Loss of synergistic transcriptional feedback loops drives diverse B-cell cancers.

BACKGROUND: The most common B-cell cancers, chronic lymphocytic leukemia/lymphoma (CLL), follicular and diffuse large B-cell (FL, DLBCL) lymphomas, have distinct clinical courses, yet overlapping "cell-of-origin". Dynamic changes to the epigenome are essential regulators of B-cell differentiation. Therefore, we reasoned that these distinct cancers may be driven by shared mechanisms of disruption in transcriptional circuitry. METHODS: We compared purified malignant B-cells from 52 patients with normal B-cell subsets (germinal center centrocytes and centroblasts, naïve and memory B-cells) from 36 donor tonsils using >325 high-resolution molecular profiling assays for histone modifications, open chromatin (ChIP-, FAIRE-seq), transcriptome (RNA-seq), transcription factor (TF) binding, and genome copy number (microarrays). FINDINGS: From the resulting data, we identified gains in active chromatin in enhancers/super-enhancers that likely promote unchecked B-cell receptor signaling, including one we validated near the immunoglobulin superfamily receptors FCMR and PIGR. More striking and pervasive was the profound loss of key B-cell identity TFs, tumor suppressors and their super-enhancers, including EBF1, OCT2(POU2F2), and RUNX3. Using a novel approach to identify transcriptional feedback, we showed that these core transcriptional circuitries are self-regulating. Their selective gain and loss form a complex, iterative, and interactive process that likely curbs B-cell maturation and spurs proliferation. INTERPRETATION: Our study is the first to map the transcriptional circuitry of the most common blood cancers. We demonstrate that a critical subset of B-cell TFs and their cognate enhancers form self-regulatory transcriptional feedback loops whose disruption is a shared mechanism underlying these diverse subtypes of B-cell lymphoma. FUNDING: National Institute of Health, Siteman Cancer Center, Barnes-Jewish Hospital Foundation, Doris Duke Foundation.

Mitigation of deleterious phenotypes in chloroplast-engineered plants accumulating high levels of foreign proteins.

BACKGROUND: The global demand for functional proteins is extensive, diverse, and constantly increasing. Medicine, agriculture, and industrial manufacturing all rely on high-quality proteins as major active components or process additives. Historically, these demands have been met by microbial bioreactors that are expensive to operate and maintain, prone to contamination, and relatively inflexible to changing market demands. Well-established crop cultivation techniques coupled with new advancements in genetic engineering may offer a cheaper and more versatile protein production platform. Chloroplast-engineered plants, like tobacco, have the potential to produce large quantities of high-value proteins, but often result in engineered plants with mutant phenotypes. This technology needs to be fine-tuned for commercial applications to maximize target protein yield while maintaining robust plant growth. RESULTS: Here, we show that a previously developed Nicotiana tabacum line, TetC-cel6A, can produce an industrial cellulase at levels of up to 28% of total soluble protein (TSP) with a slight dwarf phenotype but no loss in biomass. In seedlings, the dwarf phenotype is recovered by exogenous application of gibberellic acid. We also demonstrate that accumulating foreign protein represents an added burden to the plants' metabolism that can make them more sensitive to limiting growth conditions such as low nitrogen. The biomass of nitrogen-limited TetC-cel6A plants was found to be as much as 40% lower than wildtype (WT) tobacco, although heterologous cellulase production was not greatly reduced compared to well-fertilized TetC-cel6A plants. Furthermore, cultivation at elevated carbon dioxide (1600 ppm CO2) restored biomass accumulation in TetC-cel6A plants to that of WT, while also increasing total heterologous protein yield (mg Cel6A plant-1) by 50-70%. CONCLUSIONS: The work reported here demonstrates that well-fertilized tobacco plants have a substantial degree of flexibility in protein metabolism and can accommodate considerable levels of some recombinant proteins without exhibiting deleterious mutant phenotypes. Furthermore, we show that the alterations to protein expression triggered by growth at elevated CO2 can help rebalance endogenous protein expression and/or increase foreign protein production in chloroplast-engineered tobacco.

Field-grown tobacco plants maintain robust growth while accumulating large quantities of a bacterial cellulase in chloroplasts.

High accumulation of heterologous proteins expressed from the plastid genome has sometimes been reported to result in compromised plant phenotypes. Comparisons of transplastomic plants to wild-type (WT) are typically made in environmentally controlled chambers with relatively low light; little is known about the performance of such plants under field conditions. Here, we report on two plastid-engineered tobacco lines expressing the bacterial cellulase Cel6A. Field-grown plants producing Cel6A at ~20% of total soluble protein exhibit no loss in biomass or Rubisco content and only minor reductions in photosynthesis compared to WT. These experiments demonstrate that, when grown in the field, tobacco possesses sufficient metabolic flexibility to accommodate high levels of recombinant protein by increasing total protein synthesis and accumulation and/or by reallocating unneeded endogenous proteins. Based on current tobacco cultivation practices and readily achievable recombinant protein yields, we estimate that specific proteins could be obtained from field-grown transgenic tobacco plants at costs three orders of magnitude less than current cell culture methods.

Novel cell adhesion/migration pathways are predictive markers of HDAC inhibitor resistance in cutaneous T cell lymphoma.

BACKGROUND: Treatment for Cutaneous T Cell Lymphoma (CTCL) is generally not curative. Therefore, selecting therapy that is effective and tolerable is critical to clinical decision-making. Histone deacetylase inhibitors (HDACi), epigenetic modifier drugs, are commonly used but effective in only ~30% of patients. There are no predictive markers of HDACi response and the CTCL histone acetylation landscape remains unmapped. We sought to identify pre-treatment molecular markers of resistance in CTCL that progressed on HDACi therapy. METHODS: Purified T cells from 39 pre/post-treatment peripheral blood samples and skin biopsies from 20 patients were subjected to RNA-seq and ChIP-seq for histone acetylation marks (H3K14/9 ac, H3K27ac). We correlated significant differences in histone acetylation with gene expression in HDACi-resistant/sensitive CTCL. We extended these findings in additional CTCL patient cohorts (RNA-seq, microarray) and using ELISA in matched CTCL patient plasma. FINDINGS: Resistant CTCL exhibited high levels of histone acetylation, which correlated with increased expression of 338 genes (FDR < 0·05), including some novel to CTCL: BIRC5 (anti-apoptotic); RRM2 (cell cycle); TXNDC5, GSTM1 (redox); and CXCR4, LAIR2 (cell adhesion/migration). Several of these, including LAIR2, were elevated pre-treatment in HDACi-resistant CTCL. In CTCL patient plasma (n = 6), LAIR2 protein was also elevated (p < 0·01) compared to controls. INTERPRETATION: This study is the first to connect genome-wide differences in chromatin acetylation and gene expression to HDACi-resistance in primary CTCL. Our results identify novel markers with high pre-treatment expression, such as LAIR2, as potential prognostic and/or predictors of HDACi-resistance in CTCL. FUNDING: NIH:CA156690, CA188286; NCATS: WU-ICTS UL1 TR000448; Siteman Cancer Center: CA091842.

Examining Relationships among Choice, Affect, and Engagement in Summer STEM Programs.

Out-of-school time programs focused on science, technology, engineering and mathematics (STEM) have proliferated recently because they are seen as having potential to appeal to youth and enhance STEM interest. Although such programs are not mandatory, youth are not always involved in making the choice about their participation and it is unclear whether youth's involvement in the choice to attend impacts their program experiences. Using data collected from experience sampling, traditional surveys, and video recordings, we explore relationships among youth's choice to attend out-of-school time programs (measured through a pre-survey) and their experience of affect (i.e., youth experience sampling ratings of happiness and excitement) and engagement (i.e., youth experience sampling ratings of concentration and effort) during program activities. Data were collected from a racially and ethnically diverse sample of 10-16 year old youth (n = 203; 50% female) enrolled in nine different summer STEM programs targeting underserved youth. Multilevel analysis indicated that choice and affect are independently and positively associated with momentary engagement. Though choice to enroll was a significant predictor of momentary engagement, positive affective experiences during the program may compensate for any decrements to engagement associated with lack of choice. Together, these findings have implications for researchers, parents, and educators and administrators of out-of-school time programming.

Targeted epigenetic repression of a lymphoma oncogene by sequence-specific histone modifiers induces apoptosis in DLBCL.

Alterations to the epigenetic landscape of diffuse large B-cell lymphoma (DLBCL) play a fundamental role in deregulating genes involved in normal lymphocyte differentiation. To determine whether targeted epigenetic therapy could reverse these pathogenic chromatin changes and suppress the expression of a lymphoma oncogene, we focused on BCL6, a transcriptional repressor whose aberrant expression is tightly linked to DLBCL proliferation and survival. We fused zinc-finger (ZF) domains specific for regulatory regions in the BCL6 locus to a repressive epigenetic modifier, the Kruppel-associated box (KRAB) repressor domain. Distinct ZF-KRAB fusions repressed the local chromatin landscape, suppressed BCL6 expression, significantly impaired DLBCL growth, and caused widespread cell death in a BCL6-dependent manner. Importantly, expression of ectopic BCL6 protein rescued ZF-KRAB-induced cell death, demonstrating the modifiers' specificity. We show that sequence-specific epigenetic modifiers can alter oncogene expression and induce apoptosis in cancer cells, underscoring their potential for future development as targeted epigenetic protein therapies.

Does Mindset Intervention Predict Students' Daily Experience in Classrooms? A Comparison of Seventh and Ninth Graders' Trajectories.

One's beliefs about whether ability is fixed or malleable-also known as fixed or growth mindset-can impact academic outcomes. This quasi-experimental study investigated effects of a six-week classroom intervention targeting growth mindset on students' daily quality of experience in science classrooms. Seventh grade (N = 370) and 9th grade (N = 356) students (50 % female, 61 % Hispanic) were randomly assigned by classroom to either a mindset intervention condition or content writing task condition. Students provided self-reports on multiple aspects of their daily classroom experience 11 times across the school year. Hierarchical linear growth models indicate that 7th and 9th grade students who were not exposed to the mindset intervention showed declines in perceived control skill, interest, and learning. In contrast, 9th graders in the mindset intervention reported increased control and interest, and maintained constant levels in skill and learning. Similar effects were not observed among 7th graders. The results are discussed in terms of implications for future research and optimal developmental periods for mindset intervention.

Enhancer sequence variants and transcription-factor deregulation synergize to construct pathogenic regulatory circuits in B-cell lymphoma.

Most B-cell lymphomas arise in the germinal center (GC), where humoral immune responses evolve from potentially oncogenic cycles of mutation, proliferation, and clonal selection. Although lymphoma gene expression diverges significantly from GC B cells, underlying mechanisms that alter the activities of corresponding regulatory elements (REs) remain elusive. Here we define the complete pathogenic circuitry of human follicular lymphoma (FL), which activates or decommissions REs from normal GC B cells and commandeers enhancers from other lineages. Moreover, independent sets of transcription factors, whose expression was deregulated in FL, targeted commandeered versus decommissioned REs. Our approach revealed two distinct subtypes of low-grade FL, whose pathogenic circuitries resembled GC B or activated B cells. FL-altered enhancers also were enriched for sequence variants, including somatic mutations, which disrupt transcription-factor binding and expression of circuit-linked genes. Thus, the pathogenic regulatory circuitry of FL reveals distinct genetic and epigenetic etiologies for GC B-cell transformation.

Associations of participation in service activities with academic, behavioral, and civic outcomes of adolescents at varying risk levels.

Youth who participate in service activities differ from those who do not on a number of key demographic characteristics like socio-economic status and other indicators of risk; and most studies demonstrating positive outcomes among service participants employ small non-representative samples. Thus, there is little evidence as to whether the outcomes associated with service participation are similar among students with varying levels of risk. The National Household Education Survey of 1999, a large nationally representative cross-sectional data set that focused on community service, was analyzed to investigate associations between the risk status of 4,306 adolescent students (50.2% female; 63.3% European American, M age = 15.9), their participation in community service, and their academic adjustment, behavioral problems, and civic knowledge. Because adolescents who participate in service differ from those who do not with respect to demographic characteristics, propensity score analyses were used to correct for potential selection bias in the examination of these relationships. Analyses tested competing theoretical models of service-protective versus compensatory-among students at varying levels of risk, and suggested that service acts as a compensatory factor with respect to academic, behavioral, and civic outcomes. Propensity score analyses revealed patterns suggesting that, in some cases, students with certain demographic profiles that are themselves related to the likelihood of service participation may benefit from service participation more than others. Findings are discussed in terms of their significance for adolescent development, for planning service programs, and for educational policy.

Seborrheic dermatitis: a clinical practice snapshot.

Seborrheic dermatitis is a chronic, recurring skin disorder that has no cure.Current clinical research has implicated Malassezia yeast in the etiology. Using a clear, concise clinical picture and a thorough patient history, even the novice NP can formulate an effective treatment plan.

Regulation of the CgPdr1 transcription factor from the pathogen Candida glabrata.

Candida glabrata is an opportunistic human pathogen that is increasingly associated with candidemia, owing in part to the intrinsic and acquired high tolerance the organism exhibits for the important clinical antifungal drug fluconazole. This elevated fluconazole resistance often develops through gain-of-function mutations in the zinc cluster-containing transcriptional regulator C. glabrata Pdr1 (CgPdr1). CgPdr1 induces the expression of an ATP-binding cassette (ABC) transporter-encoding gene, CgCDR1. Saccharomyces cerevisiae has two CgPdr1 homologues called ScPdr1 and ScPdr3. These factors control the expression of an ABC transporter-encoding gene called ScPDR5, which encodes a homologue of CgCDR1. Loss of the mitochondrial genome (ρ(0) cell) or overexpression of the mitochondrial enzyme ScPsd1 induces ScPDR5 expression in a strictly ScPdr3-dependent fashion. ScPdr3 requires the presence of a transcriptional Mediator subunit called Gal11 (Med15) to fully induce ScPDR5 transcription in response to ρ(0) signaling. ScPdr1 does not respond to either ρ(0) signals or ScPsd1 overproduction. In this study, we employed transcriptional fusions between CgPdr1 target promoters, like CgCDR1, to demonstrate that CgPdr1 stimulates gene expression via binding to elements called pleiotropic drug response elements (PDREs). Deletion mapping and electrophoretic mobility shift assays demonstrated that a single PDRE in the CgCDR1 promoter was capable of supporting ρ(0)-induced gene expression. Removal of one of the two ScGal11 homologues from C. glabrata caused a major defect in drug-induced expression of CgCDR1 but had a quantitatively minor effect on ρ(0)-stimulated transcription. These data demonstrate that CgPdr1 appears to combine features of ScPdr1 and ScPdr3 to produce a transcription factor with chimeric regulatory properties.

Evidence for the bifunctional nature of mitochondrial phosphatidylserine decarboxylase: role in Pdr3-dependent retrograde regulation of PDR5 expression.

Multidrug resistance in the yeast Saccharomyces cerevisiae is sensitive to the mitochondrial genome status of cells. Cells that lose their organellar genome ([rho(0)] cells) dramatically induce transcription of multiple or pleiotropic drug resistance genes via increased expression of a zinc cluster-containing transcription factor designated Pdr3. A major Pdr3 target gene is the ATP-binding cassette transporter-encoding gene PDR5. Pdr5 has been demonstrated to act as a phospholipid floppase catalyzing the net outward movement of phosphatidylethanolamine (PE). Since the mitochondrially localized Psd1 enzyme provides a major route of PE biosynthesis, we evaluated the potential linkage between Psd1 function and PDR5 regulation. Overproduction of Psd1 in wild-type ([rho(+)]) cells was found to induce PDR5 transcription and drug resistance in a Pdr3-dependent manner. Loss of the PSD1 gene from [rho(0)] cells prevented the normal activation of PDR5 expression. Surprisingly, expression of a catalytically inactive form of Psd1 still supported PDR5 transcriptional activation, suggesting that PE levels were not the signal triggering PDR5 induction. Expression of green fluorescent protein fusions mapped the region required to induce PDR5 expression to the noncatalytic amino-terminal portion of Psd1. Psd1 is a novel bifunctional protein required both for PE biosynthesis and regulation of multidrug resistance.

Taurine monochloramine activates a cell death pathway involving Bax and Caspase-9.

Taurine is an abundant free amino acid that interacts with the potent oxidant hypochlorous acid to form the less toxic and more stable oxidant taurine monochloramine (TauNHCl). TauNHCl has diverse cellular effects ranging from inhibiting the production of proinflammatory mediators to inhibiting cell proliferation and inducing cell death. We hypothesized that TauNHCl could activate a cell death pathway involving Bcl-2 members and the activation of caspase proteases. FL5.12 cells are lymphocytic cells that undergo apoptosis following interleukin-3 (IL-3) withdrawal. Therefore, cell death following TauNHCl treatment of FL5.12 cells was compared and contrasted with IL-3 withdrawal. We found that TauNHCl treatment activates a cell death pathway with kinetics very similar to IL-3 withdrawal. TauNHCl-treated cells undergo an annexin V-positive/propidium iodide-negative phase of death consistent with apoptosis. TauNHCl treatment results in a conformational change in BAX that is associated with its activation. Both Bcl-2 and, to a lesser degree, the dominant negative form of caspase-9 inhibit cell death following TauNHCl treatment. In contrast with IL-3 withdrawal, TauNHCl treatment of FL5.12 cells results in a rapid cell cycle arrest that is cell cycle phase-independent. These results demonstrate that TauNHCl treatment induces a rapid, cell cycle-independent proliferative arrest followed by the activation of a cell death pathway involving Bcl-2 family members and caspase activation.