Abstract
Using RNA-seq (RNA sequencing) of ribosome-depleted RNA, we have identified 1,273 lncRNAs (long non-coding RNAs) in P493-6 human B-cells. Of these, 534 are either up- or downregulated in response to MYC overexpression. An increase in MYC occupancy near their TSS (transcription start sites) was observed for MYC-responsive lncRNAs suggesting these are direct MYC targets. MYC binds to the same TSS across several cell lines, but the number of TSS bound depends on cellular MYC levels and increases with higher MYC concentrations. Despite this concordance in promoter binding, a majority of expressed lncRNAs are specific for one cell line, suggesting a determinant role of additional, possibly differentiation-specific factors in the activity of MYC-bound lncRNA promoters. A significant fraction of the lncRNA transcripts lack polyadenylation. The RNA-seq data were confirmed on eight selected lncRNAs by NRO (nuclear run-on) and RT-qPCR (quantitative reverse transcription PCR).
Highlights
MYC is a basic helix-loop-helix leucine zipper protein that controls cell proliferation, differentiation, metabolism, apoptosis, and the maintenance of pluripotency
Total RNA was harvested after 24 hours and transcript expression levels determined by RNA sequencing (RNA-seq)
We observed increased levels of total RNA and nuclear RNA per cell at high where i refers to the sample, gene refers to the gene and N refers to the total number of samples (6). (b) A volcano plot of observed fold changes in ncRNA expression vs significance
Summary
MYC is a basic helix-loop-helix leucine zipper (bHLHLZ) protein that controls cell proliferation, differentiation, metabolism, apoptosis, and the maintenance of pluripotency. It is a key component of a broad transcription factor network, forming heterodimers with the bHLHLZ protein MAX [1,2,3,4]. The incidence of MYC gain of function extends from 5 to 45% [7, 8]. The traditional view of MYC-driven tumorigenesis is that the MYC protein behaves as a classical transcription factor, which regulates the expression of a specific set of downstream genes that contribute to cancer progression. It has been shown recently that MYC-mediated transcriptional regulation is so widespread that MYC can be considered an ‘amplifier’ of transcription on a global scale [10,11,12,13]
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