters or enhancers and subsequently recruiting other functional proteins to activate or repress transcription^4,5,

Protocol

1. Preparation of the lipid bilayer master mix

1. Rinse glass vials with double-distilled water (ddH₂O) and 99% ethanol and dry them in a 60 °C drying oven.
2. Make the lipid master mix by dissolving 1 g of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 100 mg of polyethylene glycol-reacted (PEGylated) lipids (18:1 of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (ammonium salt) (PEG2000 DOPE) and 25 mg of biotinylated lipids (18:1 of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (sodium salt) (Biotinyl Cap DOPE)) into 10 mL of chloroform.
3. Prepare 1 mL aliquots of the l....

Results

The schematic of DNA Curtains is shown in Figure 1A, Figure 1B, and Figure 1D. The cloned target sequence containing 25 uninterrupted repeats of GGAA is found in the NORB1 promoter in Ewing sarcoma. This target sequence is crucial for EWS-FLI1 recruitment²⁸. EWS-FLI1 molecules were visualized by detecting the mCherry-labeled EWS-FLI1 signals obtained with a 561 nm laser (Figure 1.......

Discussion

As single-molecule approaches are extremely sensitive to the contents of the reaction system, extra effort must be invested to ensure good quality of all the materials and solutions during the DNA Curtains experiments, especially the lipids prepared in sections 1 and 2 and the buffers used in section 5. Reagents of higher purity must be used to prepare buffers, and buffers must be freshly prepared for the single-molecule assay

When 500 nM mCherry-labeled EWS-FLI1 was flushed into the chamber, .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
488 nm diodepumped solid-state laser	Coherent	OBIS488LS
561 nm diodepumped solid-state laser	Coherent	OBIS561LS
Agar	Rhawn	R003215-50g
biotinylated DOPE	Avanti	870273P
Bovine Serum Albumin	Sigma	A7030
Chloroform	Amresco	1595C027
Coating Electra 92	Allresist GmbH	AR-PC 5090.02	The conductive protective coating
Deoxyribonuclease I bovine	Sigma	D5139-2MG
DOPC	Avanti	850375P
DTT	Sigma	D9779
Glass coverslip	Fisher Scientific	12-544-7
Hellmanex III	Sigma	Z805939-1EA
KCl	Sigma	60130
Lambda DNA	NEB	N3013S
Lambda Packing Extracts	Epicentre	MP5120
MgCl2	Sigma	M2670
NaCl	Sigma	s3014
Nanoport	Idex	N-333-01
NheI-HF	NEB	R3131S
Nikon Inverted Microscope	Nikon	Eclipse Ti
NZCYM Broth	Sigma	N3643-250G
PEG-2000 DOPE	Avanti	880130P-1G
PEG-8000	Amresco	25322-68-3
PMMA 200K, ETHYL LACTATE 4%	Allresist GmbH	AR-P 649.04
PMMA 950K, ANISOLE 2%	Allresist GmbH	AR-P 672.02
Prime 95B Scientific CMOS camera	PHOTOMETRICS	Prime95B
proteinase K	NEB	P8107S
Silica glass slide	G.Finkenbeiner
Six-way injection valve	Idex	MXP9900-000
Streptavidin	Thermo	S888	Diluted with ddH2O
Syringe pump	Harvard Apparatus	Pump11 Elite
T4 DNA Ligase	NEB	M0202S
Tris base	Sigma	T6066
XhoI	NEB	R0146V
YOYO-1 Iodide (491/509)	Invitrogen	Y3601	Diluted with DMSO