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Title: ATP-Dependent Chromatin Remodeling by Cockayne Syndrome Protein B and NAP1-Like Histone Chaperones Is Required for Efficient Transcription-Coupled DNA Repair}
Article:
Abstract:
The Cockayne syndrome complementation group B( CSB) protein is essential for transcription-coupled DNA repair, and mutations in CSB are associated with Cockayne syndrome—a devastating disease with complex clinical features, including the appearance of premature aging, sun sensitivity, and numerous neurological and developmental defects. CSB belongs to the SWI2/SNF2 ATP–dependent chromatin remodeler family, but the extent to which CSB remodels chromatin and whether this activity is utilized in DNA repair is unknown. Here, we show that CSB repositions nucleosomes in an ATP–dependent manner in vitro and that this activity is greatly enhanced by the NAP1-like histone chaperones, which we identify as new CSB–binding partners. By mapping functional domains and analyzing CSB derivatives, we demonstrate that chromatin remodeling by the combined activities of CSB and the NAP1-like chaperones is required for efficient transcription-coupled DNA repair. Moreover, we show that chromatin remodeling and repair protein recruitment mediated by CSB are separable activities. The collaboration that we observed between CSB and the NAP1-like histone chaperones adds a new dimension to our understanding of the ways in which ATP–dependent chromatin remodelers and histone chaperones can regulate chromatin structure. Taken together, the results of this study offer new insights into the functions of chromatin remodeling by CSB in transcription-coupled DNA repair as well as the underlying mechanisms of Cockayne syndrome.
Introduction:
Cockayne syndrome is an autosomal recessive disease associated with numerous developmental and neurological defects, sun sensitivity and the appearance of premature aging. More than 70% of Cockayne syndrome patients have mutations in the locus encoding the Cockayne syndrome complementation group B( CSB) protein. CSB is important for transcription regulation mediated by RNA polymerase I, II and III, and it is crucial for transcription-coupled DNA repair[1]–[4]. CSB is also implicated in base-excision DNA repair[5],[6]. The CSB protein belongs to the SNF2/SWI2 ATP-dependent chromatin remodeling protein family. Members of this family regulate chromatin structure non-covalently, by using ATP as energy to alter histone-DNA contacts. Depending upon the particular chromatin remodeler, these alterations can lead to changes in nucleosome position, composition or conformation[7],[8]. However, not all members of this broad protein family have been shown to possess chromatin-remodeling activity. A collection of biochemical activities has been demonstrated for the CSB protein, including DNA/nucleosome-stimulated ATP hydrolysis, DNA strand annealing and exchange, as well as DNA wrapping[9]–[12]. Furthermore, CSB was shown to alter DNase I hypersensitivity of mononucleosomal DNA and randomize a nucleosomal array[13]. However, the extent to which CSB alters nucleosome structure, the nature of the remodeled products and the biological significance of ATP-dependent chromatin by CSB remain largely undescribed. Currently, in vivo, it is only known that the DNA/nucleosome-stimulated ATPase activity of CSB is essential for the recruitment of CSB to lesion-stalled transcription, a critical and early step of transcription-coupled DNA repair[14]. ATP-dependent chromatin remodelers often complex with other proteins[8]. These non-catalytic subunits are involved in targeting the respective remodeling activity and in regulating the activity of the motor protein. Although CSB has been found to interact with several proteins, how these interactions impact CSB's activities is still unclear. Interestingly, CSB exhibits auto-regulation, in which the N-terminal region negatively regulates both the ATPase activity and stable CSB-chromatin association[14]. Histone chaperones are also important for chromatin structure regulation[15]. These proteins function as histone carriers, donating and accepting histones. In vitro, histone chaperones can coordinate their activities with ATP-dependent chromatin remodelers to facilitate chromatin assembly or disassembly. For example, ACF or CHD1 can work with the NAP1 chaperone in chromatin assembly[15],[16], while yeast RSC can cooperate with NAP1 in nucleosome disassembly[17]. The human genome encodes five NAP1-like proteins( NAP1L1–NAP1L5) that share homology with yeast NAP1, the first NAP1 cloned[18]. NAP1L1 and NAP1L4 are ubiquitously expressed, while NAP1L2, NAP1L3 and NAP1L5 are predominantly expressed in neurons[19]. These latter three proteins are believed to have evolved more recently and likely perform mammalian-specific functions( Figure S1A)[20]. NAP1L1 and NAP1L4 are more ancient and share ∼63% amino acid identity( Figure S1B)[21]. The most notable difference between NAP1L1 and NAP1L4 is that the former has three clusters of acidic residues[22] while the latter has only two[21]. Both NAP1L1 and NAP1L4 possess histone chaperone activity and are believed to have similar functions[23]. Structure studies predict that the NAP1-like proteins can heterodimerize. Other members of the NAP1 family include SET, CINAP and TSPY[19]. In this study, we used a collection of in vitro and in vivo assays to characterize CSB's remodeling activity and to dissect the functions of CSB in transcription-coupled DNA repair. We demonstrate that CSB can reposition nucleosomes and that this activity is greatly enhanced by NAP1-like histone chaperones. Moreover, we show that chromatin remodeling, orchestrated by the combined activities of CSB and the NAP1-like chaperones, is required for efficient transcription-coupled DNA repair. From our results, we present a model in which CSB has two separable functions in transcription-coupled DNA repair: one function is protein factor recruitment and that is independent of nucleosome remodeling. A second activity, which is dependent upon nucleosome remodeling, is to establish a chromatin environment that is conducive for DNA repair and/or transcription resumption after repair.
Discussion:
In this study, we found that CSB is a relatively inefficient remodeler that slides nucleosomes away from DNA ends and creates an array of remodeled products containing histone octamers at different positions( Figure 1). This weak remodeling activity is similar to that of the Drosophila ISWI remodeler, which interacts with the dAcf1 protein to form the dACF complex; in the absence of Acf1, the ISWI remodeler displays only 3% of the activity that it does when in complex with Acf1[29]. In our study, we identified the human histone chaperones NAP1L1 and NAP1L4 as new CSB-binding partners and found that each of these proteins greatly accelerated chromatin remodeling by CSB( Figure 2). Strikingly, in the presence of either of these histone chaperones, one predominant remodeled product containing a centrally localized nucleosome was created( Figure 3 and Figure S5). By mapping functional domains and analyzing CSB derivatives, we found that CSB-mediated recruitment of some critical components of the DNA repair machinery( i. e. XPA, DDB1 and ERCC3) occurs independently of chromatin remodeling by CSB( Figure 6 and Figure S7). Our finding that co-expression of the remodeling-defective CSBΔN1 protein and NAP1L4 can rescue the UV sensitivity of CS1AN-Sv cells to a level similar to wild-type CSB, supports the notion that the protein recruitment and remodeling activities of CSB are separable( Figure 7). In this case, NAP1L4 overexpression would compensate for the diminished remodeling activity of CSBΔN1, while CSBΔN1 would maintain the capacity to recruit essential repair factors. How then might the ATP-dependent chromatin remodeling activity of CSB be utilized in the DNA repair mechanism? Given that the remodeling defective CSBΔN1 protein alone can only partially rescue loss-of-CSB function, a key role for chromatin remodeling by CSB may be to make sure that the overall repair process occurs as efficiently as possible, by decreasing nucleosome barriers. During normal transcription as RNA polymerase translocates, histone octamers can be completely displaced from the DNA template or partially disassembled through loss of an H2A/H2B dimer[30]. If, however, a bulky DNA lesion is located within the bounds of a histone octamer, that lesion could conceivably stall an RNA polymerase before the polymerase has a chance to displace the nucleosome from its path. Nucleosomes that surround a stalled RNA polymerase could create barriers to any step of the repair mechanism, from RNA polymerase displacement, through nucleotide incision and excision, to DNA re-synthesis and transcription resumption[6],[31],[32]. Although in vitro assays indicate that neither RNA polymerase displacement nor CSB activity is required for the 3′ and 5′ incisions that are needed for the excision of lesion-containing DNA[33], these in vitro studies utilized naked DNA templates that do not reflect all the complexities of transcription-coupled repair that occur within the context of chromatin in vivo. Accordingly, the combined activities of CSB and NAP1-like chaperones might mobilize histones, nucleosomes and/or stalled RNA polymerase to allow efficient coupling of transcription and DNA repair. In agreement with this notion, the SWI/SNF chromatin-remodeling complex has been shown to increase the overall efficiency of global genome excision repair, revealing the importance of chromatin dynamics in nucleotide excision repair in general[34],[35]. In addition to nucleosome repositioning, CSB has been shown to possess other ATP-dependent and ATP-independent activities, such as DNA wrapping and strand annealing[11],[12]. Consequently, these other activities are also likely to play important roles in transcription-coupled DNA repair. The mutation frequency decline protein, Mfd, is the factor that couples transcription with repair in bacteria, and the critical functions of this protein are to displace stalled RNA polymerase and to initiate repair protein recruitment[36]. CSB also initiates repair protein recruitment, but CSB alone has not been found capable of displacing a stalled RNA polymerase[9]. However, a major difference between transcription-coupled DNA repair in eukaryotes and prokaryotes is that, in addition to a stalled RNA polymerase, nucleosomes can also create barriers to the repair process. Our results indicate that nucleosomes repositioning by CSB is critical to the coupling of DNA repair with transcription in eukaryotes. The enhancement of CSB-mediated chromatin remodeling by NAP1-like histone chaperones adds a new dimension to our understanding of the ways in which ATP-dependent chromatin remodelers and histone chaperones can collaborate. Histone chaperones have been found to function with ATP-dependent chromatin remodelers in the exchange of histones and histone variants as well as in chromatin assembly( ACF, CHD1) and disassembly( RSC)[8],[16],[17],[37]. Results from this study now reveal that histone chaperones can cooperate with ATP-dependent chromatin remodelers to increase the efficiency of nucleosome repositioning by inefficient remodelers. It remains to be determined whether CSB and the NAP1-like chaperones cooperate in nucleosome assembly/disassembly during normal transcription and transcription-coupled DNA repair. Several NAP1-like proteins have been identified based on sequence homology[19], and the functions of many are still unknown( Figure S1). Among them, NAP1L2, NAP1L3, NAP1L5 are predominantly expressed in the nervous system, and TSPY is testis-specific[19]. As the highly structured, central NAP domain is sufficient for CSB binding( data not shown), CSB may likely interact with other NAP1-like protein. It is tempting to speculate that aberrant interactions between mutant CSB proteins and tissue specific NAP1-like proteins might contribute to the diverse clinical features associated with Cockayne syndrome.
Cockayne syndrome is a devastating inherited disease; the average life span of those afflicted is 12 years . Cockayne syndrome patients have features of premature aging , are highly sensitive to sunlight , and suffer from numerous developmental and neurological disorders . The majority of Cockayne syndrome patients have mutations in the CSB protein; however , how these mutations can lead to Cockayne syndrome is largely unknown . CSB is essential for transcription-coupled DNA repair—a process that preferentially removes bulky DNA lesions that stall transcription , such as those created by ultraviolet light . In eukaryotes , DNA is packaged into nucleosomes , which consists of DNA wrapped around a set of core histone proteins , and nucleosomes can create barriers to the DNA repair process . In this study , we found that CSB can slide histones along DNA . We also found that histone chaperones , proteins that accept and donate histones , greatly facilitate this process . Importantly , we show that CSB derivatives that are unable to move nucleosomes or collaborate with histone chaperones cannot repair UV-induced DNA lesions . Our study reveals that nucleosome remodeling by CSB is important for transcription-coupled DNA repair and suggests that an inability to efficiently mobilize nucleosomes might contribute to the underlying mechanism of Cockayne syndrome .