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.

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.

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.

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.

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.

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.

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

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

Summer methane emissions from sewage water-fed tropical shallow aquaculture ponds characterized by different water depths.

Aquaculture practices are steadily increasing to meet the fish demand, especially in tropical countries like India. However, efforts to characterize the contribution of these aquaculture ponds towards greenhouse gas emission like CH4 are still very few. CH4 concentration in water [pCH4(water)] and air-water CH4 fluxes were estimated (during the summer months) in two sewage-fed ponds having different depths situated in the East Kolkata Wetlands, India (a Ramsar site). pCH4(water) in both of these ponds showed significant positive correlation with water temperature (R2 = 0.68 and 0.71, p < 0.05). Daily mean chlorophyll-a, turbidity, biochemical oxygen demand (BOD) and gross primary productivity (GPP) also showed positive correlation with pCH4(water). This indicated that higher primary production and presence of turbid materials acted as substrates for methanogenesis, which favoured air-water CH4 effluxes towards atmosphere. Mean air-water CH4 fluxes in the ponds having depth of 1.1 m and 0.6 m were observed to be 24.79 ± 12.02 mg m-2 h-1 and 6.05 ± 3.14 mg m-2 h-1 respectively. Higher depth facilitated net heterotrophic conditions, which led to lower dissolved oxygen levels, which, in turn, led to lower rate of CH4 oxidation. Moreover, under reduced photosynthetically active radiation (in the pond having greater depth), the pH values were comparatively lower (~7.7), which further facilitated a favourable condition for the methanogens to grow. On the whole, it was inferred that apart from pre-established physicochemical factors, depth was also found to play a decisive role in regulating the air-water CH4 fluxes from these aquaculture ponds. In future, continuous sampling should be carried out (by chamber method) to take into account the ebullition CH4 fluxes, and more number of ponds should be sampled throughout a complete annual cycle to have a more holistic understanding about this cluster of sewage-fed aquaculture ponds.

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.

Histone chaperone HIRA dictate proliferation vs differentiation of chronic myeloid leukemia cells.

Abnormal proliferation and disrupted differentiation of hematopoietic progenitors mark leukemia. Histone cell cycle regulator A (HIRA), a histone chaperone, regulates hemogenic to hematopoietic transition involved in normal hematopoiesis. But, its role remains unexplored in leukemia, a case of dysregulated hematopoiesis. Here, the Cancer Cell Line Encyclopedia database analysis showed enhanced HIRA mRNA expression in cells of hematopoietic and lymphoid origin with maximal expression in the chronic myeloid leukemia (CML) cell line, K562. This observation was further endorsed by the induced expression of HIRA in CML patient samples compared to healthy individuals and Acute Myeloid Leukemia patients. Downregulation of HIRA in K562 cells displayed cell cycle arrest, loss in proliferation, presence of polyploidy with significant increase in CD41+ population thereby limiting proliferation but inducing differentiation of leukemia cells to megakaryocyte fate. Induced megakaryocyte differentiation of mouse Hira-knockout hematopoietic progenitors in vivo further confirmed the in vitro findings in leukemia cells. Molecular analysis showed the involvement of MKL1/GATA2/H3.3 axis in dictating differentiation of CML cells to megakaryocytes. Thus, HIRA could be exploited for differentiation induction therapy in CML and in chronic pathological conditions involving low platelet counts.

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.

Nuclear PTEN Localization Contributes to DNA Damage Response in Endometrial Adenocarcinoma and Could Have a Diagnostic Benefit for Therapeutic Management of the Disease.

Endometrial adenocarcinoma (EndoCA) is the most common gynecologic cancer type in the United States, and its incidence is increasing. The majority of patients are disease-free after surgical resection of stage I tumors, which is often followed by radiotherapy, but most patients with advanced disease recur and have a poor prognosis, largely because the tumors become refractory to cytotoxic chemotherapies. PTEN, a commonly mutated tumor suppressor in EndoCAs, is well known for its ability to inhibit the AKT/mTOR signaling pathway. Nuclear functions for PTEN have been proposed as well, but whether those affect EndoCA development, progression, or outcomes is not well understood. Using immunohistochemistry, nuclear PTEN expression was observed in approximately half of EndoCA patient tumors, independent of grade and cytoplasmic PTEN expression. Higher levels of the DNA damage response (DDR) marker, γH2AX, were observed by immunohistochemistry and immunofluorescence in human EndoCA tumor sections that were PTEN-negative, in murine EndoCA tissues that were genetically modified to be PTEN-null, and in Ishikawa EndoCA cells, which do not express endogenous PTEN. Overexpression of exogenous PTEN-WT or PTEN-NLS, a modified PTEN with an added nuclear localization signal, significantly improved both DDR and G2-M transition in Ishikawa cells treated with a DNA-damaging agent. Whereas PARP inhibition with Olaparib was not as effective in Ishikawa cells expressing native or PTEN-NLS, inhibition with Talazoparib was not affected by PTEN overexpression. These results suggest that nuclear PTEN subcellular localization in human EndoCA could be diagnostic when considering DDR therapeutic intervention. Mol Cancer Ther; 17(9); 1995-2003. ©2018 AACR.

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.

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.

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.

The PTEN phosphatase functions cooperatively with the Fanconi anemia proteins in DNA crosslink repair.

Fanconi anemia (FA) is a genetic disease characterized by bone marrow failure and increased cancer risk. The FA proteins function primarily in DNA interstrand crosslink (ICL) repair. Here, we have examined the role of the PTEN phosphatase in this process. We have established that PTEN-deficient cells, like FA cells, exhibit increased cytotoxicity, chromosome structural aberrations, and error-prone mutagenic DNA repair following exposure to ICL-inducing agents. The increased ICL sensitivity of PTEN-deficient cells is caused, in part, by elevated PLK1 kinase-mediated phosphorylation of FANCM, constitutive FANCM polyubiquitination and degradation, and the consequent inefficient assembly of the FA core complex, FANCD2, and FANCI into DNA repair foci. We also establish that PTEN function in ICL repair is dependent on its protein phosphatase activity and ability to be SUMOylated, yet is independent of its lipid phosphatase activity. Finally, via epistasis analysis, we demonstrate that PTEN and FANCD2 function cooperatively in ICL repair.