title: Signature of CD45- metastatic niche cells at different stages of the metastatic disease

city: London

first: Probir

last: Chakravarty

address: 1 Midland Road

institute: Francis Crick Institute

summary: We analysed the signature of the non-immune cells from the metastatic niche and the distal lung using the breast tumour 4T1 cell line as a model of lung metastasis from breast cancer

overall_design: Syngenic Balb/c female mice were injected intravenously with 4T1 tumour cells and lung tissue were collected at day 5 and day 10. post-injection. RNA was isolated from CD45-Ter119- FACS sorted cells.

first: Luigi

last: Ombrato

first: Ilaria

last: Malanchi

first: Miriam

last: Llorian-Sopena


title: Light induced injury in mouse embryos revealed by single-cell RNA sequencing

status: Public on Aug 26 2019

city: SHANG HAI

first: Zhigang

last: Xue

address: Siping Road

country: China

institute: Tongji University

type: Expression profiling by high throughput sequencing

summary: We adopt mouse pre-implantation embryos to evaluate the effects of different exposure time and illumination intensity. The damage-causing time and intensity is chosen to treat embryos at 2-cell stage. Single-cell transcriptomes from 2-cell to blastocyst stage are analyzed under both control and high-intensity light conditions. These data show that embryo cells at each embryonic stage can be categorized into different light conditions. Further analyses of differentially expressed genes and relative GO terms reveal the light-induced injury as well as the potential repair response after high-intensity illumination treatment. Maternal-to-zygote transition is also affected through the failure of removing maternal RNAs and deactivating zygotic genomes. In conclusion, our work revealed integrated response of high-intensity light, involving the long-lasting injury effects, damage repair mechanism and blastocyst morphology changes.

overall_design: Two groups are included in this study: one is the high-intensity light exposure group that contained 34 samples, and the other is the control group that contains 33 samples.

first: Bo

last: Lv

first: Chaojie

last: Liu


title: Neuronal TORC1 modulates longevity via AMPK and cell nonautonomous regulation of mitochondrial dynamics in C. elegans

status: Public on Aug 27 2019

city: Boston

first: Meeta

last: Mistry

state: MA

address: 655 Huntington Ave

country: USA

institute: Harvard School of Public Health

summary: Evaluating gene expression changes between wildtype worms, raga-1 mutants (loss of gene), and a neuronal rescue worm model

overall_design: 4 replicates of each sample class was sequenced and transcriptional patterns across the three classes was evaluated together and by using pairiwise comaprisons

first: Will

last: Mair

first: Anne

last: Lanjuin


title: Molecular basis of cisplatin resistance in testicular germ cell tumors

status: Public on Aug 29 2019

city: Prague

first: Jan

last: Stuchly

address: V Uvalu 84

department: Department of Paediatric Hematology/Oncology

country: Czech Republic

institute: Charles University in Prague, 2nd Faculty of Medicne

type: Genome variation profiling by high throughput sequencing

summary: The emergence of cisplatin (CDDP) resistance is the main cause of treatment failure and death in patients with testicular germ cell tumors (TGCT), but its biologic background is poorly understood. To study the molecular basis of CDDP resistance in TGCT we prepared and sequenced CDDP-exposed TGCT cell lines as well as 31 primary patients’ samples. Long-term exposure to CDDP increased the CDDP resistance 10 times in the NCCIT cell line, while no major resistance was achieved in Tera-2. Development of CDDP resistance was accompanied by changes in the cell cycle (increase in G1 and decrease in S-fraction), increased number of acquired mutations, of which 3 were present within ATRX gene, as well as changes in gene expression pattern. Copy number variation analysis showed, apart from obligatory gain of 12p, several other large-scale gains (chr 1, 17, 20, 21) and losses (chr X), with additional more CNVs found in CDDP-resistant cells (e.g., further losses on chr 1, 4, 18, and gain on chr 8). In the patients’ samples, those who developed CDDP resistance and died of TGCT (2/31) showed high numbers of acquired aberrations, both SNPs and CNVs, and harbored mutations in genes potentially relevant to TGCT development (e.g., TRERF1, TFAP2C in one patient, MAP2K1 and NSD1 in another one). Among all primary tumor samples, the most commonly mutated gene was NSD1, affected in 9/31 patients. This gene encoding histone methyl transferase was also downregulated and identified among the 50 most differentially expressed genes in CDDP-resistant NCCIT cell line. Interestingly, 2/31 TGCT patients harbored mutations in the ATRX gene encoding a chromatin modifier that has been shown to have a critical function in sexual differentiation. Our research newly highlights its probable involvement also in testicular tumors. Both findings support the emerging role of altered epigenetic gene regulation in TGCT and CDDP resistance development.    This submission includes complete cell lines sequencing data and processed files. The patients data are available from the authors on request.

overall_design:
sensitive and resistant TGCT cell lines

Long-term cultured cell lines are long-term cultured cells that have never been exposed to cisplatin and are cisplatin sensitive, they were used as a control to distinguish between changes occuring during long term cultures without any relation to cisplatin exposure and those related to cisplatin exposition and resistance.

first: Ludmila

last: Boublikova

first: Violeta

last: Bakardjieva-Mihaylova

first: Michael

last: Svaton


title: Splicing factor SRSF1 controls T cell hyperactivity and systemic autoimmunity

status: Public on Aug 24 2019

first: Takayuki

last: Katsuyama

address: 3 Blackfan Circle, CLS-948

department: Rheumatology and Clinical Immunology

institute: Beth Israel Deaconess Medical Center

summary: Purpose: The goals of this study are to determine the role of the serine/ arginine-rich splicing factor 1 (SRSF1) in T cells and autoimmune disease.

overall_design: Methods: Naive CD4 T cells from spleens of 7-week old wild-type (WT) and serine/arginine-rich splicing factor 1 conditional knockout  (Srsf1-cKO) mice (n=3 each) were stimulated with anti-CD3 (0.5μg/mL) and anti-CD28 (1μg/mL) for 72 hours. Cells were re-stimulated with PMA (100ng/mL) and Ionomycin (1μM) for 4 hours. Total RNA was extracted using the RNeasy mini kit (Qiagen) and submitted for sequencing to the Molecular Biology Core Facility (MBCF) at the Dana-Farber Cancer Institute (DFCI). Libraries were prepared using Illumina TruSeq Stranded mRNA sample preparation kits according to the manufacturer’s protocols. Samples were sequenced on an Illumina NextSeq500 run with single-end 75-bp reads. Data analyses were performed by the MBCF at DFCI and the Harvard T. H. Chan School of Public Health Biostatistics Core

first: Vaishali

middle: R

last: Moulton


title: Gene expression alterations associated with acquired-resistance to the CDK4/6 inhibitor palbociclib [Palbociclib resistance_RNASeq]

status: Public on Aug 23 2019

city: Parkville

first: Karen

last: Sheppard

state: Victoria

address: 305 Grattan Street

country: Australia

institute: Peter MacCallum Cancer Center

summary:
RNA-seq was performed to  compare the transcriptional programmes of palbociclib-resistant A375 and CHL1 cells compared to their parental counterparts
Cyclin dependent kinase 4/6 (CDK4/6) inhibitors are an established treatment in estrogen receptor positive breast cancer and are currently in clinical development in melanoma; a tumour that exhibits high rates of CDK4 activation. We analyzed melanoma cells with acquired resistance to the CDK4/6 inhibitor palbociclib, and demonstrated that palbociclib-mediated inhibition of PRMT5 is essential for sensitivity to CDK4/6 inhibitors. Mechanistically, by inhibiting PRMT5 activity, palbociclib alters MDM4 pre-mRNA splicing leading to decreased MDM4 protein expression and subsequent p53 activation. In turn, p53 increases p21 leading to inhibition of CDK2, the main kinase substituting for CDK4/6 and a key driver of resistance to palbociclib.  Loss of the ability of palbociclib to regulate the PRMT5-MDM4 axis leads to resistance. Importantly, combining palbociclib with the PRMT5 inhibitor GSK3326595 enhances the efficacy of palbociclib in treatment naïve and resistant models and also delays the emergence of resistance. Our studies have uncovered a novel mechanism of action of CDK4/6 inhibitors in regulating the MDM4 oncogene and the tumor suppressor, p53. Furthermore, we have established that palbociclib inhibition of the PRMT5-MDM4 axis is essential for robust melanoma cell sensitivity and provide pre-clinical evidence that co-inhibition of CDK4/6 and PRMT5 is an effective and well tolerated therapeutic strategy. Overall our data provides a strong rationale for further investigation of novel combinations of CDK4/6 and PRMT5 inhibitors in not only melanoma but other tumour types including breast, pancreatic and esophageal carcinoma.

overall_design: Cells were treated with increasing concentrations of palbociclib for a prolonged period of time until resistance to the drug develop

first: AbuHammad

last: Shatha

first: Sheppard

last: Karen


title: Transcriptome of separated Pneumocystis life forms (asci and troph)

city: New Orleans

first: Jay

last: Kolls

state: LA

address: 1430 Tulane Ave

department: Medicine

institute: Tulane University

summary:
Pneumocystis pneumonia is the most common serious opportunistic infection in patients with HIV/AIDS. Furthermore, Pneumocystis pneumonia is a feared complication of the immunosuppressive drug regimens used to treat autoimmunity, malignancy, and post-transplantation rejection. With an increasing at-risk population, there is a strong need for novel approaches to discover diagnostic and vaccine targets. There are multiple challenges to finding these targets, however. First, Pneumocystis has a largely unannotated genome. To address this, we evaluated each protein encoded within the Pneumocystis genome to that of other fungi using NCBI Blast. Second, Pneumocystis relies on a multiphasic life cycle, as both the transmissible form, the ascus, and the replicative form, the troph, reside within the alveolar space of the host. Towards that end, we purified asci and trophs from Pneumocystis murina and utilized transcriptomics to identify differentially regulated genes. Two such genes, Arp9 and Sp, are differentially regulated in the ascus and the troph, respectively, and can be utilized to characterize the state of the Pneumocystis life cycle in vivo. Gsc1, a β-1,3-glucan synthase with a large extracellular domain previously identified using surface proteomics, was more highly expressed on the ascus form of Pneumocystis. GSC-1 ectodomain immunization generated a strong antibody response capable of recognizing the surface of the Pneumocystis asci. GSC-1 ectodomain immunization was also capable of reducing ascus burden following primary challenge with Pneumocystis murina. Finally, mice immunized with the GSC-1 ectodomain had limited burden following natural transmission of Pneumocystis using a co-housing model.
Pneumocystis asci and trophs were separated via flow cytometry and the transcriptome was sequenced, allowing to further understand the differential expression of various RNA transcripts. These data can be mined for life-form specific diagnostics and therapeutic targets.

overall_design: Trophs and asci were separated via flow cytometry and sequenced (n=2 biologic replicates of separated life forms)

middle: K

first: Taylor

last: Eddens

first: Mingquan

last: Zheng

first: William

last: Horne


title: Demonstration of CUT&RUN motif footprint analysis using key blood progenitor transcription factors

first: Stuart

last: Orkin

address: 1 Blackfan Circle

department: Hematology and Oncology

institute: Boston Childrens Hospital

type: Genome binding/occupancy profiling by high throughput sequencing

summary: We introduce CUT&RUNTools (https://bitbucket.org/qzhudfci/cutruntools/) as a flexible, general pipeline for facilitating the identification of chromatin-associated protein binding and genomic footprinting analysis from antibody-targeted CUT&RUN primary cleavage data. CUT&RUNTools extracts endonuclease cut site information from sequences of short read fragments and produces single-locus binding estimates, aggregate motif footprints, and informative visualizations to support the high-resolution mapping capability of CUT&RUN. We illustrate the functionality of CUT&RUNTools through analysis of CUT&RUN data acquired for GATA1, a master regulator in erythroid lineage cells. Results were compared initially to published GATA1 ChIP-seq data for cells under the same conditions. We performed de novo analysis of CUT&RUN peaks to retrieve not only GATA1’s primary motif, but also the GATA1-TAL1 composite motif, and co-factor motifs GCCCCGCCTC, CMCDCCC, and RTGASTCA that correspond to SP1, KLF1, and NFE2 co-factors. Cofactor binding was verified by independent TAL1 and KLF1 CUT&RUN, and other ChIP-seq experiments. CUT&RUNTools also generated base-pair resolution motif footprint for sequence-specific binding factors, and located likely direct binding sites by quantifying log-odds of binding scores. Overall, CUT&RUNTools should enable biologists to realize advantages of cleavage data provided by CUT&RUN, and make high-quality footprinting analysis accessible to a broad audience.

overall_design: We performed CUT&RUN using GATA1, TAL1, and KLF1 antibodies in primary human stem/progenitor CD34+ cells after 7 days of erythroid differentiation, and in erythroid progenitor HUDEP2 cells followed by 4 days of erythroid differentiation.

first: Qian

last: Zhu

last: Liu

first: Guo-cheng

last: Yuan


title: The strand-biased mitochondrial DNA methylome and its regulation by DNMT3A [hPFC_WGBS]

status: Public on Aug 22 2019

city: Shanghai

first: Jing-Dong

middle: Jackie

last: Han

address: 320 Yue Yang Road

institute: CAS-MPG Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

type: Methylation profiling by high throughput sequencing

summary: How individual genes are regulated from a mitochondrial polycistronic transcript to have variable expression remains an enigma. Here, through bisulfite sequencing and strand-specific mapping, we show mitochondrial genomes in human and other animals were strongly biased to light (L)-strand non-CpG methylation with conserved peak loci preferentially located at gene-gene boundaries, which was also independently validated by MeDIP and FspEI digestion. Such mtDNA methylation patterns are conserved across different species and developmental stages, but display dynamic local or global changes during development and aging. Knockout of DNMT3A alone perturbed mtDNA regional methylation patterns, but not global levels, and altered mitochondrial gene expression, copy number, and oxygen respiration. Overexpression of DNMT3A strongly increased mtDNA methylation and strand bias. Overall, methylation at gene body and boundaries was negatively associated with mitochondrial transcript abundance and also polycistronic transcript processing. Furthermore, HPLC-MS confirmed the methylation signals on mitochondria DNA. Together, these data provide high-resolution mtDNA methylation maps that revealed a strand specific non-CpG methylation, its dynamic regulation and its impact on the polycistronic mitochondrial transcript processing.

overall_design: Examination of mitochondrial DNA methylation in human brain

first: Xiaoyang

last: Dou

first: Jerome

middle: D

last: Boyd-Kirkup

first: Jing-dong

middle: J


title: Genome wide transcriptome analysis of palbociclib or GSK3326595 treated A375 cells [Palbociclib_GSK_RNASeq]

summary:
RNA-seq was performed to  compare the transcriptional programmes of A375 cells treated with Palbociclib or GSK3326595 compared to untreated cells
Cyclin dependent kinase 4/6 (CDK4/6) inhibitors are an established treatment in estrogen receptor positive breast cancer and are currently in clinical development in melanoma; a tumour that exhibits high rates of CDK4 activation. We analyzed melanoma cells with acquired resistance to the CDK4/6 inhibitor palbociclib, and demonstrated that palbociclib-mediated inhibition of PRMT5 is essential for sensitivity to CDK4/6 inhibitors. Mechanistically, by inhibiting PRMT5 activity, palbociclib alters MDM4 pre-mRNA splicing leading to decreased MDM4 protein expression and subsequent p53 activation. In turn, p53 increases p21 leading to inhibition of CDK2, the main kinase substituting for CDK4/6 and a key driver of resistance to palbociclib.  Loss of the ability of palbociclib to regulate the PRMT5-MDM4 axis leads to resistance. Importantly, combining palbociclib with the PRMT5 inhibitor GSK3326595 enhances the efficacy of palbociclib in treatment naïve and resistant models and also delays the emergence of resistance. Our studies have uncovered a novel mechanism of action of CDK4/6 inhibitors in regulating the MDM4 oncogene and the tumor suppressor, p53. Furthermore, we have established that palbociclib inhibition of the PRMT5-MDM4 axis is essential for robust melanoma cell sensitivity and provide pre-clinical evidence that co-inhibition of CDK4/6 and PRMT5 is an effective and well tolerated therapeutic strategy. Overall our data provides a strong rationale for further investigation of novel combinations of CDK4/6 and PRMT5 inhibitors in not only melanoma but other tumour types including breast, pancreatic and esophageal carcinoma.

overall_design: A375 cells were treated with 1 μΜ palbociclib (PD991) or 500 nM GSK3326595 (CTX) for 72 hrs


title: The strand-biased mitochondrial DNA methylome and its regulation by DNMT3A [WGBS_negative control]

overall_design: Negative control for examination of mitochondrial DNA methylation in human brain


title: Transgenerational self-reconstruction of disrupted chromatin organization after exposure to an environmental stressor in mice

status: Public on Aug 28 2019

city: Santa Cruz

first: Carlos

last: Diaz-Castillo

state: California

address: 487 Physical Sciences Building

institute: University of California Santa Cruz

summary: Exposure to environmental stressors is known to increase disease susceptibility in unexposed descendants in the absence of detectable genetic mutations. The mechanisms mediating environmentally-induced transgenerational disease susceptibility are poorly understood. We showed that great-great-grandsons of female mice exposed to tributyltin (TBT) throughout pregnancy and lactation were predisposed to obesity due to altered chromatin organization that subsequently biased DNA methylation and gene expression. Here we analyzed DNA methylomes and transcriptomes from tissues of animals ancestrally exposed to TBT spanning generations, sexes, ontogeny, and cell differentiation state. We found that TBT elicited concerted alterations in the expression of “chromatin organization” genes and inferred that TBT-disrupted chromatin organization might be able to self-reconstruct transgenerationally. We also found that the location of “chromatin organization” and “metabolic” genes is biased similarly in mouse and human genomes, suggesting that exposure to environmental stressors in different species could elicit similar phenotypic effects via self-reconstruction of disrupted chromatin organization.

overall_design:
We generated RNA-Seq data from mesenchymal stem cells (MSCs) from 8-week old F3 and F4, female and male mice and liver from 33-week old F4 male mice ancestrally exposed to DMSO (0.1%) or TBT (50 nM ) during in utero development and lactation. N=5 per treatment for MSCs and N=4 per treatment for liver--> 48 RNA-seq samples in total
We generated MBD-Seq data from mesenchymal stem cells (MSCs) from 8-week old F3 and F4, female and male mice and liver from 33-week old F4 male mice ancestrally exposed to DMSO (0.1%) or TBT (50 nM ) during in utero development and lactation. N=5 per treatment for MSCs and N=4 per treatment for liver--> 48 MBD-seq samples in total

first: Raquel

last: Chamorro-Garcia

first: Toshi

last: Shioda

first: Bruce

last: Blumberg


title: Expanding the regulon of the Bradyrhizobium diazoefficiens NnrR transcription factor: New insights into denitrification pathway

city: Zürich

first: Hans-Martin

last: Fischer

address: Vladimir-Prelog-Weg 4

department: Microbiology

country: Switzerland

institute: ETH Zürich

summary: Genome-wide transcription profiling of B. diazoefficiens (formerly B. japonicum) wild type and nnrR mutant strain which were grown in free-living anoxic denitrifying conditions (using nitrate as final electron aceptor). This study includes also the expression data of the wild type strain grown oxically (samples GSM210269 to GSM210283 in GEO record number GSE8478). Keywords: CRP/FNR proteins, in vitro transcription, microoxia, nitrogen oxides, Rhizobia, transcriptomics

overall_design: Comparative transcriptional profiling of nnrR mutant cells with wild-type cells cultured anoxically with nitrate

first: A

last: Jiménez-Leiva

first: J

last: Cabrera

first: E

last: Bueno

first: M

last: Torres

first: S

last: Salazar

last: Bedmar

last: Delgado

last: Mesa


title: Comparative Analysis of Antifungal Mechanism of Lauryl betaine in the Human Fungal Pathogens Cryptococcus neoformans and Malassezia restricta

city: Seoul

first: Yong-Joon

last: Cho

address: 1 Gwanak-ro, Gwanak-gu

country: South Korea

institute: Seoul National University

summary:
Purpose: Understanding the mechanism of action of lauryl betaine against Cryptococcus neoformans and Malassezia restricta.
Methods: The transcriptome profile of the lauryl betaine-treated cells compared to that of untreated cells were generated by RNA-seq Illumina Hiseq.
Results: A number of genes were differentially expressed in the lauryl betaine-treated cells, which include genes involved in ergosterol biosynthetic pathway.

sample_organism:
Malassezia restricta
Cryptococcus neoformans H99

overall_design: Transcriptome of lauryl betaine-treated M. restricta cells (3 mg/ml) vs. Transcriptome of untreated M. restricta cells. Transcriptome of lauryl betaine-treated C. neoformans cells (4.5, 9.0 ug/ml) vs. Transcriptome of untreated C. neoformans cells.

first: Won-Hee

last: Jung

first: Eunsoo

last: Do

first: MinJi

last: Park

first: Dong-Hyeun

last: Kim


title: T Cells Control Chemokine Secretion by Keratinocytes

city: Mainz

first: Matthias

last: Klein

address: Langenbeckstrasse 1

department: Inst. for Immunology

country: Germany

institute: University Medical Center Mainz

summary: By co-culturing murine keratinocytes with splenic T cells we show here that T cells affect the synthesis and secretion of chemokines by keratinocytes.

overall_design: Primary murine keratinocytes were isolated from tails of adult mice. Upon preparation of skin, skin pieces were incubated O/N in dispase solution at 4oC followed by a short digestion in trypsin and incubation for 6 h in S-MEM medium containing 1.3 mM Ca++. After 6 h cells were replenished with fresh medium with keratinocyte SFM medium containing 0.06 mM Ca++ for further culture. All cell splittings were performed with TrypsinLE. Splenic T cells were isolated using mouse CD8 microbeads kit and cultured in X-VIVOTM 15 medium. Cells were pre-activated by  CD3/CD28 (5  g CD3  and 2µg CD28) for 1 d. CD8+ T cells were co-cultivated with keratinocytes for 1h. The T cells were washed off from the keratinocytes. Both types of cells were separatedly collected, and the RNA sequenced. 2 parallel experiments.

first: Serfling

last: Edgar

first: Khalid

last: Muhammad


title: The strand-biased mitochondrial DNA methylome and its regulation by DNMT3A [HEK293T_WGBS]

first: Jing-Dong

last: Han

overall_design: Examination of mitochondrial DNA methylation changes upon DNMT3A knockout in HEK293T cells.

first: Xiaoyang

last: Dou

first: Joseph

last: McDermott

first: Jing-dong


title: Single-cell transcriptional logic of cell-fate specification and axon guidance in early born retinal neurons [C1]

city: Genève 4

first: Quentin

last: Lo Giudice

state: switzerland

address: 1 rue Michel Servet

department: Department of Basic Neurosciences

institute: University of Geneva

summary: Application of single-cell sequencing C1 to obtain neural retinal transcriptomes.

overall_design: 800 single-cell transcriptomes from E15.5 Sert-Cre developing retinas.

middle: L

first: Marion

last: Leuleu

first: Pierre

middle: F

last: Fabre


title: The strand-biased mitochondrial DNA methylome and its regulation by DNMT3A [HEK293T_RNA-seq]

overall_design: Examination of gene expression changes upon DNMT3A knockout in HEK293T cells.


title: The strand-biased mitochondrial DNA methylome and its regulation by DNMT3A [A549_WGBS]

overall_design: Examination of mitochondrial DNA methylation changes upon DNMT3A over expression in A549 cells.

first: Jerome

last: Boyd-Kirkup


title: The strand-biased mitochondrial DNA methylome and its regulation by DNMT3A [A549_RNA-seq]

overall_design: Examination of gene expression changes upon DNMT3A over expression in A549 cells.


title: The strand-biased mitochondrial DNA methylome and its regulation by DNMT3A [HEK293T_MeDIP]

summary:
How individual genes are regulated from a mitochondrial polycistronic transcript to have variable expression remains an enigma. Here, through bisulfite sequencing and strand-specific mapping, we show mitochondrial genomes in human and other animals were strongly biased to light (L)-strand non-CpG methylation with conserved peak loci preferentially located at gene-gene boundaries, which was also independently validated by MeDIP and FspEI digestion. Such mtDNA methylation patterns are conserved across different species and developmental stages, but display dynamic local or global changes during development and aging. Knockout of DNMT3A alone perturbed mtDNA regional methylation patterns, but not global levels, and altered mitochondrial gene expression, copy number, and oxygen respiration. Overexpression of DNMT3A strongly increased mtDNA methylation and strand bias. Overall, methylation at gene body and boundaries was negatively associated with mitochondrial transcript abundance and also polycistronic transcript processing. Furthermore, HPLC-MS confirmed the methylation signals on mitochondria DNA. Together, these data provide high-resolution mtDNA methylation maps that revealed a strand specific non-CpG methylation, its dynamic regulation and its impact on the polycistronic mitochondrial transcript processing.
Through bisulfite sequencing and strand-specific mapping, we show mitochondrial genomes in human and other animals were strongly biased to light (L)-strand non-CpG methylation with conserved peak loci preferentially located at gene-gene boundaries, which was also independently validated by MeDIP and FspEI digestion.

overall_design:
Examination of mtDNA methylation using MeDIP-seq in HEK293T cells.
The processed data was generated from all three replicates and is linked to the corresponding 'r1' sample records.


title: Regulation of PRMT5-MDM4 axis is critical in the response to CDK4/6 inhibitors in melanoma

first: Karen

last: Sheppard

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

overall_design: Refer to individual Series


title: Depletion of HuR in murine skeletal muscle enhances exercise endurance and prevents cancer-induced muscle atrophy

status: Public on Aug 21 2019

city: Montreal

first: Brenda

middle: Janice

last: Sanchez

state: QC

address: 3655 promenade Sir-William-Osler, Room 915

department: Bichemistry

country: Canada

institute: McGill University

summary: Here we show that, when compared to control mice, muscle specific HuR knockout (muHuR-KO) animals have higher exercise endurance that is associated with a significant increase in oxygen consumption and carbon dioxide production. Histological analysis demonstrates that the absence of HuR significantly enhances the proportion of oxidative type I fibers in several skeletal muscles, showing that under normal conditions, HuR promotes the formation of glycolytic type II fibers. HuR mediates this outcome by collaborating with the mRNA decay factor KSRP, to destabilize the PGC-1α mRNA, a well-known promoter of type I fiber formation. Furthermore, the type I fiber-enriched phenotype of muHuR-KO mice is associated with a protection against cancer cachexia-induced muscle wasting

overall_design: mRNA profiles of Control and muscle specif HuR ko mice was generated by RNA-seq from sckeletal muscle samples in duplicate using Illumina NextSeq 500 platform.

first: Imed

middle: E

last: Gallouzi

first: Sergio

last: Di-Marco


title: Characterization of eQTLs associated with androstenone by RNA sequencing in porcine testis - SNP Genotype Database

city: Kongens Lyngby

first: Haja

middle: N

last: Kadarmideen

address: Richard Petersens Plads, Building 324

department: Department of Applied Mathematics and Computer Science

country: Denmark

institute: Technical University of Denmark

type:
Genome variation profiling by SNP array
SNP genotyping by SNP array

summary: Boar taint is an offensive odour and flavour from developed in a proportion of non-castrated male pigs (boars). Surgical castration is an effective solution but is under debate due to animal welfare concerns. Gene-based selection for low boar taint has been proposed as concentrations of the main compounds responsible for boar taint (androstenone, skatole and indole [ASI]) exhibit heritability but candidate biomarkers are lacking. The aims of this study were to identify expression quantitative trait loci (eQTLs) associated with ASI in order to aid development of candidate biomarkers and to generate insights into biological pathways. Gene expression profiles were obtained by RNA-Seq from testis of Danish Duroc × Landrace/Yorkshire boars fed either a control or chicory-added diet and genotyping was conducted with a high density SNP panel. A tendency (P = 0.052) of lowered ASI concentrations was found in the chicory group. Furthermore, a high correlation was found between estimated breeding values (EBVs) of androstenone and androstenone concentrations (r2 = 0.48). A total of 213 eQTLs, representing 125 unique genes including MVP, COX1, MAP2K4 and RDH16, contained genotypes associated with summed ASI concentrations and of these, six genes were also differentially expressed across ASI/diet groups. The genes were functionally associated with membrane localised proteins, extracellular exosomes, ATP binding, MAPK cascade and oxidative stress. The highest densities of the eQTLs were on pig chromosomes (SSC) SSC18, SSC12, SSC2, SSC7 and SSC14, in that order. A total of 42 known porcine traits were enriched by the genomic coordinates, including health, meat quality and one reproduction trait. A total of 33 candidate eQTLs enriched known genomic coordinates for androstenone and odour intensity in fat. Our findings suggests that careful monitoring of important productions parameters is necessary, if gene-based selection is performed with markers located within the genomic coordinates of the candidate eQTLs or the eQTLs themselves.

overall_design: A total of 48 Danish crossbred Duroc X Landrace/Yorkshire male pigs with divergent androstenone, skatole and indole concentrations were subjected to genotyping of loin muscle. mRNA from testes of these pigs were sequenced with paired end sequencing. Furthermore, sensoric traits, carcass weights and pedigree data (EBVs) were available for each pig. Finally, a group of pigs received a chicory-additive a number of days before slaughter.

first: Markus

middle: H

last: Drag

first: Lisette

last: Kogelman

first: Hanne

last: Maribo

first: Lene

last: Meinert

first: Preben

last: Thomsen


title: Epigenetic Control of a Multifunctional Stress Regulator

status: Public on Aug 19 2019

city: La Jolla

last: Ecker

state: CA

address: 10010 North Torrey Pines Road

department: Genomic Analysis Laboratory

institute: HHMI-Salk-Institute

summary: Here we report the identification of ETHYLENE-INSENSITIVE6 (EIN6), which is a H3K27me3 demethylase also known as RELATIVE OF EARLY FLOWERING6 (REF6), and EIN6 ENHANCER (EEN), the Arabidopsis homolog of the yeast INO80 chromatin remodeling complex subunit IES6 (INO EIGHTY SUBUNIT6). By applying high-throughput profiling of histone modifications, histone variant occupancy, cytosine methylation and mRNA expression in various Arabidopsis mutants, we identified ETHYLENE-INSENSITIVE2 (EIN2) as a direct target of EIN6 (REF6) and EEN. EIN6 (REF6) and EEN as part of the INO80 complex interdependently and uniquely control the level and the localization of the repressive histone modification H3K27me3 and the histone variant H2A.Z at the 5’ untranslated region (5’UTR) intron of EIN2. Concomitant functional loss of EIN6 (REF6) and the INO80 complex shifts the chromatin landscape at EIN2 to a repressive state causing a dramatic reduction of EIN2 expression and subsequent collapse of the entire ethylene transcriptional cascade.

overall_design: ChIP-seq, ChIP-reChIP-seq, MethylC-seq and RNA-seq was applied to comprehensively profile the chromatin landscape in various Arabidopsis mutants and different tissue types.

first: Mark

last: Zander

first: Björn

middle: C

last: Willige

first: Yupeng

last: He

first: Thu

middle: A

last: Nguyen

first: Amber

last: Langford

first: Ramlah

last: Nehring

first: Elizabeth

last: Howell

first: Robert

last: McGrath

first: Anna

last: Bartlett

first: Rosa

last: Castanon

last: Nery

first: Huaming

last: Chen

first: Zhuzhu

last: Zhang

first: Florian

last: Jupe

first: Mathew

middle: G

last: Lewsey

last: Stepanova

last: Schmitz

first: Joanne

last: Chory


title: Unique Trophoblast Chromatin Environment Mediated by the PcG Protein SFMBT2

status: Public on Aug 18 2019

city: Toronto

first: Susannah

last: Varmuza

state: Ontario

address: 25 Harbord Street

institute: University of Toronto


title: Spatially clustered loci with multiple enhancers are frequent targets of HIV-1

city: Heidelberg

first: Wei

last: Wang

address: Im Neuenheimer Feld 581

department: Translational Oncology

institute: German Cancer Research Center

summary: HIV-1 recurrently targets active genes that are positioned in the outer shell of the nucleus and integrates in the proximity of the nuclear pore compartment. However, the genomic features of these genes and the relevance of their transcriptional activity for HIV-1 integration have so far remained unclear. Here we show that recurrently targeted genes are delineated with super-enhancer genomic elements and that they cluster in specific spatial compartments of the T cell nucleus. We further show that these gene clusters acquire their location at the nuclear periphery during the activation of T cells. The clustering of these genes along with their transcriptional activity are the major determinants of HIV-1 integration in T cells. Our results show for the first time the relevance of the spatial compartmentalization of the genome for HIV-1 integration, thus further strengthening the role of nuclear architecture in viral infection.

overall_design: Primary CD4 positive T cells were left in resting state or activated with Dynabeads Human T-Activator CD3/CD28. Cells were infected with the pNL4_3-envFS. Linear Amplification Mediated (LAM) PCR was performed. HIV integration sites were identified.

first: Raffaele

last: Fronza

first: Manfred

last: Schmidt


title: Mechanosensation of Cyclical Force by PIEZO1 is Essential for Innate Immunity

city: New Haven

first: Christian

last: Harman

state: Connecticut

address: 300 Cedar Street, The Anlyan Center

department: Genetics/Immunobiology

institute: Yale University

summary: Although cells of the immune system experience force and pressure throughout their lifecycle, almost nothing is known about how these mechanical processes regulate the immune response. Immune cells in highly mechanical organs, such as the lung, are constantly exposed to tonic and dynamically changing mechanical cues. Here using reverse genetics, we show that myeloid cells respond to force and alterations in cyclical hydrostatic pressure (CHP) via the mechanosensory ion channel (MSIC) PIEZO1. Unbiased RNA-sequencing from macrophages subjected to CHP reveals a striking state of proinflammatory reprogramming. We report a novel mechanosensory-immune signaling circuit which PIEZO1 initiates in response to CHP, activating c-JUN, upregulating Endothelin-1 (EDN1), and stabilizing HIF1α to facilitate a prolonged program of proinflammatory mediators. Using mice conditionally deficient of PIEZO1 in myeloid cells, and cellular depletion assays, we show infiltrating monocytes respond to cyclical force to recruit neutrophils and clear pulmonary Pseudomonas aeruginosa infection. Furthermore, myeloid PIEZO1 also drove lung pathology in a mouse model of pulmonary fibrosis. Our results demonstrate a novel environmental sensory axis that myeloid cells recognize to mount an inflammatory response, and is the first report showing a physiological role for PIEZO1 and mechanosensation in immunity.

overall_design: To observe any biological response to mechanostimulation, BMDMs from Piezo1fl/fl and Piezo1fl/fl, LysM Cre+ (ΔLysM) mice were incubated in the pressure chamber for 6 hours under either static or cyclical hydrostatic pressure and subjected to RNA-seq analysis.

first: Angel

last: Solis

first: Piotr

last: Bielecki

first: Holly

last: Steach

first: Lokesh

last: Sharma

first: Sanguk

last: Yun

first: Noah

middle: W

last: Palm

first: Marcel

last: de Zoete

first: James

last: Warnock

first: S

last: To

first: Autumn

last: York

first: Matthias

last: Mack

first: Martin

last: Schwartz

first: Charles

middle: S

last: Cruz

first: Ruaidhrí

last: Jackson

first: Richard

last: Flavell


title: Unique Trophoblast Chromatin Environment Mediated by the PcG Protein SFMBT2 [RNA-seq]

first: Susannah

last: Varmuza

summary: Sfmbt2 is a paternally imprinted gene critical for the establishment and maintenance of trophoblast stem cells in mice (Miri K et al., 2013).  Inheritance of a paternal Sfmbt2 gene trap null allele (Sfmbt2gt/gt) results in embryonic lethality by e12.5 due to reduced numbers in all trophoblast cell types.  Of note, SFMBT2 is characterized as a Polycomb group (PcG) protein and is assumed to act as a master transcriptional repressor.  Through RNA-seq analysis of extraembryonic tissues acquired from both wild-type and Sfmbt2gt/gt embryos, we aim to elucidate differentially expressed genes critical for the dramatic phenotype observed in mutant embryos.

overall_design: Sfmbt2gt/gt embryos were generated by intercrossing heterozygous Sfmbt2gt males and females.  Embryos were extracted at e7.5 and individually dissected into embryo and extraembryonic (i.e. ectoplacental cone and extraembryonic ectoderm) portions.  The embryonic portion was used for genotyping, and total RNA was extracted from extraembryonic portions of both wild-type (+/+) and mutant (gt/gt) embryos using Trizol.  Total RNA from pools of 12 embryos was extracted and processed by The Centre for Applied Genomics (TCAG) at The Hospital for Sick Children in Toronto.  The RNA-seq library was generated using the Illumina TruSeq protocol and was subject to paired-end next-generation sequencing on the Illumina HiSeq 2500 platform.  Two biological replicates of each were analyzed.

first: Priscilla

last: Tang

first: Kamelia

last: Miri


title: Genome-wide mapping of HA-tagged Rev-Erba in mice Brown Adipose Tissue (BAT) after one week at thermoneutrality (29C) or exposed to chronic cold (4C)

city: Philadelphia

first: Yann

last: Aubert

state: Pennsylvania

address: Biomedical Research Building 421 Curie Blvd

department: Penn Dermatology Research

institute: University of Pennsylvania

summary: Identification of temperature-selective RevErba binding sites in mice brown adipose tissue.

overall_design: Investigation of RevErba cistrome in chronic cold challanged mice.

first: Marine

last: Adlanmerini

first: Bryce

last: Carpenter

first: Jarrett

last: Remsberg

first: Lindey

last: Peed

first: Hannah

last: Richter

first: Mitchell

last: Lazar


title: Diverse repertoire of human adipocyte subtypes develops from transcriptionally distinct mesenchymal progenitor cells

city: Worcester

first: Silvia

last: Corvera

address: 373 Plantation St

institute: University of Massachusetts Medical School

summary: Single cell sequencing technologies are providing unexpected insights on how seemingly homogenous cell populations differ markedly in their functional properties, and how diverse cell repertoires mediate the functions of tissues and organs. Adipose tissue controls multiple key aspects of systemic energy homeostasis, but only two human adipocyte subtypes have been recognized so far. Here we developed methods to characterize single human mesenchymal progenitors and discovered four previously unknown adipocyte subtypes specialized for distinct adipose tissue functions, which are derived from distinct progenitors. Recognizing the existence of these different adipocyte subtypes and their progenitors will allow us to elucidate mechanisms that control their abundance and properties, and to better understand how dysregulation of these mechanisms can lead to metabolic disease.

overall_design: Human subcutaneous adipose tissue derived progenitor cells 52 clones under 3 treatment conditions (Control (C), MDI (M) and Forskolin (F)) are included in the analysis.

first: So

middle: Y

last: Min

first: Anand

last: Desai

first: Zinger

last: Yang

first: Agstya

last: Sharma

first: Agastya

first: Tiffany

last: DeSouza

first: Ryan

middle: M

last: Genga

first: Alper

last: Kucukural

first: Lawrence

last: Lifshitz

first: Søren

last: Nielsen

first: Camila

last: Scheele

first: René

last: Maehr

first: Manuel

last: Garber


title: Chronic cadmium exposure decreases the dependency of MCF7 breast cancer cells on ERα

city: Blacksburg

first: Mathew

last: Bloomfield

state: VA

address: 1015 Life Science Circle

department: Biological Sciences

institute: Virginia Tech

summary: To understand how chronic cadmium exposure alters the dependency of ERα in terms of gene expression, we transiently silenced ERα using ICI, an antiestrogen that promotes the degradation of ERα. MCF7 and cadmium-adapted cells (Cd7 and Cd12) were treated with ICI to mediate the degradation of ERα, and a nonbiased global gene expression analysis was conducted using RNA-seq.  MCF7 shared 67.3% and 59.5% of the DE genes with Cd7 and Cd12 cells, respectively, suggesting that ERα continues to play an important role in regulating the expression of genes following chronic cadmium exposure.  138 ERE genes (76.7%) were shared by all three cell lines, in that expression changed in the same direction (either up- or downregulated). For the estrogen-responsive genes, 428 (53.6%) of the 799 genes were altered in the same direction in all three cell lines. These findings show that while a majority of ERE genes responded in the same manner to loss of ERα, more variability existed within the estrogen-responsive genes. Collectively, these results indicate that while chronic cadmium exposure leads to genome-wide transcriptional changes, ERα remains important for regulating the expression of genes and maintaining the malignant phenotypes associated with breast cancer progression.

overall_design: Examination of mRNA expression, specifically of estrogen-regulated and estrogen-responsive genes, in breast cancer cells chronically exposed to cadmium.

middle: R

first: Maggie

last: Louie


title: Effect of ZIKV on human ESCs derived trophoblast cells

status: Public on Aug 16 2019

city: New York

first: Shuibing

last: Chen

state: NY

address: A 827B, 1300 York Ave

department: Surgery

institute: Weill Cornell Medical College

summary: To characterise the human trophoblast cells derived from human ESCs and the effects of ZIKV infection on them, we performed RNA-seq on two sets of samples: 1)human ES cell line HuES8 and the trophoblast cells differentiated. 2)trophoblast cells infected at 4 days after differentiation by MOCK and ZIKV, and harvested 72hpi. Here, we demonstrate gene expression profile of trophoblasts derived from human ESCs is similar to the trophoectoderm cells isolated from human blastocysts. And ZIKV infection on the human ESCs derived trophoblasts activated a panel of pathways, including programmed cell death.

overall_design: 1) RNA-seq of HuES8, trophoblasts differentiation to day 4 and day 9. 2) RNA-seq of day 4 trophoblast cells infected with MOCK and ZIKV.

first: Lei

last: Tan

first: Lauretta

last: Lacko


title: Effect of ZIKV on pre-implantation embryos

summary: To determine the effect of Zika virus infection on pre-implantation embryonic development, we performed single blastocyst RNA-Seq on MOCK and ZIKV infected embryos.   ZIKV infection results in an increased risk of spontaneous abortion and poor intrauterine growth  although the mechanisms underlying fetal loss remain undetermined. Little is known about the  impact of ZIKV infection during the earliest stages of pregnancy, or pre- and peri-implantation,  because most current studies of ZIKV infection in pregnancy models focus on post-implantation  stages. Here, we demonstrate that trophectoderm cells of pre-implantation human and mouse  embryos can be efficiently infected with ZIKV, and that trophectoderm can propagate virus  causing cell death of neural progenitors. These findings were corroborated by our demonstration  that hESC-derived trophectoderm cells are infected by ZIKV in a dose dependent manner. RNAseq of single blastocysts revealed key transcriptional changes in cellular and physiologic functions  upon ZIKV infection, including nervous system development and function, prior to commitment  to the neural cell lineage. Finally, the pregnancy rate of mice infected pre-implantation was > 50%  lower than females infected at E4.5. These results demonstrate that pre-implantation ZIKV  infection of trophectoderm leads to miscarriage or spontaneous abortion. Moreover, pre- and peri-implantation ZIKV infects trophectoderm cells that propagate virus over time causing cell death  in neural progenitors. Cumulatively, these data demonstrate that vertical pre- and peri-implantation  ZIKV infection of trophectoderm impairs fetal development and causes neural progenitor cell  death, elucidating a previously unappreciated association of pre- and peri-implantation ZIKV  infection and microcephaly.

overall_design: Single Blastocyst RNASeq of Mock (n=5 samples) and Zika (n=7 samples) infected mouse blastocysts


title: Unique Trophoblast Chromatin Environment Mediated by the PcG Protein SFMBT2 [ChIP-seq]

first: Susannah

last: Varmuza

summary: Sfmbt2 is a paternally imprinted gene critical for the establishment and maintenance of trophoblast stem cells in mice (Miri K et al., 2013).  Inheritance of a gene trap null allele (Sfmbt2gt/gt) results in embryonic lethality by e12.5 due to reduced numbers in all trophoblast cell types.  SFMBT2 is a mammalian Polycomb group protein (PcG) and is assumed to act as a master transcriptional regulator.  ChIP-seq was performed against endogenous and FLAG-SFMBT2 in trophoblast stem cells (TSCs) to complement the RNA-seq dataset acquired from extraembryonic tissues of wildtype and Sfmbt2gt/gt mice.  We aim to determine the binding profile of SFMBT2 in TSCs and how it contributes to the observed transcriptomic profile.

overall_design: Trophoblast stem cells derived from C57Bl6/Castaneus mice were cultured as described (Tanaka S et al., 1998).  A stable FLAG-SFMBT2 TSC line was generated through lentiviral transfection using Polybrene transfection reagent.  Endogenous SFMBT2 and FLAG-SFMBT2 ChIP-seq samples were isolated from wildtype and a stably transfected FLAG-SFMBT2 TSC cell line respectively.  To reduce cellular differentiation of TSCs resulting from dissociation of colonies, the weight of 10^7 TSCs was calculated and all subsequent TSCs to be used for ChIP-seq were aliquoted into tubes of 0.23 g.  All TSCs were crosslinked and quenched prior before they were aliquoted.  ChIP-seq fragmentation was achieved through either sonication (Raga-Iglesias et al., 2011) or micrococcal nuclease digestion (Tsankov AM et al., 2015).  To reduce technical variability between samples, all subsequent steps post-immunoprecipitation was performed as published by Raga-Iglesias et al (2011).  Of note, FLAG-SFMBT2 crosslinked complexes were immunoprecipitated using FLAG-conjugated magnetic beads (Sigma Aldrich #M8823), while endogenous complexes are immunoprecipitated using protein G Dynabeads (ThermoFisher Scientific #100009D) coated with anti-SFMBT2 generated inhouse (Miri K et al., 2013).  To reduce phenol contamination, aqueous fractions were heated at 55oC for 5 mins then cooled to room temperature for 2 mins prior to the ethanol-based DNA precipitation.  DNA quantification was performed using PicoGreen and 10 ng of input and ChIP DNA was submitted to The Centre for Applied Genomics (TCAG) for library preparation and next-generation sequencing.  All samples were prepared in duplicates except for input DNA.  Of note, the sonicated endogenous SFMBT2 ChIP and input DNA library was prepared using the Illumina TruSeq protocol and subject to paired-end sequencing (2 x 100 bp) at an earlier date on the Illumina HiSeq 2500 platform.  All other DNA libraries were prepared using the NEB Ultra DNA protocol to account for the low DNA concentration in micrococcal nuclease-digested samples, and subject to paired-end sequencing (2 x 126 bp) on the Illumina HiSeq 2500 platform.

first: Priscilla

last: Tang

first: Kamelia

last: Miri


title: The role of MLL1 in post natal myogenesis

city: Ottawa

first: Theodore

last: Perkins

state: ON

address: 501 Smyth Rd.

department: Cellular and Molecular Medicine

institute: Ottawa Hospital Research Institute

summary: The trithorax H3K4 histone methyltransferase (HMT) MLL1 has important roles for early embryonic development, hematopoiesis and neurogenesis through regulation of Hox and homeodomain factor expression. MLL1 has been previously implicated in activation of Myf5 expression through an interaction with Pax7. Here, we find that in vivo, MLL1 is necessary for efficient muscle regeneration, and for maintenance of muscle stem and progenitor cells. Loss of MLL1 in cultured myoblasts reveals an essential role for proliferation and expression of both Myf5 and Pax7. Loss of Myf5 is conditional on loss of Pax7 and exogenous Pax7 rescues Myf5 in the absence of MLL1 suggesting a role for MLL1 in Pax7 expression but not Pax7 activity. Importantly, Mll1 knockout results in a minor decrease to H3K4me3 at Pax7, a 40% decrease in Pax7 mRNA and an 85% decrease in Pax7 protein, suggesting a previously proposed non-HMT role for MLL1 may involve linking transcription to translation.

overall_design: For in-vitro analysis of loss of MLL1, myoblasts were isolated from Mll1fl/fl mice (Jude et al., 2007) kindly provided by Patricia Ernst, crossed with Rosa26-CreERT2 mice (Seibler et al., 2003). Myoblast line was established in culture for 10 days followed by treatment to excise exon 2 of MLL1 for knockout. Control and MLL1 KO samples are from the same cell line treated or not treated with 4-OHT.

first: Gregory

last: Addicks

first: Michael

last: Rudnicki


title: RNA-seq of three Ewing sarcoma cell lines (A673, SK-N-MC, RDES), transfected with either siControl or siMYBL2.

first: Thomas

middle: Georg Philipp

last: Grunewald

address: Im Neuenheimer Feld 280

department: Division of Translational Pediatric Sarcoma Research

institute: German Cancer Research Center (DKFZ)

summary: Deciphering principles of inter-individual tumor heterogeneity is essential for refinement of personalized anti-cancer therapy. Unlike cancers of adulthood, pediatric malignancies including Ewing sarcoma feature a striking paucity of somatic alterations except for pathognomonic driver-mutations that cannot explain overt variations in clinical outcome. Here we demonstrate in the Ewing sarcoma model how cooperation of a dominant oncogene and regulatory variants determine tumor growth, patient survival and drug response. We show that binding of the oncogenic EWSR1-FLI1 fusion transcription factor to a polymorphic enhancer-like DNA element controls transcription of MYBL2, whose high expression promotes poor patient outcome via activation of pro-proliferative signatures. Genetic interference with this regulatory element almost abolished MYBL2 transcription, and MYBL2 knockdown decreased proliferation and tumorigenicity of Ewing sarcoma cells. Combined RNA- and ChIP-seq experiments identified CCNF, BIRC5 and AURKB as direct MYBL2 targets and critical mediators of its phenotype. In drug-response experiments high MYBL2 levels sensitized Ewing sarcoma cells for inhibition of its activating cyclin dependent kinase, CDK2, in vitro and in vivo, suggesting MYBL2 as a predictive biomarker for targeted anti-CDK2-therapy.Collectively, our findings establish cooperation of somatic mutations and regulatory variants as a major determinant of tumor progression and indicate the importance of integrating the regulatory genome in the process of developing new diagnostic and/or therapeutic strategies to fully harness the potential of precision medicine.

overall_design: Three Ewing sarcoma cell lines (A673, SK-N-MC, RDES), transfected with either siControl or siMYBL2. Data contain three replicates of each condition.

first: Julian

last: Musa

first: Marie-Ming

last: Aynaud

first: Sandrine

last: Grossetête-Lalami

first: Olivier

last: Mirabeau

last: Delattre

last: Grünewald


title: Cooperation of dominant oncogenes with regulatory variants shapes clinical outcomes in pediatric cancer


title: Serum-free media supplements carry miRNAs that co-purify with extracellular vesicles (EVs)

status: Public on Aug 15 2019

first: Emil

last: Karaulanov

address: Ackermannweg 4

institute: Institute of Molecular Biology

type: Non-coding RNA profiling by high throughput sequencing

summary: With the goal to identify miRNAs and other small RNAs involved in EV-mediated oligodendrocyte-neuron communication, we performed deep sequencing (RNA-seq) of small RNAs derived from EVs isolated from culture supernatants of primary mouse oligodendrocytes by differential centrifugation.  Oligodendrocytes were cultured and EVs harvested under serum-free conditions in presence of B27 supplement. We found that RNA isolated from EVs harvested from cells under serum-replacement conditions contains miRNA contaminants carried into the sample by defined media components.

overall_design: Small RNA expression profiling of extracellular vesicles (EVs) derived from mouse oligodendrocytes

first: Martin

last: Auber

first: Dominik

last: Fröhlich

first: Oliver

last: Drechsel

first: Eva-Maria

last: Krämer-Albers


title: ChIP-seq of A673 Ewing sarcoma cells using a specific anti-MYBL2 antibody


title: Intestinal endocrine subtype specification analyzed by Single Cell RNA-Seq in mouse enteroids/mini-guts.

city: Illkirch-Graffenstaden

first: Gérard

last: Gradwohl

address: 1 rue Laurent Fries

country: France

institute: IGBMC

summary: The goal of this experiment is to decipher the mechanism of intestinal endocrine / enteroendocrine subtype specification, that is far from being fully understood. To this end, we used single cell genomics in mouse mini-guts. Enteroendocrine cells are derived from endocrine progenitors expressing the transcription factor Ngn3. We took advantage of the Ngn3+/eYFP mouse model -where endocrine progenitors and their descendants can be isolated and sorted by FACS on the basis of the eYFP fluorescence- and established enteroids or mini-guts from the small intestine. Enteroids were dissociated and eYFP+ single cells were directly sorted in 96 wells of the Precise WTA Single Cell Encoding Plate (BD™ Precise WTA Single Cell Kit, BD Genomics). cDNA and libraries were prepared following the BD protocol. Sequencing (paired-end, 2x100b) was performed in a HiSeq 4000 (Illumina). The bioinformatic analysis allowed to identify 8 different groups of enteroendocrine cells with specific signatures.

overall_design: Cells from Ngn3eYFP mice enteroids were sorted and captured. Their transcriptomes were analyzed by single-cell RNASeq.

first: Constance

last: Vagne

first: Florence

last: Blot

first: Christelle

last: Thibault

first: Adèle

last: De Arcangelis


title: Identification of Rfx6-dependent genes in the adult mouse ileon by RNAseq

summary: The goal of this experiment was to identify Rfx6-regulated genes in the ileum of adult mice by comparative transcriptome studies, 8 days and 3 months after Rfx6 removal .

overall_design: Rfx6f/f; Vilin-CreERT2 referred as Rfx6ΔAdIntmice were generated. Rfx6 was deleted by tamoxifen treatment. Ileum of mutant (Rfx6f/f; Vilin-CreERT2) and control mice (Rfx6f/+, or Rfx6f/f) were harvested 8 days and 3 months after tamoxifen treatment and RNA extracted.

first: Julie

last: Piccand

first: Aline

last: Meunier

first: Céline

last: Keime

last: de Arcangelis


title: Identification of Rfx6-regulated genes in mouse embryonic small intestine endocrine cells by microarrays

summary: To identify genes regulated by Rfx6 in the mouse small intestine endocrine cells we performed microarrays gene profiling

overall_design: To identify genes regulated by Rfx6 in small intestine endocrine cells we generated mice with an endocrine-specific deletion of Rfx6. Therefore we crossed Rfx6f/f mice (Piccand et al. Cell Reports 2014) with Ngn3-Cre mice (Takakura et al. Dev. Biol. 2004). The small intestine of control (Rfx6f/+; Rfx6f/f) or mutant (Rfx6f/f, Ngn3-Cre) mice were harvested at embryonic stage E18.5.

first: Anthony

last: Beucher

first: Doulaye

last: Dembele


title: Identification of Rfx6-regulated genes in mouse embryonic small intestine by microarrays

summary: To identify genes regulated by Rfx6 in the mouse small intestine, we performed microarrays gene profiling

overall_design: To identify genes regulated by Rfx6 in small intestine we generated Rfx6-/- mice. First, we crossed Rfx6f/+ mice (Piccand et al. Cell Reports 2014) with CMV-Cre mice to generate Rfx6+/- heterozygotes. Second heterozygotes were crossed to generate Rfx6-/- homozygotes. The small intestine of wild-type or Rfx6-/- mice were harvested at embryonic stage E18.5.


title: Identification of zinc and Zur-regulated genes in Corynebacterium diphtheriae

city: Silver Spring

first: Michael

last: Schmitt

state: MD

address: 10903 New Hampshire Ave

institute: US Food and Drug Administration

summary:
We assessed changes in gene expression in response to zinc availability for the human pathogen Corynebacterium diphtheriae. Expression profiles of wild-type C. diphtheriae strain 1737 grown in semi-defined metal-poor media (mPGT) without and with 5 µM zinc chloride supplementation were compared; the expression profile of wild-type C. diphtheriae strain 1737 in zinc-replete conditions was also compared against an isogenic Δzur mutant grown in zinc-replete conditions. Three biological replicates were prepared by isolating total RNA from mid-logarithmic growth cultures and eleven genes (dip0013, dip0093, dip0173, dip0438, dip1087, dip1486, dip1724, dip2114, dip2128, dip2162, and dip2325) were assessed by real-time PCR to validate the array results.
(Abstract) Corynebacterium diphtheriae is a Gram-positive bacterial pathogen and the causative agent of diphtheria, a severe disease of the upper respiratory tract of humans. Factors required for C. diphtheriae to survive in the human host are not well defined, but likely include the acquisition of essential metals such as zinc. In C. diphtheriae, zinc-responsive global gene regulation is controlled by the Zinc Uptake Regulator (Zur), a member of the Fur-family of transcriptional regulators. In this study, we use transcriptomics to identify zinc-regulated genes in C. diphtheriae by comparing gene expression of a wild-type strain grown without and with zinc supplementation. Zur-regulated genes were identified by comparing wild-type gene expression with that of an isogenic zur mutant. We observed zinc repression of several putative surface proteins, the heme efflux system hrtBA, various ABC transporters, and the non-ribosomal peptide synthetase/polyketide synthase cluster sidAB. Furthermore, increased gene expression in response to zinc was observed for the alcohol dehydrogenase, adhA. Zinc and Zur regulation were confirmed for several genes by complementing the zur deletion and subsequent qPCR analysis. We used MEME to predict Zur binding sites within the promoter regions of zinc- and Zur-regulated genes, and verified Zur binding by electrophoretic mobility shift assays. Additionally, we characterized cztA (dip1101), which encodes a putative cobalt/zinc/cadmium efflux family protein. Deletion of cztA results in increased sensitivity to zinc, but not to cobalt or cadmium. This study advances our knowledge of changes to Zur-dependent global gene expression in response to zinc in C. diphtheriae. The identification of zinc-regulated ABC transporters herein will facilitate future studies to characterize zinc transport in C. diphtheriae.

overall_design: Total RNA harvested from mid-logarithmic cultures of Corynebacterium diphtheriae strain 1737 (wild type and an isogenic zur mutant) with no (wild type only) and 5 µM zinc chloride (both strains) supplementation was assessed for differences. Three separate cultures were used per strain and condition for a total of nine samples.

first: Eric

middle: D

last: Peng

middle: P


title: Complex responses to hydrogen peroxide and hypochlorous acid by the probiotic bacterium Lactobacillus reuteri

city: Birmingham

first: Alexander

last: Rosenberg

state: AL

address: 1720 2nd Avenue South, Suite 276/11

institute: University of Alabama at Birmingham

summary: Inflammatory diseases of the gut are associated with increased intestinal oxygen concentrations and high levels of inflammatory oxidants, including hydrogen peroxide (H2O2) and hypochlorous acid (HOCl), which are antimicrobial compounds produced by the innate immune system. This contributes to dysbiotic changes in the gut microbiome, including increased populations of pro-inflammatory enterobacteria (Escherichia coli and related species) and decreased levels of health-associated anaerobic Firmicutes and Bacteroidetes. The pathways for H2O2 and HOCl resistance in E. coli have been well-studied, but little is known about how commensal and probiotic bacteria respond to inflammatory oxidants. In this work, we have characterized the transcriptomic response of the anti-inflammatory, gut colonizing Gram-positive probiotic Lactobacillus reuteri to both H2O2 and HOCl. L. reuteri mounts distinct responses to each of these stressors, and both gene expression and survival were strongly affected by the presence or absence of oxygen. Oxidative stress response in L. reuteri required several factors not found in enterobacteria, including the small heat shock protein Lo18, polyphosphate kinase 2, and RsiR, an L. reuteri-specific regulator of anti-inflammatory mechanisms. These results raise the intriguing possibility of developing treatments for inflammatory gut diseases that could sensitize pro-inflammatory enterobacteria to killing by the immune system while sparing anti-inflammatory, health-associated species.

overall_design: Triplicate mid-log phase cultures of Lactobacillus reuteri were treated with sublethal doses of H2O2 (0.12 mM) and HOCl (1.25 mM) and RNA-seq was used to assess gene expression at 0, 5, 15, and 30 minutes after treatment

first: Poulami

last: Basu Thakur

first: Abagail

last: Long

first: Benjamin

middle: J

last: Nelson

first: Ranjit

last: Kumar

last: Gray


title: The mutational landscape of a Prion-like domain

status: Public on Aug 14 2019

city: barcelona

first: benedetta

last: bolognesi

state: Barcelona

address: c comte borrell 232 5a-1a

country: Spain

institute: IBEC

summary: Specific insoluble protein aggregates are the hallmarks of many neurodegenerative diseases. For example, cytoplasmic aggregates of the RNA-binding protein TDP-43 are observed in 97% of cases of Amyotrophic Lateral Sclerosis (ALS). However, whether the protein aggregates themselves or other forms of the proteins are toxic to cells is still a very open question for many of these diseases. Here we address this question for TDP-43 by systematically mutating the protein and quantifying the effects on cellular toxicity.  In a dataset of >50,000 mutations in the intrinsically disordered prion-like domain (PRD), changes in hydrophobicity and aggregation potential are highly predictive of changes in toxicity. Surprisingly, however, increased hydrophobicity and cytoplasmic aggregation reduce toxicity. Mutations have their strongest effects in a central region of the PRD, with variants that increase toxicity promoting the formation of more dynamic liquid-like condensates. Moreover, the genetic interactions in double mutants indicate that specific secondary structures form in this region and have detectable effects on aggregation and toxicity in vivo. Our results demonstrate that deep mutagenesis is a powerful approach for probing the sequence-function relationships of intrinsically disordered proteins, as well as their in vivo structural conformations.  Moreover, they reveal that aggregation of TDP-43 is not toxic but actually protects cells, most likely by titrating the protein away from a toxic liquid-like phase.

overall_design:
Systematic measurment of the toxic effects of 50.000 mutations in the region 290-373 of TDP-43
Input 5-8 are biological replicates for library TDP aa 290-331; Input 1-4 are biological replicates for library  TDP aa 332-373. Each input corresponds to DNA extracted from cells growing after transformation and without expression of TDP-43. Outputs named with the same number are technical replicates and they originate from the same input. Each output corresponds to DNA extracted from cells growing after having expressed TDP-43 for roughly 6 generations.

first: Benedetta

last: Bolognesi

first: Ben

last: Lehner


title: Impact of G-content on Arabidopsis AtPAB binding and their role in enhancing translational efficiency

city: Beijing

first: xiaofeng

last: cao

address: West Lincui Road, Chaoyang District

department: State Key Laboratory of Plant Genomics

institute: Institute of Genetics and Developmental Biology

summary: Polyadenylation plays a key role in producing mature mRNAs in eukaryotes. It is widely believed that the poly(A)-binding proteins (PABs) uniformly bind to poly(A)-tailed mRNAs, regulating their stability and translational efficiency. Here, we characterized the RNA-binding landscape of three broadly expressed PABs in Arabidopsis thaliana. We observed substantial variation in the AtPAB-binding efficiency among mRNAs, which can be partly explained by the guanosine (G) content of poly(A) tails. AtPAB-binding efficiency of a gene was positively associated with translational efficiency rather than mRNA stability. Consistently, genes with stronger AtPAB binding exhibited a greater reduction in translational efficiency when AtPAB is depleted. Our study provides a new mechanism that translational efficiency of a gene can be regulated through G-content-dependent PAB binding, paving the way for a better understanding of poly(A) tail-associated regulation of gene expression.

overall_design: CLIP-seq and ribosome profiling were used to study direct binding genes and the functions on translational regulation of AtPABs. Poly(A)-seq was used to detect the mRNA tail information in Col. Degradtion data was used to detect mRNA degradation rate in Col.

first: Taolan

last: Zhao

first: Jing

last: Sun

first: Chunyan

last: Liu

first: Qing

last: Huan

first: Wenfeng

last: Qian

first: Xiaofeng

last: Cao


title: Addressing the repertoire of T helper Cells in the context of myocardial infarction

city: Würzburg

first: Gustavo

middle: Campos

last: Ramos

address: AM Schwarzenberg, 15

department: DZHI

institute: Universitätsklinikum Würzburg

summary: T-cells can influence the post-myocardial infarction healing process. The fundamental role of T cells in adaptive immunity relies mainly on a diverse repertoire of T cell receptor (TCR) α and β chains that display unique antigen specificities. Thus, we performed high-throughput sequencing of the TCRβ chains on CD4+ T cells purified by fluorescence-activated cell sorting (FACS) from the heart and med-LNs of MI and sham-operated mice as an unbiased approach to screen for MI-related TCR signatures.

overall_design: Experimental myocardial infarction (EMI) was induced in Balb/c WT mice by means of ligating the left anterior descending coronary artery. As a sham-operation control, animals were subjected to thoracotomy without coronary ligation. CD4+ T-cells were purified (FACS) from the mediastinal lymph nodes (med-LN, heart-draining) of from the infarcted heart at day 7 post-operation. In addition, we assessed the TCRb repertoire of CD4+ T cells purified from the axillary LNs (ax-LNs) of mice receiving subcutaneous immunization with cardiac myosin and heart extract (in adjuvants), as these repertoires were expected to be enriched in heart-specific sequences upon specific immunization. As a control, we also assessed the ax-LN TCRb repertoire in mice that received subcutaneous administration of the adjuvant alone.

first: Max

last: Rieckmann

first: Ulrich

last: Hofmann


title: NAPRT activation of macrophages

status: Public on Aug 13 2019

city: Groningen

first: Danny

last: Incarnato

state: Netherlands

address: Nijenborgh 7

department: Molecular Genetics

country: Netherlands

institute: University of Groningen

summary: Damage-associated molecular patterns (DAMPs) are molecules that can be actively or passively released by injured tissues and that activate the immune system. Here we show for the first time that the NAD biosynthetic enzyme nicotinate phosphoribosyltransferase (NAPRT), the rate-limiting enzymes in the intracellular synthesis of NAD from nicotinic acid, is physiologically present in human sera, where it acts as a novel DAMP. We detected NAPRT in plasma/sera from human donors by antibody-mediated luminex assays and mass spectrometry. Exposure of human and mouse macrophages to NAPRT triggers activation of ERK1/2, phosphorylation of IKKα/β and nuclear translocation of the p65 subunit of the NF-kB complex, with synthesis and secretion of inflammatory cytokines, including IL-1β, IL-8, TNFα and CCL3. Furthermore, NAPRT enhances monocyte differentiation into macrophages, by inducing macrophage colony-stimulating factor. These effects are independent of the NAPRT catalytic activity, but rely on the protein’s binding to TLR4, as demonstrated by showing direct in vitro interaction and by the in vivo lack of NAPRT effects in TLR4-/- macrophages. In line with the finding that NAPRT mediates endotoxin tolerance, sera from patients with sepsis or septic shock contain the highest levels of circulating NAPRT, compared to other chronic inflammatory conditions, including cancer. Importantly, patients with serum NAPRT >15 ng/ml are characterized by a worse clinical outcome, compared to the counterparts. Together, these data identify NAPRT as a novel endogenous ligand for TLR4 and a critical mediator of inflammation.

overall_design: Transcriptome analysis of human macrophages treated (6 hours) with rNAPRT, rNAMPT (1µg/ml) or LPS (2 µg/ml; Sigma-Aldrich)

first: Antonella

last: Managò

first: Valentina

last: Audrito

first: Francesca

last: Mazzola

first: Leonardo

last: Sorci

first: Federica

last: Gaudino

first: Katiuscia

last: Gizzi

first: Nicoletta

last: Vitale

first: Gabriele

last: Minazzato

first: Alice

last: Ianniello

first: Antonio

last: Varriale

first: Sabato

last: D'Auria

first: Giulio

last: Mengozzi

first: Gianfranco

last: Politano

first: Salvatore

last: Oliviero

first: Nadia

last: Raffaelli

first: Silvia

last: Deaglio


title: Rfx6-regulated genes in the mouse intestine

first: Gérard

last: Gradwohl

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

overall_design: Refer to individual Series


title: Gene expression analysis of mouse BNST and blood cells after chronic social defeat stress (CSDS)

city: Helsinki

first: Iiris

last: Hovatta

address: Haartmaninkatu 3

department: Department of Psychology and Logopedics

country: Finland

institute: University of Helsinki

summary: Chronic psychosocial stress is a risk factor for psychiatric disorders, and genetic factors interact in conferring susceptibility. The chronic social defeat stress (CSDS) mouse model allows identifying factors underlying resilience and susceptibility to chronic psychosocial stress. Following 10 days of CSDS, we collected the brain and blood tissues. We  performed (1) AGO2 RNA immunoprecipitation-sequencing (AGO2 RIP-seq) of active miRNAs and their mRNA targets in the BNST. Additionally, we carried out RNA-seq and miRNA-seq from blood cells.

overall_design: We performed ribo-zero globin depletion of RNA from blood cells. Immunoprecipitation of AGO2-bound miRNAs and their target mRNAs was done by pooling of 3 BNST collected from all the phenotypic groups in both strains. We sequenced with Illumina Next-seq 500 from (1) BNST AGO2 mRNA (C57BL/6Crl: control n = 5, resilient n = 5, susceptible n = 4), (2) BNST AGO2 miRNA (C57BL/6Crl: control n = 5 , resilient n =5 , susceptible n = 3), (3) blood cels mRNA (C57BL/6Crl: control n = 5, resilient n = 5, susceptible n = 5; DBA/2Crl: control n = 5, susceptible n = 5), (4) blood cels miRNA (C57BL/6Crl: control n = 5, resilient n = 5, susceptible n = 5; DBA/2Crl: control n = 5,  susceptible n = 5) 6-8 days after the last CSDS.

first: Zuzanna

last: Misiewicz


title: Germline-Encoded Affinity for Cognate Antigen Enables Vaccine Amplification of a Human Broadly Neutralizing Response Against Influenza Virus

city: Cambridge

first: Maya

last: Sangesland

state: MA

address: 400 Technology Square

institute: Ragon Institute of MGH, MIT, and Harvard

summary: Antibodies display antigen-binding loops comprised of a hypervariable CDRH3 and V gene-encoded CDRs, where the latter may harbor gene-endowed or ‘public’ targeting solutions. To test this, we generated transgenic mice containing human CDRH3 diversity but simultaneously constrained to individual human immunoglobulin VH genes, including IGHV1-69, which shows biased usage in human broadly neutralizing antibodies (bnAbs) targeting the hemagglutinin stalk of Group 1 influenza A viruses. We then rank-ordered the VH-contribution to bnAb-epitope targeting following exposure to viral and nanoparticle displays of hemagglutinin. Notably, sequential immunization with a stalk-only hemagglutinin nanoparticle elicited Group 1 bnAbs, but only in IGHV1-69 constrained mice. This response required minimal affinity maturation, could be elicited under pre-existing influenza immunity, and when IGHV1-69 B cells were diluted to match the frequency measured in humans. Thus, encoded CDRs can naturally endow for specific antibody targets, and a rationally designed immunogen may elicit human influenza bnAbs through this principle.

overall_design: Single cell and bulk BCR sequences from human IGHV1-69 restricted, human IGHV1-2 restricted, and WT C57BL/6 mice generated by deep sequencing using Illumina Miseq.

first: Samuel

last: Kazer

first: Daniel

last: Lingwood


title: Single-cell RNAseq characterization of anatomically-identified OLM interneurons in different transgenic mouse lines

city: Zurich

first: Csaba

last: Földy

state: Zurich

address: Winterthurerstrasse 190

department: Brain Research Institute

institute: University of Zurich

summary: Inhibitory GABAergic interneurons create different brain activity patterns that correlate with behavioral states. In this characterizing study, we used single-cell RNAseq to analyze anatomically- and electrophysiologically-identified hippocampal oriens-lacunosum moleculare (OLM) interneurons. OLMs express somatostatin (Sst), generate feedback inhibition, and play important roles in theta oscillations and fear encoding. Although an anatomically- and biophysically-homogenous population, OLMs comprise two functionally distinct types with presumed different developmental origins, inferred from the expression pattern of serotonin type-3a (5-HT3a, or Htr3a) receptor subunit and 5-HT-excitability in a set of OLMs. To broadly characterize OLM cells, we used the Sst-Cre and the BAC-transgenic Htr3a-Cre mouse lines and separately analyzed SstCre-OLM and Htr3aCre-OLM types. We found a surprisingly consistent expression of Npy in OLMs, which was previously not associated with the identity of this type. Our analyses furthermore revealed uniform expression of developmental origin-related genes, including transcription factors and neurexin isoforms, without providing support for the current view that OLMs may originate from multiple neurogenic zones. Together, we found that OLMs constitute a highly homogenous transcriptomic population. Finally, our results revealed surprisingly infrequent expression of Htr3a in only ~10% of OLMs, and an apparently specific expression of the 5-HT3b subunit-coding gene Htr3b in Htr3aCre-OLMs, but not in SstCre-OLMs. However, additional in situ hybridization experiments suggested that the differential expression of Htr3b may represent an unexpected consequence arising from the design of the Htr3a-Cre BAC transgenic line.

overall_design: mRNA profiles of 46 OLM cells (23 Sst line, 23 Htr3a line)

first: Jochen

last: Winterer

first: David

last: Lukacsovich

first: Lin

last: Que

first: Andrea

last: Sartori

first: Wenshu

last: Luo


title: Transcriptomic Signatures of Postnatal and Adult Intrinsically Photosensitive Ganglion Cells

city: Palo Alto

last: Berg

address: 1050 Arastradero Road

institute: Stanford University

summary:
Purpose: The goal of this study was to determine a “molecular parts list” for intrinsically photosensitive retinal ganglion cells (ipRGCs), which are rare mammalian photoreceptors essential for non-image-forming vision functions, such as circadian photoentrainment and the pupillary light reflex.
Methods: We conducted a thorough unbiased transcriptomic analysis of ipRGCs by purifying GFP-tagged ipRGCs through a combination of FACS and immunoaffinity and comparing this the transcriptional profile of GFP-negative RGCs. We did this in two different mouse lines, marking partially overlapping subsets of ipRGCs. One was a BAC transgenic reporter (Opn4-GFP), which fluorescently labels M1-M3 ipRGCs and the other was a Opn4-Cre;Z/EG reporter system, which labels all ipRGC subtypes, M1-M6
Results: We find dozens of novel genes highly enriched in ipRGCs. We reveal that Rasgrp1 and Tbx20 are selectively expressed in subsets of ipRGCs, though these molecularly defined groups imperfectly match established ipRGC subtypes. We demonstrate that the ipRGCs regulating circadian photoentrainment are unexpectedly diverse at the molecular level.
Conclusions: Our study represents the first detailed analysis of ipRGC transcriptomes by RNA-seq. Our findings reveal unexpected complexity in gene expression patterns across mammalian ipRGC subtypes.

last: Berson


title: ChIP-exo and ChIP-seq of human KRAB-ZNFs transduced in HEK 293T cells

status: Public on Aug 12 2019

city: Lausanne

first: Julien

last: Duc

state: VAUD

address: Station 19 CH-1015 Lausanne

department: School of Life Science

institute: EPFL

summary: Kruppel-associated box (KRAB)-containing zinc-finger proteins (KZFPs) represent the largest family of transcription factors encoded by higher vertebrates. Together with their heterochromatin-inducing cofactor KAP1, many act as repressors of endogenous retroelements (EREs) during early embryonic genome reprogramming, yet their widespread expression in adult tissues and enrichment at other genetic loci indicate additional roles. By characterizing the protein interactome of 101 of the ~350 human KZFPs, we uncovered that it is highly diversified for a subset of those. Most of placental mammal- or primate-restricted KZFPs, which primarily bind EREs and constitute 90% of human KZFPs, essentially recruit KAP1 and effectors. However, certain KZFPs conserved in placental mammals and more distant clades associated with factors related to functions such as genome architecture or RNA processing. Additionally, members of the most conserved human KZFPs did not appear to interact with KAP1. Nevertheless, KZFPs from coelacanth, our most distant KZFP-encoding relative, bind KAP1 from this species. We thus propose that KZFPs first emerged as ERE-controlling repressors, had their pool continuously renewed by turn-over of their hosts’ ERE loads, thereby yielded largely species-specific sets of transcriptional controllers, yet occasionally produced derivatives that escaped this evolutionary flushing by development of novel functions and exaptation.

overall_design: We transduced HEK 293T cells with doxycycline-inducible plasmids encoding for human KRAB-ZFPs fused with 3 HA tags at the C-terminus. We next performed ChIP-exo and ChIP-seq and prepared a barcoded Illumina library for each sample. For the ChIP-exo, the libraries were pooled and sequenced 12 per lane on an Illumina HiSeq 2500 to a minimum depth of around 15 million 100 bp single-end reads. For the ChIP-seqs, all samples were pooled and sequenced on the same lane on an NextSeq 500 to a minimum depth of around 27 million 85 bp single-end reads. The reads were mapped to the hg19 assembly of the human genome and peaks were identified using MACS. For the ChIP-exos, we used a total input derived from random sampling from all samples in the study, as it was found to perform better than either the standard total input or the exonuclease treated total input. For the ChIP-seqs, we used a total input sample that had a matching sonication profile to the IP sample.

first: Pierre-Yves

last: Helleboid

first: Moritz

last: Heusel

first: Michaël

last: Imbeault

first: Ruedi

last: Aebersold

first: Didier

last: Trono


title: CDK5-dependent phosphorylation and nuclear translocation of TRIM59 promotes macroH2A1 ubiquitination and tumorigenicity

status: Public on Aug 11 2019

city: Shanghai

first: Haizhong

last: Feng

state: Shanghai

address: Pujian Road 160

department: Ren Ji Hospital

institute: Shanghai Jiao Tong University

summary: Despite the development of adjuvant therapies, glioblastoma (GBM) patients remain incurable and justify the urgent need of new therapies. CDK5 functions a critical role in GBM and is a potential target for GBM. However, the mechanism by which CDK5 promotes GBM tumorigenicity remains largely unknown. Here we identify TRIM59 as a substrate of CDK5. EGFR-activated CDK5 directly binds to and phosphorylates TRIM59, a ubiquitin ligase at serine 308, which recruits PIN1 for cis-trans isomerization of TRIM59, leading to TRIM59 binding to importin a5 and nuclear translocation. Nuclear TRIM59 induces ubiquitination and degradation of the tumor suppressive histone variant macroH2A1, leading to enhanced STAT3 signaling activation and tumorigenicity. These findings are confirmed by inhibition of CDK5-activated TRIM59 activity that results in suppression of intracranial tumor growth. Correlative expressions of the components of this pathway are clinically prognostic. Our findings suggest targeting CDK5/TRIM59 signaling axis as a putative strategy for treating GBM.

overall_design: Examing of erlotinib and roscovitine on EGFRvIII-mediated macroH2A1 binding in genome wide.


title: Identify signature genes associated with nanoparticle carrying IRF5 mRNA treatment in bone marrow derrived macrophages

status: Public on Aug 09 2019

city: Seattle

first: Matthias

last: Stephan

state: WA

address: 1100 Fairview Ave. N., Thomas Research Buildin

institute: Fred Hutchinson Cancer Research Institute

summary: To identify signature genes associated with nanoparticles carrying IRF5 mRNA treatment on primary macrophages, we analyzed 770 genes in 19 different pathways and processes across 7 different myeloid cell types.

overall_design: In the study presented here,  we performed genome analysis on (1) M2-like(preincubated with IL4) and (2) M1-like macrophage standards (preincubated with  MPLA), (3) IRF5-NPs and (4) eGFP-NPs treated M2-like macrophages,

first: Michael

last: Coon

first: Fan

last: Zhang


title: Transcriptomics of human macrophages upon infection with Legionella pneumophila and two isogenic mutants, LamA and AnkH using RNA-seq

city: Louisville

first: Yousef

last: Abu Kwaik

state: KY

address: 505 S Hancock St

department: Microbiology and Immunology

institute: University of Louisville

summary: The objective of this study is to determine changes in the global transcriptome of human macrophages upon infection with Legionella pneumophila and two isogenic mutants, LamA and AnkH

overall_design: Human macrophages were infected with wild type L. pneumophila or its isogenic mutant AnkH, for 6h. Following 6h infection, total RNA was isolated and subjected to RNAseq analysis.

first: Christopher

last: Price

first: Juanita

last: Von Dwingelo

first: Eric

last: Rouchka


title: Gene expression profile of myosin-specific CD4+ T-cells activated after myocardial infarction

first: Gustavo

last: Ramos

summary: Pathological expansion of self-reactive T cells is a hallmark of autoimmune diseases. Notwith-standing, T cell autoreactivity can also benefit self-maintenance and foster tissue repair. Herein, we investigated whether heart-specific T cells exert salutary or detrimental effects in the context of the post myocardial infarction (MI) healing process in mice, and whether the findings can be translated to humans. Transferred cardiac-myosin-specific CD4+ T cells (TCR-M) selectively accumulate in the myocardium and mediastinal lymph nodes (med-LNs) of infarcted mice. TCR-M cells activated in the MI-context acquire a Foxp3+ regulatory phenotype, exhibit a distinct gene expression profile enriched with growth factors, and promote cardioprotection. Massive parallel sequencing of T cell receptors revealed that CD4+ T cells infiltrating the infarcted myo-cardium display a unique repertoire signature with skewed diversity and dominated by a limited set of expanded clones - hallmarks of antigen-specific responses. CD4+ Foxp3+ cells were also detected in myocardial biopsies obtained from patients who suffered MI, especially in associa-tion with the granulation tissue (i.e., healing phase). Noninvasive positron emission tomogra-phy/computed tomography (PET/CT) imaging using a CXCR4 radioligand revealed that MI pa-tients display enlarged med-LNs with increased T cellularity.  Alterations in the med-LN corre-lated with infarct size and cardiac function. Taken together, these results provide strong evidence that the MI-context induces protective T cell autoimmunity in mice, and confirm the existence of an analogous physiological heart/med-LN/T cell axis in MI patients, which offers translational potential.

overall_design: Myosin-specific CD4+ T cells (Thy1.1) were purified from naive TCR-M mice and then transferred into WT syngeneic recipients (male balb/c, 8-12 wk old, Thy1.2) one day prior to MI/sham operation. Transferred cells were later FACS-purified from the mediastinal lymph nodes of infarcted (N=5 animals) and sham-operated (N= 3) mice at 7 d post-operation. Endogenous WT CD4+ T-cells (ENDO) were also purified in parallel from the same animals.

first: Philipp

last: Steinau

first: Max

last: Rieckmann


title: Differential HITS-CLIP of miRNA binding sites in miR-29 deficient CD4 T cells.

city: San Francisco

first: Robin

last: Kageyama

state: California

address: 513 Parnassus Ave

institute: University of California San Francisco

overall_design: AGO HITS-CLIP librarires were generated from wildtype and miR-29 deficient CD4 T cells under various stimulation conditions after crosslinking immunoprecipiation of AGO2.  Libraries were generated with an 11nt index read containing a 5nt barcode and a 6nt random barcode. 2 random nucleotides were added to the 5' end of the read for ligation controls.

first: R

first: K

last: Ansel


title: β-catenin ChIP-seq, RARα ChIP-seq, and ATAC-seq datasets in the standard or altered 2C protocols.

status: Public on Aug 08 2019

city: Bunkyo-ku

first: Manabu

last: Kawata

state: Tokyo

address: 7-3-1 Hongo

country: Japan

institute: The University of Tokyo

summary: We performed β-catenin ChIP-seq, RARα ChIP-seq, and ATAC-seq on the hiPSC-derived cells in the standard or altered 2C differentiation protocols.

overall_design: Human iPS-derived cells in the standard or altered 2C protocols were analyzed in duplicate.


title: Quantitative Analysis of Wild Type and scrib mutant Drosophila wing imaginal discs Transcriptomes at different time points

city: Hong Kong

first: Yan

last: Yan

address: Clear Water Bay

department: LIFS

institute: HKUST

summary:
Purpose: There are two goals of this study. Firstly, we want to figure out the transcriptomic differences between control and scrib¹ mutant wing imaginal discs.  Secondly, we aim to investigate the transcriptomic changes as the scrib¹ mutant tumor develop over time.
Methods: Total RNA was used to generate cDNA libraries and 150bp paired-end sequencing on Illumina HiSeq platform were performed by Novogene. Cleaned raw reads were mapped to the reference genome using STAR and counts are generated by featuresCounts available in Subread package. Count normalization was performed using edgeR.

overall_design: mRNA profiles of 4, 5-day wild type and 4, 5, 6, 8, 10, 12, 14-day scrib mutant wing imaginal discs were generated by Next Generation Sequencing, in triplicate or quadruplicate, using Illumina HiSeq.

first: Tiantian

last: Ji

first: Lina

first: Mingxi

last: Deng


title: Expression data from isolated SHARPIN-deficient thymic Treg cells

city: Kyoto

first: Kazuhiro

last: Iwai

address: Yoshida Konoecho, Sakyo-ku

institute: Kyoto university

summary:
One of the ubiquitin ligases, LUBAC, is a trimetric complex composed of SHARPIN, HOIL-1, and HOIP. LUBAC works at the proximal TCR signaling pathway allowing normal Treg development.
We used microarrays to detail the global programme of gene expression underlying LUBAC-mediated signaling pathway in Treg cells.

overall_design: CD3e+CD4+CD25+Foxp3+ thymicTreg cells were isolated from the pools of splenocytes and lymph node cells in Sharpinfl/fl-Foxp3-Cre or the corresponding control mice. To reveal the difference of gene expression between the two, RNA were extracted from the cells, and hybridized on Affymetrix microarrays. Three experiments were independently performed.

first: Katsuhiro

last: Sasaki


title: Transcriptomic profiles of Pcy and Pdk2KO mice and human kidney epithelial cells treated with RGLS4326

city: San Diego

first: Sole

last: Gatto

state: CA

address: 12636 High Bluff Drive, Suite 400

institute: Monoceros Biosystems

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: Transcriptomic profile of Pcy mice treated with RGLS4326

summary: Kidney global mRNA expression profiles was carried out in the Pcy/CD1 model following RGLS4326 treatment versus PBS-treated controls. We found that RGLS4326 treatment had a significant transcriptome-wide impact and globally de-repressed mRNAs of predicted miR-17 target genes in cystic kidneys of Pcy/CD1 model

overall_design: 5-weeks old mice were randomly assigned and dosed with RGLS4326 (25 mg kg-1) or PBS by SC injections once- monthly (Q4W) for 25 weeks. All animals were euthanized, and kidney harvested at 30 weeks of age. Five untreated male WT/CD1 mice were included for comparison.

first: Tania

last: Valencia

first: Edmund

last: Lee

first: Kara

last: Kersjes


title: Gene expression during chondrocyte differentiation from human iPS cells by the 2C method

city: TOKYO

first: Taku

last: Saito

address: 7-3-1 Hongo, Bunkyo-ku

department: Orthopaedic Surgery, Faculty of Medicine

summary: To identify gene expression profiles during chondrocyte differentiation from human iPS cells by the 2C method

overall_design: human iPS cells during chondrocyte differentiation by the 2C method at day 0, 2, 3, 4, 5, and 9.


title: Transcriptomic profile of human kidney epithelial cells - derived from cysts of ADPKD donors

summary: We utilized primary cyst cultures derived from human ADPKD donors transfected with RGLS4326 to demonstrate the human translational potential of RGLS4326.  Results show that RGLS4326 treatment globally de-repressed mRNAs of predicted miR-17 target genes in primary human ADPDK cyst cells.

overall_design: Primary cyst cultures derived from human ADPKD donors were transfected with RGLS4326 at 100 nM for 24 hrs and harvested for RNA-seq analysis

first: Annelie

last: Schairer


title: RNA-seq on Fixed and Permeabilized Plasma cells

city: Atlanta

first: Chris

last: Scharer

state: GA

address: 1510 Clifton Rd, Suite 3086A

institute: Emory University

summary: Here we developed a protocol to extract total RNA from fixed and permeabilized cells that were intracellularly stained and FACS isolated.  We applied these techniques to derive the transcriptome of influenza- and BCR isotype-specific antibody secreting cells or plasma cells.  These data reveal that ASC isotypes have more distinct transcriptional signatures than previously appreciated.

overall_design: RNAseq on fixed, permeabilized, and sorted antibody secreting cells

first: C

first: J

last: Boss


title: The strand-biased mitochondrial DNA methylome and its regulation by DNMT3A

first: Jing-Dong

middle: Jackie

last: Han

address: 320 Yue Yang Road

institute: CAS-MPG Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

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

overall_design: Refer to individual Series


title: Transcriptomic profile of Pdk2KO mice treated with RGLS4326

summary: Kidney global mRNA expression profiles was carried out in the Pkd2-KO model following RGLS4326 treatment PBS-treated controls. We found that RGLS4326 treatment had a significant transcriptome-wide impact and globally de-repressed mRNAs of predicted miR-17 target genes in cystic kidneys of Pkd2-KO model

overall_design: Pkd2-KO mice were randomly assigned and dosed with RGLS4326 (20 mg kg-1) or PBS by SC injections at post natal day (P)10, 11,12 and 19.   Non-transgenic strain-matched mice were  used for comparison. All animals were euthanized, and kidney harvested at P28 .

first: Vishal

last: Patel


title: Expression profiling of colorectal cancer cell lines

status: Public on Aug 07 2019

city: Daejeon

first: Soobeom

state: Daejeon

address: E16, 709, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea

institute: Korea Advanced Institute of Science and Technology

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

These data are associated with single-cell RNA-seq data available at SRA at accession: SRP217361


title: Genome-wide analysis on the human iPS-derived cells during chondrocyte differentiation by the 2C method

first: Manabu

summary: We performed open chromatin analysis and genome-wide analysis for RAR-DNA association, on the hiPSC-derived cells in the 2C protocol. The purposes were to analyze sequential opening and closing of chromatin elements, and to elucidate the molecular roles of RAR, in each differentiation stage of the 2C protocol.

overall_design: Human iPS-derived cells at day 0, 2, 3, 4, 5, 7 and 9 in the 2C method were analyzed in duplicate.


title: Identification of overlap between KDM3A and FOXA1 regulated transcriptome in the ER positive breast cancer cell line MCF-7

city: Hull

first: Mark

last: Wade

state: Humberside

address: Room 121, Hardy Building, University of Hull, Cottingham Road

country: United Kingdom

institute: University of Hull

summary: To interrogate the extent of overlap between KDM3A and FOXA1 regulated transcriptomes in ER-positive breast cancer cells we assessed global gene expression changes in KDM3A-depleted and FOXA1-depleted MCF-7 cells by microarray analysis using the Illumina Human HT12 Version 4 BeadChip array. We identified that 43% of the KDM3A regulated transcriptome was also regulated by FOXA1 and that 43% of the FOXA1 regulated transcriptome was also regulated by KDM3A. Genes co-regulated by KDM3A and FOXA1 were involved in ER-signalling and the cell growth. We also identified that depletion of KDM3A was required for the deposition of FOXA1 on chromatin.

overall_design: MCF-7 cells were transfected with either a non-silencing scrambled control siRNA (siSCR) a KDM3A targeting siRNA (siKDM3A#1), or a FOXA1 targeting siRNA (siFOXA1#1) using Lipfectamine RNAiMAX (Invitrogen) to a final concentration of 25 nM in steroid depleted conditions. Cells were grown for 48 hours prior to treatment with 10 nM E2 for 6 hours before RNA extraction using TRIzol (Ambion, Life Technologies) and hybridization of triplicate samples to an Illumine HT-12 v4 BeadChip array.

first: Dominic

last: Jones

first: Laura

last: Wilson

first: Huw

last: Thomas

first: Luke

last: Gaughan


title: Bcl11b prevents catastrophic autoimmunity by controlling multiple aspects of regulatory T-cell gene expression program [ATAC-seq]

city: Cheonan

first: Keunsoo

last: Kang

address: 119 Dandae-ro

institute: Dankook University

summary: In order to investigate whether Bcl11b mediates any chormatin accebility in Treg cell, we performed ATAC-Seq from Bcl11b sufficient (WT) and Bcl11b deficient Treg(KO) cells.We used 100,000 cells for each experiment.

overall_design: Genome wide occupancy profiling followed by high throughput sequencing for each conditions (3 replicates for Bcl11b WT Treg, 3 replicates for Bcl11b KO Treg) was done using Illumina HiSeq 2500.

first: Nurul

last: Syed

first: Amit

last: Sharma

first: Sayantani

last: Ghosh

first: Sung-Wook

last: Hong

first: Sinchita

last: Roy-Chowdhuri

first: Sin-Hyeog

last: Im

first: Dipayan

last: Rudra