title: Transgenerational inheritance of impaired larval T cell development in zebrafish

city: Freiburg im Breisgau

first: Thomas

last: Boehm

address: Stübeweg 51

department: Immunology

institute: MPI-IE Freiburg

summary: The transgenerational inheritance of epigenetic information is an attractive mechanism by which the phenotypic consequences of exposure of parents to certain environmental conditions could be transmitted to the next generation. Although this phenomenon is well described in yeast and plants, and in some invertebrates, such as the D. melanogaster and C. elegans, the evidence for transgenerational inheritance in vertebrates is scarce. Among the mechanisms that are known or suspected to be involved in mediating the propagation of epigenetic information to subsequent generations, the process of enzymatic DNA methylation is of particular interest.  During DNA replication, the methylation pattern of the parental strand is faithfully copied onto the daughter strand by the maintenance methylase DNMT1, although methylation patterns can be changed through the action of de novo methylases and mechanisms of active and passive demethylation. In the context of epigenetic inheritance, the induction of aberrant methylation patterns in gametes may set the stage for transmission into future generations. Here, we describe a viable hypomorphic allele of dnmt1 in zebrafish that causes widespread demethylation of CpG dinucleotides in sperm and somatic tissues. Surprisingly, homozygous mutants present with an essentially normal phenotype, with the exception of drastically impaired lymphopoiesis, affecting both larval and adult phases of T cell development. Male but not female mutants are fertile. The phenotype of impaired larval (but not adult) T cell development is transmitted to subsequent generations by about 50% of fish that are genotypically wildtype. Whole genome bisulfite sequencing of sperm DNA of transmitting and non-transmitting males identified about 200 differentially methylated regions, including hypermethylated sites associated with runx3 and rptor genes. Knock-down of these two genes leads to impaired larval T cell development. Our results raise the possibility of an epigenetic origin of certain immunodeficiency syndromes in animals and humans.

overall_design: Our forward genetic screen identified a missense mutation in maintenance DNA methylase Dnmt1 specifically affecting lymphoid development. To examine the molecular consequences and transgenerational inheritance, whole genome methylome analyses were conducted for zebrafish Dnmt1 mutant.

first: Norimasa

last: Iwanami

first: Divine

middle: F

last: Lawir

first: Katarzyna

last: Sikora

first: Connor

last: O'Meara

first: Kohei

last: Takeshita

first: Michael

last: Schorpp


title: Global timeospatial map of RNA polymerase in Escherichia coli

status: Public on Jul 06 2020

city: Odense M

first: Thøger

middle: Jensen

last: Krogh

address: Campusvej 55

country: Denmark

institute: University of Southern Denmark

type: Genome binding/occupancy profiling by high throughput sequencing

type: Expression profiling by high throughput sequencing

summary: To investigate the relationship between RNA polymerase binding and transcription ChIP-seq on the common house-keeping SigmaD and RNA polymerase beta subunit were coupled with RNA-seq at various growth phases of Escherichia coli in rich media.

overall_design: ChIP-seq combined with RNA-seq to map RNA polymerase binding and transcription

middle: J

first: Jakob

last: Møller-Jensen

first: Christoph

last: Kaleta


title: DNA methylation in aging BXD mouse strains

city: Memphis

first: Khyobeni

last: Mozhui

state: TN

address: 66 N. Pauline Street, #631

department: Preventive Medicine

country: USA

institute: University of Tennessee Health Science Center

type: Methylation profiling by high throughput sequencing

summary: We performed affinity-based enrichment with methyl-CpG binding domain protein followed by high-throughput sequencing (MBD-seq) to assay DNA methylation in mouse liver tissue.

overall_design: A total of 70 liver samples from 12 BXD mouse strains maintained on either normal laboratory chow or high fat diet (60% calories from fat) were processed for next gen sequencing after affinity capture with methyl-CpG binding protein.


title: Systematic analysis of IL-17 receptor signalosome reveals a robust regulatory feedback loop

city: Vestec

first: Peter

last: Draber

state: Czech Republic

address: Průmyslová 595

department: BIOCEV

country: Czech Republic

institute: First Faculty of Medicine, Charles University

summary: We analyzed the changes in gene-expression upon IL-17 stimulation in the presence or absence of TBK1 and IKKε inhibitor MRT67307

overall_design: ST2 cells were treated with inhibitor MRT67307 and subsequently stimulated or not with IL-17 for 2 hours. Isolated RNA was subjected to RNASeq analysis

first: Giovanni

last: Stracquadanio


title: Perturbation-based gene regulatory network inference to unravel oncogenic mechanisms

city: Stockholm

first: Erik

last: Sonnhammer

address: Tomtebodavagen 23A

department: Biochemistry & Biophysics

country: Sweden

institute: SciLifeLab/ Stockholm University

summary: The project consisted of single and double knockdown experiments involving forty-five genes in the human squamous carcinoma cell culture line A431. During the knockdown phase, the cells were transfected with siRNAs which decreased target gene expression. After 72 hours the cells were deemed to have reached a new steady-state and approximate cell counts were calculated using resazurin. At this point the cells were lysed using CelluLyser, and a proprietary RNA spike was added to each sample relative to the cell count to act as a reference gene for the qPCR analysis. Then cDNA was prepared from the RNA and preamplified in preparation for the high-throughput qPCR screening. The transcript profiles, with respect to the forty-five target genes, were determined using TaqMan assays on Fluidigm's Biomark platform using 96x96 Dynamic Array IFCs.

overall_design: 384 experiments, 192 technical replicates, Experiments are replicates on the first 3 plates

first: Emelie

last: Lott

first: Jens

last: Björkman

first: Matt

last: Studham

first: Daniel

last: Morgan

first: Andreas

last: Tjarnberg


title: Single-cell RNA-seq reports growth condition-specific global transcriptomes of individual bacteria

city: Würzburg

first: Antoine-Emmanuel

last: Saliba

address: Josef-Schneider-Straße 2 / D15

country: Germany

institute: Helmholtz Institute for RNA-based Infection Research

summary: Bacteria respond to changes in their environment with specific transcriptional programmes, but even within genetically identical populations these programmes are not homogenously expressed. Such transcriptional heterogeneity between individual bacteria allows genetically clonal communities to develop a complex array of phenotypes, examples of which include persisters that resist antibiotic treatment and metabolically specialized cells that emerge under nutrient-limiting conditions. Fluorescent reporter constructs have played a pivotal role in deciphering heterogeneous gene expression within bacterial populations but have been limited to recording the activity of single genes in a few genetically tractable model species, whereas the vast majority of bacteria remain difficult to engineer and/or even to cultivate. Single-cell transcriptomics is revolutionizing the analysis of phenotypic cell-to-cell variation in eukaryotes, but technical hurdles have prevented its robust application to prokaryotes. Here, using the improved poly(A)-independent single-cell RNA-sequencing protocol MATQ-seq, we report the faithful capture of growth-dependent gene expression patterns in individual Salmonella and Pseudomonas bacteria across all RNA classes and genomic regions. These transcriptomes provide important reference points for single-cell RNA-sequencing of other bacterial species, mixed microbial communities and host–pathogen interactions.

sample_organism: Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344

overall_design: In three culture conditions (Late Stationary phase, NaCl shock and Anaerobic shock), we systematically sorted a pool of ten Salmonella (n=60) and single Salmonella (n=71). For Pseudomonas aeruginosa only anaerobic shock condition was applied and systematically sorted as a pool of ten bacteria (n=5) and single bacteria (n=13). After sorting bacteria were lysed immediately and RNA was reverse transcribed following the MATQ-seq protocol. Libraries were prepared unsing Nextera XT (Illumina) library preparation kit. Pooled libraries were sequenced using and Illumina Nextseq 500 (2 × 75 base pairs (bp)), mid-output (1×) and high output (1×) for Pseudomonas, and an Illumina Novaseq 6000 (2 × S1 and 1 × S2 full cartridges, 2 × 50 bp PE) for Salmonella. After demultiplexing, data quality was examined using FastQC (v.0.11.7). Illumina and MATQ-seq adaptors were removed using cutadapt (v.1.9). Trimmed reads were mapped to the S. enterica SL1344 (NCBI ASM21085v2) and P. aeruginosa strain PAO1 genomes using STAR aligner (v.2.5.4b) with default settings. Read counts for each gene were determined using the featureCounts programme. Genes with more than five aligned reads were considered detected. Data were then subjected to PCA, where the 300 genes with the highest variance were selected to perform dimension reduction. Five libraries (M2G3, M3C4, M2H7, M2B7 and M2C10) that were clear outliers on the PCA plot were excluded from further analysis.

first: Fabian

last: Imdahl

first: Ehsan

last: Vafadarnejad

first: Christina

last: Homberger

first: Antoine

middle: E

first: Jörg

last: Vogel


title: Gene expression data from breast ductal cells from women at normal risk or high risk for breast cancer

status: Public on Jul 05 2020

city: Bethesda

first: David

middle: N

last: Danforth

state: MD

address: 10 Center drive

department: NCI/CCR

institute: NIH

summary:
Breast cancer develops through the accumulation of genomic changes in the ductal epithelia cells of normal breast tissue. A determination of whether gene expression changes in ductal cells is associated with an increased risk for breast cancer is needed. We sought to determine if the global gene expression profiles of ductal cells of women at high risk for breast cancer or with cytologic ductal epithelial atypia differed from those of women at normal risk or without cytologic atypia.
We used microarrays to detail the gene expression profile of breast ductal cells associated with normal risk or high risk for sporadic breast cancer and with or without cytologic epithelial atypia. We did not identify any separation of the sample groups (normal risk vs high-risk, or atypia vs nonatypia) according to expression of subgroups of genes.

overall_design: Breast ductal cells were collected by ductal lavage from women at normal risk or at high risk for sporadic breast cancer, and with or without cytologic atypia on cytologic analysis of ductal cells. All subjects were premenopausal. Subjects within the respective risk groups for atypia/no atypia were matched according to age and risk. Total RNA was extracted and hybridized to Affymetrix microarrays. Gene expression microarray analysis of samples from these groups was conducted.

last: Sun


title: Gene expression analysis of mouse ID8 ovarian cancer cells stimulated with CCL6  in vitro

city: Stanford

first: Oliver

last: Dorigo

address: 300 Pasteur Dr

institute: Stanford University

summary: We used mouse Clariom-S microarrays to study the gene expression profile of ID8 cancer cells stimulated with C-C Chemokine Ligand 6 (CCL6).

overall_design: RNA extracted from ID8 mouse ovarian cancer cell lines (n=3) and ID8 cells stimulated with 200ng/mL CCL6 for 6 hours (n=3) was hybridized on Affymetrix Mouse Clariom S microarrays. We sought to identify key regulatory pathways activated in ovarian cancer cells upon stimulation with CCL6 that play a role in migration and metastasis. To understand the signaling mechanism of CCR1-CCL6 interaction in ovarian cancer, we generated biological replicates for unstimulated ID8 cells and ID8 cells stimulated with 200ng/mL CCL6 for 6 hours.


title: Whole-blood transcriptome profiling reveals signatures of metformin and its therapeutic response

city: Riga

first: Monta

last: Brīvība

address: Ratsupites 1

country: Latvia

institute: Latvian Biomedical Research and Study Centre

summary: Metformin, a biguanide agent, is the first-line treatment for type 2 diabetes mellitus due to its glucose-lowering effect. Despite its wide application in the treatment of multiple health conditions, the glycemic response to metformin is highly variable, emphasizing the need for reliable biomarkers.   We chose the RNA-Seq-based comparative transcriptomics approach to evaluate the systemic effect of metformin and highlight potential predictive biomarkers of metformin response in drug-naïve type 2 diabetes patient volunteers in vivo.  Longitudinal blood-derived transcriptome analysis revealed metformin-induced differential expression of novel and previously described genes involved in cholesterol homeostasis (SLC46A1 and LRP1), cancer development (CYP1B1, STAB1, CCR2, TMEM176B) and immune responses (CD14, CD163) after administration of metformin for three months. We demonstrate for the first time transcriptome-based molecular discrimination between metformin responders (delta HbA1c ≥ 1% or 12.6 mmol/mol) and non-responders (delta HbA1c < 1% or 12.6 mmol/mol) determined by expression levels of 56 genes, explaining 13.9% of the variance in the therapeutic efficacy of the drug.  Moreover, we found a significant upregulation of IRS2 gene (log2FC 0.89) in responders compared to non-responders before the use of metformin. Finally, we provide evidence for the mitochondrial respiratory complex I as one of the factors related to the high variability of the therapeutic response to metforminin patients with type 2 diabetes mellitus.

overall_design: Longitudinal RNA-Seq data of venous blood samples collected from 17 type 2 diabetes mellitus patients in two consecutive time points:before administration of metformin, after 3 months of metformin therapy.

last: Ustinova

first: Laura

last: Ansone

first: Ivars

last: Silamikelis

first: Vita

last: Rovite

first: Ilze

last: Elbere

first: Laila

last: Silamikele

first: Ineta

last: Kalnina

first: Davids

last: Fridmanis

first: Jelizaveta

last: Sokolovska

last: Konrade

first: Valdis

last: Pirags

first: Janis

last: Klovins


title: RNA-sequencing of FACS-sorted Prominin1+ radial glial cells of E12.5 SmadNes mutant embryos (Smad1wt/fl;Smad5wt/fl;Nestin:Cre+/0) and control littermates (Smad1wt/fl;Smad5wt/fl;Nestin:Cre0/0)

status: Public on Jul 04 2020

city: Barcelona

first: Gwenvael

last: Le Dréau

email: ledreau.g@gmail.com, gldbmc@ibmb.csic.es, gwenvael.le-dreau@inserm.fr, ledreaugwenvael@yahoo.fr

address: Parc Cientific de Barcelona, Helix building

department: Developmental Biology

country: Spain

institute: Instituto de Biologia Molecular de Barcelona - CSIC

summary: We report the transcriptomes of mouse E12.5 cortical radial glial cells and the changes caused by inhibition of the canonical BMP signalling effectors SMAD1/5, whose expression levels are reduced by 50% in the SmadNes mutants.

overall_design: Examination of the expression levels quantified by RNA-seq of transcript isoforms at the whole-genome level in mouse cortical radial glial cells at embryonic day E12.5.

first: Sonia

last: Najas

first: Isabel

last: Pijuan

first: Anna

last: Esteve-Codina

first: Susana

last: Usieto

first: Juan

middle: D

last: Martinez

first: An

last: Zwijsen

first: Maria

middle: L

last: Arbones

first: Elisa

last: Martí


title: A single cell RNA-seq resource for early sea urchin development

city: Providence

first: Gary

last: Wessel

state: RI

address: 185 Meeting St

department: Molecular Biology, Cell Biology & Biochemistry

institute: Brown University

summary: Here we leverage the sea urchin embryo for its rich history of developmental transitions, its well-established gene regulatory network, and the ability to readily dissociate embryos into single cells to interrogate the embryo by single cell RNA-seq. We tested eight developmental stages in S. purpuratus, from the eight-cell stage to late in gastrulation. The analysis revealed cell types derived from the abundant cells of the three germ layers as well as the rare cells of the germline. We used these datasets to parse out 22 major cell states of the embryo with focus on key transition stages for cell type specification of each germ layer. Sub-clustering of these major embryonic domains revealed over 50 cell states with distinct transcript profiles. Further, we identified the transcript profile of two cell states expressing germ cell factors, one we conclude are the primordial germ cells, and the other transiently present prior to gastrulation. We hypothesize that these cells of the veg2 tier of the early embryo may represent a lineage that converts to the germ line when the primordial germ cells are deleted (Voronina et al., 2008). This broad resource will hopefully enable the community to identify other cell states and genes of interest to expose the underpinning of developmental mechanisms.

overall_design: Eggs and sperm of S.purpuratus were spawned by injection of 0.5M KCl into the adult coelomic cavity. Fertilization was accomplished in sea water containing 10mM p-aminobenzoic acid to reduce cross-linking of the fertilization envelope, and which was washed out after 30 minutes. Embryos were cultured in filtered (0.2micron) sea water collected at the Marine Biological laboratories in Woods Hole MA, until the appropriate stage for dissociation. Multiple fertilizations were initiated in this study and timed such that the appropriate stages of embryonic development were reached at a common endpoint. The embryos were then collected and washed twice with calcium-free sea water, and then suspended hyalin-extraction media (HEM) for 10-15 minutes, depending on the stage of dissociation.  When cells were beginning to dissociate, the embryos were collected and washed in 0.5M NaCl, gently sheared with a pipette, run through a 40micron Nitex mesh, counted on a hemocytometer, and diluted to reach the appropriate concentration for the scmRNA-seq protocol. Equal numbers of embryos were used in each time point and at no time were cells or embryos pelleted in a centrifuge.

first: Stephany

last: Foster

first: Nathalie

last: Oulhen


title: Glioma-initiating cells at tumor edge gain signals from tumor core cells to promote their malignancy

status: Public on Jul 03 2020

city: Birmingham

first: Soniya

last: Bastola

state: AL

address: 1824 6th Ave S

department: Neurosurgery

institute: UAB

summary: Intratumor spatial heterogeneity facilitates therapeutic resistance in glioblastoma (GBM). Nonetheless, understanding of GBM is limited to the resectable tumor core lesion while the seeds for recurrence reside in the unresectable tumor edge. In this study, stratification of GBM to core and edge demonstrated clinically relevant surgical sequelae. We establish regionally derived models of GBM edge and core that retain their spatial identity in a cell autonomous manner. Edge cells show a higher capacity for infiltrative growth, while core cells demonstrate greater therapy resistance. Investigation of intercellular signaling between these two cell populations uncovered the paracrine crosstalk from tumor core that provokes malignancy and therapy resistance of edge cells. These phenotypic alterations were initiated by HDAC1 in GBM core cells which subsequently affect edge cells by secreting the soluble form of CD109 protein.  Our data reveal the role of intracellular communication between regionally different populations of GBM cells in tumor recurrence.

overall_design: Comparison of edge and core regions within human glioblastoma tumors

first: Ichiro

last: Nakano

first: Marat

middle: S

last: Pavlyukov

first: David

middle: K

last: Crossman


title: Genome-Wide CRISPR Screens for Toxoplasma Gondii Genes Related to Artemisinin Susceptibility

city: Cambridge

first: Sebastian

last: Lourido

state: MA

address: 455 Main St.

institute: Whitehead Institute

summary: We used CRISPR to generate pools of T. gondii mutants in the presence of dihydroartemisinin to identify genes that confer increased or decreased sensitivity to this drug.

sample_organism:
Toxoplasma gondii
synthetic construct

platform_organism:
Toxoplasma gondii
synthetic construct

overall_design: We performed three screens in which we looked for mutations that decrease susceptibility to DHA and one screen in which we looked for mutations that increase susceptibility.

first: Saima

last: Sidik


title: Discovery of a molecular glue promoting CDK12-DDB1 interaction to trigger Cyclin K degradation

city: Beijing

first: Zhi

last: Zeng

address: 7 Science Park Road, ZGC life science park

country: China

institute: National Institute of Biological Sciences, Beijing

type: Genome variation profiling by high throughput sequencing

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: Discovery of a molecular glue promoting CDK12-DDB1 interaction to trigger Cyclin K degradation [WES]

summary: Molecular glues are small molecules that exert their biologic or therapeutic activities by inducing gain-of-function interactions between pairs of proteins. In particular, molecular-glue degraders, which mediate interactions between target proteins and components of the ubiquitin proteasome system to cause targeted protein degradation, hold great promise as a unique modality for therapeutic targeting of proteins that are currently intractable. Here, we report a new molecular glue HQ461 discovered by high-throughput screening of small molecules that inhibited NRF2 activity. Using unbiased loss-of-function and gain-of-function genetic screening followed by biochemical reconstitution, we show that HQ461 acts by promoting interaction between CDK12 and DDB1-CUL4-RBX1 E3 ubiquitin ligase, leading to polyubiquitination and proteasomal degradation of CDK12’s interacting protein Cyclin K (CCNK). Degradation of CCNK mediated by HQ461 compromised CDK12 function, leading to reduced phosphorylation of CDK12 substrate, downregulation of DNA damage response genes, and cell death. Structure-activity relationship analysis of HQ461 revealed the importance of a 5-methylthiazol-2-amine pharmacophore and resulted in an HQ461 derivate with improved potency. Our studies reveal a new molecular glue that engages its target protein directly with DDB1 to bypass the requirement of a substrate-specific receptor, presenting a new strategy for targeted protein degradation.

overall_design: Parental HCT-116 were plated sparsely on 10-cm plates to allow the isolation of ten individual clones. Each of these ten clones were expanded to one confluent 15-cm plate to establish ten independent cell populations. One million cells from each population were then plated on one 10-cm plate and treated with 20 μM HQ461 continuously for two weeks, with media change every 3 to 4 days. HQ461 resistant clones emerged from 5 out of the 10 populations. These clones were isolated, expanded, and tested for their sensitivity to HQ461 using cell viability assay. Five HQ461-resistant clones selected from independent populations were subjected to genomic DNA isolation as described in the previous session. Their genomic DNAs were then pooled in equal amounts for whole-exome sequencing at 200x average coverage (Novogene). As a control, genomic DNAs isolated from the corresponding cell populations before HQ461 selection were also pooled for whole-exome sequencing.

first: Lu

last: Lv

first: Peihao

last: Chen

first: Longzhi

last: Cao

first: Yamei

last: Li

first: Yue

last: Cui

first: Qingcui

last: Wu

first: Jiaojiao

first: Jian

middle: H

last: Wang

first: Meng

middle: Q

last: Dong

first: Xiangbing

last: Qi

first: Ting

last: Han


title: Bulk RNA-Sequencing of Murine Normal and Tumor-Associated Omental and Mesenteric Macrophages

first: Oliver

last: Dorigo

summary: The omentum is the most common site of ovarian cancer metastasis. Immune cell clusters called milky spots are found throughout the omentum.  It is however unknown if these immune cells contribute to ovarian cancer metastasis. Here we report that omental macrophages promote the migration and colonization of ovarian cancer cells to the omentum through the secretion of chemokine ligands that interact with chemokine receptor 1 (CCR1).  We found that depletion of macrophages reduces ovarian cancer colonization of the omentum. RNA-sequencing of macrophages isolated from mouse omentum and mesenteric adipose tissue revealed a specific enrichment of CCL6 chemokine ligand in omental macrophages. CCL6 and the human homolog CCL23 were both necessary and sufficient to promote ovarian cancer migration by activating ERK1/2 and PI3K pathways. Importantly, inhibition of CCR1 reduced ovarian cancer colonization. These findings demonstrate a critical mechanism of omental macrophage induced colonization by ovarian cancer cells via CCR1 signaling.

overall_design: Omenta and Mesentery were excised from C57BL/6 mice (n=9) and processed as three separate groups (n=3 per group). Macrophages (CD45+CD11b+F4/80+) cells from dissociated tissues were flow cytometry sorted and the RNA was sequenced

first: Venkatesh

last: Krishnan


title: The Hippo pathway effector YAP controls mouse hepatic stellate cell activation

city: Brussels

first: Inge

last: Mannaerts

address: Laarbeeklaan 103

department: Liver Cell Biology Lab

country: Belgium

institute: Vrije Universiteit Brussel

summary:
We identified the Hippo pathway and its effector YAP as a key pathway that controls stellate cell activation.  YAP is a transcriptional co-activator and we found that it drives the earliest changes in gene expression during stellate cell activation.
Activation of stellate cells in vivo by CCl4 or activation in vitro caused  rapid activation of YAP as revealed by its nuclear translocation  and  the induction of YAP target genes.

overall_design: Hepatic stellate cells were isolated from BALB/c livers. Healthy mice were used for isoaltion of quiescent cells, CCl4 treated fibrotic mice were used for isolation of activated cells. In vitro activation was obtained by culturing fresh quiescent cells on plastic dishes for 10 days

first: I

first: S

middle: B

last: Leite

last: Verhulst

last: Claerhout

first: N

last: Eysackers

first: L

last: Thoen

first: A

last: Hoorens

first: H

last: Reynaert

first: G

last: Halder

middle: A

last: van Grunsven


title: Protein proximity analysis unravels a complex interplay between lymphoid transcription factors and ARID1a in T-cell development.

city: Lund

first: Mikael

last: Sigvardsson

address: Sölvegatan 19

institute: Lund University

summary: Maturation of lymphoid cells is controlled by the action of stage and lineage restricted transcription factors, which work with the general transcription and chromatin remodelling machinery to regulate gene expression. To better understand this functional interplay we used BioID to identify proximity interaction partners for GATA3, TCF7, SPI1, HLF, IKZF1, PAX5, ID1 and ID2. We found that ARID1a, a BAF complex component, interacts with a number of these transcription factors. Consistent with this observation, deletion of Arid1a in early lymphoid progenitors resulted in reduced production of IgM+ immature B-cells, a pronounced block in early T-cell development, a 10-fold reduction in thymic cellularity, a reduction in CD4+CD8+ cells, and a developmental arrest in the DN3 to DN4 transition. Our work highlights the critical importance of functional interactions between stage and lineage restricted factors and the basic transcription machinery during lymphocyte differentiation.

overall_design: T-cells of the developmental stage DN3 were sorted from Thymus of Arid1a Wt and KO mice. Sorted cells were later processed for RNA- and ATAC-seq. Differentally expressed in Arid1a WT vs. KO. Cells were identified using the RNA-seq data and Arid1a WT/KO ATAC signal was counted on DN3 and DN4 T-cell progenitor peak positions (peaks positions generated from data retrived from  GSE100738).

first: Jacob

last: Kuruvilla

first: Jonas

last: Ungerbäck

first: Tobias

last: Strid

first: Rajesh

last: Somasundaram


title: Identification and characterization of a sulfite reductase gene and new insights regarding the sulfur-containing amino acid metabolism in the basidiomycetous yeast Cryptococcus neoformans

city: Tokyo

first: Kiminori

last: Shimizu

address: Niijuku

department: Department of Biological Science and Technology

country: Japan

institute: Tokyo University of Science

summary: The amino acid biosynthetic pathway of invasive pathogenic fungi has been studied as a potential antifungal drug target. Studies of the disruption of genes involved in amino acid biosynthesis have demonstrated the importance of this pathway in the virulence of Cryptococcus neoformans. Here, we identified the MET5 (CNL05500) and MET10 (CNG03990) genes in this pathway, both encoding sulfite reductase, which catalyzes the reduction of sulfite to sulfide. The MET14 (CNE03880) gene was also identified, which is responsible for the conversion of sulfate to sulfite. The use of cysteine as a sulfur source led to the production of methionine via hydrogen sulfide synthesis mediated by CYS4 (CNA06170), CYS3 (CNN01730), and MST1 (CND03690). MST1 exhibited high homology with the TUM1 gene of Saccharomyces cerevisiae, which has functional similarity with the 3-mercaptopyruvate sulfurtransferase (3-MST) gene in humans. Although the hypothesis that hydrogen sulfide is produced from cysteine via CYS4, CYS3, and MST1 warrants further study, the new insight into the metabolic pathway of sulfur-containing amino acids in C. neoformans provided here indicates the usefulness of this system in the development of screening tools for antifungal drug agents.

overall_design: Examination of mRNA expression levels of wild type strain in liquid B mediums supplemented with four different sulfur sources

first: Phuong-Thao

last: Nguyen

first: Akio

last: Toh-e

first: Ngoc-Hung

first: Yumi

first: Akira

last: Watanabe

first: Katsuhiko

last: Kamei


title: Exaggerated autophagy as molecular characterization of Stanford type A aortic dissection: A transcriptome analysis of human ascending aortic tissues

status: Public on Jul 02 2020

city: Nanjing

first: Zeyi

last: Zhou

state: JiangSu province

address: Zhongshan Road No.321

institute: Cardiothoracic surgery department

summary:
Purpose: The aim of this study is to have a fullscape of  molecular pathology of Stanford type A aortic dissection
Methods: All TAAD patients under consideration underwent an ascending aortic replacement surgery during a cardiopulmonary bypass. The normal ascending aortic tissue samples were obtained from patients undergoing coronary artery bypass grafting surgery (CABG) without any aortic diseases. We selected 20 samples (10 TAAD and 10 normal) for the whole transcriptome sequencing. Total RNA was extracted from each sample using TRIzol Reagent (ThermoFisher) and was stored in 1 mL of 75% ethanol at -80 ℃ until further usage.
Conclusions:  We identified exaggerated autophagy as a molecular biomarker for aortic dissection. We also predicted 10 hub genes and an HIF1A-ATG3 axis which could provide new insights in understanding aortic dissection.

overall_design: transcriptome sequencing analysis of  Stanford type A aortic dissection

first: Can

last: Xu

first: Xiyu

last: Zhu

first: Xinlong

last: Tang

first: Bin

last: Gong

first: Congcong

last: Tian


title: TBL1XR1 mutations bias germinal center B-cells towards a pro-tumorigenic memory fate

city: New York

first: Matt

last: Teater

state: NY

address: 445 E 69th St

institute: Weill Cornell Medical College

summary: Diffuse large B-cell lymphomas (DLBCL) encompass a heterogeneous group of diseases derived from different stages of B-cell differentiation. The most clinically aggressive forms of DLBCL, C5/MCD, carry activating mutations in canonical B-cell signaling pathways, as well as early-occurring hits in the poorly-characterized gene TBL1XR1. Here, we investigated the role of TBL1XR1 mutations in the early steps of malignant transformation. We found that TBL1XR1 mutations disrupt germinal center development, and introduce a cell fate bias towards memory B (MB) cells. At the molecular level, TBL1XR1 mutations trigger a switch, by which the SMRT/HDAC3 co-repressor complex shuttles between the lineage-defining transcription factors BCL6 and BACH2. We further demonstrate that terminal differentiation of TBL1XR1 mutant MB is limited upon recall, and that these MB re-enter the germinal center reaction instead, suggesting a role for these cells as the cell-of-origin of C5/MCD DLBCL.

overall_design: RNAseq results of Tbl1xr1 murine model systems

first: Leandro

last: Venturutti

first: Ari

middle: M

last: Melnick


title: Discovery of a molecular glue promoting CDK12-DDB1 interaction to trigger Cyclin K degradation [CRISPR]

first: Zhi

last: Zeng

overall_design: Pooled genome-wide CRISPR Cas9 knockout screening by targeting 19,114 genes with four individual sgRNAs per gene. We treated sgRNA-transduced A549 cells with either the vehicle DMSO or a sub-lethal dose of HQ461 for three weeks to enrich for cells that were resistant to HQ461’s cytotoxic effects.

first: Lu

last: Lv

first: Peihao

last: Chen

first: Longzhi

last: Cao

first: Yamei

last: Li

first: Yue

last: Cui

first: Qingcui

last: Wu

first: Jiaojiao

first: Jian

last: Wang

first: Meng

last: Dong

first: Xiangbing

last: Qi

first: Ting

last: Han


title: Matched primary ovarian cancer, omental metastases, and non-omental metastases from 10 patients with high-grade serous ovarian carcinoma

city: Los Angeles

first: Ye

last: Hu

state: California

address: 8635 W. 3rd Street, Suite 290W

institute: Cedars Sinai Medical Center

summary: Matched high-grade serous ovarian carcinoma samples collected from the ovary (ov), omental metastasis (om-met), and non-omental intraperitoneal metastasis (met) from 10 patients at the time of primary debulking surgery were analyzed for RNA expression by RNA sequencing.

overall_design: FFPE blocks of matched high-grade serous ovarian carcinoma samples collected from the ovary (ov), omental metastasis (om-met), and non-omental intraperitoneal metastasis (met) from 10 patients were sectioned onto uncharged glass slides. One 4 µm H&E-stained section was used by a pathologist to circle the tumor areas and delineate them from the adjacent normal tissue. Depending on the tumor size, 1 to 3 unstained 10 µm sections were macrodissected (removal of non-tumor areas based on the H&E template) with a clean razor blade. Total RNA was isolated using the miRNeasy FFPE kit. RNA sequencing was done by MedGenome.

first: Barbie

last: Taylor-Harding

first: Sandra

last: Orsulic


title: Transcriptome comparison of wild-type and tnnt2a MO zebrafish hearts

status: Public on Jul 01 2020

city: Potsdam

first: Salim

last: Seyfired

address: Karl-Liebknecht-Str. 24-25

institute: University of Potsdam

summary: We performed RNA-seq analyses to characterize transcriptomic changes in non-beating tnnt2a morphant compared to wild-type hearts.

overall_design: Cardiac mRNA profile of wild-type and tnnt2a morphant zebrafish hearts at 54 hpf.

first: Timm

last: Haack

last: Seyfried


title: NanoString expression profiling in metastatic ovarian cancer

state: CA

address: 10833 Le Conte Ave

department: Obstetrics and Gynecology

institute: David Geffen School of Medicine at UCLA

summary:
Purpose: Most molecular characterizations of high-grade serous ovarian cancer (HGSOC) have been done in primary ovarian tumors. Such data may not be fully representative of HGSOC, which is characterized by widespread peritoneal metastases at the time of diagnosis and upon relapse. The most common site of HGSOC metastasis is the omentum. The omentum is characterized by strong immune and fibrotic activities and is a rich source of mesenchymal stem cells (MSCs), which have the capacity to differentiate into cancer-associated fibroblasts (CAFs) and promote ovarian cancer progression. The omentum is also rich in tertiary lymphoid structures (TLS), which are transient aggregates of leukocytes structurally and functionally comparable to secondary lymphoid tissues.

Experimental design: With the goal of elucidating expression patterns in a large number of omental metastases, we profiled expression of immune- and cancer progression-related genes in pretreatment omental metastasis samples from 152 patients diagnosed with HGSOC.

overall_design: A total of 152 specimens from patients with HGSOC were profiled for gene expression and classified by clinicopharmacological characteristics including age, PFS, OS, debulking and BRCA mutation status.


title: Reversible and irreversible PAH in a rat model for MCT+Shunt-induced PAH and hemodynamic unloading.

city: Groningen

first: Diederik

middle: Eilard

last: van der Feen

address: Antonius Deusinglaan 1

department: Pediatric and Congenital Cardiology

country: Netherlands

institute: University Medical Center Groningen

summary: PAH was induced by 60mg/kg MCT and an aorto-caval shunt. At different timepoints of PAH progression (day 14, 21 and 28 after MCT-injection), the left lung with PAH was hemodynamically unloading by unilateral orthotopic transplatation into a syngeneic, healthy recipient. All day 14 and 7/10 day 21 transplanted lungs showed reversal of PAH after LTx. All day 28 and 3/10 day 21 transplanted lungs showed PAH progression after LTx. Lung tissue of Reversible and Irreversible PAH and normal controls, acquired at LTx, was compared using RNA-seq.

overall_design: Reversible PAH: N=11. RNA extracted in 3 biological replicates. Irreversible PAH: N=7, RNA extracted in 3 biological replicates. Normal control: N=5, RNA extracted in 3 biological replicates.

first: D

first: R

last: Berger

first: M

last: Rabinovitch


title: Clonal tracing reveals the contribution of both cancer-intrinsic and -extrinsic mechanisms to the heterogeneity of responses to immune checkpoint blockade [RNA-seq_ICB]

city: Boston

first: Xiaole

last: Liu

address: 3 Blackfan Circle

institute: Dana-Farber Cancer Institute

summary: Although multiple studies have investigated the biomarkers of response to immune checkpoint blockade (ICB), the significance of each biomarker varies across clinical cohorts independent of cancer type. It remains unclear whether primary ICB response and resistance is encoded in the cancer cells (cancer-intrinsic) or driven by the immune microenvironment unique to each host (cancer-extrinsic). To answer this question, we established a unique mouse system that utilizes clonal tracing and mathematical modeling to uncouple the cancer-intrinsic and -extrinsic mechanisms of ICB resistance. We found that tumors with the same clonal constitution show heterogeneous ICB response in different hosts. Primary resistance is associated with the cancer-extrinsic immune microenvironment rather than proliferation of intrinsically ICB-resistant cancer cells. Instead, pre-existing cancer-intrinsic ICB-resistant clones with distinct transcriptional and epigenetic profiles were enriched in responders. We further identified two gene expression signatures associated with cancer-intrinsic resistance, including increased interferon response genes and glucocorticoid response genes. Our findings are supported by experiments in another mouse model and clinical data from multiple ICB treatment cohorts. Our study emphasizes the importance of optimal microenvironment in cancer immunotherapy, and implicates the value of immunotherapy biomarkers that account for both cancer-intrinsic and -extrinsic mechanisms of resistance.

overall_design: We performed RNA-seq of the in vivo bulk CT26 tumors that were control IgG-treated, anti-PD-1 responder, or anti-PD-1 non-responder.

first: Shengqing

last: Gu

first: Xiaoqing

first: Xihao

first: Peng

last: Jiang

first: Ziyi

first: Nicole

last: Traugh

first: Xia

last: Bu

first: Qin

last: Tang

first: Chenfei

first: Zexian

last: Zeng

first: Cliff

last: Meyer

first: Paloma

last: Cejas

first: Klothilda

last: Lim

first: Jingxin

last: Fu

first: Jin

first: Wubing

last: Zhang

first: Xiaofang

last: Xing

first: Yi

first: Benjamin

last: Kroger

first: Henry

last: Long

first: Gordon

last: Freeman

first: Myles

last: Brown

first: X


title: Clonal tracing reveals the contribution of both cancer-intrinsic and -extrinsic mechanisms to the heterogeneity of responses to immune checkpoint blockade [RNA-seq]

first: Xiaole

last: Liu

overall_design: We performed RNA-seq of the parental CT26 line and the two ICB-resistant CT26 clones B04 and B64.

first: Shengqing

first: Xiaoqing

last: Wang

first: Xihao

last: Hu

first: Peng

first: Ziyi

last: Li

first: Nicole

first: Xia


title: Genome-scale CRISPR-Cas9 knockout screening for identification of genes which regulate CD38 expression in RPMI 8226 cells

first: Daisuke

last: Ogiya

address: 450 Brookline Ave

department: Medical Oncology

summary: We showed that interleukin-6 (IL-6) from bone marrow stromal cells downregulated CD38 expression in multiple myeloma (MM) cells. Then we performed a genome-scale CRISPR-Cas9 knockout screening to identify key molecules mediating IL-6-induced CD38 downregulation. Although IL-6 triggered downregulation of CD38 expression on the majority of RPMI 8226 cells, a small fraction maintained relatively high CD38 expression. We therefore sorted those top 5% cells with CD38-high expression and compared these cells with non-sorted control cells. The gene rank based on p-value identified Janus kinase (JAK) 1 and signal transducer-activator of transcription (STAT) 3 as highly positively enriched genes as potential mediators of IL-6-induced CD38 downregulation. We validated STAT3 and found CD38 expression was regulated by both STAT1 (positively) and STAT3 (negatively), and that inhibition of JAK-STAT3 pathway represents  a novel therapeutic option to enhance CD38 expression and anti-CD38 monoclonal antibody-mediated MM cytotoxicity.

overall_design: CRISPR-Cas9 GeCKOv2 library containing 6 unique sgRNAs against each of 19,050 genes and 4 sgRNAs against each of 1,864 microRNAs were infected into myeloma cell line (RPMI 8226) cells. After 72 h incubation with interleukin-6, top 5 % cells with CD38-high expression were sorted and compared these cells with non-sorted control cells. Then deep sequencing of embedded sgRNA barcodes was performed. The detailed procedures were conducted according to the article of Nature Protocol (Nat Protoc. 2017;12(4):828-863.).

first: D

first: M

last: Samur

first: K

middle: C

last: Anderson


title: VRK-1 Extends Lifespan by Activation of AMPK via Phosphorylation

city: Daejeon

first: Seung-Jae

middle: V

last: Lee

address: 291 Daehak-ro

department: Biological Sciences

country: South Korea

institute: KAIST

summary: Vaccinia virus-related kinase (VRK) is an evolutionarily conserved nuclear protein kinase. VRK-1, the single Caenorhabditis elegans VRK ortholog, functions in cell division and germline proliferation. However, the role of VRK-1 in post-mitotic cells and adult lifespan remain unknown. Here, we show that VRK-1 increases organismal longevity by activating the cellular energy sensor, AMP-activated protein kinase (AMPK), via direct phosphorylation. We found that overexpression of vrk-1 in the soma of adult C. elegans increased lifespan, and conversely, inhibition of vrk-1 decreased lifespan. In addition, vrk-1 was required for longevity conferred by mutations that inhibit C. elegans mitochondrial respiration, which requires AMPK. Notably, VRK-1 directly phosphorylated and up-regulated AMPK in both C. elegans and cultured human cells. Thus, our data show that the somatic nuclear kinase, VRK-1, promotes longevity through AMPK activation, and this function appears to be conserved between C. elegans and humans.

overall_design: The investigation of the changes of gene expression in vrk-1 RNAi-treated wild-type or isp-1(qm150) mutant animals

first: Sangsoon

last: Park

first: Murat

last: Artan

first: Seung Hyun

last: Han


title: Genome accessibilty (ATACseq) data in neruonal and non-neuronal cells from the putamen of human heroin abusers

first: Yasmin

last: Hurd

address: 1470 Madison Avenue

institute: Icahn School of Medicine at Mount Sinai

summary: The putamen plays a central role in ther regulation of reward, emotion, motivation and goal-directed behavior. Genome accessibility changes in this brain region are of crucial interest to understand the neurobiological underpinnings of heroin use disorder.

overall_design: Human brains from apparent heroin overdose and normal control Caucasian subjects without head trauma were collected at autopsy with 24 hours of death. Cause of death was determined by forensic pathologist. Neuronal and non-neuronal (glial) cell populations were FACS-sorted based on NeuN expression.


title: An optogenetic switch for the Set2 methyltransferase provides evidence for transcription-dependent and independent dynamics of H3K36 methylation

city: Chapel Hill

first: Austin

last: Hepperla

state: NC

address: 7018B Mary Ellen Jones Building

department: Genetics

institute: University of North Carolina at Chapel Hill

summary: Histone H3 lysine 36 methylation (H3K36me) is a conserved histone modification associated with transcription and DNA repair. Although the effects of H3K36 methylation have been studied, the genome-wide dynamics of H3K36me deposition and removal are not known. We established rapid and reversible optogenetic control for Set2, the sole H3K36 methyltransferase in yeast, by fusing the enzyme with the light activated nuclear shuttle (LANS) domain. Early H3K36me3 dynamics identified methylation in vivo, with total H3K36me3 levels correlating with RNA abundance. Although genes exhibited disparate levels of H3K36 methylation, relative rates of H3K36me3 accumulation were largely linear and consistent across genes, suggesting that H3K36me3 deposition occurs uniformly across all genes in a directed fashion regardless of transcription frequency. Removal of H3K36me3 was highly dependent on the demethylase Rph1. However, the per-gene rate of H3K36me3 loss weakly correlated with RNA abundance and followed exponential decay, suggesting H3K36 demethylases act in a global, stochastic manner. Altogether, these data provide a detailed temporal view of H3K36 methylation and demethylation that suggest transcription-dependent and independent mechanisms for H3K36me deposition and removal, respectively.

overall_design: Examination of reversible Set2 and early dynamics of histone 3 lysine 36 methylation (H3K36me) in yeast.

first: Andrew

last: Lerner

first: Gregory

middle: R

last: Keele

first: Hashem

last: Meriesh

first: Hayretin

last: Yumerefendi

first: David

last: Restrepo

first: Seth

last: Zimmerman

first: James

last: Bear

first: Brian

last: Kuhlman

first: Ian

last: Davis

last: Strahl


title: Timeless collaborates with DDX11 to ensure processive replication of G4-forming DNA [RNA-seq]

city: Cambridge UK

first: Julian

last: Sale

address: Cambridge Biomedical Campus, Francis Crick Ave

department: PNAC

country: United Kingdom

institute: MRC Laboratory of Molecular Biology

summary: Regions of DNA with the potential to form secondary structure pose a frequent and significant impediment to DNA replication that must be actively countered by cells in order to preserve genetic and epigenetic integrity. The fork protection complex (FPC), a conserved group of replisome-associated proteins, comprising Timeless, plays an important role in maintaining efficient replisome function. A previously unappreciated DNA binding domain in the C terminus of Timeless, which exhibits specificity towards G4 structures, acts in collaboration with the DDX11 helicase to ensure replication of G4 structures in vivo and maintenance of genetic and epigenetic stability. Using RNA-seq experiments, we show that (i) Timeless and DDX11 share common gene targets, (ii) the promoter of Timeless- and DDX11-dependent genes are enriched in G4 structures in the vicinity of their promoter and (iii) both Timeless and DDX11 share a common set of gene targets with the FANCJ helicase known to maintain epigenetic stability in the vicinity of G4 structures.

overall_design: RNA-seq data of WT, TIMELESS, DDX11 and FANCJ KO DT40 cells

first: Leticia

last: Koch Lerner

first: Pierre

last: Murat


title: Clonal tracing reveals the contribution of both cancer-intrinsic and -extrinsic mechanisms to the heterogeneity of responses to immune checkpoint blockade

first: Xiaole

last: Liu

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: Clonal tracing reveals the contribution of both cancer-intrinsic and -extrinsic mechanisms to the heterogeneity of responses to immune checkpoint blockade [ATAC-seq]

overall_design: We performed ATAC-seq of the parental CT26 line and the two ICB-resistant CT26 clones B04 and B64.

first: Shengqing

last: Gu

first: Xiaoqing

last: Wang

first: Xihao

last: Hu

first: Peng

last: Jiang

first: Ziyi

last: Li

first: Nicole

last: Traugh

first: Xia

last: Bu

first: Qin

last: Tang

first: Chenfei

first: Zexian

last: Zeng

first: Cliff

last: Meyer

first: Paloma

last: Cejas

first: Klothilda

last: Lim

first: Jingxin

last: Fu

first: Jin

first: Wubing

last: Zhang

first: Xiaofang

last: Xing

first: Yi

first: Benjamin

last: Kroger

first: Henry

last: Long

first: Gordon

last: Freeman

first: Myles

last: Brown

first: X


title: RNA-seq analysis of immune responses from human blood monocyte dendritic cells from 3 donors stimulated with 5 pattern recognition receptor (PRR) ligands along with their pairwise and triplet combinations

city: Chicago

first: Nicolas

last: Chevrier

state: Illinois

address: 5640 South Ellis Avenue

department: Pritzker School of Molecular Engineering

institute: The University of Chicago

summary: The immune system makes decisions in response to combinations of multiple microbial inputs. We do not understand the combinatorial logic governing how higher-order combinations of microbial signals shape immune responses. Here, using coculture experiments and statistical analyses, we discover a general property for the combinatorial sensing of microbial signals, whereby the effects of triplet combinations of microbial signals on immune responses can be predicted by combining the effects of single and pairs. Mechanistically, we find that singles and pairs dictate the information signaled by triplets in mouse and human DCs at the levels of transcription, chromatin and protein secretion. We exploit this simplifying property to develop cell-based immunotherapies prepared with adjuvant combinations that trigger protective responses in mouse models of cancer. We conclude that the processing of multiple input signals by innate immune cells is governed by pairwise effects, which will inform the rationale combination of adjuvants to manipulate immunity.

overall_design: mRNA expression profiles of human monocytes derived dendritic cells from three independent donors stimulated with five PRR ligands in singles, their pair and triplet combinations generated by RNA-seq in triplicates using illumina nextseq 550

first: Surya

last: Pandey


title: ATAC-seq analysis of immune responses from bone marrow derived dendritic cells stimulated with 7 pattern recognition receptor (PRR) ligands along with their pairwise and triplet combinations

overall_design: Genome wide chromatin accessbility profiles of mouse bone marrow derived dendritic cells stimulated for 6 hours with  3 random triplet PRR ligand combinations and their constituting single and pair ligand combinations generated by ATAC-seq in duplicates using illumina NextSeq 550


title: Somatic mutation distributions in cancer genomes vary with three-dimensional chromatin structure

city: Houston

first: Kadir

middle: Caner

last: Akdemir

state: TX

address: 1515 Holcombe Blvd

institute: MD Anderson Cancer Center

summary: Somatic mutations arise during the life history of a cell. Mutations occurring in cancer driver genes may ultimately lead to the development of clinically detectable disease. Nascent cancer lineages continue to acquire somatic mutations throughout the neoplastic process and during cancer evolution. Extrinsic and endogenous mutagenic factors contribute to the accumulation of these somatic mutations. Understanding the underlying causes of mutations is critical for developing potential preventions and tailoring the clinical treatments. Earlier studies have revealed that DNA replication timing and chromatin modifications are associated with variations in mutational density. In order to understand the interplay between spatial genome organization and individual mutational processes, we report here a study of more than 3000 whole genome datasets from 50 different cancer studies. Our analyses revealed that different mutational processes lead to distinct somatic mutation distributions between chromatin folding domains. APOBEC- or MSI-related mutations are enriched in transcriptionally-active domains while mutations occurring due to tobacco-smoke and ultraviolet (UV) light exposure or a gastric flux-related mutational signature (signature 17) enrich predominantly in transcriptionally-inactive domains. Active mutational processes dictate the mutation distributions in cancer genomes, therefore mutational distributions could shift during cancer evolution upon mutational processes switch. Moreover, a dramatic instance of extreme chromatin structure in humans, that of the unique folding pattern of the inactive X-chromosome leads to distinct somatic mutation distribution on X chromosome in females compared to males in various cancer types. Overall, the interplay between three-dimensional genome organization and active mutational processes has a substantial influence on the large-scale mutation rate variations observed in human cancer.

overall_design: Profiling chromatin conformation dynamics in 4 cancer cell lines.

first: Jesse

last: Dixon


title: Actin-induced Endothelial Injury After Hematopoietic Stem Cell Transplant and Impact on Clinical Outcomes

city: Cincinnati

first: Stella

last: Davies

state: OH

address: 3333 Burnet Ave

department: Cancer & Blood Disease Institute

institute: Cincinnati Children's Hospital

summary: We report vitamin D binding protein levels in the serum alter macrophage function

overall_design: RNA-seq after incubation of normal peripheral blood mononuclear cells with serum collected from transplant recipients 100 days after HSCT with either high or low levels of Vitamin D Binding Protein.

first: Nathan

last: Luebbering


title: Imprecise DNMT1 activity coupled with neighbor-guided correction enables robust yet flexible epigenetic inheritance

first: Wei

last: Xie

address: Haidian District

institute: Tsinghua University

summary: The epigenome, including DNA methylation, is stably propagated during mitotic division. However, single-cell clonal expansion produces heterogeneous methylomes, raising the question of how the DNA methylome remains stable despite constant epigenetic drift. Here, we report that a clonal population of DNA (cytosine-5)-methyltransferase 1 (DNMT1)-only cells produces a heterogeneous methylome, which is robustly propagated on cell expansion and differentiation. Our data show that DNMT1 has imprecise maintenance activity and likely possesses weak de novo activity, leading to spontaneous ‘epimutations’. However, these epimutations tend to be corrected through a neighbor-guided mechanism, ikely enabled by environment-sensitive de novo activity (‘tuner’) and maintenance activity (‘stabilizer’) of DNMT1. By generating base-resolution maps of de novo and maintenance activities, we find that H3K9me2/3-marked regions show enhanced de novo activity, and CpG islands have both poor maintenance and de novo activities. The imprecise epigenetic machinery coupled with neighbor-guided correction may be a fundamental mechanism underlying robust yet flexible epigenetic inheritance.

overall_design: In this study, we showed that the epigenetic mosaicism is an intrinsic property of imprecise DNMT1 activity which leads to spontaneous DNA methylation “epimutations”. However, such “epimutations” tend to be corrected over time in a neighbor DNA methylation-guided manner. In silico modeling and base-resolution mapping of DNMT1 maintenance and de novo activity support a model that the de novo activity of DNMT1 is not simply a leaky activity, but rather a major factor in an environment-sensitive “Tuner-Stabilizer” system, to achieve robust-yet-flexible maintenance of the methylome.

first: Qiujun

last: Wang

first: Guang

last: Yu

first: Xuan

last: Ming

first: Bing

last: Zhu


title: Genes regulated by phosphorylation of serine 236 on OCT4 in embryonal carcinoma

city: Gyeonggi-do

first: Hyonchol

last: Jang

address: 323, Ilsan-ro, Ilsandong-gu, Goyang-si

institute: National Cancer Center

summary: OCT4 was knocked down  doxycycline-dependently, and then  wild type or S236 phosphorylation mimicking OCT4 were rescued.

overall_design: mRNA expression profiles of the  ' NCCIT Teton-shOCT4 DOX (-), which harbor endogenous OCT4;  NCCIT Teton-shOCT4 DOX (+), which are depleted in  OCT4;  NCCIT Teton-shOCT4 DOX (+) OCT4 WT rescue ; NCCIT Teton-shOCT4 DOX (+) OCT4 S236D rescue '   by Illumina Hi-seq 2500.

first: Bomin

last: Song


title: Acquired FGFR and FGF alterations confer resistance to estrogen receptor (ER) targeted therapy in ER+ metastatic breast cancer

status: Public on Jun 30 2020

city: Cambridge

first: Ofir

last: Cohen

state: Massachusetts

address: 415 Main St, Cambridge, MA 02142

department: Cancer Program

institute: Broad Institute

summary: Beyond acquired mutations in the estrogen receptor (ER), mechanisms of resistance to ER-directed therapies in ER+ breast cancer have not been clearly defined. We conducted a genome-scale functional screen spanning 10,135 genes to investigate genes whose overexpression confer resistance to selective estrogen receptor degraders. Pathway analysis of candidate resistance genes demonstrated that the FGFR, ERBB, insulin receptor, and MAPK pathways represented key modalities of resistance. In parallel, we performed whole exome sequencing in paired pre-treatment and post-resistance biopsies from 60 patients with ER+ metastatic breast cancer who had developed resistance to ER-targeted therapy. The FGFR pathway was altered via FGFR1, FGFR2, or FGF3/FGF4 amplifications or FGFR2 mutations in 24 (40%) of the post-resistance biopsies. In 12 of the 24 post-resistance tumors exhibiting FGFR/FGF alterations, these alterations were not detected in the corresponding pre-treatment tumors, suggesting that they were acquired or enriched under the selective pressure of ER-directed therapy. In vitro experiments in ER+ breast cancer cells confirmed that FGFR/FGF alterations led to fulvestrant resistance as well as cross-resistance to the CDK4/6 inhibitor palbociclib, through activation of the MAPK pathway. The resistance phenotypes were reversed by FGFR inhibitors and, to a lesser extent, MEK inhibitors, suggesting potential treatment strategies.

overall_design: Examination of the transcriptional output (mRNA) of  the FGFR  activation, with T47D cells perturbed to overexpress FGFR pathway activation including FGFR1, FGFR2 (WT, K660N, M538I and N550K), and FGF3, as well as GFP and parental as a control. DMSO, fulvestrant (100 nM), palbociclib (1 uM), FIIN-3 (100 nM), and trametinib (500 nM) as single agent and in combinations for 24 hours. All experimental conditions were done in 6 replicates

first: Pingping

last: Mao

first: Wagle

last: Nikhil


title: NanoString gene expression data of OCI-C5x and OCI-P5x

city: Seattle

first: Reid

last: Shaw

state: WA

address: 401 Terry Ave

institute: Cure First

summary: Gene expression data of two ovarian carcinoma patient-derived cells was analyzed with respect to high-throughput functional siRNA screens and small-molecule screens.

overall_design: Two ovarian carcinoma patient-derived cells were characterized using nanoString nCounter PanCancer Pathways Panel.


title: Tandem CAR and single CAR T cells have different fates and therapeutic potency

city: Beijing

first: Tong

last: Chuan

state: Beijing

address: No. 28 Fuxing Road

department: Molecular Biology and Immunology

institute: Chinese PLA General Hospital

summary: Chimeric antigen receptor T (CAR T) cells targeting CD19 have achieved breakthroughs in the treatment of haematological malignancies, but many clinical studies have also shown that a proportion of patients relapse after remission. In this study, we designed a series of tandem CARs (TanCARs) and found that TanCAR7 T cells retained relatively potent antitumour activity compared with single CAR T cell upon target antigen recognition. It may be associated with stable immunological synapse formation and rapid degranulation. Our transcriptional analysis underscores the potential of  scFv domains binding to direct different T cell fates.

overall_design: In order to assess the different phenotypic and functional patterns of CARs between TanCAR7 and single CAR, we compared the genome-wide transcriptional profiles of TanCAR7, CD19 CAR and CD20 CAR T cell after cocultured with Raji cells for 24 h.


title: Grad-seq shines light on unrecognized RNA and protein complexes in the model bacterium Escherichia coli

city: Cologne

first: Konrad

middle: U.

last: Förstner

state: North Rhine-Westphalia

address: Gleueler Str. 60

department: Information Services

institute: ZB MED - Information Centre for Life Sciences

summary: Stable protein complexes, including those formed with RNA, are major building blocks of every living cell. Escherichia coli has been the leading organism with respect to global bacterial protein-protein networks. However, there has been no global census of RNA/protein complexes in this model species of microbiology. Here, we performed Grad-seq to establish an RNA/protein complexome, reconstructing sedimentation profiles in a glycerol gradient for ~85% of all E. coli transcripts and ~49% of the proteins. In presenting use cases for this resource to be exploited, we show that its stable association with 30S ribosomes gives the seemingly noncoding RNA RyeG of prophage origin away as an mRNA of a toxic small protein. Similarly, we show that the broadly conserved uncharacterized protein YggL is a 50S subunit factor in assembled 70S ribosomes. Overall, this study complements existing interactome resources for the primary model bacterium E. coli with valuable RNA/protein complexome information and should facilitate functional discovery in this and many related species.

overall_design: 22 total RNA samples from E. coli wild type resolved on a glycerol gradient resulting in 20 fractions and a pellet fraction. A lysate sample is given as input control.

first: Jens

last: Hör

first: Silvia

last: Di Giorgio

middle: U

first: Milan

last: Gerovac

first: Elisa

last: Venturini

first: Jörg

last: Vogel


title: Ribosome profiling under ABCE1 depletion to study aberrant translation termination

city: Lausanne

first: Rene

last: Dreos

state: VD

address: Ecublens

department: CIG

country: Switzerland

institute: UNIL

summary: We report the effect of siRNA-mediated ABCE1 KD in HeLa cells, using a modified ribosome profiling protocol adapted from Ingolia et al., 2011 in order to specifically investigate ABCE1 role on translation termination

overall_design: Control and ABCE1 KD conditions, three replicates for condition. totalRNA samples in parallel for input control.

first: G

last: Annibaldis

first: R

first: O

last: Muhlemann


title: FosB-regulated gene expression in venral hippocampo-accumbal projection neurons

city: New York

first: Aarthi

last: Ramakrishnan

state: New York

address: 1425 Madison Avenue

department: Neuroscience

summary: We examined here the regulation of gene expression in ventral hippocampus neurons that project to nucleus accumbens (vHPC-NAc) by the transcription factor, ΔFosB. The activity of this circuit is critical to stress-induced social withdrawal. ΔFosB regulates the physiology and behavior of this circuit to produce resilience. Circuit-specific translating ribosomal affinity purification (TRAP) was conducted followed by RNASeq. A strain of floxed Fosb mice (the gene that encodes ΔFosB) was crossed with a ribosomal L10-GFP fusion protein line (Rosa26). To target vHPC-NAc neurons, a retrograde recombinant HSV-Cre was injected into NAc to retrogradely drive expression of GFP and knockout the Fosb gene in vHPC-NAc. Bilateral biopsy punches of vHPC containing GFP-labeled projections were collected, pooled, and processed by TRAP followed by 3rd-generation sequencing of actively translating mRNA. We have now identified Fosb-regulated gene expression in a specific hippocampal-to-accumbens circuit that is important for resilience to stress-induced social withdrawal.

overall_design: Circuit-specific mRNA profiles of ventral hippocampus projection neurons to NAc from wild type (WT) and FosB knockout (KO) mice were generated by deep sequencing, in triplicate.

first: Andrew

last: Eagle

first: Ryan

last: Bastle

first: Alfred

middle: J

last: Robison

first: Ian

last: Maze


title: Cathepsin D deficiency in mammary epithelium transiently stalls murine breast cancer by interference with mTORC1 signaling

city: Freiburg

first: Maria

middle: Elena

last: Hess

address: Breisacherstr. 153

department: Institute for Medical Bioinformatics and Systemsmedicine

institute: Universitaetsklinikum Freiburg

summary: Cathepsin D (CTSD) is an aspartic protease mainly located within the endosomal/lysosomal compartment where it participates in intracellular protein breakdown. In breast cancer, CTSD is overexpressed and suggested as an independent marker of poor prognosis but the cells responsible for this association as well as the mode of action remain unclear. Here, we use a conditional CTSD knockout mouse, which allowed cell type-specific inactivation of CTSD in the transgenic MMTV-PyMT (PyMT) breast cancer model. We demonstrate that CTSD deficiency in mammary epithelial cells, but not myeloid cells, blocks tumor development in a cell-autonomous manner. By comparing CTSD-expressing with CTSD-deficient tumor cells from this model, we show that lack of CTSD induces cellular quiescence and disrupts mechanistic Target of Rapamycin Complex 1 (mTORC1) signaling under starvation. While quiescence is reversed under long-term starvation by rewiring oncogenic signaling pathways and gene expression, mTORC1 signaling remains permanently disabled in CTSD-deficient PyMT cells. Together, these studies reveal a tumor cell-autonomous effect of CTSD deficiency, and establish a pivotal role of this major aspartic protease in the cellular response to oncogenic stimuli.

overall_design: RNA expression profiles were obtained of breast tumor tissue from 18-week-old control (CTSD wild type) and MMTV-cre;Ctsd-/- (CTSD-deficient) PyMT mice in 4 biological replicates each. RNA expression was also analyzed in spontaneously immortalized primary breast tumor cells obtained from one Ctsd+/+ (CTSD wild type) and one MMTV-cre;Ctsd-/- (Ctsd-/-) PyMT mouse. To this end, cells were grown in a mild starvation medium for short-term (10 days, ‘1% FCS’) or long-term (12 weeks, ‘1% FCS LT’) in 3 biological replicates each.

first: Stephanie

last: Ketterer

first: Thomas

last: Reinheckel

middle: E

first: Patrick

last: Metzger

first: Melanie

last: Boerries


title: Senescence rather than apoptosis of alveolar progenitor cells drives pulmonary fibrosis (human)

city: los angeles

first: Changfu

last: Yao

state: Ca

address: 8700 Beverly Blvd. AHSP Room A9401

department: Lung and Regenerative Medicine Institutes

institute: cedars-sinai medical center

summary: Idiopathic pulmonary fibrosis (IPF) is a fatal form of interstitial lung disease associated with progressive scarring of lung tissue and declining pulmonary function in aging individuals. Here we employ data mining and single cell RNA sequencing (scRNAseq) to define candidate drivers of AT2 senescence in IPF lung tissue. We show that AT2 cells isolated from IPF lung tissue exhibit characteristic features of cellular senescence and reduced abundance of Sin3a, critical determinant of endodermal progenitor cell maintenance in the developing lung. Conditional loss of Sin3a within AT2 cells of the adult mouse lung activated a program of p53-dependent cellular senescence and AT2 cell depletion, leading to progressive pulmonary fibrosis. In contrast, ablation of AT2 cells through conditional activation of a DTA toxin gene led to transient fibrosis that resolved over time, suggesting that senescence of AT2 cells was a critical driver of progressive fibrosis. Activation of b6 integrin was observed within a subset of AT2 cells with Sin3a loss-of-function that localized to the advancing margin of advancing fibroproliferative lesions. Systemic inhibition of TGF  signaling or delivery of senolytic drugs prevented lung fibrosis in Sin3a loss-of-function mice. Our findings establish a novel mouse model that recapitulates key pathological features of human IPA and show that p53-dependent senescence is a proximal regulator of TGF  induction and progressive pulmonary fibrosis.

overall_design: Single cell RNA-seq using 10x chromium system on enriched epithelial cells from 3 IPF patient samples ( Fibrotic region) and 3 donor control samples

first: Barry

last: Stripp


title: Transcriptomic analysis of four different subtypes of ubc22 mutant plants

city: Saskatoon

first: Sheng

last: Wang

address: 107 Wiggins Road

department: Biochemistry, Microbiology and Immunology

country: Canada

institute: University of Saskatchewan

summary: Transcriptomic analysis showed that transcript levels of genes related to several pathways were altered differently in the different subtypes of plants. In one subtype, the mutant plants had increased expression of genes related to pathogen defense and showed enhanced resistance to a plant pathogen.

overall_design: Analysis of differentiate expression genes in four subtypes of ubc22 mutant plants. Based on leaf size, shape and color, these mutant plants (pregeny plants from the same parent mutant) can be categorized into four subtypes: (1) Type I: similar to WT; (2) Type II: plants with wider and rounder leaves; (3) Type III: narrow and long leaves with pale color; (4) Type IV: small plants with serrated leaves.

first: Qiang

last: Li

first: Hong


title: Senescence rather than apoptosis of alveolar progenitor cells drives pulmonary fibrosis

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: CD103hi lung-resident regulatory T cells constrain the fibrotic responses induced by CD103low tissue-resident pathogenic CD4 T cells

city: Yachiyo-City

first: Kota

last: Kokubo

state: Chiba

address: Yachiyodai-minami 1-10-12

department: Department of Immunology

institute: Chiba University

summary: Tissue-resident memory T (TRM) cells are crucial mediators of adaptive immunity in non-lymphoid tissues.  However, the functional heterogeneity and pathogenic roles of CD4+ TRM cells that reside within chronic inflammatory lesions remain unknown.  We found that CD69hiCD103low CD4+ TRM cells produced effector cytokines and promoted the inflammation and fibrotic responses induced by chronic exposure to Aspergillus fumigatus.  Simultaneously, immunosuppressive CD69hiCD103hiFoxp3+ CD4+ regulatory T (Treg) cells were induced and constrained the ability of pathogenic CD103low TRM cells to cause fibrosis.  Thus, lung tissue-resident CD4+ T cells play crucial roles in the pathology of chronic lung inflammation, and CD103 expression defines pathogenic effector and immunosuppressive tissue-resident cell subpopulations in the inflamed lung.

overall_design: T cells in lung treated with Aspergillus antigen are assessed by RNA-seq and ATAC-Seq.

first: Tomomi

last: Ichikawa

first: Kiyoshi

last: Hirahara

first: Masahiro

last: Kiuchi

first: Ami

last: Aoki

first: Yuki

last: Morimoto

first: Jin

last: Kumagai

first: Atsushi

last: Onodera

first: Naoko

last: Mato

first: Damon

last: Tumes

first: Yoshiyuki

last: Goto

first: Koichi

last: Hagiwara

first: Yutaka

last: Inagaki

first: Tim

last: Sparwasser

first: Kazuyuki

last: Tobe

first: Toshinori

last: Nakayama


title: Somatic Gain of KRAS Function in the Endothelium is Sufficient to Cause Vascular Malformations that Require MEK but not PI3K Signaling

first: Manuel

middle: Eliezer

last: Cantu Gutierrez

address: 1 Baylor Plaza

institute: Baylor College of Medicine

summary:
Rationale – We previously identified somatic activating mutations in the KRAS gene in the endothelium of the majority of human sporadic brain arteriovenous malformations (bAVMs); a disorder characterized by direct connections between arteries and veins. However, whether this genetic abnormality alone is sufficient for lesion formation, as well as how active KRAS signaling contributes to AVM formation, remains unknown.
Objective – To establish the first in vivo models of somatic KRAS gain of function in the endothelium in both mice and zebrafish in order to directly observe the phenotypic consequences of constitutive KRAS activity at a cellular level in vivo, and to test potential therapeutic interventions for AVMs.
Methods and Results – Using both postnatal and adult mice, as well as embryonic zebrafish, we demonstrate that endothelial-specific gain of function mutations in Kras (G12D or G12V) are sufficient to induce bAVMs. Active KRAS signaling leads to altered endothelial cell morphogenesis and increased cell size, ectopic sprouting, expanded vessel lumen diameter, and direct connections between arteries and veins. Furthermore, we show that these lesions are not associated with altered endothelial growth dynamics or a lack of proper arteriovenous identity, but instead appear to feature exuberant angiogenic signaling. Finally, we demonstrate that KRAS-dependent AVMs in zebrafish are refractory to inhibition of the downstream effector PI3K, but instead require active MEK signaling.
Conclusions – We demonstrate that active KRAS expression in the endothelium is sufficient for bAVM formation, even in the setting of uninjured adult vasculature. Furthermore, the finding that KRAS-dependent lesions are reversible in zebrafish suggests that MEK inhibition may represent a promising therapeutic treatment for AVM patients.

overall_design: HUVEC were electroporated with pCS2+ (control) or pCS2-KRAS4AG12V and treated with U0126, LY294002 or DMSO (vehicle control).

first: Jason

last: Fish

first: Carlos

middle: P

last: Flores Suarez

first: Emilie

last: Boudreau

first: Alexander

last: Herman

first: Dakota

last: Gustafson

first: Peter

last: DiStefano

first: Meng

last: Cui

first: Zhiqi

last: Chen

first: Karen

last: Berman De Ruiz

first: Taylor

middle: S

last: Schexnayder

first: Christopher

last: Ward

first: Ivan

last: Radovanovic

first: Joshua

middle: D

last: Wythe


title: Senescence rather than apoptosis of alveolar progenitor cells drives pulmonary fibrosis (mouse_bulk RNAseq)

first: Changfu

last: Yao

summary: bulk RNAseq comparison of global transcription profiles of mouse lung AT2 Sin3a-LOF at different time points, evalute AT2 cellular senescecne contribution to lung fibrosis.

overall_design: Bulk RNA-seq using whole lung homogenate of Sin3a-LOF mouse lung for different time points after tamoxifen treatment, comparing transcriptome change, with at lease 3 replicates ( note, 6wk3 sample was not sequenced at the same time with all the other samples)

first: Barry

last: Stripp


title: PCB126 (3,3',4,4',5-Pentachlorobiphenyl) induced alterations in m6A RNA modifications in zebrafish embryos

status: Public on Jun 29 2020

city: Woods Hole

first: Neelakanteswar

last: Aluru

state: MA

address: 45 Water Street

department: Biology

institute: Woods Hole Oceanographic Institution

summary: Chemical modifications of proteins, DNA and RNA moieties play critical roles in regulating gene expression. Emerging evidence suggests these RNA modifications (epitranscriptomics) have substantive roles in basic biological processes. One of the most common modifications in mRNA and noncoding RNAs is N6-methyladenosine (m6A). In a subset of mammalian mRNAs, m6A sites are preferentially enriched near stop codons, in 3′ UTRs, and within exons, suggesting an important role in the regulation of mRNA processing and function including alternative splicing and gene expression. Very little is known about the effect of environmental chemical exposure on m6A modifications. As many of the commonly occurring environmental contaminants alter gene expression profiles and have detrimental effects on physiological processes, it is important to understand the effects of exposure on this important layer of gene regulation. Hence, the objective of this study was to characterize the acute effects of developmental exposure to PCB126, an environmentally relevant dioxin-like PCB, on m6A methylation patterns. We exposed zebrafish embryos to PCB126 for 6 hours starting from 72 hours post-fertilization and profiled m6A RNA using methylated RNA immunoprecipitation followed by sequencing (MeRIP-seq), as well as assessing changes in mRNA splicing. We observed several hundreds of peaks that are differentially expressed in response to PCB126 exposure (FDR 5%). The majority of the peaks are preferentially located around the 3’UTR and stop codons. Pathway analysis of m6A marked transcripts induced by PCB126 exposure revealed that these transcripts are associated with important developmental pathways (MAPK, Hedgehog, Notch and Wnt). These results suggest that PCB126 could affect developmental gene expression patterns by altering m6A levels. Gene expression analysis revealed upregulation of classical aryl hydrocarbon receptor (AHR) target genes such as cytochrome P450s in response to PCB126 exposure. Interestingly, none of the AHR target genes overlapped with the m6A altered transcripts, suggesting that xenobiotic metabolism may not be under m6A regulation. Further studies are necessary to understand the functional consequences of exposure-associated alterations in m6A levels. This work is supported by NIEHS ES024915.

overall_design: AB strain of zebrafish embryos were exposed to 10 nM PCB126 (3,3',4,4',5-pentachlorobiphenyl, 99.2% purity; UltraScientific, RI, USA) or DMSO (solvent control; 0.01%) starting at 72 hours post-fertilization (hpf) for 6 hours. This duration of exposure was chosen based on previous studies that demonstrated differential gene expression after 6 hour exposure. In addition, short term exposure allows us to characterize primary changes in the m6A patterns. Each treatment consisted of four biological replicates with 48 embryos per replicate. Embryos were maintained in glass petri dishes at 28±0.5oC at a density of 1 embryo per mL in 0.3X Danieau's solution (pH 7.2). At the end of the exposure, embryos were thoroughly rinsed with 0.3X Danieau's solution and sampled for MeRIP sequencing.  Four biological replicates per treatment condition.

first: Sibel

last: Karchner


title: Senescence rather than apoptosis of alveolar progenitor cells drives pulmonary fibrosis (mouse scRNAseq)

overall_design: Single cell RNA-seq using 10x chromium system on enriched epithelial cells from 2 time points (4 week and 6 week after tamoxifen treated mouse lungs) of either control or Sin3a-LOF lungs, cells are isloated from 2 males and 2 females for each group of each time point.


title: Inflammatory transcript signatures and activated regulatory T lymphocytes in melanoma metastases

status: Public on Jun 27 2020

city: Brussels

first: Grégoire

last: de Streel

state: Woluwe-Saint-Lambert

address: 75 Avenue Hippocrate

department: de Duve Institute

institute: UCLOUVAIN

summary:
Purpose: to study the correlations between transcriptional signatures reflecting the abundance and functional activity of tumor-infiltrating inflammatory cells from RNA-seq data on one hand, and the number of intratumoral activated regulatory T lymphocytes
Methods: Gene expression profiles of 19 snap-frozen cutaneous metastases from 19 melanoma patients were obtained by RNA-seq of poly-A RNA. Cryosections from the same tumors were stained by multiplexed immunofluorescence for GARP and FoxP3. FoxP3+GARP+ activated regulatory T lymphocytes (Treg) were quantified by computerized image analysis. Correlations between the proportion of activated Treg and gene expression signatures of inflammatory cells and pathways were analyzed by Gene Set Enrichment Analysis.
Results:  The genes whose expression level correlated the most with the proportion of activated Tregs were significantly enriched for genes expressed by T lymphocytes and for genes induced by IFN-gamma, TNF-alpha, IL-1-beta and TGF-beta, including genes induced by TGF-beta in effector T cells.
Conclusions: Our  results in this small series of melanoma samples suggest that Treg infiltration and Treg-dependent TGF-beta activity are proportional to T-cell infiltration and effector activity in tumors.

overall_design: mRNA profiles of cutaneous melanoma metastases

first: Nicolas

last: van Baren

first: Marjorie

last: Mercier

first: Gregoire


title: P2X7 receptor-dependent microRNA expression profile in the brain following status epilepticus in mice

status: Public on Jun 25 2020

city: Dublin

first: Giorgia

last: Conte

address: 123 St stephen's green

department: Physiology and medical physics

country: Ireland

institute: Royal College of surgeons in Ireland

summary: We explored the microRNA expression profile in mice lacking the P2X7 receptor before and after seizures. Genome-wide microRNA profiling was performed using hippocampi from wild-type and P2X7 knock-out mice following status epilepticus induced by intraamygdala kainic acid. This revealed that genetic deletion of the P2X7 receptor results in distinct patterns of microRNA expression. Specifically, we found that in vehicle-injected control mice the lack of P2X7 resulted in the up-regulation of 50 microRNAs and down-regulation of 35 microRNAs. Post-status epilepticus, P2X7 deficiency let to the up-regulation of 44 microRNAs while 13 microRNAs were down-regulated. Moreover, there was only limited overlap between identified P2X7-dependent microRNAs between control conditions and post-status epilepticus, suggesting P2X7 regulating the expression of different microRNAs during normal physiology and pathology. Bioinformatics analysis found that genes targeted via P2X7-dependent microRNAs were particularly overrepresented within pathways involved in intracellular signalling, inflammation and cell death, processes repeatedly linked to P2X7. Moreover, whereas genes involved in signalling pathways and inflammation were common among up-and down-regulated P2X7-dependent miRNAs during physiological and pathological conditions, genes associated with cell death seemed to be restricted to up-regulated miRNAs during both physiological conditions and post-status epilepticus. Taken together, our results demonstrate that P2X7 impacts on the expression profile of microRNAs in the brain, thereby possibly contributing to both the maintenance of normal cellular homeostasis and pathological processes.

platform_organism:
Mus musculus
Rattus norvegicus

overall_design: miRNA profiling was obtained using 1 µg of total RNA from 3 samples per experimental group (4 experimental groups) each sample was a pool of 2 hippocampi from different mice.

first: Tobias

last: Engel

first: Eva

last: Jimenez Mateos


title: Senescence rather than apoptosis of alveolar progenitor cells drives pulmonary fibrosis (mouse bulk AT2)

summary: bulk RNAseq of AT2 cells transcriptome changes comparing Sin3aLOF with control two weeks after tamoxifen treatment

overall_design: bulk RNAseq of AT2 cells transcriptome change comparing Sin3aLOF (SftpcCreER;Sin3a flox/flox; Rosa mTmG) with control (SftpcCreER; Rosa mTmG) two weeks after tamoxifen treatment, 3 replicates of each group.


title: eQTL mapping of the 12S globulin cruciferin gene PGCRURSE5 as a novel candidate associated with starch content in potato tubers

status: Public on Jun 24 2020

city: Mlochow

first: Dorota

last: Sołtys-Kalina

address: Platanowa 19

department: Mlochow Research Centre

country: Poland

institute: IHAR-PIB

summary: We prepared QTL map of tuber starch content in potato diploid population. Then, we examined expression level of enzyme ADP-glucose pyrophosphorylase (AGPase) taking part in starch biosynthesis (marker AGPaseS-a). Based on starch content and  AGPaseS-a expression, we constructed four bulks prepared from RNA isolated from tubers of F1 individuals H1 and H2 consisted of high TSC genotypes, bulks L1 and L2 were made of low TSC genotypes. Plants in bulks H1 and L1 strongly expressed AGPaseS-a, whereas those in bulks H2 and L2 exhibited low levels. Then we used RNA-seq technology for selection of genes displaying differential expression between RNA pools. For selected candidate genes we mapped expression QTL (e-QTL) and found eQTL of eAGPaseS-a and ePGRCRURSE5, were close to the corresponding loci of (AGPaseS-a) and the 12S globulin cruciferin gene (PGCRURSE5). We concluded that the cruciferin gene PGRCRURSE5 is a novel candidate involved in the regulation of starch content in potato tubers and suggests that cruciferin may be a novel PTST protein in potato tubers.

overall_design: Examination of eQTL for candidate genes involved in starch accumulation in potato tubers.

first: Katarzyna

last: Szajko

first: Emil

last: Stefańczyk

first: Paulina

last: Smyda-Dajmund

first: Jadwiga

last: Śliwka

first: Waldemar

last: Marczewski


title: MDA-MB-231_ZNF704_ChIP-seq

first: Chao

last: Yang

address: Youanmenwai rd

institute: Capital Medical University

summary: Copy number gain in chromosome 8q21 is considered as the risk factor of genetic abnormalities associated with development of breast cancer, yet the oncogenic potential underlying this amplicon in breast carcinogenesis remains to be delineated. We report here that ZNF704, a gene mapped to 8q21, is recurrently amplified in various malignancies including breast cancer. We found that ZNF704 acts as transcription repressor and interacts with the transcription corepressor SIN3A complex. Genome-wide interrogation of the transcriptional targets identifies that the ZNF704/SIN3A complex represses a panel of genes including PER2 that are critically involved in the function of circadian clock. Indeed, ZNF704 overexpression prolongs the period and dampens the amplitude of circadian clock. We showed that ZNF704 promotes the proliferation and invasion of breast cancer cells in vitro and accelerates the growth and metastasis of breast cancer in vivo. Consistently, the level of ZNF704 expression is inversely correlated with that of PER2 in breast carcinomas, and high level of ZNF704 correlates with advanced histological grades, lymph node positivity, and poor prognosis of breast cancer patients, especially those with HER2+ and basal-like subtypes. These results indicate that ZNF704 is an important regulator of circadian clock and a potential driver for breast carcinogenesis.

overall_design: Three samples were analyzed in total, including MDA-MB-231-Input, MDA-MB-231-ZNF704 and MDA-MB-231-SIN3A.


title: Expression data in 4T1 cells after co-culture with murine PI/AI BMDM macrophages

city: London

first: Valentí

last: Gómez

address: UCL Cancer Institute, 72 Huntley Street

department: UCL Cancer Institute

institute: University College London

summary:
Macrophages can exert both pro- and anti-tumorigenic features. Interaction between tumour-associated macrophages and tumour cells trigger signalling mechanisms affecting gene expression patterns in both cells. Changes in tumor gene expression affect essential physiological processes such as metabolism, proliferation or adhesion that can lead to an increased invasive and metastatic potential.
We used microarrays to detail the global program of gene expression in tumour cells after pro-inflammatory (anti-tumorigenic, M1 or PI) or anti-inflammatory (pro-tumorigenic, M2 or AI) and identified distinct classes of down-regulated genes during this process.

overall_design: Murine macrophages were differentiated after bone marrow extraction (BMDM) and polarised towards a PI or AI phenotype. Macrophages were then co-cultured for 24 hours with 4T1 (triple negative breast cancer mouse cells). Both populations were separated and RNA extraction and hybridization on Affymetrix microarrays was performed on the tumour fraction. 3 biological replicates (macrophages derived from individual mice) were used in the experiment.

first: Valenti

last: Gomez

first: Tony

last: Ng

first: Mathew

last: Arno


title: Mapping the epigenomic and transcriptomic interplay during memory formation and recall in the hippocampal engram ensemble

status: Public on Jun 23 2020

city: Rehovot

first: Asaf

last: Marco

address: Herzl 229

country: Israel

institute: The Hebrew University of Jerusalem

summary: Purpose: we utilized a mouse model that permanently labels neurons activated throughout a specific experience to decipher the interplay between chromatin accessibility, 3D-chromatin architecture and transcriptional changes across different memory phases. Non activated (basal) and activated neurons during memory encoding (early), consolidation (late) and recall (reactivated) were sorted and subjected to nuclear RNA sequencing (nRNA-seq) to determine gene expression, ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) to assess chromatin accessibility, chromosome conformation capture (Hi-C) to identify global 3D -genome architecture and promoter capture Hi-C to identify the long range promoter-enhancer interactions.  Results: We have found that during each memory phase, the same promoters interact more frequently with a distinct subset of enhancers (i.e. unique). We also identified a smaller subset of interactions in which the promoters were interacting with the same enhancers across different memory phases (i.e. common). Furthermore, Reactivated neurons presented significantly stronger interaction scores (as calculated by CHiCAGO). Hence, although the number of unique interactions was similar across early, late and reactivated states, stronger interaction scores indicate that specific promoter- enhancer interactions occur more frequently during memory recall.

overall_design: By utilizing promoter-capture Hi-C (pc-HiC) technique, we studied the precise changes that occur in long-range promoter-enhancers interactions during the process of memory encoding, consolidation and recall. For this study, we used custom-designed collection of 10,000 biotinylated RNA “baits” targeting ~5000 promoters. To obtain 10,000 nuclei per condition, we pooled the hippocampal tissue from multiple mice (10-12) and isolated each group of cells via fluorescence-activated cell sorting (FACS). Collected nuclei were subjected to pc-HiC library preparation in 3-5 biological replicates.

first: Hiruy

last: Meharena

first: Vishnu

last: Dileep


title: Rapid Generation of Regionally Specified CNS Neurons by Sequential Patterning and Conversion of Human Induced Pluripotent Stem Cells

city: Aarhus

first: Mark

last: Denham

address: Høegh-Guldbergs Gade 10

department: Biomedicine

country: Denmark

institute: Aarhus University

summary: The differentiation of patient-specific induced pluripotent stem cells (iPSCs) into specific neuronal subtypes has been exploited as an approach to modeling a variety of neurological disorders. However, achieving a highly pure population of neurons is challenging when using directed differentiation methods, especially for neuronal subtypes generated by complex and protracted protocols. In this study, we efficiently produced highly pure populations of regionally specified CNS neurons by using a modified NGN2-Puromycin direct conversion protocol. The protocol is amenable across a range of iPSC lines, with an efficiency above 97% by day 21, and above 95% of neurons positive for MAP2. This NGN2-Puromycin conversion resulted in a significant number of peripheral neurons, but by incorporating a short CNS patterning step, we eliminated these peripheral neurons. Furthermore, we used the patterning step to control the rostral-caudal identity. This approach to sequential patterning and NGN2-Puromycin conversion, when patterned with SMAD inhibitors, produced pure populations of forebrain neurons; when SMAD inhibitors and WNT agonists were applied, the approach produced anterior hindbrain excitatory neurons, resulting in a neuronal population containing VSX2/SHOX2 V2a interneurons. Overall, this sequential patterning and conversion protocol can be used for the production of a variety of CNS excitatory neurons from patient-derived iPSCs, which is a highly versatile system for investigating early disease events for a range of neurological disorders including Alzheimer's disease, motor neurons disease and spinal cord injury.

overall_design: 12 samples in total. Triplicates of 4 conditions. H9 undifferentiated human embryonic stem cells are a control sample.


summary: Purpose: we utilized a mouse model that permanently labels neurons activated throughout a specific experience to decipher the interplay between chromatin accessibility, 3D-chromatin architecture and transcriptional changes across different memory phases. Non activated (basal) and activated neurons during memory encoding (early), consolidation (late) and recall (reactivated) were sorted and subjected to nuclear RNA sequencing (nRNA-seq) to determine gene expression, ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) to assess chromatin accessibility, chromosome conformation capture (Hi-C) to identify global 3D -genome architecture and promoter capture Hi-C to identify the long range promoter-enhancer interactions.  Results: our data demonstrates that the initial phase of memory formation alters the chromatin accessibility landscape of activated neurons, with long lasting stable changes occurring predominantly within enhancer regions. Moreover, many of these enhancers did not return to their baseline state after stimulation ceased, but remain accessible and stable throughout all memory phases.

overall_design: We measured genome-wide changes of chromatin accessibility across different phases of memory.  To obtain 10,000 nuclei per condition, we pooled the hippocampal tissue from multiple mice (10-12) and isolated each group of cells via fluorescence-activated cell sorting (FACS). Collected nuclei were subjected to ATAC-seq library preparation in 3 biological replicates.

first: Ravikiran

last: Raju

first: Jose

last: Davila-Velderrain


summary: Purpose: we utilized a mouse model that permanently labels neurons activated throughout a specific experience to decipher the interplay between chromatin accessibility, 3D-chromatin architecture and transcriptional changes across different memory phases. Non activated (basal) and activated neurons during memory encoding (early), consolidation (late) and recall (reactivated) were sorted and subjected to nuclear RNA sequencing (nRNA-seq) to determine gene expression, ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) to assess chromatin accessibility, chromosome conformation capture (Hi-C) to identify global 3D -genome architecture and promoter capture Hi-C to identify the long range promoter-enhancer interactions.  Results: Our analysis revealed dynamic changes in compartments organization, where we observed re-localization of large chromatin segments from inactive to permissive environment (and vice versa) during the initial and late phase of memory formation. Interestingly, 52% of the regions in the early phase that switched from B to A maintained that state in the late phase (i.e. remained in state A. Moreover, nearly all these regions significantly overlapped with gained DARs identified in our ATAC-seq analysis, confirming the transition of sub-compartment from inactive to permissive environment. This data indicates that while some loci undergo sub-compartment switching across different memory phases, the majority are stable, correlate with chromatin accessibility and therefore might contribute to long-term changes in neuronal properties and function after initial activation.

overall_design: We were interested in delineating the precise changes that occur in 3D nuclear organization to facilitate functionally-relevant promoter-enhancer interactions and transcriptional alterations. Hi-C libraries were prepared from 100,000 FACS sorted cells/per group (collected from two animals) in two biological replicates, using DovetailTM Hi-C kit manual (v.1.03, Dovetail Genomics, Chicago, USA).