title: Hepatic transcriptomic alterations for 1,3,5,7,9,11-hexabromocyclododecane (HBCD) after 5-day exposure in rats

summary:
To identify liver transcripts differentially expressed due to treatment with 1,3,5,7,9,11-hexabromocyclododecane (HBCD), we collected RNA from male Harlan Sprague Dawley rats exposed to 0, 0.06, 0.641, 6.41, 64.1 or 641 mg/kg HBCD, 5 days after exposure for animals 7 weeks of age.  These samples were interrogated with the Affymetrix Rat Genome 230 2.0 GeneChip array.
A total of 1,2,1,23, and 247 gene transcripts were differentially expressed due to HBCD treatment after exposure to 0.06, 0.641, 6.41, 64.1 or 641 mg/kg HBCD (false discovery rate (FDR) < 0.05).

overall_design: There were 6 groups and 6x replication for each group, for 36 total samples. The groups were (1) HBCD-0mg/kg, (2) HBCD-0.06mg/kg, (3) HBCD-0.641mg/kg, HBCD-6.41mg/kg, HBCD-64.1mg/kg, and HBCD-641mg/kg. We compared vehicle control (0mg/kg) vs dose using the R/maanova package. The differentially expressed transcripts were identified based on a statistical significance threshold of FDR<5%.


title: Brominated flame retardants effects on the liver transcriptome

status: Public on Jul 15 2020

city: RTP

first: Keith

last: Shockley

state: NC

address: 111 TW Alexander Drive

country: USA

institute: NIEHS

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

overall_design: Refer to individual Series


title: Hepatic transcriptomic alterations for decabromodiphenylethane (DBDPE) after 5-day exposure in rats

summary:
To identify liver transcripts differentially expressed due to treatment with decabromodiphenylethane (DBDPE), we collected RNA from male Harlan Sprague Dawley rats exposed to 0, 0.1, 0.97, 9.71, 97.1 or 970 mg/kg DBDPE, 5 days after exposure for animals 7 weeks of age.  These samples were interrogated with the Affymetrix Rat Genome 230 2.0 GeneChip array.
A total of 0,0,0,2, and 3 gene transcripts were differentially expressed due to DBDPE treatment after exposure to 0.1, 0.97, 9.71, 97.1 or 970 mg/kg DBDPE (false discovery rate (FDR) < 0.05).

overall_design: There were 6 groups and 6x replication for each group, for 36 total samples. The groups were (1) DBDPE-0mg/kg, (2) DBDPE-0.1mg/kg, (3) DBDPE-0.97mg/kg, DBDPE-9.71mg/kg, DBDPE-97.1mg/kg, and DBDPE-970mg/kg. We compared vehicle control (0mg/kg) vs dose using the R/maanova package. The differentially expressed transcripts were identified based on a statistical significance threshold of FDR<5%.

middle: R

first: June

middle: K

last: Dunnick


title: Hepatic transcriptomic alterations for decabromodiphenyl oxide (decaBDE) after 5-day exposure in rats

summary:
To identify liver transcripts differentially expressed due to treatment with decabromodiphenyl oxide (decaBDE), we collected RNA from male Harlan Sprague Dawley rats exposed to 0, 0.1, 0.959, 9.59, 95.9 or 959 mg/kg decaBDE, 5 days after exposure for animals 7 weeks of age.  These samples were interrogated with the Affymetrix Rat Genome 230 2.0 GeneChip array.
A total of 0,11,27,25, and 30 gene transcripts were differentially expressed due to decaBDE treatment after exposure to 0.1, 0.959, 9.59, 95.9 or 959 mg/kg decaBDE (false discovery rate (FDR) < 0.05).

overall_design: There were 6 groups and 6x replication for each group, for 36 total samples. The groups were (1) decaBDE-0mg/kg, (2) decaBDE-0.1mg/kg, (3) decaBDE-0.959mg/kg, decaBDE-9.59mg/kg, decaBDE-95.9mg/kg, and decaBDE-959mg/kg. We compared vehicle control (0mg/kg) vs dose using the R/maanova package. The differentially expressed transcripts were identified based on a statistical significance threshold of FDR<5%.


title: Hepatic transcriptomic alterations for 1,2-bis(tribromophenoxy)ethane (BTBPE) (Firemaster 680) after 5-day exposure in rats

summary:
To identify liver transcripts differentially expressed due to treatment with 1,2-bis(tribromophenoxy)ethane (BTBPE) (Firemaster 680), we collected RNA from male Harlan Sprague Dawley rats exposed to 0, 0.07, 0.69, 6.88, 68 or 680 mg/kg BTBPE, 5 days after exposure for animals 7 weeks of age.  These samples were interrogated with the Affymetrix Rat Genome 230 2.0 GeneChip array.
A total of 0,0,0,0, and 0 gene transcripts were differentially expressed due to BTBPE treatment after exposure to 0.07, 0.69, 6.88, 68 or 680 mg/kg BTBPE (false discovery rate (FDR) < 0.05).

overall_design: There were 6 groups and 6x replications for each group, except there were 5 samples for the 68 mg/kg group, for a total of 35 samples. The groups were (1) BTBPE-0mg/kg, (2) BTBPE-0.07mg/kg, (3) BTBPE-0.69mg/kg, BTBPE-6.88mg/kg, BTBPE-68mg/kg, and BTBPE-680mg/kg. We compared vehicle control (0mg/kg) vs dose using the R/maanova package. The differentially expressed transcripts were identified based on a statistical significance threshold of FDR<5%.


title: Low-cost high-resolution chromatin profiling with CUT&Tag

city: Seattle

first: Jorja

last: Henikoff

state: WA

address: 1100 Fairview AV N, A1-162

department: Basic Sciences

institute: Fred Hutchinson Cancer Research Center

type: Genome binding/occupancy profiling by high throughput sequencing

summary: We recently introduced CUT&Tag, an epigenomic profiling strategy in which antibodies are bound to chromatin proteins in situ in permeabilized nuclei, and then used to tether the cut-and-paste transposase Tn5. Activation of the transposase simultaneously cleaves DNA and adds DNA sequencing adapters (“tagmentation”) for paired-end DNA sequencing. Here, we introduce a streamlined CUT&Tag protocol that suppresses exposure artifacts to ensure high-fidelity mapping of the antibody-targeted protein and improves signal-to-noise over current chromatin profiling methods. Streamlined CUT&Tag can be performed in a single PCR tube from cells to amplified libraries, providing low-cost high-resolution genome-wide chromatin maps. By simplifying library preparation, CUT&Tag requires less than a day at the bench from live cells to sequencing-ready barcoded libraries. Because of low background levels, barcoded and pooled CUT&Tag libraries can be sequenced for ~$25 per sample, enabling routine genome-wide profiling of chromatin proteins and modifications that requires no special skills or equipment.

overall_design: We used Cleavage under targets and Tagmentation (Cut-and-Tag), a chromatin profiling strategy in which antibody-targeted controlled integration of DNA sequencing adapters produces libraries for paired-end DNA sequencing.

first: Steven

first: Hatice

middle: S

last: Kaya-Okur

first: Derek

middle: H

last: Janssens

first: Kami

last: Ahmad


title: CENP-A overexpression promotes aneuploidy and karyotypic heterogeneity

status: Public on Jul 16 2020

city: Chicago

first: Daniel

last: Foltz

address: 303 E Superior St

institute: Northwestern University

type: Expression profiling by high throughput sequencing

summary: Centromeric localization of the evolutionarily conserved histone H3 variant, CENP-A. is essential for chromosomal stability. CENP-A overexpression (OE) causesd its mislocalization to non-centromeric regions resulting in chromosomal instability (CIN) in yeast, flies and human cells. CENP-A OE and mislocalization have been observed in cancers and correlates with poor prognosis. However, the molecular consequences of CENP-A OE on CIN and aneuploidy have not been defined. Here, we overexpressed YFP-CENP-A in a pseudodiploid DLD1 cell line and showed that CENP-A OE leads to its mislocalization and CIN due to defects in kinetochore integrity and kinetochore-microtubule attachments. CENP-A OE also leads to its mislocalization and CIN in a xenograft mouse model. Under these conditions, it contributes to aneuploidy with karyotypic heterogeneity in human cells and xenograft mouse model.  In summary, our studies provide a molecular link between CENP-A OE and aneuploidy, and suggest that karyotypic heterogeneity may contribute to the aggressive phenotype of CENP-A overexpressing cancers.

overall_design: Three biological replicates were analyzed for each of two conditions

first: Roshan

middle: L

last: Shrestha

first: Ann

last: Hogan

first: Munira

middle: A

last: Basrai


title: Single-Cell Analyses Elucidate the Cellular and Molecular Landscape of Ulcerative Colitis

city: La Jolla

first: John

last: Chang

state: CA

address: 9500 Gilman Drive

department: School of Medicine

institute: UCSD

summary: Inflammatory bowel disease (IBD) encompasses a spectrum of complex intestinal disorders characterized by dysregulated host innate and adaptive immune responses to gut microbiota in genetically susceptible hosts. We generated an integrated single-cell (scRNA-seq, scTCR-seq, and scBCR-seq) resource dataset that revealed key cellular components and cell states of the gastrointestinal mucosal and peripheral immune systems in health and ulcerative colitis (UC).

overall_design: Single-cell RNA-seq and BCR/TCR repertoire analyses of immune cell from 15 patients

first: Zhaoren

last: He


title: The Gene expression profiling by high throughput sequencing in adult mouse incisor

status: Public on Jul 10 2020

city: Los Angeles

first: Shuo

last: Chen

state: California

address: 2250 Alcazar Street, Clinical Science Annex, Room 103, CLINICAL SCIENCE ANNEX ROOM 103

institute: Center for Craniofacial Molecular Biology, University of Southern California


title: Insights into the global effect on Staphylococcus aureus growth arrest by induction of the endoribonuclease MazF toxin

city: Geneva

first: Julien

last: Prados

address: Rue Michel Servet 1

department: SCMU

country: Switzerland

institute: University of Geneva

summary: A crucial bacterial strategy to avoid killing by antibiotics is to enter a growth arrested state, yet the molecular mechanisms behind this process remain elusive. The conditional overexpression of MazF, the endoribonuclease toxin of the MazEF toxin-antitoxin system in Staphylococcus aureus, is one approach to induce bacterial growth arrest, but its targets remain largely unknown. We used overexpression of MazF and high-throughput sequence analysis following the exact mapping of non-phosphorylated transcriptome ends (nEMOTE) technique to reveal in vivo toxin cleavage sites on a global scale. We obtained a catalogue of MazF cleavage sites and unearthed an extended MazF cleavage specificity that goes beyond the previously reported one. We correlated transcript cleavage and abundance in a global transcriptomic profiling during mazF overexpression. We observed that MazF affects RNA molecules involved in ribosome biogenesis, cell wall synthesis, cell division and RNA turover and thus deliver a plausible explanation for how mazF overexpression induces stasis. We hypothesize that autoregulation of MazF occurs by directly modulating the MazEF operon, such as the rsbUVW genes that regulate the alternative SigB, including an observed cleavage site on the MazF mRNA that would ultimately play a role in entry and exit from bacterial stasis.

overall_design: mazF is a toxin with an endoribonuceolitic activity. To study mazF in S. aureus, we build the strain dmazEF where the toxin (mazF) and its antitoxin (mazE) are deleted. We introduced into it a plasmid (pF) with mazF under the control of anhydrotetracycline inducible promoter. At OD 0.4, and after 10 minutes induction of mazF, the RNA is extracted and prepared folowing two protocols: RNA-seq and nEMOTE-seq. While RNA-seq allow to obtain a global picture of the expressed RNA fragments in the cell, nEMOTE-seq focus specifically on mono-phosphorylated 5'-ends. As the endoribonucleic activity of mazF is expected to create new monoposphorylated 5'-ends when an RNA is cleaved, nEMOTE-seq is used to find cleavage sites of mazF by looking at 5'-ends specifically appearing when mazF is expressed.

first: Roberto

last: Sierra

first: Olesya

last: Panasenko

first: Diego

middle: O

last: Andrey

first: Betty

last: Fleuchot

first: Peter

last: Redder

first: William

last: Kelley

first: Patrick

last: Viollier

first: Adriana

last: Renzoni


title: Regulated snoRNA expression during differentiation corresponds with a change in ribosomal RNA pseudouridylation and altered pre-ribosomal RNA processing [Nano77]

status: Public on Jul 09 2020

city: Durham

first: Kathleen

last: McCann

address: 111 TW Alexander Dr

institute: National Institute of Environmental Health Sciences

summary: H/ACA small nucleolar RNAs (snoRNAs) guide pseudouridylation as part of a small nucleolar ribonucleoprotein complex (snoRNP). Disruption of H/ACA snoRNA expression in stem cells impairs pluripotency, yet it remains unclear how H/ACA snoRNAs contribute to differentiation. To determine if H/ACA snoRNA expression is dynamic during differentiation, we comprehensively profiled H/ACA snoRNA expression in multiple murine cell types and during differentiation in three cellular models, including mouse embryonic stem cells and mouse myoblasts. We determined that H/ACA snoRNA expression is cell-type specific, and we identified a subset of snoRNAs that are specifically regulated during differentiation. Additionally, we demonstrated that a decrease in Snora27 expression upon differentiation corresponds to a decrease in pseudouridylation of its target site within the E-site transfer RNA (tRNA) binding region of the 28S ribosomal RNA (rRNA) in the large ribosomal subunit. Many of the snoRNAs regulated during differentiation have target nucleotides in the 28S rRNA, and we found that pre-rRNA processing of large subunit precursors is altered during differentiation. Together, these data suggest a model in which H/ACA snoRNAs are specifically regulated during differentiation to potentially alter pseudouridylation and fine tune ribosome function.

overall_design: In this study, we measured expression of 72 H/ACA snoRNAs in primary spermatogonial stem cells, in cultured NIH3T3 cells, RAW264.7 macrophages, and in E14Tg2a mouse embryonic stem cells, and C2C12 mouse myoblasts before and after differentiation using a custom NanoString nCounter assay.

first: Sanam

last: Kavari

first: Bart

middle: T

last: Phillips

first: Adam

middle: B

last: Burkholder

first: Traci

middle: M

last: Hall


title: Single-cell analysis of gene expression variation and phenotype switching in melanoma

city: Leuven

first: k

last: spanier

address: Herestraat 49

country: Belgium

institute: KU Leuven

summary: We combine extensive profiling of single-cell gene expression (scRNA-seq of 39,263 cells) and chromatin accessibility applied to ten genetically-homogeneous, patient-derived melanoma cultures, to examine intra- and intertumoral phenotypic heterogeneity, to identify new cell states, and to track dynamic transitions at single-cell resolution.

overall_design:
scRNA-seq (10x Genomics Chromium Single Cell 3’ Solution V2 Chemistry and Drop-seq) of human melanoma cell lines with and without perturbations (SOX10-KD)
Bulk RNA-seq of human melanoma cell lines with and without perturbations (baselines, SOX10/EGR3/NFATC2 knockdown and THZ2 treatment)
ATAC-seq of human melanoma cell lines (untreated).

first: J

last: Wouters

first: Z

last: Kalender-Atak

first: V

last: Christiaens

first: K

middle: I

last: Spanier

first: S

last: Aerts


title: Single-cell RNA-seq of human CD4+ and CD8+ T cells transduced with a TAA-specific low-avidity TCR and the CD8αβ co-receptor

status: Public on Jul 13 2020

city: Lausanne

first: Benoit

middle: Manuel

last: Carreres

state: Vaud

address: Bugnon 25A

department: Departement of oncology

institute: Universite de Lausanne

summary: Most naturally occurring major histocompatibility class I restricted T cell receptors (TCRs) that target over-expressed tumor-associated self-antigens (TAAs) are of low avidity due to selection and tolerance in the host and are CD8 co-receptor dependent. Adoptive T cell transfer with TCR-engineered T cells thus rely on the function of CD8+ T cells and do not exploit beneficial CD4+ T cell functions. Hence, we developed a novel strategy that combines expression of a TAA-specific low-avidity TCR with the CD8ab co-receptor and explored the properties of purified transgenic CD8+ and CD4+ T cells separately, in vitro and in vivo. We found that CD8ab co-transfer enhanced TCR+ CD8+ T cell function by increasing their serial tumor killing capacity, indicating that limited availability of endogenous CD8 co-receptors impedes full deployment of their functional potential. Engineered CD4+ T cells were efficiently reprogrammed into hybrid multifunctional cytotoxic and helper T cells at the single-cell level: they recognized and killed cells expressing the cognate class I restricted tumor antigen, became serial killers, produced mostly TH1 and preserved some TH2 cytokines, and showed superior anti-tumor function in vivo in a leukemia xenograft model. CD8ab co-transfer restored the TCR-pMHC interaction in CD4+ T cells and enhanced early TCR signaling events. Single-cell RNA-seq profiling suggests that co-transferred CD4+ T cells acquired a cytotoxic gene expression program and at the same time retained CD4 lineage specific features. In conclusion, we present a novel approach that allows us to (1) enhance the function of TCR-transgenic CD8+ T cells and (2) to manufacture class I pMHC targeted hybrid cytotoxic and helper T cells with both CD8+ and CD4+ T cell functions readily available at the single-cell level.

overall_design:
9 samples were analyzed from three different timepoints of the experiment: freshly isolated (frozen on Day 0 and thawed on Day 21), expanded (Day 14), and cocultured (Day 21). Single-cell isolation (10XGenomics) and library preparation (10XGenomics) were performed at two timepoints (Day 14 and Day 21). Two samples per lane were loaded for samples with 3000 single cells, and 1 sample per lane was loaded for samples with 5000 single cells. The sequencing was performed in a paired-end PE100-100 configuration.
No reads data provided due to human data policies

first: Jan

last: Rath

first: Isabelle

middle: E

last: Gruber

first: David

last: Gfeller

first: Caroline

last: Arber


title: Regulated snoRNA expression during differentiation corresponds with a change in ribosomal RNA pseudouridylation and altered pre-ribosomal RNA processing

type: Non-coding RNA profiling by high throughput sequencing


title: NSD1-deposited H3K36me2 directs de novo methylation in the mouse male germline and counteracts Polycomb-associated silencing

city: Vancouver

first: Kenjiro

last: Shirane

state: British Columbia

address: 2350 Health Sciences Mall

department: Medical Genetics

country: Canada

institute: University of British Columbia

type: Methylation profiling by high throughput sequencing

summary: While de novo DNA methylation (DNAme) in mammalian germ cells is dependent upon DNMT3A and DNMT3L, oocytes and spermatozoa show distinct patterns of DNAme. In mouse oocytes, de novo DNAme requires the lysine methyltransferase (KMTase) SETD2, which deposits H3K36me3. Surprisingly, we show here that SETD2 is dispensable for de novo DNAme in the male germline. Rather, the KMTase NSD1, which broadly deposits H3K36me2 in euchromatic regions, plays a critical role in de novo DNAme in prospermatogonia, including of imprinted genes. However, males deficient in germline NSD1 show a more severe defect in spermatogenesis than Dnmt3l-/- males. Furthermore, unlike DNMT3L, NSD1 safeguards a subset of genes against H3K27me3-associated transcriptional silencing. In contrast, H3K36me2 plays only a minor role in de novo DNAme during oogenesis and females with NSD1 deficient oocytes are fertile. Thus, the sexually dimorphic pattern of DNAme in mature mouse gametes is driven by distinct profiles of H3K36 methylation.

overall_design: ChIP-seq from control and Setd2 KO, Nsd1 KO or Dnmt3l KO prospermaogonia at E16.5 and P0. WGBS (PBAT) in control and Setd2 KO or Nsd1 KO prospermaogonia at E16.5 and P0. RNA-seq from control and Nsd1 KO or Dnmt3l KO prospermatogonia at E16.5 and P0.

first: Fumihito

last: Miura

first: Takashi

last: Ito

first: Matthew

middle: C

last: Lorincz


title: Microbiota alteration is associated with the development of stress-induced despair behavior

status: Public on Jul 08 2020

city: Charlottesville

middle: Michael

last: Johanson

state: VA

address: 200 Jeanette Lancaster Way

department: Department of Neuroscience

institute: UVa School of Medicine

summary: Depressive disorders often run in families, which, in addition to the genetic component, may point to the microbiome as a causative agent. Here, we employed a combination of behavioral, molecular and computational techniques to test the role of the microbiota in mediating despair behavior. In chronically stressed mice displaying despair behavior, we found that the microbiota composition and the metabolic signature dramatically change. Specifically, we observed reduced Lactobacillus and increased circulating kynurenine levels as the most prominent changes in stressed mice. Restoring intestinal Lactobacillus levels was sufficient to improve the metabolic alterations and behavioral abnormalities. Mechanistically, we identified that Lactobacillus-derived reactive oxygen species may suppress host kynurenine metabolism, by inhibiting the expression of the metabolizing enzyme, IDO1, in the intestine. Moreover, maintaining elevated kynurenine levels during Lactobacillus supplementation diminished the treatment benefits. Collectively, our data provide a mechanistic scenario for how a microbiota player (Lactobacillus) may contribute to regulating metabolism and resilience during stress.

overall_design:
Mice were either wild type or SCID genotype and were either kept naïve or treated with an EAE or chronic stress protocol as described in doi: 10.1038/srep43859. Fecal samples were taken at multiple time points after commencement of treatment.

Sequencing performed at UVa Genome Analysis & Technology Core,
Office of Research Core Administration.
University of Virginia Health System,
P.O. Box 800741,
Charlottesville, VA 22908-0741

first: Ioana

last: Marin

first: Alban

last: Gaultier


title: Hepatic transcriptomic alterations for 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (TBB) after 5-day exposure in rats

first: Keith

last: Shockley

summary:
To identify liver transcripts differentially expressed due to treatment with 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (TBB), we collected RNA from male Harlan Sprague Dawley rats exposed to 0, 0.05, 0.55, 5.50, 55 or 550 mg/kg TBB, 5 days after exposure for animals 7 weeks of age.  These samples were interrogated with the Affymetrix Rat Genome 230 2.0 GeneChip array.
A total of 1,0,2,2, and 66 gene transcripts were differentially expressed due to TBB treatment after exposure to 0.05, 0.55, 5.50, 55 or 550 mg/kg TBB (false discovery rate (FDR) < 0.05).

overall_design: There were 6 groups and 6x replication for each group, except there were 5 samples for the 0 mg/kg group, for 35 total samples. The groups were (1) TBB-0mg/kg, (2) TBB-0.05mg/kg, (3) TBB-0.55mg/kg, TBB-5.50mg/kg, TBB-55mg/kg, and TBB-550mg/kg. We compared vehicle control (0mg/kg) vs dose using the R/maanova package. The differentially expressed transcripts were identified based on a statistical significance threshold of FDR<5%.

first: June

last: Dunnick


title: Network rewiring: physiological consequences of reciprocally exchanging the physical locations and growth-phase-dependent expression patterns of the Salmonella fis and dps genes. [ChIP-Seq]

status: Public on Jul 06 2020

city: Dublin

first: Aalap

last: Mogre

address: Moyne Institute of Preventitive Medicine

department: Department of Microbiology

country: Ireland

institute: Trinity College Dublin

summary: The Fis nucleoid-associated protein controls the expression of a large and diverse regulon of genes in Gram-negative bacteria. Fis production is normally maximal in bacteria during the early exponential phase of batch culture growth, becoming almost undetectable by the onset of stationary phase. We tested the effect of rewiring the Fis regulatory network in Salmonella by moving the complete fis gene from its usual location near the origin of chromosomal replication to the position normally occupied by the dps gene in the Right macrodomain of the chromosome, creating the strain GX. In a parallel experiment, we tested the effect of placing the fis open reading frame under the control of the stationary-phase-activated dps promoter at the dps genetic location within Ter, creating the strain OX. ChIP-seq was used to measure global Fis protein binding and gene expression patterns. Strain GX showed few changes when compared with the wild type, although we did detect increased Fis binding at Ter, accompanied by reduced binding at Ori. Strain OX displayed a more pronounced version of this distorted Fis protein-binding pattern together with numerous alterations in the expression of genes in the Fis regulon. OX, but not GX, had a reduced ability to infect cultured mammalian cells, had undergone a reduction in competitive fitness and had reduced motility compared to the wild type. These findings illustrate the inherent robustness of the Fis regulatory network to rewiring based on gene repositioning alone and emphasise the importance of fis expression signals in phenotypic determination.

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

overall_design: The locations of the fis and dps gene were exchanged, with or without their regulatory regions. Consequences were determined using RNA-seq and ChIP-seq.

first: Marina

last: Bogue

first: Michael

last: Beckett

first: Nicholas

last: Thomson

first: Charles

middle: J

last: Dorman


title: The effect of ANGPTL3 mAb on liver transcriptomes in LDLR-deficient mice

status: Public on Jul 05 2020

city: Tarrytown

first: Rene

middle: Christian

last: Adam

state: New York

address: 777 Old Saw Mill River Road

institute: Regeneron Pharmaceuticals

summary:
Purpose: The goal of this study was to evaluate the effect of ANGPTL3 inhibition on liver transcriptomes in LDLR KO mice.
Methods: Mice were treated once weekly for 3 weeks with 25mg/kg control mAb or ANGPTL3 mAb. Livers were harvested 6 days after the last mAb treatment.
Results: ANGPTL3 inhibition had no major impact on hepatic gene expression.

overall_design: Liver mRNA profiles of ~16 weeks old LDLR KO mice were generated by deep sequencing using Illumina HiSeq2000.

first: Viktoria

last: Gusarova


title: Regulated snoRNA expression during differentiation corresponds with a change in ribosomal RNA pseudouridylation and altered pre-ribosomal RNA processing [Nano104]

first: Kathleen

last: McCann

overall_design: In this study, we measured expression of 72 H/ACA snoRNAs in in C2C12 mouse myoblasts before, after differentiation using a custom NanoString nCounter assay.

first: Sanam

last: Kavari

first: Bart

last: Phillips

first: Adam

last: Burkholder

first: Traci

last: Hall


title: Next Generation Sequencing Facilitates Quantitative Analysis of C57BL/6J mice  Incisor Transcriptomes at single cell level.

first: Shuo

last: Chen

summary:
Purpose: Analysis the heterogeneity of the MSC population in the adult mouse incisor, using single cell RNA-sequence.
Results: About 9,318 cells (~63,000 reads per cells) were successfully barcoded and their transcriptomes sequenced.

overall_design: Methods: Mandibles were carefully dissected under a stereomicroscope. The surrounding jaw bones were removed by scalpel and the proximal region was then severed. Dental pulp with dental epithelium was isolated, cut into pieces, collected in cold PBS, and digested with 2mg/ml collagenase type I  dissolved in α-MEM  for 30 min at 37 ℃. After incubation, the suspension was homogenized by pipetting and digestion was terminated by adding 10% FBS. The digested tissues were centrifuged at 300g for 5 min and resuspended in 10% FBS. Twenty thousand cells were loaded into the 10X Chromium system with targeted cell recovery of 10,000 cells to be barcoded for scRNA-seq using a Single Cell 3’ Library Kit v3. Sequencing was performed on the Illumina Novaseq System.

first: Yang

last: Chai


title: Next Generation Sequencing Facilitates Quantitative Analysis of C57BL/6J mice.

summary:
Purpose: Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method for mapping chromatin accessibility genome-wide. The goals of this study are to investigate chromatin accessibility of C57BL/6J mice  through ATAC-seq.
Results: ATAC-seq reads were aligned to the UCSC mm10 reference genome with BWA-MEM. ATAC-seq peaks were called through MACS2 . Peaks were annotated and known transcription factor binding motifs were further analyzed in the ATAC-seq peaks by HOMER.

overall_design: Methods: Dental mesenchymal cells from C57BL/6J mice were process by cell lysis, transposition, and amplification and the DNA libraries were generated by deep sequencing using Illumina NextSeq500.


title: Endogenous glucocorticoid signaling regulates effector differentiation and development of dysfunction in CD8+ T cells in the tumor microenvironment

status: Public on Jul 01 2020

city: Boston

first: Asaf

last: Madi

state: MA

address: 60 Fenwood Road, BTM 10016F

department: Neurology

institute: BWH

summary: Identifying signals in the tumor microenvironment (TME) that shape CD8+ T cell phenotype can inform novel therapeutic approaches for cancer. Here, we identified a gradient of increasing glucocorticoid receptor (GR) expression and signaling from naive to dysfunctional CD8+ tumor-infiltrating lymphocytes (TILs). Conditional deletion of the GR in CD8+ TILs resulted in improved effector differentiation, reduced TCF-1 expression, and failure to develop dysfunctional phenotype, culminating in tumor growth inhibition. GR signaling transactivated the expression of multiple checkpoint receptors and promoted the induction of several dysfunction-associated genes upon T cell activation. In the TME, monocyte/macrophages produced glucocorticoids and genetic ablation of steroidogenesis in these cells as well localized pharmacologic inhibition of glucocorticoid biosynthesis dramatically improved tumor growth control. Active glucocorticoid signaling further associated with failure to respond to checkpoint blockade in both pre-clinical models and melanoma patients, highlighting the clinical relevance of targeting endogenous steroid signaling to enhance anti-tumor T cell responses.

overall_design: wild type (E8i-Cre-Nr3c1fl/fl) or E8i-Cre+Nr3c1fl/fl CD8+ T cells activated in the presence or absence of glucocorticoid (GC;dexamethasone) for 72hrs and wild type CD8+ T cell  9 days activated in the presence or absence of glucocorticoid (GC;dexamethasone), IL27 or both

first: Ana

last: Carrizosa Anderson

first: Nanadini

last: Acharya


title: Targeted Inhibition of Purine Metabolism is Effective in Suppressing Hepatocellular Carcinoma Progression

city: St. John's

first: Touati

last: Benoukraf

state: Newfoundland

address: Craig L. Dobbin Genetics Research Centre

department: Faculty of Medicine, Discipline of Genetics

institute: Memorial University of Newfoundland

summary: Tumor-specific metabolic rewiring, intended to confer a survival advantage over non-transformed cells, often offers an opportunity to target cancers. Here, we identify deregulated expression of purine biosynthetic enzymes as a metabolic hallmark in human hepatocellular carcinomas (HCC), with the extent of enzyme upregulation a predictor of clinical outcome. We demonstrate in HCC cell lines, patient-derived xenograft (PDX) organoids and mouse models that inhibition of purine biosynthesis abrogated cancer cell proliferation and tumor growth. Mechanistically, a PI3K-E2F1 axis coordinated purine biosynthetic enzyme expression. Clinically approved inhibitors against PI3K and the purine biosynthetic rate-limiting enzyme IMPDH synergistically reduced the tumor burden in a PDX mouse model. Collectively, our results support targeting purine metabolic reprogramming as a precision therapeutic strategy for HCC patients.

overall_design: RNA-seq of sh-control, sh-IMPDH1 and sh-IMPDH2 in Huh7 cell line. Each condition has three biological replicates

first: Yong

last: Chong

first: Tan

last: Toh

first: Zhiling

last: Chan

first: Quy Xiao Xuan

last: Lin

first: Lissa

last: Hooi

first: Zhaobing

last: Ding

first: Wai

last: Shuen

first: Han

first: Pierce

last: Chow

first: Yulan

last: Wang

first: Edward

first: Weiping

last: Han


title: Transcriptome profile of RCS chondrocyte wt cell line and RCS chondrocyte obtained from a clone ko for the FGF receptors FGFR3 and FGFR4

city: Pozzuoli

first: Rossella

last: De Cegli

address: Via Campi Flegrei 34

country: Italy

institute: Tigem

summary: To understand the transcriptomic effect of FGF18 treatment on Chondrocytes cells from wt rat and on Chondrocytes obtained from a clone ko for the FGF receptors FGFR3 and FGFR4, we performed Quant-seq experiments. Both cell lines were treated with Fibroblast growth factor 18 (FGF18), while untreated cells were used as control.

overall_design: total RNA was extracted from Both cell lines treated with Fibroblast growth factor 18 (FGF18), while RNA extracted from untreated cells was used as control.

first: R

first: L

last: Cinque


title: RNA-seq analysis of rat neurons treated with shRNA-mediated Trank1 knockdown

status: Public on Jul 02 2020

city: Kunming

first: Ming

last: Li

state: Yunnan

address: NO 32 Jiao-Chang Donglu

country: China

institute: Kunming Institute of Zoology, Chinese Academy of Sciences

summary: Genetic analyses for bipolar disorder (BPD) have achieved prominent success in Europeans in recent years, whereas its genetic basis in other populations remains relatively less understood. We herein report that the lead risk locus for BPD in European genome-wide association studies (GWAS), the single nucleotide polymorphism (SNP) rs9834970 near TRANK1 at 3p22 region, is also genome-wide significantly associated with BPD in 5,748 cases and 65,361 controls of East Asian origin. In this study, we performed RAN-seq analysis of cultured rat neurons treated with shRNA knockdown of Trank1.

overall_design: Lentiviral constructs of two rat Trank1 shRNAs and a negative control shRNA were established to infect the wild-type rat neurons for 72h (n=3 per condition). Infection efficiency was estimated to be >80% and the mean expression of Trank1 was reduced by 43.4% and 35.7% respectively in cells infected with Trank1-shRNA#1 and Trank1-shRNA#2 relative to those infected with control shRNA.

first: Xin

last: Cai

first: Hong

last: Chang

first: Huijuan


title: Human neonatal foreskin epidermis

city: Irvine

first: Scott

last: Atwood

address: 4340 McGaugh Hall, Ring Rd

department: Department of Developmental & Cell Biology

institute: University of California, Irvine

summary: We report transcriptomes of human neonatal foreskin epidermis.

overall_design: Examination of epidermal cell heteregeneity in human neonatal epidermis.

middle: X

first: Christian

middle: F

last: Guerrero-Juarez

first: Adam

middle: R

last: Stabell


title: The flooding resilience loci SUBMERGENCE 1 and ANAEROBIC GERMINATION 1 interact in rice seedlings established underwater

city: Riverside

first: Maureen

last: Hummel

address: 900 University Avenue

department: Botany and Plant Sciences

institute: University of California

summary: Little is known of the consequences of genetic pyramiding of abiotic stress tolerance loci in crops. In rice (Oryza sativa L.), ANAEROBIC GERMINATION1 (AG1) encodes TREHALOSE 6-PHOSPHATE PHOSPHATASE 7 (OsTPP7) that enhances mobilization of endosperm reserves to the embryo, promoting formation of a highly elongated hollow coleoptile in seeds sown directly into shallow paddies. This trait reduces labor costs and herbicide use. SUBMERGENCE1 (SUB1) includes the ethylene-responsive transcription factor SUB1A that confers tolerance to complete submergence by dampening shoot elongation of vegetative-phase plants and enhancing regrowth upon desubmergence. As OsTPP7 and SUB1A-1 mRNAs simultaneously accumulate in shoots, we evaluated the independent contribution and interactions between these loci in rice seeded under complete submergence until the four-leaf stage (14 d). Evaluating growth, carbohydrates, and the transcriptome of shoot tissue of four near-isogenic genotypes uncovered independent and interacting effects including: (a) early enhanced coleoptile elongation by IR64(AG1); (b) precocious transition to phototrophy followed by limited elongation by IR64(SUB1); and (c) delayed phototrophy by IR64(AG1, SUB1). mRNA-sequencing highlighted time-dependent and genotype-specific regulation of mRNAs associated with energy sensing, carbohydrate catabolism, photomorphogenesis, elongation, and defense responses. Although SUB1A did not antagonize AG1 in direct seeding, seedling establishment could be negatively impacted if submergence escape does not occur before the transition to photoautotrophy, due to antithetical regulation of carbohydrate catabolism by AG1 and SUB1.

overall_design: 45 samples in total - 3 replicates, 4 genotypes, 1 RNA populations, 2-14 days treatment:  Oryza sativa coleoptile 4-days-old, Oryza sativa coleoptile 8-days-old, Oryza sativa coleoptile 14-days-old, Oryza sativa embryo 2-days-old.

first: Elaine

last: Yeung

first: Rejbana

last: Alam

first: Julia

last: Bailey-Serres


title: Mechanics of a multilayer epithelium instruct tumor architecture and function [adult]

city: New York

first: Vincent

last: Fiore

state: NY

address: 1230 York St.

institute: Rockefeller University

summary: Loss of normal tissue architecture is a hallmark of oncogenic transformation. Mechanical forces sculpt tissue architectures during morphogenesis. However, their origins and consequences during tumorigenesis remain elusive. In skin, premalignant basal cell carcinomas (BCCs) form ‘buds’, while invasive squamous cell carcinomas (SCC) initiate as ‘folds’. Using computational modeling, genetic manipulations and biophysical measurements, we identify the biophysical underpinnings and biological consequences of these tumor architectures. Cell proliferation and actomyosin contractility dominate tissue architectures in monolayer, but not multilayer epithelia. In stratified epidermis, softening and enhanced remodeling of the basement membrane (BM) promote tumor budding, while BM stiffening promotes folding. We show that additional key forces stem from progenitor cell stratification and differentiation Tumor-specific suprabasal stiffness gradients are generated as oncogenic lesions progress toward malignancy, which we computationally predict alter extensile tensions on the tumor BM. The pathophysiologic ramifications of this prediction are profound. Genetically decreasing BM stiffness elevates BM tensions in silico and potentiates invasive SCC progression in vivo. Our findings suggest that mechanical forces, exerted from above and below progenitors of multilayered epithelia, are integrally linked and function to shape premalignant tumor architectures and influence tumor progression.

overall_design: Epidermal basal cells were FACS-purified from adult mice for RNA-seq analysis.

last: Fuchs


title: Mechanics of a multilayer epithelium instruct tumor architecture and function

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

overall_design: Refer to individual Series


title: Neuropilin-1(NRP1) is a T cell memory checkpoint limiting long-term anti-tumor immunity

city: Philadelphia

first: E

middle: JOHN

last: WHERRY

state: PA

address: 421 Curie Blvd,354 BRB II/III

department: Systems Pharmacology       and Translational Therapeutics,Institute for Immunology

institute: University of Pennsylvania

summary: Transcriptomic analysis of NRP1 KO and WT donor pMel-T cells recovered from B16-gp100 tumors

overall_design: Replicates were analyzed from draining(DLN) and non-draining lymph nodes(NdLN) on day12,day21,day35 & day63 post transfer and day12,day21 in tumors

first: Chang

last: Liu

first: Dario

last: Vignali


title: The conserved elongation factor Spn1 is required for normal transcription, histone modifications, and splicing in Saccharomyces cerevisiae

first: Fred

last: Winston

address: 77 Avenue Louis Pasteur

department: Genetics

institute: Harvard Medical School

summary: Spn1/Iws1 is a conserved protein involved in transcription and chromatin dynamics, yet its general in vivo requirement for these functions is unknown. Using a Spn1 depletion system in S. cerevisiae, we demonstrate that Spn1 broadly influences several aspects of gene expression on a genome-wide scale. We show that Spn1 is globally required for normal mRNA levels and for normal splicing of ribosomal protein transcripts. Furthermore, Spn1 maintains the localization of H3K36 and H3K4 methylation across the genome and is required for normal histone levels at highly expressed genes. Finally, we show that the association of Spn1 with the transcription machinery is strongly dependent on its binding partner, Spt6, while the association of Spt6 and Set2 with transcribed regions is partially dependent on Spn1. Taken together, our results show that Spn1 affects multiple aspects of gene expression in vivo and provide additional evidence that it functions as a histone chaperone.

overall_design: Data were generated from yeast strains with Spn1 non-depleted or depleted by auxin-induced degradation, and include ChIP-seq of Rpb1, Rpb1 Ser5-P, Rpb1 Ser2-P, Spn1, Set2, Spt6, histone H3, H3K4me3, H3K36me2, H3K36me3, as well as RNA-seq.

first: Natalia

last: Reim

first: James

last: Chuang

first: Dhawal

last: Jain

first: Burak

middle: H

last: Alver

first: Peter

last: Park


title: Mechanics of a multilayer epithelium instruct tumor architecture and function [E15]

overall_design: Epidermal basal cells were FACS-purified from mouse embyros for RNA-seq analysis.


title: Network rewiring: physiological consequences of reciprocally exchanging the physical locations and growth-phase-dependent expression patterns of the Salmonella fis and dps genes. [RNA-Seq]

first: Aalap

last: Mogre

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

first: Marina

last: Bogue

first: Michael

last: Beckett

first: Nicholas

last: Thomson

first: Charles

last: Dorman


title: mRNA- and RISC-sequencing of miR-106b-transfected juxtaglomerular (JG) cells

city: Indianapolis

first: Scot

last: Matkovich

state: IN

address: Lilly Corporate Center

institute: Eli Lilly and Company

summary: Chronic inflammation is known to contribute to the development of hypertension, but while the role of lymphocytes is well-established, the myeloid lineage, particularly as a possible mediator of renin-mediated hypertension, has not been studied. Vitamin D deficiency has pro-inflammatory consequences in monocytes, and it is linked to renin-mediated hypertension in mice. This prompted us to test the hypothesis that conditional knockout of the vitamin D receptor (VDR) in macrophages (KODMAC) would promote renin-dependent hypertension. We found that lack of macrophage vdr signaling was sufficient to induce high blood pressure (BP) in mice. Recipients of bone marrow from KODMAC mice experienced increased BP via activation of the renal renin-angiotensin system. Conversely BM transplants from mice with intact vdr signaling reduced BP in KODMAC mice by decreasing plasma renin and increasing urinary Na excretion. Deletion of myeloid vdr promotes vascular macrophage infiltration, increasing ROS-mediated scavenging of NO and reducing renal perfusion, resulting in increased plasma renin. Interestingly, deletion of macrophage vdr promotes their infiltration into the JG cell apparatus, regulating JG cell renin production by an unexpected communication between macrophages and JG cells through miR-106b-5p. Induction of macrophage endoplasmic reticulum stress phospho-PERK and CHOP pathways increased miR-106b-5p secretion, the uptake of which into JG cells induced renin production and release by repression of E2f1 and Pde3b transcription factors, constituting a novel mechanism by which inflammation causes hypertension.

overall_design: Cultured JG cells engineered to express YFP under the control of the renin promoter (R A Gomez) were transfected with a control (non-mammalian) microRNA precursor or miR-106b.  48 h after transfection, RNA was prepared from 4 replicate control and 4 replicate miR-106b cell cultures and mRNA (polyA-mRNA) was extracted.  In parallel, cell pellets were obtained from another 4 replicate control and miR-106b cell cultures and Ago2 was immunoprecipitated followed by RNA extraction.  Messenger RNA and Ago2-bound RNA were converted to Illumina RNA-sequencing libraries.

first: Carlos

last: Bernal-Mizrachi


title: Transcriptional landscape of fate choices in the sensory lineages

city: Stockholm

first: Saida

last: Hadjab

address: Solnav. 9

department: Neuroscience

country: Sweden

institute: Karolinska Institutet

summary: Somatic sensation is defined by the existence of a diversity of primary sensory neurons with unique biological features and response profiles to external and internal stimuli. However, there is no coherent picture about how this diversity of cell states is transcriptionally generated. Here, we used deep single cell analysis to resolve fate splits and molecular biasing processes during sensory neurogenesis. Our results revealed a complex series of successive and specific transcriptional changes in post-mitotic neurons that delineate hierarchical regulatory states leading to the generation of the main sensory neuron classes. In addition, our analysis identified previously undetected early gene modules expressed long before fate determination although being clearly associated with the final sensory subtypes. Overall, fate choice in sensory neurons involves initial co-activation of counteracting regulatory determinants of alternative lineages priming intermediate post-mitotic neuronal cells for a binary cell fate choice. Hence, the diversity of sensory neurons is generated through successive bi-potential intermediates in which synchronization of relevant gene modules and concurrent repression of competing fate programs precede cell fate stabilization and final commitment.

overall_design: Single cells were collected at different locations on the embryo using different tracings: Wnt1 traced cells were collected in the trunk at E9.5 and E10.5. Plp1 traced cells were collected in cranio/cervico/thoracic region at E12.5. Il1 traced cells were collected in the DRGs at E9.5 and E10.5. TrkC traced cells were collected in the DRGs at E11.5 and E12.5.

first: Louis

last: Faure

first: Yiqiao

last: Wang

first: Maria

last: Kastriti

first: Paula

last: Fontanet

first: Kylie

last: Cheung

last: Petipre

first: Haohao

last: Wu

first: Karen

last: Runge

first: Laura

last: Croci

first: Mark

last: Landy

first: Helen

last: Lai

first: Gian

last: Conzalez

first: Antoine

last: de Chevigny

first: François

last: Lallemend


title: Expression data from patients that has received grass pollen sublingual immunotherapy treatment for two years.

city: Madrid

first: Leticia

last: Mera

state: Madrid

address: Carretera Boadilla km 5300

country: Spain

institute: CEU San Pablo University

summary:
Prevalence and severity of allergic diseases have increased worldwide. To date, respiratory allergy phenotypes are not fully characterized and, in addition, the mechanisms underlying sublingual immunotherapy (SLIT) are still unknown.
We have used microarrays to understand the underlying mechanisms of SLIT using samples of patients under grass pollen SLIT at different timepoints (T0-before SLIT and T2-two years of treatment, Active or Placebo). We also have taken into account the sensitization of the subjects (Monosensitized patients and Polisensitiez patients)

overall_design: Our aim was to identify the underlying mechanisms and possible biomarkers associated to grass pollen SLIT response using microarrays.


title: Network rewiring: physiological consequences of reciprocally exchanging the physical locations and growth-phase-dependent expression patterns of the Salmonella fis and dps genes.

status: Public on Jul 03 2020

first: Aalap

last: Mogre


title: Selective Metabolic Redundancy of Gpi1 Allows for Specific Inhibition of Inflammatory Th17 Cells

first: Dan

last: Littman

address: 430 East 29th St Room 403

institute: New York University School of Medicine


title: E2A Specifies Adaptive Immunity by Instructing Large-Scale Topological Changes

city: Kyoto

first: Kazuko

last: Miyazaki

address: 53 Kawahara-cho, Shogoin, Sakyo-ku

country: Japan

institute: Institute for Life and Medical Sciences, Kyoto University

summary: Analysis of epigenetics and chromatin structure in lymphocyte development

overall_design: Analysis of epigenetics and chromatin structure between wild type and mutant mice containing deletion or mutantations in the regulatory region.

first: Masaki


title: Lack of NFATc1 SUMOylation prevents autoimmunity and alloreactivity

city: wuerzburg

first: Yin

last: Xiao

address: Josef schneider strasse 2

department: institute of pathology

country: Germany

institute: university of wuerzburg

summary: Post-translational modification with SUMO is known to regulate the activity of transcription factors, but how SUMOylation of individual proteins might influence immunity is mostly unexplored. The NFAT transcription factors play an essential role in antigen receptor-mediated gene regulation. SUMOylation of NFATc1 represses IL-2 in vitro, but its role in T cell-mediated immune responses in vivo is not clear. To this end, we generated a novel Nfatc1 transgenic mouse, which prevents SUMO modification of NFATc1. Avoidance of NFATc1 SUMOylation ameliorated experimental autoimmune encephalomyelitis as well as graft-versus-host disease. An elevated IL-2 production promoted Treg expansion and suppressed autoreactive or alloreactive T cells. Mechanistically, increased IL-2 secretion counteracted IL-17 and IFN-γ expression through STAT5 and Blimp-1 induction. Then, Blimp-1 repressed IL-2 itself and the as well induced, proliferation-associated survival factor Bcl2A1. Collectively, we demonstrate that prevention of NFATc1 SUMOylation fine-tunes T-cell responses towards lasting tolerance. Thus, targeting NFATc1 SUMOylation presents a novel and promising strategy to treat T cell-mediated inflammatory diseases.

overall_design: Isolated CD4+ T cells from spleen and LN of C57BL/6 mice, differentiation into Th1, Th2 and Th17 cells in vitro, check gene expression of two genotype mice (WT and NFATc1/ΔS ) in 3 cell types at day 2.5 and day 3.5, and each cell type has two different time points, total 12 samples.

first: Lena

last: Dietz

first: Musga

last: Qureischi

first: Matthias

last: Klein

first: Stefan

last: Klein-Hessling

first: Friederike

last: Berberich-Siebelt


title: Gene expression profiling of Th17, Th1*, Treg cells in the spinal cord of EAE mice , and Th17, Treg cells in the SILP of SFB colonized mice

summary: The goal of the RNAseq experiment is to compare gene expression of T cells in different microenvironments. We hypothesized that same T cell subtype (Th17/Th1*  cells, or Treg cells in this study) need to change gene expression to adapt to different microenvironments (spinal cord in EAE mice VS. SILP in SFB-colonized mice). By profiling and comparing gene expression of these cells, we were able to identify candidate genes that enable cells for tissue adaptation for functional study.

overall_design: The mice used for T cell isolation were 10-week old IL-17aGfp/+ Foxp3Rfp male littermates (two biological replicates for each sample). Each CD4+ T cell sub-population was sorted with Aria II (EAE Th17: IL-17a+ Klrc1- Foxp3-; EAE Th1*: Klrc1+ Foxp3-; EAE Treg: Foxp3+ IL-17a- Klrc1-; SFB Th17: IL-17a+ Foxp3-; SFB Treg: IL-17a- Foxp3+). EAE samples were isolated at the peak of the disease (Day15, score 5). SFB samples were isolated from stable SFB-colonized mice.

first: Lin

last: Wu

first: Lina

last: Kroehling


title: Synergistic Targeting of FLT3 Mutations in AML via Combined Menin-MLL and FLT3 Inhibition

city: Mainz

first: Margarita

last: Dzama

address: Langenbeckstr. 1

department: Department of Hematology, Medical Oncology, and Pulmonary Medicine,

institute: University Medical Center, Johannes Gutenberg-University

summary: The interaction of Menin (MEN1) and MLL (MLL1, KMT2A) is a dependency and potential therapeutic opportunity against NPM1 mutant (NPM1mut) and MLL-rearranged (MLL-r) leukemias. Concomitant activating driver mutations in the gene encoding the tyrosine kinase FLT3 occur in both leukemias and are particularly common in the NPM1mut subtype. Transcriptional profiling upon pharmacological inhibition of the Menin-MLL complex revealed specific changes in gene expression with downregulation of the MEIS1 transcription factor and its transcriptional target gene FLT3 being most pronounced. Combining Menin-MLL inhibition with specific small molecule kinase inhibitors of FLT3 phosphorylation resulted in a significantly superior reduction of phosphorylated FLT3 and transcriptional suppression of genes downstream to FLT3 signaling. The drug combination induced synergistic inhibition of proliferation as well as enhanced apoptosis and differentiation compared to single-drug treatment in models of human and murine NPM1mut and MLL-r leukemias harboring an FLT3 mutation. Primary AML cells harvested from patients with NPM1mut FLT3mut AML showed significantly better responses to combined Menin and FLT3 inhibition than to single-drug or vehicle control treatment, while AML cells with wildtype NPM1, MLL, and FLT3 were not affected by any of the two drugs. In vivo treatment of leukemic animals with MLL-r FLT3mut leukemia reduced leukemia burden significantly and prolonged survival compared to the single-drug and vehicle control groups. Our data suggest that combined Menin-MLL and FLT3 inhibition represents a novel and promising therapeutic strategy for patients with NPM1mut or MLL-r leukemia and concurrent FLT3 mutation.

overall_design: RNA sequencing of MV411 cells, treated with MI-503 for 3 days [2.5 µM], AC220 for 24 hours [3 nM], their combination [MI-503, 3 days, 2.5 µM; AC220, 24 hours, 3nM] or with DMSO, and MOLM13 and OCI-AML3 cells, treated with MI-503 [2.5 µM] and DMSO for 4 days. All experiments performed in biological triplicates.

first: Johanna

last: Rausch

first: Daniel

last: Sasca

first: Marlene

last: Steiner

first: Kerstin

last: Kunz

first: Martha

last: Taubert

first: Chun-Wei

last: Chen

first: Annalisa

last: Mupo

first: Patricia

last: Hähnel

last: Theobald

first: Thomas

last: Kindler

first: Richard

middle: P

last: Koche

first: George

middle: S

last: Vassiliou

first: Scott

middle: A

last: Armstrong

first: Michael

last: Kühn


title: β-catenin safeguards the ground state of pluripotency by strengthening the robustness of the transcriptional apparatus [GRO-seq]

city: Guangzhou

first: Yiwei

last: Lai

state: Guangdong

address: 190 Kai Yuan Avenue, Science Park

department: South China Institute for Stem Cell Biology and Regenerative Medicine Key Laboratory of Regenerative Biology, CAS

institute: Guangzhou Institutes of Biomedicine and Health,Chinese Academy of Sciences

summary: Mouse embryonic stem cells (ESCs) cultured with MEK and GSK3 inhibitors (2i) more closely resemble the inner cell mass of pre-implantation blastocysts than those cultured with serum/LIF (SL). The transcriptional mechanisms governing this pluripotent ground state are yet unresolved. Release of promoter-proximal paused RNA polymerase II (Pol2) is a multistep process necessary for pluripotency and cell cycle gene transcription in SL. Here, we show that β-catenin, stabilized by GSK3 inhibition in 2i, supplies transcriptional co-regulators including BRD4, CDK9, Mediator, Cohesin, and p300 at pluripotency loci. This selectively strengthens pluripotency loci and renders them addicted to transcription initiation for productive gene body elongation in detriment to Pol2 pause release. By contrast, cell cycle genes are not bound by β-catenin and, thus, proliferation/self-renewal are still tightly controlled by the Pol2 pause release mechanism under 2i conditions. Our findings help to explain how pluripotency is preserved in the ground state and also provide a general model for transcriptional resilience/adaptation upon network perturbation in other biological contexts.

overall_design: GRO-seq of mouse ESCs cultured in different conditions.


title: Immune Signatures and Tumor Biomarkers from Whole Transcriptome Sequencing Predict Outcome in Recurrent Resectable Stage III and IV Melanoma when Evaluated Following Treatment with Hu14.18-IL2

city: Bethesda

first: Javed

last: Khan

state: MD

address: 37 Convent Dr.

department: Genetics Branch

institute: NCI, NIH

summary: We analyzed whole transcriptome sequencing in tumors from 23 patients with stage III or IV melanoma from a pilot trial of the anti-GD2 immunocytokine, hu14.18-IL2, in melanoma patients at high risk for recurrence to identify predictive immune and/or tumor biomarkers. Patients with advanced resectable melanoma were randomized to receive the first of 3 monthly-courses of hu14.18-IL2 immunotherapy either before (Group A) or after (Group B) complete surgical resection of all known disease.  Tumors were evaluated by histology, immunohistochemistry and whole transcriptome sequencing. We report here that tumor infiltrating lymphocyte (TIL) levels directly correlate with relapse-free-survival (RFS) and overall-survival (OS). Resected tumors from Group A, but not Group B, patients demonstrated a direct correlation between TIL levels and survival. TIL levels directly correlated with a previously Estimated RNAseq-determined Immune Signature (EIS). This EIS correlated with RFS and OS, particularly in Group A tumors. Group A tumors demonstrated decreased cell cycling RNA gene transcripts, but increased RNA gene transcripts associated with repair and growth. Additionally, we found several direct correlations of immune signatures and immune related RNA transcripts as well as many inverse correlations of tumor growth-associated transcripts with RFS and OS, particularly in Group A tumors. Most of these correlations were not seen in Group B tumors. We interpret these data to signify that both immunologic and tumoral cell processes, as measured by RNAseq analyses, are associated with long term survival after hu14.18-IL2 therapy and could potentially be prognosticated using neoadjuvant treatment resection specimens obtained after initiating immunotherapy.

overall_design:
We analyzed whole transcriptome sequencing in tumors from 23 patients with stage III or IV melanoma from a pilot trial of the anti-GD2 immunocytokine, hu14.18-IL2, in melanoma patients at high risk for recurrence to identify predictive immune and/or tumor biomarkers. Patients with advanced resectable melanoma were randomized to receive the first of 3 monthly-courses of hu14.18-IL2 immunotherapy either before (Group A) or after (Group B) complete surgical resection of all known disease.  Tumors were evaluated by histology, immunohistochemistry and whole transcriptome sequencing.
FFPE tumor blocks were used to extract RNA using Qiagen RNeasy FFPE kits. RNA-seq libraries were constructed and captured using Illumina TruSeq RNA exome kits. RNA-seq libraries were sequenced on Illumina NextSeq 500 sequencers using 80bp paired-end sequencing methods.

first: Jun

last: Wei


title: Molecular Profiling of Failed Endochondral Ossification in Mucopolysaccharidosis VII

first: Lachlan

middle: James

last: Smith

state: Pennsylvania

address: 371 Stemmler Hall, 3450 Hamilton Walk

department: Department of Orthopaedic Surgery

summary: Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder characterized by deficient activity of β-glucuronidase, leading to progressive accumulation of incompletely degraded heparan, dermatan and chondroitin sulfate glycosaminoglycans (GAGs). Patients with MPS VII exhibit progressive skeletal deformity including kyphoscoliosis and joint dysplasia, which decrease quality of life and increase mortality. Previously, using the naturally-occurring canine model, we demonstrated that one of the earliest skeletal abnormalities to manifest in MPS VII is failed initiation of secondary ossification in vertebrae and long bones at the requisite postnatal developmental stage. The objective of this study was to obtain global insights into the molecular mechanisms underlying this failed initiation of secondary ossification using whole transcriptome sequencing (RNA-Seq). Epiphyseal tissue was isolated from the vertebrae of control and MPS VII affected dogs at 9 and 14 days-of-age (n=5 for each group). Differences in global gene expression across this developmental window for both cohorts were established using RNA-Seq. A targeted analysis focused on signaling pathways important in the regulation of endochondral ossification, and a subset of gene expression changes were validated using qPCR. Principal Component Analysis revealed clustering of samples from each group, indicating clear effects of both age and disease state. At 9 days-of-age, 1375 genes were significantly differentially expression between MPS VII and control, and by 14 days-of-age, this increased to 4719 genes. Osteoactivin (GPNMB) was the top upregulated gene in MPS VII at both ages. Targeted analysis revealed temporal changes in gene expression from 9 to 14 days-of-age in control samples consistent with chondrocyte maturation, cartilage resorption, and osteogenesis. In MPS VII samples, however, elements of key osteogenic pathways such as Wnt/β-catenin and BMP signaling were dysregulated. In conclusion, this study represents the first step towards identifying druggable therapeutic targets and putative biomarkers for bone disease in MPS VII patients during postnatal growth.

overall_design: For this study, we used the naturally-occurring canine model of MPS VII. MPS VII dogs have a missense mutation (R166H) in the GUSB gene and exhibit a similar skeletal phenotype to human patients. Animals were raised at the University of Pennsylvania School of Veterinary Medicine under NIH and USDA guidelines for the care and use of animals in research, and all studies were carried out with IACUC approval. Litter-matched control (heterozygous) and MPS VII (homozygous) dogs were identified via gene sequencing analysis at birth. Animals were euthanized at 9 or 14 days-of-age, defined as number of postnatal days (n=5 for both control and MPS VII animals at each time point), using 80 mg/kg of sodium pentobarbital in accordance with the American Veterinary Medical Association guidelines. The thoracic spine was dissected out immediately following euthanasia, and epiphyseal tissue from both the cranial and caudal sides of the T12 vertebrae for each animal was excised adjacent to the growth plate, with adjoining intervertebral disc tissue carefully removed. Cranial and caudal tissue for each sample was combined and flash frozen in liquid nitrogen.

first: Sun

last: Peck

first: John

middle: W

last: Tobias

first: Eileen

last: Shore

first: Neil

last: Malhotra

first: Mark

last: Haskins

first: Margret

middle: L

last: Casal


title: β-catenin safeguards the ground state of pluripotency by strengthening the robustness of the transcriptional apparatus [RNA-seq]

summary: Mouse embryonic stem cells (ESCs) cultured with inhibitors of MEK and GSK3 (2iL) more closely resemble the pre-implantation embryo inner cell mass than cultures in serum/LIF (SL). Unveiling the differences between both systems is important for understanding development and could also assist in isolating an ESC equivalent for other mammalian species. In SL, pluripotency and cell cycle gene transcription is a multistep process requiring release of promoter-proximal paused RNA polymerase II (Pol2) by the histone acetylation reader BRD4 and the Pol2 kinase CDK9. Here, we show that recruitment of co-regulators including Mediator and Cohesin by β-catenin changes the mode of transcriptional regulation at BRD4/CDK9-bound loci in 2iL. This switch renders pluripotency genes more reliant on transcriptional initiation and less on Pol2 pause release for effective gene body elongation. Conversely, cell cycle genes are not bound by β-catenin and are still dependent on Pol2 pause release. Thus, pluripotency is more resistant to BRD4/CDK9 suppression in 2iL while self-renewal remains highly sensitive. Our findings help to explain how pluripotency is shielded in the ground state and provide insight into transcriptional adaptation upon network perturbation in other contexts.

overall_design: RNA-seq of mouse embryonic stem cells (ESCs) cultured either with inhibitors of MEK and GSK3 (2iL)  or cultured in serum/LIF (SL), and treated with DMSO or JQ1 (100nM).


title: Effect of Sumo2 deletion on gene expression in the mouse brain

city: Durham

first: Wei

last: Yang

state: NC

address: 303 Research Drive

department: Anesthesiology

institute: Duke University Medical Center

summary: Mounting evidence indicates that small ubiquitin-like modifier (SUMO) conjugation regulates a wide range of neuronal functions and participates in learning and memory processes. Since many SUMO targets are transcription factors as well as other nuclear proteins modulating gene expression and SUMO2 is the predominant SUMO isoform, we aimed to identify how SUMO2 regualtes gene expression in the brain in order to better understand the role of SUMOylation in the brain fucntcion. In this study, we peformed RNA-Seq anaysis on hippocampus from SUMO2 conditional knockout mice.

overall_design: Compare gene expression between control (Sumo2 f/f) mice and SUMO2 conditional knockout (SUMO2-cKO; Sumo2 f/f; Emx1-Cre) mice.

first: Wulf

last: Paschen


title: Aortic smooth muscle cell phenotype modulation during Marfan syndrome aneurysm formation

status: Public on Jun 30 2020

city: Stanford

first: Albert

last: Pedroza

state: CALIFORNIA

address: 870 Quarry Rd, Falk CVRB

department: Cardiothoracic Surgery

institute: Stanford University

summary: We report dynamic temporal and spatial smooth muscle cell phenotype modulation using aortic single cell RNA sequencing in a murine model of Marfan syndrome (Fbn1C1041G/+) and littermate controls. Aortic root/ascending aortic tissue samples from both genotypes were studied at 4 and 24 weeks of age. The non-aneurysmal descending thoracic aorta was also studied at 24 weeks. Finally human aortic tissue from a Marfan syndrome patient undergoing aneurysm repair surgery was studied.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: 4 weeks mouse samples: aortic root/ascending aorta tissue from n=3 animals of each genotype (Fbn1C1041G/+ and littermate control) were pooled and digested into single cell pools for downstream analysis. For 24-week-old mice, 3 aortic root/asending aorta samples frmo control mice were similarly pooled. Separate pairs of Fbn1C1041G/+ mice were used to study sex differences, (total n=4 across two samples). Descending thoracic aortas from 24-week-old mice (n=3 control n=3 Fbn1C1041G/+) were pooled and processed as separate libraries. Separately, human aortic root tissue from a young patient with Marfan syndrome undergoing elective repair was digested and processed for single cell RNA sequencing.

first: Micheal

last: Fischbein


title: Bru-seq of healthy and Duchenne immortalized myotubes

city: Leiden

first: Pietro

last: Spitali

state: Zuid Holland

address: Postzone S4-P, Albinusdreef 2

department: Human Genetics

country: Netherlands

institute: Leiden University Medical Center

summary: Immortalized myoblasts obtained from healthy and Duchenne patients where induced to fuse into multi-nucleated myotubes and Bru-seq was performed

overall_design: control and reference samples n=1

first: Raquel

last: Garcia-Rodriguez

first: Annemieke

last: Aartsma-Rus


title: The environment-sensing aryl-hydrocarbon receptor inhibits the chondrogenic fate of modulated smooth muscle cells in atherosclerotic lesions

first: Juyong

middle: Brian

last: Kim

state: CA

address: 300 Pasteur Dr.

summary:
Aryl-hydrocarbon receptor protects against endochondral ossification of modulated smooth muscle cells in atherosclerosis
Introduction: Smooth muscle cells (SMC) play a critical role in atherosclerosis. The Aryl hydrocarbon receptor (AHR) is an environment-sensing transcription factor that contributes to vascular development, and has been implicated in coronary artery disease (CAD) risk. We hypothesized that AHR can affect atherosclerosis by regulating phenotypic modulation of SMC.
Methods: We combined RNA-Seq, ChIP-Seq, ATAC-Seq and in-vitro assays in human coronary artery SMC (HCASMC), with single-cell RNA-Seq (scRNA-Seq), histology, and RNAscope in an SMC-specific lineage-tracing Ahr knockout mouse model of atherosclerosis to better understand the role of AHR in vascular disease.
Results: Genomic studies coupled with functional assays in cultured HCASMC revealed that AHR modulates HCASMC phenotype and suppresses ossification in these cells. Lineage tracing and activity tracing studies in the mouse aortic sinus showed that the Ahr pathway is active in modulated SMC in the atherosclerotic lesion cap. Furthermore, scRNA-Seq studies of the SMC-specific Ahr knockout mice showed a significant increase in the proportion of modulated SMC expressing chondrocyte markers such as Col2a1 and Alpl, which localized to the lesion neointima. These cells, which we term    chondromyocytes    (CMC), were also identified in the neointima of human coronary arteries. In histological analyses, these changes manifested as larger lesion size, increased lineage-traced SMC participation in the lesion, decreased lineage-traced SMC in the lesion cap, and increased alkaline phosphatase activity in lesions in the Ahr knockout compared to wild-type mice. We propose that AHR is likely protective based on these data and inference from human genetic analyses.
Conclusion: Overall, we conclude that AHR promotes maintenance of lesion cap integrity and diminishes the disease related SMC-to-CMC transition in atherosclerotic tissues.

overall_design: We hypothesized that AHR can affect atherosclerosis by regulating phenotypic modulation of SMC. We combined RNA-Seq, ChIP-Seq, ATAC-Seq and in-vitro assays in human coronary artery SMC (HCASMC), with single-cell RNA-Seq (scRNA-Seq), histology, and RNAscope in an SMC-specific lineage-tracing Ahr knockout mouse model of atherosclerosis to better understand the role of AHR in vascular disease.

middle: B


title: Human intestinal organoid cell transcriptomic profiling

city: Cambridge

first: Fiona

last: Gribble

state: Cambridgeshire

address: Box 289 Addenbrooke's Hospital, Hills Road

department: Medical Research Laboratories

country: United Kingdom

institute: University of Cambridge

summary: We purified two populations of human ileal enteroendocrine cells (Venus+ and Venus-) from mature differentiated hGLU-Venus organoids by FACS and identified transcripts enriched in endocrine cell lineages.

overall_design: Comparative transcriptomic study of 4x cell populations by bulk RNAseq, with 5x biological replicates in IF growth medium, and 3x biological replicates in IF* growth medium.

first: Christopher

middle: M


title: Truncation of Pik3r1 Causes Severe Insulin Resistance Uncoupled From Obesity and Dyslipidaemia by Increased Energy Expenditure

first: Davide

last: Chiarugi

address: Box 289, Addenbrooke’s Hospital

department: Metabolic Research Laboratories

institute: WT-MRC Institute of Metabolic Science

summary: Both PIK3R1(Y657X) mutant and wild-type C57Bl/6J mice were maintained on chow until 16 weeks old, at which time they were fasted for 16 hours and then refed chow for 6 hours. The tissues were harvested and snap-frozen at three time points: chow diet before overnight fast (ad libitum chow, ALC), after 16 hour fast (overnight fast, ONF), and after 6 hour refeeding (refed 6 hours, RF6).

overall_design: RNAseq profiles of PIK3R1(Y657X) mutant and wild-type C57Bl/6J mice in three states: fasting, refed, and chow.

first: Robert

last: Semple

last: Kwok

first: David

last: Bulger


title: RNA-sequencing of serial biopsies from 3 patients treated with Capivasertib (AZD5363)

city: Barcelona

first: Violeta

last: Serra

address: CELLEX C/ Natzaret, 115-117

institute: Vall d Hebron Institute of Oncology

summary: First line treatment with AKT inhibitors combined with paclitaxel improves outcomes of metastatic TNBC, especially in tumors that harbor mutations in PTEN/PIK3CA/AKT1. In this work, we refine the composite response biomarker to AZD5363 in this setting, which is relevant to inform the clinical development of these drugs.

overall_design: 9 biospies from 3 patients pre-, on- and post- therapy

first: Avinash

last: Reddy

last: Gris-Oliver

first: Alan

last: Barnicle

first: Elza

last: De Bruin


title: Quantitative translation of dog-to-human aging by conserved remodeling of the DNA methylome

city: La Jolla

first: Tina

last: Wang

address: 9500 Gilman Dr

institute: University of California, San Diego

summary: We generated sequencing data for 104 dogs using targeted-bisulfite sequencing with targets selected to the human 450K methylation array. Using these data, we identify a conserved methylation signature that can be used to quantitatively translate dog years to human years and also extends to mice.

overall_design: Targetted bisulfite-sequencing of 104 dogs with DNA obtained from blood

first: Trey

last: Ideker


title: scRNA sequencing of total CD4+ T cells in spinal cord of EAE mice and SILP of SFB-colonized mice

first: Dan

last: Littman

summary: There are two aims in this study – (1) To figure out the pathogenic sub-populations of CD4+ T cells in the spinal cord of EAE model; and (2) by comparing the gene expression profile of pathogenic Th17 cells in EAE model to that of the non-pathogenic Th17 cells induced by SFB in SILP, to identify genes candidates that are potentially selectively essential to pathogenic ones.

overall_design: Il23r is essential for the pathogenicity of CD4+ T cells in the EAE model. We generated chimera mice reconstituted with Il23RGfp/+ CD45.1/2 and Il23rGfp/Gfp CD45.1/1 bone marrow cells, and induced EAE or inoculate SFB for isolating CD4+ T cells from EAE spinal cord or SFB SILP for scRNAseq analysis. We used Smart-seq2 protocol to generate scRNA library. By comparing Il23rGfp/+ and Il23rGfp/Gfp clusters, we identified the pathogenic Th17 populations in the EAE model. By comparing gene expression profile of the pathogenic EAE Th17 cells to that of the SFB Th17 cells, we identified genes only expressed in the pathogenic ones, the function of which were further studied in genetic mouse models.

first: Lin

last: Wu

first: Yuhan

last: Hao

first: Rahul

last: Satija


title: The DREAM complexes prevent genomic hypermethylation by repressing the expression of genes required for maintenance of DNA methylation [RNA-seq 2]

city: Beijing

first: Xin-Jian

last: He

address: No. 7, zhongguancun life science park road, changping district

institute: National Institute of Biological Sciences

summary: We identified TCX5, an Arabidopsis tesmin/TSO1-like CXC domain-containing transcription factor, and demonstrated that TCX5 functions redundantly with its paralog, TCX6, to prevent genomic hypermethylation and transcriptional silencing. TCX5 binds to the promoters of genes required for cell proliferation and maintenance of DNA methylation to repress the expression of these genes. Consistently, the tcx5/tcx6 double mutant showed enhanced cell proliferation, enlarged organ size, and increased genome-wide methylation. This study reveals how maintenance of DNA methylation is coordinated with cell division to prevent genomic hypermethylation. Given that the DREAM complexes are conserved from plants to animals, the prevention of DNA hypermethylation by the DREAM complexes may represent a conserved mechanism in higher eukaryotes.

overall_design: Examination of the mRNA expression in Arabidopsis


title: Mutant FOXL2C134W hijacks SMAD4 and SMAD2/3 to drive adult granulosa cell tumors

city: Copenhagen

first: Paul

middle: Andreas Compare

last: Cloos

address: Ole Maaløesvej5

department: Biotech Research Innovation Centre

country: Denmark

institute: Copenhagen University

summary: The FOXL2 mutant C134W occurs in virtually all adult ovarian granulosa cell tumors (AGCT), and is considered a driver of oncogenesis in this disease. However, the mechanism by which FOXL2C134W contributes to tumorigenesis is not known. Here, we show that mutant FOXL2C134W acquires the ability to bind SMAD4, forming a FOXL2C134W/SMAD4/SMAD2/3 complex that binds a novel hybrid DNA motif AGHCAHAA, unique to the FOXL2C134W mutant. This binding induces an enhancer-like chromatin state leading to transcription of nearby genes, many of which are characteristic of stemness and epithelial-to-mesenchymal transition. Importantly, primary AGCT tumors display a strong FOXL2C134W enrichment at hybrid loci. Ablation of SMAD4 or SMAD2/3 inhibits FOXL2C134W binding at hybrid sites and decreases transcription of associated genes. Accordingly, TGFβ-inhibition mitigates the transcriptional effect of FOXL2C134W.

overall_design:
FOXL2, SMAD4, SMAD2/3, H3K27ac, K4me1  enrichment profiles (ChIPseq) analyzed in human granulosa cells HGrC1 and KGN in the presence or absence of TGFbeta stimulation. HGrC1 cells express

A few samples lack processed data because they were replicates or controls not discussed in the manuscript.


title: Regulated snoRNA expression during differentiation corresponds with a change in ribosomal RNA pseudouridylation and altered pre-ribosomal RNA processing [RNA-seq and ncRNA-seq]

first: Kathleen

last: McCann

address: 111 TW Alexander Dr

institute: National Institute of Environmental Health Sciences

summary: H/ACA small nucleolar RNAs (snoRNAs) guide pseudouridylation as part of a small nucleolar ribonucleoprotein complex (snoRNP). Disruption of H/ACA snoRNA expression in stem cells impairs pluripotency, yet it remains unclear how H/ACA snoRNAs contribute to differentiation. To determine if H/ACA snoRNA expression is dynamic during differentiation, we comprehensively profiled H/ACA snoRNA expression in multiple murine cell types and during differentiation in three cellular models, including mouse embryonic stem cells and mouse myoblasts. We determined that H/ACA snoRNA expression is cell-type specific, and we identified a subset of snoRNAs that are specifically regulated during differentiation. Additionally, we demonstrated that a decrease in Snora27 expression upon differentiation corresponds to a decrease in pseudouridylation of its target site within the E-site transfer RNA (tRNA) binding region of the 28S ribosomal RNA (rRNA) in the large ribosomal subunit. Many of the snoRNAs regulated during differentiation have target nucleotides in the 28S rRNA, and we found that pre-rRNA processing of large subunit precursors is altered during differentiation. Together, these data suggest a model in which H/ACA snoRNAs are specifically regulated during differentiation to potentially alter pseudouridylation and fine tune ribosome function.

overall_design: In this study, we assessed expression of small RNAs in E14TG2a before and after differentiation with retinoic acid and we assessed gene expression in C2C12 cells before and after differentiation into myotubes.

first: Sanam

last: Kavari

first: Bart

middle: T

last: Phillips

first: Adam

last: Burkholder

first: Traci

last: Hall


title: The DREAM complexes prevent genomic hypermethylation by repressing the expression of genes required for maintenance of DNA methylation [Bisulfite-seq 2]

overall_design: Examination of the DNA methylation level in Arabidopsis


title: Rocks in the Auxin Stream: Wound induced auxin accumulation and ERF115 expression synergistically drive stem cell regeneration

city: Ghent

first: Bert

last: De Rybel

address: Technologiepark 71

department: Center for Plant Systems Biology

institute: VIB

summary: Plants are known for their outstanding capacity for recovering from various wounds and injury. However, it remains largely unknown how plants sense diverse forms of injury and canalize existing developmental processes into the execution of a correct regenerative response. Auxin, a cardinal plant hormone with morphogenlike properties, has been previously implicated in recovery from diverse types of wounding and organ loss. Here, through a combination of cellular imaging and in silico modelling, we demonstrate that vascular stem cell death obstructs the polar auxin flux, much alike rocks in a stream, and causes it to accumulate in the endodermis. This in turn grants the endodermal cells the capacity to undergo periclinal cell division that repopulate the vascular stem cell pool. Replenishment of the vasculature by the endodermis depends on the transcription factor ERF115, a wound inducible regulator of stem cell divisions. Although not being the primary inducer, auxin is required to maintain ERF115 expression. Reversely, ERF115 sensitizes cells to auxin by activating ARF5/MONOPTEROS, an auxin responsive transcription factor involved in the global auxin response, tissue patterning and organ formation. Combined, the wound induced auxin accumulation and ERF115 expression grant the endodermal cells stem cell activity. Our work provides a mechanistic model for wound induced stem cell regeneration in which ERF115 acts as a wound-inducible stem cell organizer that interprets wound-induced auxin maxima.

overall_design: Three biological replicates of Col-0 root tips treated with 0.5ug/ml bleomycin for 24h were compared with three biological replicates of Col-0 root tips under control conditions.

first: Balkan

last: Canher

first: Thomas

last: Eekhout

first: Lieven

last: De Veylder


title: Wapl repression by Pax5 promotes loop extrusion and V gene recombination at the Igh locus

city: Vienna

first: Moritz

last: Gaidt

address: Campus-Vienna-Biocenter 1

country: Austria

institute: IMP

summary: The generation of a diverse antibody repertoire is essential for humoral immunity and requires the participation of all V genes in V(D)J recombination, which depends on the Pax5-regulated contraction of the 2.8-Mb long immunoglobulin heavy-chain (Igh) locus. How Pax5 controls Igh contraction in pro-B cells is, however, not known. Here, we demonstrate that locus contraction is caused by cohesin-mediated chromatin loop extrusion across the entire Igh locus. Notably, expression of the cohesin-release factor Wapl is repressed by Pax5 specifically in pro-B and pre-B cells, which facilitates extended loop extrusion by increasing the residence time of cohesin on chromatin. Pax5 mediates the transcriptional repression of Wapl through a single Pax5-binding site by recruiting the Polycomb repressive complex 2 to induce bivalent chromatin at the Wapl promoter. Reduced Wapl expression causes global alterations of the three-dimensional chromatin architecture, indicating that the potential to recombine all V genes entails structural changes of the entire genome in pro-B cells.

first: Louisa

last: Hill

first: Busslinger

last: Meinrad

first: Markus

last: Jaritz

first: Gordana

last: Wutz

first: Kota

last: Nagasaka

first: Hiromi

last: Tagoh

first: Daniela

last: Kostanova-Poliakova

first: Karina

last: Schindler

first: Qiong

last: Sun

first: Peter

last: Bönelt

first: Maria

last: Fischer

first: Jan-Michael

last: Peters

first: Anja

last: Ebert


title: The DREAM complexes prevent genomic hypermethylation by repressing the expression of genes required for maintenance of DNA methylation [Bisulfite-seq 1]

first: Xin-Jian

last: He


title: The DREAM complexes prevent genomic hypermethylation by repressing the expression of genes required for maintenance of DNA methylation [ChIP-seq]

overall_design: Anti-FLAG M2 Magnetic Beads (M8823; Sigma-Aldrich) were used for the IP of TCX5–3xFlag.


title: The DREAM complexes prevent genomic hypermethylation by repressing the expression of genes required for maintenance of DNA methylation [RNA-seq 1]


title: β-catenin safeguards the ground state of pluripotency by strengthening the robustness of the transcriptional apparatus [ATAC-seq]

first: Yiwei

last: Lai

summary: Mouse embryonic stem cells (ESCs) cultured with inhibitors of MEK and GSK3 (2iL) more closely resemble the pre-implantation embryo inner cell mass than cultures in serum/LIF (SL). Unveiling the differences between both systems is important for understanding development and could also assist in isolating an ESC equivalent for other mammalian species. In SL, pluripotency and cell cycle gene transcription is a multistep process requiring release of promoter-proximal paused RNA polymerase II (Pol2) by the histone acetylation reader BRD4 and the Pol2 kinase CDK9. Here, we show that recruitment of co-regulators including Mediator and Cohesin by β-catenin changes the mode of transcriptional regulation at BRD4/CDK9-bound loci in 2iL. This switch renders pluripotency genes more reliant on transcriptional initiation and less on Pol2 pause release for effective gene body elongation.  Conversely, cell cycle genes are not bound by β-catenin and are still dependent on Pol2 pause release. Thus, pluripotency is more resistant to BRD4/CDK9 suppression in 2iL while self-renewal remains highly sensitive. Our findings help to explain how pluripotency is shielded in the ground state and provide insight into transcriptional adaptation upon network perturbation in other contexts.

overall_design: ATAC-seq of mouse ESCs cultured in different conditions.


title: Lipophilic-Signals Lead to Organ-Specific Gene Expression Changes in Arabidopsis Seedlings

city: Denton

first: Ashley

middle: Elisabeth

last: Cannon

state: TX

address: 1155 Union Circle #305220

department: Biological Sciences

institute: The University of North Texas

summary:
Purpose: Using RNA-seq and differential expression analysis, we examined the NAE-type and organ-specific  genetic pathways involved in transducing ear;y signals into downstream physiological changes involved in Arabidopsis seedling growth.
Methods: Intact seedlings and dissected cotyledon and root mRNA profiles of  3-day-old Arabidopsis seedlings treated with DMSO, 40 µM NAE 18:2 or 80 µM NAE 18:3  were generated by deep sequencing, in triplicate, using the Illumina Next-Seq 500 system. The sequence reads that passed quality filters were analyzed using STAR followed by DESeq2. qRT–PCR validation was performed using  SYBR Green assays
Results: Using RNA-seq and differential expression analysis, we identified early (1 h) transcriptional changes induced by the exogenous treatment of NAE 18:2 and NAE 18:3 in cotyledons, roots and intact seedlings. These two treatments led to a significant enrichment in ABA-response and chitin-response genes in organs where the treatments led to changes in development. In Arabidopsis seedlings, NAE 18:2 treatment led to the repression of genes involved in cell wall biogenesis and organization in roots and seedlings. In addition, cotyledons, roots, and seedlings treated with NAE 18:3 also showed a decrease in transcripts that encode proteins involved in growth processes. NAE 18:3 also led to changes in the abundance of transcripts involved in the modulation of chlorophyll biosynthesis and catabolism in cotyledons. Overall, NAE 18:2 and NAE 18:3 treatment led to lipid-type and organ-specific gene expression changes that include overlapping and non-overlapping gene sets. These data will provide future, rich opportunities to examine the genetic pathways involved in transducing early signals into downstream physiological changes in seedling growth.
Conclusions: A detailed transcriptional analyses provided insight into the early organ-specific molecular responses to NAE 18:2 and NAE 18:3. Using this experimental and computational approach, we gained an unbiased view of the NAE 18:2- and NAE 18:3-modulated transcriptome changes that are activated and lead to downstream changes in seedling development. NAE 18:2 and NAE 18:3 induced the expression of several genes also induced by ABA or chitin treatment. The overlap with ABA-response genes corroborates previously work; however, this is the first time chitin-response genes have been identified as part of the NAE-modulation of seedling growth. Overall, the bioinformatic analyses presented here supports the hypothesis that NAE 18:2 and NAE 18:3 elicit organ-specific and signal- specific molecular changes that precede developmental changes in Arabidopsis seedlings. Further, these data provide novel insights into the genetic programs that are modulated by NAE 18:2 and NAE 18:3 at the organ-specific level in Arabidopsis seedlings, and will facilitate future studies of the corresponding signaling networks.

middle: E

first: Chengshi

last: Yan

first: David

middle: J

last: Burks

first: Xiaolan

last: Rao

first: Rajeev

middle: K

last: Azad

first: Kent

middle: D

last: Chapman


title: Expression data of BRAF inhibitor resistant melanoma cells and spheroids

city: Debrecen

first: Viktoria

last: Koroknai

address: 26/B Kassai str.

department: Department of Public Health and Epidemiology

country: Hungary

institute: University of Debrecen

summary: In vitro cell cultures are frequently used to define the molecular background of drug resistance. In this study our major aim was to compare the gene expression signature of 2D and 3D cultured BRAFV600E mutant melanoma cell lines. We successfully developed BRAF-drug resistant cell lines from paired primary/metastatic melanoma cell lines in both 2D and 3D in vitro cultures. Using Affymetrix Human Gene 1.0 ST arrays, we determined the gene expression pattern of all cell lines. Our study highlights gene expression alterations that might help to understand the development of acquired resistance in melanoma cells in tumour tissue.

overall_design: We generated spheroids from the BRAFi sensitive (WM983A, WM983B) and BRAFi resistant (WM983ARES, WM983BRES primary/metastatic melanoma cell lines. To analyze the expression profile of the BRAFi resistant cells and BRAFi resistant spheroids, we used for Affymetrix Human Gene 1.0 ST array.

first: Margit

last: Balázs


title: β-catenin safeguards the ground state of pluripotency by strengthening the robustness of the transcriptional apparatus

address: 190 Kai Yuan Avenue, Science Park

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

overall_design: Refer to individual Series


title: RNA-seq and ChIP-seq for Lon

city: Hong Kong

first: Xin

last: DENG

address: 1/F, Block 1, To Yuen Building, City University of Hong Kong, 31 To Yuen Street, Kowloon Tong

department: Department of Biomedical Sciences

country: Hong Kong

institute: City University of Hong Kong

summary: Lon protease plays vital roles in many biological processes in Pseudomonas syringae, including type III secretion systems (T3SS), transcription regulation, protein synthesis and energy metabolism. Lon also functions as a transcriptional regulator in other bacterial species (e.g., Escherichia coli and Brevibacillus thermoruber). Therefore, we hypothesise that Lon has dual functions in P. syringae. To reveal the molecular mechanisms of Lon as a transcriptional regulator and protease under different environmental conditions, we used a combination of transcriptome sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-seq) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the genes or proteins regulated by Lon. As a transcriptional regulator, Lon bound to the promoter regions of PSPPH_4788, gacA, fur, gntR, clpS, lon and glyA and consequently regulated 1-dodecanol oxidation activity, motility, pyoverdine production, gluconokinase activity, N-end rule pathway, lon expression and serine hydroxymethyltransferase (SHMT) activity in King’s B medium (KB). In minimal medium (MM), Lon regulated SHMT activity and lon expression by binding to the promoter regions of glyA and lon, respectively. As a protease, Lon regulated the T3SS and metabolic pathways (e.g., amino acid metabolism). In MM, Lon regulated the polysaccharide metabolic process by controlling PSPPH_0514, AlgA, CysD and PSPPH_4991. Taken together, these data demonstrate that Lon acts as a transcriptional regulator or protease in different environments and tunes its virulence and metabolic functions accordingly.

sample_organism: Pseudomonas savastanoi pv. phaseolicola 1448A

overall_design:
In order to identify the direct binding loci in P. syringae, ChIP-seq assay was employed in KB or MM in vivo, by expressing lon with its native promoter fused with HA-tag in the pHM2 plasmid in P. syringae pv. phaseolicola (also named P. savastanoi pv. phaseolicola). Four sets of ChIP-seq samples were performed: (1) the pHM2-Lon-HA plasmid in the wild-type (WT) strain in KB, (2) the empty pHM2 plasmid in WT strain in KB, (3) the pHM2-Lon-HA plasmid in WT strain in MM, and (4) the empty pHM2 plasmid in WT strain in MM. By comparing with the control strains carrying the empty plasmid, the present work identified a total of 926 and 545 Lon-enriched loci in KB and MM, respectively. These results indicated that Lon functions as a transcriptional regulator in a nutritional-dependent manner in P. syrinage. Notably, nearly half of the Lon-binding loci were located in the upstream or the overlapping the start regions, suggesting the potential regulatory functions of Lon on these genes. The lon locus was deleted by homologous recombination with its upstream and downstream flank DNA fragments. Compared to the WT, 1260 and 354 genes were up-regulated in Δlon in KB (≥ 2-fold and p < 0.05) and MM, respectively. Meanwhile, the expression level of 1334 and 389 genes was down-regulated (≥ 2-fold and p <0.05) in Δlon in KB and MM, respectively. Taken together, the Venn diagram illuminated that 2187 KB-dependent genes and 336 MM-dependent genes in the Lon regulon, suggesting a more crucial role of Lon in KB than MM.
RNA-seq and ChIP-seq for Lon

last: Deng


title: Integrated analysis of genome-wide profiling of lncRNA and mRNA expression and functional networks after botulinum toxin type A injection into submandibular gland of rats.

first: Mao

last: Qianying

address: No. 22 Zhong Guan Cun South Street, Haidian District,

department: Oral and Maxillofacial Surgery

institute: Peking University School and Hospital of Stomatology

type:
Non-coding RNA profiling by array
Expression profiling by array

summary: Abstract:  Backgroud:  This study was to explore long noncoding RNA (lncRNA) and messenger RNA (mRNA) expression profiles and construct functional networks to analyze their biological functions when botulinum toxin type A (BTXA) inhibited salivary secretion.  Methods: BTXA and saline were injected into the submandibular gland of rats as the BTXA group and control group, respectively. Morphological and secretory function tests validated the animal models. Microarray analysis was applied to identify the expression variation of lncRNA and mRNA and were confirmed by qRT-PCR. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to explicit the biological functions. The construction of functional networks including lncRNA-mRNA co-expression network and competing endogenous RNA (ceRNA) network was to reveal the interaction between coding and noncoding genes.  Results: A total of 254 lncRNAs and 631 mRNAs were differentially expressed (DE) between the control group and the BTXA group. Ten lncRNAs and eleven mRNAs were confirmed by qRT-PCR and the results were consistent with the microarray analysis. The bioinformatic analysis found that most of mRNAs were closely related to transmembrane transporter activity. LncRNA-mRNA co-expression network and ceRNA network were constructed and identified several critical mRNA-lncRNA axes and key microRNAs related to salivary secretion, respectively.   Conclusions: Our study identified DE lncRNAs and mRNAs through microarray analysis and explored the biological functions and interaction between coding and noncoding genes through bioinformatics analysis. These findings may provide new directions for the mechanism of BTXA to inhibit salivary secretion.

overall_design: Detection of expression profiles of lncRNA and mRNA and construct functional networks to analyze biological functions in submandibular glands of control group and BTXA group.

first: Ruolan

last: xiang

first: Qianying

last: Mao


title: Identification of differentially expressed genes in the mutant tail regression failed (trf) in Ciona intestinalis.

city: Setouchi

first: Mayuko

last: Hamada

state: Okayama

address: 130-17, Kashino, Ushimado

institute: Okayama University

summary: Tunicate ascidians exhibit metamorphosis that converts tadpole, swimming larva into immotile adult. In ascidian Ciona intestinalis, the mutant tail regression failed (trf)  which shows defects in the metamorphosis was previously reported (Nakayama-Ishimura et al., 2009). In the metamorphosis process, trf larvae settle normally with their adhesive papillae, but do not start tail regression, papillae retraction and sensory vesicle retraction, while development of adult organs proceed. To understand the molecular mechanism of the metamorphosis, microarray analysis of trf mutant was performed.

overall_design: Gene expressions in trf mutants was compared to the normal larvae and juveniles around the metamorphosis stage by Two-color detection method. Dye-swap analysis was carried out.

first: Nori

last: Satoh

first: Yasunori

last: Sasakura


title: Tissue-specific transcription reprogramming promotes liver metastasis of colorectal cancer

first: Yang

middle: Eric

last: Li

state: Beijing

address: Room 2-110, Biotechnology Building, School of Life Sciences, Tsinghua University

department: School of Life Sciences

institute: Tsinghua University