| Oncoprotein MYC is one of the major drivers of human tumorigenesis and has critical roles in the regulation of a variety of cellular processes,including cell cycle,transcription,transformation,cell reprogramming,growth,and proliferation.MYC was long considered as a classical transcription factor that regulates the transcription of specific target genes by binding E-box(CACGTG)or its variants via forming a heterodimer with Max.Subsequent studies have found that MYC can also act as a general transcription amplifier to augment the level of global transcription.Currently,there are three different models to describe the role of MYC in global transcriptional regulation.The first model holds that the effects of MYC on global transcription depend on the affinity between MYC and promoters.Genes with MYC high-affinity binding sites are bound and up-regulated by MYC at physiological levels.However,MYC at oncogenic levels can up-regulate the expression of genes with low MYC binding affinity sites and thus increases the levels of global transcription.The second model posits that MYC binds to all active promoters and promotes the release of RNA polymerase II(RNAPII)at the transcriptional pause site by recruiting P-TEFb complex and thus directly amplifies the global transcription.In this model,genes with high expression are amplified disproportionately compared to those with low expression.The third model was proposed based on our previous study,in which MYC was found to enhance global transcription elongation by antagonizing CDK9 SUMOylation.Thus,exactly how MYC promotes global transcription remains to be further investigated.In this study,we aim to elucidate how MYC regulates CDK9 SUMOylation and determine whether MYC controls global transcription in an DNA-binding dependent manner.We constructed a series of MYC mutants with deletion of various functional domains.We then compared the ability for these mutants to antagonizing CDK9 SUMOylation,promoting phosphorylation of the second serine residue(Ser2P)of the heptapeptide(YSPTSPS)repeats within the C-terminal domain(CTD)of the largest subunit of RNA Pol II and enhancing global transcription.We found that MYC antagonized CDK9 SUMOylation and promoted Ser2 P independent of MYC’s DNA-binding and transcriptional activation domains.In addition,we found that the mutants with deletion of MYC DNA binding or activation domains are partially active in promoting global transcription.However,the mutant inactive for antagonizing CDK9 SUMOylation is also inactive in promoting both Pol II CTD Ser2 P and global transcription,a result in agreement with our previous working model.We also carried out a set of experiments to dissect the mechanism by which MYC antagonizes CDK9 SUMOylation.Our results demonstrated that MYC antagonizes CDK9 SUMOylation by inhibiting the interaction of CDK9 with UBC9,the unique SUMO E2 conjugating enzyme,and the SUMO E3 ligase PIAS1.In addition,the interaction between MYC and CDK9 also protects CDK9 from SUMOylation.We further show MYC promotes the interaction between CDK9 with Cyclin T1 and thus the formation of active P-TEFb complex.Finally,we showed that MYC’s ability in promoting global transcription positively correlates with its effects on cell transformation and proliferation.Taken together,our study demonstrated that MYC can antagonize CDK9 SUMOylation in an DNA-binding independent manner.Furthermore,MYC can also stimulate Pol II CTD Ser2 phosphorylation as well as global transcription in the absence of its DNA binding activity.We also unraveled the mechanism by which MYC antagonizes CDK9 SUMOylation. |
