Dynamics of polycomb proteins-mediated histone modifications during UV irradiation-induced DNA damage
Graphical abstract
Introduction
Cellular functions depend on the maintenance and inheritance of genetic and epigenetic information. DNA as the carrier for this information is, however, frequently challenged either by intracellular agents such as reactive oxygen species and errors during DNA replication or by extracellular agents like radiations and radiomimetic chemicals (Bergink et al., 2006, Falk et al., 2007, Price and D'Andrea, 2013). The consequences of these events thus lead to various DNA damages and subsequent genomic instability. Kinds of mechanisms for DNA damage response (DDR) have been developed in cells to protect the integrity of their genomes, including damage-induced cell cycle checkpoints and DNA repair pathways (Bergink et al., 2006, Hoeijmakers, 2001, Peterson and Cote, 2004). For example, DNA double-strand breaks (DSBs) that are induced primarily by ionizing radiation (IR) can be repaired through homologous recombination (HR) or non-homologous end joining (NHEJ) pathway in cells (Madigan et al., 2002). In contrast, DNA lesions caused by ultraviolet (UV) light radiation, such as cyclobutane pyrimidine dimer (CPD) and 6-4 pyrimidine-pyrimidone photoproduct (6-4 PP) are normally removed from the genome by nucleotide excision repair (NER) mechanism in most organisms (Hoeijmakers, 2001, Nakajima et al., 2004). The common response to these DNA damages, however, comes from the earliest event of phosphorylation of a conserved residue in the histone variant H2AX in mammals that is referred to as γ-H2AX, or γ-H2A in yeast, or γ-H2AV in Drosophila melanogaster (Madigan et al., 2002, Rogakou et al., 1998, Shi and Oberdoerffer, 2012). The phosphorylated form of these histones then provides a high-affinity binding site for further recruitment of functionally distinct DNA damage-associated repair factors according to the different repair pathways (Bekker-Jensen and Mailand, 2010, Shi and Oberdoerffer, 2012).
Although the elaborate molecular mechanisms for these pathways in which DNA repair factors are involved have been widely investigated, the structural organization of chromosomes that may either promote or inhibit the access of repair factors to DNA damage sites still needs to be exploited in detail. Indeed, the increasing evidence has emphasized the crucial roles of chromatin modifiers in the response to DNA damage. The modifications for histones, including phosphorylation, ubiquitylation, methylation, and acetylation, were shown to participate in DNA repair processes (Lukas et al., 2011, Price and D'Andrea, 2013, Shi and Oberdoerffer, 2012, Zhu and Wani, 2010). It has been demonstrated that chromatin modifiers via histone modifications modulate the structure of chromatins during DNA repair and help restore the original state (Palomera-Sanchez and Zurita, 2011, Zhu and Wani, 2010). In addition to the phosphorylation of H2AX mentioned above, for instance, ubiquitylation of histone H2A at lysine 119 induced by DSBs was found to promote transcriptional repression near the break sites (Ginjala et al., 2011, Shanbhag et al., 2010), whereas histone H2B at lysine 120 ubiquitylation was shown to facilitate DSB repair largely by mediating chromatin accessibility (Moyal et al., 2011, Nakamura et al., 2011).
Polycomb group (PcG) complexes, as the important chromatin modifiers and also transcriptional repressors, control numerous biological processes including the maintenance of cellular identity and proliferation (Simon and Kingston, 2009, Vissers et al., 2012). More recently, it has begun to elucidate the involvement of the specific PcG proteins in the local DDR at DSBs (Vissers et al., 2012). Polycomb repressive complex 1 (PRC1), one complex in the PcG complexes, can function as an E3-ubuiquitin ligase for ubiquitylating histone H2A (H2Aub) as well as H2AX (H2AXub) (Ismail et al., 2010, Pan et al., 2011). This E3-ubuiquitin ligase activity is accompanied with PRC1 components of RING finger protein (RING) and B lymphoma Mo-MLV insertion region 1 (BMI1) in mammals, or the orthologues of Sex combs extra (Sce) and Posterior sex combs (Psc) in Drosophila (Ismail et al., 2010, Pan et al., 2011). Loss of BMI1 increased the sensitivity of human U2OS cells to IR damage and decreased the efficiency of DSB repair (Ismail et al., 2010). The further experiment revealed that ubiquitylation of H2AX by the PRC1 complex plays a critical role in initiating DNA damage signaling (Ismail et al., 2010, Liu et al., 2012, Pan et al., 2011). Besides PRC1, another PcG complex PRC2 responsible for the bulk of trimethylated lysine 27 on histone H3 (H3K27me3) could be recruited to the damaged chromatin in response to DNA damage, suggesting that this complex may be also involved in the DNA damage repair (Chou et al., 2010, Liu et al., 2012, O'Hagan et al., 2008, Seiler et al., 2011).
It has been previously identified the conserved components of PcG complexes in the silkworm Bombyx mori, a model lepidopteran insect with holocentric chromosomes (Li et al., 2012b, Murakami and Imai, 1974). Different from monocentric chromosome of which kinetochores assemble on a localized region, holocentric chromosome has diffuse kinetochores that form along the entire length of the chromosomes (Maddox et al., 2004). This feature of holocentric chromosome organization may help to avoid the drastic consequences of unrepaired DNA breakages because chromosome fragments caused by the break events have a higher possibility of being maintained during mitosis owing to the multiple sites on each chromosome for spindle microtubule attaching (Maddox et al., 2004, Pimpinelli and Goday, 1989). In agreement with this, the cultured silkworm cells were shown to be highly resistant to the irradiations (Takahashi et al., 2006). Our previous work also demonstrated the conserved repair pathways including NHEJ and HR that are existed in the silkworm system (Mon et al., 2011). The detailed resistant mechanism of silkworm genome to irradiation and how the DNA damage repair factors involved, however, remain to be uncovered. Moreover, there is no available marker for monitoring the process of DNA repair in silkworm, like γ-H2AX in mammals. In order to provide potential insight into these questions and understand the early response of silkworm holocentric chromosomes to DNA damage, we here focused on one of the best-characterized histone-modifying complexes that are responsible for multiple histone modifications, the PcG complexes that direct H3K27me3 and H2Aub by its subcomplex of PRC2 and PRC1, respectively. Our present data showed the time-course-dependent response of PcG complexes-mediated ubiquitylation and methylation on distinct histones in response to UV–C irradiation and revealed a crucial role of these remodeling events in creating an open chromatin structure for the efficient DNA repair.
Section snippets
Cell lines
For the present research we used the silkworm BmN4-SID1 cell line that expresses a Caenorhabditis elegans SID-1 (CeSID-1) transmembrane protein with the ability to soaking RNA interference (RNAi) (Mon et al., 2012) and BmN4-Luc5′3′DR18 cell line that carries two truncated luciferase genes (Luc5′ΔC and Luc3′ΔA) to detect HR repair efficiency (Mon et al., 2011). Cells were cultured at 27 °C in IPL-41 medium (Sigma–Aldrich, St. Louis, USA) containing 10% fetal bovine serum (FBS) (Life
PcG proteins are required for efficient DNA repair and confer protection against UV–C exposure
We first tested whether the silkworm PcG proteins are also involved in the local DSB repair event in cultured silkworm cells using an HR reporter assay system established by our previous work (Mon et al., 2011). BmN4-Luc5′3′DR18 cell line where the pBac-Luc5′3′ plasmid was stably integrated into the chromosome was able to trigger HR repair by the introduction of I-SceI endonuclease to cleave the I-SceI site between two inactive luciferase genes (Luc5′ΔC and Luc3′ΔA) in pBac-Luc5′3′ plasmid,
Discussion
Maintenance of genomic integrity in cells upon DNA damage relies on a complex molecular network that coordinates numerous repair factors and chromatin modifiers to achieve the repair of DNA breaks. Although the identification and functional analysis of those proteins involved in DNA repair pathway have been widely elucidated in many model species, the silkworm Bombyx, a lepidopteran model insect possessing holocentric chromosomes, is still largely unknown. One reason for this might be owing to
Acknowledgments
We would like to thank two anonymous reviewers for their constructive comments that have improved our manuscript. This work was supported by JSPS KAKENHI Grant Number 24-02396 to ZL. The authors have declared that no conflict of interest exists.
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