Myb Binding Sites Research Articles

Peptidomimetic blockade of MYB in acute myeloid leukemia

Aberrant gene expression is a hallmark of acute leukemias. MYB-driven transcriptional coactivation with CREB-binding protein (CBP)/P300 is required for acute lymphoblastic and myeloid leukemias, including refractory MLL-rearranged leukemias. Using structure-guided molecular design, we developed a peptidomimetic inhibitor MYBMIM that interferes with the assembly of the molecular MYB:CBP/P300 complex and rapidly accumulates in the nuclei of AML cells. Treatment of AML cells with MYBMIM led to the dissociation of the MYB:CBP/P300 complex in cells, its displacement from oncogenic enhancers enriched for MYB binding sites, and downregulation of MYB-dependent gene expression, including of MYC and BCL2 oncogenes. AML cells underwent mitochondrial apoptosis in response to MYBMIM, which was partially rescued by ectopic expression of BCL2. MYBMIM impeded leukemia growth and extended survival of immunodeficient mice engrafted with primary patient-derived MLL-rearranged leukemia cells. These findings elucidate the dependence of human AML on aberrant transcriptional coactivation, and establish a pharmacologic approach for its therapeutic blockade.

Cloning and Characterization of Promoters of the Fungal Immunomodulatory Protein Genes from Ganoderma spp. (Agaricomycetes) and Their Response to Methyl Jasmonate and Salicylic Acid.

Ganoderma mushrooms for medicinal use contain various bioactive compounds, but the genetic elements available for these medicinal mushrooms are still limited. In this study we cloned and analyzed the promoters of fungal immunomodulatory protein (FIP) genes from G. lucidum and G. atrum. FIP gene expression was induced by different concentrations of methyl jasmonate (MeJA) and salicylic acid (SA), and messenger RNA expression was detected by quantitative reverse-transcription polymerase chain reaction. The results provided 5' upstream sequences of FIP genes from G. lucidum and G. atrum. Sequence analysis showed that the FIP-glu promoter sequence contained 11 CAAT boxes, 3 TATA boxes, 3 MeJA-responsive elements, 3 MYB binding site (MBS) motifs, 1 abscisic acid responsive element, 1 TGA, 1 anaerobic inducible element, 2 circadian elements, 1 fungal elicitor, 1 meristem-specific activation element, 3 Skn-1 motifs, and several light-responsive elements. The 5' flanking region of FIP-gat included 9 CAAT boxes, 4 TATA boxes, 3 MeJA-responsive elements, 1 AuxRR core, 1 GC motif, 1 MBS, 1 fungal elicitor, 1 meristem-specific activation element, 3 Skn-1 motifs, and several light-responsive elements. On the transcriptional level, both FIP-glu and FIP-gat reached their highest expression after treatment with MeJA at 500 μmol/L. FIP-glu expression depended on the concentration of SA (0-1000 mg/L); the expression of the FIP-gat gene was highest at a concentration of 100 mg MeJA/L. This research lays the foundation to use Ganoderma mycelia as bioreactors for producing FIPs.

Multiple Copies of a Simple MYB-Binding Site Confers Trans-regulation by Specific Flavonoid-Related R2R3 MYBs in Diverse Species.

In apple, the MYB transcription factor MYB10 controls the accumulation of anthocyanins. MYB10 is able to auto-activate its expression by binding its own promoter at a specific motif, the R1 motif. In some apple accessions a natural mutation, termed R6, has more copies of this motif within the MYB10 promoter resulting in stronger auto-activation and elevated anthocyanins. Here we show that other anthocyanin-related MYBs selected from apple, pear, strawberry, petunia, kiwifruit and Arabidopsis are able to activate promoters containing the R6 motif. To examine the specificity of this motif, members of the R2R3 MYB family were screened against a promoter harboring the R6 mutation. Only MYBs from subgroups 5 and 6 activate expression by binding the R6 motif, with these MYBs sharing conserved residues in their R2R3 DNA binding domains. Insertion of the apple R6 motif into orthologous promoters of MYB10 in pear (PcMYB10) and Arabidopsis (AtMY75) elevated anthocyanin levels. Introduction of the R6 motif into the promoter region of an anthocyanin biosynthetic enzyme F3′5′H of kiwifruit imparts regulation by MYB10. This results in elevated levels of delphinidin in both tobacco and kiwifruit. Finally, an R6 motif inserted into the promoter the vitamin C biosynthesis gene GDP-L-Gal phosphorylase increases vitamin C content in a MYB10-dependent manner. This motif therefore provides a tool to re-engineer novel MYB-regulated responses in plants.

OsARM1, an R2R3 MYB Transcription Factor, Is Involved in Regulation of the Response to Arsenic Stress in Rice.

Bioaccumulation of arsenic (As) in rice (Oryza sativa) increases human exposure to this toxic, carcinogenic element. Recent studies identified several As transporters, but the regulation of these transporters remains unclear. Here, we show that the rice R2R3 MYB transcription factor OsARM1 (ARSENITE-RESPONSIVE MYB1) regulates As-associated transporters genes. Treatment with As(III) induced OsARM1 transcript accumulation and an OsARM1-GFP fusion localized to the nucleus. Histochemical analysis of OsARM1pro::GUS lines indicated that OsARM1 was expressed in the phloem of vascular bundles in basal and upper nodes. Knockout of OsARM1 (OsARM1-KO CRISPR/Cas9-generated mutants) improved tolerance to As(III) and overexpression of OsARM1 (OsARM1-OE lines) increased sensitivity to As(III). Measurement of As in As(III)-treated plants showed that under low As(III) conditions (2 μM), more As was transported from the roots to the shoots in OsARM1-KOs. By contrast, more As accumulated in the roots in OsARM1-OEs in response to high As(III) exposure (25 μM). In particular, the As(III) levels in node I were significantly higher in OsARM1-KOs, but significantly lower in OsARM1-OEs, compared to wild-type plants, implying that OsARM1 is important for the regulation of root-to-shoot translocation of As. Moreover, OsLsi1, OsLsi2, and OsLsi6, which encode key As transporters, were significantly downregulated in OsARM1-OEs and upregulated in OsARM1-KOs compared to wild type. Chromatin immunoprecipitation-quantitative PCR of OsARM1-OEs indicated that OsARM1 binds to the conserved MYB-binding sites in the promoters or genomic regions of OsLsi1, OsLsi2, and OsLsi6 in rice. Our findings suggest that the OsARM1 transcription factor has essential functions in regulating As uptake and root-to-shoot translocation in rice.

MdMYB4 enhances apple callus salt tolerance by increasing MdNHX1 expression levels

Exposure to high salt concentrations is one of the main stresses adversely affecting crop growth, development, and yield. In this study, a putative salt stress-responsive gene (MdMYB4) encoding an R2R3-MYB transcription factor was cloned and characterized. This gene belongs to the SG2 subgroup, and its C-terminal contains a conserved SG2 motif. The relative MdMYB4 expression levels in Malus × domestica Borkh. ‘Orin’ calli cultured under various NaCl concentrations were analyzed. Saline conditions upregulated the expression of MdMYB4, and the effects of the encoded transcription factor on salt tolerance were determined in MdMYB4-overexpressing (OE-MYB4) ‘Orin’ calli. The OE-MYB4 calli grew significantly better than the wild-type calli under salt stress conditions. Additionally, an analysis of the cis-acting elements in the MdNHX1 and MdSOS1 promoters revealed MYB-binding sites. However, the electrophoretic mobility shift assay results indicated that MdMYB4 can bind only to the MdNHX1 promoter. Moreover, the MdNHX1 expression levels were higher in OE-MYB4 calli than in wild-type calli at 150, 200, and 250 mM NaCl. In contrast, there were no significant changes to the MdSOS1 expression levels in OE-MYB4 calli. In conclusion, MdMYB4 is an R2R3-MYB transcription factor that mediates salt tolerance by activating the MdNHX1 promoter. Our findings may provide a theoretical basis for enriching the genetic resources associated with stress tolerance and for improving stress resistance in apple cultivars.

Characterization and functional analysis of a MYB gene (GbMYBFL) related to flavonoid accumulation in Ginkgo biloba.

Flavonoids are a group of metabolites in Ginkgo biloba thought to provide health benefits. R2R3-MYB transcription factors (TFs) play key roles in the transcriptional regulation of the flavonoid biosynthesis in plants. In this study, an R2R3-MYB transcription factor gene, GbMYBFL, was isolated from G. biloba and characterized. Results of bioinformatic analysis indicated that GbMYBFL is more closely related to the R2R3-MYB involved in flavonoid biosynthesis and displayed high similarity to MYB from other plants. The genmomic sequence of GbMYBFL had three exons and two introns, with its upstream sequence containing cis-acting regulatory elements Myb binding site, Myc recognition sites, and light, SA, MeJA responsive elements. Subcellular localization analysis indicates that GbMYBFL was located in the nucleus. Quantitative real-time PCR revealed that GbMYBFL was expressed in leaves, stems, roots, young fruits, male flower and female flower, and the level of transcription in male flower and leaves were higher than that in female flower, stems, roots, and young fruits. During G. biloba leaf growth, the transcription of GbMYBFL is positively correlated with the flavonoid content, suggesting that the GbMYBFL is involved in the flavonoid biosynthesis. Overexpression of GbMYBFL under the control of the CaMV35S promoter in Ginkgo callus notably enhanced the accumulation of flavonoids and anthocyanin compared with non-transformed callus. This finding suggested that GbMYBFL positively related to flavonoid biosynthesis, and the overexpression of GbMYBFL was sufficient to induce flavonoids and anthocyanin accumulation.

QTL mapping and loci dissection for leaf epicuticular wax load and canopy temperature depression and their association with QTL for staygreen in Sorghum bicolor under stress

Plant waxes and staygreen are distinct phenotypic traits that have been independently implicated in heat and drought tolerance among grasses. The association between these two traits has not been fully explored, which makes the exploitation of synergy between them difficult. This study assessed the association between QTL regulating the staygreen (Stg) trait in sorghum and those regulating epicuticular wax load (WL) and associated canopy temperature depression (TD). Using a recombinant inbred line (RIL) population derived from Tx642 and Tx7000, phenotypic data were collected in three replicated field trials and one greenhouse trial. High absolute TD generally corresponded to high WL. The r2 of TD against WL was highest under non-stress conditions in the greenhouse while it was much larger in the cooler and irrigated field conditions compared to hotter, drier field trials. The genetic correlations between the two traits also followed this pattern. Composite interval mapping identified a total of 28 QTL, 15 of which had significant overlaps between different traits. Most of the wax QTL were associated with pre-anthesis drought tolerant Tx7000. However, one QTL for WL overlapped with a QTL for staygreen (Stg2) and was represented by a single, isolated marker near the centromeric region on the short arm of SBI-01. The marker is identified by a Cis-acting regulatory module and is part of a 2-kb multifunctional motif-rich region which includes core promoter and enhancer regions and transcription elements, including a drought-responsive MYB binding site. We suggest that this QTL may be pleiotropic for important stress tolerance mechanisms regulating both staygreen and leaf wax in sorghum.

Molecular cloning, characterization, and promoter analysis of the isochorismate synthase (AaICS1) gene from Artemisia annua.

Isochorismate synthase (ICS) is a crucial enzyme in the salicylic acid (SA) synthesis pathway. The full-length complementary DNA (cDNA) sequence of the ICS gene was isolated from Artemisia annua L. The gene, named AaICS1, contained a 1710-bp open reading frame, which encoded a protein with 570 amino acids. Bioinformatics and comparative study revealed that the polypeptide protein of AaICS1 had high homology with ICSs from other plant species. Southern blot analysis suggested that AaICS1 might be a single-copy gene. Analysis of the 1470-bp promoter of AaICS1 identified distinct cis-acting regulatory elements, including TC-rich repeats, MYB binding site (MBS), and TCA-elements. An analysis of AaICS1 transcript levels in multifarious tissues of A. annua using quantitative real-time polymerase chain reaction (qRT-PCR) showed that old leaves had the highest transcription levels. AaICS1 was up-regulated under wounding, drought, salinity, and SA treatments. This was corroborated by the presence of the predicted cis-acting elements in the promoter region of AaICS1. Overexpressing transgenic plants and RNA interference transgenic lines of AaICS1 were generated and their expression was compared. High-performance liquid chromatography (HPLC) results from leaf tissue of transgenic A. annua showed an increase in artemisinin content in the overexpressing plants. These results confirm that AaICS1 is involved in the isochorismate pathway.

The wheat salinity-induced R2R3-MYB transcription factor TaSIM confers salt stress tolerance in Arabidopsis thaliana

MYB transcription factors are a large family of proteins involved in plant development and responses to stress. In this study, the wheat salinity-induced R2R3-MYB transcription factor TaSIM was functionally characterized, with a focus on its role in salt stress tolerance. TaSIM protein enters the nucleus and binds to the MYB-binding site II motif. Expression analysis revealed that TaSIM was induced by drought, high salinity, low temperature, and abscisic acid treatment. Overexpression of TaSIM improved salt stress tolerance in transgenic plants. Furthermore, the transcript levels of genes involved in abscisic acid (ABA)-dependent (RD22) and ABA-independent (RD29A) signaling were higher in TaSIM-overexpressing plants than in the wild type. These results suggest that TaSIM positively modulates salt stress tolerance and has potential applications in molecular breeding to enhance salt tolerance in crops.

Overexpression of the OsIMP Gene Increases the Accumulation of Inositol and Confers Enhanced Cold Tolerance in Tobacco through Modulation of the Antioxidant Enzymes' Activities.

Inositol is a cyclic polyol that is involved in various physiological processes, including signal transduction and stress adaptation in plants. l-myo-inositol monophosphatase (IMPase) is one of the metal-dependent phosphatase family members and catalyzes the last reaction step of biosynthesis of inositol. Although increased IMPase activity induced by abiotic stress has been reported in chickpea plants, the role and regulation of the IMP gene in rice (Oryza sativa L.) remains poorly understood. In the present work, we obtained a full-length cDNA sequence coding IMPase in the cold tolerant rice landraces in Gaogonggui, which is named as OsIMP. Multiple alignment results have displayed that this sequence has characteristic signature motifs and conserved enzyme active sites of the phosphatase super family. Phylogenetic analysis showed that IMPase is most closely related to that of the wild rice Oryza brachyantha, while transcript analysis revealed that the expression of the OsIMP is significantly induced by cold stress and exogenous abscisic acid (ABA) treatment. Meanwhile, we cloned the 5’ flanking promoter sequence of the OsIMP gene and identified several important cis-acting elements, such as LTR (low-temperature responsiveness), TCA-element (salicylic acid responsiveness), ABRE-element (abscisic acid responsiveness), GARE-motif (gibberellin responsive), MBS (MYB Binding Site) and other cis-acting elements related to defense and stress responsiveness. To further investigate the potential function of the OsIMP gene, we generated transgenic tobacco plants overexpressing the OsIMP gene and the cold tolerance test indicated that these transgenic tobacco plants exhibit improved cold tolerance. Furthermore, transgenic tobacco plants have a lower level of hydrogen peroxide (H2O2) and malondialdehyde (MDA), and a higher content of total chlorophyll as well as increased antioxidant enzyme activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), when compared to wild type (WT) tobacco plants under normal and cold stress conditions.

Abstract 3005: Whole-genome sequencing identified novel non-coding mutations causal of oncogene activation in T-cell acute lymphoblastic leukemia

Abstract There is growing evidence that non-coding sequences in human genome often function as transcriptional regulatory elements of protein-coding genes. In fact, germline polymorphisms and somatically acquired mutations within regulatory DNA can profoundly alter chromatin structure and modify gene transcription, directly contributing to tumorigenesis. However, there is a paucity of unbiased genome-wide characterization of somatic non-coding mutations in cancer. Using T-cell acute lymphoblastic leukemia (T-ALL) as a model disease, we herein report a systematic interrogation of driver non-coding genomic alterations by paired whole-genome and transcriptome sequencing of 31 children with T-ALL. To identify non-coding mutations with potential regulatory impact in a genome-wide fashion, our analytical pipeline consisted of 3 approaches: 1) the “hotspot analysis” for recurring mutations at the nearby positions, 2) the “regional recurrence analysis” for predefined regulatory regions with significant enrichment of non-coding mutations, 3) the “transcriptional factor analysis” for mutations that potentially result in gain/loss of transcription factor binding sites and alter expression of adjacent genes. Remarkably, T-ALL oncogenes LMO1 and TAL1 emerged as loci with the most significant recurring non-coding mutations. At the LMO1 locus, 3 patients (9.7%) showed an identical single-nucleotide mutation proximal to the transcription start site of the long isoform of LMO1. This recurring mutation resulted in the gain of a canonical Myb binding site (AACGG) and ~120-fold increase in LMO1 transcription compared to patients with wildtype genotype. TAL1 overexpression was observed in 15 patients, of whom 11 had intrachromosomal rearrangement (STIL-TAL1 fusion). The remaining 4 patients had somatic insertion that created a MYB-mediated super enhancer, consistent with recent reports. LMO1 enhancer mutation was further confirmed in an independent validation cohort (N=26), in which we additionally identified a novel intrachromosomal rearrangement between MED17 and LMO1 resulting in transcriptional activation of the latter. In a panel of T-ALL cell lines, LMO1 enhancer mutation was again associated with dramatic overexpression of LMO1, an active enhancer histone mark (H3K27ac), Dnase hypersensitivity, and allele-specific binding of MYB. Interestingly, we also observed robust binding of TAL1, CREBBP, RUNX1, ETS1, ELF1 and RNA Polymerase II at this site. Reporter gene assay confirmed the MYB-mediated transcription activation effects of this LMO1 enhancer mutation in vitro. In this genome-wide investigation of non-coding mutations in T-ALL, we identified novel enhancer mutations with drastic effects on oncogene activation. Our findings expand the understanding of how genomic alterations in regulatory DNA contribute to cancer pathogenesis. Citation Format: Maoxiang Qian, Shaoyan Hu, Hui Zhang, Yu Guo, Jin Yang, Xujie Zhao, Lin Wan, Jun Lu, Jian Pan, Meimei Chang, Shirley K. Kham, Yong Cheng, Chunliang Li, Allen E. Yeoh, Anders Skanderup, Jun J. Yang. Whole-genome sequencing identified novel non-coding mutations causal of oncogene activation in T-cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3005. doi:10.1158/1538-7445.AM2017-3005

Activation of the LMO2 oncogene through a somatically acquired neomorphic promoter in T-cell acute lymphoblastic leukemia

AbstractSomatic mutations within noncoding genomic regions that aberrantly activate oncogenes have remained poorly characterized. Here we describe recurrent activating intronic mutations of LMO2, a prominent oncogene in T-cell acute lymphoblastic leukemia (T-ALL). Heterozygous mutations were identified in PF-382 and DU.528 T-ALL cell lines in addition to 3.7% of pediatric (6 of 160) and 5.5% of adult (9 of 163) T-ALL patient samples. The majority of indels harbor putative de novo MYB, ETS1, or RUNX1 consensus binding sites. Analysis of 5′-capped RNA transcripts in mutant cell lines identified the usage of an intermediate promoter site, with consequential monoallelic LMO2 overexpression. CRISPR/Cas9-mediated disruption of the mutant allele in PF-382 cells markedly downregulated LMO2 expression, establishing clear causality between the mutation and oncogene dysregulation. Furthermore, the spectrum of CRISPR/Cas9-derived mutations provides important insights into the interconnected contributions of functional transcription factor binding. Finally, these mutations occur in the same intron as retroviral integration sites in gene therapy–induced T-ALL, suggesting that such events occur at preferential sites in the noncoding genome.

The MTP1 promoters from Arabidopsis halleri reveal cis-regulating elements for the evolution of metal tolerance.

In the hyperaccumulator Arabidopsis halleri, the zinc (Zn) vacuolar transporter MTP1 is a key component of hypertolerance. Because protein sequences and functions are highly conserved between A.halleri and Arabidopsis thaliana, Zn tolerance in A.halleri may reflect the constitutively higher MTP1 expression compared with A.thaliana, based on copy number expansion and different cis regulation. Three MTP1 promoters were characterized in A.halleri ecotype I16. The comparison with the A.thaliana MTP1 promoter revealed different expression profiles correlated with specific cis-acting regulatory elements. The MTP1 5' untranslated region, highly conserved among A.thaliana, Arabidopsis lyrata and A.halleri, contains a dimer of MYB-binding motifs in the A.halleri promoters absent in the A.thaliana and A.lyrata sequences. Site-directed mutagenesis of these motifs revealed their role for expression in trichomes. A. thaliana mtp1 transgenic lines expressing AtMTP1 controlled by the native A.halleri promoter were more Zn-tolerant than lines carrying mutations on MYB-binding motifs. Differences in Zn tolerance were associated with different distribution of Zn among plant organs and in trichomes. The different cis-acting elements in the MTP1 promoters of A.halleri, particularly the MYB-binding sites, are probably involved in the evolution of Zn tolerance.

Molecular Characterization of a MYB Protein from Oryza sativa for its Role in Abiotic Stress Tolerance

ABSTRACT The MYB family represents one of the most abundant classes of transcriptional regulators that perform pivotal role under different developmental processes and abiotic stresses. In present study, a MYB gene from Oryza sativa was selected for functional characterization. Bioinformatics analysis revealed that OsMYB1 cDNA encodes R2-R3 type DNA binding domain consisting of 413 amino acids having size of 44 kDa and pI of 6.24. DNA binding domain containing region was cloned and over-expressed in E. coli. Then, the survival of pGEX-OsMYB1 transformed E. coli cells was compared with control plasmid under different concentrations of NaCl, mannitol, high and low temperature. pGEX-OsMYB1 enhanced the survival of cells at high temperature and salinity. Electrophoretic mobility shift assays (EMSAs) have shown that recombinant OsMYB1 protein was able to bind with DIG labeled probe containing MYB binding site. RT-qPCR analysis revealed high MYB1 expression under wounding, salt, drought and heat stresses in rice. Expression was 23 fold higher in response to wounding demonstrating the worth of OsMYB1 up-regulation in wounding. Intrinsic disorder profile predicted that OsMYB1 exhibits 60% degree of intrinsic disorder proposing that these regions might be involved in DNA binding specificity and protein-protein interaction. The positive response of OsMYB1 suggests that its over-expression in crop plants may help in providing protection to plants to grow under abiotic stresses.

Identification of the Oncogene SKI As a New Target Gene of the Myeloid Transcription Factor c-MYB in AML

Background:Acute myeloid leukemia (AML) accounts for 80% of acute leukemias and arises through clonal expansion and arrest of differentiation of hematopoietic progenitor cells in the bone marrow. Depending on the AML subtype and age of the patient, AML patients have a 5-year survival rate of about 25%. AML is a genetically heterogenous disease, where chromosomal aberrations, point mutations in critical oncogenes as well as aberrant expression of key regulatory factors of hematopoiesis collude during transformation. c-MYB is an important hematopoietic transcription factor involved in proliferation and differentiation of progenitor cells of the myeloid and lymphoid lineages. It was first described as a viral oncogene of avian leukemia viruses and is upregulated or mutated in many leukemic subtypes as well as solid tumors. c-MYB interacts with other transcription factors or co-factors, which are essential for its transcriptional activity. In this regard, c-MYB transactivation ability is inhibited by a histone deacetylases recruiting corepressor complex containing SKI, TIF1BETA, NCOR and mSIN3A. c-SKI is a proto-oncogene and an inhibitor of TGFβ signalling. However, it acts not only as a transcriptional co-repressor but also as a transcriptional co-activator. Like c-MYB, c-SKI is upregulated in different solid tumors and leukemias. Though SKI activity is well described, transcriptional regulation of the SKI gene itself still remains unknown. Here, we deliver insight into the transcriptional regulation of the human SKI gene via the transcription factor c-MYB.Methods:In silico analyses were performed to identify potential MYB binding sites in the SKI regulatory region. Chromatin immunoprecipitation (ChIP) assays were used to validate the interaction between MYB and the potential SKI regulatory regions. Reporter gene assays were engaged to analyze MYB regulatory potential regarding SKI expression. RNAi experiments were performed to further examine transcriptional regulation of SKI by MYB. Since MYB is known to be downregulated by the histone deacetylase inhibitor (HDACi) valproic acid (VPA), MYB and SKI protein levels were analyzed in AML cell lines treated with VPA via Western Blot. Correlated protein levels of MYB and SKI were examined in the myeloid leukemia cell lines HL60, U937, THP1, NB4 and K562 as well as in primary cells of AML patients (n=27). Correlation of MYB and SKI transcript levels were performed in three different data sets of primary AML samples. The first cohort (n=7) was analyzed via RT-qPCR. Cohort 2 consists of cDNA microarray data of AML patients (n=17). Furthermore, principal component analysis (PCA) of the gene expression profile of MYB and SKI of AML patients (n=542) were performed with the leukemia gene atlas (LGA).Results:In silico analyses revealed four putative MYB binding sites (MBS1-4) in the SKI regulatory region. Direct binding of MYB to the regulatory sites MBS2-4 of the SKI gene could be confirmed via ChIP experiments. Dual luciferase reporter gene assays comprising c-MYB binding sites present in the SKI gene locus further show c-MYB-dependent transcriptional activation of the reporter. RNAi experiments depleting c-MYB in leukemic cell lines resulted in the decrease of SKI protein levels and thereby reveal that c-MYB is essential for the induction of SKI gene expression. Accordingly, treatment of the AML cell lines with the HDACi VPA led to a decrease of MYB and consequently SKI protein levels. Consistently, we observed a positive correlation of MYB and SKI protein expression in leukemic cell lines and in samples of AML patients. Moreover, a highly positive correlation of MYB and SKI transcript levels could be observed in three different cohorts of AML patients, further confirming regulation of SKI expression by the transcription factor MYB.Conclusion:Our findings provide new insights in the transcriptional regulation of the proto-oncogene c-SKI by the oncogenic transcription factor c-MYB during leukemogenesis. c-MYB and c-SKI expression and functions are highly positively correlated in human AML suggesting that c-SKI is a mediator of c-MYB oncogenic potential. So far, various therapeutic approaches targeting MYB failed to be transferred to patients. In this regard, c-MYB and c-SKI represent promising marker and target proteins for novel HDACi-based therapeutic approaches in AML. DisclosuresNo relevant conflicts of interest to declare.

Activation of the LMO2 Oncogene in T-ALL through a Somatically Acquired Neomorphic Promoter

LMO2is a crucial regulator of normal hematopoiesis but is progressively silenced from the early T-cell progenitor stage of thymic development. Aberrant LMO2 expression leads to a block in differentiation, increased self-renewal and aggressive T-cell acute lymphoblastic leukemia (T-ALL). Although ≈50% of T-ALL patients overexpress LMO2, this can be attributed to a cytogenetic lesion in only ≈10% of patients, leaving a significant portion mechanistically unaccounted for. We recently discovered somatic mutations that create an oncogenic super-enhancer driving TAL1 expression (Mansour et al., 2014, Science). Therefore, we investigated whether comparable mutations may be causing dysregulated LMO2 expression.Aberrant H3K27ac marks indicative of active chromatin were identified prior to and encompassing the non-coding exon 2 of the LMO2 gene by ChIP-seq in DU.528 and PF-382 T-ALL cell lines, both of which exhibit upregulated LMO2 expression but lack chromosomal lesions at this locus. Sequencing across this peak revealed a heterozygous 20bp duplication in PF-382 cells and a heterozygous 1bp deletion in DU.528 cells, located close to a region recently described as an intermediate promoter. Both indels generated a de novo MYB consensus motif. MYB ChIP-seq showed that ≥96% of reads aligned to the mutant rather than the wild-type allele, suggesting that MYB was preferentially recruited to the mutant allele. Heterozygous mutations were also detected in diagnostic samples from 3% (5/159) of pediatric and 6% (10/164) of adult T-ALL patients. Absence of the mutations in 7 available patient-matched remission samples confirmed that they were somatic. The mutations were densely distributed around highly conserved native ETS1, MYB and GATA motifs; 5 patients had an additional MYB site, 3 both a MYB and an ETS1 site, 1 an ETS1 site alone, and 4 had new RUNX1 binding sites. This suggests that this region may have a regulatory role that is exploited by the mutations to constitutively activate LMO2.All 6 mutant-positive patients with available RNA showed LMO2 overexpression as determined by qRT-PCR, consistent with the hypothesis that these mutations activate gene expression. Furthermore, monoallelic LMO2 expression could be demonstrated in DU.528 cells and 3 of 4 informative T-ALL samples using a heterozygous germline SNP. Expression of the mutations in luciferase reporter assays also indicated that they all markedly activated luciferase activity to between 2x and 57x compared to the wild-type sequence.To assess causality between the mutations and LMO2 dysregulation, we used CRISPR/Cas9 genome-editing with a guide RNA designed to target the mutant MYB site in PF-382 cells. Reduction of LMO2 expression to ≤10% of PF-382 mutant activity was observed in a clone where the mutant allele had been reverted to wild-type and in another clone with a single T>C substitution disrupting the mutant MYB binding site. Interestingly, 2 clones that increased the distance between the native and the mutant MYB sites also resulted in a reduction in LMO2 expression to 19% and 25% of PF-382 activity, suggesting that there are functionally limiting spatial constraints on the mutant MYB site in relation to other neighboring transcription factor binding sites. This was further validated by the lack of reduction in LMO2 expression in a clone where the sequence between the two MYB sites was altered but the spacing distance was unchanged.In conclusion, we have identified and functionally validated a novel recurrent mutation hotspot occurring in a non-coding site whereby introduction of additional binding sites for a number of different transcription factors drives monoallelic LMO2 overexpression from a neomorphic promoter in a substantial proportion of both adult and pediatric T-ALL patients. This mechanism of oncogene activation may be relevant to a wide variety of human cancers. DisclosuresFielding:Baxalta: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees.

Peptidomimetic Blockade of MYB in Acute Myeloid Leukemia

Aberrant gene expression is a hallmark of acute leukemias. However, therapeutic strategies for its blockade are generally lacking, in large part due to the pharmacologic challenges of drugging transcription factors. MYB-driven gene trans-activation with CREB-binding protein (CBP) is required for the initiation and maintenance of a variety of acute lymphoblastic and myeloid leukemias, including refractory MLL-rearranged leukemias. Using structure-guided molecular design, we developed a prototypical peptidomimetic inhibitor MYBMIM that interferes with the assembly of the molecular MYB:CBP complex at μM concentrations and rapidly accumulates in the nuclei of AML cells. We found that treatment of AML cells with MYBMIM, but not with its inactive near-isosteric analogue TG3, led to the displacement and dissociation of MYB:CBP complex in cells, causing rapid downregulation of MYB-dependent gene expression including MYC and BCL2 oncogenes. This was associated with obliteration of H3K27Ac-driven oncogenic enhancers induced by CBP and enriched for MYB binding sites (Figure 1A; p=1e-118). Both human MLL-rearranged and non-rearranged AML cells, but not normal CD34+ umbilical cord blood progenitor cells, underwent sustained mitochondrial apoptosis in response to MYBMIM treatment, an effect that could be partially blocked by ectopic expression of BCL2. We observed that MYBMIM treatment (50 mg/kg/day) impeded leukemia progression and extended survival of immunodeficient mice engrafted with primary patient-derived MLL-rearranged leukemia cells (Figure 1B; p=3.8e-3). These findings emphasize the exquisite dependence of human AML on MYB:CBP transcriptional dysregulation, and establish a pharmacologic approach for its therapeutic blockade. [Display omitted] DisclosuresArmstrong:Epizyme, Inc: Consultancy; Vitae Pharmaceuticals: Consultancy; Imago Biosciences: Consultancy; Janssen Pharmaceutical: Consultancy.

Genome-wide analysis and characterization of molecular evolution of the HCT gene family in pear (Pyrus bretschneideri)

Lignin is a major component of stone cells in pear fruit, which significantly affects fruit quality. Hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT), a recently discovered enzyme in plants, is an important gene that participates in the formation of lignin. Although HCT gene cloning and expression patterns have been studied in several species, including pear, there is still no extensive genome-wide bioinformatics analysis on the whole gene family, and the evolutionary history of HCT gene family is still unknown. A total of 82 HCT genes were identified in pear, most of which have one or two exons, and all with the conserved HXXXD motif and transferase domains. Based on the structural characteristics and phylogenetic analysis of these sequences, the HCT gene family genes could be classified into four main groups. Structural analysis also revealed that 25 % of HCT genes share a MYB binding site. Expansion of the HCT gene family mostly occurred before the divergence between Arabidopsis and Rosaceae, with whole-genome duplication or segmental duplication events playing the most important role in the expansion of the HCT gene family in pear. At the same time, purifying selection also played a critical role in the evolution of HCT genes. Five of the 82 HCT genes were verified by qRT-PCR to correspond to the pattern of stone cell formation during pear fruit development. The genome-wide identification, chromosome localization, gene structures, synteny, and expression analyses of pear HCT genes provide an overall insight into HCT gene family and their potential involvement in growth and development of stone cells.

Phylogeny and Expression Analyses Reveal Important Roles for Plant PKS III Family during the Conquest of Land by Plants and Angiosperm Diversification.

Polyketide synthases (PKSs) utilize the products of primary metabolism to synthesize a wide array of secondary metabolites in both prokaryotic and eukaryotic organisms. PKSs can be grouped into three distinct classes, types I, II, and III, based on enzyme structure, substrate specificity, and catalytic mechanisms. The type III PKS enzymes function as homodimers, and are the only class of PKS that do not require acyl carrier protein. Plant type III PKS enzymes, also known as chalcone synthase (CHS)-like enzymes, are of particular interest due to their functional diversity. In this study, we mined type III PKS gene sequences from the genomes of six aquatic algae and 25 land plants (1 bryophyte, 1 lycophyte, 2 basal angiosperms, 16 core eudicots, and 5 monocots). PKS III sequences were found relatively conserved in all embryophytes, but not exist in algae. We also examined gene expression patterns by analyzing available transcriptome data, and identified potential cis-regulatory elements in upstream sequences. Phylogenetic trees of dicots angiosperms showed that plant type III PKS proteins fall into three clades. Clade A contains CHS/STS-type enzymes coding genes with diverse transcriptional expression patterns and enzymatic functions, while clade B is further divided into subclades b1 and b2, which consist of anther-specific CHS-like enzymes. Differentiation regions, such as amino acids 196-207 between clades A and B, and predicted positive selected sites within α-helixes in late appeared branches of clade A, account for the major diversification in substrate choice and catalytic reaction. The integrity and location of conserved cis-elements containing MYB and bHLH binding sites can affect transcription levels. Potential binding sites for transcription factors such as WRKY, SPL, or AP2/EREBP may contribute to tissue- or taxon-specific differences in gene expression. Our data shows that gene duplications and functional diversification of plant type III PKS enzymes played a critical role in the ancient conquest of the land by early plants and angiosperm diversification.

The wheat R2R3-MYB transcription factor TaRIM1 participates in resistance response against the pathogen Rhizoctonia cerealis infection through regulating defense genes

The necrotrophic fungus Rhizoctonia cerealis is a major pathogen of sharp eyespot that is a devastating disease of wheat (Triticum aestivum). Little is known about roles of MYB genes in wheat defense response to R. cerealis. In this study, TaRIM1, a R. cerealis-induced wheat MYB gene, was identified by transcriptome analysis, then cloned from resistant wheat CI12633, and its function and preliminary mechanism were studied. Sequence analysis showed that TaRIM1 encodes a R2R3-MYB transcription factor with transcription-activation activity. The molecular-biological assays revealed that the TaRIM1 protein localizes to nuclear and can bind to five MYB-binding site cis-elements. Functional dissection results showed that following R. cerealis inoculation, TaRIM1 silencing impaired the resistance of wheat CI12633, whereas TaRIM1 overexpression significantly increased resistance of transgenic wheat compared with susceptible recipient. TaRIM1 positively regulated the expression of five defense genes (Defensin, PR10, PR17c, nsLTP1, and chitinase1) possibly through binding to MYB-binding sites in their promoters. These results suggest that the R2R3-MYB transcription factor TaRIM1 positively regulates resistance response to R. cerealis infection through modulating the expression of a range of defense genes, and that TaRIM1 is a candidate gene to improve sharp eyespot resistance in wheat.