Methylation status of Dnmt1 promoter depends on poly(ADP-ribosy)lation

Research is focused on CpG islands and on the mechanism that poly(ADP-ribosyl)ation uses to defend the unmethylated state of these important DNA sequences which are located in the promoter regions of the housekeeping genes having a role of transcription regulators. Data here reported show that inhibition of PARP activity allows the diffuse insertion of methyl groups onto some CpG islands and in particular on the CpG island which is located in the promoter region of Dnmt1 gene. Hence, following inhibition of PARPs activity, this promoter loses its protection against methylation becoming silenced through methylation as shown by analyses with Methylation Sensitive PCR (MS-PCR) and sequencing after bisulphite treatment. Analyses of Western Blotting, RT-PCR and Real-time RT-PCR confirm that the gene has undergone silencing. The role of ADP-ribose polymers in silencing Dnmt1 has been demonstrated by additional experiments in which overexpression of poly(ADP-ribose) glycohydrolase leads to reduction of ADP-ribose polymers in nuclei associated to a sharp decrease of Dnmt1 level respect to control. A parallel genome-wide methyl-sensitive restriction assay demonstrates that the variation of Dnmt1 level is followed by a bimodal alteration of DNA methylation pattern. In fact, the inhibition of poly(ADP-ribosyl)ation initially causes an increase in methyl-group insertion onto DNA while this phenomenon is reversed after prolonged treatments and demethylation is detected within Alu sequences. Considering the important role played by Dnmt1 in the epigenetic scenario, these data lead us to think about what happens in tumor cells where both anomalous methylation of some CpG islands and diffuse hypomethylation are present. These findings open up a new path into epigenetic research in tumors. What is remarkable is that the demethylated pattern found in Alu sequences after treatment of cells with 3-ABA for 96 hours is very similar to the one found on DNA from cells treated with 5-AZA for the same time. The discovery of a DNA demethylating activity dependent on the use of inhibitors of poly(ADP-ribosyl)ation process increases the knowledge of mechanism by which these inhibitors enhance the cytotoxicity of other anticancer agents

Methylation status of Dnmt1 promoter depends on poly(ADP-ribosy)lation

Research is focused on CpG islands and on the mechanism that poly(ADP-ribosyl)ation uses to defend the unmethylated state of these important DNA sequences which are located in the promoter regions of the housekeeping genes having a role of transcription regulators. Data here reported show that inhibition of PARP activity allows the diffuse insertion of methyl groups onto some CpG islands and in particular on the CpG island which is located in the promoter region of Dnmt1 gene. Hence, following inhibition of PARPs activity, this promoter loses its protection against methylation becoming silenced through methylation as shown by analyses with Methylation Sensitive PCR (MS-PCR) and sequencing after bisulphite treatment. Analyses of Western Blotting, RT-PCR and Real-time RT-PCR confirm that the gene has undergone silencing. The role of ADP-ribose polymers in silencing Dnmt1 has been demonstrated by additional experiments in which overexpression of poly(ADP-ribose) glycohydrolase leads to reduction of ADP-ribose polymers in nuclei associated to a sharp decrease of Dnmt1 level respect to control. A parallel genome-wide methyl-sensitive restriction assay demonstrates that the variation of Dnmt1 level is followed by a bimodal alteration of DNA methylation pattern. In fact, the inhibition of poly(ADP-ribosyl)ation initially causes an increase in methyl-group insertion onto DNA while this phenomenon is reversed after prolonged treatments and demethylation is detected within Alu sequences. Considering the important role played by Dnmt1 in the epigenetic scenario, these data lead us to think about what happens in tumor cells where both anomalous methylation of some CpG islands and diffuse hypomethylation are present. These findings open up a new path into epigenetic research in tumors. What is remarkable is that the demethylated pattern found in Alu sequences after treatment of cells with 3-ABA for 96 hours is very similar to the one found on DNA from cells treated with 5-AZA for the same time. The discovery of a DNA demethylating activity dependent on the use of inhibitors of poly(ADP-ribosyl)ation process increases the knowledge of mechanism by which these inhibitors enhance the cytotoxicity of other anticancer agents

Effects of niacin restriction on sirtuin and PARP responses to photodamage in human skin.

Sirtuins (SIRTs) and poly(ADP-ribose) polymerases (PARPs), NAD(+)-dependent enzymes, link cellular energy status with responses to environmental stresses. Skin is frequently exposed to the DNA damaging effects of UV irradiation, a known etiology in skin cancer. Thus, understanding the defense mechanisms in response to UV, including the role of SIRTs and PARPs, may be important in developing skin cancer prevention strategies. Here, we report expression of the seven SIRT family members in human skin. SIRTs gene expressions are progressively upregulated in A431 epidermoid carcinoma cells (SIRTs1 and 3), actinic keratoses (SIRTs 2, 3, 5, 6, and 7) and squamous cell carcinoma (SIRTs 1-7). Photodamage induces dynamic changes in SIRT expression with upregulation of both SIRT1 and SIRT4 mRNAs. Specific losses of SIRT proteins occur early after photodamage followed by accumulation later, especially for SIRT4. Niacin restriction, which decreases NAD(+), the sirtuin substrate, results in an increase in acetylated proteins, upregulation of SIRTs 2 and 4, increased inherent DNA damage, alterations in SIRT responses to photodamage, abrogation of PARP activation following photodamage, and increased sensitivity to photodamage that is completely reversed by repleting niacin. These data support the hypothesis that SIRTs and PARPs play important roles in resistance to photodamage and identify specific SIRTs that respond to photodamage and may be targets for skin cancer prevention

Structural Implications for Selective Targeting of PARPs.

Poly(ADP-ribose) polymerases (PARPs) are a family of enzymes that use NAD(+) as a substrate to synthesize polymers of ADP-ribose (PAR) as post-translational modifications of proteins. PARPs have important cellular roles that include preserving genomic integrity, telomere maintenance, transcriptional regulation, and cell fate determination. The diverse biological roles of PARPs have made them attractive therapeutic targets, which have fueled the pursuit of small molecule PARP inhibitors. The design of PARP inhibitors has matured over the past several years resulting in several lead candidates in clinical trials. PARP inhibitors are mainly used in clinical trials to treat cancer, particularly as sensitizing agents in combination with traditional chemotherapy to reduce side effects. An exciting aspect of PARP inhibitors is that they are also used to selectivity kill tumors with deficiencies in DNA repair proteins (e.g., BRCA1/2) through an approach termed synthetic lethality. In the midst of the tremendous efforts that have brought PARP inhibitors to the forefront of modern chemotherapy, most clinically used PARP inhibitors bind to conserved regions that permits cross-selectivity with other PARPs containing homologous catalytic domains. Thus, the differences between therapeutic effects and adverse effects stemming from pan-PARP inhibition compared to selective inhibition are not well understood. In this review, we discuss current literature that has found ways to gain selectivity for one PARP over another. We furthermore provide insights into targeting other domains that make up PARPs, and how new classes of drugs that target these domains could provide a high degree of selectivity by affecting specific cellular functions. A clear understanding of the inhibition profiles of PARP inhibitors will not only enhance our understanding of the biology of individual PARPs, but may provide improved therapeutic options for patients

A systematic analysis of the PARP protein family identifies new functions critical for cell physiology

The poly(ADP-ribose) polymerase (PARP) family of proteins use NAD[superscript +] as their substrate to modify acceptor proteins with ADP-ribose modifications. The function of most PARPs under physiological conditions is unknown. Here, to better understand this protein family, we systematically analyse the cell cycle localization of each PARP and of poly(ADP-ribose), a product of PARP activity, then identify the knockdown phenotype of each protein and perform secondary assays to elucidate function. We show that most PARPs are cytoplasmic, identify cell cycle differences in the ratio of nuclear to cytoplasmic poly(ADP-ribose) and identify four phenotypic classes of PARP function. These include the regulation of membrane structures, cell viability, cell division and the actin cytoskeleton. Further analysis of PARP14 shows that it is a component of focal adhesion complexes required for proper cell motility and focal adhesion function. In total, we show that PARP proteins are critical regulators of eukaryotic physiology.Rita Allen FoundationSidney Kimmel Foundation (Cancer Research Scholar)Howard S. and Linda B. Stern Career Development Assistant ProfessorNational Cancer Institute (U.S.) (Cancer Center Support (Core) Grant P30-CA14051)National Institutes of Health (U.S.) (Grant RO1GM087465)National Institutes of Health (U.S.) (Grant 1F32GM103089-01)Jeptha H. and Emily V. Wade FundKathy and Curt Marble Cancer Research Fun

Poly(ADP-ribose) polymerases regulate cell division and development in Arabidopsis roots

Root organogenesis involves cell division, differentiation and expansion. The molecular mechanisms regulating root development are not fully understood. In this study, we identified poly (ADP-ribose) polymerases (PARPs) as new players in root development. PARP catalyzes poly (ADP-ribosyl)ation of proteins by repeatedly adding ADP-ribose units onto proteins using nicotinamide adenine dinucleotide (NAD+) as the donor. We found that inhibition of PARP activities by 3-aminobenzomide (3-AB) increased the growth rates of both primary and lateral roots, leading to a more developed root system. The double mutant of Arabidopsis PARPs, parp1parp2, showed more rapid primary and lateral root growth. Cyclin genes regulating G1-to-S and G2-to-M transition were up-regulated upon treatment by 3-AB. The proportion of 2C cells increased while cells with higher DNA ploidy cells declined in the roots of treated plants, resulting in an enlarged rootmeristematic zone. The expression level of PARP2 was very low in the meristematic zone but high in the maturation zones, consistent with a role of PARP in inhibiting mitosis and promoting cell differentiation. Our results suggest that PARPs play an important rolein root development by negatively regulating root cell division

It takes two to tango: NAD+ and sirtuins in aging/longevity control

AbstractThe coupling of nicotinamide adenine dinucleotide (NAD+) breakdown and protein deacylation is a unique feature of the family of proteins called ‘sirtuins.’ This intimate connection between NAD+ and sirtuins has an ancient origin and provides a mechanistic foundation that translates the regulation of energy metabolism into aging and longevity control in diverse organisms. Although the field of sirtuin research went through intensive controversies, an increasing number of recent studies have put those controversies to rest and fully established the significance of sirtuins as an evolutionarily conserved aging/longevity regulator. The tight connection between NAD+ and sirtuins is regulated at several different levels, adding further complexity to their coordination in metabolic and aging/longevity control. Interestingly, it has been demonstrated that NAD+ availability decreases over age, reducing sirtuin activities and affecting the communication between the nucleus and mitochondria at a cellular level and also between the hypothalamus and adipose tissue at a systemic level. These dynamic cellular and systemic processes likely contribute to the development of age-associated functional decline and the pathogenesis of diseases of aging. To mitigate these age-associated problems, supplementation of key NAD+ intermediates is currently drawing significant attention. In this review article, we will summarize these important aspects of the intimate connection between NAD+ and sirtuins in aging/longevity control.</jats:p

Evolutionary history of the poly(ADP-ribose) polymerase gene family in eukaryotes

Abstract Background The Poly(ADP-ribose)polymerase (PARP) superfamily was originally identified as enzymes that catalyze the attachment of ADP-ribose subunits to target proteins using NAD+ as a substrate. The family is characterized by the catalytic site, termed the PARP signature. While these proteins can be found in a range of eukaryotes, they have been best studied in mammals. In these organisms, PARPs have key functions in DNA repair, genome integrity and epigenetic regulation. More recently it has been found that proteins within the PARP superfamily have altered catalytic sites, and have mono(ADP-ribose) transferase (mART) activity or are enzymatically inactive. These findings suggest that the PARP signature has a broader range of functions that initially predicted. In this study, we investigate the evolutionary history of PARP genes across the eukaryotes. Results We identified in silico 236 PARP proteins from 77 species across five of the six eukaryotic supergroups. We performed extensive phylogenetic analyses of the identified PARPs. They are found in all eukaryotic supergroups for which sequence is available, but some individual lineages within supergroups have independently lost these genes. The PARP superfamily can be subdivided into six clades. Two of these clades were likely found in the last common eukaryotic ancestor. In addition, we have identified PARPs in organisms in which they have not previously been described. Conclusions Three main conclusions can be drawn from our study. First, the broad distribution and pattern of representation of PARP genes indicates that the ancestor of all extant eukaryotes encoded proteins of this type. Second, the ancestral PARP proteins had different functions and activities. One of these proteins was similar to human PARP1 and likely functioned in DNA damage response. The second of the ancestral PARPs had already evolved differences in its catalytic domain that suggest that these proteins may not have possessed poly(ADP-ribosyl)ation activity. Third, the diversity of the PARP superfamily is larger than previously documented, suggesting as more eukaryotic genomes become available, this gene family will grow in both number and type.</p