Time-Dependent c-Myc Transactomes Mapped by Array-Based Nuclear Run-On Reveal Transcriptional Modules in Human B Cells

Abstract

BackgroundThe definition of transcriptional networks through measurements of changes in gene expression profiles and mapping of transcription factor binding sites is limited by the moderate overlap between binding and gene expression changes and the inability to directly measure global nuclear transcription (coined “transactome”).Methodology/Principal FindingsWe developed a method to measure nascent nuclear gene transcription with an Array-based Nuclear Run-On (ANRO) assay using commercial microarray platforms. This strategy provides the missing component, the transactome, to fully map transcriptional networks. ANRO measurements in an inducible c-Myc expressing human P493-6 B cell model reveals time-dependent waves of transcription, with a transactome early after c-Myc induction that does not persist at a late, steady-state phase, when genes that are regulated by c-Myc and E2F predominate. Gene set matrix analysis further uncovers functionally related groups of genes putatively regulated by waves of transcription factor motifs following Myc induction, starting with AP1 and CREB that are followed by EGR1, NFkB and STAT, and ending with E2F, Myc and ARNT/HIF motifs.Conclusions/SignificanceBy coupling ANRO with previous global mapping of c-Myc binding sites by chromatin immunoprecipitation (ChIP) in P493-6 cells, we define a set of transcriptionally regulated direct c-Myc target genes and pave the way for the use of ANRO to comprehensively map any transcriptional network.

Overexpression of the c-myc Oncogene Inhibits Nonsense-mediated RNA Decay in B Lymphocytes

“…have Myc bound at their promoters (18). In addition, a recent study noted that many of the transcripts up-regulated by Myc are not induced at the transcriptional level, suggesting that post-transcriptional processes may play an important role in their up-regulation (20). Consistent with these findings, Myc has recently been appreciated to regulate microRNAs that can then affect mRNA stability (21).…”

Egr1 mediates p53-independent c-Myc–induced apoptosis via a noncanonical ARF-dependent transcriptional mechanism

“…In contrast, we have now established that ARF is necessary for c-Myc to directly induce transcription of a noncanonical target gene, Egr1. In support of this finding, Egr1 was recently found to be induced by c-Myc in a microarray using B cells (27). Several previously identified canonical c-Myc target genes can induce apoptosis, including ODC and MT-MC1 (28,29); however, it has not been shown conclusively that loss of a canonical direct target gene disrupts cMyc-induced apoptosis independently of p53.…”

Regulation of gene transcription by the oncoprotein MYC

“…Because in all these analyses RNA levels are measured, effects on RNA processing, in particular RNA stability, may also contribute to the observed differences. In another approach nuclear run-on assays were combined with array analyses to assess the effect of MYC on gene-bound active polymerases (Fan et al, 2010). The data reveal that a small number of genes is rapidly activated or repressed by MYC, evidence that these genes are indeed direct targets.…”

“…The genes identified in the nuclear run-on assays/array analyses described above were further classified using Gene Set Matrix Analysis (Fan et al, 2010). A time dependent correlation to certain TF binding sites could be established, suggesting that MYC cooperates preferentially with different factors during specific windows of time.…”