Bifunctional Transcription Factor Research Articles

Bifunctional transcription factors SlERF.H5 and H7 activate cell wall and repress gibberellin biosynthesis genes in tomato via a conserved motif

The plant cell wall is a dynamic structure that plays an essential role in development, but the mechanism regulating cell wall formation remains poorly understood. We demonstrate that two transcription factors, SlERF.H5 and SlERF.H7, control cell wall formation and tomato fruit firmness in an additive manner. Knockout of SlERF.H5, SlERF.H7, or both genes decreased cell wall thickness, firmness, and cellulose contents in fruits during early development, especially in double-knockout lines. Overexpressing either gene resulted in thicker cell walls and greater fruit firmness with elevated cellulose levels in fruits but severely dwarf plants with lower gibberellin contents. We further identified that SlERF.H5 and SlERF.H7 activate the cellulose biosynthesis gene SlCESA3 but repress the gibberellin biosynthesis gene GA20ox1. Moreover, we identified a conserved LPL motif in these ERFs responsible for their activities as transcriptional activators and repressors, providing insight into how bifunctional transcription factors modulate distinct developmental processes.

Genetic distinction between functional tissue-resident and conventional natural killer cells

Tissue-residential natural killer (trNK) cells act as pioneering responders during infectious challenges. However, their discrimination with conventional NK (cNK) cells is still an issue. Through an integrative transcriptome comparison of the two NK subgroups from different tissues, we have defined two genesets capable of efficiently distinguishing them. Based on the two genesets, a fundamental difference between the activation of trNK and cNK is identified and further confirmed. Mechanistically, we have discovered a particular role of chromatin landscape in regulating the trNK activation. In addition, IL-21R and IL-18R are respectively highly expressed by trNK and cNK, indicating a role of cytokine milieu in determining their differential activation. Indeed, IL-21 is particularly critical in accessorily promoting trNK activation using a bunch of bifunctional transcription factors. Together, this study sheds light on the bona fide difference between trNK and cNK, which will further expand our knowledge about their distinct functionalities during immune responses.

Further Delineation of Duplications of ARX Locus Detected in Male Patients with Varying Degrees of Intellectual Disability.

The X-linked gene encoding aristaless-related homeobox (ARX) is a bi-functional transcription factor capable of activating or repressing gene transcription, whose mutations have been found in a wide spectrum of neurodevelopmental disorders (NDDs); these include cortical malformations, paediatric epilepsy, intellectual disability (ID) and autism. In addition to point mutations, duplications of the ARX locus have been detected in male patients with ID. These rearrangements include telencephalon ultraconserved enhancers, whose structural alterations can interfere with the control of ARX expression in the developing brain. Here, we review the structural features of 15 gain copy-number variants (CNVs) of the ARX locus found in patients presenting wide-ranging phenotypic variations including ID, speech delay, hypotonia and psychiatric abnormalities. We also report on a further novel Xp21.3 duplication detected in a male patient with moderate ID and carrying a fully duplicated copy of the ARX locus and the ultraconserved enhancers. As consequences of this rearrangement, the patient-derived lymphoblastoid cell line shows abnormal activity of the ARX-KDM5C-SYN1 regulatory axis. Moreover, the three-dimensional (3D) structure of the Arx locus, both in mouse embryonic stem cells and cortical neurons, provides new insight for the functional consequences of ARX duplications. Finally, by comparing the clinical features of the 16 CNVs affecting the ARX locus, we conclude that—depending on the involvement of tissue-specific enhancers—the ARX duplications are ID-associated risk CNVs with variable expressivity and penetrance.

DkMYB14 is a bifunctional transcription factor that regulates the accumulation of proanthocyanidin in persimmon fruit

SUMMARYProanthocyanidins (PAs) are phenolic secondary metabolites that contribute to the protection of plant and human health. Persimmon (Diospyros kaki Thunb.) can accumulate abundant PAs in fruit, which cause a strong sensation of astringency. Proanthocyanidins can be classified into soluble and insoluble PAs; the former cause astringency but the latter do not. Soluble PAs can be converted into insoluble PAs upon interacting with acetaldehydes. We demonstrate here that DkMYB14, which regulates the accumulation of PA in persimmon fruit flesh, is a bifunctional transcription factor that acts as a repressor in PA biosynthesis but becomes an activator when involved in acetaldehyde biosynthesis. Interestingly, both functions contribute to the elimination of astringency by decreasing PA biosynthesis and promoting its insolubilization. We show that the amino acid Gly39 in the R2 domain and the ethylene response factor‐associated amphiphilic repression‐like motif in the C‐terminal of DkMYB14 are essential for the regulation of both PA and acetaldehyde synthesis. The repressive function of DkMYB14 was lost after the mutation of either motif, and all activities of DkMYB14 were eliminated following the mutation of both motifs. Our results demonstrate that DkMYB14 functions as both a transcriptional activator and a repressor, directly repressing biosynthesis of PA and promoting its insolubilization, resulting in non‐astringency in persimmon.

The miR166–SlHB15A regulatory module controls ovule development and parthenocarpic fruit set under adverse temperatures in tomato

Fruit set is inhibited by adverse temperatures, with consequences on yield. We isolated a tomato mutant producing fruits under non-permissive hot temperatures and identified the causal gene as SlHB15A, belonging to class III homeodomain leucine-zipper transcription factors. SlHB15A loss-of-function mutants display aberrant ovule development that mimics transcriptional changes occurring in fertilized ovules and leads to parthenocarpic fruit set under optimal and non-permissive temperatures, in field and greenhouse conditions. Under cold growing conditions, SlHB15A is subjected to conditional haploinsufficiency and recessive dosage sensitivity controlled by microRNA 166 (miR166). Knockdown of SlHB15A alleles by miR166 leads to a continuum of aberrant ovules correlating with parthenocarpic fruit set. Consistent with this, plants harboring an Slhb15a–miRNA166-resistant allele developed normal ovules and were unable to set parthenocarpic fruit under cold conditions. DNA affinity purification sequencing and RNA-sequencing analyses revealed that SlHB15A is a bifunctional transcription factor expressed in the ovule integument. SlHB15A binds to the promoters of auxin-related genes to repress auxin signaling and to the promoters of ethylene-related genes to activate their expression. A survey of tomato genetic biodiversity identified pat and pat-1, two historical parthenocarpic mutants, as alleles of SlHB15A. Taken together, our findings demonstrate the role of SlHB15A as a sentinel to prevent fruit set in the absence of fertilization and provide a mean to enhance fruiting under extreme temperatures.

Evidence That Runt Acts as a Counter-Repressor of Groucho During Drosophila melanogaster Primary Sex Determination

Runx proteins are bifunctional transcription factors that both repress and activate transcription in animal cells. Typically, Runx proteins work in concert with other transcriptional regulators, including co-activators and co-repressors to mediate their biological effects. In Drosophila melanogaster the archetypal Runx protein, Runt, functions in numerous processes including segmentation, neurogenesis and sex determination. During primary sex determination Runt acts as one of four X-linked signal element (XSE) proteins that direct female-specific activation of the establishment promoter (Pe) of the master regulatory gene Sex-lethal (Sxl). Successful activation of SxlPe requires that the XSE proteins overcome the repressive effects of maternally deposited Groucho (Gro), a potent co-repressor of the Gro/TLE family. Runx proteins, including Runt, contain a C-terminal peptide, VWRPY, known to bind to Gro/TLE proteins to mediate transcriptional repression. We show that Runt’s VWRPY co-repressor-interaction domain is needed for Runt to activate SxlPe. Deletion of the Gro-interaction domain eliminates Runt-ability to activate SxlPe, whereas replacement with a higher affinity, VWRPW, sequence promotes Runt-mediated transcription. This suggests that Runt may activate SxlPe by antagonizing Gro function, a conclusion consistent with earlier findings that Runt is needed for Sxl expression only in embryonic regions with high Gro activity. Surprisingly we found that Runt is not required for the initial activation of SxlPe. Instead, Runt is needed to keep SxlPe active during the subsequent period of high-level Sxl transcription suggesting that Runt helps amplify the difference between female and male XSE signals by counter-repressing Gro in female, but not in male, embryos.

An NAC domain transcription factor ATAF2 acts as transcriptional activator or repressor dependent on promoter context.

The ARABIDOPSIS THALIANA ACTIVATION FACTOR 2 (ATAF2) protein has been demonstrated to be involved in various biological processes including biotic stress responses, photo morphogenesis, and auxin catabolism. However, the transcriptional function of ATAF2 currently remains elusive. Therefore, to further understand the molecular function of ATAF2, we evaluated the transcriptional activities of ATAF2 using a transient assay system in this study. We used an effector consisting of a GAL4-DNA binding domain (GAL4-BD) fused to ATAF2, and observed upregulated reporter gene expression, suggesting that ATAF2 potentially has transcriptional activation activity. ATAF2 has been shown to activate reporter gene expression under the control of the ORE1 promoter. By contrast, ATAF2 significantly repressed reporter gene expression driven by the NIT2 promoter. These data suggest that ATAF2 is a bifunctional transcription factor that can alter target gene expression depending on the promoter sequences.

Transcriptional profiling of Zygosaccharomyces bailii early response to acetic acid or copper stress mediated by ZbHaa1

The non-conventional yeast species Zygosaccharomyces bailii is remarkably tolerant to acetic acid, a highly important microbial inhibitory compound in Food Industry and Biotechnology. ZbHaa1 is the functional homologue of S. cerevisiae Haa1 and a bifunctional transcription factor able to modulate Z. bailii adaptive response to acetic acid and copper stress. In this study, RNA-Seq was used to investigate genomic transcription changes in Z. bailii during early response to sublethal concentrations of acetic acid (140 mM, pH 4.0) or copper (0.08 mM) and uncover the regulatory network activated by these stresses under ZbHaa1 control. Differentially expressed genes in response to acetic acid exposure (297) are mainly related with the tricarboxylic acid cycle, protein folding and stabilization and modulation of plasma membrane composition and cell wall architecture, 17 of which, directly or indirectly, ZbHaa1-dependent. Copper stress induced the differential expression of 190 genes mainly involved in the response to oxidative stress, 15 ZbHaa1-dependent. This study provides valuable mechanistic insights regarding Z. bailii adaptation to acetic acid or copper stress, as well as useful information on transcription regulatory networks in pre-whole genome duplication (WGD) (Z. bailii) and post-WGD (S. cerevisiae) yeast species, contributing to the understanding of transcriptional networks’ evolution in yeasts.

Hunchback is counter-repressed to regulate even-skipped stripe 2 expression in Drosophila embryos.

Hunchback is a bifunctional transcription factor that can activate and repress gene expression in Drosophila development. We investigated the regulatory DNA sequence features that control Hunchback function by perturbing enhancers for one of its target genes, even-skipped (eve). While Hunchback directly represses the eve stripe 3+7 enhancer, we found that in the eve stripe 2+7 enhancer, Hunchback repression is prevented by nearby sequences—this phenomenon is called counter-repression. We also found evidence that Caudal binding sites are responsible for counter-repression, and that this interaction may be a conserved feature of eve stripe 2 enhancers. Our results alter the textbook view of eve stripe 2 regulation wherein Hb is described as a direct activator. Instead, to generate stripe 2, Hunchback repression must be counteracted. We discuss how counter-repression may influence eve stripe 2 regulation and evolution.

CRABS CLAW Acts as a Bifunctional Transcription Factor in Flower Development.

One of the crucial steps in the life cycle of angiosperms is the development of carpels. They are the most complex plant organs, harbor the seeds, and, after fertilization, develop into fruits and are thus an important ecological and economic trait. CRABS CLAW (CRC), a YABBY protein and putative transcription factor, is one of the major carpel developmental regulators in A. thaliana that includes a C2C2 zinc finger and a domain with similarities to an HMG box. CRC is involved in the regulation of processes such as carpel fusion and growth, floral meristem termination, and nectary formation. While its genetic interactions with other carpel development regulators are well described, its biochemical properties and molecular way of action remain unclear. We combined Bimolecular Fluorescence Complementation, Yeast Two-Hybrid, and Yeast One-Hybrid analyzes to shed light on the molecular biology of CRC. Our results showed that CRC dimerizes, also with other YABBY proteins, via the YABBY domain, and that its DNA binding is mainly cooperative and is mediated by the YABBY domain. Further, we identified that CRC is involved in floral meristem termination via transcriptional repression while it acts as a transcriptional activator in nectary development and carpel fusion and growth control. This work increases our understanding on how YABBY transcription factors interact with other proteins and how they regulate their targets.

Distinct pathways of RNA polymerase regulation by a phage-encoded factor

Transcription antitermination is a common strategy of gene expression regulation, but only a few transcription antitermination factors have been studied in detail. Here, we dissect the transcription antitermination mechanism of Xanthomonas oryzae virus Xp10 protein p7, which binds host RNA polymerase (RNAP) and regulates both transcription initiation and termination. We show that p7 suppresses intrinsic termination by decreasing RNAP pausing and increasing the transcription complex stability, in cooperation with host-encoded factor NusA. Uniquely, the antitermination activity of p7 depends on the ω subunit of the RNAP core and is modulated by ppGpp. In contrast, the inhibition of transcription initiation by p7 does not require ω but depends on other RNAP sites. Our results suggest that p7, a bifunctional transcription factor, uses distinct mechanisms to control different steps of transcription. We propose that regulatory functions of the ω subunit revealed by our analysis may extend to its homologs in eukaryotic RNAPs.

Driving with the Brakes on: An Incoherent Transcriptional Circuit Patterns the Drosophila Embryo

We used modeling and quantitative experiments to uncover a case where a bifunctional transcription factor regulates the same expression pattern in opposite ways. I will discuss the functional implications of this type of transcriptional circuit, and how our results highlight the need for new methods to determine how promoters integrate information from multiple enhancers.

A Predictive Model of Bifunctional Transcription Factor Signaling during Embryonic Tissue Patterning

Hedgehog signaling controls pattern formation in many vertebrate tissues. The downstream effectors of the pathway are the bifunctional Gli transcription factors, which, depending on hedgehog concentration, act as either transcriptional activators or repressors. Quantitatively understanding the interplay between Gli activator and repressor forms for patterning complex tissues is an open challenge. Here, we describe a reductionist mathematical model for how Gli activators and repressors are integrated in space and time to regulate transcriptional outputs of hedgehog signaling, using the pathway readouts Gli1 and Ptch1 as a model system. Spatially resolved measurements of absolute transcript numbers for these genes allow us to infer spatiotemporal variations of Gli activator and repressor levels. We validate our model by successfully predicting expression changes of Gli1 and Ptch1 in mutants atdifferent developmental stages and in different tissues. Our results provide a starting point for understanding gene regulation by bifunctional transcription factors during mammalian development.

A Positive Regulator of Nodule Organogenesis, NODULE INCEPTION, Acts as a Negative Regulator of Rhizobial Infection in Lotus japonicus.

Legume-rhizobium symbiosis occurs in specialized root organs called nodules. To establish the symbiosis, two major genetically controlled events, rhizobial infection and organogenesis, must occur. For a successful symbiosis, it is essential that the two phenomena proceed simultaneously in different root tissues. Although several symbiotic genes have been identified during genetic screenings of nonsymbiotic mutants, most of the mutants harbor defects in both infection and organogenesis pathways, leading to experimental difficulty in investigating the molecular genetic relationships between the pathways. In this study, we isolated a novel nonnodulation mutant, daphne, in Lotus japonicus that shows complete loss of nodulation but a dramatically increased numbers of infection threads. Characterization of the locus responsible for these phenotypes revealed a chromosomal translocation upstream of NODULE INCEPTION (NIN) in daphne. Genetic analysis using a known nin mutant revealed that daphne is a novel nin mutant allele. Although the daphne mutant showed reduced induction of NIN after rhizobial infection, the spatial expression pattern of NIN in epidermal cells was broader than that in the wild type. Overexpression of NIN strongly suppressed hyperinfection in daphne, and daphne phenotypes were partially rescued by cortical expression of NIN. These observations suggested that the daphne mutation enhanced the role of NIN in the infection pathway due to a specific loss of the role of NIN in nodule organogenesis. Based on these results, we provide evidence that the bifunctional transcription factor NIN negatively regulates infection but positively regulates nodule organogenesis during the course of the symbiosis.

Nitric Oxide and KLF4 Protein Epigenetically Modify Class II Transactivator to Repress Major Histocompatibility Complex II Expression during Mycobacterium bovis Bacillus Calmette-Guérin Infection

Pathogenic mycobacteria employ several immune evasion strategies such as inhibition of class II transactivator (CIITA) and MHC-II expression, to survive and persist in host macrophages. However, precise roles for specific signaling components executing down-regulation of CIITA/MHC-II have not been adequately addressed. Here, we demonstrate that Mycobacterium bovis bacillus Calmette-Guérin (BCG)-mediated TLR2 signaling-induced iNOS/NO expression is obligatory for the suppression of IFN-γ-induced CIITA/MHC-II functions. Significantly, NOTCH/PKC/MAPK-triggered signaling cross-talk was found critical for iNOS/NO production. NO responsive recruitment of a bifunctional transcription factor, KLF4, to the promoter of CIITA during M. bovis BCG infection of macrophages was essential to orchestrate the epigenetic modifications mediated by histone methyltransferase EZH2 or miR-150 and thus calibrate CIITA/MHC-II expression. NO-dependent KLF4 regulated the processing and presentation of ovalbumin by infected macrophages to reactive T cells. Altogether, our study delineates a novel role for iNOS/NO/KLF4 in dictating the mycobacterial capacity to inhibit CIITA/MHC-II-mediated antigen presentation by infected macrophages and thereby elude immune surveillance.

Loss of Krüppel-Like Factor 4 (KLF4) Impairs the Self-Renewal of Leukemic Stem Cells

Abstract 911The identification of pathways that differentially regulate the self-renewal in normal hematopoietic stem cells (HSC) and leukemic stem cells (LSC) is critical for effective treatment of relapsed leukemia without affecting normal hematopoiesis. Krüppel-like factor 4 (KLF4) is a bi-functional transcription factor that can either activate or repress transcription and thus function as an oncogene or a tumor suppressor depending on the cellular context. We have previously shown that loss of KLF4 leads to increased self-renewal of HSC in serial bone marrow transplantation. In this work, we investigated whether KLF4 also regulates self-renewal in LSCs using a mouse model of somatic deletion of KLF4 in hematopoietic cells (Klf4fl/fl Vav-iCre+) and the BCR-ABL-induced chronic myeloid leukemia (CML) model. Mice transplanted with wild type bone marrow (BM) cells transduced with retrovirus carrying BCR-ABL (p210) (n=18) showed an expansion of myeloid cells (GFP+ Gr1+) with a median survival of 19 days. In contrast, mice transplanted with KLF4-deficient BM cells transduced with BCR-ABL retrovirus (n=12) exhibited prolonged survival with a median survival of 27 days (p<0.0011). Progression of leukemia was monitored in peripheral blood, BM and the spleen by flow cytometry. Mice transplanted with KLF4-deficient cells showed expansion of myeloid leukemic cells (GFP+ Gr1+) in the first two weeks after BM transplant, which was followed by a progressive loss of myeloid cells and an expansion of B cells (GFP+ B220+). In control group, 90% of leukemic mice succumbed from CML whereas more than 50% of KLF4-deficient leukemic mice developed mixed CML and B-ALL leukemia. These results suggest that loss of KLF4 impairs the maintenance of BCR-ABL-induced CML, while allowing expansion of BCR-ABL positive B cells (GFP+ B220+). Since KLF4 deletion prevented continuous expansion of myeloid leukemic cells (GFP+ Gr1+), we postulated that KLF4 may be critical to LSC maintenance. Thus, we analyzed the frequency of LSCs (GFP+ Lin–c-Kit+ Sca-1+and GFP+ Lin– c-Kit+ Sca-1+Flt3–) in BM and the spleen. We found that LSCs were significantly reduced in recipients of BCR-ABL-transduced KLF4-deficient BM cells 18 days after transplantation (n=6, p <0.001). These studies demonstrate that KLF4 has different roles in the maintainenance of LSCs and normal HSCs and that inactivation of KLF4 provides a therapeutic strategy for eradicating LSCs without damaging normal HSC. Disclosures:No relevant conflicts of interest to declare.

FILAMENTOUS FLOWER controls lateral organ development by acting as both an activator and a repressor

BackgroundThe YABBY (YAB) family of transcription factors participate in a diverse range of processes that include leaf and floral patterning, organ growth, and the control of shoot apical meristem organisation and activity. How these disparate functions are regulated is not clear, but based on interactions with the LEUNIG-class of co-repressors, it has been proposed that YABs act as transcriptional repressors. In the light of recent work showing that DNA-binding proteins associated with the yeast co-repressor TUP1 can also function as activators, we have examined the transcriptional activity of the YABs.ResultsOf the four Arabidopsis YABs tested in yeast, only FILAMENTOUS FLOWER (FIL) activated reporter gene expression. Similar analysis with Antirrhinum YABs identified the FIL ortholog GRAMINIFOLIA as an activator. Plant-based transactivation assays not only confirmed the potential of FIL to activate transcription, but also extended this property to the FIL paralog YABBY3 (YAB3). Subsequent transcriptomic analysis of lines expressing a steroid-inducible FIL protein revealed groups of genes that responded either positively or negatively to YAB induction. Included in the positively regulated group of genes were the polarity regulators KANADI1 (KAN1), AUXIN RESPONSE FACTOR 4 (ARF4) and ASYMMETRIC LEAVES1 (AS1). We also show that modifying FIL to function as an obligate repressor causes strong yab loss-of-function phenotypes.ConclusionsCollectively these data show that FIL functions as a transcriptional activator in plants and that this activity is involved in leaf patterning. Interestingly, our study also supports the idea that FIL can act as a repressor, as transcriptomic analysis identified negatively regulated FIL-response genes. To reconcile these observations, we propose that YABs are bifunctional transcription factors that participate in both positive and negative regulation. These findings fit a model of leaf development in which adaxial/abaxial patterning is maintained by a regulatory network consisting of positive feedback loops.

Molecular basis for the specification of floral organs by APETALA3 and PISTILLATA

How different organs are formed from small sets of undifferentiated precursor cells is a key question in developmental biology. To understand the molecular mechanisms underlying organ specification in plants, we studied the function of the homeotic selector genes APETALA3 (AP3) and PISTILLATA (PI), which control the formation of petals and stamens during Arabidopsis flower development. To this end, we characterized the activities of the transcription factors that AP3 and PI encode throughout flower development by using perturbation assays as well as transcript profiling and genomewide localization studies, in combination with a floral induction system that allows a stage-specific analysis of flower development by genomic technologies. We discovered considerable spatial and temporal differences in the requirement for AP3/PI activity during flower formation and show that they control different sets of genes at distinct phases of flower development. The genomewide identification of target genes revealed that AP3/PI act as bifunctional transcription factors: they activate genes involved in the control of numerous developmental processes required for organogenesis and repress key regulators of carpel formation. Our results imply considerable changes in the composition and topology of the gene network controlled by AP3/PI during the course of flower development. We discuss our results in light of a model for the mechanism underlying sex-determination in seed plants, in which AP3/PI orthologues might act as a switch between the activation of male and the repression of female development.

Upregulation of KLF4 Expression in Pediatric B-NHL Lymphomas and Its Association with Low EFS and Poor Survival Following Treatment with Chemotherapy

Abstract 5184Krüppel-like factor 4 (KLF4) is a transcription factor expressed in a variety of tissues in humans and has been implicated in several physiologic processes including development, differentiation, and tissue homeostasis. KLF4 is a bi-functional transcription factor that can either activate or repress transcription depending on the target gene. Human KLF4 is a protein of 470 amino acids with a 55 kDa. It contains three C-terminal C2H2-type zinc fingers that bind DNA. It is divided into three separate domains, namely, an N-terminal activation domain, a central repression domain and a C-terminal DNA-binding domain. For instance, KLF4 acts as a tumor suppressor gene in several cancers (colon, gastric, esophageal, bladder, and NSCLC) or as an oncogene (laryngeal carcinoma, squamous cell carcinoma, ductal carcinoma of the breast). However, the role of KLF4 in hematologic malignancies is still poorly understood. Reported studies in leukemia suggested that KLF4 may be a tumor suppressor. The goal of this study was to investigate the expression and the clinical significance of KLF4 in B-Non-Hodgkin’s lymphomas (B-NHLs). Both B-NHL cell lines and patient-derived tumor tissues (TMA) were examined by western blot and immunohistochemistry, respectively. The expression of KLF4 was calculated based on the intensity and the percentage of the area stained, and scoring was corroborated by two pathologists. The complete absence of KLF4 expression was considered as KLF4 negative. Normal peripheral blood mononuclear cells expressed low levels of KLF4, in contrast, there was a significant overexpression of KLF4 in Ramos and Raji (Burkitt’s lymphoma) and 2F7 (AIDS lymphoma) B-NHL cell lines. However, the DHL4 (DBLCL) cell line showed similar expression to normal cells. Among the 73 childhood lymphomas studied, 13/23 (57%) of lymphoblastic lymphoma, 7/20 (35%) of large B-cell lymphoma, 4/4 (100%) of anaplastic large cell lymphoma and 5/6 NHL not otherwise specified were KLF4 positive. Noteworthy, 18/18 (100%) Burkitt’s lymphoma was KLF4 positive. In addition, the nuclear expression of KLF4 was significantly higher in Burkitt’s lymphoma (n=18) compared to the remaining subtypes (lymphoblastic lymphoma, n=23, large B-cell lymphoma n=20 and others). All patients were treated with chemotherapy and the majority of the patients that were KLF4 positive had a stage 3–4 disease. Analysis of the EFS demonstrated that patients’ tumors that were KLF4 negative had significantly higher EFS as compared to tumors that were KLF4 positive. Likewise, there was significant prolongation of survival in patients with tumors that were KLF4 negative. We suggest that the expression of KLF4 and poor response to chemotherapy may be attributed to its role in resistance via its regulation by the resistance factor Notch31. In contrast, the absence of KLF4 and good response to chemotherapy may be due to shifting p53 activity from cellular repair to cell death2. The present findings demonstrate that KLF4 may be considered as an oncogene in Burkitt’s lymphoma and subsets of other types of lymphoma. The findings also suggest that the expression of KLF4 may be a potential prognostic factor, though, this need to be validated in a large cohort of patients. We propose that KLF4 may be a therapeutic target in patients with B-NHL lymphomas. Disclosures:No relevant conflicts of interest to declare.

Peroxisome Proliferator-activated Receptor γ (PPARγ) Mediates a Ski Oncogene-induced Shift from Glycolysis to Oxidative Energy Metabolism

Overexpression of the Ski oncogene induces oncogenic transformation of chicken embryo fibroblasts (CEFs). However, unlike most other oncogene-transformed cells, Ski-transformed CEFs (Ski-CEFs) do not display the classical Warburg effect. On the contrary, Ski transformation reduced lactate production and glucose utilization in CEFs. Compared with CEFs, Ski-CEFs exhibited enhanced TCA cycle activity, fatty acid catabolism through β-oxidation, glutamate oxidation, oxygen consumption, as well as increased numbers and mass of mitochondria. Interestingly, expression of PPARγ, a key transcription factor that regulates adipogenesis and lipid metabolism, was dramatically elevated at both the mRNA and protein levels in Ski-CEFs. Accordingly, PPARγ target genes that are involved in lipid uptake, transport, and oxidation were also markedly up-regulated by Ski. Knocking down PPARγ in Ski-CEFs by RNA interference reversed the elevated expression of these PPARγ target genes, as well as the shift to oxidative metabolism and the increased mitochondrial biogenesis. Moreover, we found that Ski co-immunoprecipitates with PPARγ and co-activates PPARγ-driven transcription.