The Arabidopsis PHD-finger protein EDM2 has multiple roles in balancing NLR immune receptor gene expression.

Plant NLR-type receptors serve as sensitive triggers of host immunity. Their expression has to be well-balanced, due to their interference with various cellular processes and dose-dependency of their defense-inducing activity. A genetic "arms race" with fast-evolving pathogenic microbes requires plants to constantly innovate their NLR repertoires. We previously showed that insertion of the COPIA-R7 retrotransposon into RPP7 co-opted the epigenetic transposon silencing signal H3K9me2 to a new function promoting expression of this Arabidopsis thaliana NLR gene. Recruitment of the histone binding protein EDM2 to COPIA-R7-associated H3K9me2 is required for optimal expression of RPP7. By profiling of genome-wide effects of EDM2, we now uncovered additional examples illustrating effects of transposons on NLR gene expression, strongly suggesting that these mobile elements can play critical roles in the rapid evolution of plant NLR genes by providing the "raw material" for gene expression mechanisms. We further found EDM2 to have a global role in NLR expression control. Besides serving as a positive regulator of RPP7 and a small number of other NLR genes, EDM2 acts as a suppressor of a multitude of additional NLR genes. We speculate that the dual functionality of EDM2 in NLR expression control arose from the need to compensate for fitness penalties caused by high expression of some NLR genes by suppression of others. Moreover, we are providing new insights into functional relationships of EDM2 with its interaction partner, the RNA binding protein EDM3/AIPP1, and its target gene IBM1, encoding an H3K9-demethylase.

Nascent RNA sequencing reveals mechanisms of gene regulation in the human malaria parasite Plasmodium falciparum.

Gene expression in Plasmodium falciparum is tightly regulated to ensure successful propagation of the parasite throughout its complex life cycle. The earliest transcriptomics studies in P. falciparum suggested a cascade of transcriptional activity over the course of the 48-hour intraerythrocytic developmental cycle (IDC); however, the just-in-time transcriptional model has recently been challenged by findings that show the importance of post-transcriptional regulation. To further explore the role of transcriptional regulation, we performed the first genome-wide nascent RNA profiling in P. falciparum. Our findings indicate that the majority of genes are transcribed simultaneously during the trophozoite stage of the IDC and that only a small subset of genes is subject to differential transcriptional timing. RNA polymerase II is engaged with promoter regions prior to this transcriptional burst, suggesting that Pol II pausing plays a dominant role in gene regulation. In addition, we found that the overall transcriptional program during gametocyte differentiation is surprisingly similar to the IDC, with the exception of relatively small subsets of genes. Results from this study suggest that further characterization of the molecular players that regulate stage-specific gene expression and Pol II pausing will contribute to our continuous search for novel antimalarial drug targets.

Analysis of nucleosome positioning landscapes enables gene discovery in the human malaria parasite Plasmodium falciparum.

BACKGROUND: Plasmodium falciparum, the deadliest malaria-causing parasite, has an extremely AT-rich (80.7 %) genome. Because of high AT-content, sequence-based annotation of genes and functional elements remains challenging. In order to better understand the regulatory network controlling gene expression in the parasite, a more complete genome annotation as well as analysis tools adapted for AT-rich genomes are needed. Recent studies on genome-wide nucleosome positioning in eukaryotes have shown that nucleosome landscapes exhibit regular characteristic patterns at the 5'- and 3'-end of protein and non-protein coding genes. In addition, nucleosome depleted regions can be found near transcription start sites. These unique nucleosome landscape patterns may be exploited for the identification of novel genes. In this paper, we propose a computational approach to discover novel putative genes based exclusively on nucleosome positioning data in the AT-rich genome of P. falciparum. RESULTS: Using binary classifiers trained on nucleosome landscapes at the gene boundaries from two independent nucleosome positioning data sets, we were able to detect a total of 231 regions containing putative genes in the genome of Plasmodium falciparum, of which 67 highly confident genes were found in both data sets. Eighty-eight of these 231 newly predicted genes exhibited transcription signal in RNA-Seq data, indicative of active transcription. In addition, 20 out of 21 selected gene candidates were further validated by RT-PCR, and 28 out of the 231 genes showed significant matches using BLASTN against an expressed sequence tag (EST) database. Furthermore, 108 (47%) out of the 231 putative novel genes overlapped with previously identified but unannotated long non-coding RNAs. Collectively, these results provide experimental validation for 163 predicted genes (70.6%). Finally, 73 out of 231 genes were found to be potentially translated based on their signal in polysome-associated RNA-Seq representing transcripts that are actively being translated. CONCLUSION: Our results clearly indicate that nucleosome positioning data contains sufficient information for novel gene discovery. As distinct nucleosome landscapes around genes are found in many other eukaryotic organisms, this methodology could be used to characterize the transcriptome of any organism, especially when coupled with other DNA-based gene finding and experimental methods (e.g., RNA-Seq).

Babesia duncani multi-omics identifies virulence factors and drug targets.

Babesiosis is a malaria-like disease in humans and animals that is caused by Babesia species, which are tick-transmitted apicomplexan pathogens. Babesia duncani causes severe to lethal infection in humans, but despite the risk that this parasite poses as an emerging pathogen, little is known about its biology, metabolic requirements or pathogenesis. Unlike other apicomplexan parasites that infect red blood cells, B. duncani can be continuously cultured in vitro in human erythrocytes and can infect mice resulting in fulminant babesiosis and death. We report comprehensive, detailed molecular, genomic, transcriptomic and epigenetic analyses to gain insights into the biology of B. duncani. We completed the assembly, 3D structure and annotation of its nuclear genome, and analysed its transcriptomic and epigenetics profiles during its asexual life cycle stages in human erythrocytes. We used RNA-seq data to produce an atlas of parasite metabolism during its intraerythrocytic life cycle. Characterization of the B. duncani genome, epigenome and transcriptome identified classes of candidate virulence factors, antigens for diagnosis of active infection and several attractive drug targets. Furthermore, metabolic reconstitutions from genome annotation and in vitro efficacy studies identified antifolates, pyrimethamine and WR-99210 as potent inhibitors of B. duncani to establish a pipeline of small molecules that could be developed as effective therapies for the treatment of human babesiosis.

Identification of Novel Regulatory Regions Induced by Intrauterine Growth Restriction in Rat Islets.

Intrauterine growth restriction (IUGR) leads to the development of type 2 diabetes in adulthood, and the permanent alterations in gene expression implicate an epigenetic mechanism. Using a rat model of IUGR, we performed TrueSeq-HELP Tagging to assess the association of DNA methylation changes and gene dysregulation in islets. We identified 511 differentially methylated regions (DMRs) and 4377 significantly altered single CpG sites. Integrating the methylome and our published transcriptome data sets resulted in the identification of pathways critical for islet function. The identified DMRs were enriched with transcription factor binding motifs, such as Elk1, Etv1, Foxa1, Foxa2, Pax7, Stat3, Hnf1, and AR. In silico analysis of 3-dimensional chromosomal interactions using human pancreas and islet Hi-C data sets identified interactions between 14 highly conserved DMRs and 35 genes with significant expression changes at an early age, many of which persisted in adult islets. In adult islets, there were far more interactions between DMRs and genes with significant expression changes identified with Hi-C, and most of them were critical to islet metabolism and insulin secretion. The methylome was integrated with our published genome-wide histone modification data sets from IUGR islets, resulting in further characterization of important regulatory regions of the genome altered by IUGR containing both significant changes in DNA methylation and specific histone marks. We identified novel regulatory regions in islets after exposure to IUGR, suggesting that epigenetic changes at key transcription factor binding motifs and other gene regulatory regions may contribute to gene dysregulation and an abnormal islet phenotype in IUGR rats.

Modulation of BCL-2 in Both T Cells and Tumor Cells to Enhance Chimeric Antigen Receptor T-cell Immunotherapy against Cancer.

Chimeric antigen receptor T-cell (CART) immunotherapy led to unprecedented responses in patients with refractory/relapsed B-cell non-Hodgkin lymphoma (NHL); nevertheless, two thirds of patients experience treatment failure. Resistance to apoptosis is a key feature of cancer cells, and it is associated with treatment failure. In 87 patients with NHL treated with anti-CD19 CART, we found that chromosomal alteration of B-cell lymphoma 2 (BCL-2), a critical antiapoptotic regulator, in lymphoma cells was associated with reduced survival. Therefore, we combined CART19 with the FDA-approved BCL-2 inhibitor venetoclax and demonstrated in vivo synergy in venetoclax-sensitive NHL. However, higher venetoclax doses needed for venetoclax-resistant lymphomas resulted in CART toxicity. To overcome this limitation, we developed venetoclax-resistant CART by overexpressing mutated BCL-2(F104L), which is not recognized by venetoclax. Notably, BCL-2(F104L)-CART19 synergized with venetoclax in multiple lymphoma xenograft models. Furthermore, we uncovered that BCL-2 overexpression in T cells intrinsically enhanced CART antitumor activity in preclinical models and in patients by prolonging CART persistence. SIGNIFICANCE: This study highlights the role of BCL-2 in resistance to CART immunotherapy for cancer and introduces a novel concept for combination therapies-the engineering of CART cells to make them resistant to proapoptotic small molecules, thereby enhancing the therapeutic index of these combination therapies. This article is highlighted in the In This Issue feature, p. 2221.

Strand-Specific RNA-Seq Applied to Malaria Samples.

Over the past few years only, next-generation sequencing technologies became accessible and many applications were rapidly derived, such as the development of RNA-seq, a technique that uses deep sequencing to profile whole transcriptomes. RNA-seq has the power to discover new transcripts and splicing variants, single nucleotide variations, fusion genes, and mRNA levels-based expression profiles. Preparing RNA-seq libraries can be delicate and usually obligates buying expensive kits that require large amounts of stating materials. The method presented here is flexible and cost-effective. Using this method, we prepared high-quality strand-specific RNA-seq libraries from RNA extracted from the human malaria parasite Plasmodium falciparum. The libraries are compatible with Illumina®'s sequencers Genome Analyzer and Hi-Seq. The method can however be easily adapted to other platforms.

The Transcriptome and Epigenome Reveal Novel Changes in Transcription Regulation During Pancreatic Rat Islet Maturation.

Islet function is critical for normal glucose homeostasis. Unlike adult ß cells, fetal and neonatal islets are more proliferative and have decreased insulin secretion in response to stimuli. However, the underlying mechanisms governing functional maturity of islets have not been completely elucidated. Pancreatic islets comprise different cell types. The microenvironment of islets and interactions between these cell types are critical for ß-cell development and maturation. Thus, the study of intact islets is optimal to identify novel molecular mechanisms controlling islet functional development. Transcriptomes and genome-wide histone landscapes of H3K4me3, H3K27me3, and H3K27Ac from intact islets isolated from 2- and 10-week-old Sprague-Dawley rats were integrated to elucidate genes and pathways modulating islet development, as well as the contribution of epigenetic regulation. A total of 4489 differentially expressed genes were identified; 2289 and 2200 of them were up- and down-regulated in 10-week islets, respectively. Ingenuity Pathway Analysis revealed critical pathways regulating functional maturation of islets, including nutrient sensing, neuronal function, immune function, cell replication, and extracellular matrix. Furthermore, we identified significant changes in enrichment of H3K4me3, H3K27me3, and H3K27Ac marks, which correlated with expression changes of genes critical for islet function. These histone marks were enriched at critical transcription factor-binding motifs, such as Hoxa9, C/EBP-ß, Gata1, Foxo1, E2f1, E2f3, and Mafb. In addition, our chromatin immunoprecipitation sequencing data revealed multiple potential bivalent genes whose poised states changed with maturation. Collectively, our current study identified critical novel pathways for mature islet function and suggested a role for histone modifications in regulating islet development and maturation.

Antigen-independent activation enhances the efficacy of 4-1BB-costimulated CD22 CAR T cells.

While CD19-directed chimeric antigen receptor (CAR) T cells can induce remission in patients with B cell acute lymphoblastic leukemia (ALL), a large subset relapse with CD19- disease. Like CD19, CD22 is broadly expressed by B-lineage cells and thus serves as an alternative immunotherapy target in ALL. Here we present the composite outcomes of two pilot clinical trials ( NCT02588456 and NCT02650414 ) of T cells bearing a 4-1BB-based, CD22-targeting CAR in patients with relapsed or refractory ALL. The primary end point of these studies was to assess safety, and the secondary end point was antileukemic efficacy. We observed unexpectedly low response rates, prompting us to perform detailed interrogation of the responsible CAR biology. We found that shortening of the amino acid linker connecting the variable heavy and light chains of the CAR antigen-binding domain drove receptor homodimerization and antigen-independent signaling. In contrast to CD28-based CARs, autonomously signaling 4-1BB-based CARs demonstrated enhanced immune synapse formation, activation of pro-inflammatory genes and superior effector function. We validated this association between autonomous signaling and enhanced function in several CAR constructs and, on the basis of these observations, designed a new short-linker CD22 single-chain variable fragment for clinical evaluation. Our findings both suggest that tonic 4-1BB-based signaling is beneficial to CAR function and demonstrate the utility of bedside-to-bench-to-bedside translation in the design and implementation of CAR T cell therapies.

Altered Transcription Factor Binding and Gene Bivalency in Islets of Intrauterine Growth Retarded Rats.

Intrauterine growth retardation (IUGR), which induces epigenetic modifications and permanent changes in gene expression, has been associated with the development of type 2 diabetes. Using a rat model of IUGR, we performed ChIP-Seq to identify and map genome-wide histone modifications and gene dysregulation in islets from 2- and 10-week rats. IUGR induced significant changes in the enrichment of H3K4me3, H3K27me3, and H3K27Ac marks in both 2-wk and 10-wk islets, which were correlated with expression changes of multiple genes critical for islet function in IUGR islets. ChIP-Seq analysis showed that IUGR-induced histone mark changes were enriched at critical transcription factor binding motifs, such as C/EBPs, Ets1, Bcl6, Thrb, Ebf1, Sox9, and Mitf. These transcription factors were also identified as top upstream regulators in our previously published transcriptome study. In addition, our ChIP-seq data revealed more than 1000 potential bivalent genes as identified by enrichment of both H3K4me3 and H3K27me3. The poised state of many potential bivalent genes was altered by IUGR, particularly Acod1, Fgf21, Serpina11, Cdh16, Lrrc27, and Lrrc66, key islet genes. Collectively, our findings suggest alterations of histone modification in key transcription factors and genes that may contribute to long-term gene dysregulation and an abnormal islet phenotype in IUGR rats.

Plasmodium Condensin Core Subunits SMC2/SMC4 Mediate Atypical Mitosis and Are Essential for Parasite Proliferation and Transmission.

Condensin is a multi-subunit protein complex regulating chromosome condensation and segregation during cell division. In Plasmodium spp., the causative agent of malaria, cell division is atypical and the role of condensin is unclear. Here we examine the role of SMC2 and SMC4, the core subunits of condensin, during endomitosis in schizogony and endoreduplication in male gametogenesis. During early schizogony, SMC2/SMC4 localize to a distinct focus, identified as the centromeres by NDC80 fluorescence and chromatin immunoprecipitation sequencing (ChIP-seq) analyses, but do not form condensin I or II complexes. In mature schizonts and during male gametogenesis, there is a diffuse SMC2/SMC4 distribution on chromosomes and in the nucleus, and both condensin I and condensin II complexes form at these stages. Knockdown of smc2 and smc4 gene expression reveals essential roles in parasite proliferation and transmission. The condensin core subunits (SMC2/SMC4) form different complexes and may have distinct functions at various stages of the parasite life cycle.

Impaired Death Receptor Signaling in Leukemia Causes Antigen-Independent Resistance by Inducing CAR T-cell Dysfunction.

Primary resistance to CD19-directed chimeric antigen receptor T-cell therapy (CART19) occurs in 10% to 20% of patients with acute lymphoblastic leukemia (ALL); however, the mechanisms of this resistance remain elusive. Using a genome-wide loss-of-function screen, we identified that impaired death receptor signaling in ALL led to rapidly progressive disease despite CART19 treatment. This was mediated by an inherent resistance to T-cell cytotoxicity that permitted antigen persistence and was subsequently magnified by the induction of CAR T-cell functional impairment. These findings were validated using samples from two CAR T-cell clinical trials in ALL, where we found that reduced expression of death receptor genes was associated with worse overall survival and reduced T-cell fitness. Our findings suggest that inherent dysregulation of death receptor signaling in ALL directly leads to CAR T-cell failure by impairing T-cell cytotoxicity and promoting progressive CAR T-cell dysfunction. SIGNIFICANCE: Resistance to CART19 is a significant barrier to efficacy in the treatment of B-cell malignancies. This work demonstrates that impaired death receptor signaling in tumor cells causes failed CART19 cytotoxicity and drives CART19 dysfunction, identifying a novel mechanism of antigen-independent resistance to CAR therapy.See related commentary by Green and Neelapu, p. 492.

Human chimeric antigen receptor macrophages for cancer immunotherapy.

Chimeric antigen receptor (CAR) T cell therapy has shown promise in hematologic malignancies, but its application to solid tumors has been challenging1-4. Given the unique effector functions of macrophages and their capacity to penetrate tumors5, we genetically engineered human macrophages with CARs to direct their phagocytic activity against tumors. We found that a chimeric adenoviral vector overcame the inherent resistance of primary human macrophages to genetic manipulation and imparted a sustained pro-inflammatory (M1) phenotype. CAR macrophages (CAR-Ms) demonstrated antigen-specific phagocytosis and tumor clearance in vitro. In two solid tumor xenograft mouse models, a single infusion of human CAR-Ms decreased tumor burden and prolonged overall survival. Characterization of CAR-M activity showed that CAR-Ms expressed pro-inflammatory cytokines and chemokines, converted bystander M2 macrophages to M1, upregulated antigen presentation machinery, recruited and presented antigen to T cells and resisted the effects of immunosuppressive cytokines. In humanized mouse models, CAR-Ms were further shown to induce a pro-inflammatory tumor microenvironment and boost anti-tumor T cell activity.