Polycomb repressive complex 2 accessory factors: rheostats for cell fate decision?

Epigenetic reprogramming during development is key to cell identity and the activities of the Polycomb repressive complexes are vital for this process. We focus on polycomb repressive complex 2 (PRC2), which catalyzes H3K27me1/2/3 and safeguards cellular integrity by ensuring proper gene repression. Notably, various accessory factors associate with PRC2, strongly influencing cell fate decisions, and their deregulation contributes to various illnesses. Yet, the exact role of these factors during development and carcinogenesis is not fully understood. Here, we present recent progress toward addressing these points and an analysis of the expression levels of PRC2 accessory factors in various tissues and developmental stages to highlight their abundance and roles. Last, we evaluate their contribution to cancer-specific phenotypes, providing insight into novel anticancer therapies.

Beta Amyloid Oligomers with Higher Cytotoxicity have Higher Sidechain Dynamics.

The underlying biophysical principle governing the cytotoxicity of the oligomeric aggregates of ß-amyloid (Aß) peptides has long been an enigma. Here we show that the size of Aß40 oligomers can be actively controlled by incubating the peptides in reverse micelles. Our approach allowed for the first time a detailed comparison of the structures and dynamics of two Aß40 oligomers of different sizes, viz., 10 and 23 nm, by solid-state NMR. From the chemical shift data, we infer that the conformation and/or the chemical environments of the residues from K16 to K28 are different between the 10-nm and 23-nm oligomers. We find that the 10-nm oligomers are more cytotoxic, and the molecular motion of the sidechain of its charged residue K16 is more dynamic. Interestingly, the residue A21 exhibits unusually high structural rigidity. Our data raise an interesting possibility that the cytotoxicity of Aß40 oligomers could also be correlated to the motional dynamics of the sidechains.

The Evaluation of the Effectiveness of Biomineralization Technology in Improving the Strength of Damaged Fiber-Reinforced LWAC.

Concrete cracks and local damage can affect the bond performance between concrete and steel bars, thereby reducing the durability of reinforced concrete structures. Compared with general concrete crack repair methods, biomineralization repair not only has effective bonding capabilities but is also particularly environmentally friendly. Therefore, this study aimed to apply biomineralization technology to repair damaged fiber-reinforced lightweight aggregate concrete (LWAC). Two groups of LWAC specimens were prepared. The experimental group used lightweight aggregates (LWAs) containing bacterial spores and nutrient sources, while the control group used LWAs without bacterial spores and nutrient sources. These specimens were first subjected to compression tests and pull-out tests, respectively, and thus were damaged. After the damaged specimen healed itself in different ways for 28 days, secondary compression and pull-out tests were conducted. The self-healing method of the control group involved placing the specimens in an incubator. The experimental group was divided into experimental group I and experimental group II according to the self-healing method. The self-healing method of experimental group I was the same as that of the control group. The self-healing method of experimental group II involved soaking the specimen in a mixed solution of urea and calcium acetate for two days, and then taking it out and placing it in an incubator for two days, with a cycle of four days. The test results show that in terms of the relative bond strength ratio, the experimental group II increased by 17.9% compared with the control group. Moreover, the precipitate formed at the cracks in the sample was confirmed to be calcium carbonate with the EDS and XRD analysis results, which improved the compressive strength and bond strength after self-healing. This indicates that the biomineralization self-healing method used in experimental group II is more effective.

Site specific NMR characterization of abeta-40 oligomers cross seeded by abeta-42 oligomers.

Extracellular accumulation of ß amyloid peptides of 40 (Aß40) and 42 residues (Aß42) has been considered as one of the hallmarks in the pathology of Alzheimer's disease. In this work, we are able to prepare oligomeric aggregates of Aß with uniform size and monomorphic structure. Our experimental design is to incubate Aß peptides in reverse micelles (RMs) so that the peptides could aggregate only through a single nucleation process and the size of the oligomers is confined by the physical dimension of the reverse micelles. The hence obtained Aß oligomers (AßOs) are 23 nm in diameter and they belong to the category of high molecular-weight (MW) oligomers. The solid-state NMR data revealed that Aß40Os adopt the structural motif of ß-loop-ß but the chemical shifts manifested that they may be structurally different from low-MW AßOs and mature fibrils. From the thioflavin-T results, we found that high-MW Aß42Os can accelerate the fibrillization of Aß40 monomers. Our protocol allows performing cross-seeding experiments among oligomeric species. By comparing the chemical shifts of Aß40Os cross seeded by Aß42Os and those of Aß40Os prepared in the absence of Aß42Os, we observed that the chemical states of E11, K16, and E22 were altered, whereas the backbone conformation of the ß-sheet region near the C-terminus was structurally invariant. The use of reverse micelles allows hitherto the most detailed characterization of the structural variability of Aß40Os.

N-Terminal Tails of Histones H2A and H2B Differentially Affect Transcription by RNA Polymerase II In Vitro.

Histone N-terminal tails and their post-translational modifications affect various biological processes, often in a context-specific manner; the underlying mechanisms are poorly studied. Here, the role of individual N-terminal tails of histones H2A/H2B during transcription through chromatin was analyzed in vitro. spFRET data suggest that the tail of histone H2B (but not of histone H2A) affects nucleosome stability. Accordingly, deletion of the H2B tail (amino acids 1-31, but not 1-26) causes a partial relief of the nucleosomal barrier to transcribing RNA polymerase II (Pol II), likely facilitating uncoiling of DNA from the histone octamer during transcription. Taken together, the data suggest that residues 27-31 of histone H2B stabilize DNA-histone interactions at the DNA region localized ~25 bp in the nucleosome and thus interfere with Pol II progression through the region localized 11-15 bp in the nucleosome. This function of histone H2B requires the presence of the histone H2A N-tail that mediates formation of nucleosome-nucleosome dimers; however, nucleosome dimerization per se plays only a minimal role during transcription. Histone chaperone FACT facilitates transcription through all analyzed nucleosome variants, suggesting that H2A/H2B tails minimally interact with FACT during transcription; therefore, an alternative FACT-interacting domain(s) is likely involved in this process.

Human PARP1 Facilitates Transcription through a Nucleosome and Histone Displacement by Pol II In Vitro.

Human poly(ADP)-ribose polymerase-1 (PARP1) is a global regulator of various cellular processes, from DNA repair to gene expression. The underlying mechanism of PARP1 action during transcription remains unclear. Herein, we have studied the role of human PARP1 during transcription through nucleosomes by RNA polymerase II (Pol II) in vitro. PARP1 strongly facilitates transcription through mononucleosomes by Pol II and displacement of core histones in the presence of NAD+ during transcription, and its NAD+-dependent catalytic activity is essential for this process. Kinetic analysis suggests that PARP1 facilitates formation of "open" complexes containing nucleosomal DNA partially uncoiled from the octamer and allowing Pol II progression along nucleosomal DNA. Anti-cancer drug and PARP1 catalytic inhibitor olaparib strongly represses PARP1-dependent transcription. The data suggest that the negative charge on protein(s) poly(ADP)-ribosylated by PARP1 interact with positively charged DNA-binding surfaces of histones transiently exposed during transcription, facilitating transcription through chromatin and transcription-dependent histone displacement/exchange.

Helicobacter pylori Neutrophil-Activating Protein Directly Interacts with and Activates Toll-like Receptor 2 to Induce the Secretion of Interleukin-8 from Neutrophils and ATRA-Induced Differentiated HL-60 Cells.

Helicobacter pylori neutrophil-activating protein (HP-NAP)-induced production of reactive oxygen species (ROS) by neutrophils and monocytes is regulated by pertussis toxin (PTX)-sensitive G proteins, whereas HP-NAP-induced cytokine secretion by monocytes is mediated by Toll-like receptor 2 (TLR2). However, it is unclear whether TLR2 participates in HP-NAP-induced cytokine secretion by neutrophils. Here, all-trans retinoic acid (ATRA)-induced differentiated HL-60 cells were first employed as a neutrophil model to investigate the molecular mechanisms underlying neutrophil responses to HP-NAP. HP-NAP-induced ROS production in ATRA-induced differentiated HL-60 cells is mediated by the PTX-sensitive heterotrimeric G protein-dependent activation of extracellular signal-regulated kinase 1/2 and p38-mitogen-activated protein kinase, which is consistent with the findings reported for human neutrophils. Next, whether TLR2 participated in HP-NAP-induced secretion of interleukin-8 (IL-8) was investigated in neutrophils and ATRA-induced differentiated HL-60 cells. In both cells, TLR2 participated in HP-NAP-induced IL-8 secretion but not HP-NAP-induced ROS production. Interestingly, PTX-sensitive G proteins also contributed to the HP-NAP-induced secretion of IL-8 from neutrophils and the differentiated HL-60 cells. Our ELISA-based binding assay further revealed the competitive binding of Pam3CSK4, a TLR2 agonist, and HP-NAP to TLR2, which suggests the presence of specific and direct interactions between HP-NAP and TLR2. Thus, HP-NAP directly interacts with and activates TLR2 to induce IL-8 secretion in neutrophils and ATRA-induced differentiated HL-60 cells.

Proceedings from the CIH-LMU 2021 Symposium: "Global Health Perspectives: Climate Change & Migration".

Climate change shapes human migration through the interaction of environmental changes with political, social, economic, and demographic drivers of mobility. Low-and middle-income countries bear the brunt of the health impacts of climate change and migration, despite their overall low contribution to greenhouse gas emissions. The CIHLMU Symposium 2021 aimed to explore the complex interconnections between climate change, migration and health from diverse global perspectives. A number of themes, such as the relationship between climate and trade, the role of technology, and the issue of responsibility were tackled. The speakers also highlighted the need for climate resilient health-systems, gender mainstreaming in climate strategies, collaboration between the Global North and South and urgently defining the 'climate refugee'. It is crucial that the narrative around climate change moves from an environmental framing to encompass human health and migration within climate discussions and strategies.

Sensory cilia as the Achilles heel of nematodes when attacked by carnivorous mushrooms.

Fungal predatory behavior on nematodes has evolved independently in all major fungal lineages. The basidiomycete oyster mushroom Pleurotus ostreatus is a carnivorous fungus that preys on nematodes to supplement its nitrogen intake under nutrient-limiting conditions. Its hyphae can paralyze nematodes within a few minutes of contact, but the mechanism had remained unclear. We demonstrate that the predator-prey relationship is highly conserved between multiple Pleurotus species and a diversity of nematodes. To further investigate the cellular and molecular mechanisms underlying rapid nematode paralysis, we conducted genetic screens in Caenorhabditis elegans and isolated mutants that became resistant to P. ostreatus We found that paralysis-resistant mutants all harbored loss-of-function mutations in genes required for ciliogenesis, demonstrating that the fungus induced paralysis via the cilia of nematode sensory neurons. Furthermore, we observed that P. ostreatus caused excess calcium influx and hypercontraction of the head and pharyngeal muscle cells, ultimately resulting in rapid necrosis of the entire nervous system and muscle cells throughout the entire organism. This cilia-dependent predatory mechanism is evolutionarily conserved in Pristionchus pacificus, a nematode species estimated to have diverged from C. elegans 280 to 430 million y ago. Thus, P. ostreatus exploits a nematode-killing mechanism that is distinct from widely used anthelmintic drugs such as ivermectin, levamisole, and aldicarb, representing a potential route for targeting parasitic nematodes in plants, animals, and humans.

Compatible solutes adaptive alterations in Arthrobacter simplex during exposure to ethanol, and the effect of trehalose on the stress resistance and biotransformation performance.

Ethanol-tolerant Arthrobacter simplex is desirable since ethanol facilitates hydrophobic substrates dissolution on an industrial scale. Herein, alterations in compatible solutes were investigated under ethanol stress. The results showed that the amount of trehalose and glycerol increased while that of glutamate and proline decreased. The trehalose protectant role was verified and its concentration was positively related to the degree of cell tolerance. otsA, otsB and treS, three trehalose biosynthesis genes in A. simplex, also enhanced Escherichia coli stress tolerance, but the increased tolerance was dependent on the type and level of the stress. A. simplex strains accumulating trehalose showed a higher productivity in systems containing more ethanol and substrate because of better viability. The underlying mechanisms of trehalose were involved in better cell integrity, higher membrane stability, stronger reactive oxygen species scavenging capacity and higher energy level. Therefore, trehalose was a general protectant and the upregulation of its biosynthesis by genetic modification enhanced cell stress tolerance, consequently promoted productivity.

Histone Chaperone FACT and Curaxins: Effects on Genome Structure and Function.

The histone chaperone FACT plays important roles in essentially every chromatin-associated process and is an important indirect target of the curaxin class of anti-cancer drugs. Curaxins are aromatiс compounds that intercalate into DNA and can trap FACT in bulk chromatin, thus interfering with its distribution and its functions in cancer cells. Recent studies have provided mechanistic insight into how FACT and curaxins cooperate to promote unfolding of nucleosomes and chromatin fibers, resulting in genome-wide disruption of contact chromatin domain boundaries, perturbation of higher order chromatin organization, and global disregulation of gene expression. Here, we discuss the implications of these insights for cancer biology.

Chinese Herbal Medicine Ganoderma tsugae Displays Potential Anti-Cancer Efficacy on Metastatic Prostate Cancer Cells.

Resistance to the current therapies is the main clinical challenge in the treatment of lethal metastatic prostate cancer (mPCa). Developing novel therapeutic approaches with effective regimes and minimal side effects for this fatal disease remain a priority in prostate cancer study. In the present study, we demonstrated that a traditional Chinese medicine, quality-assured Ganoderma tsugae ethanol extract (GTEE), significantly suppressed cell growth and metastatic capability and caused cell cycle arrest through decreasing expression of cyclins in mPCa cells, PC-3 and DU145 cells. GTEE also induced caspase-dependent apoptosis in mPCa cells. We further showed the potent therapeutic efficacy of GTEE by inhibiting subcutaneous PC-3 tumor growth in a xenograft model. The in vitro and in vivo efficacies on mPCa cells were due to blockade of the PI3K/Akt and MAPK/ERK signaling pathways associated with cancer cell growth, survival and apoptosis. These preclinical data provide the molecular basis for a new potential therapeutic approach toward the treatment of lethal prostate cancer progression.

Time-resolved analysis of transcription through chromatin.

During transcription along nucleosomal DNA, RNA polymerase II (Pol II) pauses at multiple positions and induces formation of multiple intermediates that aid in maintaining proper chromatin structure. To describe the kinetics of this multiple-step reaction, we utilized a computational model-based approach and KinTek Explorer software to analyze the time courses. Here we describe the stepwise protocol for analysis of the kinetics of transcription through a nucleosome that provides the rate constants for each step of this complex process. We also present an example where this time-resolved approach was applied to study the mechanism of histone chaperone FACT action during Pol II transcription through a single nucleosome by comparing the rate constants derived in the presence or in the absence of FACT.

Mechanism of FACT removal from transcribed genes by anticancer drugs curaxins.

Human FACT (facilitates chromatin transcription) is a multifunctional protein complex that has histone chaperone activity and facilitates nucleosome survival and transcription through chromatin. Anticancer drugs curaxins induce FACT trapping on chromatin of cancer cells (c-trapping), but the mechanism of c-trapping is not fully understood. Here, we show that in cancer cells, FACT is highly enriched within the bodies of actively transcribed genes. Curaxin-dependent c-trapping results in redistribution of FACT from the transcribed chromatin regions to other genomic loci. Using a combination of biochemical and biophysical approaches, we have demonstrated that FACT is bound to and unfolds nucleosomes in the presence of curaxins. This tight binding to the nucleosome results in inhibition of FACT-dependent transcription in vitro in the presence of both curaxins and competitor chromatin, suggesting a mechanism of FACT trapping on bulk nucleosomes (n-trapping).

Structure and function of the histone chaperone FACT - Resolving FACTual issues.

FAcilitates Chromatin Transcription (FACT) has been considered essential for transcription through chromatin mostly based on cell-free experiments. However, FACT inactivation in cells does not cause a significant reduction in transcription. Moreover, not all mammalian cells require FACT for viability. Here we synthesize information from different organisms to reveal the core function(s) of FACT and propose a model that reconciles the cell-free and cell-based observations. We describe FACT structure and nucleosomal interactions, and their roles in FACT-dependent transcription, replication and repair. The variable requirements for FACT among different tumor and non-tumor cells suggest that various FACT-dependent processes have significantly different levels of relative importance in different eukaryotic cells. We propose that the stability of chromatin, which might vary among different cell types, dictates these diverse requirements for FACT to support cell viability. Since tumor cells are among the most sensitive to FACT inhibition, this vulnerability could be exploited for cancer treatment.

Chromatin replication: TRANSmitting the histone code.

Efficient overcoming of the nucleosomal barrier and accurate maintenance of associated histone marks during chromatin replication are essential for normal functioning of the cell. Recent studies revealed new protein factors and histone modifications contributing to overcoming the nucleosomal barrier, and suggested an important role for DNA looping in survival of the original histones during replication. These studies suggest new possible mechanisms for transmitting the histone code to next generations of cells.

Overcoming a nucleosomal barrier to replication.

Efficient overcoming and accurate maintenance of chromatin structure and associated histone marks during DNA replication are essential for normal functioning of the daughter cells. However, the molecular mechanisms of replication through chromatin are unknown. We have studied traversal of uniquely positioned mononucleosomes by T7 replisome in vitro. Nucleosomes present a strong, sequence-dependent barrier for replication, with particularly strong pausing of DNA polymerase at the +(31-40) and +(41-65) regions of the nucleosomal DNA. The exonuclease activity of T7 DNA polymerase increases the overall rate of progression of the replisome through a nucleosome, likely by resolving nonproductive complexes. The presence of nucleosome-free DNA upstream of the replication fork facilitates the progression of DNA polymerase through the nucleosome. After replication, at least 50% of the nucleosomes assume an alternative conformation, maintaining their original positions on the DNA. Our data suggest a previously unpublished mechanism for nucleosome maintenance during replication, likely involving transient formation of an intranucleosomal DNA loop.

High yield purification of Helicobacter pylori neutrophil-activating protein overexpressed in Escherichia coli.

BACKGROUND: Helicobacter pylori neutrophil-activating protein (HP-NAP) is involved in H. pylori-induced gastric inflammation. Due to its immunogenic and immunomodulatory properties, HP-NAP has been used for developing vaccines against H. pylori infection and new drugs for cancer therapy. RESULTS: Here, we provide a simple process for high-yield production of HP-NAP by applying one-step negative chromatography to purify recombinant HP-NAP expressed in Escherichia coli (E. coli). In our E. coli expression system, recombinant HP-NAP constitutes nearly 70% of the total protein. Overexpressed recombinant HP-NAP is almost completely soluble upon cell lysis at pH 9.5. Under the optimal condition at pH 8.0, recombinant HP-NAP with purity higher than 95% can be obtained from E. coli by collecting the unbound fraction using diethylaminoethyl (DEAE) Sephadex resin in batch mode. The overall yield of HP-NAP from a 50-ml E. coli culture is ~19 mg. The purified HP-NAP folds into a multimer with a secondary structure of α-helix and is able to trigger the production of reactive oxygen species by neutrophils. CONCLUSIONS: Purification of recombinant HP-NAP overexpressed in E. coli using DEAE Sephadex negative mode batch chromatography is an efficient method for high-yield production of highly pure HP-NAP in its native state. The purified HP-NAP is useful for various clinical applications including vaccine development, diagnosis, and new drug development.

DNA topoisomerase III alpha regulates p53-mediated tumor suppression.

PURPOSE: Human DNA topoisomerase III alpha (hTOP3α) is involved in DNA repair surveillance and cell-cycle checkpoints possibly through formatting complex with tumor suppressors. However, its role in cancer development remained unsolved. EXPERIMENTAL DESIGN: Coimmunoprecipitation, sucrose gradient, chromatin immunoprecipitation (ChIP), real time PCR, and immunoblotting analyses were performed to determine interactions of hTOP3α with p53. Paired cell lines with different hTOP3α levels were generated via ectopic expression and short hairpin RNA (shRNA)-mediated knockdown approaches. Cellular tumorigenic properties were analyzed using cell counting, colony formation, senescence, soft agar assays, and mouse xenograft models. RESULTS: The hTOP3α isozyme binds to p53 and cofractionizes with p53 in gradients differing from fractions containing hTOP3α and BLM. Knockdown of hTOP3α expression (sh-hTOP3α) caused a higher anchorage-independent growth of nontumorigenic RHEK-1 cells. Similarly, sh-hTOP3α and ectopic expression of hTOP3α in cancer cell lines caused increased and reduced tumorigenic abilities, respectively. Genetic and mutation experiments revealed that functional hTOP3α, p53, and p21 are required for this tumor-suppressive activity. Mechanism-wise, ChIP data revealed that hTOP3α binds to the p53 and p21 promoters and positively regulates their expression. Two proteins affect promoter recruitments of each other and collaborate in p21 expression. Moreover, sh-hTOP3α and sh-p53 in AGS cells caused a similar reduction in senescence and hTOP3α mRNA levels were lower in gastric and renal tumor samples. CONCLUSION: We concluded that hTOP3α interacts with p53, regulates p53 and p21 expression, and contributes to the p53-mediated tumor suppression.