The Intertwined Role of 8-oxodG and G4 in Transcription Regulation.

The guanine base in nucleic acids is, among the other bases, the most susceptible to being converted into 8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) when exposed to reactive oxygen species. In double-helix DNA, 8-oxodG can pair with adenine; hence, it may cause a G > T (C > A) mutation; it is frequently referred to as a form of DNA damage and promptly corrected by DNA repair mechanisms. Moreover, 8-oxodG has recently been redefined as an epigenetic factor that impacts transcriptional regulatory elements and other epigenetic modifications. It has been proposed that 8-oxodG exerts epigenetic control through interplay with the G-quadruplex (G4), a non-canonical DNA structure, in transcription regulatory regions. In this review, we focused on the epigenetic roles of 8-oxodG and the G4 and explored their interplay at the genomic level.

8-oxodG accumulation within super-enhancers marks fragile CTCF-mediated chromatin loops.

8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a major product of the DNA oxidization process, has been proposed to have an epigenetic function in gene regulation and has been associated with genome instability. NGS-based methodologies are contributing to the characterization of the 8-oxodG function in the genome. However, the 8-oxodG epigenetic role at a genomic level and the mechanisms controlling the genomic 8-oxodG accumulation/maintenance have not yet been fully characterized. In this study, we report the identification and characterization of a set of enhancer regions accumulating 8-oxodG in human epithelial cells. We found that these oxidized enhancers are mainly super-enhancers and are associated with bidirectional-transcribed enhancer RNAs and DNA Damage Response activation. Moreover, using ChIA-PET and HiC data, we identified specific CTCF-mediated chromatin loops in which the oxidized enhancer and promoter regions physically associate. Oxidized enhancers and their associated chromatin loops accumulate endogenous double-strand breaks which are in turn repaired by NHEJ pathway through a transcription-dependent mechanism. Our work suggests that 8-oxodG accumulation in enhancers-promoters pairs occurs in a transcription-dependent manner and provides novel mechanistic insights on the intrinsic fragility of chromatin loops containing oxidized enhancers-promoters interactions.

The genomic landscape of 8-oxodG reveals enrichment at specific inherently fragile promoters.

8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) is the most common marker of oxidative stress and its accumulation within the genome has been associated with major human health issues such as cancer, aging, cardiovascular and neurodegenerative diseases. The characterization of the different genomic sites where 8-oxodG accumulates and the mechanisms underlying its formation are still poorly understood. Using OxiDIP-seq, we recently derived the genome-wide distribution of 8-oxodG in human non-tumorigenic epithelial breast cells (MCF10A). Here, we identify a subset of human promoters that accumulate 8-oxodG under steady-state condition. 8-oxodG nucleotides co-localize with double strand breaks (DSBs) at bidirectional and CG skewed promoters and their density correlate with RNA Polymerase II co-occupancy and transcription. Furthermore, by performing OxiDIP-seq in quiescent (G0) cells, we found a strong reduction of oxidatively-generated damage in the majority of 8-oxodG-positive promoters in the absence of DNA replication. Overall, our results suggest that the accumulation of 8-oxodG at gene promoters occurs through DNA replication-dependent or -independent mechanisms, with a possible contribution to the formation of cancer-associated translocation events.

Inhibition of lysine-specific demethylase LSD1 induces senescence in Glioblastoma cells through a HIF-1α-dependent pathway.

Senescence is a stress-responsive cellular program that leads to cell cycle arrest. In cancer cells, senescence has profound implications for tumor aggressiveness and clinical outcome, but the molecular events that provoke cancer cells to undergo senescence remain unclear. Herein, we provide evidence that the histone demethylase LSD1/KDM1A supports the growth of Glioblastoma tumor cells and its inhibition triggers senescence response. LSD1 is a histone modifier that participates in key aspects of gene transcription as well as in the regulation of methylation dynamics of non-histone proteins. We found that down-regulation of LSD1 inhibits Glioblastoma cell growth, impairs mTOR pathway and cell migration and induces senescence. At mechanistic level, we found that LSD1 regulates HIF-1α protein stability. Pharmacological inhibition or siRNA-mediated silencing of LSD1 expression effectively reduces HIF-1α protein levels, which suffices for the induction of senescence. Our findings elucidate a mechanism whereby LSD1 controls senescence in Glioblastoma tumor cells through the regulation of HIF-1α, and we propose the novel defined LSD1/HIF-1α axis as a new target for the therapy of Glioblastoma tumors.

Genome-wide mapping of 8-oxo-7,8-dihydro-2'-deoxyguanosine reveals accumulation of oxidatively-generated damage at DNA replication origins within transcribed long genes of mammalian cells.

8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) is one of the major DNA modifications and a potent pre-mutagenic lesion prone to mispair with 2'-deoxyadenosine (dA). Several thousand residues of 8-oxodG are constitutively generated in the genome of mammalian cells, but their genomic distribution has not yet been fully characterized. Here, by using OxiDIP-Seq, a highly sensitive methodology that uses immuno-precipitation with efficient anti-8-oxodG antibodies combined with high-throughput sequencing, we report the genome-wide distribution of 8-oxodG in human non-tumorigenic epithelial breast cells (MCF10A), and mouse embryonic fibroblasts (MEFs). OxiDIP-Seq revealed sites of 8-oxodG accumulation overlapping with γH2AX ChIP-Seq signals within the gene body of transcribed long genes, particularly at the DNA replication origins contained therein. We propose that the presence of persistent single-stranded DNA, as a consequence of transcription-replication clashes at these sites, determines local vulnerability to DNA oxidation and/or its slow repair. This oxidatively-generated damage, likely in combination with other kinds of lesion, might contribute to the formation of DNA double strand breaks and activation of DNA damage response.

LSD1 mediates MYCN control of epithelial-mesenchymal transition through silencing of metastatic suppressor NDRG1 gene.

Neuroblastoma (NB) with MYCN amplification is a highly aggressive and metastatic tumor in children. The high recurrence rate and resistance of NB cells to drugs urgently demands a better therapy for this disease. We have recently found that MYCN interacts with the lysine-specific demethylase 1 (LSD1), a histone modifier that participates in key aspects of gene transcription. In cancer cells, LSD1 contributes to the genetic reprogramming that underlies to Epithelial-Mesenchymal Transition (EMT) and tumor metastasis. Here, we show that LSD1 affects motility and invasiveness of NB cells by modulating the transcription of the metastasis suppressor NDRG1 (N-Myc Downstream-Regulated Gene 1). At mechanistic level, we found that LSD1 co-localizes with MYCN at the promoter region of the NDRG1 gene and inhibits its expression. Pharmacological inhibition of LSD1 relieves repression of NDRG1 by MYCN and affects motility and invasiveness of NB cells. These effects were reversed by overexpressing NDRG1. In NB tissues, high levels of LSD1 correlate with low levels of NDRG1 and reduced patients survival. Collectively, our findings elucidate a mechanism of how MYCN/LSD1 control motility and invasiveness of NB cells through transcription regulation of NDRG1 expression and suggest that pharmacological targeting of LSD1 represents a valuable approach for NB therapy.

Cell cycle-dependent resolution of DNA double-strand breaks.

DNA double strand breaks (DSBs) elicit prompt activation of DNA damage response (DDR), which arrests cell-cycle either in G1/S or G2/M in order to avoid entering S and M phase with damaged DNAs. Since mammalian tissues contain both proliferating and quiescent cells, there might be fundamental difference in DDR between proliferating and quiescent cells (or G0-arrested). To investigate these differences, we studied recruitment of DSB repair factors and resolution of DNA lesions induced at site-specific DSBs in asynchronously proliferating, G0-, or G1-arrested cells. Strikingly, DSBs occurring in G0 quiescent cells are not repaired and maintain a sustained activation of the p53-pathway. Conversely, re-entry into cell cycle of damaged G0-arrested cells, occurs with a delayed clearance of DNA repair factors initially recruited to DSBs, indicating an inefficient repair when compared to DSBs induced in asynchronously proliferating or G1-synchronized cells. Moreover, we found that initial recognition of DSBs and assembly of DSB factors is largely similar in asynchronously proliferating, G0-, or G1-synchronized cells. Our study thereby demonstrates that repair and resolution of DSBs is strongly dependent on the cell-cycle state.

Chemical characterization, antioxidant and cytotoxic activities of the methanolic extract of Hymenocrater longiflorus grown in Iraq.

Hymenocrater longiflorus was collected from northern Iraq, and the chemical composition and antioxidant and cytotoxic activities of this plant were investigated. Ten compounds detected by HPLC-ESI/MS were identified as flavonoids and phenolic acids. The free radical scavenging activity of the 70% methanol extract was evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The antioxidant activities of the extract may be attributed to its polyphenolic composition. The cytotoxicity of the plant extract against the osteosarcoma (U2OS) cell line was assessed with the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The extract significantly reduced the viability of cells in a concentration- and time-dependent manner. Cells were arrested during the S-phase of the cell cycle, and DNA damage was revealed by antibodies against histone H2AX. The apoptotic features of cell shrinkage and decrease in cell size were also observed. Western blot analysis revealed cleavage of poly (ADP-ribose)-polymerase 1 (PARP-1), in addition to increases in the proteins p53, p21, and γ-H2AX. Collectively, our findings demonstrate that the H. longiflorus extract is highly cytotoxic to U2OS cells, most likely due to its polyphenolic composition.

Lysine-specific demethylase (LSD1/KDM1A) and MYCN cooperatively repress tumor suppressor genes in neuroblastoma.

The chromatin-modifying enzyme lysine-specific demethylase 1, KDM1A/LSD1 is involved in maintaining the undifferentiated, malignant phenotype of neuroblastoma cells and its overexpression correlated with aggressive disease, poor differentiation and infaust outcome. Here, we show that LSD1 physically binds MYCN both in vitro and in vivo and that such an interaction requires the MYCN BoxIII. We found that LSD1 co-localizes with MYCN on promoter regions of CDKN1A/p21 and Clusterin (CLU) suppressor genes and cooperates with MYCN to repress the expression of these genes. KDM1A needs to engage with MYCN in order to associate with the CDKN1A and CLU promoters. The expression of CLU and CDKN1A can be restored in MYCN-amplified cells by pharmacological inhibition of LSD1 activity or knockdown of its expression. Combined pharmacological inhibition of MYCN and LSD1 through the use of small molecule inhibitors synergistically reduces MYCN-amplified Neuroblastoma cell viability in vitro. These findings demonstrate that LSD1 is a critical co-factor of the MYCN repressive function, and suggest that combination of LSD1 and MYCN inhibitors may have strong therapeutic relevance to counteract MYCN-driven oncogenesis.

MYC impairs resolution of site-specific DNA double-strand breaks repair.

Although it is established that when overexpressed, the MYC family proteins can cause DNA double-stand breaks (DSBs) and genome instability, the mechanisms involved remain unclear. MYC induced genetic instability may result from increased DNA damage and/or reduced DNA repair. Here we show that when overexpressed, MYC proteins induce a sustained DNA damage response (DDR) and reduce the wave of DSBs repair. We used a cell-based DSBs system whereby, upon induction of an inducible restriction enzyme AsiSI, hundreds of site-specific DSBs are generated across the genome to investigate the role of MYC proteins on DSB. We found that high levels of MYC do not block accumulation of γH2AX at AsiSI sites, but delay its clearance, indicating an inefficient repair, while the initial recognition of DNA damage is largely unaffected. Repair of both homologous and nonhomologous repair-prone segments, characterized by high or low levels of recruited RAD51, respectively, was delayed. Collectively, these data indicate that high levels of MYC proteins delay the resolution of DNA lesions engineered to occur in cell cultures.

Role of noncoding RNAs in the regulation of P-TEFb availability and enzymatic activity.

P-TEFb is a transcriptional factor that specifically regulates the elongation step of RNA polymerase II-dependent transcription and its activity strictly required for Human Immunodeficiency Virus (HIV) infection and during cardiac differentiation. P-TEFb role has emerged as a crucial regulator of transcription elongation and its activity found finely tuned in vivo at transcriptional level as well as posttranscriptionally by dynamic association with different multisubunit molecular particles. Both physiological and pathological cellular signals rapidly converge on P-TEFb regulation by modifying expression and activity of the complex to allow cells to properly respond to different stimuli. In this review we will give a panoramic view on P-TEFb regulation by noncoding RNAs in both physiological and pathological conditions.

RNA polymerase II CTD modifications: how many tales from a single tail.

Eukaryote's RNA polymerases II (RNAPII) have the feature to contain, at the carbossi-terminal region of their largest subunit Rpb1, a unique CTD domain. Rpb1-CTD is composed of an increasing number of repetitions of the Y1 S2 P3 T4 S5 P6 S7 heptad that goes in parallel with the developmental level of organisms. Because of its composition, the CTD domain has a huge structural plasticity; virtually all the residues can be subjected to post-translational modifications and the two prolines can either be in cis or trans conformations. In light of these features, it is reasonable to think that different specific nuances of CTD modification and interacting factors take place not only on different gene promoters but also during different stages of the transcription cycle and reasonably might have a role even if the polymerase is on or off the DNA template. Rpb1-CTD domain is involved not only in regulating transcriptional rates, but also in all co-transcriptional processes, such as pre-mRNA processing, splicing, cleavage, and export. Moreover, recent studies highlight a role of CTD in DNA replication and in maintenance of genomic stability and specific CTD-modifications have been related to different CTD functions. In this paper, we examine results from the most recent CTD-related literature and give an overview of the general function of Rpb1-CTD in transcription, transcription-related and non transcription-related processes in which it has been recently shown to be involved in.

Sequence-specific double strand breaks trigger P-TEFb-dependent Rpb1-CTD hyperphosphorylation.

Double strand DNA breaks (DSBs) are one of the most challenging forms of DNA damage which, if left unrepaired, can trigger cellular death and can contribute to cancer. A number of studies have been focused on DNA-damage response (DDR) mechanisms, and most of them rely on the induction of DSBs triggered by chemical compounds or radiations. However, genotoxic drugs and radiation treatments of cultured cell lines induce random DSBs throughout the genome, thus heterogeneously across the cell population, leading to variability of the cellular response. To overcome this aspect, we used here a recently described cell-based DSBs system whereby, upon induction of an inducible restriction enzyme, hundreds of site-specific DSBs are generated across the genome. We show here that sequence-specific DSBs are sufficient to activate the positive transcription elongation factor b (P-TEFb), to trigger hyperphosphorylation of the largest RNA polymerase II carboxyl-terminal-domain (Rpb1-CTD) and to induce activation of p53-transcriptional axis resulting in cell cycle arrest.

The histone LSD1 demethylase in stemness and cancer transcription programs.

DNA and histone chromatin modifying enzymes play a crucial role in chromatin remodeling in several biological processes. Lysine-specific demethylase 1 (LSD1), the first identified histone demethylase, is a relevant player in the regulation of a broad spectrum of biological processes including development, cellular differentiation, embryonic pluripotency and cancer. Here, we review recent insights on the role of LSD1 activity in chromatin regulatory complexes, its functional role in the epigenetic changes during embryonic development, in the establishment and maintenance of stemness and during cancer progression.

Epigenetic reprogramming of Myc target genes.

Myc protein plays a fundamental role in regulation of cell cycle, proliferation, differentiation and apoptosis by modulating the expression of a large number of targets. Myc binding to its targets depends on the presence of the E-box binding sequence and by a chromatin context in which histone H3K4me3 lysine methylation favors Myc binding. Myc role in transcription is still an open question since Myc is able to either activate or repress target genes and the molecular mechanisms by which it exerts these functions span from chromatin remodeling to processive RNAPII elongation. Since the types and number of enzymes able to reversibly modify histones is recently growing, some of the acquisitions regarding Myc chromatin remodeling properties are being revaluated. Here, we summarize recent findings regarding the function of Myc in epigenetic reprogramming of its targets in transcription of differentiated as well as pluripotent cells.

Myc and PI3K/AKT signaling cooperatively repress FOXO3a-dependent PUMA and GADD45a gene expression.

Growth factor withdrawal inhibits cell cycle progression by stimulating expression of growth-arresting genes through the activation of Forkhead box O transcription factors such as FOXO3a, which binds to the FHRE-responsive elements of a number of target genes such as PUMA and GADD45a. Following exposure of cells to growth factors FOXO3a-mediated transcription is rapidly repressed. We determined that repression correlates with activation of PI3K/AKT pathway leading to FOXO3a phosphorylation and release of FOXO3a protein from PUMA and GADD45a chromatin. We show here that Myc significantly and selectively contributes to repression of FOXO-mediated expression of PUMA and GADD45a. We found that in Myc deprived cells inhibition of PUMA and GADD45a following serum stimulation is impaired and that Myc does not interfere with p53 induction of PUMA transcription. We observed that following activation, Myc is rapidly recruited to PUMA and GADD45a chromatin, with a concomitant switch in promoter occupancy from FOXO3a to Myc. Myc recruitment stimulates deacetylation of Histone H3 and H4 and methylation of lysine 9 in H3 (H3K9me2) on both PUMA and GADD45 chromatin. These data highlight a Myc role on cell growth by selectively inhibiting FOXO3a induced transcription of PUMA and GADD45.

DNA oxidation drives Myc mediated transcription.

Myc oncogene is a transcription factor that contributes to the genesis of a wide variety of tumors by regulating proliferation, differentiation and apoptosis. Despite being one of the first isolated oncogene, the biochemical mechanisms of Myc mediated transcriptional regulation remain unclear. Myc has been found to govern different aspects of gene expression, from chromatin remodeling to basal transcription and processive RNAPII elongation. Myc binding to targets genes depends on the presence of the E-box binding motif and the presence of histone H3K4me3 lysines. Here, we summarize recent findings regarding the function of Myc in orchestrating different steps in transcription, and we propose a model that links histone H3 methylation code to Myc target genes. Myc upon binding to the E box triggers a series of events that assembles the transcription initiation complex, recruits the demethylating enzyme LSD1, induces DNA oxidation and chromating looping. Once started RNAPII still needs Myc assistance during transcription elongation. Myc seems to modulate at least two crucial steps in transcription. i.e., chromatin modifications for initiation and RNAPII pause release for productive elongation.

Caffeine prevents transcription inhibition and P-TEFb/7SK dissociation following UV-induced DNA damage.

BACKGROUND: The mechanisms by which DNA damage triggers suppression of transcription of a large number of genes are poorly understood. DNA damage rapidly induces a release of the positive transcription elongation factor b (P-TEFb) from the large inactive multisubunit 7SK snRNP complex. P-TEFb is required for transcription of most class II genes through stimulation of RNA polymerase II elongation and cotranscriptional pre-mRNA processing. METHODOLOGY/PRINCIPAL FINDINGS: We show here that caffeine prevents UV-induced dissociation of P-TEFb as well as transcription inhibition. The caffeine-effect does not involve PI3-kinase-related protein kinases, because inhibition of phosphatidylinositol 3-kinase family members (ATM, ATR and DNA-PK) neither prevents P-TEFb dissociation nor transcription inhibition. Finally, caffeine prevention of transcription inhibition is independent from DNA damage. CONCLUSION/SIGNIFICANCE: Pharmacological prevention of P-TEFb/7SK snRNP dissociation and transcription inhibition following UV-induced DNA damage is correlated.

Camptothecin releases P-TEFb from the inactive 7SK snRNP complex.

An immediate effect of DNA Topoisomerase I inhibitors camptothecin (CPT) and its derivates is the inhibition of transcription. These fast-acting drugs are believed to inhibit transcription by blocking topoisomerase-mediated relief of DNA supercoiling that occurs during transcription elongation. The CPT effects are commonly considered to be due to a collision between the drug-trapped enzyme on the DNA template and the elongating RNAPII. Here we present evidences that CPT treatment induces an early affect on the positive elongation factor b (P-TEFb). The P-TEFb activity is tightly and dynamically regulated, and a reservoir of P-TEFb is kept in an inactive state in the multisubunit 7SK snRNP. We found that, shortly after treatment, CPT disrupts the large inactive P-TEFB complex, and such effect is reversible and independent from DNA replication. Thus, CPT modulates P-TEFb equilibrium in a manner similar to Flavopiridol (FP), a pan-Cdk inhibitor proposed as chemotherapeutic agents against cancers. We determined that while FP inhibits Cdk9 leading to hypo-phosphorylation of RNA polymerase II, CPT-mediated release of free P-TEFb correlates with a concomitant hyper-phosphorylation of RNAPII, which in turn alters the levels and distribution of the RNAPII along transcribed genes. The findings that CPT affects P-TEFb activity provide a direct evidence of the mechanism of this drug to inhibit transcription.

p14ARF is capable of promoting HIV-1 tat degradation.

The p14(ARF) tumor suppressor functions as 'oncogenic checkpoint' that prevents unrestricted cellular proliferation in response to oncogenic signaling. Albeit, the major pathway through which ARF operates is the ARF-Mdm2-p53 axis, ARF directly binds to and inactivates transcription function of a number of DNA-bound activators. In the present study we show that p14(ARF) inhibits transcription activation of HIV-1 LTR promoter activity by Tat protein. Tat protein is a RNA-bound transcriptional activator whose function is strictly required for HIV-1 replication. We determined that p14(ARF) inhibits Tat transactivation of HIV-1 LTR by promoting Tat degradation via an ubiquitin-independent pathway.