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Figure S6. Proposed pathway of H1.3 inhibiting H19 and ovarian cancer cell growth. Proposed pathway of H1.3 inhibiting H19 and ovarian cancer cell growth.

Figure S1. Overexpression and knockdown of H1.3. A) Generation of OV-3/H1.3 stable cell lines. Western blotting of H1.3 overexpressing clones. OVCAR-3 cells were transfected with a pcDNA-FLAG-H1.3 construct. G418 resistant clones were screened by Western blotting using an anti-FLAG antibody. Coomassie staining shows equal loading of the cell lysates. Lane 1: OVCAR-3; Lane 2: OVCAR-3 transfected with pcDNA3 vector only (designated as OV-3/V.O); Lanes 3-9: OVCAR-3/H1.3 clones. Clones marked with "*" and "#" were used as representative high and low FLAG-H1.3 expressing clones (OV-3/H1.3(H) and OV-3/H1.3(L)). B) Knockdown of H1.3 levels by shRNA in OV-3/H1.3(H) cells. Whole cell lysates were analyzed by Western blotting using an anti-H1.3 antibody. Western blots using an anti-ACTIN antibody were included as loading controls.

Figure S2. Quantitate RT-PCR analysis of selected genes. Upper and lower panels show representative genes downregulated and upregulated, respectively, in OV-3/H1.3(H) as compared with OVCAR-3 cells. Bar: SD.

Table S2. Gene Ontology Categories of Genes with Altered Expression in OV3/H1.3(H) Cells. Gene Ontology Categories of Genes with Altered Expression in OV3/H1.3(H) Cells.

Figure S5. ChIP-seq analysis of FLAG-H1.3 in OV-3/H1.3(H) cells. A) Metagene analysis of H1.3 distribution on genes partitioned by gene expression levels (each group containing 20% of genes in genome) over a 10 kb region covering -5 kb to +5 kb of transcription start sites (TSS). On average, H1.3 exhibits the deepest dip at TSS of highly expressed genes (red) but no or minimal dip at TSS of inactive genes (blue). ChIP-seq was performed using anti-FLAG antibody in OV-3/H1.3(H) cells. B) Distribution of H1.3 along H19 (upper) and GAPDH (lower) loci in OV-3/H1.3(H) cells. Y axis: tag counts per 1000 bp window per 10 million mappable reads. IP-IN: normalized signal intensity of ChIP-seq subtracted by that of input-seq.

Ovarian cancer is a deadly gynecologic malignancy for which novel biomarkers and therapeutic targets are imperative for improving survival. Previous studies have suggested the expression pattern of linker histone variants as potential biomarkers for ovarian cancer. To investigate the role of histone H1 in ovarian cancer cells, we characterize individual H1 variants and overexpress one of the major somatic H1 variants, H1.3, in the OVCAR-3 epithelial ovarian cancer cell line. We find that overexpression of H1.3 decreases the growth rate and colony formation of OVCAR-3 cells. We identify histone H1.3 as a specific repressor for the noncoding oncogene H19. Overexpression of H1.3 suppresses H19 expression, and knockdown of H1.3 increases its expression in multiple ovarian epithelial cancer cell lines. Furthermore, we demonstrate that histone H1.3 overexpression leads to increased occupancy of H1.3 at the H19 regulator region encompassing the imprinting control region (ICR), concomitant with increased DNA methylation and reduced occupancy of the insulator protein CTCF at the ICR. Finally, we demonstrate that H1.3 overexpression and H19 knockdown synergistically decrease the growth rate of ovarian cancer cells. Our findings suggest that H1.3 dramatically inhibits H19 expression, which contributes to the suppression of epithelial ovarian carcinogenesis. Cancer Res; 74(22); 6463–73. ©2014 AACR.

Figure S4. Characterization of OVCAR-3/H1.1 cells. A) qChIP analysis of FLAG-H1.1 along H19 regulatory regions in OV-3/H1.1 and OVCAR-3 cells. Dashed line: overlay of FLAG-H1.3 occupancy in OV-3/H1.3 cells (Fig. 4). Bars: SD. B) qChIP analysis of CTCF in OVCAR-3 and OV-3/H1.1(H) cells. C-E) Growth curves (C), MTT assay (D), and colony formation assays (E) of OVCAR-3 and OV-3/H1.1(H) cells. Data are presented as mean {plus minus} SD.

Table S1. Genes with Altered Expression in OV3/H1.3(H) Cells. Genes upregulated or downregulated ({greater than or equal to}2-fold) are shown.

Figure S3. H1.3 suppresses H19 expression and cell growth in OV-90 and SK-OV-3 cells. A) Overexpression of H1.3 in OV-90 cells suppresses H19 expression and cell growth. B) Knockdown of H1.3 in SK-OV-3 cells augments H19 expression and cell growth. Ai, Bi) Reverse phase HPLC and mass spectrometry analysis of linker histone H1 variants in OV-90 cells (Ai) and SK-OV-3 cells (Bi). Insets: ESI-TOF mass spectrometry profiles of the indicated peak eluted from HPLC. Aii, Bii) Western blots of histone extracts (Aii) and whole cell lysates (Bii) from indicated cell lines using an anti-H1.3 antibody. Western blots using anti-H3 and anti-ACTIN antibodies were included as the loading controls. Aiii, Biii) Relative expression of the H19 mRNA transcript level in each indicated cell line analyzed by qRT-PCR. H19 expression unit was normalized over GAPDH, and H19 relative expression of each indicated cell line was normalized over respective OV-90 (Aiii) and SK-OV-3 (Biii) cells. Bar: SD. Overexpressed exogenous FLAG-H1 levels were analyzed by immunoblotting using an anti-FLAG antibody (Aiii middle). Equal loading of histone extracts is indicated by anti-H3 Western blots (Aiii lower). Aiv, Biv) Growth curves of indicated cells. Data are presented as mean {plus minus} SD. P

The nanostructured two-dimensional (2D) materials for humidity sensing applications have become increasingly attractive. However, 2D materials have negative aspects of easily stacking and agglomerating multilayers. Here, a novel resistive-type humidity sensor utilizing multi-layer titanium carbide (Ti3C2Tx) films as sensitive material is demonstrated. The humidity sensor exhibits an ultrasensitive and reversible sensing performance in a wide relative humidity range. Furthermore, the ultrafast response and recovery properties are achieved at room temperature. The Fourier transform infrared spectroscopy is used to investigate the adsorption of water vapor on Ti3C2Tx surfaces. A rapid capillary condensation of water vapor on the unique accordion-like microstructures and the hierarchical nanostructures of hydroxyl-riched Ti3C2Tx surface are supposed to be responsible for the super adsorption capability for water vapor. The electrostatic field induced by adsorbed water molecules is proposed to explain the resistance change of the titanium carbide humidity sensor. This work highlights the unique advantages of humidity sensor with layered 2D materials of Ti3C2Tx MXenes, including ultrasensitive, good reversibility and fast recovery at room temperature.

Macrophage-rich atherosclerotic arteries are highly active in glycolysis. PFKFB3, a key glycolytic enzyme, has emerged as a potential therapeutic target in atherosclerosis. Small-molecule inhibitors of PFKFB3, such as 3PO and PFK158, have demonstrated efficacy in hampering atherogenesis in preclinical models. However, genetic studies elucidating the role of Pfkfb3 in atherogenesis need to be conducted to validate pharmacological findings and to unveil potential pharmacological side effects.ApoeGlobal heterozygous or myeloid cell-specific Pfkfb3 deficiency reduced atherogenesis in ApoeCollectively, these findings suggest the existence of a double-edged sword effect of myeloid PFKFB3 on the pathogenesis of atherosclerosis and highlight the need for caution in developing anti-atherosclerotic strategies that target PFKFB3.

Acne was treated by combining the N-terminal of HPA3NT3 (where Lys in position 13 was replaced by Asn) and the C-terminal antibacterial peptide HAG of GLP-1 (32–36) amide pentapeptide after amino replacement. The minimal inhibitory concentration (MIC) was measured using the broth dilution method to evaluate HAG’s antibacterial activity. HAG’s cytotoxicity was determined using water-soluble tetrazolium salt (WST-1). Interleukin-8 (IL-8) expression in human immortalized keratinocytes (HaCaT) cells stimulated by Propionibacterium acnes and treated with HAG was measured by Enzyme-linked immunosorbent assay (ELISA). IL-8 and toll-like receptor 2 (TLR2) expression was detected using real-time reverse transcriptase polymerase chain reaction (qRTPCR) to analyze HAG’s anti-inflammatory effect in vitro. The total RNA was extracted using SiO2 nanoparticles. Oil red O staining was used to detect the intracellular lipid drop of Sebaceous Gland Cell Line (SZ95), and ELISA was used to detect triglyceride levels. Hematoxylin-eosin (HE) staining was used to evaluate ear edema induced by Propionibacterium acne in mice. The MIC of HAG in Propionibacterium acnes was 6.3 μg/mL, and 50 μg/mL of HAG showed cytotoxicity. HAG significantly reduced TLR2 and IL-8 expression in HaCaT cells. Oil red O staining showed that the lipid distribution of HAG-treated SZ95 and triglyceride secretion decreased. HAG reduced ear swelling induced by Propionibacterium acnes. HAG has anti-bacterial and anti-inflammatory effects, excellent hypolipidemic function, and low cytotoxicity. Further development of HAG could be a promising and effective reagent for acne therapy.

Embryonic stem cells (ESCs) are defined by their ability to self-renew and the potential to differentiate into all tissues of the developing organism. We previously demonstrated that deleting the catalytic SET domain of the Set1A/complex of proteins associated with SET1 histone methyltransferase (Set1A/COMPASS) in mouse ESCs does not impair their viability or ability to self-renew; however, it leads to defects in differentiation. The precise mechanisms by which Set1A executes these functions remain to be elucidated. In this study, we demonstrate that mice lacking the SET domain of Set1A are embryonic lethal at a stage that is unique from null alleles. To gain insight into Set1A function in regulating pluripotency, we conducted a CRISPR/Cas9-mediated dropout screen and identified the MOZ/MORF (monocytic leukaemia zinc finger protein/monocytic leukaemia zinc finger protein-related factor) and HBO1 (HAT bound to ORC1) acetyltransferase complex member ING5 as a synthetic perturbation to Set1A. The loss of Ing5 in Set1AΔSET mouse ESCs decreases the fitness of these cells, and the simultaneous loss of ING5 and in Set1AΔSET leads to up-regulation of differentiation-associated genes. Taken together, our results point toward Set1A/COMPASS and ING5 as potential coregulators of the self-renewal and differentiation status of ESCs.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells which are immunosuppressive and glycolytically inactive in inflammatory diseases. However, it is unknown whether MDSCs contribute to ischemic stroke and how glycolysis regulates MDSC function in such a context. Here, we showed that MDSCs arise in the blood of patients at early phase of stroke. Similar results were observed in temporary middle cerebral artery occlusion-induced cerebral ischemic mice. Pharmaceutical exhaustion of MDSCs aggravated, while adoptive transfer of MDSCs rescued the ischemic brain injury. However, the differentiation of MDSCs into immunopotent myeloid cells which coincides with increased glycolysis was observed in the context of ischemic stroke. Mechanistically, the glycolytic product lactate autonomously induces MDSC differentiation through activation of mTORC1, and paracrinely activates Th1 and Th17 cells. Moreover, gene knockout or inhibition of the glycolytic enzyme PFKFB3 increased endogenous MDSCs by blocking their differentiation, and improved ischemic brain injury. Collectively, these results revealed that glycolytic switch decreases the immunosuppressive and neuroprotective role of MDSCs in ischemic stroke and pharmacological targeting MDSCs via glycolysis inhibition constitutes a promising therapeutic strategy for ischemic stroke.

Sun, W.; Zhang, J.; Meng, J.; Li, Y., and Cao, K., 2020. An arctic gridded sea surface temperature product constructed from spaceborne radiometer data. In: Jung, H.-S.; Lee, S.; Ryu, J.-H., and Cui, T. (eds.), Advances in Geospatial Research of Coastal Environments. Journal of Coastal Research, Special Issue No. 102, pp. 280-286. Coconut Creek (Florida), ISSN 0749-0208.A sea surface temperature (SST) record with a grid size of 0.1° for the Arctic region is presented by fusing satellite-derived SST products from three passive-microwave (WindSat, AMSR-E and ASMR2) and four infrared (AVHRR, MODIS onboard Terra and Aqua, and VIIRS) radiometers (RMs). In this study, optimum interpolation (OI) is used to construct the dataset. The fused SST data of Arctic covering the regime north of 60°N span from 2010 to 2018, available with the format of NetcDF. The accuracy of the gridded Arctic SST product was assessed with the drifting buoy measurements. Comparisons with the measurements from Argo buoys show that the Root Mean Squared Errors (RMSE) of the fusion SST data obtained in this study is relatively stable with values ranging from 0.54 to 0.75 °C. This gridded SST data set is characterized with a high spatiotemporal resolution and an assured accuracy.

Actively transcribed genes in mammals are decorated by H3K79 methylation, which is correlated with transcription levels and is catalyzed by the histone methyltransferase DOT1L. DOT1L is required for mammalian development, and the inhibition of its catalytic activity has been extensively studied for cancer therapy; however, the mechanisms underlying DOT1L's functions in normal development and cancer pathogenesis remain elusive. To dissect the relationship between H3K79 methylation, cellular differentiation, and transcription regulation, we systematically examined the role of DOT1L and its catalytic activity in embryonic stem cells (ESCs). DOT1L is dispensable for ESC self-renewal but is required for establishing the proper expression signature of neural progenitor cells, while catalytic inactivation of DOT1L has a lesser effect. Furthermore, DOT1L loss, rather than its catalytic inactivation, causes defects in glial cell specification. Although DOT1L loss by itself has no major defect in transcription elongation, transcription elongation defects seen with the super elongation complex inhibitor KL-2 are exacerbated in DOT1L knockout cells, but not in catalytically dead DOT1L cells, revealing a role of DOT1L in promoting productive transcription elongation that is independent of H3K79 methylation. Taken together, our study reveals a catalytic-independent role of DOT1L in modulating cell-fate determination and in transcriptional elongation control.

The COMPASS family members Set1A, Set1B, and Mll2 differentially regulate H3K4me3 peak breadth in embryonic stem cells., Set1A and Set1B, two members of the COMPASS family of methyltransferases that methylate the histone H3 lysine 4 (H3K4) residue, have been accredited as primary depositors of global H3K4 trimethylation (H3K4me3) in mammalian cells. Our previous studies in mouse embryonic stem cells (ESCs) demonstrated that deleting the enzymatic SET domain of Set1A does not perturb bulk H3K4me3, indicating possible compensatory roles played by other COMPASS methyltransferases. Here, we generated a series of ESC lines harboring compounding mutations of COMPASS methyltransferases. We find that Set1B is functionally redundant to Set1A in implementing H3K4me3 at highly expressed genes, while Mll2 deposits H3K4me3 at less transcriptionally active promoters. While Set1A-B/COMPASS is responsible for broad H3K4me3 peaks, Mll2/COMPASS establishes H3K4me3 with narrow breadth. Additionally, Mll2 helps preserve global H3K4me3 levels and peak breadth in the absence of Set1A-B activity. Our results illustrate the biological flexibility of such enzymes in regulating transcription in a context-dependent manner to maintain stem cell identity.

Histone H3 lysine 4 monomethylation (H3K4me1) is an evolutionarily conserved feature of enhancer chromatin catalyzed by the COMPASS-like methyltransferase family, which includes Trr in Drosophila melanogaster and MLL3 (encoded by KMT2C) and MLL4 (encoded by KMT2D) in mammals. Here we demonstrate that Drosophila embryos expressing catalytically deficient Trr eclose and develop to productive adulthood. Parallel experiments with a trr allele that augments enzyme product specificity show that conversion of H3K4me1 at enhancers to H3K4me2 and H3K4me3 is also compatible with life and results in minimal changes in gene expression. Similarly, loss of the catalytic SET domains of MLL3 and MLL4 in mouse embryonic stem cells (mESCs) does not disrupt self-renewal. Drosophila embryos with trr alleles encoding catalytic mutants manifest subtle developmental abnormalities when subjected to temperature stress or altered cohesin levels. Collectively, our findings suggest that animal development can occur in the context of Trr or mammalian COMPASS-like proteins deficient in H3K4 monomethylation activity and point to a possible role for H3K4me1 on cis-regulatory elements in specific settings to fine-tune transcriptional regulation in response to environmental stress.

Histone H3 Lys4 (H3K4) methylation is a chromatin feature enriched at gene cis-regulatory sequences such as promoters and enhancers. Here we identify an evolutionarily conserved factor, BRWD2/PHIP, which colocalizes with histone H3K4 methylation genome-wide in human cells, mouse embryonic stem cells, and Drosophila. Biochemical analysis of BRWD2 demonstrated an association with the Cullin-4–RING ubiquitin E3 ligase-4 (CRL4) complex, nucleosomes, and chromatin remodelers. BRWD2/PHIP binds directly to H3K4 methylation through a previously unidentified chromatin-binding module related to Royal Family Tudor domains, which we named the CryptoTudor domain. Using CRISPR–Cas9 genetic knockouts, we demonstrate that COMPASS H3K4 methyltransferase family members differentially regulate BRWD2/PHIP chromatin occupancy. Finally, we demonstrate that depletion of the single Drosophila homolog dBRWD3 results in altered gene expression and aberrant patterns of histone H3 Lys27 acetylation at enhancers and promoters, suggesting a cross-talk between these chromatin modifications and transcription through the BRWD protein family.

Aim Adenosine receptors and extracellular adenosine have been demonstrated to modulate vascular smooth muscle cell (VSMC) proliferation and neointima formation. Adenosine kinase (ADK) is a major enzyme regulating intracellular adenosine levels, but is function in VSMC remains unclear. Here, we investigated the role of ADK in vascular injury-induced smooth muscle proliferation and delineated the mechanisms underlying its action. Methods and results We found that ADK expression was higher in the neointima of injured vessels and in PDGF-treated VSMCs. Genetic and pharmacological inhibition of ADK was enough to attenuate arterial injury-induced neointima formation due to inhibition of VSMC proliferation. Mechanistically, using infinium methylation assays and bisulfite sequencing, we showed that ADK metabolized the intracellular adenosine and potentiated the transmethylation pathway, then induced the aberrant DNA hypermethylation. Pharmacological inhibition of aberrant DNA hypermethylation increased KLF4 expression and suppressed VSMC proliferation as well as the neointima formation. Importantly, in human femoral arteries, we observed increased ADK expression and DNA hypermethylation as well as decreased KLF4 expression in neointimal VSMCs of stenotic vessels suggesting that our findings in mice are relevant for human disease and may hold translational significance. Conclusions Our study unravels a novel mechanism by which ADK promotes VSMC proliferation via inducing aberrant DNA hypermethylation, thereby downregulating KLF4 expression and promoting neointima formation. These findings advance the possibility of targeting ADK as an epigenetic modulator to combat vascular injury. Translational perspective Abnormal proliferation of vascular smooth muscle cell (VSMC) is key to abundant occlusive vascular diseases in humans, such as atherosclerosis and intimal hyperplasia associated with restenosis. Adenosine has been shown to combat abnormal smooth muscle proliferation. Here, we demonstrate that increased catabolism of adenosine by adenosine kinase (ADK) promotes abnormal VSMC proliferation. The pathological ADK overexpression in both mice and humans with vascular disease promotes VSMC proliferation via inducing aberrant DNA hypermethylation and KLF4 downregulation. Our study suggests that pharmacological augmentation of endogenous adenosine by targeting ADK represents a promising therapeutic strategy for occlusive vascular diseases.

The COMPASS protein family catalyzes histone H3 lysine 4 (H3K4) methylation and its members are essential for regulating gene expression. MLL2/COMPASS methylates H3K4 on many developmental genes and bivalent clusters. To understand MLL2-dependent transcriptional regulation, we performed a CRISPR-based screen with an MLL2-dependent gene as a reporter in mouse embryonic stem cells (mESCs) and found that MLL2 functions in gene expression by protecting developmental genes from repression via repelling PRC2 and DNA methylation machineries. Accordingly, repression in the absence of MLL2 is relieved by inhibition of PRC2 and DNA methyltransferases. Furthermore, recruitment of DNA demethylation machineries on such loci leads to reactivation of MLL2-dependent genes not only by removing DNA methylation but also by opening up previously CpG methylated regions for PRC2 recruitment, diluting PRC2 at Polycomb-repressed genes. These findings reveal how the context and function of these three epigenetic modifiers of chromatin can orchestrate transcriptional decisions and demonstrate that prevention of active repression by the context of the enzyme and not H3K4me3 underlies transcriptional regulation on MLL2/COMPASS targets.

The counterbalance between coactivator and co-repressor complexes at enhancers instructs stem cell pluripotency transition., Chromatin regulators control cellular differentiation by orchestrating dynamic developmental gene expression programs, and hence, malfunctions in the regulation of chromatin state contribute to both developmental disorders and disease state. Mll4 (Kmt2d), a member of the COMPASS (COMplex of Proteins ASsociated with Set1) protein family that implements histone H3 lysine 4 monomethylation (H3K4me1) at enhancers, is essential for embryonic development and functions as a pancancer tumor suppressor. We define the roles of Mll4/COMPASS and its catalytic activity in the maintenance and exit of ground-state pluripotency in murine embryonic stem cells (ESCs). Mll4 is required for ESC to exit the naive pluripotent state; however, its intrinsic catalytic activity is dispensable for this process. The depletion of the H3K4 demethylase Lsd1 (Kdm1a) restores the ability of Mll4 null ESCs to transition from naive to primed pluripotency. Thus, we define an opposing regulatory axis, wherein Lsd1 and associated co-repressors directly repress Mll4-activated gene targets. This finding has broad reaching implications for human developmental syndromes and the treatment of tumors carrying Mll4 mutations.

Multitalented enhancers Productive transcription from DNA demands initiation, elongation, and termination. Enhancers are DNA sequences that loop with promoters to initiate transcription. Chen et al. show that enhancers also regulate gene expression by modulating transcription elongation. PAF1, a RNA polymerase II–associated factor, sits on enhancers. This prevents the full activation of the enhancer required for the release of paused polymerase at promoters to achieve successful transcription elongation. Science , this issue p. 1294

The homeotic (Hox) genes are highly conserved in metazoans, where they are required for various processes in development, and misregulation of their expression is associated with human cancer. In the developing embryo, Hox genes are activated sequentially in time and space according to their genomic position within Hox gene clusters. Accumulating evidence implicates both enhancer elements and noncoding RNAs in controlling this spatiotemporal expression of Hox genes, but disentangling their relative contributions is challenging. Here, we identify two cis-regulatory elements (E1 and E2) functioning as shadow enhancers to regulate the early expression of the HoxA genes. Simultaneous deletion of these shadow enhancers in embryonic stem cells leads to impaired activation of HoxA genes upon differentiation, while knockdown of a long noncoding RNA overlapping E1 has no detectable effect on their expression. Although MLL/COMPASS (complex of proteins associated with Set1) family of histone methyltransferases is known to activate transcription of Hox genes in other contexts, we found that individual inactivation of the MLL1-4/COMPASS family members has little effect on early Hox gene activation. Instead, we demonstrate that SET1A/COMPASS is required for full transcriptional activation of multiple Hox genes but functions independently of the E1 and E2 cis-regulatory elements. Our results reveal multiple regulatory layers for Hox genes to fine-tune transcriptional programs essential for development.

Cao, K.; Sun, W.; Chen, L.; Meng, J., and Zhang, J., 2020. Retrieval and analysis of sea surface salinity in the adjacent waters of the Yangtze River Estuary based on multisource satellite data. Journal of Coastal Research, 36(3), 590–599. Coconut Creek (Florida), ISSN 0749-0208.Moderate Resolution Imaging Spectroradiometer, Sea-Viewing Wide Field-of-View Sensor, and Visible Infrared Imaging Radiometer Suite multisource satellite remote sensing reflectance data are used with in situ sea surface salinity (SSS) measurements in the Yellow Sea and East China Sea. A linear algorithm is developed for the retrieval of summer SSS in adjacent waters of the Yangtze Estuary in the Yellow Sea and East China Sea. The accuracy of SSS retrieval results is validated using independent in situ measurements. Results show that the mean bias of the model is –0.34 psu, the root-mean-square error is 1.55 psu, and the correlation coefficient is 0.80. The linear model is extended to the retrieval of multisource satellite monthly remote sensing reflectance data to obtain monthly SSS products of the Yangtze Estuary. The spatial coverage of fused satellite SSS data is about 10% greater than that of single-satellite monthly retrieved SSS data. The SSS in the Yangtze Estuary research area has lower values nearshore and higher values offshore. The SSS gradually rises from nearshore areas to open water, and an obvious low-salinity water tongue forms in the Yangtze Estuary. The eastward expansion of freshwater flows of the Yangtze River into the sea is mainly affected by the runoff of the Yangtze River, while a southerly wind and northeastward flow in the research area are the main factors responsible for the eastward expansion of the Yangtze River freshwater flows to the NE. The effect of freshwater on the Yellow Sea and East China Sea extends to 125.5° E, and the low-salinity freshwater tongue distributes northeastward. The expansion types of the Yangtze River freshwater include NE expansion, N-NE expansion, E-NE expansion, bidirectional expansion, and nondirectional expansion.

Understanding how to specify rapid differentiation of human neural progenitor towards enriched non-transformed human astrocyte progenitors will provide a critical cell source to further our understanding of how astrocytes play a pivotal role in neural function and development. Human neural progenitors derived from pluripotent embryonic stem cells and propagated in adherent serum-free cultures provide a fate restricted renewable source for quick production of neural cells; however, such cells are highly refractive to astrocytogenesis and show a strong neurogenic bias, similar to neural progenitors from the early embryonic central nervous system (CNS). We found that several astrocytic genes are hypermethylated in such progenitors potentially preventing generation of astrocytes and leading to the proneuronal fate of these progenitors. However, epigenetic modification by Azacytidine (Aza-C) and Trichostatin A (TSA), with concomitant signaling from BMP2 and LIF in neural progenitor cultures shifts this bias, leading to expression of astrocytic markers as early as 5days of differentiation, with near complete suppression of neuronal differentiation. The resultant cells express major astrocytic markers, are amenable to co-culture with neurons, can be propagated as astrocyte progenitors and are cryopreservable. Although previous reports have generated astrocytes from pluripotent cells, the differentiation required extensive culture or selection based on cell surface antigens. The development of a label free and rapid differentiation process will expedite future derivation of astrocytes from various sources pluripotent cells including, but not limited to, human astrocytes associated with various neurological diseases.

Ovarian cancer is a deadly gynecologic malignancy for which novel biomarkers and therapeutic targets are imperative for improving survival. Previous studies have suggested the expression pattern of linker histone variants as potential biomarkers for ovarian cancer. To investigate the role of histone H1 in ovarian cancer cells, we characterize individual H1 variants and overexpress one of the major somatic H1 variants, H1.3, in the OVCAR-3 epithelial ovarian cancer cell line. We find that overexpression of H1.3 decreases the growth rate and colony formation of OVCAR-3 cells. We identify histone H1.3 as a specific repressor for the noncoding oncogene H19. Overexpression of H1.3 suppresses H19 expression, and knockdown of H1.3 increases its expression in multiple ovarian epithelial cancer cell lines. Furthermore, we demonstrate that histone H1.3 overexpression leads to increased occupancy of H1.3 at the H19 regulator region encompassing the imprinting control region (ICR), concomitant with increased DNA methylation and reduced occupancy of the insulator protein CTCF at the ICR. Finally, we demonstrate that H1.3 overexpression and H19 knockdown synergistically decrease the growth rate of ovarian cancer cells. Our findings suggest that H1.3 dramatically inhibits H19 expression, which contributes to the suppression of epithelial ovarian carcinogenesis. Cancer Res; 74(22); 6463–73. ©2014 AACR.

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure. H1 is essential for mammalian development and regulates specific gene expression in vivo. Among the highly conserved histone proteins, the family of H1 linker histones is the most heterogeneous group. There are 11 H1 subtypes in mammals that are differentially regulated during development and in different cell types. These H1 subtypes include 5 somatic H1s (H1a-e), the replacement H1(0), 4 germ cell specific H1 subtypes, and H1x. The presence of multiple H1 subtypes that differ in DNA binding affinity and chromatin compaction ability provides an additional level of modulation of chromatin function. Thus, quantitative expression analysis of individual H1 subtypes, both of mRNA and proteins, is necessary for better understanding of the regulation of higher order chromatin structure and function. Here we describe a set of assays designed for analyzing the expression levels of individual H1 subtypes. mRNA expression of various H1 variant genes is measured by a set of highly sensitive and quantitative reverse transcription-PCR (qRT-PCR) assays, which are faster, more accurate and require much less samples compared with the alternative approach of Northern blot analysis. Unlike most other cellular mRNA messages, mRNAs for most histone genes, including the majority of H1 genes, lack a long polyA tail, but contain a stem-loop structure at the 3' untranslated region (UTR). Therefore, cDNAs are prepared from total RNA by reverse transcription using random primers instead of oligo-dT primers. Realtime PCR assays with primers specific to each H1 subtypes are performed to obtain highly quantitative measurement of mRNA levels of individual H1 subtypes. Expression of housekeeping genes are analyzed as controls for normalization. The relative abundance of proteins of each H1 subtype and core histones is obtained through reverse phase high-performance liquid chromatography (RP-HPLC) analysis of total histones extracted from mammalian cells. The HPLC method and elution conditions described here give optimum separations of mouse H1 subtypes. By quantifying the HPLC profile, we calculate the relative proportion of individual H1 subtypes within H1 family, as well as determine the H1 to nucleosome ratio in the cells.

Tumor oncogenes include transcription factors that co-opt the general transcriptional machinery to sustain the oncogenic state1, but direct pharmacological inhibition of transcription factors has thus far proven difficult2. However, the transcriptional machinery contains various enzymatic co-factors that can be targeted for development of new therapeutic candidates3, including cyclin-dependent kinases (CDKs)4. Here we present the discovery and characterization of the first covalent CDK7 inhibitor, THZ1, which has the unprecedented ability to target a remote cysteine residue located outside of the canonical kinase domain, providing an unanticipated means of achieving selectivity for CDK7. Cancer cell line profiling indicates that a subset of cancer cell lines, including T-ALL, exhibit exceptional sensitivity to THZ1. Genome-wide analysis in Jurkat T-ALL shows that THZ1 disproportionally affects transcription of RUNX1 and suggests that sensitivity to THZ1 may be due to vulnerability conferred by the RUNX1 super-enhancer and this transcription factor’s key role in the core transcriptional regulatory circuitry of these tumor cells. Pharmacological modulation of CDK7 kinase activity may thus provide an approach to identify and treat tumor types exhibiting extreme dependencies on transcription for maintenance of the oncogenic state.

The spatiotemporal regulation of gene expression is central for cell-lineage specification during embryonic development and is achieved through the combinatorial action of transcription factors/co-factors and epigenetic states at cis-regulatory elements. Here, we show that in addition to implementing H3K4me3 at promoters of bivalent genes, Mll2 (KMT2B)/COMPASS can also implement H3K4me3 at a subset of non-TSS regulatory elements, a subset of which shares epigenetic signatures of active enhancers. Our mechanistic studies reveal that association of Mll2's CXXC domain with CpG-rich regions plays an instrumental role for chromatin targeting and subsequent implementation of H3K4me3. Although Mll2/COMPASS is required for H3K4me3 implementation on thousands of loci, generation of catalytically mutant MLL2/COMPASS demonstrated that H3K4me3 implemented by this enzyme was essential for expression of a subset of genes, including those functioning in the control of transcriptional programs during embryonic development. Our findings suggest that not all H3K4 trimethylations implemented by MLL2/COMPASS are functionally equivalent. D.H. was supported by the Robert H. Lurie Comprehensive Cancer Center - Translational Bridge Program Fellowship in Lymphoma Research. G.M. received the support of Ia Convocatoria de Ayudas Fundación BBVA a Investigadores, Innovadores y Creadores Culturales. A.V. was supported by NIH training grant T32CA080621. These studies were further supported by grants from the Spanish “Ministerio de Educación y Ciencia” (SAF2013-48926-P) and AGAUR to L.D.C., and NIH grants R01CA101774 to J.D.C., R50CA211428 to E.R.S., and R35CA197569 to A.S.