Joint changes in RNA, RNA polymerase II, and promoter activity through the cell cycle identify non-coding RNAs involved in proliferation

Siv Anita Hegre,Konika Chawla,Kristin G. Nørsett,Endre B. Stovner,Pål Sætrom,Per Arne Aas,Lene Christin Olsen,Nina Beate Liabakk,Antonin Klima,Helle Samdal

doi:10.1038/s41598-021-97909-w

Abstract

Proper regulation of the cell cycle is necessary for normal growth and development of all organisms. Conversely, altered cell cycle regulation often underlies proliferative diseases such as cancer. Long non-coding RNAs (lncRNAs) are recognized as important regulators of gene expression and are often found dysregulated in diseases, including cancers. However, identifying lncRNAs with cell cycle functions is challenging due to their often low and cell-type specific expression. We present a highly effective method that analyses changes in promoter activity, transcription, and RNA levels for identifying genes enriched for cell cycle functions. Specifically, by combining RNA sequencing with ChIP sequencing through the cell cycle of synchronized human keratinocytes, we identified 1009 genes with cell cycle-dependent expression and correlated changes in RNA polymerase II occupancy or promoter activity as measured by histone 3 lysine 4 trimethylation (H3K4me3). These genes were highly enriched for genes with known cell cycle functions and included 57 lncRNAs. We selected four of these lncRNAs—SNHG26, EMSLR, ZFAS1, and EPB41L4A-AS1—for further experimental validation and found that knockdown of each of the four lncRNAs affected cell cycle phase distributions and reduced proliferation in multiple cell lines. These results show that many genes with cell cycle functions have concomitant cell-cycle dependent changes in promoter activity, transcription, and RNA levels and support that our multi-omics method is well suited for identifying lncRNAs involved in the cell cycle.

Highlights

Genome-wide gene expression studies have revealed that several genes are regulated in a cell cycle-specific manner[1,2,3,4,5]
Cyclin B1 (CCNB1) was one of the well-known cell cycle genes we identified, and we observed RNA sequencing (RNA-seq) reads for all exons and Polymerase II (Pol II) Chromatin immunoprecipitation (ChIP)-seq signals were captured around the transcription start sites (TSSs) (Fig. 2E)
By sequencing total RNA through the cell cycle we identified 99 Long non-coding RNAs (lncRNAs) with cell cycle-dependent gene expression

Summary

Introduction

Genome-wide gene expression studies have revealed that several genes are regulated in a cell cycle-specific manner[1,2,3,4,5]. LncRNAs are classified into five main categories according to where they are encoded in the genome in relation to mRNAs: sense, antisense, bi-directional, intergenic, and intronic They are able to regulate the gene expression at the transcriptional level by acting as signals, guides, scaffolds, or decoys[12]. In prostate cancer GAS5 functions as a tumor suppressor that inhibits proliferation by targeting the CDK inhibitor p2717 Another known cell cycle-associated lncRNA is the zinc finger antisense 1 (ZFAS1). As lncRNAs can bind DNA, RNAs, and proteins, they can regulate the gene expression at the transcriptional, post-transcriptional, and epigenetic level They can serve as signaling molecules, as sponges by binding microRNAs (miRNAs), and inhibit miRNA-induced degradation of mRNAs, as guides by recruiting transcription regulators, or act as scaffolds by binding proteins to regulate gene expression[21]. A higher cell type specificity means that targeting lncRNAs supposedly have less side-effects than targeting protein coding g enes[24]

Methods

Results

Conclusion