Histone chaperone APLF level dictates the implantation of mouse embryos.

Our recent findings demonstrated that the histone chaperone and DNA repair factor aprataxin and PNK-like factor (APLF) could regulate epithelial to mesenchymal transition (EMT) during the reprogramming of murine fibroblasts and in breast cancer metastasis. Therefore, we investigated the function of APLF in EMT associated with mouse development. Here, we show that APLF is predominantly enhanced in trophectoderm (TE) and lineages derived from TE in pre- and post-implantation embryos. Downregulation of APLF induced the hatching of embryos in vitro, with a significant increase in Cdh1 and Cdx2 expression. Aplf short hairpin RNA-microinjected embryos failed to implant in vivo Rescue experiments neutralized the knockdown effects of APLF both in vitro and in vivo Reduced expression of Snai2 and Tead4, and the gain in Cdh1 and sFlt1 (also known as Flt1) level, marked the differentiation of APLF-knocked down trophoblast stem cells that might contribute towards the impaired implantation of embryos. Hence, our findings suggest a novel role for APLF during implantation and post-implantation development of mouse embryos. We anticipate that APLF might contribute to the establishment of maternal-fetal connection, as its fine balance is required to achieve implantation and thereby attain proper pregnancy.

PTEN-negative endometrial cancer cells protect their genome through enhanced DDB2 expression associated with augmented nucleotide excision repair.

BACKGROUND: Endometrial cancer (EC) arises from uterine endometrium tissue and is the most prevalent cancer of the female reproductive tract in developed countries. It has been predicted that the global prevalence of EC will increase in part because of its positive association with economic growth and lifestyle. The majority of EC presented with endometrioid histology and mutations in the tumor suppressor gene PTEN, resulting in its loss of function. PTEN negatively regulates the PI3K/Akt/mTOR axis of cell proliferation and thus serves as a tumorigenesis gatekeeper. Through its chromatin functions, PTEN is also implicated in genome maintenance procedures. However, our comprehension of how DNA repair occurs in the absence of PTEN function in EC is inadequate. METHODS: We utilized The Cancer Genome Atlas (TCGA) data analysis to establish a correlation between PTEN and DNA damage response genes in EC, followed by a series of cellular and biochemical assays to elucidate a molecular mechanism utilizing the AN3CA cell line model for EC. RESULTS: The TCGA analyses demonstrated an inverse correlation between the expression of the damage sensor protein of nucleotide excision repair (NER), DDB2, and PTEN in EC. The transcriptional activation of DDB2 is mediated by the recruitment of active RNA polymerase II to the DDB2 promoter in the PTEN-null EC cells, revealing a correlation between increased DDB2 expression and augmented NER activity in the absence of PTEN. CONCLUSION: Our study indicated a causal relationship between NER and EC that may be exploited in disease management.

Correction: Telomere length-dependent transcription and epigenetic modifications in promoters remote from telomere ends.

[This corrects the article DOI: 10.1371/journal.pgen.1007782.].

Non-duplex G-Quadruplex Structures Emerge as Mediators of Epigenetic Modifications.

The role of non-duplex DNA, the guanine-quadruplex structure in particular, is becoming widely appreciated. Increasing evidence in the last decade implicates quadruplexes in important processes such as transcription and replication. Interestingly, more recent work suggests roles for quadruplexes, in association with quadruplex-interacting proteins, in epigenetics through both DNA and histone modifications. Here, we review the effect of the quadruplex structure on post-replication epigenetic memory and quadruplex-induced promoter DNA/histone modifications. Furthermore, we highlight the epigenetic state of the telomerase promoter where quadruplexes could play a key regulatory role. Finally, we discuss the possibility that DNA structures such as quadruplexes, within a largely duplex DNA background, could act as molecular anchors for locally induced epigenetic modifications.

Telomere length-dependent transcription and epigenetic modifications in promoters remote from telomere ends.

Telomere-binding proteins constituting the shelterin complex have been studied primarily for telomeric functions. However, mounting evidence shows non-telomeric binding and gene regulation by shelterin factors. This raises a key question-do telomeres impact binding of shelterin proteins at distal non-telomeric sites? Here we show that binding of the telomere-repeat-binding-factor-2 (TRF2) at promoters ~60 Mb from telomeres depends on telomere length in human cells. Promoter TRF2 occupancy was depleted in cells with elongated telomeres resulting in altered TRF2-mediated transcription of distal genes. In addition, histone modifications-activation (H3K4me1 and H3K4me3) as well as silencing marks (H3K27me3)-at distal promoters were telomere length-dependent. These demonstrate that transcription, and the epigenetic state, of telomere-distal promoters can be influenced by telomere length. Molecular links between telomeres and the extra-telomeric genome, emerging from findings here, might have important implications in telomere-related physiology, particularly ageing and cancer.

Telomere repeat-binding factor 2 binds extensively to extra-telomeric G-quadruplexes and regulates the epigenetic status of several gene promoters.

The role of the telomere repeat-binding factor 2 (TRF2) in telomere maintenance is well-established. However, recent findings suggest that TRF2 also functions outside telomeres, but relatively little is known about this function. Herein, using genome-wide ChIP-Seq assays of TRF2-bound chromatin from HT1080 fibrosarcoma cells, we identified thousands of TRF2-binding sites within the extra-telomeric genome. In light of this observation, we asked how TRF2 occupancy is organized within the genome. Interestingly, we found that extra-telomeric TRF2 sites throughout the genome are enriched in potential G-quadruplex-forming DNA sequences. Furthermore, we validated TRF2 occupancy at several promoter G-quadruplex motifs, which did adopt quadruplex forms in solution. TRF2 binding altered expression and the epigenetic state of several target promoters, indicated by histone modifications resulting in transcriptional repression of eight of nine genes investigated here. Furthermore, TRF2 occupancy and target gene expression were also sensitive to the well-known intracellular G-quadruplex-binding ligand 360A. Together, these results reveal an extensive genome-wide association of TRF2 outside telomeres and that it regulates gene expression in a G-quadruplex-dependent fashion.

Insights about genome function from spatial organization of the genome.

Over the last 15 years, development of chromosome conformation capture (3C) and its subsequent high-throughput variants in conjunction with the fast development of sequencing technology has allowed investigators to generate large volumes of data giving insights into the spatial three-dimensional (3D) architecture of the genome. This huge data has been analyzed and validated using various statistical, mathematical, genomics, and biophysical tools in order to examine the chromosomal interaction patterns, understand the organization of the chromosome, and find out functional implications of the interactions. This review summarizes the data generated by several large-scale high-throughput chromosome conformation capture studies and the functional implications obtained from the data analyses. We also discuss emerging results on factors (both CCCTC binding factor (CTCF) related and CTCF independent) that could contribute to looping interactions.

Extratelomeric Binding of the Telomere Binding Protein TRF2 at the PCGF3 Promoter Is G-Quadruplex Motif-Dependent.

Telomere repeat binding factor 2 (TRF2) is critical for the protection of chromosome ends. Mounting evidence suggests that TRF2 associates with extratelomeric sites and TRF2 functions may not be limited to telomeres. Here, we show that the PCGF3 promoter harbors a sequence capable of forming the DNA secondary structure G-quadruplex motif, which is required for binding of TRF2 at the PCGF3 promoter. We demonstrate that promoter binding by TRF2 mediates PCGF3 promoter activity, and both the N-terminal and C-terminal domains of TRF2 are necessary for promoter activity. Altogether, this shows for the first time that a telomere binding factor may regulate a component of the polycomb group of proteins.

Enhanced expression of histone chaperone APLF associate with breast cancer.

DNA damage-specific histone chaperone Aprataxin PNK-like factor (APLF) regulates mesenchymal-to-epithelial transition (MET) during cellular reprogramming. We investigated the role of APLF in epithelial-to-mesenchymal transition (EMT) linked to breast cancer invasiveness and metastasis. Here, we show that a significant manifestation of APLF is present in tumor sections of patients with invasive ductal carcinoma when compared to their normal adjacent tissues. APLF was significantly induced in triple negative breast cancer (TNBC) cells, MDAMB-231, in comparison to invasive MCF7 or normal MCF10A breast cells and supported by studies on invasive breast carcinoma in The Cancer Genome Atlas (TCGA). Functionally, APLF downregulation inhibited proliferative capacity, altered cell cycle behavior, induced apoptosis and impaired DNA repair ability of MDAMB-231 cells. Reduction in APLF level impeded invasive, migratory, tumorigenic and metastatic potential of TNBC cells with loss in expression of genes associated with EMT while upregulation of MET-specific gene E-cadherin (CDH1). So, here we provided novel evidence for enrichment of APLF in breast tumors, which could regulate metastasis-associated EMT in invasive breast cancer. We anticipate that APLF could be exploited as a biomarker for breast tumors and additionally could be targeted in sensitizing cancer cells towards DNA damaging agents.

Histone chaperone APLF regulates induction of pluripotency in murine fibroblasts.

Induction of pluripotency in differentiated cells through the exogenous expression of the transcription factors Oct4, Sox2, Klf4 and cellular Myc involves reprogramming at the epigenetic level. Histones and their metabolism governed by histone chaperones constitute an important regulator of epigenetic control. We hypothesized that histone chaperones facilitate or inhibit the course of reprogramming. For the first time, we report here that the downregulation of histone chaperone Aprataxin PNK-like factor (APLF) promotes reprogramming by augmenting the expression of E-cadherin (Cdh1), which is implicated in the mesenchymal-to-epithelial transition (MET) involved in the generation of induced pluripotent stem cells (iPSCs) from mouse embryonic fibroblasts (MEFs). Downregulation of APLF in MEFs expedites the loss of the repressive MacroH2A.1 (encoded by H2afy) histone variant from the Cdh1 promoter and enhances the incorporation of active histone H3me2K4 marks at the promoters of the pluripotency genes Nanog and Klf4, thereby accelerating the process of cellular reprogramming and increasing the efficiency of iPSC generation. We demonstrate a new histone chaperone (APLF)-MET-histone modification cohort that functions in the induction of pluripotency in fibroblasts. This regulatory axis might provide new mechanistic insights into perspectives of epigenetic regulation involved in cancer metastasis.

Interplay between autophagy and apoptosis mediated by copper oxide nanoparticles in human breast cancer cells MCF7.

BACKGROUND: Metal oxide nanoparticles are well known to generate oxidative stress and deregulate normal cellular activities. Among these, transition metals copper oxide nanoparticles (CuO NPs) are more compelling than others and able to modulate different cellular responses. METHODS: In this work, we have synthesized and characterized CuO NPs by various biophysical methods. These CuO NPs (~30nm) induce autophagy in human breast cancer cell line, MCF7 in a time- and dose-dependent manner. Cellular autophagy was tested by MDC staining, induction of green fluorescent protein-light chain 3 (GFP-LC3B) foci by confocal microscopy, transfection of pBABE-puro mCherry-EGFP-LC3B plasmid and Western blotting of autophagy marker proteins LC3B, beclin1 and ATG5. Further, inhibition of autophagy by 3-MA decreased LD50 doses of CuO NPs. Such cell death was associated with the induction of apoptosis as revealed by FACS analysis, cleavage of PARP, de-phosphorylation of Bad and increased cleavage product of caspase 3. siRNA mediated inhibition of autophagy related gene beclin1 also demonstrated similar results. Finally induction of apoptosis by 3-MA in CuO NP treated cells was observed by TEM. RESULTS: This study indicates that CuO NPs are a potent inducer of autophagy which may be a cellular defense against the CuO NP mediated toxicity and inhibition of autophagy switches the cellular response into apoptosis. CONCLUSIONS: A combination of CuO NPs with the autophagy inhibitor is essential to induce apoptosis in breast cancer cells. GENERAL SIGNIFICANCE: CuO NP induced autophagy is a survival strategy of MCF7 cells and inhibition of autophagy renders cellular fate to apoptosis.

Attenuation of PTEN perturbs genomic stability via activation of Akt and down-regulation of Rad51 in human embryonic kidney cells.

To address the involvement of PTEN/Akt signaling in DNA repair and genomic stability, we developed a shRNA-mediated PTEN knockdown cell line from HEK293T cells and evaluated its response to etoposide by analyzing γH2AX and Rad51 foci formation, cell cycle analysis, and chromosome damage. HEK PTEN knockdown cells were impaired in DNA repair associated with loss of G2/M checkpoint and reduced Rad51 foci formation. Furthermore, inhibition of Akt did not restore etoposide-induced G2/M arrest in PTEN knockdown cells, suggesting that loss of G2/M checkpoint in PTEN knockdown cells is Akt-independent. On the other hand, these cells become sensitive to etoposide when Akt was inhibited. Thus, loss of G2/M checkpoint and reduction of Rad51-mediated homologous recombination is responsible for the genomic instability of PTEN knockdown cells where activated Akt additionally contribute to strong survival signal.

Multinucleation regulated by the Akt/PTEN signaling pathway is a survival strategy for HepG2 cells.

Hepatocellular carcinoma (HCC) is non-responsive to many chemotherapeutic agents including etoposide. The aim of this study was to examine the survival strategy of the HCC cell line HepG2 after etoposide treatment. Here we analyzed and compared spontaneous and etoposide-induced DNA damage in HepG2 (α-fetoprotein (AFP)-positive) and Chang Liver (AFP-negative) cell lines. Compared to Chang Liver cells, HepG2 cells exhibited a significantly higher degree of micronucleation and a higher nuclear division index, as determined by the cytokinesis-block micronucleus assay, following exposure to etoposide. HepG2 cells were also more resistant to etoposide-induced cytotoxicity compared to Chang Liver cells. We also establish that increased etoposide-induced multinucleation in HepG2 cells is dependent on the catalytic activity of Akt, as phosphatidylinositol-3-kinase inhibitors as well as the overexpression of kinase-defective Akt reversed this phenotype. Moreover, ectopic expression of wild type PTEN reduced the frequency of etoposide-induced multinucleated HepG2 cells, and restored HepG2 etoposide sensitivity. Taken together, these results implicate the Akt/PTEN cellular axis as a major determinant of the etoposide resistance of HCC cells.

Characterization of a Distinct State in the Continuum of Pluripotency Facilitated by Inhibition of PKCζ in Mouse Embryonic Stem Cells.

Inhibition of PKC (PKCi) signaling maintains pluripotency of embryonic stem cells (ESCs) across different mammalian species. However, the position of PKCi maintained ESCs in the pluripotency continuum is largely unknown. Here we demonstrate that mouse ESCs when cultured continuously, with PKCi, for 75 days are retained in naïve state of pluripotency. Gene expression analysis and proteomics studies demonstrated enhanced naïve character of PKCi maintained ESCs in comparison to classical serum/LIF (S/L) supported ESCs. Molecular analysis revealed that activation of PKCζ isoform associate with primed state of pluripotency, present in epiblast-like stem cells generated in vitro while inhibition of PKCζ phosphorylation associated with naïve state of pluripotency in vitro and in vivo. Phosphoproteomics and chromatin modification enzyme array based studies showed loss in DNA methyl transferase 3B (DNMT3B) and its phosphorylation level upon functional inhibition of PKCζ as one of the crucial components of this regulatory pathway. Unlike ground state of pluripotency maintained by MEK/GSK3 inhibitor in addition to LIF (2i/LIF), loss in DNMT3B is a reversible phenomenon in PKCi maintained ESCs. Absence of phosphorylation of c-MYC, RAF1, SPRY4 while presence of ERF, DUSP6, CIC and YAP1 phosphorylation underlined the phosphoproteomics signature of PKCi mediated maintenance of naïve pluripotency. States of pluripotency represent the developmental continuum and the existence of PKCi mediated mouse ESCs in a distinct state in the continuum of pluripotency (DiSCo) might contribute to the establishment of stages of murine embryonic development that were non-permissible till date.

Neutrophil nucleus: shaping the past and the future.

Neutrophils are innate immune cells that are key to protecting the host against infection and maintaining body homeostasis. However, if dysregulated, they can contribute to disease, such as in cancer or chronic autoinflammatory disorders. Recent studies have highlighted the heterogeneity in the neutrophil compartment and identified the presence of immature neutrophils and their precursors in these pathologies. Therefore, understanding neutrophil maturity and the mechanisms through which they contribute to disease is critical. Neutrophils were first characterized morphologically by Ehrlich in 1879 using microscopy, and since then, different technologies have been used to assess neutrophil maturity. The advances in the imaging field, including state-of-the-art microscopy and machine learning algorithms for image analysis, reinforce the use of neutrophil nuclear morphology as a fundamental marker of maturity, applicable for objective classification in clinical diagnostics. New emerging approaches, such as the capture of changes in chromatin topology, will provide mechanistic links between the nuclear shape, chromatin organization, and transcriptional regulation during neutrophil maturation.

Antibacterial activities of polyethylene glycol, tween 80 and sodium dodecyl sulphate coated silver nanoparticles in normal and multi-drug resistant bacteria.

Antibacterial activity of silver nanoparticles coated with different functionalizing agents i.e., polyethylene glycol, tween 80 and sodium dodecyl sulphate were evaluated on both normal and multi-drug resistant strains of bacteria. Under the same reaction conditions, these functionalizing agents were added separately to coat silver nanoparticles. Among these, polyethylene glycol coated nanoparticles were most effective in killing all the bacterial strains which includes Escherichia coli DH5a, Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus and multi-drug resistant clinical isolates of Shigella spp. (flexneri, boydii, sohnea) and Vibrio cholerae. The minimum inhibitory concentration of polyethylene glycol coated silver nanoparticles was also less compared to the other two sets of nanoparticles. Consistence with that polyethylene glycol coated nanoparticles produced more intracellular reactive oxygen species in bacteria. Moreover, when human cell lines MCF7 and Chang Liver were incubated in presence of these nanoparticles for 18 h with same concentrations as used for bacteria, no toxicity was observed. But significant increase in cell killing was observed with longer incubation time. Thus our present investigation implicates the potential therapeutic use of silver nanoparticles as antibacterial agent particularly the polyethylene glycol coated one.

Both phosphorylation and phosphatase activity of PTEN are required to prevent replication fork progression during stress by inducing heterochromatin.

PTEN is a tumor suppressor protein frequently altered in various cancers. PTEN-null cells have a characteristic of rapid proliferation with an unstable genome. Replication stress is one of the causes of the accumulation of genomic instability if not sensed by the cellular signaling. Though PTEN-null cells have shown to be impaired in replication progression and stalled fork recovery, the association between the catalytic function of PTEN regulated by posttranslational modulation and cellular response to replication stress has not been studied explicitly. To understand molecular mechanism, we find that PTEN-null cells display unrestrained replication fork progression with accumulation of damaged DNA after treatment with aphidicolin which can be rescued by ectopic expression of full-length PTEN, as evident from DNA fiber assay. Moreover, the C-terminal phosphorylation (Ser 380, Thr 382/383) of PTEN is essential for its chromatin association and sensing replication stress that, in response, induce cell cycle arrest. Further, we observed that PTEN induces HP1α expression and H3K9me3 foci formation in a C-terminal phosphorylation-dependent manner. However, phosphatase dead PTEN cannot sense replication stress though it can be associated with chromatin. Together, our results suggest that DNA replication perturbation by aphidicolin enables chromatin association of PTEN through C-terminal phosphorylation, induces heterochromatin formation by stabilizing and up-regulating H3K9me3 foci and augments CHK1 activation. Thereby, PTEN prevents DNA replication fork elongation and simultaneously causes G1-S phase cell cycle arrest to limit cell proliferation in stress conditions. Thus PTEN act as stress sensing protein during replication arrest to maintain genomic stability.

Inactivation of PTEN is responsible for the survival of Hep G2 cells in response to etoposide-induced damage.

The chemo-resistance character of human hepatocellular carcinoma cells is well known but the anomalies associated with such resistance character are not completely understood. In this study, etoposide-induced signaling events in human hepatocellular carcinoma cell line, Hep G2 has been compared with Chang Liver cells, a normal human liver cell line. Hep G2 cells are resistant to etoposide when compared with Chang Liver cells. Etoposide-induced γH2AX foci in Hep G2 cells are persisted for a longer time without affecting cell cycle, indicating that Hep G2 cells are able to maintain its growth with damaged DNA. Further, Akt signaling pathway is deregulated in Hep G2 cells. The upstream negative regulator of Akt, PTEN remains inactive, as it is hyperphosphorylated in Hep G2 cells. Inhibition of PI-3K pathway by wortmannin partially reverses the etoposide-resistance character of Hep G2 cells. Either Hep G2 or Chang Liver cells when transfected with plasmid carrying active Akt (myr-Akt) become resistance towards etoposide compared to the cells transfected with empty vectors or kinase defective Akt. Transient transfection of wild type PTEN in Hep G2 cells does not change its response towards etoposide whereas Chang Liver cells become sensitive after transfection with same plasmid. These results suggest that inactivation of PTEN, which renders activation of Akt, may contribute largely for the etoposide-resistance character of Hep G2 cells.

Human telomerase is directly regulated by non-telomeric TRF2-G-quadruplex interaction.

Human telomerase reverse transcriptase (hTERT) remains suppressed in most normal somatic cells. Resulting erosion of telomeres leads eventually to replicative senescence. Reactivation of hTERT maintains telomeres and triggers progression of >90% of cancers. However, any direct causal link between telomeres and telomerase regulation remains unclear. Here, we show that the telomere-repeat-binding-factor 2 (TRF2) binds hTERT promoter G-quadruplexes and recruits the polycomb-repressor EZH2/PRC2 complex. This is causal for H3K27 trimethylation at the hTERT promoter and represses hTERT in cancer as well as normal cells. Two highly recurrent hTERT promoter mutations found in many cancers, including ∼83% glioblastoma multiforme, that are known to destabilize hTERT promoter G-quadruplexes, showed loss of TRF2 binding in patient-derived primary glioblastoma multiforme cells. Ligand-induced G-quadruplex stabilization restored TRF2 binding, H3K27-trimethylation, and hTERT re-suppression. These results uncover a mechanism of hTERT regulation through a telomeric factor, implicating telomere-telomerase molecular links important in neoplastic transformation, aging, and regenerative therapy.