Quantification of protein enrichment at site-specific DNA double-strand breaks by chromatin immunoprecipitation in cultured human cells.

Here, we present a chromatin-immunoprecipitation-based protocol to quantify the recruitment of proteins adjacent to site-specific DNA double-strand breaks (DSBs), such as proteins involved in DSB repair. We describe steps to induce DSBs in U2OS osteosarcoma cells stably expressing the restriction endonucleases FokI or AsiSI. We then detail the procedures of chromatin isolation and immunoprecipitation, followed by protein elution and quantitative-PCR-based quantification of DNA. This protocol cannot be used on DSBs generated at random loci by DNA damaging agents. For complete details on the use and execution of this protocol, please refer to Fitieh et al. (2022).1.

BMI-1 regulates DNA end resection and homologous recombination repair.

BMI-1 is an essential regulator of transcriptional silencing during development. Recently, the role of BMI-1 in the DNA damage response has gained much attention, but the exact mechanism of how BMI-1 participates in the process is unclear. Here, we establish a role for BMI-1 in the repair of DNA double-strand breaks by homologous recombination (HR), where it promotes DNA end resection. Mechanistically, BMI-1 mediates DNA end resection by facilitating the recruitment of CtIP, thus allowing RPA and RAD51 accumulation at DNA damage sites. Interestingly, treatment with transcription inhibitors rescues the DNA end resection defects of BMI-1-depleted cells, suggesting BMI-1-dependent transcriptional silencing mediates DNA end resection. Moreover, we find that H2A ubiquitylation at K119 (H2AK119ub) promotes end resection. Taken together, our results identify BMI-1-mediated transcriptional silencing and promotion of H2AK119ub deposition as essential regulators of DNA end resection and thus the progression of HR.

Protocol to measure end resection intermediates at sequence-specific DNA double-strand breaks by quantitative polymerase chain reaction using ER-AsiSI U2OS cells.

DNA end resection is a critical step in the homologous recombination pathway of repairing DNA double-strand breaks (DSBs) that can be visualized in cells by detecting the generation of single-stranded DNA (ssDNA) intermediates formed during the resection of the DSBs. Here, we describe quantitative polymerase-chain-reaction-based procedures to quantitatively measure ssDNA intermediates formed during the DNA end resection. Using the ER-AsiSI system, we use differential digestion patterns by restriction endonucleases that digest unresected double-stranded DNA at DSB sites. For complete details on the use and execution of this protocol, please refer to Fitieh et al. (2022).1.

The relationship between histone posttranslational modification and DNA damage signaling and repair.

PURPOSE: The cellular response to DNA damage occurs in the context of an organized chromatin environment in order to maintain genome integrity. Immediately after DNA damage, an array of histone modifications are induced to relieve the physical constraints of the chromatin environment, mark the site as damaged, and function as a platform for the assembly of mediator and effector proteins of DNA damage response signaling pathway. Changes in chromatin structure in the vicinity of the DNA double-strand break (DSB) facilitates the efficient initiation of the DNA damage signaling cascade. Failure of induction of DNA damage responsive histone modifications may lead to genome instability and cancer. Here we will discuss our current understanding of the DNA damage responsive histone modifications and their role in DNA repair as well as their implications for genome stability. We further discuss recent studies which highlight not only how histone modification has involved in the signaling and remodeling at the DSB but also how it influences the DNA repair pathway choice. CONCLUSIONS: Histone modifications pattern alter during the induction of DNA DSBs induction as well as during the repair and recovery phase of DNA damage response. It will be interesting to understand more precisely, how DSBs within chromatin are repaired by HR and NHEJ. The emergence of proteomic and genomic technologies in combination with advanced microscopy and imaging methods will help in better understanding the role of chromatin environment in the regulation of genome stability.

Emerging roles of eraser enzymes in the dynamic control of protein ADP-ribosylation.

Protein ADP-ribosylation is essential for the regulation of several cellular pathways, enabling dynamic responses to diverse pathophysiological conditions. It is modulated through a dynamic interplay between ADP-ribose readers, writers and erasers. While ADP-ribose synthesis has been studied and reviewed extensively, ADP-ribose processing by erasing enzymes has received comparably less attention. However, major progress in the mass spectrometric identification of ADP-ribosylated residues and the biochemical characterization of ADP-ribose erasers has substantially expanded our knowledge of ADP-ribosylation dynamics. Herein, we describe recent insights into the biology of ADP-ribose erasers and discuss the intricately orchestrated cellular processes to switch off ADP-ribose-dependent mechanisms.

Poly(ADP-ribose) polymerase-1 antagonizes DNA resection at double-strand breaks.

PARP-1 is rapidly recruited and activated by DNA double-strand breaks (DSBs). Upon activation, PARP-1 synthesizes a structurally complex polymer composed of ADP-ribose units that facilitates local chromatin relaxation and the recruitment of DNA repair factors. Here, we identify a function for PARP-1 in DNA DSB resection. Remarkably, inhibition of PARP-1 leads to hyperresected DNA DSBs. We show that loss of PARP-1 and hyperresection are associated with loss of Ku, 53BP1 and RIF1 resection inhibitors from the break site. DNA curtains analysis show that EXO1-mediated resection is blocked by PARP-1. Furthermore, PARP-1 abrogation leads to increased DNA resection tracks and an increase of homologous recombination in cellulo. Our results, therefore, place PARP-1 activation as a critical early event for DNA DSB repair activation and regulation of resection. Hence, our work has direct implications for the clinical use and effectiveness of PARP inhibition, which is prescribed for the treatment of various malignancies.

Techniques to access histone modifications and variants in cancer.

Recent years have witnessed an explosion of epigenetic research on the role of histone variants and modifications in cancer. To understand the global dynamics of chromatin structure and function, analysis of histone variants incorporated into the nucleosome and their covalent modifications, is required. The nucleosome is the fundamental structural unit of chromatin, contains an octamer of core histones H3, H4, H2A, and H2B. The differential alterations in diverse histone variants and their accompanying modifications patterns will provide a deeper insight into their biological role in structural and functional properties of chromatin. Here we provide a step-by-step protocol to investigate these aspects, the histone modifications and variants, their localization and dynamics within specific regions of chromatin under distinct condition and the recruitment/retention of epigenetic regulators at their target sites in chromatin to influence cell growth and differentiation.

MKP1 phosphatase mediates G1-specific dephosphorylation of H3Serine10P in response to DNA damage.

Histone mark, H3S10 phosphorylation plays a dual role in a cell by maintaining relaxed chromatin for active transcription in interphase and condensed chromatin state in mitosis. The level of H3S10P has also been shown to alter on DNA damage; however, its cell cycle specific behavior and regulation during DNA damage response is largely unexplored. In the present study, we demonstrate G1 cell cycle phase specific reversible loss of H3S10P in response to IR-induced DNA damage is mediated by opposing activities of phosphatase, MKP1 and kinase, MSK1 of the MAP kinase pathway. We also show that the MKP1 recruits to the chromatin in response to DNA damage and correlates with the decrease of H3S10P, whereas MKP1 is released from chromatin during recovery phase of DDR. Furthermore, blocking of H3S10 dephosphorylation by MKP1 inhibition impairs DNA repair process and results in poor survival of WRL68 cells. Collectively, our data proposes a pathway regulating G1 cell cycle phase specific reversible reduction of H3S10P on IR induced DNA damage and also raises the possibility of combinatorial modulation of H3S10P with specific inhibitors to target the cancer cells in G1-phase of cell cycle.

Dynamic alteration in H3 serine 10 phosphorylation is G1-phase specific during ionization radiation induced DNA damage response in human cells.

Chromatin acts as a natural barrier in DNA-damage recognition and repair. Histones undergo differential post-translational modification(s) to facilitate DNA damage response (DDR). Importance of modifications like phosphorylation of histone variant H2A.X in DNA repair is very well understood, however, ambiguous results exist in literature regarding the levels of certain histone modifications and their possible role in repair. In the present study, we have investigated in depth the alteration in the level of the highly dynamic histone mark H3S10P as it plays a dual role in different phases of the cell cycle. We show here that H3S10P decreases specifically from irradiated G1-enriched cells irrespective of the damaging agent or the cell line used in the study. Interestingly, the loss occurs predominantly from H3.3 variant which is a transcription activation mark like H3S10P itself, suggesting that the alteration might be implicated in transcription repression. The decrease in other transcription marks like H3K9Ac, H3K14Ac, H3K56Ac and H3S28P along with the occurrence of chromatin condensation in response to DNA damage in G1 phase strengthens the hypothesis. In addition, the alteration in the level of H3S10P shows an inverse correlation with that of γH2AX in a dose-dependent manner and probably occurs from the same mononucleosome. We propose that the drop in the levels of histone H3S10 phosphorylation is a universal phenomenon in response to DNA damage and is a trigger to induce transcription repressive state to facilitate repair.

Cell-type specificity of ß-actin expression and its clinicopathological correlation in gastric adenocarcinoma.

AIM: To investigate cell type specific distribution of ß-actin expression in gastric adenocarcinoma and its correlation with clinicopathological parameters. METHODS: ß-actin is a housekeeping gene, frequently used as loading control, but, differentially expresses in cancer. In gastric cancer, an overall increased expression of ß-actin has been reported using tissue disruptive techniques. At present, no histological data is available to indicate its cell type-specific expression and distribution pattern. In the present study, we analyzed ß-actin expression and distribution in paired normal and tumor tissue samples of gastric adenocarcinoma patients using immunohistochemistry (IHC), a tissue non-disruptive technique as well as tissue disruptive techniques like reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting. Correlation of ß-actin level with clinicopathological parameters was done using univariate analysis. RESULTS: The results of this study showed significant overexpression, at both mRNA and protein level in tumor tissues as confirmed by RT-PCR (1.47 ± 0.13 vs 2.36 ± 0.16; P < 0.001) and western blotting (1.92 ± 0.26 vs 2.88 ± 0.32; P < 0.01). IHC revealed that ß-actin expression is majorly distributed between epithelial and inflammatory cells of the tissues. Inflammatory cells showed a significantly higher expression compared to epithelial cells in normal (2.46 ± 0.13 vs 5.92 ± 0.23, P < 0.001), as well as, in tumor tissues (2.79 ± 0.24 vs 6.71 ± 0.14, P < 0.001). Further, comparison of immunostaining between normal and tumor tissues revealed that both epithelial and inflammatory cells overexpress ß-actin in tumor tissues, however, significant difference was observed only in inflammatory cells (5.92 ± 0.23 vs 6.71 ± 0.14, P < 0.01). Moreover, combined expression in epithelial and inflammatory cells also showed significant increase (4.19 ± 0.15 vs 4.75 ± 0.14, P < 0.05) in tumor tissues. In addition, univariate analysis showed a positive correlation of ß-actin level of inflammatory cells with tumor grade (P < 0.05) while epithelial cells exhibited negative correlation (P > 0.05). CONCLUSION: In gastric cancer, ß-actin showed an overall higher expression predominantly contributed by inflammatory or tumor infiltrating immune cells of the tissue microenvironment and correlates with tumor grade.

Preparation of novel carbon microfiber/carbon nanofiber-dispersed polyvinyl alcohol-based nanocomposite material for lithium-ion electrolyte battery separator.

A novel nanocomposite polyvinyl alcohol precursor-based material dispersed with the web of carbon microfibers and carbon nanofibers is developed as lithium (Li)-ion electrolyte battery separator. The primary synthesis steps of the separator material consist of esterification of polyvinyl acetate to produce polyvinyl alcohol gel, ball-milling of the surfactant dispersed carbon micro-nanofibers, mixing of the milled micron size (~500 nm) fibers to the reactant mixture at the incipience of the polyvinyl alcohol gel formation, and the mixing of hydrophobic reagents along with polyethylene glycol as a plasticizer, to produce a thin film of ~25 µm. The produced film, uniformly dispersed with carbon micro-nanofibers, has dramatically improved performance as a battery separator, with the ion conductivity of the electrolytes (LiPF6) saturated film measured as 0.119 S-cm(-1), approximately two orders of magnitude higher than that of polyvinyl alcohol. The other primary characteristics of the produced film, such as tensile strength, contact angle, and thermal stability, are also found to be superior to the materials made of other precursors, including polypropylene and polyethylene, discussed in the literature. The method of producing the films in this study is novel, simple, environmentally benign, and economically viable.

Molecular Modeling of Differentially Phosphorylated Serine 10 and Acetylated lysine 9/14 of Histone H3 Regulates their Interactions with 14-3-3ζ, MSK1, and MKP1.

Histone modifications occur in precise patterns, with several modifications known to affect the binding of proteins. These interactions affect the chromatin structure, gene regulation, and cell cycle events. The dual modifications on the H3 tail, serine10 phosphorylation, and lysine14 acetylation (H3Ser10PLys14Ac) are reported to be crucial for interaction with 14-3-3ζ. However, the mechanism by which H3Ser10P along with neighboring site-specific acetylation(s) is targeted by its regulatory proteins, including kinase and phosphatase, is not fully understood. We carried out molecular modeling studies to understand the interaction of 14-3-3ζ, and its regulatory proteins, mitogen-activated protein kinase phosphatase-1 (MKP1), and mitogen- and stress-activated protein kinase-1 (MSK1) with phosphorylated H3Ser10 alone or in combination with acetylated H3Lys9 and Lys14. In silico molecular association studies suggested that acetylated Lys14 and phosphorylated Ser10 of H3 shows the highest binding affinity towards 14-3-3ζ. In addition, acetylation of H3Lys9 along with Ser10PLys14Ac favors the interaction of the phosphatase, MKP1, for dephosphorylation of H3Ser10P. Further, MAP kinase, MSK1 phosphorylates the unmodified H3Ser10 containing N-terminal tail with maximum affinity compared to the N-terminal tail with H3Lys9AcLys14Ac. The data clearly suggest that opposing enzymatic activity of MSK1 and MKP1 corroborates with non-acetylated and acetylated, H3Lys9Lys14, respectively. Our in silico data highlights that site-specific phosphorylation (H3Ser10P) and acetylation (H3Lys9 and H3Lys14) of H3 are essential for the interaction with their regulatory proteins (MKP1, MSK1, and 14-3-3ζ) and plays a major role in the regulation of chromatin structure.