Project description:The epigenetic dysregulation of tumor suppressor genes is a major driver of human carcinogenesis. We have combined genome-wide methylation analyses with functional screening to identify novel candidate tumor suppressor genes in diffuse large B-cell lymphoma (DLBCL). We find that the dual-specificity phosphatase DUSP4 is aberrantly silenced in nodal and extranodal DLBCL due to promoter hypermethylation; ectopic expression of wild type DUSP4, but not of a phosphatase-deficient mutant, dephosphorylates c-JUN N-terminal kinase (JNK) and induces apoptosis in DLBCL cells. JNK inhibition prevents DLBCL survival in vitro and in vivo, and synergizes strongly with inhibitors of chronic active B-cell receptor signaling. Our results provide a mechanistic basis for the clinical development of JNK inhibitors in DLBCL, alone or in synthetic lethal combinations. A methylation profiling data set related to this experiment was also deposited at ArrayExpress under accession number E-MTAB-2926: http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-2926/
Project description:Background: Inducing apoptosis of autoreactive lymphocytes is part of the therapeutic strategy for Crohn’s disease (CD) patients. Failure to respond to medical therapies upon inflammatory bowel disease could result from insufficient apoptosis. To date there are no useful molecular factors for the prediction of clinical relapse. Study Aims: Characterization of the BCL-2 family-related risk of therapy resistance and verifying its usefulness as parameter for the prediction of clinical relapse could be helpful in deciding which medical therapy to recommend. The project is directly targeted to develop rapidly therapeutic improvement for patients with IBD. A long-term goal is the development of new therapeutic options for the treatment of IBD via a physiologic homeostasis and turnover of lymphocytes. Study Design: We propose to characterize the BCL-2 family-related risk of therapy resistance and its usefulness as parameter for the prediction of clinical relapse upon medical therapy. Samples will be used for next generation sequencing, qPCR, WB, immunohistochemistry and immunofluorescence. The hypothesis is considered confirmed if expression of BCL-2 family members in human peripheral blood is significantly changed between patient groups and correlates with the number of lymphocyte subpopulations.
Project description:The aim of the study was to investigate which genes are up- and down-regulated in response to hyperthermia and combined thermoradiotherapy in HSP70 proficient and deficient canine osteosarcoma cell line. Canine osteosarcoma cell line Abrams was transfected with negative control siRNA or siRNA targeting HSP70. Cells were treated with hyperthermia (42C, 1 hour, HT), radiotherapy (6Gy, RT) and thermoradiotherapy (HTRT). RNA was extracted 24 hours after treatment and used for RNA sequencing. Three independent experiments were performed, each with negative control siRNA (Neg) and HSP70 knockdown (HSP70kd) cells. Cells were treated with radiotherapy (RT), hyperthermia (HT), thermoradiotherapy (HTRT) or untreated (ctrl). One experiment consists of 8 samples (ctrl, RT, HT, HTRT in neg and HSP70kd cells), total 24 samples were sequenced.
Project description:Flower phenotypes in the species Diplacus aurantiacus in Southern California along an east west transect range from large, yellow, insect-pollinated flowers through orange flowers to small, red, bird-pollinated flowers. Until now, intermediate forms were attributed to recurrent hybridization at the (sub)-species level. However, by monitoring the flower phenotypes of these populations in field studies over the past 20 years, Rolf Baumberger observed that the transition in flower phenotype occurs during the lifespan of individual long-lived plants, thus ruling out a hybrid origin of intermediate forms. Further research has revealed that this transition bears the hallmark of an epigenetic transition. The small, red, bird-pollinated state is stable and heritable but reverts at frequencies of 1-2 %, much higher than that of genetic alterations. In our first approach a comparative transcriptome analysis of individuals of both stable morphotypes, we like to unravel candidate genes involved in floral colour and morphology determination.
Project description:Arabidopsis thaliana seeds that maternally inherit a medea (mea) mutant allele abort before completing embryogenesis. However, mea seeds can be rescued by pollen from several natural ecotypes of A. thaliana, including the Cape Verdian accession Cvi-0. We developed a method for the mapping of parent-of-origin effects using whole-genome sequencing of segregant bulks. The strategy is to create an F2 population that contains one set of chromosomes from the maternal parent (mea, in a Ler background) but inherits two segregating sets (Ler and Cvi-0) from the other parent. The two paternally segregating sets have opposite effects in mea penetrance: Ler fathers allow full mea seed abortion, while Cvi fathers rescue 90% of medea seeds. Therefore, the two segregating paternal sets are not equally transmitted to the next generation. DNA extracted from pools of viable F2 seedlings was sequenced on an Illumina HiSeq 2000 platform, mapped to the reference TAIR10 A. thaliana genome and the ratio between Ler and Cvi-0 SNPs used to identify chromosomal regions enriched in Cvi-0 sequences. As a control, DNA pools extracted from crosses between a wild-type Ler mother and a hybrid Cvi-0:Ler father were also sequenced. Three biological replicates were made for each pool. Ler-1 x Ler-1:Cvi-0 hybrid viable seedlings pools (each is a biological replicate): WT_pool_1, WT_pool_2, WT_pool_3 mea-1/mea-1; MEA-GR x Ler-1:Cvi-0 hybrid viable seedlings pool (each is a biological replicate): mea_pool_1, mea_pool_2, mea_pool_3
Project description:ChIP-Sequencing of 4 diffuse large B-cell lymphoma cell lines expressing different amounts of FOXP1 was performed in order to identify target genes bound by the transcription factor FOXP1.
Project description:Organoids derived from small intestine epithelial cells of C57BL/6 wild-type mice were cultured in the presence or absence of IL-28 (1000 ng/ml) for 24 hours. The experimental setup included 8 samples in a pairwise design (4 unstimulated vs 4 stimulated). Profiling of whole-genome transcriptomic patterns was performed by RNA-Seq on an Illumina HiSeq 2500 platform using total RNA at the Next Generation Sequencing core unit of the University Hospital Erlangen, Germany. Demultiplexed reads were quality filtered against rRNAs, tRNAs, mt-rRNAs and mt-tRNAs. Alignment against the mus musculus reference genome (Ensemble V.84 for GRCm38) was performed with RNA-seq aligner STAR (V.2.5.1b) and statistical analysis of unique mappings (HTseq count) was computed in R using the DESeq2 1.10.1.
Project description:Genome wide transcript and target gene profiling reveal that FOXP1 acts directly and indirectly by enforcing known ABC-DLBCL hallmarks, including Chronically Activated B cell receptor Signaling (CABS) and the classical NF-κB survival pathway. Our data further suggest that FOXP1 maintains ABC-subtype distinction by repressing gene expression programs dominant in GCB-DLBCL and support a model in which the normally transitory B cell plasmablast is the target of ABC-DLBCL transformation. ChIP sequenicng was performed for the FOXP1 transcription factor in DLBCL cell lines. Input was sequenced and used as a control.
Project description:Mouse embryonic stem cells (mESCs), derived from pre-implantation blastocyst cells, can be maintained in vitro in defined N2B27 medium supplemented with two chemical inhibitors for GSK3 and MEK (2i) and the cytokine leukemia inhibitory factor (LIF), which act synergistically to promote self-renewal and pluripotency. Many efforts have been devoted to identify genes that promote exit from the pluripotent state and the transition to a primed state of differentiation. One of the first identified players in this process was the Wnt/b-catenin effector TCF7L1 (previously referred to as TCF3), belonging to the family of four TCF/LEF transcription factors, which acts as pro-differentiation factor by repressing pluripotency genes. Of note, there is little evidence that the genetic abrogation of the mechanisms required for the exit from the pluripotent state is sufficient to enable self-renewal in the absence of 2iL. Here, we found that complete loss-of-function of Tcf7, Lef1, Tcf7l1 and Tcf7l2, the genes encoding for the four TCF/LEF transcription factors, (refered to as qKO) allows mESCs to become fully 2iL-independent and to propagate in basal N2B27. To understand the genetic program that allows qKO cells to achieve 2iL-independent self-renewal, we performed RNA sequencing (RNA-seq) of qKO and wild type mESCs.
Project description:To provide insight into the role of and target genes of the transcription factor FOXP1 in mature human B cells and in B cell non-Hodkgin lymhomas, we performed gene expression microarray studies, upon ectopic overexpression or silencing of FOXP1 in these cells. human memory B cells from 2 separate donors were transduced with LZRS-FOXP1-IRES-YFP or LZRS-IRES-YFP (negative control); DLBCL cell lines OCI-Ly1, OCI-Ly7, and OCI-Ly10 were transduced with LZRS-FOXP1-IRES-YFP or LZRS-IRES-YFP (negative control); DLBCL cell lines OCI-Ly1, OCI-Ly7, and OCI-Ly10 were transiently transfected with siRNA targeting FOXP1 or sigenome non-targeting siRNA (negative control), using the Lonza nucleofection system.