Human cytomegalovirus (hCMV) immediate early 1 (IE1) protein associates with condensed chromatin of the host cell during mitosis. We have determined the structure of the chromatin-tethering domain (CTD) of IE1 bound to the nucleosome core particle, and discovered that IE1-CTD specifically interacts with the H2A-H2B acidic patch and impairs the compaction of higher-order chromatin structure. Our results suggest that IE1 loosens up the folding of host chromatin during hCMV infections.

The H3 histone variant CENP-A is an epigenetic marker critical for the centromere identity and function. However, the precise regulation of the spatiotemporal deposition and propagation of CENP-A at centromeres during the cell cycle is still poorly understood. Here, we show that CENP-A is phosphorylated at Ser68 during early mitosis by Cdk1. Our results demonstrate that phosphorylation of Ser68 eliminates the binding of CENP-A to the assembly factor HJURP, thus preventing the premature loading of CENP-A to the centromere prior to mitotic exit. Because Cdk1 activity is at its minimum at the mitotic exit, the ratio of Cdk1/PP1α activity changes in favor of Ser68 dephosphorylation, thus making CENP-A available for centromeric deposition by HJURP. Thus, we reveal that dynamic phosphorylation of CENP-A Ser68 orchestrates the spatiotemporal assembly of newly synthesized CENP-A at active centromeres during the cell cycle.

Studies have begun to focus on the emerging function of platelets as immune and inflammatory cells that initiate and accelerate vascular inflammation. Phosphoinositide 3-kinase gamma (PI3Kγ) is critically involved in a number of inflammatory and autoimmune diseases. This study aims to investigate the contribution of platelet PI3Kγ to vascular remodeling under flow severely reduced conditions. Mouse partial left carotid artery ligation with adoptive transfer of activated, washed wild-type or PI3Kγ-/- platelets was used as the model. Intima-media area, leukocyte recruitment, and proinflammatory mediator expression were assessed. In vitro PI3Kγ-/- platelets were used to verify the effect of PI3Kγ on platelet activation, interaction with leukocytes, and endothelial cells. Mice injected with activated platelets showed a significant increase in intima-media thickening, recruitment of neutrophils (at 3 d) and macrophages (at 21 d), and intercellular adhesion molecule-1, vascular cell adhesion molecule-1, tumor necrosis factor alpha, and interleukin-6 expression (at 3 d) in the flow-reduced area. These effects were abrogated by platelet PI3Kγ deficiency. Circulating platelet-leukocyte aggregates were reduced in PI3Kγ-/- mice after partial ligation. In vivo data confirmed that PI3Kγ mediated Adenine di-Phosphate -induced platelet activation through the Akt and p38 MAP kinase signaling pathways. Moreover, platelet PI3Kγ deficiency reduced platelet-leukocyte aggregation and platelet-endothelial cell (EC) interaction. These findings indicate that platelet PI3Kγ contributes to platelet-mediated vascular inflammation and carotid intima-media thickening after flow severely reduced. Platelet PI3Kγ may be a new target in the treatment of vascular diseases.

Astrocytes are critical for the development and function of the central nervous system. In developing brains, immature astrocytes undergo morphological, molecular, cellular, and functional changes as they mature. Although the mechanisms that regulate the maturation of other major cell types in the central nervous system such as neurons and oligodendrocytes have been extensively studied, little is known about the cellular and molecular mechanisms that control astrocyte maturation. Here, we identified molecular markers of astrocyte maturation and established an in vitro assay for studying the mechanisms of astrocyte maturation. Maturing astrocytes in vitro exhibit similar molecular changes and represent multiple molecular subtypes of astrocytes found in vivo. Using this system, we found that astrocyte-to-astrocyte contact strongly promotes astrocyte maturation. In addition, secreted signals from microglia, oligodendrocyte precursor cells, or endothelial cells affect a small subset of astrocyte genes but do not consistently change astrocyte maturation. To identify molecular mechanisms underlying astrocyte maturation, we treated maturing astrocytes with molecules that affect the function of tumor-associated genes. We found that a positive feedback loop of heparin-binding epidermal growth factor-like growth factor (HBEGF) and epidermal growth factor receptor (EGFR) signaling regulates astrocytes maturation. Furthermore, HBEGF, EGFR, and tumor protein 53 (TP53) affect the expression of genes important for cilium development, the circadian clock, and synapse function. These results revealed cellular and molecular mechanisms underlying astrocytes maturation with implications for the understanding of glioblastoma.

Polycomb group proteins are critical to maintaining gene repression established during Drosophila development. Part of this group forms the PRC2 complex containing Ez that catalyzes di- and trimethylation of histone H3 lysine 27 (H3K37me2/3), marks repressive to transcription. We report that the mammalian homologs Ezh1 and Ezh2 form similar PRC2 complexes but exhibit contrasting repressive roles. While PRC2-Ezh2 catalyzes H3K27me2/3 and its knockdown affects global H3K27me2/3 levels, PRC2-Ezh1 performs this function weakly. In accordance, Ezh1 knockdown was ineffectual on global H3K27me2/3 levels. Instead, PRC2-Ezh1 directly and robustly represses transcription from chromatinized templates and compacts chromatin in the absence of the methyltransferase cofactor SAM, as evidenced by electron microscopy. Ezh1 targets a subset of Ezh2 genes, yet Ezh1 is more abundant in nonproliferative adult organs while Ezh2 expression is tightly associated with proliferation, as evidenced when analyzing aging mouse kidney. These results might reflect subfunctionalization of a PcG protein during evolution.

Oxidative stress is an important contributor to cancer development. Consistent with that, antioxidant enzymes have been demonstrated to suppress tumorigenesis when being elevated both in vitro and in vivo, making induction of these enzymes a more potent approach for cancer prevention. Protandim, a well-defined combination of widely studied medicinal plants, has been shown to induce superoxide dismutase (SOD) and catalase activities and reduce superoxide generation and lipid peroxidation in healthy human subjects. To investigate whether Protandim can suppress tumor formation by a dietary approach, a two-stage mouse skin carcinogenesis study was performed. At the end of the study, the mice on a Protandim-containing basal diet had similar body weight compared with those on the basal diet, which indicated no overt toxicity by Protandim. After three weeks on the diets, there was a significant increase in the expression levels of SOD and catalase, in addition to the increases in SOD activities. Importantly, at the end of the carcinogenesis study, both skin tumor incidence and multiplicity were reduced in the mice on the Protandim diet by 33% and 57% respectively, compared with those on basal diet. Biochemical and histological studies revealed that the Protandim diet suppressed tumor promoter-induced oxidative stress (evidenced by reduction of protein carbonyl levels), cell proliferation (evidenced by reduction of skin hyperplasia and suppression of PKC/JNK/Jun pathway), and inflammation (evidenced by reduction of ICAM-1/VCAM-1 expression, NF-kappaB binding activity, and nuclear p65/p50 levels). Overall, induction of antioxidant enzymes by Protandim may serve as a practical and potent approach for cancer prevention.

Apoptosis is a common pathway that finally mediated the killing functions of anticancer drugs, which is an important cause of multidrug resistance (MDR). The aim of this study was to investigate the potential benefit of combination therapy with magnetic nanoparticle of Fe(3)O(4) (MNP(Fe(3)O(4))) and 5-bromotetrandrin (BrTet). Analysis of the apoptosis percentage showed that combination of daunorubicin (DNR) with either MNP(Fe(3)O(4)) or BrTet exerted a potent cytotoxic effect on K562/A02 cells, while MNP(Fe(3)O(4)) and BrTet cotreatment can synergistically enhance DNR-induced apoptosis. Importantly, we confirmed that the distinct synergism effect of that composite on reverse multidrug resistance may owe to the regulation of various proliferative and antiapoptotic gene products, including P53 and caspase-3. Thus our in vitro data strongly suggests a potential clinical application of MNP(Fe(3)O(4)) and BrTet combination on CML.

Coactosin-like protein 1 (Cotl1), a member of the actin-depolymerizing factor (ADF)/cofilin family, was first purified from a soluble fraction of Dictyostelium discoideum cells. Neuronal migration requires cytoskeletal remodeling and actin regulation. Although Cotl1 strongly binds to F-actin, the role of Cotl1 in neuronal migration remains undescribed. In this study, we revealed that Cotl1 overexpression impaired migrationof both early- and late-born neurons during mouse corticogenesis. Moreover, Cotl1 overexpression delayed, rather than blocked, neuronal migration in late-born neurons. Cotl1 expression disturbed the morphology of migrating neurons, lengthening the leading processes. This study is the first to investigate the function of Cotl1, and the results indicate that Cotl1 is involved in the regulation of neuronal migration and morphogenesis.

H3.3 is an ancient and conserved H3 variant and plays essential roles in transcriptional regulation. HIRA complex, which is composed of HIRA, UBN1 or UBN2, and Cabin1, is a H3.3 specific chaperone complex. However, it still remains largely uncharacterized how HIRA complex specifically recognizes and deposits H3.3 to the chromatin, such as promoters and enhancers.

Chromatin dynamics play a critical role in cell fate determination and maintenance by regulating the expression of genes essential for development and differentiation. In mouse embryonic stem cells (mESCs), maintenance of pluripotency coincides with a poised chromatin state containing active and repressive histone modifications. However, the structural features of poised chromatin are largely uncharacterized. By adopting mild time-course MNase-seq with computational analysis, the low-compact chromatin in mESCs is featured in two groups: one in more open regions, corresponding to an active state, and the other enriched with bivalent histone modifications, considered the poised state. A parameter called the chromatin opening potential index (COPI) is also devised to quantify the transcription potential based on the dynamic changes of MNase-seq signals at promoter regions. Use of COPI provides effective prediction of gene activation potential and, more importantly, reveals a few developmental factors essential for mouse neural progenitor cell (NPC) differentiation.

Phosphoinositide 3-kinase beta (PI3Kβ) plays an important role in platelet activation and thrombosis, but its role in stroke pathology remains unknown. In this study, we investigated whether inhibition of PI3Kβ protects against cerebral ischemia/reperfusion (I/R) injury by preventing circulating platelet activation and downstream microvascular thrombosis. We used a rat intraluminal filament model of transient middle cerebral artery occlusion (tMCAO) because the rapid restoration of cerebral blood flow to the ischemic area in both tMCAO and endovascular thrombectomy provides clinical relevance for this model. The results showed that TGX221, a selective PI3Kβ inhibitor, treatment immediately before the onset of reperfusion dose-dependently reduced infarct volume and improved neurological function. The protective effects were associated with blocking platelet activation and thrombotic response, thereby reducing downstream microvascular thrombosis, and maintaining reperfusion efficiency. These results suggest that PI3Kβ might be a promising target for treating downstream microvascular thrombosis induced by cerebral I/R injury and offer a novel adjunctive treatment to improve reperfusion therapy for acute ischemic stroke.

This study aimed to develop an integrated approach of deep eutectic solvent-based ultrasound-assisted extraction (DES-UAE) to simultaneously extract five major bioactive macamides from the roots of Lepidium meyenii Walp. Ten different DESs containing choline chloride and selected hydrogen-bond donors were prepared and evaluated based on the extracted macamide content determination using high-performance liquid chromatography (HPLC). Choline chloride/1,6-hexanediol in a 1:2 molar ratio with 20% water exhibited the most promising extraction efficiencies under the optimized parameters verified using single-factor optimization as well as Box-Behnken design. Using the optimized DES-UAE method, the extraction efficiencies of the five macamides were up to 40.3% higher compared to those using the most favorable organic solvent petroleum ether and were also superior to those of the other extraction methods, such as heating and combination of heating and stirring. Furthermore, using the macroporous resin HPD-100, the recoveries of the five target macamides from the DES extraction reached 85.62-92.25%. The 20 μg/mL group of the five macamide extracts showed superior neuroprotective activity against PC12 cell injury than that of the positive drug nimodipine. The macamide extracts also showed higher NO inhibition in LPS-stimulated RAW264.7 cells. Thus, the developed approach was a green and potential alternative that can be used to extract bioactive macamide constituents from L. meyenii in the pharmaceutical and food industries.

The transcription factor hypoxia-inducible factor HIF1A induces cardioprotection from ischemia and reperfusion injury. Here, we investigate tissue-specific pathways that are critical for HIF1A-elicited tissue protection. Initial studies showed that mice with induced global Hif1a deletion (Hif1aloxP/loxP UbiquitinCre+) have exaggerated myocardial injury during in situ ischemia and reperfusion. Surprisingly, this phenotype was mirrored only in mice with myeloid-specific Hif1a deletion (Hif1a loxP/loxP LysM Cre+). In contrast, mice with myocardial specific (Hif1aloxP/loxP Myosin Cre+), or vascular Hif1a deletion (Hif1aloxP/loxP VEcadherin Cre+) experienced similar levels of injury as controls. Subsequent studies using adoptive transfer of Hif1a-deficient polymorphonuclear neutrophils (PMNs) prior to myocardial injury demonstrated increased reperfusion injury. On the contrary, the adoptive transfer of PMNs treated ex vivo with the hypoxia inducible factor (HIF) stabilizer dimethyloxalylglycine (DMOG) was associated with attenuated myocardial injury. Furthermore, DMOG-mediated cardioprotection was abolished in Hif1aloxP/loxP LysM Cre+ mice, but not in Hif2aloxP/loxP LysM Cre+ mice. Finally, studies of PMN-dependent HIF1A target genes implicated the neuronal guidance molecule netrin-1 in mediating the cardioprotective effects of myeloid HIF1A. Taken together, the present studies identified a functional role for myeloid-expressed HIF1A in providing cardioprotection during ischemia and reperfusion injury, which is mediated, at least in part, by the induction of the netrin-1 neuronal guidance molecule in neutrophils.

Metazoan cells only utilize a small subset of the potential DNA replication origins to duplicate the whole genome in each cell cycle. Origin choice is linked to cell growth, differentiation, and replication stress. Although various genetic and epigenetic signatures have been linked to the replication efficiency of origins, there is no consensus on how the selection of origins is determined.

LSH, a SNF2 family DNA helicase, is a key regulator of DNA methylation in mammals. How LSH facilitates DNA methylation is not well defined. While previous studies with mouse embryonic stem cells (mESc) and fibroblasts (MEFs) derived from Lsh knockout mice have revealed a role of Lsh in de novo DNA methylation by Dnmt3a/3b, here we report that LSH contributes to DNA methylation in various cell lines primarily by promoting DNA methylation by DNMT1. We show that loss of LSH has a much bigger effect in DNA methylation than loss of DNMT3A and DNMT3B. Mechanistically, we demonstrate that LSH interacts with UHRF1 but not DNMT1 and facilitates UHRF1 chromatin association and UHRF1-catalyzed histone H3 ubiquitination in an ATPase activity-dependent manner, which in turn promotes DNMT1 recruitment to replication fork and DNA methylation. Notably, UHRF1 also enhances LSH association with the replication fork. Thus, our study identifies LSH as an essential factor for DNA methylation by DNMT1 and provides novel insight into how a feed-forward loop between LSH and UHRF1 facilitates DNMT1-mediated maintenance of DNA methylation in chromatin.

The chromatin-based rule governing the selection and activation of replication origins in metazoans remains to be investigated. Here we report that NFIB, a member of Nuclear Factor I (NFI) family that was initially purified in host cells to promote adenoviral DNA replication but has since mainly been investigated in transcription regulation, is physically associated with the pre-replication complex (pre-RC) in mammalian cells. Genomic analyses reveal that NFIB facilitates the assembly of the pre-RC by increasing chromatin accessibility. Nucleosome binding and single-molecule magnetic tweezers shows that NFIB binds to and opens up nucleosomes. Transmission electron microscopy indicates that NFIB promotes nucleosome eviction on parental chromatin. NFIB deficiency leads to alterations of chromosome contacts/compartments in both G1 and S phase and affects the firing of a subset of origins at early-replication domains. Significantly, cancer-associated NFIB overexpression provokes gene duplication and genomic alterations recapitulating the genetic aberrance in clinical breast cancer and empowering cancer cells to dynamically evolve growth advantage and drug resistance. Together, these results point a role for NFIB in facilitating replication licensing by acting as a genome organizer, shedding new lights on the biological function of NFIB and on the replication origin selection in eukaryotes.

Lysine succinylation is one of the major post-translational modifications occurring on histones and is believed to have significant roles in regulating chromatin structure and function. Currently, histone desuccinylation is widely believed to be catalyzed by members of the SIRT family deacetylases. Here, we report that histone desuccinylation is in fact primarily catalyzed by the class I HDAC1/2/3. Inhibition or depletion of HDAC1/2/3 resulted in a marked increase of global histone succinylation, whereas ectopic expression of HDAC1/2/3 but not their deacetylase inactive mutants downregulated global histone succinylation. We demonstrated that the class I HDAC1/2/3 complexes have robust histone desuccinylase activity in vitro. Genomic landscape analysis revealed that histone succinylation is highly enriched at gene promoters and inhibition of HDAC activity results in marked elevation of promoter histone succinylation. Furthermore, our integrated analysis revealed that promoter histone succinylation positively correlates with gene transcriptional activity. Collectively, we demonstrate that the class I HDAC1/2/3 but not the SIRT family proteins are the major histone desuccinylases particularly important for promoter histone desuccinylation. Our study thus sheds new light on the role of histone succinylation in transcriptional regulation.

In vivo, the complex process of drugs metabolism alters the change in drug composition and determines the final pharmacological properties of oral drugs. Ginsenosides are primary constituents of ginseng, whose pharmacological activities are greatly affected by liver metabolism. However, the predictive power of existing in vitro models is poor due to their inability to mimic the complexity of drug metabolism in vivo. The advance of organs-on-chip-based microfluidics system could provide a new in vitro drug screening platform by recapitulating the metabolic process and pharmacological activity of natural product. In this study, an improved microfluidic device was employed to establish an in vitro co-culture model by culturing multiple cell types in compartmentalized microchambers. Different cell lines were seeded on the device to examine the metabolites of ginsenosides from the hepatocytes in top layer and its resulting efficacy on the tumors in bottom layer. Metabolism dependent drug efficacy of Capecitabine in this system demonstrated the model is validated and controllable. High concentrations of CK, Rh2 (S), and Rg3 (S) ginsenosides showed significant inhibitory effects on two types of tumor cells. In addition, apoptosis detection showed that Rg3 (S) through liver metabolism promoted early apoptosis of tumor cells and displayed better anticancer activity than prodrug. The detected ginsenoside metabolites indicated that some protopanaxadiol saponins were converted into other anticancer aglycones in varying degrees due to orderly de-sugar and oxidation. Ginsenosides exhibited different efficacy on target cells by impacting their viabilities, indicating hepatic metabolism plays an important role in determining ginsenosides efficacy. In conclusion, this microfluidic co-culture system is simple, scalable, and possibly widely applicable in evaluating anticancer activity and metabolism of drug during the early developmental phases of natural product.

DNA replication ensures the accurate transmission of genetic information during the cell cycle. Histone variant H2A.Z is crucial for early replication origins licensing and activation in which SUV420H1 preferentially recognizes H2A.Z-nucleosome and deposits H4 lysine 20 dimethylation (H4K20me2) on replication origins. Here, we report the cryo-EM structures of SUV420H1 bound to H2A.Z-nucleosome or H2A-nucleosome and demonstrate that SUV420H1 directly interacts with H4 N-terminal tail, the DNA, and the acidic patch in the nucleosome. The H4 (1-24) forms a lasso-shaped structure that stabilizes the SUV420H1-nucleosome complex and precisely projects the H4K20 residue into the SUV420H1 catalytic center. In vitro and in vivo analyses reveal a crucial role of the SUV420H1 KR loop (residues 214-223), which lies close to the H2A.Z-specific residues D97/S98, in H2A.Z-nucleosome preferential recognition. Together, our findings elucidate how SUV420H1 recognizes nucleosomes to ensure site-specific H4K20me2 modification and provide insights into how SUV420H1 preferentially recognizes H2A.Z nucleosome.

Whole-genome duplication (WGD), or polyploidy, events are widespread and significant in the evolutionary history of angiosperms. However, empirical evidence for rediploidization, the major process where polyploids give rise to diploid descendants, is still lacking at the genomic level. Here we present chromosome-scale genomes of the mangrove tree Sonneratia alba and the related inland plant Lagerstroemia speciosa. Their common ancestor has experienced a whole-genome triplication (WGT) approximately 64 million years ago coinciding with a period of dramatic global climate change. Sonneratia, adapting mangrove habitats, experienced extensive chromosome rearrangements post-WGT. We observe the WGT retentions display sequence and expression divergence, suggesting potential neo- and sub-functionalization. Strong selection acting on three-copy retentions indicates adaptive value in response to new environments. To elucidate the role of ploidy changes in genome evolution, we improve a model of the polyploidization-rediploidization process based on genomic evidence, contributing to the understanding of adaptive evolution during climate change.