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The 3’-UTR of MYC couples RNA polymerase II function to ribonucleotide levels

ABSTRACT: Deregulated expression of MYC enhances glutamine utilization and renders cell survival dependent on an external supply of glutamine, an observation termed “glutamine addiction”. Surprisingly, human colon cancer cells, which express high levels of MYC due to mutations in the WNT pathway, are not glutamine-addicted but undergo a reversible cell cycle arrest upon glutamine deprivation. We show here that glutamine deprivation suppresses endogenous MYC expression via the 3’-UTR of the MYC mRNA, enabling escape from apoptosis. The glutamine-dependent metabolites that mediate this regulation are adenosine nucleotides. Glutamine deprivation causes a global reduction in promoter association of RNA Polymerase II (RNAPII) and reduces the RNAPII traveling ratio, indicative of defects in transcriptional elongation. While RNAPII function is restored by activation of MYC on most genes, restoration of elongation is imperfect and activation of MYC in the absence of glutamine enhances stalling of RNAPII in the body of individual genes, promoting the formation of R-loops. Since stalling of RNAPII and R-loop formation are potential causes of DNA damage, our data argue that the MYC 3’-UTR has a critical role in maintaining genome instability when ribonucleotide levels are low.

ORGANISM(S): Homo sapiens

PROVIDER: GSE86556 | GEO | 2017/04/13

SECONDARY ACCESSION(S): PRJNA342303

REPOSITORIES: GEO

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The 3’-UTR of MYC couples RNA polymerase II function to ribonucleotide levels

Project description:Deregulated expression of MYC enhances glutamine utilization and renders cell survival dependent on glutamine, inducing “glutamine addiction”. Surprisingly, colon cancer cells that express high levels of MYC due to WNT pathway mutations, are not glutamine-addicted but undergo a reversible cell cycle arrest upon glutamine deprivation. We show here that glutamine deprivation suppresses translation of endogenous MYC via the 3’-UTR of the MYC mRNA, enabling escape from apoptosis. This regulation is mediated by glutamine-dependent changes in adenosine nucleotide levels. Glutamine deprivation causes a global reduction in promoter association of RNA Polymerase II (RNAPII) and slows transcriptional elongation. While activation of MYC restores binding of MYC and RNAPII function on most promoters, restoration of elongation is imperfect and activation of MYC in the absence of glutamine causes stalling of RNAPII on multiple genes, correlating with R-loop formation. Stalling of RNAPII and R-loop formation can cause DNA damage, arguing that the MYC 3’-UTR is critical for maintaining genome stability when ribonucleotide levels are low.

2022-02-28 | PXD005829 | Pride

Ubiquitin-dependent turnover of MYC promotes loading of the PAF complex on RNA Polymerase II to drive transcriptional elongation (ChIP-seq)

Project description:The MYC transcription factor is an unstable protein and its turnover is controlled by the ubiquitin system. Ubiquitination enhances MYC-dependent transactivation, but the underlying mechanism remains unresolved. Here we show that proteasomal turnover of MYC is dispensable for recruitment of RNA polymerase II (RNAPII), but is required to promote transcriptional elongation at MYC target genes. Degradation of MYC stimulates histone acetylation and recruitment of BRD4 and P-TEFb to target promoters, leading to phosphorylation of RNAPII CTD and the release of elongating RNAPII. In the absence of degradation, the RNA polymerase II-associated factor (PAF) complex associates with MYC via interaction of its CDC73 subunit with a conserved domain in the amino-terminus of MYC ("MYC box I"), suggesting that a MYC/PAF complex is an intermediate in transcriptional activation. Since histone acetylation depends on a second highly conserved domain in MYCs amino-terminus ("MYC box II"), we propose that both domains co-operate to transfer elongation factors onto paused RNAPII. ChIP-Seq experiments for MYC-HA (HA-IP) and RNAPII (total,Ser2p,Ser5p) performed in IMEC primary breast epithelial cells. Input-samples were sequenced as controls. The following antibodies were used: HA (Abcam; ab 9110)/ total RNAPII (Santa Cruz; sc-899x)/ Ser2p RNAPII (Abcam; ab 5095)/ Ser5p RNAPII (Covance; MMS-128P)

2016-01-04 | E-GEOD-70001 | biostudies-arrayexpress

Ubiquitin-dependent turnover of MYC promotes loading of the PAF complex on RNA Polymerase II to drive transcriptional elongation (RNA-seq)

Project description:The MYC transcription factor is an unstable protein and its turnover is controlled by the ubiquitin system. Ubiquitination enhances MYC-dependent transactivation, but the underlying mechanism remains unresolved. Here we show that proteasomal turnover of MYC is dispensable for recruitment of RNA polymerase II (RNAPII), but is required to promote transcriptional elongation at MYC target genes. Degradation of MYC stimulates histone acetylation and recruitment of BRD4 and P-TEFb to target promoters, leading to phosphorylation of RNAPII CTD and the release of elongating RNAPII. In the absence of degradation, the RNA polymerase II-associated factor (PAF) complex associates with MYC via interaction of its CDC73 subunit with a conserved domain in the amino-terminus of MYC ("MYC box I"), suggesting that a MYC/PAF complex is an intermediate in transcriptional activation. Since histone acetylation depends on a second highly conserved domain in MYCs amino-terminus ("MYC box II"), we propose that both domains co-operate to transfer elongation factors onto paused RNAPII. RNA-Seq Experiments were performed in a primary breast epithelial cell line (IMEC).The cell line expressed doxycycline-inducible versions of MYC (WT;Kless,Swap=WTN-KC). Where indicated cells were transfected with siRNAs (siCtrl;siSKP2). Where indicated cells were treaed with the proteasome inhibitor MG132 or EtOH as solvent control. DGE was performed by comparing Dox-treated populations expressing either Dox-inducible MYC or a vector control or comparing Dox-induced cells with EtOH (solvent control) treated cells.

2016-01-04 | E-GEOD-70000 | biostudies-arrayexpress

Ubiquitin-dependent turnover of MYC promotes loading of the PAF complex on RNA Polymerase II to drive transcriptional elongation (ChIP-seq)

2016-01-04 | GSE70001 | GEO

Ubiquitin-dependent turnover of MYC promotes loading of the PAF complex on RNA Polymerase II to drive transcriptional elongation (RNA-seq)

2016-01-04 | GSE70000 | GEO

Ubiquitin-dependent transfer of PAF1c from MYC onto RNA Polymerase controls double-strand break repair at active promoters

Project description:MYC family proteins are oncogenic transcription factors that can globally affect the function of RNA Polymerase II (RNAPII). The ability of MYC proteins to promote transcription elongation depends on their ubiquitination, but the underlying mechanism and its biological relevance are unknown. Here we show that MYC and the Polymerase II associated factor, PAF1c, interact directly and their function is mutually dependent, since the specific binding of MYC to active promoters depends on PAF1c and, conversely, PAF1c is required for MYC-dependent pause release. Upon binding, PAF1c is rapidly transferred from MYC onto RNAPII and this transfer is driven by the HUWE1 ubiquitin ligase. Both MYC and HUWE1 globally control histone H2B ubiquitylation, which promotes transcriptional elongation and alters chromatin structure for double-strand break repair. Consistently, MYC suppresses the accumulation of double-strand breaks at promoters in response to topoisomerase II inhibition. While depletion of PAF1c has only minor effects on MYC-dependent gene expression, MYC induces rampant transcription-dependent DNA damage in PAF1c-depleted cells. We propose that the HUWE1-dependent transfer of PAF1c from MYC onto RNAPII is critical for absorbing the topological stress accompanied with deregulated and oncogenic transcription.

2021-02-15 | PXD023242 | Pride

Transcription profiling of Zebrafish 24hpf mutant sk8

Project description:The production of functional mRNA involves multiple steps including transcription initiation, elongation, and termination. spt5 encodes a conserved essential transcription elongation factor that controls RNAPII processivity in vitro and co-localizes with RNAPII in vivo. We used microarrays to detail the global expression of gene expression at 24 hours post fertilization in wild-type and Spt5 mutant (fog-sk8) to identify Spt5 transcriptionally dependent genes and affected pathways. Here we report that a mutation in zebrafish spt5, sk8, deregulates <5% of ~10,000 genes examined. These findings reveal a small but diverse set of developmentally regulated genes, whose expression is critically dependent on Spt5-regulated RNAPII stalling in vertebrate development. Experiment Overall Design: RNA from zebrafish embryos were harvested at 24 hours post fertilization for hybridization to Affymetrix gene arrays.

2008-10-25 | E-GEOD-12826 | biostudies-arrayexpress

Glutamine deprivation enhances directed differentiation of renal organoids

Project description:Glutamine deprivation enhances directed differentiation of renal organoids

| PRJNA529412 | ENA

Association with Aurora-A controls N-MYC-dependent promoter escape and pause release of RNA polymerase II during the cell cycle

Project description:MYC proteins bind globally to active promoters and promote transcriptional elongation by RNA polymerase II (RNAPII). To identify effector proteins that mediate this function, we performed mass spectrometry on N-MYC complexes in neuroblastoma cells. The analysis shows that N-MYC forms complexes with TFIIIC, TOP2A and RAD21, a subunit of cohesin. N-MYC and TFIIIC bind to overlapping sites in thousands of RNAPII promoters and intergenic regions. TFIIIC promotes association of RAD21 with N-MYC target sites and is required for N-MYC-dependent promoter escape and pause release of RNAPII. Aurora-A competes with binding of TFIIIC and RAD21 to N-MYC in vitro and antagonizes association of TOP2A, TFIIIC and RAD21 with N-MYC during S-phase, blocking N-MYC-dependent release of RNAPII from the promoter. Inhibition of Aurora-A in S-phase restores RAD21 and TFIIIC binding to chromatin and partially restores N-MYC-dependent transcriptional elongation. We propose that complex formation with Aurora-A controls N-MYC function during the cell cycle.

2017-12-20 | GSE78957 | GEO

Zebrafish 24hpf mutant sk8 gene expression

2008-09-18 | GSE12826 | GEO

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