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CD39 (ENTPD1) is an ecto-nucleoside triphosphate diphosphohydrolase expressed widely in the tumor microenvironment (TME) responsible for catalyzing the conversion of ATP to AMP. Inhibition of CD39 enzymatic activity can promote anti-tumor immune responses by increasing the immunostimulatory substrate ATP and decreasing the formation of the product AMP, a precursor to immunoinhibitory adenosine. CD39 inhibition has been shown to have an anti-tumor effect by activating dendritic cells, macrophages, and NK cells in the TME to promote antigen presentation and inflammatory cytokine release. AB598 is a novel antibody, highly specific for human and cynomolgus CD39. AB598 potently binds to CD39 expressed on human primary myeloid cells and tumor cells and potently inhibits both soluble and membrane-bound CD39 enzymatic activity. CD39 inhibition results in increased extracellular ATP (eATP) in the TME and subsequent activation of P2X and P2Y receptors. Myeloid cells highly express both CD39 and P2X7, the P2X receptor with the weakest ligand affinity for ATP (greater than 100 μM), and most likely to be specifically activated by very high levels of eATP resulting from CD39 inhibition. Thus, the myeloid compartment has emerged as a key target associated with the therapeutic effects of CD39 inhibition. AB598 retains the ability to bind and inhibit membrane bound CD39 in the presence of high ATP (400 μM). AB598 promotes maturation of human dendritic cells in the presence of ATP, as determined by increased expression of CD83 and CD86 and decreased expression of CD14. In human macrophages treated with ATP, AB598 activates the inflammasome, resulting in the secretion of pro-inflammatory IL-18 and IL-1β. The ATP-dependent effects of AB598 support a therapeutic rationale of combining it with immunogenic therapies that induce release of ATP, such as chemotherapy and radiation, which might represent a new paradigm in the treatment of advanced solid tumors. Citation Format: Christine E. Bowman, Ada Chen, Damie Juat, Kaustubh Parashar, Julie Clor, Hema Singh, Ritu Kushwaha, Bryan Handlos, Suan Liu, Janine Kline, Xiaoning Zhao, Hyock Joo Kwon, David Green, Stephen W. Young, Ester Fernandez-Salas, Matthew J. Walters, Nigel P. Walker. Inhibition of CD39 results in elevated ATP and activation of myeloid cells to promote anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 321.

Summary Human male germ cell tumors (GCTs) are derived from primordial germ cells (PGCs). The master pluripotency regulator and neuroectodermal lineage effector transcription factor SOX2 is repressed in PGCs and the seminoma (SEM) subset of GCTs. The mechanism of SOX2 repression and its significance to GC and GCT development currently are not understood. Here, we show that SOX2 repression in SEM-derived TCam-2 cells is mediated by the Polycomb repressive complex (PcG) and the repressive H3K27me3 chromatin mark that are enriched at its promoter. Furthermore, SOX2 repression in TCam-2 cells can be abrogated by recruitment of the constitutively expressed H3K27 demethylase UTX to the SOX2 promoter through retinoid signaling, leading to expression of neuronal and other lineage genes. SOX17 has been shown to initiate human PGC specification, with its target PRDM1 suppressing mesendodermal genes. Our results are consistent with a role for SOX2 repression in normal germline development by suppressing neuroectodermal genes., Graphical Abstract, Highlights • SOX2 is repressed in hPGC, germ cell neoplasia in situ, and seminoma • SOX2 repression is mediated by PcG and H3K27me3 enrichment at its promoter • Retinoid signaling recruits UTX to SOX2 promoter leading to reactivation of SOX2 • These studies shed light on the role of SOX2 in germline development, In this article, Chaganti and colleagues show that SOX2 repression in human germ cell tumors and possibly also their precursor hPGC is mediated by PcG and H3Kme27 at its promoter. The repression can be reversed by recruitment of UTX, a H3K27 demethylase, leading to upregulation of neuronal lineage genes.

MicroRNAs (miRNAs) regulate myriad biological processes; however, their role in cell fate choice is relatively unexplored. Pluripotent NT2/D1 embryonal carcinoma cells differentiate into an epithelial/smooth muscle phenotype when treated with bone morphogenetic protein-2 (BMP-2). To identify miRNAs involved in epithelial cell development, we performed miRNA profiling of NT2/D1 cells treated with BMP-2 at 6, 12, and 24 h, and on days 6 and 10. Integration of the miRNA profiling data with previously obtained gene expression profiling (GEP) data of NT2/D1 cells treated with BMP-2 at the same time points identified miR-18b and miR-518b as the top two miRNAs with the highest number of up-regulated predicted targets with known functions in epithelial lineage development. Silencing of miR-18b and miR-518b in NT2/D1 cells revealed several up-regulated TFs with functions in epithelial lineage development; among these, target prediction programs identified FOXN1 as the only direct target of both miRNAs. FOXN1 has previously been shown to play an important role in keratinocyte differentiation and epithelial cell proliferation. NT2/D1 and H9 human embryonic stem cells with silenced miR-18b and miR-518b showed up-regulation of FOXN1 and the epithelial markers CDH1, EPCAM, KRT19, and KRT7. A 3'UTR luciferase assay confirmed FOXN1 to be a target of the two miRNAs, and up-regulation of FOXN1 in NT2/D1 cells led to the expression of epithelial markers. Overexpression of the two miRNAs in BMP-2-treated NT2/D1 cells led to down-regulation of FOXN1 and epithelial lineage markers. These results show that miR-18b and miR-518b are upstream controllers of FOXN1-directed epithelial lineage development.

Fluorescence probes NA1 and NA2 derived from 1-naphthylamine ( NA ) as fluorophore have been synthesized and characterized by different spectroscopic studies. Identification behaviour of these probes towards various metal ions has been investigated. Both the fluorescent probes are selective as well as sensitive towards Fe(III) ion. Novel fluorescence probe NA2 afforded turn-on fluorescence behaviour for Fe(III) ion over other metal ions such as Ca(II), Mg(II), Mn(II), Fe(II), Co(II), Fe(III), Ni(II), Cu(II), Zn(II) and Hg(II).

Environmental factors can stably perturb the epigenome of exposed individuals and even that of their offspring, but the pleiotropic effects of these factors have posed a challenge for understanding the determinants of mitotic or transgenerational inheritance of the epigenetic perturbation. To tackle this problem, we manipulated the epigenetic states of various target genes using a tetracycline-dependent transcription factor. Remarkably, transient manipulation at appropriate times during embryogenesis led to aberrant epigenetic modifications in the ensuing adults regardless of the modification patterns, target gene sequences or locations, and despite lineage-specific epigenetic programming that could reverse the epigenetic perturbation, thus revealing extraordinary malleability of the fetal epigenome, which has implications for ‘metastable epialleles’. However, strong transgenerational inheritance of these perturbations was observed only at transgenes integrated at the Col1a1 locus, where both activating and repressive chromatin modifications were heritable for multiple generations; such a locus is unprecedented. Thus, in our inducible animal models, mitotic inheritance of epigenetic perturbation seems critically dependent on the timing of the perturbation, whereas transgenerational inheritance additionally depends on the location of the perturbation. In contrast, other parameters examined, particularly the chromatin modification pattern and DNA sequence, appear irrelevant.

Although calmodulin (CaM) is known to play multiple regulatory roles in eukaryotes, its direct function as transcriptional regulator is unknown. Furthermore, the physiological functions of CaM are largely unknown in plants. Here, we show that one of the four Arabidopsis thaliana CaM isoforms, CAM7, is a transcriptional regulator that directly interacts with the promoters of light-inducible genes and promotes photomorphogenesis. CAM7 overexpression causes hyperphotomorphogenic growth and an increase in the expression of light-inducible genes. Mutations in CAM7 produce no visible effects on photomorphogenic growth, indicating likely redundant gene functions. However, cam7 mutants display reduced expression of light-inducible genes, and cam7 hy5 double mutants show an enhancement of the hy5 phenotype. Moreover, overexpression of CAM7 can partly suppress the hy5 phenotype, indicating that the two factors work together to control light-induced seedling development. The mutational and transgenic studies, together with physiological analyses, illustrate the concerted function of CAM7 and HY5 basic leucine zipper transcription factor in Arabidopsis seedling development.

Light is an important factor for plant growth and development. We have identified and functionally characterized a regulatory gene SHORT HYPOCOTYL IN WHITE LIGHT1 (SHW1) involved in Arabidopsis (Arabidopsis thaliana) seedling development. SHW1 encodes a unique serine-arginine-aspartate-rich protein, which is constitutively localized in the nucleus of hypocotyl cells. Transgenic analyses have revealed that the expression of SHW1 is developmentally regulated and is closely associated with the photosynthetically active tissues. Genetic and molecular analyses suggest that SHW1 acts as a negative regulator of light-mediated inhibition of hypocotyl elongation, however, plays a positive regulatory role in light-regulated gene expression. The shw1 mutants also display shorter hypocotyl in dark, and analyses of shw1 cop1 double mutants reveal that SHW1 acts nonredundantly with COP1 to control hypocotyl elongation in the darkness. Taken together, this study provides evidences that SHW1 is a regulatory protein that is functionally interrelated to COP1 and plays dual but opposite regulatory roles in photomorphogenesis.

Background Chromatin immunoprecipitation followed by sequencing of protein-bound DNA fragments (ChIP-Seq) is an effective high-throughput methodology for the identification of context specific DNA fragments that are bound by specific proteins in vivo. Despite significant progress in the bioinformatics analysis of this genome-scale data, a number of challenges remain as technology-dependent biases, including variable target accessibility and mappability, sequence-dependent variability, and non-specific binding affinity must be accounted for. Results and discussion We introduce a nonparametric method for scoring consensus regions of aligned immunoprecipitated DNA fragments when appropriate control experiments are available. Our method uses local models for null binding; these are necessary because binding prediction scores based on global models alone fail to properly account for specialized features of genomic regions and chance pull downs of specific DNA fragments, thus disproportionally rewarding some genomic regions and decreasing prediction accuracy. We make no assumptions about the structure or amplitude of bound peaks, yet we show that our method outperforms leading methods developed using either global or local null hypothesis models for random binding. We test prediction performance by comparing analyses of ChIP-seq, ChIP-chip, motif-based binding-site prediction, and shRNA assays, showing high reproducibility, binding-site enrichment in predicted target regions, and functional regulation of predicted targets. Conclusions Given appropriate controls, a direct nonparametric method for identifying transcription-factor targets from ChIP-Seq assays may lead to both higher sensitivity and higher specificity, and should be preferred or used in conjunction with methods that use parametric models for null binding.

The predominant view of pluripotency regulation proposes a stable ground state with coordinated expression of key transcription factors (TFs) that prohibit differentiation. Another perspective suggests a more complexly regulated state involving competition between multiple lineage-specifying TFs that define pluripotency. These contrasting views were developed from extensive analyses of TFs in pluripotent cells in vitro. An experimentally validated, genome-wide repertoire of the regulatory interactions that control pluripotency within the in vivo cellular contexts is yet to be developed. To address this limitation, we assembled a TF interactome of adult human male germ cell tumors (GCTs) using the Algorithm for the Accurate Reconstruction of Cellular Pathways (ARACNe) to analyze gene expression profiles of 141 tumors comprising pluripotent and differentiated subsets. The network (GCTNet) comprised 1,305 TFs, and its ingenuity pathway analysis identified pluripotency and embryonal development as the top functional pathways. We experimentally validated GCTNet by functional (silencing) and biochemical (ChIP-seq) analysis of the core pluripotency regulatory TFs POU5F1, NANOG, and SOX2 in relation to their targets predicted by ARACNe. To define the extent of the in vivo pluripotency network in this system, we ranked all TFs in the GCTNet according to sharing of ARACNe-predicted targets with those of POU5F1 and NANOG using an odds-ratio analysis method. To validate this network, we silenced the top 10 TFs in the network in H9 embryonic stem cells. Silencing of each led to downregulation of pluripotency and induction of lineage; 7 of the 10 TFs were identified as pluripotency regulators for the first time. Stem Cells 2015;33:367–377

Upon toll-like receptor 4 (TLR4) signaling in macrophages, the mammalian Swi/Snf-like BAF chromatin remodeling complex is recruited to many TLR4 target genes where it remodels their chromatin to promote transcription. Here, we show that, surprisingly, recruitment is not sufficient for chromatin remodeling; a second event, dependent on calcium/calmodulin (CaM), is additionally required. Calcium/CaM directly binds the HMG domain of the BAF57 subunit within the BAF complex. Calcium/CaM antagonists, including a CaM-binding peptide derived from BAF57, abolish BAF-dependent remodeling and gene expression without compromising BAF recruitment. BAF57 RNAi and BAF57 dominant negative mutants defective in CaM binding similarly impair the induction of BAF target genes. Our data implicate calcium/CaM in TLR4 signaling, and reveal a previously undescribed, recruitment-independent mode of regulation of the BAF complex that is probably achieved through a direct CaM-BAF interaction.

Pluripotent embryonal carcinoma (EC) cells derived from germ cell tumors (GCTs) are an excellent model system to study the interface between malignancy, pluripotency, and lineage differentiation. We previously characterized the gene expression profile (GEP) of a panel of 135 germ cell tumor (GCT) biopsies comprising all described differentiation lineages and 6 normal testes as controls, using the Affymetrix U133A+B arrays (Korkola et al. Cancer Res., 66: 820-7, 2006 and J. Clin. Oncol., 27: 5240-7, 2009). We applied the ARACNE reverse engineering algorithm to this GEP to obtain the first de novo and context-specific transcriptional interactome (GCTNet) comprising 1,310 transcription factors (TFs). GCTnet recapitulated 830 regulatory targets together of POU5F1, NANOG, and SOX2, the core pluripotency TFs. ChIP-seq and ChIP-PCR analysis of NT2/D1 embryonal carcinoma and H9 ES cells provided significant validation of ARACNe-inferred targets, besides providing the first detailed mapping of DNA binding sites of these 3 genes in human cells. Functional validation of ARACNe-inferred targets by stable knockdown of the POU5F1, NANOG and SOX2 in NT2/D1 cells also showed a significant enrichment of ARACNe targets. Analysis of GEP in a time-course of SOX2 knockdown by principal component analysis (PCA) identified 3 orthogonal gene expression programs: (1) genes with monotonic change (PC1), (2) genes showing a strong and focal gene expression at day 5 (PC2), and (3) genes which show oscillatory pattern of expression (PC3). Ingenuity and GO annotation analysis of PCA provided new insights into the pathways regulated by this TF. Our results provide a novel, comprehensive, and accurate regulatory networks responsible for pluripotency and lineage differentiation in GCTs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3410. doi:10.1158/1538-7445.AM2011-3410

Micro RNAs (miRNAs) are small non-coding RNAs that regulate gene expression by targeting the complementary mRNA sequence for post-transcriptional modification, or mRNA cleavage. MiRNAs regulate a wide range of biological processes such as nervous system and cardiac development, as well as stem cell maintenance and differentiation. However, their role in regulating cell fate choice still remains largely unexplored. Cell lines derived from human embryonal carcinoma (EC) comprise a valuable in vitro resource for study of molecular events regulating cell fate and lineage decisions. NT2/D1 EC cells differentiate into an epithelial/smooth muscle phenotype when induced with BMP-2 (Chadalavada R et al., Stem Cells 25: 771-8, 2007). We used this experimental system to discover miRNAs likely to regulate epidermal cell specification. In a time-course experiment treating NT2/D1 cells with BMP-2, we performed gene expression profiling (GEP) using Affymetrix HG-U133A+B arrays and miRNA expression profiling using Agilent Human V.2 arrays. A set of miRNAs were identified that were up- or down-regulated as cells differentiated towards the epidermal lineage. 53 miRNAs were specifically down-regulated (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5162.