PtKTI12 genes influence wobble uridine modifications and drought stress tolerance in hybrid poplar

ABSTRACT The multisubunit Elongator complex plays key roles in transcription by interacting with RNA polymerase II and chromatin modeling. Kti proteins have been identified as the auxiliary protein for the Elongator complex. However, our knowledge of Kti proteins in woody plants remains limited. In this study, in total 16 KTI gene homologs were identified in Populus trichocarpa. Among them, the two KTI12 candidates were named PtKTI12A and PtKTI12B. Although PtKTI12A and PtKTI12B were largely different in gene expression level and tissue specificity, both genes were induced by heat and drought stresses. PtKTI12A and PtKTI12B RNAi transgenic poplar plants showed reduced levels of modified nucleosides, in particular 5-carbamoylmethyluridine and 5-methoxycarbonylmethyl-2-thiouridine. Meanwhile, their tolerance to drought was improved when subjected to withdrawal of watering. Also, the protein products of PtKTI12A and PtKTI12B had similar subcellular localization and predicted tertiary structure. The results suggest that Kti12 proteins are involved in tRNA wobble uridine modification, stress response and drought stress tolerance in hybrid poplar.

HMGA Proteins in Hematological Malignancies

The high mobility group AT-Hook (HMGA) proteins are a family of nonhistone chromatin remodeling proteins known as “architectural transcriptional factors”. By binding the minor groove of AT-rich DNA sequences, they interact with the transcription apparatus, altering the chromatin modeling and regulating gene expression by either enhancing or suppressing the binding of the more usual transcriptional activators and repressors, although they do not themselves have any transcriptional activity. Their involvement in both benign and malignant neoplasias is well-known and supported by a large volume of studies. In this review, we focus on the role of the HMGA proteins in hematological malignancies, exploring the mechanisms through which they enhance neoplastic transformation and how this knowledge could be exploited to devise tailored therapeutic strategies.

G-Dash: A Genome Dashboard Integrating Modeling and Informatics

Genomics is a sequence based informatics science and a structure based molecular material science. There are few tools available that unite these approaches in a scientifically robust manner. Here we describe G-Dash, a web based prototype of a genomics dashboard, specifically designed to integrate informatics and 4D material studies of chromatin. G-Dash unites our Interactive Chromatin Modeling(ICM) tools with the Biodalliance genome browser and the JSMol molecular viewer to rapidly fold any DNA sequence into atomic or coarse-grained models of DNA, nucleosomes or chromatin. As a chromatin modeling tool, G-Dash enables users to specify nucleosome positions from various experimental or theoretical sources, interactively manipulate nucleosomes, and assign different conformational states to each nucleosome. As an informatics tool, data associated with 3D structures are displayed as tracks in a genome browser. The exchange of data between informatics and structure is bi-directional so any informatics track can inform a molecular structure (e.g. color by function) and structure features can be displayed as informatics tracks in a genome browser(e.g. Roll, Slide, or Twist). As a sample application, models of the CHA1 promoter based on experimentally determined nucleosome positions are explored with G-Dash. Steric clashes and DNA knotting are observed but can be resolved with G-Dash's minimal coarse-grained model without significant variation in structure. Results raise questions about the interpretation of nucleosome positioning data and promoter structures. In this regard, G-Dash is a novel tool for investigating structure-function relationships for regions of the genome ranging from base pairs to chromosomes and for generating, validating and testing mechanistic hypotheses.

T(6;9)-DEK/CAN-Positive Leukemia: Role of FLT3-ITD for the Determination of the Leukemic Phenotype

Abstract 1316 In acute myeloid leukemia (AML), translocations and the resulting fusion proteins (FPs) such as PML/RAR, AML1/ETO and DEK/CAN represent the leukemia initiating event. t(6;9)(DEK/CAN)-positive AML is classified as a separate clinical entity, because of its early onset and poor prognosis. We recently have shown that DEK/CAN is a leukemia-inducing oncogene, which targets a very small subpopulation of primitive hematopoietic stem cells (HSC) for leukemic transformation. Like other FPs, DEK/CAN also interferes with the epigenetic regulation of transcription by modifying key processes of chromatin modeling such as histone acetylation and methylation as well as DNA methylation. In the DEK/CAN fusion protein, all the chromatin binding domains of DEK are conserved and we recently showed that DEK/CAN is associated to chromatin and strongly interferes with chromatin modeling by inhibiting the decondensation of chromatin and accessibility to transcription. As a “Class 1 mutation”, the oncogenic internal tandem duplication (ITD) of the receptor tyrosine kinase Flt3 (Flt3-ITD) is found in 88% of the t(6;9)-positive AML-patients, which otherwise is present in about 30% of other AML cases. The simultaneous presence of Flt3-ITD and DEK/CAN in AML is correlated with a high WBC and significantly lower rates of complete remission. Aim of the study was to determine the effect of Flt3-ITD on the DEK/CAN-induced leukemic phenotype. Therefore we expressed Flt3-ITD and DEK/CAN from a single vector as p2A fusion protein in order to obtain an equimolar expression of the two proteins. We investigated the capacity to mediate factor-independent growth of the single factors and in combination in the myeloid progenitor cell line 32D upon IL-3 withdrawal. The leukemic phenotype was studied in primary Sca1+/Lin- murine hematopoietic stem and progenitor cells (mHSPC) retrovirally transduced with DEK/CAN, FLT3-ITD and FLT3-ITD-p2a-DEK/CAN. These cells were tested for their differentiation potential in liquid culture, for their serial replating capacity in semi-solid medium, their stem cell capacity in colony-forming unit spleen - day12 (CFU-S12) assays, and their potential to induce leukemia in sublethally irradiated recipients. Here we show that I.) Flt3-ITD mediated factor-independent growth alone and in presence of DEK/CAN, but the onset of factor-independent growth was delayed by DEK/CAN; II.) FLT3-ITD did not influence the differentiation potential of DEK/CAN-positive HPSCs; III.) Flt3-ITD increased the colony-number of DEK/CAN-positive, but not the overall serial replating efficiency of DEK/CAN-positive HPSCs; IV.) FLT3-ITD accelerated and increased efficiency of leukemia induction by DEK/CAN in vivo, without modifying either the morphological or the immunological phenotype of DEK/CAN-induced leukemia. Finally we investigated whether FLT3-ITD influences the known capacity of histone-deacetylase (HDAC) inhibitors (HDACi) to revert the leukemogenic potential of DEK/CAN. Therefore we employed a xenograft model based on the patient derived FKH-1 cell line expressing both FLT3-ITD and DEK/CAN. We found that exposure to the HDACi Dacinostat prevented the leukemia-induction in this model. Taken together these findings strongly suggest that DEK/CAN drives the transformation of immature HPSCs which is supported by the presence FLT3-ITD regarding proliferation, without strong effects on the leukemic phenotype induced by DEK/CAN. Disclosures: Bug: Novartis Oncology: Honoraria, Travel grants Other.