Integrative analysis of RNA polymerase II and transcriptional dynamics upon MYC activation

Abstract

Overexpression of the MYC transcription factor causes its widespread interaction with regulatory elements in the genome but leads to the up-and down-regulation of discrete sets of genes. The molecular determinants of these selective transcriptional responses remain elusive. Here, we present an integrated time-course analysis of transcription and mRNA dynamics following MYC activation in proliferating mouse fibroblasts, based on chromatin immunoprecipitation, metabolic labeling of newly synthesized RNA, extensive sequencing, and mathematical modeling. Transcriptional activation correlated with the highest increases in MYC binding at promoters. Repression followed a reciprocal scenario, with the lowest gains in MYC binding. Altogether, the relative abundance (henceforth, "share") of MYC at promoters was the strongest predictor of transcriptional responses in diverse cell types, predominating over MYC's association with the corepressor ZBTB17 (also known as MIZ1). MYC activation elicited immediate loading of RNA polymerase II (RNAPII) at activated promoters, followed by increases in pause-release, while repressed promoters showed opposite effects. Gains and losses in RNAPII loading were proportional to the changes in the MYC share, suggesting that repression by MYC may be partly indirect, owing to competition for limiting amounts of RNAPII. Secondary to the changes in RNAPII loading, the dynamics of elongation and pre-mRNA processing were also rapidly altered at MYC regulated genes, leading to the transient accumulation of partially or aberrantly processed mRNAs. Altogether, our results shed light on how overexpressed MYC alters the various phases of the RNAPII cycle and the resulting transcriptional response.[Supplemental material is available for this article.]The MYC transcription factor is overexpressed and acts as an oncogenic driver in numerous tumor types. Shedding light on the transcriptional programs driven by MYC is thus a critical area of investigation, with important translational implications. Indeed, numerous studies focused on the analysis of MYC-induced transcriptional responses, typically measuring its binding through ChIP-seq and profiling transcriptional maps by RNA-seq . A series of papers proposed that, rather than acting as a gene-specific regulator, MYC acts as a general amplifier of transcriptional activity (Lin et al. 2012;Nie et al. 2012). However, our re-analysis of the available data led us to reconsider this model and to conclude that the primary activity of MYC lies in the up-and down-regulation of selected sets of genes, RNA "amplification"-when occurring-being best explained as a secondary consequence (Sabò et al. 2014;Walz et al. 2014;Kress et al. 2015Kress et al. , 2016.At the mechanistic level, how MYC activates and represses transcription remains to be largely addressed. In particular, a unifying view on the role of MYC in the recruitment and progression of RNA polymerase II (RNAPII) within the transcriptional units of regulated genes and the consequent dynamics of transcriptiona...

An early Myc‐dependent transcriptional program orchestrates cell growth during B‐cell activation

“…Among the Myc‐dependent induced genes, those of CL1 also showed the highest increases in Myc share (Fig G). Myc‐dependent repressed genes, instead (CL3, 6, 7, 8), were characterized by a relative loss of Myc binding (decreased share) and by decreased RNAPII recruitment (Fig B and G, Appendix Fig S2), consistent with the concept that Myc‐dependent repression may largely be a passive process —albeit not excluding the existence of active repressive mechanisms at select loci . Altogether, Myc appears to primarily drive RNAPII recruitment at activated loci , with differential contributions of other regulatory steps, in particular pause‐release or elongation .…”

“…Myc-dependent repressed genes, instead (CL3, 6,7,8), were characterized by a relative loss of Myc binding (decreased share) and by decreased RNAPII recruitment ( Fig 4B and G, Appendix Fig S2), consistent with the concept that Myc-dependent repression may largely be a passive process [34]-albeit not excluding the existence of active repressive mechanisms at select loci [28,44]. Altogether, Myc appears to primarily drive RNAPII recruitment at activated loci [34], with differential contributions of other El-myc mice (P), or tumors (T) [4]. Each row represents a promoter-associated Myc peak within a 6-kb genomic interval (centered on the midpoint of the peak).…”

“…Together with the findings that Myc deletion only impacts a subset of the genes regulated by either LPS (this work) or serum [8], we infer that chromatin association cannot be systematically equated with productive regulatory engagement of the transcription factor onto genomic DNA [24,31,34]. One parameter predicting transcriptional responses in other cell types was the relative efficiency in Myc binding at promoters [29][30][31]34], which we expressed as the "Myc share" (obtained by normalizing the signal at each promoter by the total amount of promoter-bound Myc in the genome) [34]: indeed, those genes for which the RNA synthesis rate increased upon LPS stimulation also showed the highest gains in Myc binding (i.e., increasing share), while those with a reduced synthesis showed the lowest gains (decreasing share) ( Fig 3A and B). This correlation was the strongest for Myc-dependent genes, although still present at Myc-independent loci, as also observed in Myc-driven liver cancer [30,34]: albeit paradoxical at first sight, this observation is consistent with the notion that promoter activity and Myc binding are mutually dependent, owing to the interaction of Myc with other chromatin-associated factors, RNAPII, and components of the basal transcription machinery [39,42,43].…”

“…acute accumulation of Myc upon LPS treatment led to its widespread association with pre-existing promoters and enhancers: this phenomenon, also termed "invasion" [3,4,25], most probably occurs through low-affinity, non-sequence-specific interactions with genomic DNA [38]. Together with the findings that Myc deletion only impacts a subset of the genes regulated by either LPS (this work) or serum [8], we infer that chromatin association cannot be systematically equated with productive regulatory engagement of the transcription factor onto genomic DNA [24,31,34]. One parameter predicting transcriptional responses in other cell types was the relative efficiency in Myc binding at promoters [29][30][31]34], which we expressed as the "Myc share" (obtained by normalizing the signal at each promoter by the total amount of promoter-bound Myc in the genome) [34]: indeed, those genes for which the RNA synthesis rate increased upon LPS stimulation also showed the highest gains in Myc binding (i.e., increasing share), while those with a reduced synthesis showed the lowest gains (decreasing share) ( Fig 3A and B).…”

“…Scheme of the different steps of RNAPII regulation for which the corresponding rates were modeled [34]. B, C Boxplots reporting LPS-induced changes (log 2 FC) for each of the four RNAPII kinetic rates in c-myc wt/wt (wt) and c-myc Δ/Δ cells for Myc-dependent (B) and Mycindependent (C) induced and repressed genes.…”