The extent of cyclin C promoter occupancy directs changes in stress-dependent transcription

Abstract

The Cdk8 kinase module (CKM) is a detachable Mediator subunit composed of cyclin C and one each of paralogs Cdk8/Cdk19, Med12/Med12L, and Med13/Med13L. Our previous RNA-Seq studies demonstrated that cyclin C represses a subset of hydrogen peroxide–induced genes under normal conditions but is involved in activating other loci following stress. Here, we show that cyclin C directs this transcriptional reprograming through changes in its promoter occupancy. Following peroxide stress, cyclin C promoter occupancy increased for genes it activates while decreasing at loci it represses under normal conditions. Promoter occupancy of other CKM components generally mirrored cyclin C, indicating that the CKM moves as a single unit. It has previously been shown that some cyclin C leaves the nucleus following cytotoxic stress to induce mitochondrial fragmentation and apoptosis. We observed that CKM integrity appeared compromised at a subset of repressed promoters, suggesting a source of cyclin C that is targeted for nuclear release. Interestingly, mTOR inhibition induced a new pattern of cyclin C promoter occupancy indicating that this control is fine-tuned to the individual stress. Using inhibitors, we found that Cdk8 kinase activity is not required for CKM movement or repression but was necessary for full gene activation. In conclusion, this study revealed that different stress stimuli elicit specific changes in CKM promoter occupancy correlating to altered transcriptional outputs. Finally, although CKM components were recruited or expelled from promoters as a unit, heterogeneity was observed at individual promoters, suggesting a mechanism to generate gene- and stress-specific responses. The Cdk8 kinase module (CKM) is a detachable Mediator subunit composed of cyclin C and one each of paralogs Cdk8/Cdk19, Med12/Med12L, and Med13/Med13L. Our previous RNA-Seq studies demonstrated that cyclin C represses a subset of hydrogen peroxide–induced genes under normal conditions but is involved in activating other loci following stress. Here, we show that cyclin C directs this transcriptional reprograming through changes in its promoter occupancy. Following peroxide stress, cyclin C promoter occupancy increased for genes it activates while decreasing at loci it represses under normal conditions. Promoter occupancy of other CKM components generally mirrored cyclin C, indicating that the CKM moves as a single unit. It has previously been shown that some cyclin C leaves the nucleus following cytotoxic stress to induce mitochondrial fragmentation and apoptosis. We observed that CKM integrity appeared compromised at a subset of repressed promoters, suggesting a source of cyclin C that is targeted for nuclear release. Interestingly, mTOR inhibition induced a new pattern of cyclin C promoter occupancy indicating that this control is fine-tuned to the individual stress. Using inhibitors, we found that Cdk8 kinase activity is not required for CKM movement or repression but was necessary for full gene activation. In conclusion, this study revealed that different stress stimuli elicit specific changes in CKM promoter occupancy correlating to altered transcriptional outputs. Finally, although CKM components were recruited or expelled from promoters as a unit, heterogeneity was observed at individual promoters, suggesting a mechanism to generate gene- and stress-specific responses. Cells exhibit multiple adaptive responses following exposure to cytotoxic agents. The failure to mitigate the cellular damage caused by these stressors can initiate regulated cell death (RCD) pathways (1Fulda S. Gorman A.M. Hori O. Samali A. Cellular stress responses: cell survival and cell death.Int J. Cell Biol. 2010; 2010 (20182529): 21407410.1155/2010/214074Crossref PubMed Scopus (870) Google Scholar). One common cause of cellular damage is reactive oxygen that can be derived either endogenously (e.g. elevated respiration, oxidase overexpression) or by environmental exposure to pro-oxidants (2Avery S.V. Molecular targets of oxidative stress.Biochem. J. 2011; 434 (21309749): 201-21010.1042/BJ20101695Crossref PubMed Scopus (334) Google Scholar), such as H2O2 or chemotherapeutics (3Martins N.M. Santos N.A. Curti C. Bianchi M.L. Santos A.C. Cisplatin induces mitochondrial oxidative stress with resultant energetic metabolism impairment, membrane rigidification and apoptosis in rat liver.J. Appl. Toxicol. 2008; 28 (17604343): 337-34410.1002/jat.1284Crossref PubMed Scopus (154) Google Scholar). An important early adaptation is transcriptional reprogramming, which directs many molecular processes within the cell. The transcriptional adaptations induced by oxidative stress include the up-regulation of both pro-death and prosurvival genes. For example, oxidative stress stimulates the tumor suppressor p53-dependent activation of genes that promote intrinsic RCD (iRCD) (4Siauciunaite R. Foulkes N.S. Calabrò V. Vallone D. Evolution shapes the gene expression response to oxidative stress.Int. J. Mol. Sci. 2019; 20 (31234431): 204010.3390/ijms20082040Crossref Scopus (24) Google Scholar). In addition, genes encoding proteins mitigating the effects of oxidative stress are also induced (4Siauciunaite R. Foulkes N.S. Calabrò V. Vallone D. Evolution shapes the gene expression response to oxidative stress.Int. J. Mol. Sci. 2019; 20 (31234431): 204010.3390/ijms20082040Crossref Scopus (24) Google Scholar). This system allows the cell to balance the amount of damage incurred with its ability to facilitate repairs. In contrast to oxidative stress, amino acid starvation inhibits mTOR, resulting in induction of several genes necessary for cell survival pathways including autophagy (5Schworer C.M. Mortimore G.E. Glucagon-induced autophagy and proteolysis in rat liver: mediation by selective deprivation of intracellular amino acids.Proc. Natl. Acad. Sci. U.S.A. 1979; 76 (290994): 3169-317310.1073/pnas.76.7.3169Crossref PubMed Scopus (177) Google Scholar). However, prolonged mTOR inhibition represses cell growth eventually inducing cell death (6Hua H. Kong Q. Zhang H. Wang J. Luo T. Jiang Y. Targeting mTOR for cancer therapy.J. Hematol. Oncol. 2019; 12 (31277692): 7110.1186/s13045-019-0754-1Crossref PubMed Scopus (261) Google Scholar). Therefore, the balance between survival and death is influenced by the type, intensity, and duration of the stress that is relayed to the nucleus to impact transcription. Our previous RNA-Seq studies identified the oxidative stress cyclin C-regulated transcriptome in mouse (Mus musculus) embryonic fibroblast (MEF) cells (7Stieg D.C. Chang K.-T. Cooper K.F. Strich R. Cyclin C regulated oxidative stress responsive transcriptome in Mus musculus embryonic fibroblasts.G3 (Bethesda). 2019; 9 (31036676): PubMed Scopus Google Scholar). Cyclin C was identified as a transcriptional of and genes in the R. of genes that the expression of early genes in Natl. Acad. Sci. U.S.A. PubMed Scopus Google K.F. Strich R. and of the cyclin J. PubMed Scopus Google Strich R. The gene the of in response to Biol. PubMed Scopus Google the of a PubMed Scopus Google Scholar). However, cyclin C as both a transcriptional and a with in C. for Mediator Cdk8 module in J. PubMed Scopus Google A. J. H. Wang H. S. A. A. T. T. C is a Cell Biol. PubMed Scopus Google T. Cdk8 is for mouse Biol. PubMed Scopus Google Scholar). Cyclin C is a of the Cdk8 kinase module which is a detachable and subunit of the Mediator a for the transcriptional 2008; PubMed Scopus Google Scholar). The other of the CKM include Cdk8 or or and or T. R. H. of the and transcriptional proteins from Biol. PubMed Scopus Google Scholar). Cdk8/Cdk19, Med12/Med12L, and are paralogs with but not that are with cyclin C, in a The Mediator a of Mol. Cell Biol. PubMed Scopus Google Scholar). this in it not be that and studies that the CKM can either or Mediator kinase module and Biol. Google Scholar). For example, stimulates and cancer A. J. D. H. S. C. A. J. J. A. of in cancer at PubMed Scopus Google R. S. S. Y. is a cancer that 2008; PubMed Scopus Google Scholar). Cdk8 been and in a impact within C. A. A. for the of PubMed Scopus Google Scholar). stimulates in and mouse Mediator kinase module and Biol. Google J. Wang R. A. Cooper K.F. Strich R. repression of by cyclin C and Cell Sci. 2019; PubMed Google Scholar). Interestingly, is that the CKM in both a and Zhang T. A. R. A. Y. for in 2019; PubMed Scopus Google Scholar). studies that the CKM a in transcriptional control to However, the CKM is to this transcriptional activity is In to its nuclear of cyclin C, but not to the in response to oxidative stress R. Cooper K.F. Strich R. Cyclin C mitochondrial and Biol. PubMed Scopus Google Scholar). In to Cdk8 to cyclin C both the and the to promote mitochondrial fragmentation and R. Cooper K.F. Strich R. Cyclin C mitochondrial and Biol. PubMed Scopus Google V. Chang S. Cooper K.F. Strich R. Cyclin C stimulates to mitochondrial Biol. 2019; PubMed Scopus Google J. Chang A.M. Strich R. of cyclin C stimulates intrinsic apoptosis through 2019; 20 PubMed Scopus Google Scholar). This mitochondrial is in and demonstrated that is required for cyclin C nuclear S. Cooper K.F. Strich R. mitochondrial and cell death in through nuclear of cyclin Biol. PubMed Scopus Google Scholar). cyclin C nuclear in response to oxidative stress in is the system in a regulated multiple pathways and Cdk8 activity D.C. V. J. J. Strich R. Cooper K.F. molecular directs cyclin C nuclear through of Biol. PubMed Scopus Google D.C. R. Strich Cooper K.F. with the to the of following oxidative PubMed Scopus Google Scholar). studies revealed that the CKM to the Mediator suggesting that its in cyclin C in the nucleus is S. C. kinase module Mol. Biol. 20 PubMed Scopus Google Scholar). However, it is not the of cyclin C is for nuclear in cells and is Here, studies revealed that promoter or of cyclin C and the CKM with its as a transcriptional or This following starvation stress, indicating that cyclin C and CKM are by the Finally, CKM integrity was at a subset of repressed promoters suggesting a source for cyclin C nuclear release. these are with a that the CKM is a that directs transcriptional repression and activation through changes in promoter occupancy at genes. Our transcriptome revealed that cyclin C both and transcriptional control of genes induced by H2O2 exposure (7Stieg D.C. Chang K.-T. Cooper K.F. Strich R. Cyclin C regulated oxidative stress responsive transcriptome in Mus musculus embryonic fibroblasts.G3 (Bethesda). 2019; 9 (31036676): PubMed Scopus Google Scholar). this we D. H. A. S. for the of the PubMed Scopus Google to processes in the for genes induced by H2O2 We to study genes and required for autophagy as to cell death and The genes nuclear kinase and and in in The genes were and kinase subunit in in In addition, subunit and were that were induced by H2O2 but are the p53-dependent genes in in Finally, and were as cyclin and under but are not by H2O2 stress. We the transcriptional changes observed with RNA-Seq by the gene of induction in response to H2O2 and were not of were observed for cyclin genes. For the p53-dependent cyclin C is required for of and but for H2O2 induction The transcriptional of genes by identified that required cyclin C for activation and that were repressed normal growth The were observed in that cyclin C is required for both and full pattern was observed for and in that cyclin C was required for both and For the cyclin were observed in a of that was not by H2O2 However, cells a in suggesting that the cyclin to repressed genes and elevated in the of cyclin C that induced by H2O2 in cells that H2O2 not elicit the full activation of these was observed for and repressed genes and loci in that cells the of H2O2 not in that H2O2 was to cyclin was to cyclin C promoter occupancy at these promoters and H2O2 For cyclin C demonstrated promoter in cells suggesting that its control of the at each is are shown in for the Following H2O2 was a in promoter occupancy for the p53-dependent genes by cyclin C and demonstrated increased promoter occupancy at the promoter by the CKM cyclin C, and following but not S. is a of PubMed Scopus Google Scholar). In was a in promoter occupancy of cyclin C following oxidative stress for the repressed gene that cyclin C is recruited to promoters its ability is but its repression is in occupancy were observed for the autophagy and genes. in cyclin C occupancy was observed for the repressed genes and with CKM changes in promoter occupancy were observed for the genes and these are with a that cyclin C normal gene its is important for transcriptional this is we cyclin C promoter occupancy for a of expression that cyclin C for its expression but is in response to H2O2 to this gene exhibit of cyclin C occupancy from its promoter following stress. We one of this and found that both cyclin C and Cdk8 were from the promoter following H2O2 Therefore, changes in CKM occupancy be from the transcriptional and the transcriptional stress Our that cyclin C occupancy is increased it a in but to its studies the CKM that it is found as a for the transcriptional 2008; PubMed Scopus Google Scholar). Therefore, we other components of the CKM also changes in promoter occupancy. were of the CKM and and were individual not been The promoter occupancy by each of the for each gene in cells for both cyclin and genes. Following H2O2 promoter occupancy of the CKM generally mirrored that observed for cyclin C for each of the induced genes and in promoter occupancy for of these proteins was observed for For the repressed genes and was a in promoter occupancy of each CKM in response to oxidative stress to that observed for cyclin C However, CKM promoter occupancy at and although cyclin C were to at these promoters, of the CKM were that cyclin C was from other CKM components at a subset of the or Interestingly, that Cdk8 occupancy at the promoter was at the of in cells a different for Cdk8 promoter occupancy cyclin C is in response to stress. these that promoter by cyclin C and other CKM components are that the CKM moves as a single to and from to this is the repressed promoters, and in which cyclin C and CKM studies revealed between the oxidative stress and D. stress and the between damage and PubMed Scopus Google Scholar). it has been that oxidative stress induces autophagy S. the is the for autophagy 2010; PubMed Scopus Google Scholar), amino acid starvation a oxidative stress response Y. is the reactive oxygen PubMed Scopus Google Scholar). cyclin C of and were with the mTOR S. is a of PubMed Scopus Google Scholar). to H2O2 stress, cyclin C was required for both and of and In addition, repressed and following that was to observed in suggesting that cyclin repression was for induction Interestingly, one gene repressed by cyclin C, following that and H2O2 is cyclin C although to H2O2 stress, following exposure revealed increased cyclin C promoter occupancy for the induced genes and In addition, cyclin C promoter occupancy was at both and are with that cyclin C from promoters is of the the impact of cyclin C is these the that a common is to induce these loci following both H2O2 exposure and mTOR However, following to that observed for H2O2 stress. that cyclin C is recruited to promoters transcriptional we that its promoter occupancy also following to this with a in cyclin C promoter occupancy following with cells of the genes and found or in between control and cells with this cyclin C promoter occupancy also Our from the repressed and promoters a source for nuclear cyclin C following H2O2 cyclin C is from these promoters in response to However, H2O2 cyclin C nuclear following and the Finally, was in cyclin C or induction in response to that not is CKM promoter occupancy altered in response to a its integrity also be the the cell has in cyclin C to and mitochondrial responses to normal growth and exhibit a by the J. The and and module with PubMed Scopus Google Scholar). This study found that or with J. The and and module with PubMed Scopus Google Scholar). In in response to oxidative stress is also by the S. Cooper K.F. Strich R. mitochondrial and cell death in through nuclear of cyclin Biol. PubMed Scopus Google Scholar). Therefore, we cyclin C promoter and transcriptional required the cells were with the to H2O2 of the from the cyclin genes different responses. The of the gene were with H2O2 and was elevated H2O2 suggesting that both stressors to increased transcription. Finally, induction was This that full for the genes and a in promoter occupancy following exposure to the with that of oxidative stress However, although cyclin C occupancy was at the promoter following with both the of was observed in are with a that at of the CKM and transcriptional we that be in response to but this was by with this of and in from with H2O2 revealed of in with the in However, this was of these are with the previous from and cells that cyclin C promoter is with not promoters demonstrated elevated cyclin C following is with previous that the of cyclin C occupancy and CKM integrity are in a Our studies revealed that Cdk8 is required for and cyclin C nuclear the D.C. V. J. J. Strich R. Cooper K.F. molecular directs cyclin C nuclear through of Biol. PubMed Scopus Google Scholar). Cdk8 activity is required for the changes observed for the CKM promoter occupancy in we and promoter occupancy in and cells with the Cdk8 kinase D.C. S. Chang R. S. J. kinase Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar). Following of the of cyclin genes and were with the cells that stress, of these genes Cdk8 kinase the p53-dependent genes and were induced to to that observed in following H2O2 and the cyclin and following the that Cdk8 kinase activity is required for both and induction following H2O2 although for the The of cyclin C promoter occupancy for these genes revealed that cyclin C to these promoters following H2O2 not Cdk8 kinase activity the cyclin and were following with This is in contrast to that of both genes. that transcriptional repression of and is of Cdk8 kinase The up-regulation of and following exposure to oxidative stress is with the promoter of cyclin C. with of cyclin C occupancy with However, were observed with cyclin C in cells at the the gene cyclin C at its promoter under these conditions However, the observed in cyclin C promoter occupancy for the gene was not that although in cyclin C promoter occupancy are of Cdk8 kinase its following H2O2 stress be are not to changes in cyclin C following Interestingly, was a in in cells following exposure to oxidative stress that Cdk8 kinase activity was required for This was not as as that observed in cells suggesting between and for This be to the of cyclin C at the in cells these that Cdk8 kinase activity is required for full transcriptional repression of this The CKM with the Mediator to induce the of genes responsive to several stress pathways including S. is a of PubMed Scopus Google Scholar), oxidative (7Stieg D.C. Chang K.-T. Cooper K.F. Strich R. Cyclin C regulated oxidative stress responsive transcriptome in Mus musculus embryonic fibroblasts.G3 (Bethesda). 2019; 9 (31036676): PubMed Scopus Google Scholar), or Wang to in response to PubMed Scopus Google Scholar). In addition, we found that cyclin C represses of genes that are also induced by H2O2 (7Stieg D.C. Chang K.-T. Cooper K.F. Strich R. Cyclin C regulated oxidative stress responsive transcriptome in Mus musculus embryonic fibroblasts.G3 (Bethesda). 2019; 9 (31036676): PubMed Scopus Google Scholar). However, the CKM both and in response to the stress the CKM is a of this its or from promoters a mechanism to this Using the CKM cyclin C as a to promoter we both and repressed loci that to oxidative stress and starvation through mTOR observed in previous S. is a of PubMed Scopus Google Wang to in response to PubMed Scopus Google Scholar), we found that cyclin C is recruited to promoters that the CKM for activation. In addition, we found that cyclin C occupancy also repression is cyclin C is from genes repressed by the CKM under normal conditions. of cyclin C occupancy are specific to H2O2 or starvation stress was suggesting that changes in CKM promoter occupancy a to this Our also revealed that the other CKM cyclin C promoter or indicating that this as a unit, although were the of CKM in the these that the CKM from a promoter has the CKM is recruited to a promoter this for transcriptional activation this early that of the CKM in the cell was with the Mediator and not of a R. H. R. S. D. with and PubMed Scopus Google Scholar). studies are required to the CKM from one promoter can be for by occupancy at The other of the CKM to cyclin C to to the it with proteins at the to induce and this genes to be that are repressed by cyclin C and induced following H2O2 these that the CKM movement to and from promoters its transcriptional This heterogeneity in response observed at individual promoters that this system the cell a of with which to transcriptional control with CKM CKM is in a of and Mediator kinase module and Biol. Google Scholar). The that to early embryonic A. Zhang Y. M.L. Mediator activation and is required for in the PubMed Scopus Google that and at However, of cyclin C, and promoter occupancy found that the CKM generally moves as a both to and from promoters, although some were observed In addition, with the of the repressed and were found at promoters and with to promoter or at for this gene and transcription. Therefore, of the is that or a gene For example, studies found that or in several but not A. T. O. T. in the gene encoding a of the cause a J. 2019; PubMed Scopus Google A. H. S. C. O. A. the J. PubMed Scopus Google of the J. PubMed Scopus Google R. H. in a of the Mediator are with a PubMed Scopus Google T. with PubMed Scopus Google V. R. R. A. O. C. in encoding a subunit of the kinase are for with transcriptional 2019; PubMed Scopus Google Scholar). Therefore, transcriptional control by and be to gene both of or be to embryonic transcriptional control to cause the of for both and this Our previous from revealed that is required for the nuclear of cyclin C following oxidative stress S. Cooper K.F. Strich R. mitochondrial and cell death in through nuclear of cyclin Biol. PubMed Scopus Google Scholar). with studies indicating that the CKM to the Mediator The Mediator a of Mol. Cell Biol. PubMed Scopus Google Scholar). studies found that the CKM is also in response to many stressors including starvation is a of transcriptional within the response Mol. Biol. 2010; PubMed Scopus Google Scholar). However, revealed that the of the CKM components the stress. For example, in response to oxidative stress, the is by the cyclin C the to mitochondrial fragmentation and RCD to its D.C. V. J. J. Strich R. Cooper K.F. molecular directs cyclin C nuclear through of Biol. PubMed Scopus Google Scholar). In response to cyclin C has a different nuclear cyclin C is the cell from its mitochondrial regulated cell death T. Cooper K.F. cyclin C cell survival following Biol. PubMed Google Scholar). to the we found that in response to H2O2 or cyclin C is from promoters that it represses under normal growth conditions However, several were observed between and cyclin C not following H2O2 or starvation stress in In addition, in which the CKM is this to as it moves from one promoter to the transcriptional the CKM has in In to stress or responsive RNA-Seq also revealed a for cyclin C in the transcriptional activation of genes involved in and (7Stieg D.C. Chang K.-T. Cooper K.F. Strich R. Cyclin C regulated oxidative stress responsive transcriptome in Mus musculus embryonic fibroblasts.G3 (Bethesda). 2019; 9 (31036676): PubMed Scopus Google Scholar). Therefore, the for cyclin C nuclear with the increased of the CKM in transcription. revealed that CKM was not For example, H2O2 in of cyclin C at the and promoters, but in or This pattern was not observed for other repressed and that the CKM was at the and for these is of the mitochondrial of cyclin C In the cyclin C mitochondrial fragmentation and cell death in cells through its with mitochondrial proteins and V. Chang S. Cooper K.F. Strich R. Cyclin C stimulates to mitochondrial Biol. 2019; PubMed Scopus Google J. Chang A.M. Strich R. of cyclin C stimulates intrinsic apoptosis through 2019; 20 PubMed Scopus Google K.F. S. Strich R. of cyclin C mitochondrial in 28 PubMed Scopus Google Scholar). that the CKM at the and promoters is to cyclin C nuclear release. In this is by the a is in we that CKM promoter release. In addition, failure to the CKM of its resulting in transcription. were with in which cyclin C was and activation was observed following This response be to the heterogeneity of at these repressed promoters or a of not the Our that were increased that at proteolysis was However, not repressed genes in this and at the promoter cyclin C that cyclin C from promoters is by one mechanism that be by at promoters that exhibit be heterogeneity in the that the CKM movement and integrity are and of and promoter Our are with of the for the CKM through one or targets of including or is a of transcriptional within the response Mol. Biol. 2010; PubMed Scopus Google The is a kinase that for activity and can of Biol. PubMed Scopus Google Scholar). The observed in the of cyclin C for promoter activation be to the of For example, the p53-dependent response cyclin C for full gene activation in and cyclin C for both and transcription. of the genes induced by cyclin C following H2O2 with the that Cdk8 kinase activity is required for it is not that in response to oxidative stress. Interestingly, in a in in response to oxidative stress The between the impact of and Cdk8 be to the of cyclin C. to However, the of cyclin C at the the impact Therefore, the transcriptional of cyclin We previously demonstrated that cyclin C nuclear is not Cdk8 activity R. Cooper K.F. Strich R. Cyclin C mitochondrial and Biol. PubMed Scopus Google Scholar). Therefore, both resulting in cellular from H2O2 This be a also the of the The of the of cyclin C to has to be from its other these the of cyclin C at multiple of the oxidative and R. Cooper K.F. Strich R. Cyclin C mitochondrial and Biol. 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