Roles of Enhancer RNAs in RANKL-induced Osteoclast Differentiation Identified by Genome-wide Cap-analysis of Gene Expression using CRISPR/Cas9

Scientific Reports
Yukako SakaguchiMasaru Ishii

Abstract

Bidirectional transcription has been proposed to play a role associated with enhancer activity. Transcripts called enhancer RNAs (eRNAs) play important roles in gene regulation; however, their roles in osteoclasts are unknown. To analyse eRNAs in osteoclasts comprehensively, we used cap-analysis of gene expression (CAGE) to detect adjacent transcription start sites (TSSs) that were distant from promoters for protein-coding gene expression. When comparing bidirectional TSSs between osteoclast precursors and osteoclasts, we found that bidirectional TSSs were located in the 5'-flanking regions of the Nrp2 and Dcstamp genes. We also detected bidirectional TSSs in the intron region of the Nfatc1 gene. To investigate the role of bidirectional transcription in osteoclasts, we performed loss of function analyses using the CRISPR/Cas9 system. Targeted deletion of the DNA regions between the bidirectional TSSs led to decreased expression of the bidirectional transcripts, as well as the protein-coding RNAs of Nrp2, Dcstamp, and Nfatc1, suggesting that these transcripts act as eRNAs. Furthermore, osteoclast differentiation was impaired by targeted deletion of bidirectional eRNA regions. The combined results show that eRNAs play important r...Continue Reading

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Citations

Sep 11, 2020·Stem Cells International·Yi ZhaoWeiran Li
May 1, 2021·International Journal of Molecular Sciences·Costantino ParisiCecilia Lanny Winata
Jul 6, 2021·Frontiers in Cell and Developmental Biology·Kazuki InoueBaohong Zhao

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Datasets Mentioned

BETA
SRP137592
SRP137597

Methods Mentioned

BETA
transfection

Software Mentioned

JASPAR
JMP Pro
Bioconductor package
Bowtie2
edgeR
CAGEr
ChIPseeker
FANTOM5
BSgenome
Bioconductor

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