Published online 16 October 2013 Nucleic Acids Research, 2014, Vol. 42, No. 2 893–905 doi:10.1093/nar/gkt912 High-resolution characterization of CPD hotspot formation in human fibroblasts Anamaria G. Zavala*, Robert T. Morris, John J. Wyrick and Michael J. Smerdon Biochemistry and Biophysics, School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-7520, USA Received December 30, 2012; Revised August 25, 2012; Accepted September 12, 2013 ABSTRACT factors, such as reactive oxygen species, as well as physical and chemical agents in the environment (1). Repair of DNA lesions must occur within the Ultraviolet (UV) radiation is the most common physical chromatin landscape and is associated with alter- carcinogen in our environment and the major etiological ations in histone modifications and nucleosome factor in the development of skin cancer in humans. rearrangement. To directly associate these chroma- Cyclobutane pyrimidine dimers (CPDs) are the predomin- tin features with DNA damage and repair, it is ne- ant lesions formed during UV exposure. UV-induced cessary to be able to map DNA adducts. We have photoproducts form exclusively at dipyrimidines and pref- developed a cyclobutane pyrimidine dimer (CPD)- erentially at TT sites. CPD formation at dipyrimidines specific immunoprecipitation method and mapped varies, with induction at TT>TC>CT>CC at a ratio of ultraviolet damage hotspots across human chromo- 55:33:11:1 (2). CPD formation is influenced by sequence somes 1 and 6. CPD hotspots occur almost equally context (3) as well as by chromatin environment (4). The highly dynamic and condensed structure of chro- in genic and intergenic regions. However, these matin is composed of nucleosomes separated by linker hotspots are significantly more prevalent adjacent regions. The nucleosome core consists of 147 bp of DNA to repeat elements, especially Alu repeats. wrapped around an octameric core of histone proteins Nucleosome mapping studies indicate that nucleo- H2A, H2B, H3 and H4. Our laboratory has previously somes are consistently positioned at Alu elements demonstrated a small bias in CPD formation in nucleo- where CPD hotspots form, but by 2 h post- some linker regions compared with nucleosome core DNA irradiation, these same regions are significantly (1.5–2-fold) [reviewed in (5)]. In addition, we have depleted of nucleosomes. These results indicate shown that CPD formation is highly influenced by the that nucleosomes associated with hotspots of orientation of the DNA on the core histone surface, CPD formation are readily rearranged, potentially with CPDs forming at a 10.3 base periodicity (4). CPD making them accessible to DNA repair machinery. formation in the core DNA is as much as 10-fold higher when the phosphate backbone is furthest from the histone Our results represent the first chromosome scale surface compared with when it is in contact with the map of ultraviolet-induced DNA lesions in the histones. human genome, and reveal the sequence features A cell’s ability to correctly repair DNA damage is and dynamic chromatin changes associated with essential for the continued vitality of virtually all organ- CPD hotspots. isms. Eukaryotic cells repair DNA lesions through two main pathways: base excision repair (BER) and nucleotide excision repair (NER). BER removes small non–helix-dis- INTRODUCTION torting damage to bases, for instance oxidation or methy- Exposure of cells to genotoxic agents, both chemical and lation. NER is responsible for the removal of helix- physical in nature, is responsible for the initiation and pro- distorting lesions in DNA such as CPDs. Xeroderma motion of the majority of human cancers. Despite the vast pigmentosum, a severe genetic disorder characterized by array of DNA-damaging agents, the single unifying prin- an exceptionally high incidence of UV-induced skin cipal is their ability to interact with cellular DNA cancer, results from disruption of the NER pathway and producing potentially mutagenic and heritable lesions. indicates a crucial role for NER in preventing cancer DNA damage can result from interaction with endogenous development (6). *To whom correspondence should be addressed. Tel: +1 208 885 9237; Fax: +1 208 885 7905; Email: [email protected] Present address: Anamaria G. Zavala, Department of Biological Sciences, University of Idaho, University of Idaho, Moscow, ID, 83844-3051, USA. Robert T. Morris, Massachusetts General Hospital Cancer Center, Boston, MA 02114, USA. ß The Author(s) 2013. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 894 Nucleic Acids Research, 2014, Vol. 42, No. 2 In eukaryotic cells, NER must take place within the MATERIALS AND METHODS complex landscape of chromatin. The presence of DNA irradiation and fragmentation histones decreases the rate of repair of DNA damage compared with naked DNA by reducing the accessibility Telomerase (tert)-immortalized normal human fibroblast of damaged DNA to repair enzymes (7,8). However, the monolayers (NHF1-tert) (18) were cultured in Dulbecco’s rate of CPD removal in Xenopus oocyte nuclear extracts is modified Eagle’s medium (Gibco Invitrogen) supple- not influenced by the rotational setting of the adduct on mented with 10% fetal bovine serum and 1% penicillin/ the histone octamer (9). For efficient DNA repair to take streptomycin at 37C in a humidified incubator at 10% place, nucleosome rearrangement must occur. Our labora- CO2. DNA was isolated from unirradiated NHF1 cells tory and others have shown that NER is facilitated by using a previously described protocol (19,20) and histone modifications as well as by ATP-dependent chro- irradiated to the indicated doses with 254-nm UVC. matin remodeling complexes (10–12). UVC was used to induce CPDs in this study as well as Determining histone modification states and nucleo- in most of the studies referenced. It is commonly used in some positioning at sites of DNA adduct formation will laboratories despite the fact that UVC does not reach the allow us to determine nucleosome level changes that may earth’s surface. However, both UVC and UVB, which is be related to repair initiation and progression. Changes in more relevant to human exposure, efficiently form both histone modifications and nucleosome positioning over CPDs and 6-4 photoproducts, the two most common the course of DNA damage and repair in human cells UV-induced DNA lesions (21). can be mapped using ChIP-chip or ChIP-seq techniques. Ten micrograms of isolated DNA was diluted in 400 ml However, without the corresponding map of DNA of 10 mM Tris-Cl (pH 8) and sonicated to an average adducts it is impossible to determine which histone modi- fragment size of 300 bp using a Fisher dismembrator 500 fications may be associated with and potentially facilitate fitted with a microtip and set at 40% power. Samples were DNA damage recognition and repair. Therefore, it is sonicated for four cycles of 10 s on and 30 s off in ice necessary to develop a high resolution map of DNA water. adducts in human cells that can be correlated to nucleo- some position and DNA sequence data to further charac- Cell irradiation and repair terize adduct formation. The ability to map DNA damage NHF1-tert cells were grown to confluence before UV across the genome would provide an unprecedented irradiation. The media was replaced with phosphate snapshot of where CPDs form without bias toward genic buffered saline (PBS) before irradiating with 254-nm and promoter regions, the typical targets of previous UVC to a dose of 12 J/m2 at room temperature. After studies (10,13,14). In addition, such data would enable irradiation, the cells were allowed to repair at 37Cin the identification of hotspots of CPD formation and de- conditioned medium for the indicated period. Cells were termine the sequence and chromatin similarities that may harvested by scraping into PBS. permit increased lesion formation. Strickland and Gentile (15) first described immunopre- CPD immunoprecipitation cipitation of UV-induced DNA adducts over two decades ago. More recently, Rochette et al. (16) used CPD-specific Ten micrograms of aCPD antibody (clone TDM2, Cosmo immunoprecipitation and polymerase chain reaction Bio Co., Ltd, Japan) was bound to 100 ml of Protein G Dynabeads (Invitrogen) in 1Â PBS with 0.5% bovine (PCR) to determine rates of CPD repair in telomeres. Teng et al. (17) used CPD immunoprecipitation (cpdIP) serum albumin overnight at 4 C while rotating. Before to map UV-induced DNA damage across the immunoprecipitation, sonicated samples were brought to Saccharomyces cerevisiae genome. By probing microarrays final buffer conditions (1Â PBS, 0.025% Triton X-100, containing 6256 probes spaced at an average of 290 bp 25 mg/ml yeast tRNA), boiled for 5 min and flash cooled covering the 12 Mb yeast genome, these authors were in an ice water bath before adding the sample to the protein G-bound antibodies. This mixture was incubated able to show CPD adduct formation and repair across the yeast genome. at 4 C overnight while rotating. Beads were washed four times in wash buffer (1Â PBS, 0.025% Triton X-100) We have developed a similar immunoprecipitation and microarray-based technique to map UV-induced DNA before eluting at 65 C in TE with 1% sodium dodecyl adducts within the human genome. We mapped CPDs sulphate. After treatment with RNase A (0.2 mg/ml) and across chromosomes 1 and 6 using high-resolution tiling proteinase K (0.2 mg/ml), DNA was ethanol precipitated arrays. These arrays cover 416 Mb of the human genome and resuspended in 10 mM Tris, pH 8.0. with 6.5 million probes spaced at an average of 35 bp.