A critical role for topoisomerase IIb and DNA double strand breaks in transcription

ABSTRACT

Recent studies have indicated a novel role for topoisomerase IIb in transcription. Transcription of heat shock genes, serum-induced immediate early genes and nuclear receptor-activated genes, each required DNA double strands generated by topoisomerase IIb. Such strand breaks seemed both necessary and sufficient for transcriptional activation. In addition, such transcription was associated with initiation of the DNA damage response pathways, including the activation of the enzymes: ataxia-telangiectasia mutated (ATM), DNA-dependent protein kinase and poly (ADP ribose) polymerase 1. DNA damage response signaling was involved both in transcription and in repair of DNA breaks generated by topoisomerase IIb.

Introduction

In order to maintain the genetic integrity, the sequence of the genome must be faithfully maintained throughout the lifetime of the host. However, genomic DNA is highly dynamic in nature, undergoing complete replication during each cell cycle, continuous transcription of coding sequences into RNA and reversible folding into higher order structures. These processes are more or less error prone, and thus the existence of complex and highly effective systems for DNA repair.^1,2 DNA damage is closely associated with the exposure to oxidative stresses that can occur as byproducts of normal metabolism but which are amplified when cells are exposed to ultraviolet rays or ionizing radiation.³ In this review, we will discuss the unexpected involvement of mechanisms, thought originally to be utilized exclusively in the repair of DNA double strands breaks, in gene transcription induced by physiological prompts and by stress-responsive transcription factors.

The changes in topology involved when genes are undergoing transcription may be generally conducive to DNA damage. For instance, genomic DNA may be at increased risk for incurring damage when chromatin de-condenses close to the transcriptional start site (TSS), a normal occurrence in transcription required for the transcription factors to access the cognate DNA sequences.⁴ Thus, the resultant “naked DNA”, which has been freed from the nucleosomal proteins becomes more likely to interact with reactive oxygen species (ROS). DNA damage could also occur, when the DNA double helix unwinds within the general transcription factor (GTF)-RNA polymerase II (Pol II) complexes that accumulate on gene promoters during transcription.^3,4 R-loop formation may also become more likely upon initiation of transcription; this process involves the ability of the growing RNA strand to hybridize with the complementary single-stranded template DNA sequence.⁵ The displaced DNA strand, no longer incorporated in ds DNA, can then become sensitive to acquisition of chemical damage on exposure to ROS and the formation of intra-strand secondary structures prone to recombination and mutation. Melting of the DNA double strand by Pol II also results in torsion which generates negative supercoiling in the upstream DNA, with further potential for oxidative damage and positive supercoiling downstream that may physically retard Pol II processivity.⁶

A role for scheduled DNA damage in transcription

In addition to the sporadic DNA damage that may occur as a byproduct of gene transcription as discussed above, recent experiments have also suggested that cells may deploy DNA damage in a more calculated, “scheduled” way in order to trigger transcription. We discuss below the emerging mechanisms involved in transcription-scheduled DNA damage.

Topoisomerase II-generated DNA breaks and transcription

Recent publications have highlighted a key role for topoisomerase-IIb-mediated DNA double strand breaks (DSB) in the transcription of mammalian genes.^7-9 Topoisomerases are essential enzymes involved in resolving the topological problems in duplex DNA, such as positive supercoiling and knotting.¹⁰ The topoisomerase II family catalyze the resolution of such DNA topologies by creating transient DSBs, followed by re-ligation of the breaks when the topological problems are resolved. Topoisomerase II exists in two closely related isoforms, the products of different genes—topoisomerase IIa and IIb.^11,12,13 The topoisomerase IIb isoform is expressed independently of the cell cycle and appears to be important in transcriptional activation by agents as diverse as heat shock, serum induction and responses to nuclear receptors.^7,8,14-16 Previous studies in yeast had shown roles for topoisomerase IIb, particularly in 3′ coding sequences where the enzyme appeared to be involved in relaxing positive supercoiling that occurred downstream of elongating RNA polymerase II, leading to reduced histone density.^17,18 These effects of topoisomerase IIb on RNA elongation during transcription seemed particularly pronounced in longer genes.¹⁹ In eukaryotes, a detailed study of the role of topoisomerase IIb in estrogen-induced transcription was carried out by Ju et al.¹⁴ They found that, in the uninduced state, topoisomerase IIb was located in a repressor complex on the estrogen-dependent pS2 gene promoter, in association with proteins such as nucleolin, nucleophosmin, Hsp70, PARP1 (poly (ADP ribose) polymerase 1), HDAC3 and the gene repressor N-Cor. Treatment with the estrogen 17-estradiol led to estrogen receptor (ER) binding to the promoter, eviction of the majority of the components of the repressor complex (apart from an increase in PARP1 and topoisomerase IIb) as well as loss of nucleosomes from the promoter. The chromatin-associated topoisomerase IIb then catalyzed the production of a DNA DSB at the promoter, a lesion which in turn led to the recruitment of components of the non-homologous end joining repair (NHEJ) pathway. These proteins included subunits of DNA-dependent kinase (DNA-PK) such as the catalytic domain (DNA-PKc), as well as Ku70 (XRCC6) and Ku80 (XRCC5) that associate with DNA-PKc when bound to the ends of ds DNA. Thus, although the DSBs created by topoisomerase IIb activity during transcription may be transient, they are still evidently detected by the DNA DSB repair pathway, and lead to the induction of NHEJ. In the pS2 gene, DSB generation by topoisomerase IIb was followed by the dissociation of the suppressive linker histone H1 from the promoter and recruitment of the high mobility group B1 (HMGB1) protein, as well as association of the activating histone acetylase CBP. Indeed a role for “creative DNA damage”, resulting from topoisomerase IIb activation in transcription seems to be a theme running through a number of subsequent studies. Topoisomerase IIb activity was shown to be required for transcription of serum-inducible immediate early genes in mammalian cells and recruitment of Pol II was prevented by topoisomerase IIb inhibitor ICRF193.⁸ Serum led to the increases in the non-canonical histone gamma-H2AX on IE genes such Fos, Myc, Jun and EGR1 and Hsp70, an effect inhibited by ICRF193.⁸ As with the pS2 gene, activation led to the recruitment of NHEJ proteins DNA-PKc and Ku70, an effect that appeared essential for transcription. Similar findings were also reported for neuronal IE genes, in which induction led to the generation of DSB within the promoter and accumulation of gamma-H2AX.⁷ Of even more significance, the artificial generation of targeted DSB in the promoters of IE genes Fos and Npas4 led to their transcription even in the absence of activation signals, strongly indicating the significance of topoisomerase-IIb-induced DSB as an initiating event in transcription.⁷ These findings strongly suggested that topoisomerase-IIb-mediated DSB in promoter DNA is an initiating event in activator-induced transcription. In addition, the glucocorticoid receptor was shown to activate transcription by recruiting topoisomerase IIb, Ku70 and Ku80 through a mechanism involving BRG1, a component of SWI/SWF chromatin remodeling complexes.²⁰ It may be significant that in the hsp70 and IE genes, gamma-H2AX was found in the promoter, promoter proximal and ORF regions after activation.⁸ It was not clear whether DSB were generated throughout the activated gene or whether a DSB produced in the promoter led to spreading of gamma-H2AX to distant locations away from the break, as has been observed in responses to DNA-damaging agents.²¹ It would appear that the DSB generated by topoisomerase IIb might have more than one role in transcription. The breaks appeared to be generated in the promoters of a number of genes, initiating DDR signaling events and leading to remodeling of histones essential for transcription.^7,14 Topoisomerase IIb may also have been required to resolve the supercoiling and torsion generated by elongating Pol II. Such Pol-II-generated torsion is thought to stall Pol II on transcribing genes and effective elongation may thus require the intervention of topoisomerase IIb to resolve inhibitory supercoiling (Fig. 1).^6,18

Figure 1.

Role of Topoisomerase IIb in Transcription.

Although topoisomerase IIb is necessary for many positive events in cells and seems required for growth, its deployment in transcription is hazardous to such cells.¹² Unrepaired strand breaks have the capacity to lead to programmed cell death or chromosomal translocations.^12,22 Indeed, androgen-mediated and topoisomerase-IIb-dependent transcription in prostate cancer can lead to carcinogenic gene rearrangements, indicating the hazards involved in utilizing this pathway in transcriptional activation.¹⁶ Translocations involve the generation of DSB, followed by the search by the broken strand for partner sequences in the genome.²³ Re-ligation of broken DNA ends involves recombination repair, usually with adjacent sequences.²³ However, broken DNA may recombine with sequences that are not adjacent in linear sequences due to the extensive looping that can occur as regulatory motifs are bound by transcription factors and architectural factors anchoring intra- and inter-chromosomal DNA loops.^24,25 Thus, regularly occurring oncogenic rearrangements can be generated between non-adjacent genes, potentially leading to multiple fusion proteins, as seen in ALL and prostate cancer.^26,27 The involvement of topoisomerase IIb-mediated DNA breaks in transcription can thus exacerbate this problem and has been shown to promote the TMPRSS2-ERG rearrangement in prostate cancer.¹⁶

DNA repair kinases activated during transcription

Transcriptional activation has been shown recently to involve the activities of protein kinases associated previously with DNA repair.^8,14,28 These included DNA-PKc as mentioned above as well as the kinase ataxia-telangiectasia mutated (ATM). Both kinases are members of the Phosphatidyl inositol-3-kinase-like protein kinase (PIKK) family, are of high molecular weight and phosphorylate overlapping substrates to regulate the DNA damage response.^29-31 ATM is the principle coordinator of the DNA damage response and can initiate phosphorylation signaling, leading to both cell cycle arrest after damage and repair of damaged DNA lesions.³⁰ DNA-PK is a key regulator of NHEJ recombination repair as mentioned above and acts as a primary factor in this mechanism as well as in signaling downstream of ATM.^29,31 DNA-PK is recruited to the sites of DSB by its association with Ku70 and Ku80, proteins that bind to DNA proximal to the strand break (Fig. 2). ATM is recruited to DSB in association with the MRN (RAD50/MRE11/NBS1) complex that can directly sense DNA damage.^30,32 Both ATM and DNA-PK phosphorylate H2AX, lead to gamma-H2AX increases on chromatin after ionizing radiation and appear to operate in a redundant manner.³³ Similar overlapping effects of ATM and DNA-PK were observed in activation of the mammalian HSP70 gene by heat shock.²⁸

Figure 2.

PIKK Kinases and DNA Damage.

Using ChIP-PCR and ChIP-seq approaches, it was shown that gamma-H2Ax accumulates on the chromatin of transcribing heat shock and IE genes coordinately with DNA-PK, suggesting a role for this histone modification in transcription. In the DNA damage response, gamma-H2Ax recruits mediator of DNA damage checkpoint1 (MDC1) to chromatin, resulting in further accumulation of MRN complexes and ATM signaling.²¹ Next, gamma-H2Ax spreads from the original DSB site, likely due to phosphorylation by kinases downstream of ATM such as CHK2 and may, thus, interact with distant targets.³⁴ It is notable that spreading of gamma-H2Ax throughout the ORFs of IE genes activated by serum was observed and that this effect was inhibited by DNA-PK and ATM inhibitors.⁸ However roles for downstream kinases in H2Ax phosphorylation were not investigated.

Transcriptional activation of heat shock and IE genes also involved the phosphorylation of KRAB-associated protein 1 (KAP1), also known as Tripartate Motif Containing 28 (TRIM28) by ATM and DNA-PK.^8,28,35 KAP1 is a regulator of heterochromatin and may be deleterious to DNA repair.^21,36 In the DDR, KAP1 is phosphorylated by ATM after DNA damage and this effect appeared to loosen its association with heterochromatin and lead to chromatin de-condensation and accession of repair proteins to the site.³⁷ It is tempting to imagine a similar role for KAP1 phosphorylation by ATM and or DNA-PK, triggered by topoisomerase IIb nicking during transcription, in removing nucleosome obstacles ahead of the elongating Pol II, although evidence to support this possibility is not available. Studies on pS2 gene activation by estrogens had suggested a role for HMG1 association at the promoter in transcriptional activation downstream of topoisomerase-IIb-mediated DSB.¹⁴ During the DDR response, HMGA1 has been shown to be phosphorylated by ATM, a modification leading to its recruitment to chromatin.^38,39 It will be interesting, in further studies to discover additional PIKK substrates that may play significant roles in transcription after DNA damage generated by this process.

The role of PARP1

A recurring theme in the studies linking DNA repair to transcription has been the involvement of PARP1 in the mechanism of trans activation.^14-16,40 PARP1 is an enzyme that modifies substrate proteins in the nucleus by adding poly (ADP-ribose) (PAR) residues to glutamate or aspartate residues to its substrates. Further residues can be added to the PAR chain, which can also form branched polymers, leading to a large cloud-like structure with a strong negative charge. For DNA-bound proteins, this addition of chains of bulky, negatively charged residues can lead to dissociation of the proteins from DNA.⁴¹ PARP1 can interact with other proteins on chromatin, either non-covalently or covalently by poly (ADP-ribosylation) or PARylation and these contrasting interactions appear to be important in transcriptional regulation.⁴² In DNA repair pathways, PARP1 plays key roles in repair of single strand breaks as well as, in combination with BRCA in homologous recombination.^43,44 DNA strand breaks powerfully activate the PARylation activity of PARP1 and this leads to modification of multiple proteins involved in the repair.⁴² Most notably, PARP1 leads to modification of histones in damaged DNA, eviction of such histones and nucleosome relaxation.

PARP1 appears to play roles both in transcriptional repression and activation.⁴² In the DNA damage response, PARylation of activating factors and GTF s may inhibit their association, preventing transcription of damaged DNA.^42,45-47 However, PARP1 itself appears to be an activator of multiple genes, an effect that does not seem to require its enzymatic activity.⁴² The presence of PARP1 within multiple genes may actually be a bonus under conditions of cell stress when its enzyme activity is switched on by stress-induced signaling and transcription can be shut down rapidly.

However, under some circumstances such PARylation activity can directly activate transcription. Studies using Drosophila polytene chromosomes indicated that activated developmental and heat shock genes expand into puffs, which are areas of de-condensed chromatin, and this property was ascribed to the induction of histone PARylation by a Drosophila PARP1 homolog.^48,49 Likewise during heat shock, binding of the factor HSF to the Drosophila Hsp70 gene led to PARP1 release from structures where it had been stored at the promoter and its processive movement along the gene undergoing transcriptional elongation. PARP1 modification by PARylation, and the resulting extended poly-ADP-ribose chains were envisaged as playing roles in releasing histones from the transcribed ORF, dislodgement of nucleosomes and escape from Pol II promoter proximal pausing. The histone acetylase TIP60 was shown to be recruited by HSF to the promoter and to play a decisive role in release of PARP1 from the promoter.^50,51 It may be significant that TIP60 also plays a key role in activation of ATM by DNA damage.³⁰ In these studies therefore, the role of PARP1 in transcriptional activation is seen as being decisive in the de-condensation of chromatin in the activated genes.^48-51 In neuronal differentiation, PARP1 was seen to play a different trans-activating role by PARylation, by inactivating a repressive complex containing itself, nucleolin, nucleophospmin and RAD50 on exposure to inducing stimuli.¹⁵

Finally, the seminal studies of Ju et al. in 2006, on estrogen-inducible transcription bring together many of the threads explored in this review, including the roles of topoisomerase IIb, PIKK kinases and their substrates and PARP1 in transcription (Fig. 3). This group was able to demonstrate estrogen-induced recruitment of topoisomerase IIb, PARP1, DNA-PK, Ku70 to the pS2 gene (along with dissociation of a repressor complex containing nucleolin, nucleophosmin, N-Cor and HDAC3).¹⁴ Activation involved the production of DNA DSB within the promoter by topoisomerase IIb and the exchange of repressive histone H1 for HMGB1. Causal roles in transcription were demonstrated for both topoisomerase IIb and PARP1. The transcriptional mechanisms explored in this study appeared to be general in nature and were involved in a range of inducible genes including the PSA, RAR-α, Dio1 and MMP12 genes.¹⁴

Figure 3.

Combinatorial Roles of Topoisomerase IIb, PARP1 and PIKK kinases in Transcription.

DSB-mediated transcriptional arrest

There is a considerable body of evidence to also indicate that DNA damage can inhibit transcription largely at the elongation level, with Pol II arresting upstream from the lesions.^52,53 The degree of Pol II pausing appeared to depend on the nature and severity of the damage.⁵² DNA DSB generated by radiation were associated with a profound delay in transcription due to reduced rates of RNA elongation by Pol II.⁵³ Delays in transcription after DNA damage were dependent on the activities of PIKK kinase ATM and with PARP1 activity.^54,55 These activities of ATM and PARP1 have thus been associated with both inhibition and activation of transcription (as discussed above), dependent on context. Clearly, the influence of DNA damage in terms of transcription may be quite different depending on whether DSB are generated in a scheduled manner at discrete locations as with transcription-regulated topoisomerase IIb nicking, or whether the areas of DNA damage are extended in nature as in responses to ROS during radiation.

Conclusions

Evidence from multiple sources thus seems to concur on a central role for topoisomerase IIb as a key initiating factor in the transcription of multiple genes in prokaryotic and eukaryotic cells.^7,8,14,56 Most of the available evidence has suggested that the enzyme becomes activated during transcriptional initiation, leading to DNA DSB within the promoters of target genes.^7,14 However, the nature of the transcriptional mechanisms that require the activity of topoisomerases in transcription is not clear. Dynamic supercoiling has been observed upstream of the TSS in active genes, using psoralen photobinding as a probe. Handling of torsion associated with this activity involved topoisomerases, particularly at high-output promoters.⁵⁷ In addition, topoisomerase IIb was shown to be recruited to locations in promoters of neuronal early-response genes at consensus sites for the architectural factor CCCTC-binding factor (CTCF).⁷ This factor has been shown in 3C-based studies to play a diverse array of properties in regulating the three-dimensional properties of DNA in the nucleus, often in association with the structural protein cohesin.^25,58 CTCF can thus facilitate the long-range DNA looping interactions in the nucleus, bringing together enhancer sequences and promoters through intra- and inter-chromosomal interactions.⁵⁸ CTCF can also bind to RNA polymerase II.⁵⁹ Topoisomerase-IIb-catalyzed breaks might permit rapid resolution/recycling of loop structures arising in the promoter that may be involved in accessing RNA Polymerase II after transcriptional activation.⁶⁰

These findings suggested therefore the possibility that topological factors within DNA sequences in unactivated promoters may strongly influence transcription. This scenario is somewhat reminiscent of recent findings regarding the mechanisms of AR-regulated gene enhancers which, in this case require topoisomerase I for transcriptional activity.⁶¹ The nature of the topological events involved is however, in each case unclear. One possibility could be that the restricted levels of nucleosomes found in many promoters and enhancers may influence relative levels of supercoiling. In addition, some gene promoters have been shown to contain G-quadruplex (G4) structures that may retard transcription and require topological resolution.⁶² Dynamic cycles of DNA looping and release, involving topoisomerase IIb within promoters, as mentioned above may also play a crucial regulatory role. Clearly, more precise evidence will be required to ascertain whether promoters and enhancers are characterized by a particular DNA topology and whether topoisomerase-IIb-mediated DSB are needed to revert these structures to forms more compatible with transcription.

Topoisomerase-mediated transcription also generates DSB that are recognized and resolved by the DNA repair pathways.^7,8,14,61 However, these DSB repair mechanisms may be required for functions over-and-above assuring the sealing of topoisomerase-induced breaks. PIKK kinases and PARP1 appear to play regulatory roles in transcriptional activation.^8,14,28,51 PIKK kinases may be required to phosphorylate substrates such as Kap1 and H2Ax thus influencing trans activation and elongation.^8,28,35 PIKK kinases may restrain the rate of transcription by mediating DSB repair,⁷ while being essential to reduce accumulation of unrepaired breaks. PARP1 appears to play multiple roles in transcription and is required for the relaxation of chromatin in activated transcription units.^14,48,50,51

We depict the enzyme playing two potential roles in transcriptional activation, including (i) the generation of an initiating DSB at the promoter when activating factors are bound, and (ii) the resolution of Pol II-induced positive supercoiling downstream in the ORF as the induced RNA is elongated.

DNA-PK becomes activated when the Ku70 and Ku80 subunits bind to the ends of DSBs and recruit DNA-PKc. Activated DNA-PKc is then able to catalyze phosphorylation of histone H2Ax depicted within a nucleosome, as well as other substrates on DNA. ATM is recruited to DSB by association with the MRN complex and can then catalyze phosphorylation of multiple substrates, many of which overlap with those phosphorylated by DNA-PK.

We depict in (a) an inducible gene that might be hsp70, an IE gene, or nuclear receptor-activated gene, as containing PARP1 and topoisomerase IIb in the promoter and downstream nucleosomes as well as paused Pol II in the gene body. (b) On activation, a transcription factor dimer (blue symbol) binds the promoter leading to the accumulation of the PIC at the TSS and generation of DSB by topoisomerase IIb at a promoter site. (c) In addition, PARP1 is released from its promoter site, becomes modified by PARylation and leads to chromatin de-condensation along the transcribing gene in a processive manner. (d) The DSBs created by topoisomerase IIb are recognized by the DNA damage response pathway, triggering recruitment of complexes containing ATM and DNA-PK, and leading to modification of histone H2Ax (indicated by yellow circles) and other chromatin bound proteins and a cascade of DNA damage signaling. The combination of the processes in (b), (c) and (d) leads to transcription as depicted by Pol II elongation in (d).

Abbreviations

ADP

ribose poly

PARP 1

polymerase 1

PIKK

phosphatidyl-inositol-3 kinase like protein kinase

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by NIH research grants, RO-1CA119045 and RO-1CA094397.