Jesper Svejstrup: Projects

Background and objectives

Transcription of mRNA-encoding genes underpins all life. Indeed, most processes in cells and organisms are regulated primarily at the level of transcription. DNA damage in genes can directly give rise to harmful mutations, but it can also obstruct the progress of transcribing RNA polymerase II, thereby blocking gene expression. Indeed, repair pathways have evolved that specifically target lesions that stop RNA polymerase II (RNAPII) during its journey across a gene(1,2), so-called transcription-coupled repair pathways. Damage-stalled RNAPII can also be removed by ubiquitylation/degradation, clearing the gene for repair by other means(3).

Conversely, while transcription is essential and therefore protected by a variety of mechanisms, it also itself comes at a cost for genome integrity. For example, high levels of transcription are correlated with breaks at fragile chromosome sites(4), mutagenesis(5), and elevated levels of DNA recombination. Research into how the genome-destabilising effects of transcription are minimised is still at a very early stage, but insight into this research area is essential for our general appreciation of the regulatory mechanisms at play in the interface between transcription and other DNA-related processes, as well as for the understanding of processes underlying genome instability.

Our laboratory has over the last two decades focused on elucidating mechanisms that couple transcription, chromatin remodeling, and DNA repair, initially using the yeast Saccharomyces cerevisiae as our primary model system. However, for the last 5-10 years the yeast system has been supplemented with several exciting projects in human cells, so that research in mammalian cells now comprises the vast majority of our activity. The text below outlines the background for our interest in transcript elongation and the basic biochemical mechanisms underlying the transcription-related cellular response to DNA damage.

RNA polymerase II transcript elongation

During transcript elongation RNAPII moves by Brownian motion rather than the ATP-driven power-strokes typical of DNA helicases. Interestingly, this means that although it moves rapidly forward on average, the polymerase can also perform retrograde motion (backtracking). Indeed, transcript elongation by RNAPII in vitro is known to be a highly discontinuous process, involving frequent pausing, backtracking, and transcriptional arrest, and a plethora of elongation factors are required to stimulate its progression (reviewed in 2). We study the mechanism of transcript elongation and gene traffic in general(6-17), and actively seek to isolate new elongation factors and characterise their activity, in vivo and in vitro.

Transcription and chromatin

Transcription and DNA occur in the context of chromatin. Unfortunately, when we study these reactions in vitro, the present biochemical methods are arguably very sub-optimal. Indeed, the reconstituted chromatin used by most laboratories is extremely un-physiological, and it is reconstituted on what can only be described as very artificial DNA templates. We are working towards establishing techniques which make it possible to produce natural chromatin templates, isolated in large scale from living cells, which we believe should, with time, become the new standard for studies of biochemical reactions that occur in chromatin.

Our lab also spends a large proportion of our time trying to discover new protein factors involved in the interface between the DNA-related processes described above. Here, we have realised that it is sub-optimal to isolate proteins from the soluble nucleoplasm, as most people do, rather than from the compartment where they are actually active, namely chromatin(15,18). For example, we are engaged in isolating factors, which are important for transcription-related response to DNA damage, from human chromatin.

Transcription-coupled DNA repair, and the transcription-related damage response:

UV-induced DNA damage elicits a complex cellular response, including induction of genes required for repair, signalling to checkpoint proteins, and direct activation of various repair pathways. It is obvious that repair of damage in active genes needs to be particularly efficient. Indeed, because they block the progress of RNA polymerase II, bulky DNA lesions (such as those generated by UV-irradiation) pose a particular threat to genome integrity and cellular survival, and multiple mechanisms have therefore evolved to minimise the detrimental effects of such obstacles.

Transcription-coupled nucleotide excision repair (TC-NER) ensures that the transcribed strand of an active gene is repaired much faster than the non-transcribed strand and the genome in general(2). In humans, an inability to perform TC-NER is associated with the severe disorder called Cockayne's syndrome (CS)(19). CS patients are characterised by cutaneous photosensitivity, and CS cells are sensitive to a number of DNA-damaging agents including UV-irradiation, due to their defect in TC-NER.

Surprisingly, CS patients are not predisposed to developing skin cancer, but CS is a very pleiotropic disorder with associated physical and mental retardation (19). The vast majority of CS patients have defects in either the CSA or CSB gene, and cells carrying mutations in these genes are sensitive to UV-irradiation, lack TC-NER, and display a dramatic delay in the recovery of RNA synthesis after DNA damage. Even though the existence of TC-NER and the transcription-repair coupling proteins CSA and CSB have been known for over three decades, the mechanism underlying TC-NER remains obscure. Elucidating the mechanism of transcription-coupled repair is thus one of the last frontiers of research into the mechanisms of DNA repair.

Research in several different laboratories have shown that the CSB protein is recruited to damage-stalled RNAPII and is instrumental in recruiting basal nucleotide excision repair (NER) factors, as well as proteins required for subsequent repair-dependent DNA synthesis (Figure 1).

Figure 1

Figure 1. Stepwise model for TC-NER. Note that RNAPII is likely to be present during the whole reaction and to restart transcription after repair, but it has been omitted from the cartoon in later steps for simplicity. (Click to view larger image)

Without CSB, no TC-NER complex is assembled, and CSA is not recruited either. Interestingly, however, CSA is dispensable for both CSB recruitment and TC-NER complex assembly(20). It is, however, still absolutely required for TC-NER to take place. Our recent unpublished data show that even factors that are required for gap synthesis only after the DNA damage has been excised, are recruited already prior to dual DNA incision (Figure 1)(21).

The function of CSB (a DNA-dependent ATPase (translocase) of the SNF/SWI family) appears to be conserved from yeast to man, and in general, it is highly likely that TC-NER in yeast and mammalian cells occurs via similar basic mechanisms (reviewed in2). Interestingly, we have found that CSB contains a ubiquitin-binding domain (UBD), which is dispensable for repair complex assembly, but essential for activation of the DNA incision(21).

Importantly, the CSA protein is part of a cullin-based ubiquitin ligase complex, but the functional importance of this activity in TC-NER is not known. With the discovery that CSB contain a functionally important UBD, it is an obvious possibility that CSA-Cullin mediated ubiquitylation of a factor in the repair complex is recognised by CSB via its UBD, and that this in turn activates the repair reaction(21). It is obviously a major goal for our research into transcription-coupled repair to understand the biochemical function of the CSA-cullin complex and CSB translocase in human cells.

Ubiquitylation of RNA polymerase II: a last resort

Another very important, transcription-relevant response to bulky DNA damage in active genes is ubiquitylation and degradation of RNAPII. Ubiquitylation ultimately results in proteasome-dependent polymerase degradation. Like TCR, it is triggered by the arrest of RNAPII at obstacles (and specifically targets this polymerase form), rather than by DNA damage per se(3). Our work in the yeast model system first uncovered the intriguing connection between TCR and RNAPII ubiquitylation: the main factor required for TCR in yeast, Rad26 (homologue of CSB), interacts with a protein called Def1, which is not involved inTC-NER but is instead required for ubiquitylation and degradation of RNAPII in response to DNA damage(22).

Our work has led to the idea that TCR and RNAPII ubiquitylation/degradation represent interconnected, but distinct cellular pathway for contending with DNA damage in active genes(14,16,21-28). However, the molecular function of Def1 and the mechanism underlying ubiquitylation and degradation of RNAPII remain unclear. For example, we have recently reported that RNAPII is ubiquitylated in a two-step process, requiring distinct ubiquitin ligases(27,28) (Figure 2).

Figure 2

Figure 2. Two-step ubiquitylation of RNAPII by distinct ubiquitin ligases.When RNAPII stalls at a bulky DNA lesion, TC-NER allows rapid damage removal and continued transcription. Alternatively, if the lesion for some reason cannot be removed by TC-NER, RNAPII becomes subject to polyubiquitylation (and subsequent degradation by the proteasome). This occurs via the two-step mechanism outlined. Question mark indicates that involvement of a human homologue of the yeast protein has not been shown. Note that the function, if any, of RNAPII K63 poly-ubiquitylation is unknown. (Click to view larger image)

First, Rsp5 (Nedd4 in humans) mono-ubiquitylates RNAPII. This triggers the activity of an Elc1-Cul3 (Elongin-cullin) ubiquitin ligase complex which then produces a lysine 48-linked poly-ubiquitin chain, and triggers polymerase proteolysis. The biochemical mechanism underlying this two-step process remains to be established.

Recently, the fascinating regulation mechanism involving Def1, an unusual protein required for poly-ubiquitylation but not mono-ubiquitylation of RNAPII(22), was resolved(28). Moreover, we have made good progress in isolating what we believe are functional analogues of Def1 in human cells. We believe these proteins help to make decisions about the cellular response to transcription-blocking DNA lesions: DNA repair, RNAPII ubiquitylation, or apoptosis. Work on these proteins, isolated through targeted proteomics, presently represents an important activity in the laboratory.

Transcription-associated genome instability:

DNA helicases such as Senataxin (Sen1 in yeast) and RECQL5 are connected to RNA polymerase II and the maintenance of genome stability. Sen1/Senataxin is required for suppressing the occurrence of genome-destabilising R-loops (RNA-DNA hybrids) in yeast and humans, for example(29,30). RECQL5 is one of five human helicases in the highly conserved RECQ family of proteins.

Genes encoding RECQ helicases such as WRN, BLM, and RECQ4 are mutated in severe human disorders with cancer pre-disposition (Werner's syndrome, Bloom's syndrome, and Rothmund-Thomson Syndrome, respectively)(31,32), and mouse recq5-/- cells show elevated levels of chromosome crossovers and a higher incidence of cancer(33,34). However, whereas much progress has been made on WRN, BLM, and RECQ4, the cellular role of RECQL5 remains very poorly understood.

In proteomic screens for proteins associated with RNAPII in human chromatin, we isolated the RECQL5 protein and showed that it is the only RECQ family member that associates with RNAPII(17). The direct connection between RECQ5 and the RNAPII transcription apparatus suggests a very exciting and completely unexpected role for RECQ5 helicase at the interface of transcription and genomic stability. The functional consequence of this intriguing interaction is unknown, but it is noteworthy that recq5-/- mouse cells have elevated levels of recombination between direct repeats, and are prone to gross chromosomal rearrangements in response to replication stress(34). This is potentially telling in light of the fact that high transcription levels give rise to dramatically elevated levels of DNA recombination, and that such Transcription-Associated Recombination (TAR) requires DNA replication(35,36).

Intriguingly, we have recently reported that RECQL5 potently inhibits RNAPII transcription in vitro(12), but the nature of the connection between RECQL5 and transcription in vivo was until recently unknown. We then showed that RECQL5 is a general elongation factor, important for preserving genome stability during transcription(17). Depletion or overexpression of RECQL5 results in corresponding shifts in the genome-wide RNAPII density profile. Transcript elongation is particularly affected, with RECQL5 depletion causing a striking increase in the average rate. Concurrently, increased transcription stress is observed, together indicating that RECQL5 controls the movement of RNAPII across genes. Loss of RECQL5 also results in the loss or gain of genomic regions, with the breakpoints of lost regions located in genes and common fragile sites. Importantly, the chromosomal breakpoints overlap with areas of elevated RNAPII transcription stress, suggesting that RECQL5 suppresses the detrimental effects of such stress and thereby prevents genome instability in the transcribed region of genes(17).

References:

1 Selth, L. A., Sigurdsson, S. & Svejstrup, J. Q. (2010). Transcript Elongation by RNA Polymerase II. Annu Rev Biochem 79, 271-293.

2 Svejstrup, J. Q. (2002). Mechanisms of transcription-coupled DNA repair. Nat Rev Mol Cell Biol 3, 21-29.

3 Wilson, M. D., Harreman, M. & Svejstrup, J. Q. (2013). Ubiquitylation and degradation of elongating RNA polymerase II: the last resort. Biochim Biophys Acta 1829, 151-157.

4 Yu, A., Fan, H. Y., Liao, D., Bailey, A. D. & Weiner, A. M. (2000). Activation of p53 or loss of the Cockayne syndrome group B repair protein causes metaphase fragility of human U1, U2, and 5S genes. Mol Cell 5, 801-810.

5 Saxowsky, T. T. & Doetsch, P. W. (2006). RNA polymerase encounters with DNA damage: transcription-coupled repair or transcriptional mutagenesis? Chem Rev 106, 474-488.

6 Kristjuhan, A. et al. (2002). Transcriptional inhibition of genes with severe histone h3 hypoacetylation in the coding region. Mol Cell 10, 925-933.

7 Winkler, G. S., Kristjuhan, A., Erdjument-Bromage, H., Tempst, P. & Svejstrup, J. Q. (2002). Elongator is a histone H3 and H4 acetyltransferase important for normal histone acetylation levels in vivo. Proc Natl Acad Sci USA 99, 3517-3522.

8 Gilbert, C., Kristjuhan, A., Winkler, G. S. & Svejstrup, J. Q. (2004). Elongator interactions with nascent mRNA revealed by RNA immunoprecipitation. Mol Cell 14, 457-464.

9 Kristjuhan, A. & Svejstrup, J. Q. (2004). Evidence for distinct mechanisms facilitating transcript elongation through chromatin in vivo. Embo J 23, 4243-4252.

10 Close, P. et al. (2006). Transcription impairment and cell migration defects in elongator-depleted cells: implication for familial dysautonomia. Mol Cell 22, 521-531.

11 Uhler, J. P., Hertel, C. & Svejstrup, J. Q. (2007). A role for noncoding transcription in activation of the yeast PHO5 gene. Proc Natl Acad Sci U S A 104, 8011-8016.

12 Aygun, O. et al. (2009). Direct Inhibition of RNA Polymerase II Transcription by RECQL5. J Biol Chem 284, 23197-23203.

13 Saeki, H. & Svejstrup, J. Q. (2009). Stability, Flexibility, and Dynamic Interactions of Colliding RNA Polymerase II Elongation Complexes. Mol Cell 35, 191-205.

14 Sigurdsson, S., Dirac-Svejstrup, A. B. & Svejstrup, J. Q. (2010). Evidence that transcript cleavage is essential for RNA polymerase II transcription and cell viability. Mol Cell 38, 202-210.

15 Close, P. et al. (2012). DBIRD complex integrates alternative mRNA splicing with RNA polymerase II transcript elongation. Nature 484, 386-389.

16 Hobson, D. J., Wei, W., Steinmetz, L. M. & Svejstrup, J. Q. (2012). RNA polymerase II collision interrupts convergent transcription. Mol Cell 48, 365-374.

17 Saponaro, M. et al. (2014). RECQL5 Controls Transcript Elongation and Suppresses Genome Instability Associated with Transcription Stress. Cell 157, 1037-1049.

18 Aygun, O., Svejstrup, J. & Liu, Y. (2008). A RECQ5-RNA polymerase II association identified by targeted proteomic analysis of human chromatin. Proc Natl Acad Sci U S A 105, 8580-8584.

19 Brooks, P. J. (2013). Blinded by the UV light: how the focus on transcription-coupled NER has distracted from understanding the mechanisms of Cockayne syndrome neurologic disease. DNA Repair (Amst) 12, 656-671.

20 Fousteri, M., Vermeulen, W., van Zeeland, A. A. & Mullenders, L. H. (2006). Cockayne syndrome A and B proteins differentially regulate recruitment of chromatin remodeling and repair factors to stalled RNA polymerase II in vivo. Mol Cell 23, 471-482.

21 Anindya, R. et al. (2010). A ubiquitin-binding domain in Cockayne syndrome B required for transcription-coupled nucleotide excision repair. Mol Cell 38, 637-648.

22 Woudstra, E. C. et al. (2002). A Rad26-Def1 complex coordinates repair and RNA pol II proteolysis in response to DNA damage. Nature 415, 929-933.

23 Reid, J. & Svejstrup, J. Q. (2004). DNA damage-induced Def1-RNA polymerase II interaction and Def1 requirement for polymerase ubiquitylation in vitro. J Biol Chem 279, 29875-29878.

24 Somesh, B. P. et al. (2005). Multiple Mechanisms Confining RNA Polymerase II Ubiquitylation to Polymerases Undergoing Transcriptional Arrest. Cell 121, 913-923.

25 Anindya, R., Aygun, O. & Svejstrup, J. Q. (2007). Damage-Induced Ubiquitylation of Human RNA Polymerase II by the Ubiquitin Ligase Nedd4, but Not Cockayne Syndrome Proteins or BRCA1. Mol Cell 28, 386-397.

26 Somesh, B. P. et al. (2007). Communication between distant sites in RNA polymerase II through ubiquitylation factors and the polymerase CTD. Cell 129, 57-68.

27 Harreman, M. et al. (2009). Distinct ubiquitin ligases act sequentially for RNA polymerase II polyubiquitylation. Proc Natl Acad Sci U S A 106, 20705-20710.

28 Wilson, M. D. et al. (2013). Proteasome-mediated processing of Def1, a critical step in the cellular response to transcription stress. Cell 154, 983-995.

29 Mischo, H. E. et al. (2011). Yeast Sen1 helicase protects the genome from transcription-associated instability. Mol Cell 41, 21-32.

30 Skourti-Stathaki, K., Proudfoot, N. J. & Gromak, N. (2011). Human senataxin resolves RNA/DNA hybrids formed at transcriptional pause sites to promote Xrn2-dependent termination. Mol Cell 42, 794-805.

31 Hickson, I. D. (2003). RecQ helicases: caretakers of the genome. Nat Rev Cancer 3, 169-178.

32 Bachrati, C. Z. & Hickson, I. D. (2008). RecQ helicases: guardian angels of the DNA replication fork. Chromosoma 117, 219-233.

33 Hu, Y. et al. (2005). Recql5 and Blm RecQ DNA helicases have nonredundant roles in suppressing crossovers. Mol Cell Biol 25, 3431-3442.

34 Hu, Y. et al. (2007). RECQL5/Recql5 helicase regulates homologous recombination and suppresses tumor formation via disruption of Rad51 presynaptic filaments. Genes Dev 21, 3073-3084.

35 Prado, F. & Aguilera, A. (2005). Impairment of replication fork progression mediates RNA polII transcription-associated recombination. Embo J 24, 1267-1276.

36 Gottipati, P., Cassel, T. N., Savolainen, L. & Helleday, T. (2008). Transcription-associated recombination is dependent on replication in Mammalian cells. Mol Cell Biol 28, 154-164.

Jesper Svejstrup

jesper.svejstrup@crick.ac.uk
+44 (0)20 379 62045

  • Qualifications and history
  • 1993 PhD, Aarhus University, Denmark
  • 1993 Postdoctoral Fellow, Stanford School of Medicine, USA
  • 1996 Established lab at the Imperial Cancer Research Fund, UK (in 2002 the Imperial Cancer Research Fund became Cancer Research UK)
  • 2011 Adjunct Professor, University of Copenhagen, Denmark
  • 2015 Group Leader, the Francis Crick Institute, London, UK