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ATP-dependent chromatin remodeling by Cockayne syndrome protein B and NAP1-like histone chaperones is required for efficient transcription-coupled DNA repair - PubMed

ATP-dependent chromatin remodeling by Cockayne syndrome protein B and NAP1-like histone chaperones is required for efficient transcription-coupled DNA repair

Iltaeg Cho et al. PLoS Genet. 2013 Apr.

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. ATP–Dependent Chromatin Remodeling Activity of CSB.

(A) Schematic representation of CSB and CSBΔN proteins used in the remodeling assays shown in D–E. (B) Coomassie-stained gel showing purified proteins and complexes used in this study. (C) Representation of the centrally localized mononucleosome with two 45 bp DNA overhangs used in assays shown in D–E. The position of the Pst I site used to assay restriction enzyme accessibility is as noted. (D–E) Restriction enzyme accessibility assays to examine the kinetics of remodeling. Rate constants for ACF at 12.5 and 50 nM were 0.1 and 0.16 min−1, respectively. (D) Rate constants for CSB at 25 and 100 nM were 2.5 and 17×10−3 min−1, respectively. Rate constant for CSB in the absence of ATP was 4×10−7 min−1. (E) Rate constants for CSBΔN at 50 and 200 nM were 3 and 22×10−4 min−1, respectively. In the absence of ATP, the rate constant for CSBΔN was 2.5×10−4 min−1. (F) Electrophoretic mobility-shift assays to analyze remodeled nucleosome structure. End-positioned mononucleosomes with a single 91-bp DNA overhang were used as substrate. Reactions were carried out for 15 minutes at 30°C, as previously described . ACF created a centrally localized nucleosome, as previously reported . CSB and CSBΔN were used at two concentrations, as indicated. Migration positions of centrally- and end-positioned nucleosomes are as noted.

Figure 2
Figure 2. Histone Chaperones NAP1L1 and NAP1L4 Interact with CSB and Enhance ATP–Dependent Chromatin Remodeling by CSB.

(A) Silver stained gel resolving proteins from UV-irradiated CS1AN-Sv cell lysates that bound to immobilized CSB, CSBΔN (CSB 455–1493), CSBΔC (CSB1-1009) or an M2-beads-only control (−). Asterisks denote bands excised for analysis by mass spectrometry. (B) Western blot analyses of samples shown in (A), using anti-NAP1L1 or NAP1L4 antibodies. (C) Co-immunoprecipitation of CSB, NAP1L1 and NAP1L4 from chromatin-enriched fractions of UV-irradiated HeLa cells (S is the soluble fraction, and C is the chromatin-enriched fraction). Antibodies were crosslinked to beads and co-immunoprecipitating proteins were eluted. The first two elutions are shown (lane 4–5). Western blots were performed with antibodies indicated to the right. (D) Mononucleosome used in assays E–F. (E–F) Restriction enzyme accessibility assays with 280 nM CSB. (E) Effect of NAP1L1 on CSB remodeling activity. Rate constants for CSB with 0, 100, 300 and 900 nM NAP1L1 were 1, 1, 2 and 6, ×10−2 min−1, respectively. In the absence of ATP or CSB, no appreciable stimulation of activity was detected. (F) Effect of NAP1L4 on CSB remodeling. Rate constants for CSB with 0, 70, 200 and 600 nM NAP1L4 were 1, 2, 2, and 4, ×10−2 min−1, respectively. In the absence of ATP or CSB, no appreciable stimulation of activity was detected.

Figure 3
Figure 3. CSB and NAP1-like Chaperones Efficiently Slide Nucleosomes to the Center of a DNA Fragment.

(A) Electrophoretic mobility-shift assays to analyze remodeled nucleosome structure. An end-positioned mononucleosome with a 91 bp DNA overhang was used as substrate. Varying amounts of NAP1L1 were used in CSB-mediated nucleosome remodeling assays. NAP1L1 facilitates chromatin remodeling by CSB to form centralized nucleosomes. Nucleosome positions are illustrated to the left. (B) MNase mapping of protected DNA before and after remodeling reactions. Schematic representation of the assay is to the left. (C) Results from MNase mapping. After CSB remodeling, ∼60% of the nucleosomes were similar to the end-positioned nucleosomal substrate. (D) After remodeling by CSB and NAP1L1, ∼80% of the nucleosomes were centrally positioned. (E) Restriction enzyme accessibility assays using a 202 bp nucleosomal substrate with a Pst I site at position 93.

Figure 4
Figure 4. The N1 Region of CSB Is Critical for NAP1L1 Interaction.

(A) Schematic illustration of CSB and CSBΔN1 used in assays. (B) Western blot analysis of GST pull-down assays to examine CSB and NAP1L1 interactions. Purified CSB or CSBΔN1 were mixed with immobilized GST or GST-NAP1L1. Western blots showing levels of input CSB and CSBΔN1 as well as their binding to GST-NAP1L1. (C) Immunoprecipitation using an anti-NAP1L1 antibody to examine the CSB- and CSBΔN1-NAP1L1 interactions in cells, with or without UV treatment (100 J/m2).

Figure 5
Figure 5. The N1 Region of CSB Is Critical for Chromatin Remodeling.

(A) Coomassie stained gel of purified CSBΔN1 used in B–C. (B–C) Restriction enzyme accessibility assays were carried out in the presence of ∼70 nM of 240 bp nucleosomes and limiting amounts of CSB (B) or CSBΔN1 (C). (B) CSB remodeled nucleosomes in an ATP-dependent manner with a rate of 0.12 nM Pst I site cutting/min at 20 nM CSB and 0.42 nM Pst I site cutting/min at 60 nMCSB. (C) Using the same concentrations of CSBΔN1, no ATP-dependent increase in Pst I accessibility was detected.

Figure 6
Figure 6. CSB Has Nucleosome Remodeling-Dependent and -Independent Functions in Transcription-Coupled DNA Repair.

(A) Western blot showing transgene expression levels of cell lines used in B–E. GAPDH was used as a loading control. Cell lines expressing different transgenes are as indicated at the top of blots, and antibodies used for probing are indicated to the left. (B) UV-induced chromatin association of CSB and CSB derivatives. Cells were irradiated with different UV doses. After 1-hour recovery, lysates were prepared and divided into soluble and chromatin-enriched fractions. Antibodies used in western blot analyses are as indicated to the left. The upper three rows in the boxed area contain samples from CSBΔN1 expressing cells. The lower two rows contain samples from CSB and CSBR670W expressing CS1AN-Sv, used as positive and negative controls, respectively . (C) Quantification of the data in (B), showing the percentage of CSB (black, left Y-axis) and CSBΔN1 (grey, right Y-axis) that co-fractionated with chromatin as a function of UV dose. Shown are averages from three independent experiments +/− SEM. (D) Clonogenic cell-survival assays. CS1AN-Sv cell lines expressing the indicated proteins were treated with 0, 5, 8 or 12 J/m2 of UV light. Plotted is the percent survival versus UV dose. Shown are averages +/− SEM. n is number of experiments performed for each cell line. (E) ChIP-western analysis of CSB- and CSBΔN1-interacting proteins. CS1AN-Sv cells expressing CSB or CSBΔN1 were irradiated with 25 J/m2 of UV light, allowed to recover for one hour and then subjected to ChIP using anti-CSB antibodies. Immunoprecipitated material was reverse-crosslinked and analyzed by western blot using the indicated antibodies.

Figure 7
Figure 7. Model Depicting the Distinct Activities of CSB in Transcription-Coupled DNA Repair.

The targeting of CSB to lesion-stalled transcription (Top) is a critical and early step in the process of transcription-coupled DNA repair . Once recruited, CSB has two separable activities. Lower left: one activity is protein factor recruitment that occurs independently of nucleosome repositioning. Recruitment includes components of the nucleotide excision repair complex, the CSA/CSN E3 ligase complex and the TFIIH transcription elongation complex . Direct interactions between CSB and the NAP1-like chaperones suggest that CSB also recruits NAP1L1 and NAP1L4 to sites of lesion-stalled transcription. Lower right: a second activity is dependent upon nucleosome repositioning. In conjunction with the NAP1-like histone chaperones, CSB slides nucleosomes to allow efficient DNA repair and/or resumption of transcription after repair.

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