Sirt1 suppresses RNA synthesis after UV irradiation in combined xeroderma pigmentosum group D/Cockayne syndrome (XP-D/CS) cells

Renier Vélez-Cruz; Anton S. Zadorin; Frédéric Coin; Jean-Marc Egly

doi:10.1073/pnas.1213076110

Edited by Philip C. Hanawalt, Stanford University, Stanford, CA, and approved November 28, 2012 (received for review August 23, 2012)

Abstract

Specific mutations in the XPD subunit of transcription factor IIH result in combined xeroderma pigmentosum (XP)/Cockayne syndrome (CS), a severe DNA repair disorder characterized at the cellular level by a transcriptional arrest following UV irradiation. This transcriptional arrest has always been thought to be the result of faulty transcription-coupled repair. In the present study, we showed that, following UV irradiation, XP-D/CS cells displayed a gross transcriptional dysregulation compared with “pure” XP-D cells or WT cells. Furthermore, global RNA-sequencing analysis showed that XP-D/CS cells repressed the majority of genes after UV, whereas pure XP-D cells did not. By using housekeeping genes as a model, we demonstrated that XP-D/CS cells were unable to reassemble these gene promoters and thus to restart transcription after UV irradiation. Furthermore, we found that the repression of these promoters in XP-D/CS cells was not a simple consequence of deficient repair but rather an active heterochromatinization process mediated by the histone deacetylase Sirt1. Indeed, RNA-sequencing analysis showed that inhibition of and/or silencing of Sirt1 changed the chromatin environment at these promoters and restored the transcription of a large portion of the repressed genes in XP-D/CS cells after UV irradiation. Our work demonstrates that a significant part of the transcriptional arrest displayed by XP-D/CS cells arises as a result of an active repression process and not simply as a result of a DNA repair deficiency. This dysregulation of Sirt1 function that results in transcriptional repression may be the cause of various severe clinical features in patients with XP-D/CS that cannot be explained by a DNA repair defect.

The human genome is exposed to a variety of endogenous and exogenous insults that can alter the genetic information and physically interfere with critical cellular processes such as DNA replication and transcription (1, 2). The inability to remove these DNA alterations can lead to mutations or the halt of transcription and/or DNA replication. Mutations can ultimately cause cancers, and the arrest of cellular processes can induce cell death, which can result in premature aging (3, 4).

The nucleotide excision repair (NER) pathway is responsible for the removal of a variety of bulky DNA lesions, such as those induced by UV irradiation, and is subdivided into two subpathways. Global genome repair (GGR) is responsible for the removal of adducts from the whole genome and transcription-coupled repair (TCR) is responsible for the accelerated removal of lesions located on the transcribed strand of active genes (5, 6). Whereas GGR is initiated by the damage-recognition protein XPC-RAD23B, TCR is initiated by an RNA polymerase II (Pol II) stalled in front of a transcription-blocking lesion, and does not require XPC-RAD23B. Upon the stalled Pol II, the TCR-specific factors CSA and CSB are recruited to the site and recruit other chromatin remodeling factors (7, 8). The remaining steps of these mechanisms are thought to be identical for both subpathways (9, 10).

The absence of these repair mechanisms leads to severe genetic disorders such as xeroderma pigmentosum (XP), trichothiodystrophy (TTD), and Cockayne syndrome (CS), which present an array of clinical symptoms, including segmental progeria (Table 1) (3, 11 ⇓–13). Patients with CS in particular display a complex list of clinical features that are hard to reconcile with a sole defect in DNA repair, which argues for the involvement of CSA and CSB proteins in other cellular processes, such as transcription.

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Table 1.

Clinical features of patients with XP, TTD, CS, or XP/CS

A limited number of specific mutations in NER genes (XPB, XPD, and XPG) have resulted in patients with a combined XP/CS phenotype (14 ⇓⇓⇓–18). The clinical severity of combined XP/CS probably arises as a result of an inherent link between transcription and NER. For instance, XPB and XPD are both helicase subunits of the transcription/repair factor transcription factor IIH (TFIIH) (19). Furthermore, XPG was recently identified as a protein required for maintaining the integrity of the TFIIH complex, and therefore also engaged in the transcription process (20). Even though the contribution of the DNA repair deficiency to the clinical features of patients with XP, CS, or XP/CS is irrefutable, studies have shown a clear dysregulation of a variety of transcriptional pathways, which may also contribute to the clinical phenotype of these patients (21 ⇓⇓⇓⇓⇓–27). Interestingly, at the cellular level, XP/CS cells share with CS cells a sustained global transcriptional arrest after UV irradiation, which has been always explained by the inability of these cells to perform TCR (17, 28). The fact that the so-called global transcriptional arrest displayed by XP/CS and CS cells excludes genes that are activated upon DNA damage, such as p53-dependent genes, suggests that there must be an active transcriptional repression process, rather than a physical blocking of transcription. In this regard, CSB and other NER factors have been shown to affect chromatin remodeling for optimal transcription initiation (24, 29 ⇓–31).

In the present study, we showed that XP-D/CS cells display a gross transcriptional dysregulation upon UV irradiation, whereas WT and XP-D cells displayed no and a mild dysregulation, respectively. XP-D/CS cells (XPD-G675R and XPD-G602D) were never able to restart transcription of housekeeping (HK) genes after UV irradiation, identical to the CS phenotype. We showed that the histone deacetylase (HDAC) Sirt1 was responsible for the establishment of a heterochromatin environment at these promoters after UV irradiation. Treatment of XP-D/CS cells with a Sirt1-specific inhibitor or down-regulation of Sirt1 by siRNA resulted in the restoration of the expression of a large proportion of the repressed genes in these cells. Many of the genes whose expression was restored play a role in multiple pathways, including DNA repair and genomic stability, and may help explain the severe phenotype of XP-D/CS patients.

Results

XP-D/CS Cells Display Global Transcriptional Dysregulation After UV Irradiation.

The inability to restart transcription after UV (measured by [³H]uridine incorporation) is the hallmark characteristic of CS cells (28). However, rRNA synthesis (which is rather high in growing cells) represents a large fraction of the incorporated [³H]uridine in these studies, and thus results may not be representative of global transcription of type II genes (32). Furthermore, a more recent analysis showed HK genes were repressed after UV in CS-B cells, whereas p53-inducible genes were not (24). XP-D/CS cells also displayed a global transcriptional arrest after UV irradiation (17, 28).

To study the global transcriptional response of XP-D/CS cells upon UV irradiation, we first performed RNA-sequencing (RNA-seq) analysis in WT, XP-D/CS (XPD-G675R), and “pure” XP-D (XPD-R683W) primary fibroblasts untreated and 24 h after UV irradiation (10 J/m²; Fig. 1 A–C). The scatter plots and correlation analysis in WT cells showed that, at 24 h after UV irradiation, these cells had reestablished overall transcriptional equilibrium (i.e., the expression of most genes had returned to basal levels; note the close distribution of the genes along the black diagonal line in Fig. 1 A). On the contrary, XP-D/CS cells displayed a gross transcriptional dysregulation after UV irradiation (Fig. 1B; note the spread of all of the data points away from the black diagonal line). The UV treatment in XP-D/CS cells resulted in a total of ∼2,000 genes whose expression changed more than threefold and was reproducible and statistically significant. The majority of the genes that changed in XP-D/CS cells were repressed (70%; dots below the black diagonal line in Fig. 1B); however, ∼30% of the genes were overexpressed at 24 h post-UV irradiation (∼600 genes; dots above the black diagonal line in Fig. 1B), thus challenging the global transcriptional arrest described for XP-D/CS. Interestingly, the same analysis on XPD-R683W fibroblasts resulted in a scatter plot that more resembles that of the WT cells (Fig. 1C). Additionally, XP-D cells only repressed 26% of genes (compared with 70% repressed in XP-D/CS cells). Patients with XP-D/CS display a variety of severe clinical features associated with XP and CS phenotypes, whereas the patient with pure XP displayed only UV hypersensitivity (Tables 1 and 2). Interestingly, these differences in transcriptional dysregulation parallel the clinical severity of the XP-D/CS vs. XP-D cases, thus underscoring their importance (Table 1) (13, 17, 33, 34).

Fig. 1.

XP-D/CS cells display a gross gene dysregulation after UV irradiation. RNA-seq analysis scatter plots show the transcription of all genes read at 24 h after 10 J/m² UV-C vs. untreated conditions for (A) WT, (B) XP-D/CS (XPD-G675R), and (C) XP-D (XPD-R683W). Each gray dot represents a gene. The black dot represents DHFR. (D) XP-D/CS and (E) XP-D cells pretreated with the Sirt1-specific inhibitor EX-527 (50 μM) for 10 h before UV irradiation. Red dots represent the 484 genes that were repressed more than threefold in XP-D/CS cells and whose expression was restored by the Sirt1-specific inhibitor EX-527. Red line represents the best linear fit for the genes in red. k is the slope for the red line. Axes presented as fragments per kilobase of exon per million fragment mapped (FPKM). Treatment of XP-D/CS cells resulted in significant change in expression (statistically significant ± threefold change) of 2,024 genes (600 genes overexpressed and 1,424 genes repressed).

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Table 2.

Clinical and molecular features of XP-D/CS patients and proteins

XP-D/CS Cells Cannot Restart Transcription of HK Genes After UV Irradiation.

To further dissect the mechanism by which XP-D/CS (XPD-G675R and XPD-G602D) cells repress transcription after UV irradiation, we used HK genes as a model. WT cells displayed a slight decrease followed by the reestablishment of the mRNA levels of the HK gene dihydrofolate reductase (DHFR; which was identified in our RNA-seq analysis) upon UV irradiation (Fig. 2A). On the contrary, upon UV irradiation, both XP-D/CS fibroblasts displayed a progressive decrease in the mRNA levels of DHFR (Fig. 2 B and C). Interestingly, UV irradiation of XPD-R683W cells caused an initial decrease in the mRNA of this HK gene, followed by an increase in transcription (Fig. 2D). These results are in agreement with our RNA-seq results in which WT and XP-D recovered the expression of many genes (97% for WT and 74% genes for XP-D), whereas XP-D/CS cells did not (Fig. 1 A–C, black dot for DHFR). Additionally, XP-C cells (devoid of GGR) displayed a decrease in the mRNA levels of DHFR upon UV irradiation (10 J/m²), which was recovered within 12 h (Fig. 2E). Finally, XP-A cells (devoid of NER) displayed also a decrease in the levels of DHFR mRNA after UV irradiation, which slowly recovered by 24 h (Fig. 2F). Similar results were observed for the GAPDH HK gene (Fig. S1).

Fig. 2.

XP-D/CS cells cannot restart transcription of HK genes after UV irradiation. (A–F) Relative mRNA expression of DHFR after UV irradiation (10 J/m²). DHFR mRNA was normalized to the amount of 18S rRNA and results are presented as fold expression, which represents the ratio of each time point relative to the nonirradiated cells. Error bars represent the SEM of three independent experiments. (G–L) ChIP monitoring the occupancy of general transcription factor TFIIB, RNA Pol II, vitamin D receptor (VDR; as a negative control), (M–R) TFIIH subunits XPB and XPD, and the TCR factor CSB, at the promoter of the DHFR gene in WT and XP-D/CS (G675R and G602D), XP-D (R683W), XP-C, and XP-A cells. All results are presented as fold recruitment, which represents the ratio of the percent input of each time point relative to that of the nonirradiated cells (t = 0 h). Each point represents the average of three real-time PCR reactions of three independent experiments, and error bars represent SEM.

We next monitored the recruitment of the transcriptional machinery to the DHFR promoter by using ChIP coupled to real-time PCR. In WT cells, the transcriptional machinery reassembled on the promoter of the DHFR gene at 6 h, as shown by the enrichment of Pol II and the transcription initiation factor IIB (TFIIB; Fig. 2G). Furthermore, we also observed at this time the presence of the TFIIH transcription/repair factor, and the TCR factor CSB, which is recruited and required for the reassembly of the transcriptional machinery at the promoters of activated genes (24). In WT cells, we observed agreement between the recruitment of Pol II, TFIIB, TFIIH, and CSB, i.e., the reassembly of the promoter 6 h (and 16 h) after UV irradiation (Fig. 2 G and M), which correlated with the restoration of the mRNA levels.

When we monitored the reassembly of the DHFR promoter in XPD-G675R and XPD-G602D cells, neither of the two cell lines was able to reassemble the transcriptional machinery at this promoter (Fig. 2 H, I, N, and O). The amount of Pol II at the DHFR promoter decreased progressively to less than 20% of the initial amount at 24 h for both XP-D/CS cells, and did not recover. Furthermore, none of the transcription initiation factors, including TFIIB, or the repair factor CSB, were recruited to a significant extent or with a particular profile/pattern to these promoters (Fig. 2 H, I, N, and O). Importantly, the basal protein levels of these transcription and repair factors are similar between the different cell lines (Fig. S1O). Interestingly, and in agreement with the mRNA expression data (Fig. 2D) and RNA-seq data (Fig. 1C), cells from a patient with pure XP-D were able to reassemble the promoters of HK after UV irradiation (Fig. 2 D, J, and P). Thus, mutations in XPD that result in XP-D/CS do not allow the reassembly of the transcriptional machinery on the DHFR promoter after UV irradiation, in agreement with the decreased mRNA levels of this gene after UV irradiation (Fig. 2 B and C). These results show that XP-D/CS cells, as CS-B cells, are unable to reassemble the promoters of HK genes after UV irradiation (24). Furthermore, the reassembly of the HK gene promoters after UV irradiation seems to differentiate an XP-D from an XP-D/CS phenotype.

We next asked whether proficient NER was required for the reassembly of the transcriptional machinery on the promoters of HK genes. XP-C cells recovered basal levels of Pol II at the DHFR promoter starting at 12 h after UV irradiation (Fig. 2E). The progressive increase (albeit low) in the recruitment of Pol II was concomitant with a similarly progressive increase in the recruitment of the CSB protein and TFIIH (Fig. 2 K and Q). Similarly, in XP-A cells, we also observed the slow but progressive increase in the recruitment of the CSB protein and Pol II on the promoter of this gene (Fig. 2 F, L, and R). Additionally, very similar results were observed for the GAPDH HK gene (Fig. S1 E, F, K, and L). It is important to note that, in XP-C and XP-A cells, we observed a statistically significant (P < 0.05) progressive increase in the levels of mRNA of these HK genes as well as the levels of Pol II recruited at these promoters, in complete contrast to what we observed with XP-D/CS cells, which was a progressive decrease of mRNA and Pol II at these promoters (compare panels in Fig. 2 and also Fig. S1 A–F, M and N). Although the removal of the DNA lesions per se may not be required for the reassembly of the transcriptional machinery on these promoters, the presence of functional NER seems to aide the process.

XP-D/CS Cells Elicit Transcriptional Stress Response upon UV Irradiation.

As XP-D/CS cells were unable to reassemble the promoters of HK genes after UV irradiation, we investigated whether these cells were able to launch a p53-induced transcriptional response upon DNA damage. WT, both XP-D/CS, and XP-D cells displayed an accumulation of the p53 protein as soon as 1 h after UV irradiation (Fig. 3 A–D). We analyzed the transcriptional response of the p53-inducible growth arrest and DNA damage inducible-α (GADD45α) gene upon UV irradiation. In agreement with the increasing p53 protein levels observed for all four cell lines, we observed an accumulation of the GADD45α mRNA immediately after UV irradiation (Fig. 3 E–H). ChIP analysis on the promoter of the GADD45α gene showed increased levels of the transcriptional machinery, Pol II, TFIIH, and p53 (Fig. 3 I–L). In addition to GADD45α, other DNA damage-inducible genes were also transcribed (ATF3, p21; Fig. S1 P–S). Taken together, these results demonstrate that both XP-D/CS cell lines are capable of launching a transcriptional response upon UV irradiation in the presence of mutated TFIIH, and thus establishes different requirements for the reassembly of promoters of these two gene families (HK and stress-induced genes). Finally, it is clear that the “global” transcriptional arrest does not include DNA damage-inducible genes.

Fig. 3.

XP-D/CS cells transcribe GADD45α after UV irradiation. (A–D) p53 protein accumulates upon UV irradiation (10 J/m²) in WT, XP-D/CS (G675R and G602D), and XP-D (R683W) cells. Fifty micrograms of whole cell extracts were separated by SDS/PAGE, transferred to nitrocellulose membrane, and probed with the indicated antibodies. Tubulin-α was used as a loading control. A representative blot of three independent experiments is shown. (E–H) GADD45α mRNA is expressed upon UV irradiation (10 J/m²). GADD45α mRNA was normalized to the amount of 18S rRNA, and results are presented as fold expression as previously described. Error bars represent the SEM of three independent experiments. (I–L) ChIP monitoring occupancy of RNA Pol II, XPD, and p53 at the promoter of the GADD45α gene of WT, XP-D/CS (G675R and G602D), and XP-D (R683W) cells. All results are presented as fold recruitment as previously described. Each point represents the average of three real-time PCR reactions of three independent experiments, and error bars represent SEM.

XP-D/CS Cells Acquire Heterochromatin Marks on HK Genes.

Euchromatin allows transcription and is characterized by acetylated (H3K9-Ac and H4K16-Ac) and methylated (H3K4me3, and H3K79me2) histone H3 and H4 (35). Heterochromatin, on the contrary, inhibits RNA synthesis and is characterized by a different set of chromatin marks such as di- and trimethylated H3K9 (H3K9me2-3) and H3K27 (H3K27me2), the recruitment of histone H1, in addition to the loss of euchromatic acetylation and methylation marks (36 ⇓⇓–39). As XP-D/CS cells were unable to restart the transcription of HK genes after UV irradiation, we thus monitored these promoters at the chromatin level.

ChIP analysis of WT cells revealed that the DHFR promoter displayed increased levels of H3K9-Ac, H4K16-Ac, H3K4me3, and H3K79me2 upon UV irradiation (Fig. 4 A, E, I, and M). In striking difference, the DHFR promoter in XPD-G675R and XPD-G602D cells displayed no significant increase in H3K9-Ac, H4K16-Ac, H3K4me3, or H3K79me2, but rather a decrease in some of these chromatin marks (Fig. 4 B, C, F, G, J, K, N, and O). Furthermore, we observed a remarkable agreement between the lower levels of these euchromatic marks and the repression of these promoters (i.e., the decreasing levels of mRNA, absence of Pol II, TFIIH, and CSB at this promoter; Fig. 2 B, C, H, I, N, and O), therefore suggesting the establishment of facultative heterochromatin. Interestingly, in XPD-R683W cells, we observed the maintenance of euchromatic marks on the DHFR promoter (Fig. 4 D, H, L, and P), in agreement with the presence of the transcription machinery, and the resumption of mRNA synthesis after UV irradiation shown by RNA-seq and quantitative PCR (qPCR) analysis (Fig. 1C and 2D, respectively).

Fig. 4.

XP-D/CS cells lose euchromatin marks and acquire heterochromatin marks on the promoter of DHFR after UV irradiation. ChIP monitoring of occupancy of (A–D) H3K9-Ac, (E–H) H4K16-Ac, (I–L) H3K4me3, (M–P) H3K79me2, (Q–T) Sirt1, (U–X) H1, and (Y–Z, 3) H3K9me2 on the promoter of the DHFR gene in WT, XP-D/CS (G675R and G602D), and XP-D (R683W) cells. All results are presented as fold recruitment as previously described. Each point represents the average of three real-time PCR reactions of at three independent ChIP experiments, and error bars represent SEM.

As we observed a marked decrease in the amount of H3K9-Ac and H4K16-Ac, both of which are substrates for the type III family of HDACs known as sirtuins, we decided to determine whether Sirt1, a member of the sirtuin family and responsible for the formation of facultative heterochromatin, was recruited to these promoters (38, 40). Although we detected only background levels of Sirt1 on this promoter in WT and XP-D cells, XP-D/CS cells displayed increased levels of Sirt1 recruited to these promoters (Fig. 4 Q–T). Furthermore, when we looked at other marks of facultative heterochromatin such as histone H1 and H3K9me2, we found that, just like Sirt1, they were absent in WT and XP-D cells but present in XP-D/CS cells (Fig. 4 U–Z3), confirming the existence of facultative heterochromatin on these promoters. Interestingly, the lack of active chromatin marks of transcription and the appearance of heterochromatin marks on HK genes was in agreement with the inability of the XP-D/CS cells to recruit the transcriptional machinery to these loci (Fig. 2 H, I, N, and O). In addition to the DHFR gene, we observed a very similar pattern of heterochromatin formation and Sirt1 recruitment on the GAPDH HK gene (Fig. S1 G–J).

Altogether, our results show that, in XP-D/CS cells, HK genes, such as DHFR and GAPDH, acquire specific heterochromatic marks, whereas, in WT and XP-D cells, these genes maintain euchromatic marks and are expressed. Moreover, this UV-induced transcriptional repression is not global, as p53-inducible genes, such as GADD45α, are expressed after UV irradiation in all cells studied, and this promoter displays marks of active transcription (Fig. 3 and Fig. S2). Importantly, these differences in the recruitment of Sirt1 did not arise as a result of different expression of this protein in different cell lines, as the basal levels of Sirt1 are very similar among the cell lines studied (Fig. S3).

Sirt1 Mediates Repression of HK Genes in XP-D/CS Cells After UV Irradiation.

As histone acetylation seems to be important for restarting transcription after UV irradiation in XP-D/CS cells, we reasoned that inhibition of HDACs might relieve the transcriptional repression of these genes. Pretreatment of XP-D/CS cells with sodium burtyrate, which inhibits type I, II, and IV HDACs, did not relieve the progressive decrease in DHFR mRNA upon UV irradiation of XP-D/CS cells (Fig. S3 T–V). To further confirm the involvement of Sirt1, a type III HDAC, in the transcriptional regulation of HK genes after UV irradiation, we first used a pan-inhibitor for all sirtuins, nicotinamide (NAM). Pretreatment of XP-D/CS cells with NAM resulted in the reestablishment of the mRNA levels of DHFR, whereas it had no effect in WT cells (Fig. 5 A and B). Furthermore, ChIP analysis showed that NAM pretreatment in XP-D/CS cells resulted in the recovery of the recruitment of Pol II and H4K16-Ac levels on the DHFR promoter, whereas no significant changes were observed in WT cells (Fig. 5 G, H, M, and N), further supporting the idea that a member of the type III HDAC family plays a role in the repression of HK genes after UV irradiation in XP-D/CS cells. Similar effects were observed with the GAPDH gene (Fig. S3 A–O).

Fig. 5.

Inhibition of Sirt1 restores transcription of HK genes in XP-D/CS cells. DHFR mRNA expression at the indicated times after UV irradiation (10 J/m²) of WT of XP-D/CS (G675R) cells (A and B) pretreated for 12 h with 15 mM NAM, (C and D) pretreated for 10 h with 50 μM EX-527, or (E and F) previously transfected with siRNA targeting SIRT1 (siSIRT1) or a nontargeting control (siCTRL). Expression levels of the Sirt1 protein are shown on immunoblot (Inset) at the top of E and F. ChIP monitoring of occupancy of (G–L) RNA Pol II and (M–R) H4K16-Ac on the promoter of the DHFR gene in WT and XP-D/CS cells pretreated with NAM, EX-527, or siRNA targeting SIRT1. All results are presented as fold recruitment as previously described. Each point represents the average of three real-time PCR reactions of three independent ChIP experiments, and error bars represent SEM (*P < 0.05, **P < 0.01, Student t test).

Because NAM inhibits all sirtuins and also other enzymes such as poly(ADP ribose) polymerases, we used a Sirt1-specific inhibitor, EX-527 (41, 42). Pretreatment of XP-D/CS cells with EX-527 also resulted in the restoring of the transcription of DHFR in these cells (Fig. 5 C and D). Importantly, EX-527 pretreatment also resulted in increased levels of Pol II and H4K16-Ac on the DHFR promoter, again supporting the idea that Sirt1 mediates the repression of HK genes upon UV in XP-D/CS cells. No significant changes were observed in WT cells (Fig. 5 C, D, I, J, O, and P).

Finally, to confirm that Sirt1 is responsible for the repression of DHFR upon UV irradiation in XP-D/CS cells, we depleted cells of Sirt1 by transfecting them with siRNA targeting Sirt1 or a nonspecific control. At 72 h after siSIRT1 transfection, Sirt1 levels were undetectable by Western blot (Fig. 5 E and F). Although XP-D/CS cells transfected with the nonspecific control displayed progressive decrease in DHFR mRNA upon UV irradiation, cells transfected with siSIRT1 restored the transcription of DHFR (Fig. 5 E and F). Importantly, XP-D/CS cells transfected with siSIRT1 also displayed reestablished levels of Pol II and H4K16-Ac, thus confirming that Sirt1 is responsible for the repression of DHFR in XP-D/CS cells after UV irradiation (Fig. 5 K, L, Q, and R). It is important to note that neither the inhibitors nor the siSIRT1 affected the basal expression of DHFR or GAPDH in the absence of UV irradiation, thus suggesting that, under normal conditions, these genes are not under the regulation of Sirt1.

Most importantly, our RNA-seq analysis showed that pretreatment of XP-D/CS cells with the Sirt1 inhibitor EX-527 significantly ameliorated the transcriptional dysregulation of these cells after UV irradiation. Among the 1,400 genes down-regulated more than threefold upon UV irradiation in XP-D/CS cells, 10 h pretreatment of cells with 50 μM of the Sirt1-specific inhibitor reestablished the transcription of 484 genes in these cells (i.e., ∼35% of the genes; Fig. 1 B and D, red dots). This amelioration can be easily appreciated by comparing the slope of the red line (i.e., k values) for these genes (k = 0.12 vs. k = 0.49, nontreated vs. EX-527-treated XP-D/CS cells). This effect was not observed in XP-D cells (Fig. 1 C and E). Our results thus demonstrate that Sirt1 is responsible for the transcriptional repression of a significant number of the genes repressed upon UV irradiation in XP-D/CS cells. We would like to note that, even though RNA-seq analysis was not performed in the XPD-G602D cells, we have found very little difference between both XP-D/CS cell lines in terms of transcriptional repression after UV irradiation [HK genes, p53-inducible genes, nuclear receptor (NR)-inducible genes (Fig. S4)], and validation of the RNA-seq studies with EX-527 by qPCR was done in both cell lines and confirm very similar results.

Discussion

Discriminated Transcription After UV Irradiation in XP-D/CS Cells.

XP-D/CS cells cannot reassemble the promoters or restart transcription of HK genes, such as DHFR or GAPDH, after UV irradiation (Figs. 1 and 2), similar to CS-B cells (24). It is important to note that, through all our studies, we have not observed significant differences between the two XP-D/CS cells we studied (XPD-G675R and XPD-G602D). On the contrary, repair-deficient cells (XP-C, XP-A, and XP-D) were able to slowly reassemble the transcriptional machinery and restart transcription of these genes after UV irradiation (Fig. 2). In the unlikely case that there were lesions in every HK gene promoter, our results thus suggest that the reinitiation of transcription after UV irradiation does not depend (exclusively) on the removal of DNA lesion.

The reassembly of HK gene promoters did seem to occur concomitant with the recruitment of the CSB chromatin-remodeling factor (24). This SWI2/SNF2 ATPase is involved in transcription elongation and chromatin remodeling after UV irradiation. At the present time, we do not know exactly why XP-D cells display faster recovery or restoration of transcription than XP-C or XP-A cells. These differences could simply be a result of residual repair, as XP-D cells still have some residual NER (∼30–40% unscheduled DNA synthesis), whereas XP-A and XP-C cells do not. In addition to repair, the reassembly of the HK gene promoters in XP-A, XP-C, and XP-D cells might indicate that these proteins (or mutation) do not play a critical role in the initiation of transcription of HK genes after UV irradiation, whereas other proteins such as CSB are required for this reassembly (24). It is possible that, because XP-C (and XP-A) cells have only TCR, most of the CSB is at sites of damage and thus unavailable to mediate the reassembly of the HK gene promoters early on, thus explaining the slower kinetics of promoter reassembly. The absence of CSB at the HK gene promoters in XP-D/CS cells (even though this protein was expressed; Fig. S3S), suggests that the XP-D/CS mutations do not support the recruitment and/or the function(s) of CSB. The potential modulation of the function(s) of CSB by TFIIH pinpoints the importance of CSB in transcription initiation, and may be the reason behind the inability of XP-D/CS cells to reassemble the promoters of HK genes after UV irradiation. Similarly, the slow recruitment of the CSB protein to the promoters of HK genes in XP-A and XP-C cells may explain the slower kinetics for the reassembly of these promoters in these cells.

Interestingly, we also observed that XP-D/CS cells were unable to transactivate NR-inducible genes after UV irradiation (Fig. S4), whereas other repair-deficient cells transactivated NR genes under the same conditions. A deficiency in NR transactivation could be explained by the weakened interaction between XPD and p44 that results from the XPD-G675R and XPD-R683W mutations, but not for XPD-G602D; thus, it is likely that the inability to restart the transcription of HK and NR genes after UV irradiation stems from another problem (25). In stark contrast with the transcription of HK and NR-inducible genes, the transcription of the p53-inducible GADD45α gene, as well as of other stress-inducible genes (such as ATF3 and p21), was not impaired in XP-D/CS cells even though these genes may have also been damaged (Fig. 3 and Fig. S1 P–S). Previous work showed that the transcription of these genes does not require CSB (24). The difference between these gene families may lie in the fact that stress-response gene promoters are preassembled, awaiting for a stimulus to start elongation, whereas NR-inducible and HK genes must undergo cycles or assembly and disassembly (24, 29, 30, 43 ⇓–45).

Finally, our study unveils a difference between the cellular XP, CS, and XP-D/CS phenotypes. It seems that the inability to restart the transcription of HK genes after UV irradiation is strongly linked to the CS phenotype, whereas the exact mechanisms or reason for the repression mechanism for these genes may be different (Table 3).

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Table 3.

Characteristics of NER-deficient cells

Sirt1-Mediated Heterochromatinization of HK Genes.

CS-B cells displayed impaired recruitment of the histone acetyltransferase p300 and thus lower levels of H3K9-Ac, which caused impaired recruitment of the transcriptional machinery and transcription of the promoters of HK genes upon UV irradiation (24, 46, 47). We tested whether the mechanism behind the transcriptional repression in CS-B cells was the same as the one we report here for XP-D/CS cells, but it was not the case. Although we did observe lower levels of euchromatin marks (H3K9-Ac, H4K16-Ac, H3K4me3) at the promoters of HK genes in CS-B cells after UV irradiation, we did not observe marks of facultative heterochromatin, and, in agreement with this, pretreatment of cells with HDAC inhibitor NAM did not restore the transcription of HK genes in CS-B cells (Fig. S3W). Our results therefore suggest that histone acetylation at these promoters is very important for the restart of transcription after UV irradiation, and, thus, faulty histone acetylation (impaired histone acetyltransferase recruitment for CS-B cells, uncontrolled HDAC recruitment for XP-D/CS cells, or a combination of both) can lead to transcriptional dysregulation and the inability of cells to restart transcription after UV irradiation. It is possible that no one single chromatin modification may be responsible for the inability of CS and XP/CS cells to restart transcription, but, rather, a combination of several deficiencies may create a “perfect storm” for the subsequent heterochromatinization of these promoters. It is not surprising that XPD mutations result in a chromatin dysregulation, as more recent studies place NER factors at the intersection between transcription and repair by regulating chromatin structure (24, 29 ⇓–31).

The impact that the inhibition of Sirt1 had on the chromatin modifications of the promoters of HK genes strongly suggests that Sirt1 is directly changing the chromatin environment at these promoters and not altering repair indirectly through the deacetylation of another factor (48).

Why is Sirt1 mediating the heterochromatinization of HK genes? Interestingly, upon DNA damage and during normal aging, Sirt1 undergoes a redistribution, thus abandoning (and thereby allowing the transcription of) typically repressed loci, and regulating another set of genes. This shift was named redistribution of chromatin modifiers response, and had been observed for oxidative damage and DNA double strand breaks (49 ⇓–51). The type of DNA damage may be what finally sets apart XP-D/CS from other types of combined XP/CS cells (XP-B/CS and XP-G/CS) and even CS cells, as XP-D/CS cells are the only type of XP/CS cells that have been shown to induce double strand breaks upon UV irradiation (18) (Table 3) and preliminary studies showed that XP-B/CS and XP-G/CS cells did not respond to the inhibition of Sirt1. We propose that the redistribution of chromatin modifiers response is also responsible for the repression of constitutively expressed loci, such as those of HK genes in XP-D/CS cells after UV irradiation. In support of this model, our RNA-seq analysis showed that pretreatment with the Sirt1-specific inhibitor EX-527 corrected the expression of a large fraction (at least ∼35%) of the genes down-regulated in XP-D/CS cells upon UV irradiation (Fig. 1 B and D). Which factor(s) is responsible for the recruitment of Sirt1 to specific genes is unclear. The fact that the promoters we studied became rather depleted from general transcription factors, including Pol II, TFIIH, and CSB, made it difficult to identify a factor that recruits Sirt1 (we did not detect any interactions between these factors and Sirt1 either) to these promoters, and suggest that maybe the substrates for this enzyme (e.g., H3K9-Ac, H4K16-Ac) and the absence of chromatin modifications that would normally block Sirt1 recruitment such as the H3K79me2 (52 ⇓–54) contribute to the recruitment of Sirt1 to these promoters.

XP-D/CS Phenotype and Sirt1.

XP-D/CS cells display genomic instability, higher cancer incidence, and a multitude of severe clinical features (Table 2) (14, 17, 34, 55). Although it would be unthinkable to try to explain all the different clinical features of these patients, studying the list of genes that are repressed upon UV irradiation, we observed many genes involved in DNA repair (FANCA, FANCI, RAD51L1, RAD54L, POLQ), cell cycle control (RB1), and neuronal development (OPTN, BDNF), among others that could potentially be responsible for these features. Importantly, the expression of these genes is restored when cells are treated with the Sirt1 inhibitor EX-527, thus opening the door for potential therapeutic avenues. Furthermore, in our study, inhibition of Sirt1 had a modest enhancement of the survival of XP-D/CS upon UV irradiation (Fig. S3 P –R), thus suggesting that the silencing of HK genes may thwart the ability of these cells to cope with DNA damage. Finally, the link between Sirt1 and XP-D/CS mutations has important implications, as this dysregulation of Sirt1 may contribute to the severe early-onset progeria, metabolic problems, and other clinical features observed in these patients that cannot be explained by a DNA repair defect (Table 2).

Materials and Methods

Cell Culture.

Human primary fibroblasts [WT, XP-D/CS (XP8BR, G675R and XPCS2, G602D), XP-D (XP34BE), CS-B (CS1PV), XP-C (GM11847), XP-A (XP39OS)] were cultured under standard conditions. For sirtuin inhibitor experiments, cells were pretreated with NAM (15 mM; Sigma) for 12 h before the experiment (56, 57), UV-irradiated, and incubated again with media containing 15 mM NAM. Similarly, experiments with EX-527 (Sigma) were conducted by incubating cells with 50 μM EX-527 for 10 h, irradiating cells (UV-C, 254 nm), and incubating cells again with media containing EX-527 (41).

mRNA Expression.

mRNA was extracted by using the GeneElute Kit (Sigma). The reverse transcription reaction was done with random primers and SuperScript II (Invitrogen), followed by qPCR (SYBR Green; Qiagen) for the indicated genes. The gene expression was normalized to that of 18S. Primer sequences are available upon request. For the RNA-seq analysis, Tag library preparation and high-throughput sequencing were conducted on an Illumina Genome Analyzer II sequencing system with sequencing depth of 72 nt. Image analysis and base calling were done with CASAVA 1.8.2 (Illumina). Tag alignment, transcript assembly, differential expression analysis, and statistical significance calculation were performed in the Galaxy Web-based environment by using a pipeline of TopHat–Cufflinks–Cuffdiff with hg19 human genome (false discovery rate, 0.05; minimal alignment count, 1,000, i.e., -c parameter for Cuffdiff).

ChIP.

Experiments were carried out as previously described (24, 29). Briefly, cells were crosslinked with 1% formaldehyde for 10 min at room temperature, rinsed with 200 mM glycine and cold PBS solution with protease inhibitors, and harvested. Nuclear extracts were sonicated (Bioruptor; Diagenode). ChIP experiments were performed with the indicated antibodies [RNA pol II, XPB, XPD, CSB (IGBMC antibody facility, 7C2, 1B3, 1B5, 1A11/3H8, respectively), TFIIB, p53, Sirt1 (Santa Cruz Biotechnology), H3K9-Ac, H3K9me2, H3K4me3, H3K79me2 (Cell Signaling), H4K16-Ac (Epigenetek), H1 (Millipore)] followed by qPCR on the indicated gene promoters. Primer sequences are available upon request. All results are presented as fold recruitment and represent the ratio of the percentage of input at each time point relative to the nonirradiated cells (0 h). Each point represents the average of three real-time PCR reactions of three independent ChIP experiments. Statistical significance was determined by the Student t test.

Immunoblots.

Cells were UV-irradiated (10 J/m²) and harvested at the indicated times in RIPA buffer (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, and protease inhibitor mixture), and whole cell lysates were sonicated. Lysate were separated by SDS/PAGE, transferred to nitrocellulose membrane, and probed with the indicated antibodies: p53, CSB (Santa Cruz Biotechnology), tubulin-α (Abcam), and Sirt1 (Upstate). Tubulin was used as a loading control.

siRNA Transfections.

Cells were plated at 30% confluence 24 h before transfection. Cells were transfected with 50 nM nontargeting siCTRL or siSIRT1 using Lipofectamine 2000 (Invitrogen) and Opti-MEM media for 24 h. After 24 h, regular media containing FCS was added to cells and incubated for an additional 48 h before exposing cells to UV irradiation.

Acknowledgments

We thank E. Compe and N. Le May for their critical comments on the manuscript. This study was supported by l’Agence National de la Recherche Grant ANR-08-GENOPAT-042, Association pour la Recherche sur le Cancer, European Research Council (ERC) Advanced Scientists Grants (to J.-M.E.), Institut National du Cancer Grant INCA-2008-041 (to F.C.), a Chateaubriand postdoctoral fellowship (to R.V.-C.), an Association pour la Recherche sur le Cancer postdoctoral fellowship (to R.V.-C.) and predoctoral fellowship (to A.S.Z.), and an ERC predoctoral fellowship (to A.S.Z.).

Footnotes

↵¹Present address: Department of Molecular Carcinogenesis, University of Texas MD Anderson Cancer Center, Science Park Research Division, Smithville, TX 78657.
↵²R.V.-C. and A.S.Z. contributed equally to this work.
↵³To whom correspondence should be addressed. E-mail: egly{at}igbmc.fr.

Author contributions: R.V.-C. and J.-M.E. designed research; R.V.-C. and A.S.Z. performed research; F.C. contributed new reagents/analytic tools and discussions; R.V.-C., A.S.Z., and J.-M.E. analyzed data; and R.V.-C. and J.-M.E. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
See Author Summary on page 814 (volume 110, number 3).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1213076110/-/DCSupplemental.

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[1] ↵
Jackson SP,
Bartek J
(2009) The DNA-damage response in human biology and disease. Nature 461(7267):1071–1078.
OpenUrl CrossRef PubMed

[2] Jackson SP,

[3] Bartek J

[4] ↵
Tornaletti S
(2009) DNA repair in mammalian cells: Transcription-coupled DNA repair: directing your effort where it’s most needed. Cell Mol Life Sci 66(6):1010–1020.
OpenUrl CrossRef PubMed

[5] Tornaletti S

[6] ↵
Cleaver JE,
Lam ET,
Revet I
(2009) Disorders of nucleotide excision repair: The genetic and molecular basis of heterogeneity. Nat Rev Genet 10(11):756–768.
OpenUrl CrossRef PubMed

[7] Cleaver JE,

[8] Lam ET,

[9] Revet I

[10] ↵
Schumacher B,
Hoeijmakers JH,
Garinis GA
(2009) Sealing the gap between nuclear DNA damage and longevity. Mol Cell Endocrinol 299(1):112–117.
OpenUrl CrossRef PubMed

[11] Schumacher B,

[12] Hoeijmakers JH,

[13] Garinis GA

[14] ↵
Mellon I,
Bohr VA,
Smith CA,
Hanawalt PC
(1986) Preferential DNA repair of an active gene in human cells. Proc Natl Acad Sci USA 83(23):8878–8882.
OpenUrl Abstract/FREE Full Text

[15] Mellon I,

[16] Bohr VA,

[17] Smith CA,

[18] Hanawalt PC

[19] ↵
Bohr VA,
Smith CA,
Okumoto DS,
Hanawalt PC
(1985) DNA repair in an active gene: removal of pyrimidine dimers from the DHFR gene of CHO cells is much more efficient than in the genome overall. Cell 40(2):359–369.
OpenUrl CrossRef PubMed

[20] Bohr VA,

[21] Smith CA,

[22] Okumoto DS,

[23] Hanawalt PC

[24] ↵
Fousteri M,
Vermeulen W,
van Zeeland AA,
Mullenders LHF
(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(4):471–482.
OpenUrl CrossRef PubMed

[25] Fousteri M,

[26] Vermeulen W,

[27] van Zeeland AA,

[28] Mullenders LHF

[29] ↵
Lainé J-P,
Egly J-M
(2006) Initiation of DNA repair mediated by a stalled RNA polymerase IIO. EMBO J 25(2):387–397.
OpenUrl CrossRef PubMed

[30] Lainé J-P,

[31] Egly J-M

[32] ↵
Mocquet V,
et al.
(2008) Sequential recruitment of the repair factors during NER: The role of XPG in initiating the resynthesis step. EMBO J 27(1):155–167.
OpenUrl CrossRef PubMed

[33] Mocquet V,

[34] et al.

[35] ↵
Staresincic L,
et al.
(2009) Coordination of dual incision and repair synthesis in human nucleotide excision repair. EMBO J 28(8):1111–1120.
OpenUrl CrossRef PubMed

[36] Staresincic L,

[37] et al.

[38] ↵
Lehmann AR
(2001) The xeroderma pigmentosum group D (XPD) gene: One gene, two functions, three diseases. Genes Dev 15(1):15–23.
OpenUrl FREE Full Text

[39] Lehmann AR

[40] ↵
Cleaver JE
(2005) Cancer in xeroderma pigmentosum and related disorders of DNA repair. Nat Rev Cancer 5(7):564–573.
OpenUrl CrossRef PubMed

[41] Cleaver JE

[42] ↵
Kraemer KH,
et al.
(2007) Xeroderma pigmentosum, trichothiodystrophy and Cockayne syndrome: A complex genotype-phenotype relationship. Neuroscience 145(4):1388–1396.
OpenUrl CrossRef PubMed

[43] Kraemer KH,

[44] et al.

[45] ↵
Ellison AR,
Nouspikel T,
Jaspers NG,
Clarkson SG,
Gruenert DC
(1998) Complementation of transformed fibroblasts from patients with combined xeroderma pigmentosum-Cockayne syndrome. Exp Cell Res 243(1):22–28.
OpenUrl CrossRef PubMed

[46] Ellison AR,

[47] Nouspikel T,

[48] Jaspers NG,

[49] Clarkson SG,

[50] Gruenert DC

[51] ↵
van Hoffen A,
et al.
(1999) Cells from XP-D and XP-D-CS patients exhibit equally inefficient repair of UV-induced damage in transcribed genes but different capacity to recover UV-inhibited transcription. Nucleic Acids Res 27(14):2898–2904.
OpenUrl Abstract/FREE Full Text

[52] van Hoffen A,

[53] et al.

[54] ↵
Nouspikel T,
Lalle P,
Leadon SA,
Cooper PK,
Clarkson SG
(1997) A common mutational pattern in Cockayne syndrome patients from xeroderma pigmentosum group G: Implications for a second XPG function. Proc Natl Acad Sci USA 94(7):3116–3121.
OpenUrl Abstract/FREE Full Text

[55] Nouspikel T,

[56] Lalle P,

[57] Leadon SA,

[58] Cooper PK,

[59] Clarkson SG

[60] ↵
Broughton BC,
et al.
(1995) Molecular and cellular analysis of the DNA repair defect in a patient in xeroderma pigmentosum complementation group D who has the clinical features of xeroderma pigmentosum and Cockayne syndrome. Am J Hum Genet 56(1):167–174.
OpenUrl PubMed

[61] Broughton BC,

[62] et al.

[63] ↵
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Sirt1 suppresses RNA synthesis after UV irradiation in combined xeroderma pigmentosum group D/Cockayne syndrome (XP-D/CS) cells

Abstract

Results

XP-D/CS Cells Display Global Transcriptional Dysregulation After UV Irradiation.

XP-D/CS Cells Cannot Restart Transcription of HK Genes After UV Irradiation.

XP-D/CS Cells Elicit Transcriptional Stress Response upon UV Irradiation.

XP-D/CS Cells Acquire Heterochromatin Marks on HK Genes.

Sirt1 Mediates Repression of HK Genes in XP-D/CS Cells After UV Irradiation.

Discussion

Discriminated Transcription After UV Irradiation in XP-D/CS Cells.

Sirt1-Mediated Heterochromatinization of HK Genes.

XP-D/CS Phenotype and Sirt1.

Materials and Methods

Cell Culture.

mRNA Expression.

ChIP.

Immunoblots.

siRNA Transfections.

Acknowledgments

Footnotes

References

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Abstract

Results

XP-D/CS Cells Display Global Transcriptional Dysregulation After UV Irradiation.

XP-D/CS Cells Cannot Restart Transcription of HK Genes After UV Irradiation.

XP-D/CS Cells Elicit Transcriptional Stress Response upon UV Irradiation.

XP-D/CS Cells Acquire Heterochromatin Marks on HK Genes.

Sirt1 Mediates Repression of HK Genes in XP-D/CS Cells After UV Irradiation.

Discussion

Discriminated Transcription After UV Irradiation in XP-D/CS Cells.

Sirt1-Mediated Heterochromatinization of HK Genes.

XP-D/CS Phenotype and Sirt1.

Materials and Methods

Cell Culture.

mRNA Expression.

ChIP.

Immunoblots.

siRNA Transfections.

Acknowledgments

Footnotes

References

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