Mono-methylated histones control PARP-1 in chromatin and transcription.

PARP-1 is central to transcriptional regulation under both normal and stress conditions, with the governing mechanisms yet to be fully understood. Our biochemical and ChIP-seq-based analyses showed that PARP-1 binds specifically to active histone marks, particularly H4K20me1. We found that H4K20me1 plays a critical role in facilitating PARP-1 binding and the regulation of PARP-1-dependent loci during both development and heat shock stress. Here, we report that the sole H4K20 mono-methylase, pr-set7, and parp-1 Drosophila mutants undergo developmental arrest. RNA-seq analysis showed an absolute correlation between PR-SET7- and PARP-1-dependent loci expression, confirming co-regulation during developmental phases. PARP-1 and PR-SET7 are both essential for activating hsp70 and other heat shock genes during heat stress, with a notable increase of H4K20me1 at their gene body. Mutating pr-set7 disrupts monomethylation of H4K20 along heat shock loci and abolish PARP-1 binding there. These data strongly suggest that H4 monomethylation is a key triggering point in PARP-1 dependent processes in chromatin.

Histone Demethylase Modulation: Epigenetic Strategy to Combat Cancer Progression.

Epigenetic modifications are heritable, reversible changes in histones or the DNA that control gene functions, being exogenous to the genomic sequence itself. Human diseases, particularly cancer, are frequently connected to epigenetic dysregulations. One of them is histone methylation, which is a dynamically reversible and synchronously regulated process that orchestrates the three-dimensional epigenome, nuclear processes of transcription, DNA repair, cell cycle, and epigenetic functions, by adding or removing methylation groups to histones. Over the past few years, reversible histone methylation has become recognized as a crucial regulatory mechanism for the epigenome. With the development of numerous medications that target epigenetic regulators, epigenome-targeted therapy has been used in the treatment of malignancies and has shown meaningful therapeutic potential in preclinical and clinical trials. The present review focuses on the recent advances in our knowledge on the role of histone demethylases in tumor development and modulation, in emphasizing molecular mechanisms that control cancer cell progression. Finally, we emphasize current developments in the advent of new molecular inhibitors that target histone demethylases to regulate cancer progression.

Synergetic effect of high dose rate radiations (10× FFF/2400 MU/min/10 MV x-rays) and paclitaxel selectively eliminates melanoma cells.

BACKGROUND: Melanoma is one of the most aggressive cancers, with 1.6% of total cancer deaths in the United States. In recent years treatment options for metastatic melanoma have been improved by the FDA approval of new therapeutic agents. However, these inhibitors-based therapies are non-specific and have severe toxicities, including hyperkeratosis, photosensitivity, hepatitis, arthralgia, and fatigue. AIMS: The aim of this study is to determine the synthetic lethal effect (paclitaxel and radiations) on melanoma cells and reduce the total radiation doses by increasing the dose rates up to 2400 MU/min. METHODS AND RESULTS: We previously reported a radiation treatment (10 MV x-rays, 10X-FFF, dose rate 2400MU/min, low total dose 0.5 Gy) that kills melanoma cells with 80% survival of normal HEM in vitro. In this study, we extended the radiation cycle up to four and included paclitaxel treatment to study the synthetic lethal effect on melanoma and two other normal primary cells, HDF and HEK. Cells were treated with paclitaxel prior to the radiation at a dose rate of 400 and 2400 MU/min with a total radiation dose of only 0.5 Gy. Mitochondrial respiration assay, DNA damage assay, and colony formation assays were performed to study apoptosis and cell death induction. Four days of consequent radiation treatment with paclitaxel significantly reduces the survival of melanoma cells by inducing apoptosis and mitochondrial damage. After treatment, excessive DNA damage in melanoma cells leads to an increase in the expression of pro-apoptotic genes (Caspase-3) and a decrease in the expression of DNA repair gene (PARP1) and anti-apoptotic gene (Bcl-2) to activate the apoptosis pathway. The combination of paclitaxel and radiation reduces the survival of melanoma cells colonies compared to radiation alone. CONCLUSION: Our study indicates that radiations with paclitaxel have a potential synthetic lethal effect on melanoma cells and can be developed as a melanoma therapy without toxicities or harmful effects on normal primary skin cells.

Epigenetic malleability at core promoter initiates tobacco PR-1a expression post salicylic acid treatment.

BACKGROUND: Tobacco's PR-1a gene is induced by pathogen attack or exogenous application of salicylic acid (SA). Nucleosome mapping and chromatin immunoprecipitation assay were used to delineate the histone modifications on the PR-1a promoter. However, the epigenetic modifications of the inducible promoter of the PR-1a gene are not fully understood yet. METHODS AND RESULTS: Southern approach was used to scan the promoter of PR-1a to identify presence of nucleosomes, ChIP assays were performed using anti-histones antibodies of repressive chromatin by di- methylated at H3K9 and H4K20 or active chromatin by acetylated H3K9/14 and H4K16 to find epigenetic malleability of nucleosome over core promoter in uninduced or induced state post SA treatment. Class I and II mammalian histone deacetylase (HDAC) inhibitor TSA treatment was used to enhance the expression of PR-1a by facilitating the histone acetylation post SA treatment. Here, we report correlated consequences of the epigenetic modifications correspond to disassembly of the nucleosome (spans from - 102 to + 55 bp, masks TATA and transcription initiation) and repressor complex from core promoter, eventually initiates the transcription of PR-1a gene post SA treatment. While active chromatin marks di and trimethylation of H3K4, acetylation of H3K9 and H4K16 are increased which are associated to the transcription initiation of PR-1a following SA treatment. However, in uninduced state constitutive expression of a negative regulator (SNI1) of AtPR1, suppresses AtPR1 expression by six-fold in Arabidopsis thaliana. Further, we report 50-to-1000-fold increased expression of AtPR1 in uninduced lsd1 mutant plants, up to threefold increased expression of AtPR1 in uninduced histone acetyl transferases (HATs) mutant plants, SNI1 dependent negative regulation of AtPR1, all together our results suggest that inactive state of PR-1a is indeed maintained by a repressive complex. CONCLUSION: The study aimed to reveal the mechanism of transcription initiation of tobacco PR-1a gene in presence or absence of SA. This is the first study that reports nucleosome and repressor complex over core promoter region maintains the inactivation of gene in uninduced state, and upon induction disassembling of both initiates the downstream gene activation process.

DNA Methylation Malleability and Dysregulation in Cancer Progression: Understanding the Role of PARP1.

Mammalian genomic DNA methylation represents a key epigenetic modification and its dynamic regulation that fine-tunes the gene expression of multiple pathways during development. It maintains the gene expression of one generation of cells; particularly, the mitotic inheritance of gene-expression patterns makes it the key governing mechanism of epigenetic change to the next generation of cells. Convincing evidence from recent discoveries suggests that the dynamic regulation of DNA methylation is accomplished by the enzymatic action of TET dioxygenase, which oxidizes the methyl group of cytosine and activates transcription. As a result of aberrant DNA modifications, genes are improperly activated or inhibited in the inappropriate cellular context, contributing to a plethora of inheritable diseases, including cancer. We outline recent advancements in understanding how DNA modifications contribute to tumor suppressor gene silencing or oncogenic-gene stimulation, as well as dysregulation of DNA methylation in cancer progression. In addition, we emphasize the function of PARP1 enzymatic activity or inhibition in the maintenance of DNA methylation dysregulation. In the context of cancer remediation, the impact of DNA methylation and PARP1 pharmacological inhibitors, and their relevance as a combination therapy are highlighted.

Age-Related Changes of Gene Expression Profiles in Drosophila.

An individual's gene expression profile changes throughout their life. This change in gene expression is shaped by differences in physiological needs and functions between the younger and older organism. Despite intensive studies, the aging process is not fully understood, and several genes involved in this process may remain to be identified. Here we report a transcriptomic analysis of Drosophila melanogaster using microarrays. We compared the expression profiles of two-day-old female adult flies with those of 45-day-old flies. We identified 1184 genes with pronounced differences in expression level between young and old age groups. Most genes involved in muscle development/maintenance that display different levels of expression with age were downregulated in older flies. Many of these genes contributed to sarcomere formation and function. Several of these genes were functionally related to direct and indirect flight muscles; some of them were exclusively expressed in these muscles. Conversely, several genes involved in apoptosis processes were upregulated in aging flies. In addition, several genes involved in resistance to toxic chemicals were upregulated in aging flies, which is consistent with a global upregulation of the defense response system in aging flies. Finally, we randomly selected 12 genes among 232 genes with unknown function and generated transgenic flies expressing recombinant proteins fused with GFP protein to determine their subcellular expression. We also found that the knockdown of some of those 12 genes can affect the lifespan of flies.

SARS-CoV-2: Understanding the Transcriptional Regulation of ACE2 and TMPRSS2 and the Role of Single Nucleotide Polymorphism (SNP) at Codon 72 of p53 in the Innate Immune Response against Virus Infection.

Human ACE2 and the serine protease TMPRSS2 of novel SARS-CoV-2 are primary entry receptors in host cells. Expression of these genes at the transcriptional level has not been much discussed in detail. The ISRE elements of the ACE2 promoter are a binding site for the ISGF3 complex of the JAK/STAT signaling pathway. TMPRSS2, including IFNß, STAT1, and STAT2, has the PARP1 binding site near to TSS either up or downstream promoter region. It is well documented that PARP1 regulates gene expression at the transcription level. Therefore, to curb virus infection, both promoting type I IFN signaling to boost innate immunity and prevention of virus entry by inhibiting PARP1, ACE2 or TMPRSS2 are safe options. Most importantly, our aim is to attract the attention of the global scientific community towards the codon 72 Single Nucleotide Polymorphism (SNP) of p53 and its underneath role in the innate immune response against SARS-CoV-2. Here, we discuss codon 72 SNP of human p53's role in the different innate immune response to restrict virus-mediated mortality rate only in specific parts of the world. In addition, we discuss potential targets and emerging therapies using bioengineered bacteriophage, anti-sense, or CRISPR strategies.

The circuitry of the tumor microenvironment in adult and pediatric Hodgkin lymphoma: cellular composition, cytokine profile, EBV, and exosomes.

BACKGROUND: Classical Hodgkin lymphoma (cHL) is a unique lymphoid malignancy with a tumor microenvironment (TME) consisting of a small number of neoplastic-Hodgkin and Reed-Sternberg (H-RS) cells (<1%), surrounded by a large number of nonneoplastic infiltrating immune cells (>90%). The TME of cHL critically depends on immune cells to support tumor growth as H-RS cells cannot survive and proliferate in isolation. RECENT FINDINGS: Programmed cell death protein 1 (PD-1) ligand expressed on H-RS cells inhibits the clearance of tumor by causing T-cell exhaustion. Nivolumab and pembrolizumab, PD-1 inhibitors, have been proven to be effective in treating adult and pediatric patients with R/R cHL. Tumor-associated macrophages (TAMs) are a central component of TME and are known to cause poor prognosis in adult HL. However, the prognostic impact of CD68+ TAMs in pediatric HL remains ambiguous. EBV modulates the tumor milieu of HL and plays a strategic role in immune escape by enrichment of the TME with Treg cells and associated immunosuppressive cytokines in adult HL. In contrast, EBV+ pediatric patients have increased infiltration of CD8+ T-cells and show a better therapeutic response suggesting viral-related TME is distinct in childhood HL. The role of CASP3 in apoptosis of H-RS cells and its correlation with response prediction in adult and pediatric HL suggest it may serve as a potential biomarker. In cHL, CD30, EBV, and NF-κB signaling employ exosomes for cell-cell communication that triggers the migration capacity of fibroblasts, stimulate to produce proinflammatory cytokines, and help to create a tumor-supportive microenvironment. CONCLUSION: The cHL microenvironment is distinct in adult and pediatric HL. Future studies are required to understand the role of interplay between H-RS cells and EBV-associated microenvironment and their clinical outcome. They may present novel therapeutic targets for the development of antilymphoma therapy.

Poly(ADP-ribose) polymerase 1 in genome-wide expression control in Drosophila.

Poly(ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme involved in DNA repair and transcription regulation, among other processes. Malignant transformations, tumor progression, the onset of some neuropathies and other disorders have been linked to misregulation of PARP-1 activity. Despite intensive studies during the last few decades, the role of PARP-1 in transcription regulation is still not well understood. In this study, a transcriptomic analysis in Drosophila melanogaster third instar larvae was carried out. A total of 602 genes were identified, showing large-scale changes in their expression levels in the absence of PARP-1 in vivo. Among these genes, several functional gene groups were present, including transcription factors and cytochrome family members. The transcription levels of genes from the same functional group were affected by the absence of PARP-1 in a similar manner. In the absence of PARP-1, all misregulated genes coding for transcription factors were downregulated, whereas all genes coding for members of the cytochrome P450 family were upregulated. The cytochrome P450 proteins contain heme as a cofactor and are involved in oxidoreduction. Significant changes were also observed in the expression of several mobile elements in the absence of PARP-1, suggesting that PARP-1 may be involved in regulating the expression of mobile elements.

Biomarkers and novel therapeutic approaches for diffuse large B-cell lymphoma in the era of precision medicine.

Despite the great efforts for better treatment options for diffuse large B-cell lymphoma (DLBCL) (most common form of non-Hodgkin lymphoma, NHL) to treat and prevent relapse, it continues to be a challenge. Here, we present an overview of DLBCL and address the diagnostic assays and molecular techniques used in its diagnosis, role of biomarkers in detection, treatment of early and advanced stage DLBCL, and novel drug regimens. We discuss the significant biomarkers that have emerged as essential tools for stratifying patients according to risk factors and for providing insights into the use of more targeted and individualized therapeutics. We discuss techniques such as gene expression studies, including next-generation sequencing, which have enabled a more understanding of the complex pathogenesis of DLBCL and have helped determine molecular targets for novel therapeutic agents. We examine current treatment approaches, outline the findings of completed clinical trials, and provide updates for ongoing clinical trials. We highlight clinical trials relevant to the significant fraction of DLBCL patients who present with complex cases marked by high relapse rates. Supported by an increased understanding of targetable pathways in DLBCL, clinical trials involving specialized combination therapies are bringing us within reach the promise of an effective cure to DLBCL using precision medicine. Optimization of therapy remains a crucial objective, with the end goal being a balance between high survival rates through targeted and personalized treatment while reducing adverse effects in DLBCL patients of all subsets.

Therapeutic Targeting of Vasculature in the Premetastatic and Metastatic Niches Reduces Lung Metastasis.

In the metastasis-targeted organs, angiogenesis is essential for the progression of dormant micrometastases to rapidly growing and clinically overt lesions. However, we observed changes suggesting angiogenic switching in the mouse lungs prior to arrival of tumor cells (i.e., in the premetastatic niche) in the models of breast carcinoma. This angiogenic switching appears to be caused by myeloid-derived suppressor cells recruited to the premetastatic lungs through complement C5a receptor 1 signaling. These myeloid cells are known to secrete several proangiogenic factors in tumors, including IL-1ß and matrix metalloproteinase-9, and we found upregulation of these genes in the premetastatic lungs. Blockade of C5a receptor 1 synergized with antiangiogenic Listeria monocytogenes-based vaccines to decrease the lung metastatic burden by reducing vascular density and improving antitumor immunity in the lungs. This was mediated even when growth of primary breast tumors was not affected by these treatments. This work provides initial evidence that angiogenesis contributes to the premetastatic niche in rapidly progressing cancers and that inhibiting this process through immunotherapy is beneficial for reducing or even preventing metastasis.

The Role of Poly(ADP-Ribose) Polymerase-1 in Cutaneous Wound Healing.

Critical Issue: Chronic nonhealing wounds of the lower extremities resulting in major amputations are a major health problem worldwide. Significance: Diabetes and ischemia are two major etiologies of nonhealing wounds of the lower extremities. Hyperglycemia from diabetes and oxidative stress from ischemia activate polyadenosine diphosphate (ADP)-ribose polymerase-1 (PARP-1), which is a nuclear enzyme that is best known for its role in DNA repair. However, the exact function of PARP-1 in ischemic/diabetic wound healing has not been well studied. Recent Advances: Poly-ADP-ribose (PAR) polymer has been detected in the wound bed and many of the PARylation-related reactions (oxidative stress response, expression of inflammatory cytokines and chemokines, cell proliferation, and migration) are important in the wound healing process. However, the role of PARP-1 in wound healing and the potential of targeting PARP-1 therapeutically in wounds are only recently being elucidated, with much still unknown. This review summarizes the recent advances in this field, highlighting some of the mechanisms through which PARP-1 may affect normal wound closure. Future Directions: The review also presents a perspective on some of the downstream targets of PARP-1 that may be explored for their role in wound healing and discusses about the therapeutic potential of PARP inhibitors for wound healing.

Hit and run versus long-term activation of PARP-1 by its different domains fine-tunes nuclear processes.

Poly(ADP-ribose) polymerase 1 (PARP-1) is a multidomain multifunctional nuclear enzyme involved in the regulation of the chromatin structure and transcription. PARP-1 consists of three functional domains: the N-terminal DNA-binding domain (DBD) containing three zinc fingers, the automodification domain (A), and the C-terminal domain, which includes the protein interacting WGR domain (W) and the catalytic (Cat) subdomain responsible for the poly(ADP ribosyl)ating reaction. The mechanisms coordinating the functions of these domains and determining the positioning of PARP-1 in chromatin remain unknown. Using multiple deletional isoforms of PARP-1, lacking one or another of its three domains, as well as consisting of only one of those domains, we demonstrate that different functions of PARP-1 are coordinated by interactions among these domains and their targets. Interaction between the DBD and damaged DNA leads to a short-term binding and activation of PARP-1. This "hit and run" activation of PARP-1 initiates the DNA repair pathway at a specific point. The long-term chromatin loosening required to sustain transcription takes place when the C-terminal domain of PARP-1 binds to chromatin by interacting with histone H4 in the nucleosome. This long-term activation of PARP-1 results in a continuous accumulation of pADPr, which maintains chromatin in the loosened state around a certain locus so that the transcription machinery has continuous access to DNA. Cooperation between the DBD and C-terminal domain occurs in response to heat shock (HS), allowing PARP-1 to scan chromatin for specific binding sites.

Non-NAD-Like poly(ADP-Ribose) Polymerase-1 Inhibitors effectively Eliminate Cancer in vivo.

The clinical potential of PARP-1 inhibitors has been recognized >10years ago, prompting intensive research on their pharmacological application in several branches of medicine, particularly in oncology. However, natural or acquired resistance of tumors to known PARP-1 inhibitors poses a serious problem for their clinical implementation. Present study aims to reignite clinical interest to PARP-1 inhibitors by introducing a new method of identifying highly potent inhibitors and presenting the largest known collection of structurally diverse inhibitors. The majority of PARP-1 inhibitors known to date have been developed as NAD competitors. NAD is utilized by many enzymes other than PARP-1, resulting in a trade-off trap between their specificity and efficacy. To circumvent this problem, we have developed a new strategy to blindly screen a small molecule library for PARP-1 inhibitors by targeting a highly specific rout of its activation. Based on this screen, we present a collection of PARP-1 inhibitors and provide their structural classification. In addition to compounds that show structural similarity to NAD or known PARP-1 inhibitors, the screen identified structurally new non-NAD-like inhibitors that block PARP-1 activity in cancer cells with greater efficacy and potency than classical PARP-1 inhibitors currently used in clinic. These non-NAD-like PARP-1 inhibitors are effective against several types of human cancer xenografts, including kidney, prostate, and breast tumors in vivo. Our pre-clinical testing of these inhibitors using laboratory animals has established a strong foundation for advancing the new inhibitors to clinical trials.

Charon Mediates Immune Deficiency-Driven PARP-1-Dependent Immune Responses in Drosophila.

Regulation of NF-κB nuclear translocation and stability is central to mounting an effective innate immune response. In this article, we describe a novel molecular mechanism controlling NF-κB-dependent innate immune response. We show that a previously unknown protein, termed as Charon, functions as a regulator of antibacterial and antifungal immune defense in Drosophila Charon is an ankyrin repeat-containing protein that mediates poly(ADP-ribose) polymerase-1 (PARP-1)-dependent transcriptional responses downstream of the innate immune pathway. Our results demonstrate that Charon interacts with the NF-κB ortholog Relish inside perinuclear particles and delivers active Relish to PARP-1-bearing promoters, thus triggering NF-κB/PARP-1-dependent transcription of antimicrobial peptides. Ablating the expression of Charon prevents Relish from targeting promoters of antimicrobial genes and effectively suppresses the innate immune transcriptional response. Taken together, these results implicate Charon as an essential mediator of PARP-1-dependent transcription in the innate immune pathway. Thus, to our knowledge, our results are the first to describe the molecular mechanism regulating translocation of the NF-κB subunit from cytoplasm to chromatin.

Mitotic bookmarking: maintaining post-mitotic reprogramming of transcription reactivation.

Restoring chromatin structure with high fidelity after mitosis is critical for cell survival. Transcriptional reactivation of genes is the first step toward establishing identity of the daughter cell. During mitosis, chromatin bookmarking factors associated with specific chromatin regions ensure the restoration of the original gene expression pattern in daughter cells. Recent findings have provided new insights into the mechanisms, regulation, and biological significance of gene bookmarking in eukaryotes. In this review, we discuss how epigenetic factors, such as Poly(ADP-ribose) Polymerase-1, establish epigenetic memory in mitotic chromatin.

Bookmarking promoters in mitotic chromatin: poly(ADP-ribose)polymerase-1 as an epigenetic mark.

Epigenetics are the heritable changes in gene expression or cellular phenotype caused by mechanisms other than changes in the underlying DNA sequence. After mitosis, it is thought that bookmarking transcription factors remain at promoters, regulating which genes become active and which remain silent. Herein, we demonstrate that poly(ADP-ribose)polymerase-1 (PARP-1) is a genome-wide epigenetic memory mark in mitotic chromatin, and we further show that the presence of PARP-1 is absolutely crucial for reactivation of transcription after mitosis. Based on these findings, a novel molecular model of epigenetic memory transmission through the cell cycle is proposed.

Analysis of histones and histone variants in plants.

Histone proteins are the major protein components of chromatin - the physiologically relevant form of the genome (or epigenome) in all eukaryotic cells. For many years, histones were considered passive structural components of eukaryotic chromatin. In recent years, it has been demonstrated that dynamic association of histones and their variants to the genome plays a very important role in gene regulation. Histones are extensively modified during posttranslation viz. acetylation, methylation, phosphorylation, ubiquitylation, etc., and the identification of these covalent marks on canonical and variant histones is crucial for the understanding of their biological significance. Different biochemical techniques have been developed to purify and separate histone proteins; here, we describe techniques for analysis of histones from plant tissues.

Analysis of chromatin structure in plant cells.

A vast body of evidence in the literature indicates that nucleosomes can act as barriers to transcriptional initiation. The nucleosome at the promoter inhibits association of transcription factors disallowing active transcription of the gene. We have found a nucleosome on tobacco pathogenesis-related gene-1a (PR-1a) core promoter and mapped its boundaries and extension to find its span. The nucleosome covers the TATA box and Inr region of the core promoter and gets disassembled upon induction. Prior to its removal, modifications (i.e., acetylation and methylation of histones) occur at the nucleosome, proving a role of epigenetic modifications in transcriptional regulation. We summarize here various methodologies to analyze promoter chromatin structure in plants using the PR-1a core promoter as an example.

PARP1 genomics: chromatin immunoprecipitation approach using anti-PARP1 antibody (ChIP and ChIP-seq).

Poly(ADP-ribose) polymerase1 (PARP1) is a global regulator of different cellular mechanisms, ranging from DNA damage repair to control of gene expression. Since PARP1 protein and pADPr have been shown to persist in chromatin through cell cycle, they may both act as epigenetic markers. However, it is not known how many loci are occupied by PARP1 protein during mitosis genome-wide. To reveal the genome-wide PARP1 binding sites, we used the ChIP-seq approach, an emerging technique to study genome-wide PARP1 protein interaction with chromatin. Here, we describe how to perform ChIP-seq in the context of PARP1 binding sites identification in chromatin, using human embryonic kidney cell lines.