CSB is recruited to chromatin in a UV-dependent manner
To dissect the mechanism by which CSB associates with chromatin and determine how this association may be regulated, we employed a protein fractionation scheme to examine the subcellular distribution of CSB before and after UV treatment. Cells were lysed in an isotonic buffer containing non-ionic detergent (), and proteins were separated into soluble and chromatin-containing fractions by centrifugation. Fractionation efficiency was monitored by probing for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (soluble fraction), and total histones, histone H3 or elongating RNA polymerase II (chromatin-containing fraction). Additionally, CSB-PGBD3, an endogenously expressed fusion protein composed of N-terminal CSB sequence and sequence derived from a PiggyBac transposase, was used as a common probe for normalization between soluble and chromatin-containing fractions (Newman et al., 2008
). One third of CSB-PGBD3 co-fractionated with chromatin while the remainder was soluble, and this distribution was independent of UV irradiation ().
UV irradiation induces stable CSB-chromatin association
Using this protocol, we studied the dynamics of CSB's association with chromatin. MRC5 cells (normal human fibroblasts) were heavily UV-irradiated (100 J/m2
or less than 10-6
survival), lysed at different times after irradiation, and CSB partitioning was determined by protein fractionation and Western blot analysis. As shown in , CSB was mainly in the soluble fraction during normal growth (lane 1 vs. 7). In contrast, by one hour after UV irradiation, about 90% of total CSB relocated to the chromatin-containing fraction (lane 2 vs. 8). Four hours post UV irradiation, more than 60% of total CSB once again became soluble and by seven hours, the vast majority of CSB was soluble (). As expected, GAPDH was always soluble and histones were always chromatin associated (). A similar trend in the partitioning of CSB was observed when MRC5 cells were more lightly UV-irradiated with a dose of 10 J/m2
(~ 60% survival; data not shown). Data quantification revealed insignificant changes in total CSB levels (soluble + chromatin-containing) within the first four hours after UV irradiation, while an approximately 50% reduction in total CSB was detected by seven hours post irradiation (, lanes 4 and 10 vs. lanes 5 and 11). The reduction in CSB levels could result form CSA-mediated degradation (Groisman et al., 2006
) or from lethality due to the heavy UV dose that was used. The partitioning of CSB to the chromatin-containing fraction after UV irradiation likely represents stable chromatin-associated CSB, as chromatin immunoprecipitation (ChIP) experiments had revealed that CSB is recruited to sites of DNA lesion-stalled transcription one hour after UV irradiation (Fousteri et al., 2006
). To test directly if UV-irradiation promotes stable CSB-chromatin association, we performed ChIP with anti-histone H3 antibodies. Western blot analysis demonstrated that CSB became associated with histone H3 after UV-irradiation (, lane 3 vs. 5), while the total amount of CSB remained unchanged (, lane 1 vs. 2). These observations indicate that, under normal growth conditions, CSB is either not associated with chromatin or the association is unstable; upon UV-irradiation, a stable CSB-chromatin association is induced.
We next determined if the amount of CSB co-fractionating with chromatin was directly proportional to UV dose. As the number of DNA lesions increases with increasing UV dose, such an analysis will determine whether the stable CSB-chromatin association is mediated through DNA damage. Accordingly, MRC5 cells were irradiated with a series of UV doses. Cells were lysed and proteins fractionated one-hour post UV irradiation. As the UV dose increased, a greater amount of CSB became associated with chromatin (). The fraction of chromatin-associated CSB reached a plateau at ~25 J/m2
(which introduces ~2 DNA lesions per 10 Kb of DNA (Mathonnet et al., 2003
)); a dose similar to that required to detect maximally immobilized CSB by FRAP (van den Boom et al., 2004
). We also examined the partitioning of two other proteins involved in nucleotide excision DNA repair: the endonuclease ERCC4/XP-F and the ERCC3/XPB helicase. ERCC4 also displayed UV dose-dependent, chromatin association, as did ERCC3 but to a lesser degree (). Notably, UV-irradiation did not alter the chromatin association of two other ATP-dependent chromatin remodelers, BRG1 and SNF2h ( and data not shown). The vast majority of BRG1 and SNF2h were consistently in the chromatin-containing fraction along with histone H3 and elongating RNA polymerase II, while GAPDH was exclusively soluble (). We conclude that stable CSB-chromatin association occurs after UV irradiation.
Some CSB mutations found in CS patients disrupt UV-induced CSB-chromatin association
We next assessed whether the UV-induced, chromatin association of CSB is compromised by CS-associated CSB mutations. Four mutations were examined: R670W, W851R, V957G and P1042L (). All these mutant proteins fail to complement the UV sensitivity of a mutant CSB cell line (Mallery et al., 1998
). Transgenes encoding these mutant proteins were introduced into CSB-deficient CS1AN-Sv cells to generate stable lines, and we assayed for their ability to associate with chromatin in response to UV irradiation (). At least two independent clonal cell lines were examined for each protein. Similar to endogenous CSB in MRC5 cells, CSBwt
reintroduced into CS1AN-Sv cells displayed UV dose-dependent chromatin association ( and ). Due to CSB overexpression, the saturating fraction of chromatin-bound CSB was less than that of endogenous CSB in MRC5 cells (~40% vs. ~90% respectively, and ). Interestingly, unlike CSBwt
, CSB proteins harboring the R670W, W851R or V957G mutation were impaired in their ability to associate with chromatin in response to UV irradiation, while the P1042L mutation did not interfere with this response. These results revealed that one molecular defect, associated with some forms of CS, is a failure of mutant CSB proteins to stably associate with chromatin in response to UV irradiation. However, additional mechanisms also contribute to this disease as evidenced by the P1042L mutation (see discussion).
Some CS-associated CSB mutations disrupt UV-induced chromatin association and ATPase activity
ATP hydrolysis activity is essential for the UV-induced association of CSB with chromatin
Three mutant CSB proteins we examined have mutations in the ATPase domain (R670W, W851R and V957G), while one mutant protein has a mutation (P1042L) within a putative nuclear localization signal (). Our fractionation data revealed that nuclear localization of CSBP1042L
is, however, unaffected (). To compare the biological properties of CSB to its biochemical activities, we examined the effect of these mutations on DNA-stimulated ATP-hydrolysis activity. CSBwt
and the mutant CSB proteins were expressed in and affinity purified from SF9 cells (). As previously reported, CSBwt
had robust DNA-stimulated ATPase activity (). Interestingly, CSBR670W
had no DNA-stimulated ATPase activity ( and S1
), and the V957G mutation reduced ATPase activity to 22% ( and ). In contrast, CSBP1042L
displayed a level of DNA-stimulated ATPase activity similar to CSBwt
( and ). We next examined the DNA binding activity of CSBV957G
using ATPase assays to determine their affinity for DNA (KM
, the DNA concentration necessary to achieve half-maximal rates of ATPase activity). As shown in , the KM
for DNA of CSBV957G
was 2.3-fold of CSBwt
, suggesting that the CSBV957G
protein has a lower DNA affinity than CSBwt
. In contrast, the KM
for DNA of CSBP1042L
was unaffected. These results suggest that the DNA-stimulated ATPase activity of CSB is important for UV-induced, stable CSB-chromatin association.
Effects of CSB ATPase activity on UV-induced CSB-chromatin association
To test further the requirement of DNA-stimulated ATPase activity for stable CSB-chromatin association, we generated four additional proteins containing point mutations in conserved residues predicted to contact DNA and analyzed their effects in vitro and in vivo (). N653I lies in domain 1 and is predicted to disrupt contacts made with the DNA backbone; R894E, R950E and K979E lie in domain 2 and are predicted to interfere with DNA binding and/or ATP hydrolysis (Durr et al., 2005
). As shown in , R894E abolished DNA-stimulated ATPase activity. Direct, side-by-side measurements of ATPase activity and apparent DNA affinity (KM
) demonstrated that N653I, R950E and K979E significantly attenuated the interaction of CSB with DNA, reducing DNA affinity at least 5 fold, and decreased DNA-stimulated ATPase activity to 75%, 24% and 46% of wild-type level, respectively ().
By transient expression, we next examined the in vivo consequences of these four mutant proteins in CS1AN-Sv cells, as well as CSBR670W
(a Walker A motif mutation disrupting ATP hydrolysis) (). As shown in , CSBwt
, transiently expressed in CS1AN-Sv cells, localized to the soluble fraction in the absence of UV treatment (lane 1 vs. 2), and the fraction of chromatin-bound CSBwt
increased one hour after UV irradiation (lanes 3-4). In contrast, the R670W mutation disrupted the ability of CSB to stably associate with chromatin after UV irradiation (lanes 11-12 vs. 3-4), consistent with our observations made in stable cell lines (). Interestingly, CSBK538A
, which is devoid of ATPase activity (Citterio et al., 1998
), failed to stably associate with chromatin after UV irradiation (lanes 7-8). Similarly, CSBN653I
also failed to stably associate with chromatin after UV irradiation (). These results support the hypothesis that ATP hydrolysis stimulated by DNA is essential for UV-induced, stable CSB-chromatin association.
The N- and C-terminal regions of CSB differentially regulate CSB-chromatin association
To define the structural elements of CSB that regulate chromatin association, three regions of CSB were independently expressed in CS1AN-Sv cells: CSB-N (1-507), CSB-C (1010-1493) and CSB-M (455-1009) (). Interestingly, CSB-M (ATPase domain alone) did not stably associate with chromatin after UV treatment (, lane 8), nor did CSB-N and CSB-C (, lanes 4 and 12). These observations suggest that the ATPase domain functions in concert with either the N- or C-terminal region to mediate UV-induced chromatin association.
Primary structural determinants directing CSB-chromatin association
To test this hypothesis, we characterized the chromatin association of N- and C-terminally truncated CSB proteins: CSBΔN (455-1493) and CSBΔC (1-1009) (). Deleting the C-terminal 484 amino acids disrupted the ability of CSB to become stably associated with chromatin one hour after UV treatment (, lane 8), indicating that the C-terminal domain of CSB cooperates with the ATPase domain to stabilize chromatin association. This notion was confirmed by our observation that CSBΔN readily associated with chromatin one hour after UV irradiation (, lane 12), demonstrating that the N-terminal 454 amino acids are dispensable for stable CSB-chromatin association.
Intriguingly, in contrast to CSB, a significant amount of CSBΔN was present in the chromatin-containing fraction even without UV treatment (, lane 10), indicating that the N-terminal region negatively regulates CSB-chromatin association and dictates substrate specificity. To confirm that CSBΔN stably associates with chromatin independently of UV irradiation, we performed ChIP-Western analysis with anti-histone H3 antibodies using CS1AN-Sv cells stably expressing CSBΔN. As shown in , CSBΔN co-purified with histone H3, independent of UV, indicating that CSBΔN can bind to chromatin in the absence of DNA lesions. Together, these observations suggest that both the ATPase and C-terminal domains are required for UV-induced, stable chromatin association and that the N-terminal region acts as a negative regulatory element preventing CSB from stably associating with chromatin in the absence of lesion-stalled transcription.
To dissect how the different regions contribute to CSB's biochemical activities, we measured ATP hydrolysis rates and DNA binding affinities of CSBΔN, CSB-M and CSBΔC. As shown in , CSBΔN displayed an increased KM for DNA (decreased DNA affinity) but elevated ATP hydrolysis activity. This was in sharp contrast to all other mutant CSB proteins examined (-), which displayed both decreased DNA affinity and ATPase activity in vitro. The increased ATPase activity of CSBΔN suggests that the N-terminal region negatively regulates the enzymatic activity of CSB in addition to chromatin association (). CSB-M exhibited a further decrease in DNA affinity and a concomitant decrease in ATPase activity (). Examination of CSBΔC revealed that this protein had no significant DNA-stimulated ATPase activity. These results suggest that the N-terminal region, the ATPase domain and the C-terminal regions of CSB all directly contribute to DNA association and catalytic activity.
The results from our in vivo and in vitro assays support the hypothesis that the UV-induced, stable chromatin association of CSB depends upon its DNA-stimulated ATPase activity. Moreover, these results reveal that specific targeting of CSB in vivo is ensured by a complex mechanism involving both positive (C-terminal) and negative (N-terminal) regulatory elements ().
A model for the regulation of CSB-chromatin association
CSB-N inhibits the ATPase activity of CSBΔN in trans
To verify that the N-terminal region functions as a negative regulatory element, we asked whether CSB-N could inhibit the ATPase activity of CSBΔN in trans (). Interestingly, while CSBΔN displayed significant DNA-stimulated ATPase activity, CSB-N could reduce this activity. The inhibitory effect was proportional to CSB-N concentration, reaching a maximal inhibition of 63% at an equal or greater molar ratio of CSB-N/CSBΔN (, orange). Furthermore, the inhibitory effect was specifically caused by CSB-N and not a contaminating activity, as CSB-N did not inhibit the ATPase activity of another ATP-dependent chromatin remodeler BRG1 ().
CSB-N inhibits the ATPase activity of CSBΔN in trans
In principle, CSB-N could inhibit the ATPase activity of CSBΔN by competing with CSBΔN for binding to DNA or by interacting directly with CSBΔN. To distinguish between these possibilities, we asked if increasing DNA concentrations could reverse the inhibitory effect. As shown in and Table S1
, 7 nM of CSB-N inhibited ~ 60 % of the ATPase rate of 3 nM CSBΔN in the presence of 0.123 ng/μl DNA (green with filled square). Increasing DNA concentration by 3-, 10- or 30-fold did not reverse the inhibition of ATPase activity by CSB-N (, and Table S1
). Additionally, inhibition was observed over a wide range of DNA concentrations, as low as 0.04 ng/μl. These results argue against a model whereby CSB-N prevents CSBΔN from binding to DNA and support a model in which CSB-N directly inhibits the ATPase activity of CSBΔN, consistent with our side-by-side comparisons of CSB and CSBΔN activities described in . In agreement with this model, in vitro binding assays revealed that CSB-N directly interacted with CSBΔN and CSB-C (Figure S2A
). Furthermore, CSB-N failed to inhibit the ATPase activity of CSB (), suggesting that the N-terminal region of full-length CSB normally occupies the site of interaction. Interestingly, CSB-N decreases both the Vmax
of CSBΔN to values observed for CSB ( and S2B
). Importantly, the ratios of Vmax
of CSBΔN in the presence or absence of CSB-N remained constant (). This observation is consistent with a notion that the N-terminal region of CSB can act as an uncompetitive inhibitor of CSBΔN, implying that CSB-N increases the nonreactive interaction of CSBΔN with DNA (which does not stimulate ATP hydrolysis).
ATP hydrolysis is dispensable for stable CSBΔN-chromatin association
Our results indicate that ATP hydrolysis is necessary for the UV-induced association of CSB with chromatin. In addition, the N-terminal region negatively regulates CSB-chromatin association in absence of UV irradiation. Furthermore, the N-terminal region inhibits the ATPase activity of CSBΔN, likely by increasing non-reactive interactions of CSB and DNA. Together, these results suggest that a function of ATP hydrolysis might be to remove the inhibitory effect imposed by the N-terminal region. If this were true, then a CSBΔN mutant protein that is also defective in ATP hydrolysis should still retain its ability to associate with chromatin, due to the absence of the negative regulatory region.
To test this hypothesis, we removed the N-terminal 454 amino acids from the ATPase-defective and CS-associated mutant CSB proteins, and we determined if these doubly mutant proteins interacted with chromatin in the absence of UV irradiation. As shown in , each of these proteins stably associated with chromatin regardless of UV treatment. The stable chromatin association was confirmed by ChIP-Western analysis of CS1AN-Sv cells stably expressing ATPase-defective CSBK538A ΔN (). Together, these results support a model in which the N-terminal region of CSB prevents the protein from stably associating with chromatin under normal growth conditions; upon UV irradiation, in the presence of lesion-stalled transcription, the energy from DNA-stimulated ATP hydrolysis can be used to overcome this inhibition ().
ATP hydrolysis is not required for CSBΔN-chromatin association