Enforced Activation of Enhancer RNAs In Situ through the dCas9 Synergistic Activation Mediator System

Summary

Enhancer RNAs (eRNAs) are non-coding RNAs produced from active enhancers. An optimal approach to study eRNA functions is to manipulate their levels in the native chromatin regions. Here we introduce a robust system for eRNA studies by using CRISPR-dCas9-fused transcriptional activators to induce the expression of eRNAs of interest.

Abstract

Enhancers are pivotal genomic elements scattered through the mammalian genome and dictate tissue-specific gene expression programs. Increasing evidence has shown that enhancers not only provide DNA binding motifs for transcription factors (TFs) but also generate non-coding RNAs that are referred to as eRNAs. Studies have demonstrated that eRNA transcripts can play significant roles in gene regulation in both physiology and disease. Commonly used methods to investigate the function of eRNAs are constrained to “loss-of-function” approaches by knockdown of eRNAs, or by chemical inhibition of the enhancer transcription. There has not been a robust method to conduct “gain-of-function” studies of eRNAs to mimic specific disease conditions such as human cancer, where eRNAs are often overexpressed. Here, we introduce a method for precisely and robustly activating eRNAs for functional interrogation of their roles by applying the dCas9 mediated Synergistic Activation Mediators (SAM) system. We present the entire workflow of eRNA activation, from the selection of eRNAs, the design of gRNAs to the validation of eRNA activation by RT-qPCR. This method represents a unique approach to study the roles of a particular eRNA in gene regulation and disease development. In addition, this system can be employed for unbiased CRISPR screening to identify phenotype-driving eRNA targets in the context of a specific disease.

Introduction

The human genome contains a constellation of regulatory elements^1,2,3. Among these, enhancers emerge to be one of the most critical categories^4,5,6. Enhancers play essential roles in regulating development, and are responsible for generating spatial-temporal gene expression programs to determine cell identity^5,6,7. Conventionally, enhancers are only considered to be DNA elements that provide....

Protocol

The entire workflow of this protocol is shown in Figure 1.

1. Enhancer RNA (eRNA) selection

1. Identify a putative enhancer region of interests by using binding peaks of chromatin immunoprecipitation sequencing (ChIP-Seq) data, i.e., of histone modifications (e.g., H3K4me1 and H3K27ac), or of transcription coactivators (e.g., p300).
2. Identify the eRNA of interest by intersecting the ChIP-Seq peak with RNA-seq signals (e.g., from total RNA-seq or from nascent RNA-seq such as Global Run-On Sequencing (GRO-Seq).
   NOTE: The region chosen for designing gRNAs should be usually limited to the &....

Results

Figure 1 illustrates the overall workflow of this protocol. Our focus was on a representative eRNA, NET1e¹⁵, which is overexpressed in breast cancer, for which SAM system was used to activate and study it’s biological role in regulating gene expression, cell proliferation and cancer drug response. For this NET1 enhancer, several p300 ChIP-Seq peaks, flanked by transcribed eRNA transcripts (Figure 2A,B) w.......

Discussion

Based on our data, we conclude that the SAM system is suitable for studying the role of eRNAs in regulating cellular phenotypes, e.g., cell growth or drug resistance. However, careful gRNA designing is required for robust eRNA activation, due to the following reasons. First of all, the transcription start site (TSS) of eRNA in each specific cell lines/types remains less clearly annotated. Due to this, epigenomic information (e.g., ChIP-Seq of H3K27ac, of transcription factors, or of p300), transcriptional activity depict.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Blasticidin	Goldbio	B-800-100
BsmBI restriction enzyme	New England BioLabs Inc.	R0580S
Cas9 mAb	Active Motif	61757	Lot: 10216001
Deoxynucleotide (dNTP) Solution Mix	New England BioLabs Inc.	N0447S
Dulbecco’s Modified Eagle Medium	Corning	10-013-CM
Dynabeads Protein G	Thermo Fisher Scientific	65002
EDTA	Thermo Fisher Scientific	BP118-500
EGTA	Sigma	E3889
Fetal Bovine Serum	GenDEPOT	F0900-050
Glycogen	Thermo Fisher Scientific	10814010
Hepes-KOH	Thermo Fisher Scientific	BP310-100
Hexadimethrine Bromide	Sigma	H9268
Hygromycin B	Goldbio	H-270-25
IGEPAL CA630	Sigma	D6750
IncuCyte live-cell imager	Essen BioScience	IncuCyte S3 Live-Cell Analysis System
lenti_dCAS-VP64_Blast	Addgene	61425
lenti_gRNA(MS2)_zeo backbone	Addgene	61427
lenti_MS2-p65-HSF1_Hygro	Addgene	61426
LiCL	Sigma	L9650
Lipofectamine 2000	Thermo Fisher Scientific	11668-500
NaCl	Sigma	S3014
Na-Deoxycholate	Sigma	D6750
NaHCO3	Thermo Fisher Scientific	BP328-500
N-lauroylsarcosine	Sigma	97-78-9
Opti-MEM Reduced Serum Medium	Thermo Fisher Scientific	31985070
PES syringe filter	BASIX	13-1001-07
Protease Inhibitor Cocktail Tablet	Roche Diagnostic	11836145001
pSpCas9(BB)-2A-Puro	Addgene	62988
Q5 High-Fidelity DNA Polymerase	New England BioLabs Inc.	M0491S
Q5 Reaction Buffer	New England BioLabs Inc.	B9027S
Quick-DNA Miniprep	ZYMO Research	D3025
Quick-RNA Miniprep	ZYMO Research	R1054
Restriction enzyme buffer	New England BioLabs Inc.	B7203S
RT-qPCR Detection System	Thermo Fisher Scientific	Quant Studio3
SDS	Thermo Fisher Scientific	BP359-500
Sonicator	Qsonica	Q800R2
Sso Advanced Universal SYBR Green Supermix	Bio-Rad Laboratories	1725274
Stbl3 competent cell	Thermo Fisher Scientific	C7373-03
Superscript IV reverse transcript	Thermo Fisher Scientific	719000
Surveyor Mutation Detection Kits	Integrated DNA Technologies	706020
T4 DNA Ligase	New England BioLabs Inc.	M0202S
T4 DNA Ligase Reaction Buffer	New England BioLabs Inc.	B0202S
TE buffer	Thermo Fisher Scientific	46009CM
Thermal cycler	Bio-Rad Laboratories	T100
Thermomixer	Sigma	5384000020
Zeocin	Thermo Fisher Scientific	ant-zn-1p