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 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.

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.

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.

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.

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.

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.

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.

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.

p14ARF interacts with N-Myc and inhibits its transcriptional activity.

In this study, we report that the human p14(ARF) associates in vivo with the N-Myc and inhibits N-Myc mediated transcriptional activation. We have determined that the region (aa 140-300) encompassing the N-Myc BoxIII is required for efficient interaction in vivo. Furthermore, we demonstrate that in the SK-N-BE neuroblastoma cell line p14(ARF) over-expression delocalized N-Myc from the nucleoplasm into nucleoli and that N-Myc regions required for interaction with p14(ARF) are also important for nucleoli co-localization. Finally, we determine that the N-terminal region of the p14(ARF) protein is involved in binding to c-Myc and N-Myc proteins.

MAQ1 and 7SK RNA interact with CDK9/cyclin T complexes in a transcription-dependent manner.

Positive transcription elongation factor b (P-TEFb) comprises a cyclin (T1 or T2) and a kinase, cyclin-dependent kinase 9 (CDK9), which phosphorylates the carboxyl-terminal domain of RNA polymerase II. P-TEFb is essential for transcriptional elongation in human cells. A highly specific interaction among cyclin T1, the viral protein Tat, and the transactivation response (TAR) element RNA determines the productive transcription of the human immunodeficiency virus genome. In growing HeLa cells, half of P-TEFb is kinase inactive and binds to the 7SK small nuclear RNA. We now report on a novel protein termed MAQ1 (for ménage à quatre) that is also present in this complex. Since 7SK RNA is required for MAQ1 to associate with P-TEFb, a structural role for 7SK RNA is proposed. Inhibition of transcription results in the release of both MAQ1 and 7SK RNA from P-TEFb. Thus, MAQ1 cooperates with 7SK RNA to form a novel type of CDK inhibitor. According to yeast two-hybrid analysis and immunoprecipitations from extracts of transfected cells, MAQ1 binds directly to the N-terminal cyclin homology region of cyclins T1 and T2. Since Tat also binds to this cyclin T1 N-terminal domain and since the association between 7SK RNA/MAQ1 and P-TEFb competes with the binding of Tat to cyclin T1, we speculate that the TAR RNA/Tat lentivirus system has evolved to subvert the cellular 7SK RNA/MAQ1 system.

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.

p14ARF directly interacts with Myc through the Myc BoxII domain.

Myc is a well known proto-oncogene encoding for a transcription factor whose activity is tightly regulated in the cellular context. Myc was the first oncogene recognized to activate the ARF tumor suppressor gene which suppresses cell proliferation partly through stabilization of the p53 tumor suppressor protein but which also has p53-independent growth-suppressive functions. Recent studies have indicated that mouse p19ARF negatively regulates Myc's transcriptional activity. We here show that the human p14ARF directly associates with Myc and relocates Myc from the nucleoplasm to the nucleolus. We found that p14ARF interacts with the Myc-Max complex and the binding of p14ARF does not interfere with Myc-Max interaction in vitro. Protein interaction assays define the Myc BoxII as a critical domain required for interaction with p14ARF. Moreover, we identify 30 amino acids encompassing Myc BoxII domain required for p14ARF interaction and colocalization in vivo. Finally, we show that p14ARF down regulates Myc activated transcription and that this activity cannot be addressed to an intrinsic p14ARF repressor domain.

The FCP1 phosphatase interacts with RNA polymerase II and with MEP50 a component of the methylosome complex involved in the assembly of snRNP.

RNA polymerase II transcription is associated with cyclic phosphorylation of the C-terminal domain (CTD) of the large subunit of RNA polymerase II. To date, FCP1 is the only specific CTD phosphatase, which is required for general transcription and cell viability. To identify FCP1-associated proteins, we constructed a human cell line expressing epitope-tagged FCP1. In addition to RAP74, a previously identified FCP1 interacting factor, we determined that FCP1-affinity purified extracts contain RNAPII that has either a hyper- or a hypo-phosphorylated CTD. In addition, by mass spectrometry of affinity purified FCP1-associated factors, we identified a novel FCP1-interacting protein, named MEP50, a recently described component of the methylosome complex that binds to the snRNP's Sm proteins. We found that FCP1 specifically interacts with components of the spliceosomal U small nuclear ribonucleoproteins. These results suggest a putative role of FCP1 CTD-phosphatase in linking the transcription elongation with the splicing process.

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.

Functional inactivation of Cdk9 through oligomerization chain reaction.

The oligomerization chain reaction (OCR) strategy is a recently described technique for inactivation of target proteins that function as homoassociate complexes. This novel strategy is based on the fusion of self-associating coiled-coil (CC) domain of the nuclear factor promyelocytic leukemia (PML) to target proteins. Here, we present the successful application of the OCR strategy for inactivation of the heterodimeric Cdk9/cyclin T1 complex. Cyclin T1/Cdk9 (P-TEFb) complex is a positive regulator of gene transcription, whose function is underlined by the ability to phosphorylate the carboxyl-terminal domain (CTD) of the RNA polymerase II conferring productive transcript elongation. Fusion of the CC domain to Cdk9 leads to the formation of high molecular complexes to which the endogenous cyclin T1 is recruited. The CC-Cdk9 chimera effectively inhibits HIV-1 Tat activation, whose transcription activity is exquisitely dependent upon cyclin T1/Cdk9 function. Furthermore, expression of CC-Cdk9 protein inhibits cell proliferation, as shown by colony-formation assay. Collectively, our findings add further support to the OCR strategy for functional inactivation of hetero-associated factors such as the Cdk9/cyclin T1 complex, and highlight a putative function of Cdk9 in cell growth control.

Inhibition of Tat activity by the HEXIM1 protein.

BACKGROUND: The positive transcription elongation factor b (P-TEFb) composed by CDK9/CyclinT1 subunits is a dedicated co-factor of HIV transcriptional transactivator Tat protein. Transcription driven by the long terminal repeat (LTR) of HIV involves formation of a quaternary complex between P-TEFb, Tat and the TAR element. This recruitment is necessary to enhance the processivity of RNA Pol II from the HIV-1 5' LTR promoter. The activity of P-TEFb is regulated in vivo and in vitro by the HEXIM1/7SK snRNA ribonucleic-protein complex. RESULTS: Here we report that Tat transactivation is effectively inhibited by co-expression of HEXIM1 or its paralog HEXIM2. HEXIM1 expression specifically represses transcription mediated by the direct activation of P-TEFb through artificial recruitment of GAL4-CycT1. Using appropriate HEXIM1 mutants we determined that effective Tat-inhibition entails the 7SK snRNA basic recognition motif as well as the C-terminus region required for interaction with cyclin T1. Enhanced expression of HEXIM1 protein modestly affects P-TEFb activity, suggesting that HEXIM1-mediated repression of Tat activity is not due to a global inhibition of cellular transcription. CONCLUSION: These results point to a pivotal role of P-TEFb for Tat's optimal transcription activity and suggest that cellular proteins that regulate P-TEFb activity might exert profound effects on Tat function in vivo.

Identification of proteins interacting with the RNAPII FCP1 phosphatase: FCP1 forms a complex with arginine methyltransferase PRMT5 and it is a substrate for PRMT5-mediated methylation.

FCP1, a phosphatase specific of the carboxyl-terminal-domain of the large subunit of the RNA polymerase II (RNAPII), stimulates transcription elongation and it is required for general transcription and cell viability. To identify novel interacting proteins of FCP1, we used a human cell line expressing an epitope flagged FCP1 and proteins, which formed complexes with FCP1, were identified by mass spectrometry. We identified four proteins: RPB2 subunit of the RNAPII, the nuclear kinase, NDR1, the methyltransferase PRMT5 and the enhancer of rudimentary homologue (ERH) proteins. Intriguingly, both the PRMT5 and ERH proteins are interacting partners of the SPT5 elongation factor. Interactions of RPB2, ERH, NDR1 and PRMT5 with FCP1 were confirmed by co-immunoprecipitation or in vitro pull-down assays. Interaction between PRMT5 and FCP1 was further confirmed by co-immunoprecipitation of endogenous proteins. We found that FCP1 is a genuine substrate of PRMT5-methylation both in vivo and in vitro, and FCP1-associated PRMT5 can methylate histones H4 in vitro.

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.