title: Remodeling of the m6A landscape in the heart reveals conserved post-transcriptional events underlying cardiomyocyte hypertrophy

city: Columbus

first: Federica

last: Accornero

state: OH

address: 473 W 12th Ave.

institute: Ohio State University Medical Center

summary: Regulation of gene expression plays a fundamental role in cardiac stress-responses. Modification of coding transcripts by adenosine methylation (m6A) has recently emerged as a critical post-transcriptional mechanism underlying heart disease. Thousands of mammalian mRNAs are known to be m6A-modified, suggesting that remodeling of the m6A landscape may play an important role in cardiac pathophysiology. Here we found an increase in m6A content in human heart failure samples. We then adopted genome-wide analysis to define all m6A-regulated sites in human failing compared to non-failing hearts and identified targeted transcripts involved in histone modification as enriched in heart failure. Further, we compared all m6A sites regulated in human hearts with the ones occurring in isolated rat hypertrophic cardiomyocytes to define cardiomyocyte-specific m6A events conserved across species. Our results identified 38 shared transcripts targeted by m6A during stress conditions, and 11 events that are unique to unstressed cardiomyocytes. Of these, Coronin-6 (CORO6) was further evaluated for mRNA and protein abundances, demonstrating the potential impact of m6A on post-transcriptional regulation of gene expression in the heart.

overall_design: RNA-seq and ME-RIP-seq analysis of Human heart samples undergoing cardiac hypertrophy

first: Scott

last: Hinger

first: Jiangbo

last: Wei

first: Chuan

last: He


title: Tobacco smoke exposure exacerbates silica-induced pulmonary toxicity in rats

status: Public on Nov 01 2020

city: Morgantown

first: Christina

middle: M

last: Umbright

state: WV

address: 1095 Willowdale Road

department: HELD

country: USA

institute: DHHS/PHS/CDC/NIOSH

type: Expression profiling by high throughput sequencing

summary: Previous studies have shown that smoking induces oxidative stress and inflammation, known factors that coincide with the development and progression of silicosis.  Nevertheless, the precise role of cigarette smoke exposure in silicosis and the underlying mechanisms are not clearly understood. Therefore, the objective of the present study was to determine the effect of smoking, if any, on silica-induced pulmonary response and the underlying mechanisms. Pulmonary toxicity and lung gene expression profiles were determined in male Fischer 344 rats exposed to air, crystalline silica, cigarette smoke or cigarette smoke plus crystalline silica. Silica exposure resulted in significant pulmonary toxicity which was further exacerbated by cigarette smoke exposure in the rats. Significant differences in the gene expression profiles were detected in the lungs of the rats exposed to cigarette smoke, silica or a combination of both compared with the control rats.

overall_design: Lung RNA profiles of rat exposed to air, crystalline silica, cigarette smoke, or cigarette smoke and crystalline silica were generated by deep sequencing, in sextuplate, using Illumina HiSeq.

first: Tina

last: Sager

first: Pius

last: Joseph


title: Comparing the transcriptomes of islets in high fat induced obesity mice and normal mice

city: Not Hispanic or Latino

first: Sheng

last: Zhao

address: #87, Dingjiaqiao, Gulouqu

department: Biochemistry and Molecular biology

country: China

institute: Southeast University

summary: Islets mRNA profiles of 27 weeks high fat induced obesity mice and normal mice were generated by RNA-sequencing, in triplicate, using Illumina

first: Ruifeng

last: Shi


title: Differential gene expression profile of FOLR2+ vs. FOLR2- tumor-associated macrophages

city: Philadelphia

first: Alba

last: Rodriguez Garcia

state: Pennsylvania

address: 3400 civic center boulevard

institute: Upenn

summary:
Tumor-associated macrophages (TAMs) are key components of the tumor microenvironment (TME) which can either promote tumor progression (M2) or induce antitumor immunity (M1). The hypothesis of our study is that the expression of FOLR2 is associated with an M2-like macrophage profile. The main goal of this study is to compare the transcriptomic profile of FOLR2 positive and FOLR2 negative TAMs obtained from the ascites of C57BL/6 mice bearing ovarian ID8 intraperitoneal tumors.
Abstract: The immunosuppressive tumor microenvironment (TME) represents a major barrier for effective immunotherapy. Tumor-associated macrophages (TAMs) are highly heterogeneous and plastic cell components of the TME which can either promote tumor progression (M2-like) or boost antitumor immunity (M1-like). Selective targeting of the pro-tumorigenic subset of TAMs represents an attractive therapeutic strategy. Here, we demonstrate that a subset of TAMs that expressing folate receptor β (FRβ) possess an immunosuppressive, tumor-promoting M2-like profile, while FRβ negative TAMs feature pro-inflammatory M1 macrophage properties. In syngeneic tumor mouse models, the administration of FRβ-targeted chimeric antigen receptor (CAR) T-cells mediated elimination of FRβ+ TAMs in the TME, which resulted in an enrichment of pro-inflammatory monocytes, an influx of endogenous tumor-specific CD8+ T-cells, delayed tumor progression, and prolonged survival. Preconditioning of the TME with FRβ-specific CAR T-cells also improved the effectiveness of tumor-directed anti-mesothelin CAR T-cells, while simultaneous co-administration of both CAR products did not. Thus, CAR T-cell-mediated depletion of immunosuppressive M2-like TAMs incites a pro-inflammatory TME that broadens endogenous antitumor immunity and limits tumor progression, highlighting the pro-tumor role of FRβ+ TAMs in the TME and the therapeutic implications of TAM-depleting agents as preparative adjuncts to conventional immunotherapies that directly target tumor antigens.

overall_design: Examination of 8 samples obtained from 4 different mouse individuals was performed. From each mouse, ID8 ascites were collected and CD11b+ cells isolated by using magnetic beads. TAMs were subsequently stained with antibodies and sorted based on FOLR2 expression by flow. Therefore, 2 samples were obtained from each mouse: a FOLR2 positive and a FOLR2 negative.

last: Rodriguez-Garcia

first: Daniel

middle: J

last: Powell Jr.


title: Discovery and evaluation of CRISPR/Cas-mediated systems for site-specific RNA editing

city: La Jolla

first: Gene

last: Yeo

state: CA

address: 2880 Torrey Pines Scenic Dr. Room 3805/Yeo Lab

institute: UCSD

summary: In this study we report a comparative, transcriptome-wide analysis of CRISPR/Cas-based RNA-editing technologies in HEK293T cells. Targeted RNA editing technologies hold great process in the area of post-transcriptional genomic engineering and impermanent therapeutic correction of disease-causing variants, though off-target editing events still remain a concern. Here, we perform paired-end poly(A)+ purified RNA-seq to characterize transcriptome-wide in HEK293T cells transfected with catalytically-dead Cas9-, Cas13b, or Cas13d fused to human ADAR2 deaminase domain containing a hyperactive editing mutation (E488Q) along with either CYFIP2¬-targeting or non-targeting gRNA, in duplicate. We find that all technologies tested introduced widespread but distinct off-target mutations in the transcriptome, with the majority of edits occurring in 3’UTR and CDS regions of genes. Many of these edits, however, were in transcripts with low coverage, with the majority of editing rates being below 20%. This study is the first to characterize Cas-based RNA editing technologies in a direct manner, and reveals the relative similarity of currently available methods both in terms of on-target and off-target efficacy.

overall_design: Examination of mRNA profiles of HEK293T cells 48 hours after transfection with dCas9-, dCas13b, or dCas13d-ADAR2(E488Q) fusions with either CYFIP2¬-targeting or NT gRNA

first: Ryan

last: Marina

first: Kristopher

middle: W

last: Brannan

first: Kevin

middle: D

last: Dong

first: Brian

middle: A

last: Yee

first: Eugene


title: Brachyury controls Ciona notochord fate as part of a feedforward network [IV]

status: Public on Oct 31 2020

city: Manhattan

first: Michael

last: Veeman

state: KS

address: 116 Ackert Hall

department: Division of Biology

institute: Kansas State University

summary: The notochord is a defining feature of the chordates. The transcription factor Brachyury (Bra) is a key regulator of notochord fate but here we show that it is not a unitary master regulator in the model chordate Ciona. Ectopic Bra expression only partially reprograms other cell types to a notochord-like transcriptional profile and a subset of notochord-enriched genes are unaffected by CRISPR Bra disruption. We identify Foxa.a and Mnx as potential co-regulators and find that combinatorial cocktails are more effective at reprograming other cell types than Bra alone. We reassess the network relationships between Foxa.a, and other components of the notochord gene regulatory network and find that Foxa.a expression in the notochord is regulated by vegetal FGF signaling. It is a direct activator of Bra expression and has a binding motif that is significantly enriched in the regulatory regions of notochord-enriched genes. These and other results indicate that Bra and Foxa.a act together in a regulatory network dominated by positive feed-forward interactions, with neither being a classically-defined master regulator.er

overall_design: Fertilized dechorionated eggs were electroporated with either control plasmid (Etr>Venus), or with plasmids expressing Bra, Mnx and/or Foxa.a under the control of the Etr enhancer. Triple biological replicates of purified RNA were analyzed by RNAseq and transcript enrichment was calculated using DeSeq.

middle: T

first: Wendy

last: Reeves

first: Kotaro

last: Shimai

first: Konner

last: Winkley


title: Brachyury controls Ciona notochord fate as part of a feedforward network [I]

summary: The notochord is a defining feature of the chordates. The transcription factor Brachyury (Bra) is a key regulator of notochord fate but here we show that it is not a unitary master regulator in the model chordate Ciona. Ectopic Bra expression only partially reprograms other cell types to a notochord-like transcriptional profile and a subset of notochord-enriched genes are unaffected by CRISPR Bra disruption. We identify Foxa.a and Mnx as potential co-regulators and find that combinatorial cocktails are more effective at reprograming other cell types than Bra alone. We reassess the network relationships between Foxa.a, and other components of the notochord gene regulatory network and find that Foxa.a expression in the notochord is regulated by vegetal FGF signaling. It is a direct activator of Bra expression and has a binding motif that is significantly enriched in the regulatory regions of notochord-enriched genes. These and other results indicate that Bra and Foxa.a act together in a regulatory network dominated by positive feed-forward interactions, with neither being a classically-defined master regulator.

overall_design: Fertilized dechorionated eggs were electroporated with eithercontrol plasmid (Bra<GFP) or an enhancer>Bra fusion, expressing Bra in ectopic tissues. Triple biological replicates of purified RNA were analyzed by RNAseq and transcript enrichment was calculated using DeSeq.


title: Brachyury controls Ciona notochord fate as part of a feedforward network [II]

overall_design: Unfertilized eggs were injected with RNP mix (crRNA, tracrRNA, Cas9 protein) containing guide RNA specific to either Brachyury or GFP (control). Eggs were fertilized and grown to Hotta stage 14. Triple biological replicates of purified RNA were analyzed by RNAseq and transcript enrichment was calculated using DeSeq.


title: A histone H3.3K36M mutation in mice causes imbalance of histone modifications and defects in chondrocyte differentiation [ChIP-seq]

city: Fukuoka

first: Takashi

last: Ishiuchi

address: 3-1-1 Maidashi, Higashi-ku

department: Medical Institute of Bioregulation

country: Japan

institute: Kyushu University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Histone lysine-to-methionine (K-to-M) mutations have been identified as driver mutations in human cancers. Interestingly, these ‘oncohistone’ mutations inhibit the activity of histone methyltransferases, and, therefore, they can potentially be used as versatile tools to investigate the roles of histone modifications. In this study, we created a conditional knock-in mouse line in which a H3.3K36M mutation can be induced in the endogenous H3.3 gene. Since the H3.3K36M mutation has been identified as a causative mutation of human chondroblastoma, we induced this mutation in the chondrocyte lineage in mouse embryonic limbs. We found that H3.3K36M causes global reduction of H3K36me2 and defects in chondrocyte differentiation. Importantly, the reduction of H3K36me2 was accompanied by a collapse of normal H3K27me3 distribution. Furthermore, the changes in H3K27me3, especially the loss of H3K27me3 at gene regulatory elements, were associated with misregulated expression of a set of genes important for limb development including HoxA cluster genes. Thus, taking advantage of the in vivo induction of H3.3K36M mutation, we reveal the importance of a counterbalance between H3K36me2 and H3K27me3 for chondrocyte differentiation and limb development.

overall_design: ChIP-seq for H3K36me2, H3K36me3, and H3K27me3 were performed in mouse E14.5 tibial midshafts. mRNA-seq was performed in mouse E14.5 tibial midshafts and ends.

first: Shusaku

last: Abe


title: Effective ribosomal RNA depletion for single-cell total RNA-seq by scDASH

city: Hong Kong

first: Danson

middle: Shek Chun

last: Loi

address: HKUST, Clear Water Bay

department: Division of Life Science

institute: Hong Kong University of Science and Technology

summary: A decade since its invention, single-cell RNA sequencing (scRNA-seq) has become a mainstay technology for profiling transcriptional heterogeneity in individual cells. Yet, most existing scRNA-seq methods capture only polyadenylated mRNA to avoid expending resources on profiling the types of transcripts that are usually not-of-interest, such as ribosomal RNA (rRNA). Hence, protocols that enable analysis of the whole transcriptome remain scarce. We adapted a method called DASH (Depletion of Abundant Sequences by Hybridisation) for rRNA depletion from single-cell total RNA-seq libraries. Our analyses show that with our single-cell DASH (scDASH), rRNAs are effectively depleted with minimal non-specificity. Importantly, the rest of the transcriptome is significantly enriched for detection as a result of liberated sequencing quota from depleted rRNA.

overall_design: There are 8 single-cell and 4 min-bulk RNA-seq samples, with half of which subjected to rRNA depletion using scDASH.

middle: S

first: Lei

last: Yu

first: Angela

middle: R

last: Wu


title: A molecular calcium integrator reveals a striatal cell-type driving aversion

city: Stanford

first: Christina

middle: K

last: Kim

address: 318 Campus Drive West, Clark Center W080

institute: Stanford University

summary: The ability to record transient cellular events in the DNA or RNA of cells would enable precise, large-scale analysis, selection, and reprogramming of heterogeneous cell populations. Here we report a molecular technology for stable genetic tagging of cells that exhibit activity-related increases in intracellular calcium concentration (FLiCRE). We used FLiCRE to transcriptionally label activated neural ensembles in the nucleus accumbens of the mouse brain during brief stimulation of aversive inputs. Using single-cell RNA sequencing, we detected FLiCRE transcripts among the endogenous transcriptome, providing simultaneous readout of both cell-type and calcium activation history. We identified a cell-type in the nucleus accumbens activated downstream of long-range excitatory projections. Taking advantage of FLiCRE’s modular design, we expressed an optogenetic channel selectively in this cell-type, and showed that direct recruitment of this otherwise genetically-inaccessible population elicits behavioral aversion. The specificity and minute-resolution of FLiCRE enables molecularly-informed characterization, manipulation, and reprogramming of activated cellular ensembles.

overall_design: Single-cell RNA sequencing data from the nucleus accumbens of Fli-CRE-labeled Thy1::ChR2 mice

first: Karl

last: Deisseroth

first: Alice

middle: Y

last: Ting


title: Blue light perception by epiphytic Pseudomonas syringae drives chemoreceptor expression enabling efficient plant infection

city: Pozuelo de Alarcon

first: Emilia

last: López Solanilla

state: Madrid

address: Parque Científico y Tecnológico UPM, Campus de Montegancedo, Ctra. M-40, km 38

country: Spain

institute: CBGP-Universidad Politécnica de Madrid (UPM)

sample_organism: Pseudomonas syringae pv. tomato str. DC3000

overall_design: Four different replicates of each light treatment were performed.

first: Saray

last: Santamaria-Hernando

first: Jean-Paul

last: Cerna-Vargas

first: Pedro

last: Martinez-Garcia

first: Sofía-deFrancisco

last: de Polanco

first: Jose

last: Rodriguez-Herva

first: Pablo

last: Rodriguez-Palenzuela

last: Lopez-Solanilla


title: Brachyury controls Ciona notochord fate as part of a feedforward network [III]

first: Michael

last: Veeman

overall_design: Fertilized dechorionated eggs were treated with 4mM U0126 or DMSO control at the 32 cell stage, then collected for expression analysis at either the 64 cell stage or the 112 cell stage. Triple biological replicates of purified RNA were analyzed by RNAseq and transcript enrichment was calculated using DeSeq.

first: Wendy

last: Reeves

first: Kotaro

last: Shimai

last: Winkley


title: Impact of Zika Virus Infection on Human Neural Stem Cell MicroRNA Signatures

city: Bonn

first: Denna

last: Tabari

address: Kurt-Georg-Kiesinger Allee 3

country: Germany

institute: Federal Institute for Drugs and Medical Devices

type: Non-coding RNA profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Reprogramming of the histone H3.3 landscape in the early mouse embryo

first: Takashi

last: Ishiuchi

summary: Epigenetic reprogramming of the zygote involves dynamic incorporation of the histone variant, H3.3. However, the genome-wide distribution and dynamics of H3.3 during early development remain unknown. Here, we delineate the H3.3 landscapes in mouse oocytes and early embryos. We unexpectedly identify a non-canonical H3.3 pattern in mature oocytes and zygotes, in which local enrichment of H3.3 at active chromatin is suppressed and H3.3 is relatively evenly distributed across the genome. Interestingly, while the non-canonical H3.3 pattern forms gradually during oogenesis, it quickly switches to a canonical pattern at the 2-cell stage in a transcription-independent and replication-dependent manner. We find that incorporation of H3.1/H3.2 mediated by CAF-1 is a key process for the de novo establishment of the canonical pattern. Our data suggest that the presence of the non-canonical pattern and its timely transition toward a canonical pattern support the developmental program of early embryos.

overall_design: H3.3 ChIP-seq was performed with mouse oocytes, early embryos and mESCs. H3.1/H3.2 CUT&RUN was performed with 2-cell embryos, and H3.3 CUT&RUN was performed with mESCs. RNA-seq was performed with 8-cell embryos or blastocysts.


title: Disruption of Actin Dynamics Regulated by Rho Effector mDia1 Attenuates Pressure Overload-Induced Cardiac Hypertrophic Responses and Exacerbates Dysfunction

city: Yufu

first: Ichitaro

last: Abe

state: Oita

address: 1-1 Idaigaoka, Hasama

department: Department of Cardiology and Clinical Examination

institute: Oita University

summary:
Aims: Cardiac hypertrophy is a compensatory response to pressure overload, leading to heart failure. Recent studies have demonstrated that Rho is immediately activated in left ventricles after pressure overload, and that Rho signaling plays crucial regulatory roles in actin cytoskeleton rearrangement during cardiac hypertrophic responses. However, the mechanisms by which Rho and its downstream proteins control actin dynamics during hypertrophic responses remain not fully understood. In this study, we identified the pivotal roles of mammalian homologue of Drosophila diaphanous (mDia) 1, a Rho-effector molecule, in pressure overload-induced ventricular hypertrophy.
Methods and Results: Male wild-type (WT) and mDia1-knockout (mDia1KO) mice (10–12 weeks old) were subjected to a transverse aortic constriction (TAC) or sham operation. The heart weight/tibia length ratio, cardiomyocyte cross-sectional area, left ventricular wall thickness, and expression of hypertrophy-specific genes were significantly decreased in mDia1KO mice 3 weeks after TAC, and the mortality rate was higher at 12 weeks. Echocardiography indicated that mDia1 deletion increased the severity of heart failure 8 weeks after TAC. Importantly, we could not observe apparent defects in cardiac hypertrophic responses in mDia3-knockout mice. Microarray analysis revealed that mDia1 was involved in the induction of hypertrophy related genes, including immediate early genes (IEGs), in pressure overloaded hearts. Loss of mDia1 attenuated activation of the mechanotransduction pathway in TAC-operated mice hearts. We also found that mDia1 was involved in stretch-induced activation of the mechanotransduction pathway and gene expression of c-fos in neonatal rat ventricular cardiomyocytes (NRVMs). mDia1 regulated the F/G-actin ratio in response to pressure overload in mice. Additionally, increases in nuclear myocardin-related transcription factors (MRTFs) and serum response factor (SRF) were perturbed in response to pressure overload in mDia1KO mice and to mechanical stretch in mDia1 depleted NRVMs.
Conclusions: mDia1, through actin dynamics, is involved in compensatory cardiac hypertrophy in response to pressure overload.

overall_design: The left ventricle of mDia1KO mice 3 days after TAC compared with the left ventricle of WT mice 3 days after TAC

first: Takeshi

last: Terabayashi

first: Katsuhiro

last: Hanada

first: Hidekazu

last: Kondo

first: Yasushi

last: Teshima

first: Yumi

last: Ishii

first: Miho

last: Miyoshi

first: Shintaro

last: Kira

first: Shotaro

last: Saito

first: Hirotsugu

last: Tsuchimochi

first: Mikiyasu

last: Shirai

first: Kunio

last: Yufu

first: Motoki

last: Arakane

first: Tsutomu

last: Daa

first: Dean

last: Thumkeo

first: Shuh

last: Narumiya

first: Naohiko

last: Takahashi

first: Toshimasa

last: Ishizaki


title: CD24+ and CD24- myeloma-derived cancer stem cell lines

city: Iowa City

first: Fenghuang

last: Zhan

state: IA

address: Newton rd. CBRB 3269

department: Department of Internal Medicine

institute: University of Iowa

summary:
Expression profiling of CD24+ and CD24- population cells from myeloma cell lines. Results provide insight into the role of CD24+ cells in myeloma development.
Keywords: multiple myeloma, cancer stem cell, CD24

overall_design: The CD24 compartment in multiple myeloma is enriched in side population cells, however the functional heterogeneity within this subpopulation remains poorly defined. Myeloma cancer stem cell populations (CD24+) from ARP1, OCI-MY5 and OPM2 were isolated by  flow cytometry using  CD24 antibodies (Biolegend). The total RNA was isolated from the CSC populations using the RNeasy RNA isolation kit (Qiagen). The RNA was then converted to cDNA and they were hybridized to the Human Genome U133Plus 2.0 GeneChip microarrays(Affymetrix). The data was normalized using the MAS5 algorithm of the Affymetrix expression console version1.1 software (Affymetrix). A comparison of transcriptional profiles was then performed in CD24+ cell lines compared to the CD24- cell lines.


title: Impact of Zika Virus Infection on Human Neural Stem Cell MicroRNA Signatures [array]

first: Denna

last: Tabari

summary: We used whole genome expression profiling to analyze the effects of Zika virus (ZIKV) strains Uganda and French Polynesia on the mRNA transcriptome of induced pluripotent stem cell-derived human neural stem cells (long-term self-renewing neuroepithelial-like stem (lt-NES®) cells) - particularly to identify differentially expressed genes which are associated with microcephaly phenotype.

overall_design: Gene expression profiles of Zika virus-infected lt-NES® cells were analyzed 24, 48 and 72 hours post infection. At the same timepoints gene expression profiles of uninfected lt-NES® cells were analyzed. 3 biological replicates were used.

first: Catharina

last: Scholl

first: Michael

last: Steffens

first: Sandra

last: Weickhardt

first: Fabian

last: Elgner

first: Daniela

last: Bender

first: Marie-Luise

last: Herrlein

first: Catarina

last: Sabino

first: Andreas

last: Till

first: Vesselina

last: Semkova

first: Oliver

last: Brüstle

first: Eberhard

last: Hildt

first: Julia

last: Stingl


title: Brachyury controls Ciona notochord fate as part of a feedforward network

last: Veeman


title: Paraneoplastic Secretion of Multiple Phosphatonins from a Deep Fibrous Histiocytoma Causing Oncogenic Osteomalacia

status: Public on Oct 30 2020

city: Townsville

first: Craig

last: McFarlane

state: Queensland

address: 1 James Cook Drive, Building 142-309

department: Molecular & Cell Biology

country: Australia

institute: James Cook University

summary:
This data describes the first case of oncogenic osteomalacia due to a plexiform fibrohistiocytic tumor which coincidentally has been found for the first time to induce profound muscle weakness via the overexpression of three phosphatonins which resolved fully upon radical resection of the tumor.
Much of the literature suggests that oncogenic osteomalacia is usually caused by a benign mesenchymal tumor secreting fibroblast growth factor subtype-23 (FGF23) but other phosphatonins are rarely implicated.We have previously reported a seemingly typical case of oncogenic osteomalacia. Following curative neoplasm resection, we now report unique molecular characteristics and biology of this tumor never described before. A 25-year-old man had been diagnosed with severe oncogenic osteomalacia that gradually crippled him over 6 years. 68Ga-DOTA-TATE PET-CT scan localized the culprit tumor to his left sole which on resection revealed a plexiform fibrohistiocytic tumor positive for CD34 and CD163 with osteoclast-like giant cells and scattered stromal dendritic cells within the tumor. Circulating FGF23 which was elevated more than 2-folds declined to undetectable levels 24 hours after surgery. Microarray analysis showed over 150-fold upregulation in FGF23, matrix extracellular phosphoglycoprotein (MEPE) and secreted frizzled-related protein subtype 4 (sFRP4) genes which corresponded to the same proteins overexpressed on Western blots

overall_design: Total RNA was obtained from a total of six samples, which consisted of three pairs of technical replicates each for tumour, adjacent and skin samples

first: Melvin

last: Leow

first: Shaillay

last: Dogra

first: Xiaojia

last: Ge

first: Khoon

middle: L

last: Chuah

first: Huiling

last: Liew

first: Kelvin

last: Loke


title: A histone H3.3K36M mutation in mice causes imbalance of histone modifications and defects in chondrocyte differentiation

first: Takashi

last: Ishiuchi

summary: Histone lysine-to-methionine (K-to-M) mutations have been identified as driver mutations in human cancers. Interestingly, these ‘oncohistone’ mutations inhibit the activity of histone methyltransferases. Therefore, they can potentially be used as versatile tools to investigate the roles of histone modifications. In this study, we generated a genetically engineered mouse line in which an H3.3K36M mutation could be induced in the endogenous H3f3b gene. Since H3.3K36M has been identified as a causative mutation of human chondroblastoma, we induced this mutation in the chondrocyte lineage in mouse embryonic limbs. We found that H3.3K36M causes a global reduction in H3K36me2 and defects in chondrocyte differentiation. Importantly, the reduction of H3K36me2 was accompanied by a collapse of normal H3K27me3 distribution. Furthermore, the changes in H3K27me3, especially the loss of H3K27me3 at gene regulatory elements, were associated with the mis-regulated expression of a set of genes important for limb development, including HoxA cluster genes. Thus, through the in vivo induction of the H3.3K36M mutation, we reveal the importance of maintaining the balance between H3K36me2 and H3K27me3 during chondrocyte differentiation and limb development.


title: Impact of Zika Virus Infection on Human Neural Stem Cell MicroRNA Signatures [miRNA-Seq]

first: Denna

last: Tabari

summary: We used next generation sequencing to analyze the effects of Zika virus (ZIKV) strains Uganda and French Polynesia on intracellular and extracellular vesicle (EV) derived miRNA profiles of induced pluripotent stem cell-derived human neural stem cells (long-term self-renewing neuroepithelial-like stem (lt-NES®) cells) - particularly to identify dysregulated miRNAs which are associated with the microcephaly phenotype.

overall_design: MiRNAs, which were derived from EVs and intracellular miRNAs of Zika virus-infected lt-NES® cells were analyzed 24, 48 and 72 hours post infection. At the same timepoints intracellular and EV-associated miRNA profiles of uninfected lt-NES® cells were analyzed. 3 biological replicates were used.

first: Catharina

last: Scholl

first: Michael

last: Steffens

first: Sandra

last: Weickhardt

first: Fabian

last: Elgner

last: Bender


title: ZFP423 regulates early patterning and multiciliogenesis in the hindbrain choroid plexus

status: Public on Oct 29 2020

city: Milano

first: Michela

last: Riba

address: via Olgettina 60

department: COSR, Center for Omics Sciences

country: Italy

institute: IRCCS, Ospdale San Raffaele

summary: The choroid plexus (ChP) is a secretory tissue that produces cerebrospinal fluid (CSF) secreted into the ventricular system. It is a monolayer of secretory, multiciliated epithelial cells derived from neuroepithelial progenitors and overlying a stroma of mesenchymal cells of mesodermal origin. Zfp423, encoding a Kruppel-type zinc finger transcription factor essential for cerebellar development and mutated in rare cases of cerebellar vermis hypoplasia / Joubert syndrome and other ciliopathies, is expressed in the hindbrain roof plate (RP), from which the IV ventricleChP arises, and, later, in mesenchymal cells giving rise to the stroma and leptomeninges.Zfp423mutants display a marked reduction of the hindbrain ChP (hChP), which 1) fails to express established markers of its secretory function and genes implicated in its development and maintenance (Lmx1a, Otx2); 2) shows a perturbed expression of signaling pathways previously unexplored in hChP patterning (Wnt3); 3) displays a lack of multiciliated epithelial cells and a profound dysregulation of master genes of multiciliogenesis (Gmnc). Our results propose Zfp423as a master gene and one of the earliest known determinants of hChP development.

overall_design: 3x E9.5 wt hindbrains, 3x E9.5 d28-30 hindbrains, and 3x E9.5 d9-20 hindbrains

first: Luca

last: Massimino

first: Laura

last: Croci

first: Gian

middle: G

last: Consalez

first: Filippo

last: Casoni


title: Bisulfite-free epigenomics and genomics of single cells analyzed by methylation sensitive restriction (epi-gSCAR)

status: Public on Oct 28 2020

city: Freiburg

first: Heiko

last: Becker

state: Baden-Württemberg

address: Breisacher Straße 115

department: Department of Medicine I

institute: University Medical Center

type: Methylation profiling by high throughput sequencing

summary: Single-cell multi-omics are powerful means to study cell-to-cell heterogeneity. Here, we present a single-tube, bisulfite-free method for the simultaneous, genome-wide analysis of DNA methylation and genetic variants in single cells: epigenomics and genomics of single cells analyzed by restriction (epi-gSCAR). By applying this method, we obtained DNA methylation measurements of up to 506,063 CpGs and up to 1,244,188 single-nucleotide variants from single leukemia-derived cells. We demonstrate that epi-gSCAR generates accurate and reproducible measurements of DNA methylation and allows to differentiate between two cell lines based on the DNA methylation and genetic profiles.

overall_design: DNA methylation and gene variant analysis of 27 single Kasumi-1 and 20 single OCI-AML3 AML cells

first: Christoph

last: Niemöller

first: Julius

last: Wehrle


title: Loss of Maenli lncRNA expression causes engrailed-1 dependent congenital limb malformations [scRNA-seq]

status: Public on Oct 25 2020

city: Berlin

first: Lila

last: Allou

address: 63-73 Ihnestrasse

department: FG Development and Disease

institute: Max Planck Institute For Molecular Genetics

summary: Long non-coding RNAs (lncRNAs) can be important components in gene regulatory networks1, but we are only beginning to understand the nature and extent of their involvement in human Mendelian disease. Here we show that deletions of an unannotated lncRNA locus on human chromosome 2 cause a severe congenital limb malformation. Using exome sequencing and array CGH, we identified homozygous 27-63 kb deletions located 300 kb upstream of the engrailed-1 (EN1) gene in patients with a complex limb malformation, featuring mesomelic shortening, syndactyly, and ventral nails (dorsal dimelia). Re-engineering of the human deletions in mice resulted in a complete loss of limb-specific En1 expression and a double dorsal limb phenotype, recapitulating the human malformation. Genome-wide analysis in the developing mouse limb revealed the presence of a 4-exon long non-coding transcript within the deleted region, which we named Maenli (for Master activator of En1 in the limb). Functional dissection of the Maenli locus showed that limb-specific En1 expression depends on transcription of Maenli and its loss resulted in the double dorsal limb phenotype. Concomitant monoallelic inactivation of En1 and Maenli in double heterozygous mice did not rescue the limb phenotype, indicating that En1 activation in the limb requires the cis-acting regulatory element Maenli. Moreover, our results strongly suggest that En1 activation is dependent on Maenli transcription, but not on the Maenli RNA itself. Thus, Maenli expression in the limb acts in cis to promote En1 transcriptional activation; its loss results in congenital malformation of the limbs, a subset of the full En1 associated phenotype. Together, our findings provide evidence that mutations involving lncRNAs loci can result in human Mendelian disease.

overall_design: We used exome sequencing and array CGH to analyze patients' samples, CRISPR experiments to investigate the effect of the potential pathogenic variants, genome wide analysis to identify a new lncRNA locus, and functional studies to characterize the effect of the knockout lncRNA locus.


title: A. thaliana WT and ICT1 mutant treated vs. non treated with I3C

status: Public on Oct 27 2020

city: Tel - Aviv

first: Alin

last: Finkelshtein

state: Israel

address: Haim levanon st. 30, Britania building, room 407

department: Plant sciences

country: Israel

institute: Tel Aviv university

summary:
We work with Arabidopsis thaliana. One of the things we focus on is a secondary metabolite named Indol 3 carbinol (I3C). This molecule is synthesized following insect attack (wounding). We have noticed that addition of I3C to the growth medium causes inhibition of plant/bacterial/fungal growth. We have discovered that I3C also affects different pathways in the plant. By screening overexpression library we have found Arabidopsis mutants that overexpress S30 gene (ribosomal subunit of 40s) are tolerant to the addition of I3C (their growth is less affected by the addition of I3C).
To understand better I3C mode of action we have planned the following transcriptome experiment:   1. To see what are the pathways activated following wounding, we took the wt Arabidopsis thaliana (Col-0) and kept one group of plants untreated (control) and the other was wounded (8 hours).   2. To see what are the pathways activated following incubation with 2 concentrations of I3C, we took the wt Arabidopsis thaliana (Col-0) and kept one group of plants untreated (control) and the other was incubated for 2 hours with 200µM (physiological concentration) and 400µM I3C.  3. To see if there are any differences in gene expression between the wt and the mutants, we took wt and 2 I3C tolerant mutants. Neither of them was treated.  4. To see if in the mutant other pathways are activated following incubation with I3C, both mutants were incubated in I3C.   In all cases we have used the roots only for RNA extraction.

overall_design:
The samples:
• Colombia 0– wt (used as reference)
• psx08098 (3) homozygous line (FOX overexpression library)
• 2(5) homozygous line (overexpressing transgene made in the lab)
Treatments:
• no treatment (DMSO)
• I3C 200uM
• I3C 400uM
• Wounding  Triplicate of each sample were used
Colombia no treatment (DMSO) X3
Colombia I3C 200uM X3
Colombia I3C 400uM X3
Colombia Wounding X3
Psx08098 (3) no treatment (DMSO) X3
Psx08098 (3) I3C 200uM X3
2(5) no treatment (DMSO) X3
2 (5) I3C 200uM X3
2 (5) I3C 400uM X3

first: Daniel

last: Chaimovitz


title: Loss of Maenli lncRNA expression causes engrailed-1 dependent congenital limb malformations [Derived Virtual 4C]

overall_design:
We used exome sequencing and array CGH to analyze patients' samples, CRISPR experiments to investigate the effect of the potential pathogenic variants, genome wide analysis to identify a new lncRNA locus, and functional studies to characterize the effect of the knockout lncRNA locus.

These derived virtual 4C samples are based on the same raw files used for Capture Hi-C (GSE137333) but were used for an independent analysis.


title: Loss of Maenli lncRNA expression causes engrailed-1 dependent congenital limb malformations [RNA-seq]


title: Loss of Maenli lncRNA expression causes engrailed-1 dependent congenital limb malformations [ATAC-seq]


title: Loss of Maenli lncRNA expression causes engrailed-1 dependent congenital limb malformations [Capture Hi-C]


title: Loss of Maenli lncRNA expression causes engrailed-1 dependent congenital limb malformations [ChIP-seq]


title: Non-coding deletions identify Maenli lncRNA as a limb-specific En1 regulator


title: Loss of Maenli lncRNA expression causes engrailed-1 dependent congenital limb malformations [DamID-seq]


title: The LysR-type transcriptional regulator VtlR/LsrB controls expression of multiple small RNA and small protein genes in Agrobacterium tumefaciens

status: Public on Oct 24 2020

city: Bochum

first: Franz

last: Narberhaus

state: NRW

address: Universitätsstraße 150

department: Microbial Biology

institute: Ruhr University Bochum

summary: We investigated the role of the LysR-type transcriptional regulator VtlR/LsrB (Atu2186) from Agrobacterium tumefaciens C58 (alias A. fabrum C58), which regulates the expression of small-regulatory-RNA (sRNA) genes such as AbcR1 and the expression of genes encoding small proteins with a domain of unknown function DUF1127. For this purpose, we constructed a vtlR/lsrB deletion mutant. Total RNA was isolated from this mutant as well as from an AbcR1 deletion mutant (Wilms et al., 2011) at optical densities (OD) of 0.5 or 0.5 and 1.5, respectively. Sequencing was done on the Illumina NovaSeq 6000 platform. In addition to annotated genes (NCBI genome: 13606), almost 700 previously described sRNA genes (Wilms et al., 2012; Lee et al., 2013; Möller et al., 2014) were also considered within our data analysis. Wild-type samples that derived from the same experiment were published previously (Kraus et al., 2020) (GEO accession: GSE150941). In the vtlR/lsrB mutant a total of 542 genes were downregulated and 665 were upregulated at least three-fold (p value ≤ 0.05). Among those were 25 downregulated sRNA genes (including AbcR1) and 77 upregulated sRNA genes. In the AbcR1 mutant 8 or 20 genes were downregulated at ODs 0.5 or 1.5, respectively, and 72 or 31 were upregulated. A total of 39 differentially expressed genes from the vtlR/lsrB mutant also displayed identical regulation in the AbcR1 mutant and thus are controlled indirectly by VtlR/LsrB via the sRNA AbcR1. Overall, the results demonstrate the complex network of VtlR/LsrB and regulatory sRNAs, which has a great impact on the transcriptome of A. tumefaciens.      Kraus, A., Weskamp, M., Zierles, J., Balzer, M., Busch, R., Eisfeld, J., et al. (2020) Arginine-rich small proteins with a domain of unknown function DUF1127 play a role in phosphate and carbon metabolism of Agrobacterium tumefaciens. J Bacteriol 202: e00309-20.  Lee, K., Huang, X., Yang, C., Lee, D., Ho, V., Nobuta, K., et al. (2013) A genome-wide survey of highly expressed non-coding RNAs and biological validation of selected candidates in Agrobacterium tumefaciens. PLoS One 8: e70720.  Möller, P., Overlöper, A., Förstner, K.U., Wen, T.N., Sharma, C.M., Lai, E.M., and Narberhaus, F. (2014) Profound impact of Hfq on nutrient acquisition, metabolism and motility in the plant pathogen Agrobacterium tumefaciens. PLoS One 9: e110427.  Wilms, I., Overlöper, A., Nowrousian, M., Sharma, C.M., and Narberhaus, F. (2012) Deep sequencing uncovers numerous small RNAs on all four replicons of the plant pathogen Agrobacterium tumefaciens. RNA Biol 9: 446–57.  Wilms, I., Voss, B., Hess, W.R., Leichert, L.I., and Narberhaus, F. (2011) Small RNA-mediated control of the Agrobacterium tumefaciens GABA binding protein. Mol Microbiol 80: 492–506.

overall_design: Illumina RNA sequencing of the transcriptomes of Agrobacterium fabrum C58 wild type, a vtlR/lsrB deletion mutant (∆atu2186) and an AbcR1 deletion mutant.

first: Jessica

last: Eisfeld

first: Alexander

last: Kraus

first: Christian

last: Ronge

first: Michelle

last: Jagst

first: Vivian

middle: B

last: Brandenburg


title: LIGHT/LTβR signaling regulates self-renewal and differentiation of hematopoietic and leukemia stem cells

status: Public on Oct 23 2020

city: Bern

first: Ramin

last: Radpour

address: Murtenstrasse 35

department: Department for BioMedical Research

country: Switzerland

institute: University of Bern

summary:
Purpose: The goal of this study is to compare NGS-derived transcriptome profiling (RNA-seq) of Ltbr-deficient and -proficient LT/ST-HSCs isolated from chimeric mice.
Methods: Transcriptomic profiles of Ltbr-/- and Ly5.1 LT/ST-HSCs isolated from chimeric mice 6 weeks after reconstitution were assessed in triplicate by deep sequencing, using Illumina NextSeq 500. qRT–PCR validation was performed using TaqMan and SYBR Green assays.
Results: We mapped about 60 million sequence reads per sample to the mouse genome (GRCm38 - mm10) and identified expressed transcripts in Ltbr-/- and Ly5.1 LT/ST-HSCs isolated from chimeric mice. RNA-seq. data confirmed stable expression of known housekeeping genes. Differentially expressed genes between the Ltbr-/- and Ly5.1 LT/ST-HSCs were identified with a fold change ≥1.5 and FDR p-value <0.05.
Conclusions: Our study represents the first detailed transcriptome analysis of Ltbr-deficient and -proficient LT/ST-HSCs, with biologic replicates, generated by RNA-seq. technology. Our results show that Ltbr signaling regulates HSC proliferation and differentiation. Evaluation of mRNA content in Ltbr-/- LT/ST-HSCs revealed that Ltbr-deficiency enhances HSC proliferation, differentiation, cell cycle and reduces the activity of canonical NFkB signaling.

overall_design: mRNA of Ltbr-/- and Ly5.1 LT/ST-HSCs isolated from bone marrow of chimeric mice using Illumina NextSeq 500.

first: Sabine

last: Höpner

first: Ana

last: Raykova

first: Adrian

middle: F

last: Ochsenbein


title: Evaluation of WNT pathway transcriptomes in T-cell Acute Lymphoblastic Leukemia by targeted RNA-Seq

status: Public on Oct 22 2020

first: Alessandro

last: Beghini

address: Via A.Di Rudinì

department: Dept. of Health Sciences

institute: University of Milan

summary: Wnt/Fzd signaling has been implicated in hematopoietic stem cell maintenance and in acute leukemia establishment. We previously described a recurrent rearrangement involving the WNT10B locus (WNT10BR), characterized by expression of the WNT10BIVS1 transcript variant, in acute myeloid leukemia. To determine the occurrence of WNT10BR in T-cell acute lymphoblastic leukemia (T-ALL), we retrospectively analyzed an italian cohort of patients (n=20) and detected the WNT10BIVS1 with a high prevalence (14/20). To address genes involved in WNT10B molecular response, we designed a Wnt targeted RNA sequencing panel. We identify Wnt agonists and antagonists, from which the expression of FZD6, PLCB4, and PROM1 stand out in WNT10BIVS1-positive patients compared to negative ones. Using MOLT4 and MUTZ-2 as cell models, characterized by the expression of WNT10BIVS1, we found that WNT10B drives major Wnt activation through receipt of ligand to the FZD6 receptor complex. The WNT10B/FZD6 interaction were interfered with by short hairpin RNAs (shRNAs)-mediated gene silencing and by small molecules-mediated inhibition of WNTs secretion. We found that WNT10BIVS1 knockdown, or pharmacological interference by the LGK974 porcupine (PORCN) inhibitor, reduces WNT10B/FZD6 protein complex formation and significantly impairs intracellular effectors and leukemic expansion. These results describe the molecular circuit induced by WNT10B and suggest WNT10B/FZD6 as new targets in the T-ALL treatment strategy.

overall_design: We used Ion AmpliSeq RNA WNT Signaling Pathway Research Panel targeting 178 genes that regulate the production of WNT signaling molecules, interactions with receptors on target cells, and signal transduction, to analyse mononuclear cells from T-ALL patients (n=5), healthy donors (n=2), MOLT4, and MUTZ2 cell lines.


title: RNA-sequencing analysis gene expression changes in response to neonatal pulmonary artery banding

city: Shanghai

first: Xiaoning

last: Ding

state: Shanghai

address: 1678 Dongfang Rd.

department: Shanghai Children's Medical Center

institute: Shanghai Jiao Tong University School of Medicine

summary: The goal of this study is to investigate the molecular mechanisms of cardiomyocyte hyperplasia and hypertrophy in response to the pressure overload

overall_design: Heart mRNA profiles of 3-day-old and 7-day old wild type Sham and wild type pulmonary artery banding mice

first: Zhen

last: Zhang

first: Min


title: Persistent STAT5 activation reprograms the epigenetic landscape in CD4+ T cells to drive polyfunctionality and antitumor immunity [scRNA-Seq]

city: Augusta

first: Huidong

last: Shi

state: GA

department: Georgia Cancer Center

institute: Augusta University

summary: We report here that persistent activation of signal transducer and activator of transcription 5 (STAT5) in tumor-specific CD4+ T cells drives the development of polyfunctional T cells with superior antitumor activities. We showed that ectopic expression of a constitutively active form of murine STAT5A (CASTAT5) in tumor-specific CD4+ T cells established a distinct epigenetic and transcriptional landscape, endowing CD4+ T cells polyfunctionality, exhaustion-resistance and tumor-infiltrating capability. Single cell RNA sequencing analysis (scRNAseq) identified a subset of cells with the molecular signature indicative of their role as progenitor polyfunctional T cells. Importantly, T cells engineered to co-express CD19-targeting chimeric antigen receptors (CD19CAR) and CASTAT5 gave rise to polyfunctional CD4+ CAR T cells capable of providing optimal help to CD8+ T cells to achieve durable and curative outcomes in mice with advanced B-cell lymphoma. Evidence of CASTAT5 functional activities in primary human CD4+ T cells underscores its potential clinical relevance.

overall_design: The objective of this study was to investigate the functional fate and therapeutic effectiveness of tumor-specific CD4+ T cells expressing a constitutively active form of Stat5a (CASTAT5) via retroviral transduction.  To achieve this goal, we designed a CD4+ T-cell adoptive transfer model system using a murine B-cell lymphoma model and performed ATACseq and bulk RNAseq to interrogate how CASTAT5 reshaped CD4+ T cell phenotypic and functional features using FACS-sorted tumor-specific CD4+ T cells recovered from the spleens and tumors of mice. Our datasets revealed that CASTAT5 induced genome-wide transcriptional and epigenetic remodeling in tumor-specific CD4+ T cells. Subsequent single-cell RNAseq analysis revealed the heterogeneity of CASTAT5-transduced CD4+ T cells and identified a subset of cells bearing a gene signature indicative of progenitor polyfunctional cells. We validated the beneficial effects of CASTAT5-transduced CD4+ T cells in CD19CAR T cell therapy mouse tumor models. We demonstrated that CASTAT5 was also functional in transduced primary human T cells. Mice with established tumors were randomly grouped before receiving the indicated treatment. Sample sizes for the in vivo experiments were determined based on previous studies performed in our lab, and the suitability for statistical analysis and independent repeats. Information regarding the sample size and number of replicates for each experiment is provided in the relevant figure legend.

first: Zhichun

first: Gang

last: Zhou


title: Persistent STAT5 activation reprograms the epigenetic landscape in CD4+ T cells to drive polyfunctionality and antitumor immunity [ATAC-Seq]


title: Differences in microbiome populations following hematopoietic stem cell transplantation in Crohn?s disease patients

city: Barcelona

first: Azucena

last: Salas

address: Rosselló, 149-153

department: Gastroenterology

institute: IDIBAPS

summary: Treatment of severely refractory Crohn’s disease (CD) patients remains a clinical challenge. Recent studies show the efficacy of autologous hematopoietic stem cell transplant (HSCT) in these severely compromised patients. HSCT is thought to eliminate auto-reactive cells; however, no specific studies of immune reconstitution in CD patients are available. We studied a group of CD patients (n=18) receiving autologous HSCT. We studied changes in the microbiome induced by HSCT and some non-CD samples.

overall_design: Biopsy 16S profiles of CD patients undergoing HSCT were generated by deep sequencing, using MiSeq (Illumina, San Diego, CA)

last: Corraliza

first: Lluís

last: Sancho


title: DNA methylomes of murine B and T cells from the BLUEPRINT Epigenome project

city: Cambridge

first: Anne

last: Ferguson-Smith

address: Downing Site

department: Genetics

country: United Kingdom

institute: University of Cambridge

summary: This data release is part of the EU BLUEPRINT reference epignome project (http://www.blueprint-epigenome.eu/) and consists of genome wide gene expression, histone modifications and DNA methylation profiles from murine Naive CD4+ T-cells and Resting B cells.

overall_design: DNA methylation profiled on Naive CD4+ T cells and Resting B cells derived from pooled murine male and female samples using whole genome oxidative/bisulfite sequencing. Two replicates of each combination of cell type (B cell, T cell); sex (male, female); and sequencing type (bisulfte-seq, oxidative bisulfite-seq); 16 samples in total. This series contains the same samples and raw data as in GSE94674/GSE94675; however, here the two replicates for each cell type/sex/sequencing method are not pooled.

first: Noah

last: Kessler

middle: C


title: FAM83H-AS1 is a non-coding oncogenic driver and therapeutic target of lung adenocarcinoma

status: Public on Oct 20 2020

city: Nanjing

first: Rong

last: Yin

address: Baiziting 42

department: Thoracic Surgery

institute: Nanjing Medical University Affiliated Jiangsu Cancer Hospital

summary: Our study suggested that FAM83H-AS1 was a potential oncogenic driver due to chromosome 8q24 amplification in lung adenocarcinoma. To investigate the molecular mechanism of FAM83H-AS1, we performed high-thoughput RNA sequencing (RNA-Seq) assays after the silence of FAM83H-AS1 in A549 cell lines.

overall_design: In human lung adenocarcinoma A549 cells, we downregulated the expression of FAM83H-AS1 utilizing small interference RNA (siRNA) and used scrumble as negative control. Then we used RNA-Seq to reveal the change of mRNA profiles after the silence of FAM83H-AS1.

first: Siwei

last: Wang

first: Zhifei

last: Ma

first: Wenjia

last: Xia

first: Mantang

last: Qiu


title: Differential transcriptional expression by Babesia bovis during parasite development within bovine erythrocytes and tick hemolymph

status: Public on Oct 21 2020

city: Pullman

first: Massaro

last: Ueti

state: WA

address: 3003 ADBF

department: Animal Disease Research Unit

institute: United States Department of Agriculture

summary: Babesia parasites transition between a mammalian host, where they cause babesiosis, and the tick vector that transmits them. This transition provides an environmental signal resulting in altered gene expression allowing the completion of the parasite’s life cycle. A comparison of the different life stages that occur within mammalian and tick hosts can provide insight into the adaptation of Babesia to these different environments. In this study, we used RNA-Seq to compare gene expression between Babesia bovis blood stages and tick derived kinetes.

overall_design: Parasite mRNA profiling of three biological replicates from bovine host and tick vector

first: Wendell

last: Johnson

first: David

last: Herndon

first: Lowell

last: Kappmeyer

first: Kelly

last: Brayton


title: Persistent STAT5 activation reprograms the epigenetic landscape in CD4+ T cells to drive polyfunctionality and antitumor immunity [RNA-Seq]

first: Huidong

last: Shi

last: Ding


title: HDAC inhibitors result in widespread alteration of the histone acetylation landscape and BRD4 targeting to gene bodies

city: Chapel Hill

first: Austin

last: Hepperla

state: NC

address: 7018B Mary Ellen Jones Building

institute: University of North Carolina at Chapel Hill

summary: Histone acetylation is associated with active transcription and serves as a binding site for reader proteins that function in transcriptional initiation and elongation. Histone acetylation levels are regulated through the actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs) that antagonistically control the overall balance of this posttranslational modification. HDAC inhibitors (HDACi) are potent agents that disrupt this balance and are used clinically to treat a range of human diseases including cancer.  Despite their clinical applications, little is known about their chromatin effects. By applying quantitative genomic and proteomic approaches, we demonstrate that HDACi robustly increase a low abundance histone acetylation state (H4 K5ac/K8ac/12ac/K16ac), which serves as a preferred binding substrate for a variety of human bromodomain-containing proteins, including BRD4. This H4 polyacetylation signature observed after HDACi treatment accumulates in the transcribed regions of genes and correlates with the targeting of BRD4 to genes with increased gene expression. Collectively, these results suggest that HDAC inhibition functions, at least in part, through expansion of a rare chromatin acetylation state, which then retargets lysine-acetyl reader proteins associated with changes in gene expression.

overall_design: Examination of H4ac and BRD4 in HL60 cells before and after SAHA treatment

first: Mariesa

last: Slaughter

first: Erin

last: Shanle

first: Abid

last: Khan

first: Katrin

last: Chua

first: Tao

last: Hong

first: Lisa

last: Boxer

first: C

last: Allis

first: Steven

middle: Z

last: Josefowicz

first: Benjamin

last: Garcia

first: Scott

last: Rothbart

first: Brian

last: Strahl

first: Ian

last: Davis


title: Persistent or Transient Human β-cell Dysfunction Induced by Metabolic Stress Associated with Specific Signatures and Shared Gene Expression of Type 2 Diabetes

city: Bruxelles

first: Anthony

last: Piron

address: Route de Lennik 808 CP618

department: Faculté de Medecine

country: Belgium

institute: Université Libre de Bruxelles

summary: Pancreatic β-cell failure is key to type 2 diabetes (T2D) onset and progression. We assessed whether human β-cell dysfunction induced by metabolic stress is reversible, evaluated the molecular pathways underlying persistent or transient damage, and explored the relationships with T2D islet traits. Twenty-six human islet preparations were exposed to several lipo- and/or glucotoxicity conditions, some of which impaired insulin release depending on stressor type, concentration and combination. Interestingly, reversal of dysfunction occurred after washout for some, but not for all, of the lipoglucotoxic insults. Islet transcriptomes assessed by RNA-sequencing and eQTL analysis identified specific pathways underlying β-cell failure and recovery. Notably, comparison of a large number of human T2D islet transcriptomes with those of persistent or reversible β-cell lipoglucotoxicity showed shared gene expression signatures. The identification of mechanisms associated with human β-cell dysfunction and recovery, and their overlap with T2D islet traits provide novel insights into T2D pathogenesis and should foster the development of improved β-cell targeted therapeutic strategies.

overall_design: Human islets from 26 non-diabetic donors were exposed to 0.5 mM palmitate (P), 22.2 mM glucose (G), P+G, 1 mM palmitate+oleate, (1:2 ratio, P+O) and P+O+G for 2 days (n=2-5/condition) (D4), followed by 4 days of washout (D8). Glucose-stimulated insulin secretion and RNA-seq (llumina HiSeq 2500 instrument) transcriptomes of P, G, P+G conditions were assessed. The reads were assigned to reference and quantified by Salmon v.0.13.2. We run the bioconductor R package Deseq2 to generate a paired differential analysis between metabolic stress condition vs matched control. Data were compared to RNA-seq of islets from 29 type 2 diabetic and 59 non-diabetic donors by rank-rank hypergeometric overlap (RRHO)

first: Piero

last: Marchetti

first: Mara

last: Suleiman

first: Maike

last: Colli

first: Xiaoyan

last: Yi

first: Amna

last: Khamis

first: Guy

last: Rutter

first: Marco

last: Bugliani

first: Laura

last: Giusti

first: Maurizio

last: Ronci

first: Mark

last: Ibberson

first: Jean-Valry

last: Turatsinze

first: Ugo

last: Boggi

first: Paolo

last: De Simone

first: Vincenzo

last: De Tata

first: Carmela

last: De Luca

first: Daniela

last: Campani

first: Miguel

last: Lopes

last: Nasteska

first: Anke

last: Shulte

first: Michele

last: Solimena

first: Peter

last: Hecht

last: Rady

first: Ivon

last: Bakaj

first: Alessandro

last: Pocai

last: Norquay

first: Bernar

last: Thorens

first: Michael

last: Canouil

first: Philippe

last: Frogues

first: Decio

last: Eizirik

first: Miriam

last: Cnop

first: Lorella

last: Marselli


title: RNA-seq of Tregs

city: Xi'an

first: Yanghe

last: Liu

state: China

address: Xijing  Hospital, Fourth Military Medical University, 127 Changle West Road, Xi’an

institute: Fourth Military Medical University

summary: RNA-seq of normal control Tregs and IL-17A treated Tregs

first: Gang

last: Wang

first: Luting

last: Yang


title: Niche Derived Netrin-1 Regulates Hematopoietic Stem Cell Dormancy via its Receptor Neogenin-1

city: Groningen

first: Leonid

last: Bystrykh

state: Groningen

address: A. Deusinglaan 1

department: European Research Institute for the Biology of Ageing - ERIBA

country: Netherlands

institute: University Medical Center Groningen

summary: We have performed RNA-seq in highly purified Hematopoeitic Stem cells (HSC, LSK/SLAM) from young (4-5mo) and old (20-24mo) C57BL/6J in order to investigate differences in gene expression between these groups.

overall_design: Examination of differently expressed genes in LSK/SLAM cells

first: Seka

last: Lazare

first: Arthur

last: Svendsen


title: Female human primordial germ cells display X-chromosome dosage compensation despite the absence of X-inactivation [Female]

status: Public on Oct 19 2020

city: Los Angeles

first: Tsotne

last: Chitiashvili

address: 615 Charles E Young Dr S

institute: University of California - Los Angeles

summary: X chromosome dosage compensation is one of the most important epigenetic events in female mammalian development. Although the mouse has served as the primary model to study how genetic equality is established between male and female mammals, it is now appreciated that these mechanisms are not fully conserved in humans.  Using human primordial germ cells (hPGCs) as a model together with single cell RNA-sequencing, in vitro differentiation of hPGC like cells and human embryo attachment culture, we show that X-linked gene expression in female hPGCs is compensated to levels found in female somatic cells though a mechanism related to X chromosome dampening rather than X chromosome inactivation. Loss of dampening and decompensation of both X chromosomes occurs when female hPGCs enter meiosis and is coincident with loss of the long non-coding RNAs XIST and XACT. In contrast, differentiation of male hPGCs results in no change in compensation despite loss of XACT.  Taken together, future studies of X-linked gene regulation and function in human germ cell development and fertility must take into account the unique and sexually dimorphic dosage compensation occurring during in the prenatal human germline prior to birth.

overall_design: In our work, we performed single cell RNA-sequencing (scRNA-seq) with 10x Genomics on 5 different female human prenatal ovaries from 5 different developmental timepoints: Week 7, 9, 10, 13 and 16 post fertilization.  In total, we sequenced ~25,000 gonadal somatic and germ cells to uncover X chromosome dynamics during female germline development.

first: Kathrin

last: Plath

first: Amander

last: Clark


title: Persistent STAT5 activation reprograms the epigenetic landscape in CD4+ T cells to drive polyfunctionality and antitumor immunity

first: Huidong

last: Shi

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

overall_design: Refer to individual Series


title: Female human primordial germ cells display X-chromosome dosage compensation despite the absence of X-inactivation [Male]

overall_design: In our work, we performed single cell RNA-sequencing (scRNA-seq) with 10x Genomics on 6 different male human prenatal testes from 6 different developmental timepoints: Week 6, 7, 8, 12, 15 and 16 post fertilization. In total, we sequenced ~30,000 gonadal somatic and germ cells to uncover X chromosome dynamics during female and male germline development.


title: Single-cell sequencing of peripheral blood and tumor-infiltrating immune cells in renal clear cell carcinoma [5' RNA expression sequencing]

city: Iowa City

first: Weizhou

last: Zhang

address: 100 Medical Laboratories

department: Pathology

institute: The University of Iowa

summary:
We generated a comprehensive immune profile using single-cell RNA-seq data from 25,000 immune cells from three renal cell carcinoma along with matched peripheral blood. In addition, we performed VDJ sequencing on the isolated single T cells.
In order to investigate heterogeneity and dynamics of cancer-associated immune cells, we performed single-cell sequencing on peripheral and tumor-infiltrating immune cells in three renal clear cell carcinoma patients. We chose renal clear cell carcinoma tumors based on the responsive of these tumors to immune checkpoint blockade in the context of low mutational loads, which implies a strong influence from the tumor microenvironment. Using the 10x Genomic 5    expression platform a total of 25,672 immune cells were isolated and passed filtering for quality control, with 13,433 cells from peripheral blood and 12,239 tumor-infiltrating cells. In addition, we used the Chromium Single Cell V(D)J kit to enrich for T cell receptor sequences for nearly 10,000 T cells in with accompanying expression information, allowing for the investigation of clonality and transcriptional phenotypic diversity.

overall_design:
5' single-cell RNA sequencing performed on peripheral blood and tumor-infiltrating immune cells
Single-cell RNA sequencing was performed on immune cells from three patients using the 10x genomics 5-prime expression profiling kits and TCR seauences for T cells.

first: Nicholas

last: Borcherding


title: Single-cell sequencing of peripheral blood and tumor-infiltrating immune cells in renal clear cell carcinoma [VDJ sequencing]

summary:
In order to investigate heterogeneity and dynamics of cancer-associated immune cells, we performed single-cell sequencing on peripheral and tumor-infiltrating immune cells in three renal clear cell carcinoma patients. We chose renal clear cell carcinoma tumors based on the responsive of these tumors to immune checkpoint blockade in the context of low mutational loads, which implies a strong influence from the tumor microenvironment. Using the 10x Genomic 5    expression platform a total of 25,672 immune cells were isolated and passed filtering for quality control, with 13,433 cells from peripheral blood and 12,239 tumor-infiltrating cells. In addition, we used the Chromium Single Cell V(D)J kit to enrich for T cell receptor sequences for nearly 10,000 T cells in with accompanying expression information, allowing for the investigation of clonality and transcriptional phenotypic diversity.
We generated a comprehensive immune profile using single-cell RNA-seq data from 25,000 immune cells from three renal cell carcinoma along with matched peripheral blood. In addition, we performed VDJ sequencing on the isolated single T cells.

overall_design: T cell receptor sequences of the subset of T cells in the 5' RNA expression sequencing dataset.


title: Single-cell sequencing of peripheral blood and tumor-infiltrating immune cells in renal clear cell carcinoma


title: Co-inhibition of CD73 and a2bar improves long-term cigarette smoke exposure impaired airway wound repair

status: Public on Oct 08 2020

city: Tampa

first: Feng

last: Cheng

state: FL

address: 12901 Bruce B Downs Blvd MDC 30

department: Department of Pharmaceutical Science

institute: University of South Florida

summary: Adenosine (ADO) involvement in lung injury depends on the activation of its receptors (A1, A2A, A2B, A3). The A2A receptor (A2AAR) and A2B receptor (A2BAR) are best described to have both tissue-protective and tissue-destructive processes. However, no approach has been effective in delineating the mechanism(s) involved with ADO shifting from its tissue-protective to tissue-destructive properties in chronic airway injury. Using cigarette smoke (CS) as our model of injury, we hypothesized that chronic CS exposure increases CD73-generated ADO which, activates low affinity A2BAR, subsequently impairing airway wound repair. To test this hypothesis, we chronically exposed human bronchial epithelial cell line, Nuli-1 cells, to 5% CS extract (CSE) for three years establishing a long-term CSE exposure model (LTC). We found significant morphological changes, decreased proliferation, and migration resulting in impaired airway wound closure in LTC. Further investigations showed that CSE exposure upregulates CD73 and A2BAR expression, increases ADO production, inhibits PKC alpha activity and phosphorylation of MEK, ERK, p90RSK and CREB. Moreover, when both or either of A2BAR and CD73 are knocked down in LTC, PKC alpha is activated and there is an increase in phosphorylation of MEK, ERK, p90RSK and CREB. Collectively, long-term CSE exposure upregulates CD73 expression and increases ADO production, which promotes A2BAR activation and subsequent diminution of PKC alpha activity and ERK signaling pathway, and inhibition of airway wound repair. Moreover, the data suggested that targeting both A2BAR and CD73 as potential therapeutic targets may be more efficacious in improving chronic CS impaired wound repair than either alone.

overall_design: Nuli-1 cells were treated 1 day in normal media, and then two days in media containing 5% CSE, cells (designated LTC) were passaged every 3 days. Control Nuli-1 cell (designated LTM) culture in normal media and passaged at same time as LTC. Both LTM and LTC are cultured for more than 3 years.

first: Zhi

last: Tian

first: Jendayi

last: Dixon

first: Qianjin

last: Liao

first: Yujuan

last: Zhou

first: Diane

last: Allen-Gipson


title: Arg C and Linezolid on Th17 cells

status: Public on Oct 15 2020

city: Mainz

first: Tim

middle: X

last: Sparwasser

state: Mainz

address: Langenbeckstr. 1

institute: University Medical Center of the Johannes Gutenberg-University Mainz

summary: Arg C and Linezolid were added to differentiating Th17 cells for 96h. Cells were harvested and sorted according to proliferation cycle before being lysed and having their RNA extracted.

overall_design: Arg C (P2A7) and Linezolid were added to differentiating Th17 cells for 96h. Cells were harvested and sorted according to proliferation cycle before being lysed and having their RNA extracted. Samples named e1 correspond to one experiment and sampled named e2 correspond to another experiment. The following 4 characters indicate the drug (DMSO for vehicle, P2A7 for Arg C, and Line for Linezolid). The last character indicates the proliferation cycle (2 for 1 cellular division, 3 for 2 divisions, and 4 for 3 or more divisions).

first: Luis

last: Almeida

first: Ayesha

last: Dhillon-LaBrooy

first: Nigatu

last: Adossa


title: A discrete subset of epigenetically primed human NK cells mediates antigen-specific immune responses

city: Vienna

first: Georg

last: Stary

address: Spitalgasse 23

department: Dermatology

country: Austria

institute: Medical University of Vienna

summary: Adaptive features of natural killer (NK) cells have been reported in various species with different underlying mechanisms. It is unclear, however, which NK cell populations are capable of mounting antigen-specific recall responses, and how such functions are regulated at the molecular level. Here we identify and characterize a discrete population of CD49a+CD16- NK cells in the human liver that display increased epigenetic potential to elicit memory responses and has the functional properties to exert antigen-specific immunity on the skin as an effector site. Integrated chromatin-based epigenetic and transcriptomic profiling revealed unique characteristics of hepatic CD49a+CD16- NK cells when compared to conventional CD49a-CD16+ NK cells thereby defining active genomic regions and molecules underpinning distinct NK cell reactivity. In contrast to conventional NK cells, our results suggest adaptive CD49a+CD16- NK cells to be able to bypass the KIR receptor-ligand system upon antigen-specific stimulation. Furthermore, these cells were highly migratory towards chemokine gradients expressed in epicutaneous patch test lesions as an effector site of adaptive immune responses of the skin. These results define pathways operative in human antigen-specific memory NK cells and provide a roadmap for harnessing this NK cell subset for specific therapeutic or prophylactic vaccine strategies.

overall_design:
Bulk RNA sequencing and ATAC-seq sequencing of human liver-derived CD49a+CD16- NK cells and CD49a-CD16+ NK cells upon stimulation with IL-2 (100Ui/ml) and IL-15 (10ng/ml) for 4 hours. RNA sequencing data of CD45+ cells isolated from human liver.

This series includes three liver samples enriched for CD45+ cells included, all from different donors. Human liver tissue was flushed with PBS, cut into small pieces with a sterile scalpel, passed through 70-um mesh filters and rinsed with 10% FBS in PBS. Leukocytes of the liver were isolated by density gradient centrifugation using Ficoll gradients. For flow cytometry analysis or cell sorting, cells were stained with fluorescence antibodies.

first: R

middle: V

last: Pandey

first: V

first: M

last: Farlik


title: Rescue of Male Infertility through Correcting a Genetic Mutation Causing Meiotic arrest in Spermatogonial Stem Cells

status: Public on Oct 05 2020

first: Jinsong

last: Li

address: 320 岳阳路，上海

institute: 中国科学院上海分子细胞科学卓越创新中心


title: Rescue of Male Infertility through Correcting a Genetic Mutation Causing Meiotic arrest in Spermatogonial Stem Cells [WGS]

institute: 中国科学院上海分子细胞科学卓越创新中心

summary: Azoospermia patients who carry a monogenetic mutation that causes meiotic arrest may have their biological child through genetic correction in spermatogonial stem cells (SSCs). However, such therapy for infertility has not been experimentally investigated yet. In this study, a mouse model with X-linked TEXT11 mutation (Tex11PM/Y) identified in azoospermia patients exhibited meiotic arrest due to aberrant chromosome segregation. Text11PM/Y SSCs could be isolated and expanded in vitro normally and the mutation was corrected by CRISPR-Cas9, leading to generation of repaired SSC lines. Whole-genome sequencing demonstrated that mutation rate in repaired SSCs are comparable with autonomous mutation in untreated Text11PM/Y SSCs, and no predicted off-target sites are modified. Repaired SSCs could restore spermatogenesis in infertility males and give rise to fertile offspring at a high efficiency. In summary, our study establishes a paradigm for the treatment of male azoospermia by combining in vitro expansion of SSCs and gene therapy.

overall_design: Whole genome sequencing (WGS) analysis of the corrected SSCs

first: Ying

middle: H

last: Wang

first: Meng

last: Yan

first: Xi


title: Transciptional profile analysis of the gonadal (peri-uterine) white adipose tissue of pregnant mice

first: Magdalena

last: Paolino

address: Dr.-Bohr-Gasse 9

institute: Institute of Molecular Biotechnology

summary: In this study we performed RNA sequencing to determine the transcriptome of the gonadal white adipose tissue of pregnant wild type mice. We further assessed, using conditional RankΔFoxn1 knockout mice (which lack expression of Rank in the thymic epithelia), how the expression of the receptor Rank in the thymic medullary epithelia affects the transcriptional program of this tissue during pregnancy, and whether adoptive transfer of natural regulatory Tregs can rescue the alterations observed in the RankΔFoxn1 dams. For this study, we isolated at day E17.5 of pregnancy, the gonadal (peri-uterine) white adipose tissue of wildtype RankWt as well as of RankΔFoxn1 dams pregnant females that were treated at days E0.5-2.5 of pregnancy with either vehicle (PBS) or with 1x105 sorted-purified wild-type natural Tregs isolated from RankWt pregnant E7.5-8.5 donors (denoted as RankΔFoxn1_Treg). For the isolation of viable Tregs for the transplant, we generated RankWt dams that express GFP from the Foxp3 promoter (by crossing to reporter mice), and use the expression of GFP to mark and isolate bona fide Tregs and, in addition, Neuropilin1 to denote the thymic origin of the Tregs. Of note, RankWt as well as of RankΔFoxn1 dams mice also carried the GFP-Foxp3 transgene and where treated with vehicle solution (PBS) at day E0.5-2.5. Total RNA was extracted from the isolated gonadal white adipose tissue of the three described cohorts (RankWt_veh, RankΔFoxn1_veh, and RankΔFoxn1_Treg) and analyzed by Quant-seq. Our study shows how the presence of Rank receptors in the thymic epithelium, functioning via natural Tregs, regulate the inflammatory and metabolic pathways implicated in adipose tissue biology during pregnancy.

overall_design: 3' RNA-Seq (Quantseq) analysis of gonadal (peri-uterine) adipose tissue of E17.5 pregnant mice

first: Josef

middle: M

last: Penninger


title: Mechanisms underlying growth inhibition of a denitrifier by reduced sulfur compounds

status: Public on Oct 01 2020

city: Seattle

first: Alex

last: Carr

address: 401 Terry Ave N

institute: Institute for Systems Biology (ISB)

summary: Competition among nitrate reducing bacteria (NRB) and sulfate reducing bacteria (SRB) for resources in anoxic environments is generally thought to be governed largely by thermodynamics. It is now recognized that intermediates of nitrogen and sulfur cycling (e.g., hydrogen sulfide, nitrite, etc.) can also directly impact NRB and SRB activities in freshwater, wastewater and sediment, and therefore may play important roles in competitive interactions. Here, using Intrasporangium calvum C5 as a model NRB, we performed comparative transcriptomic and metabolomic analyses to demonstrate that the reduced sulfur compounds cysteine and sulfide differentially inhibit respiratory growth on nitrate, and that inhibition by each can be selectively relieved by a specific carbon source.  These findings provide mechanistic insights into the interplay and stratification of NRBs and SRBs in diverse environments.

overall_design: Time series mRNA profiling of a bacterial strain subjected to reduced sulfur stress. Three conditions were tested, a reference (Regular) and four treatments. Each consists of measurements at four growth phases and were made in triplicate. Multiple sequencing runs were performed to obtain enough reads (e.g., L001, L002, L003, L004).

first: Annie

last: Otwell


title: Abnormal neocortex arealization and Sotos-like syndrome-associated behavior in Setd2 mutant mice

city: Wuhan

first: Lichao

last: Xu

address: luojiashan

institute: Wuhan University

summary: Epigenetic regulation of gene expression through histone modifications like methylation of various lysine residues are essential for embryonic development. Here we removed SETD2, a methyltransferase for histone 3 lysine 36 trimethylation (H3K36me3), in the developing dorsal forebrain in mice and show it is required for proper area patterning (arealization) of the neocortex and the formation of thalamo-cortico-thalamic circuits by maintaining the expression of clustered protocadherin (Pcdh) genes in an H3K36me3 methyltransferase-dependent manner. Moreover, the Setd2 mutant mice exhibit defects in social interaction, motor endurance and spatial memory, reminiscent of patients with the Sotos-like syndrome bearing SETD2 mutations.

overall_design: Cortex mRNA profiles of E13.5 control and Emx1-Cre;Setd2 fl/fl mice were generated by deep sequencing, in triplicate, using Illumina NovaSeq 6000.

first: Yue

last: Zheng

first: Xuejing

first: Andi

first: Dawei

last: Huo

first: Qinglan

first: Shikang

first: Zhiyuan

last: Luo

last: Liu

first: Fuqiang

first: Xudong

last: Wu

first: Min

first: Yan


title: Werner Helicase Prevents Cell Death in Cancers with Microsatellite Instability (END-Seq)

city: Bethesda

first: Andre

last: Nussenzweig

state: MD

address: 9000 Rockville Pike, Building 37

department: National Cancer Institute

institute: National Institutes of Health

summary: Our study uncovers a distinct biomarker for MSI that underlies the synthetic lethal dependence on WRN, thereby supporting the development of WRN-based therapeutics

overall_design: ENDseq was performed under different experimental condition in MSI and MSS cells to uncovers sites of recurrent DNA damage genome-wide

first: Niek

last: Van Wietmarschen

first: Sriram

last: Sridharan


title: Werner Helicase Prevents Cell Death in Cancers with Microsatellite Instability [PacBio]

overall_design: Pacbio CLR was performed under different experimental condition in MSI and MSS cells to uncover expansions/deletions at TA dinucleontide repeats genome-wide


title: Rescue of Male Infertility through Correcting a Genetic Mutation Causing Meiotic arrest in Spermatogonial Stem Cells [RNA-seq]

city: Shanghai

first: Jinsong

institute: 中国科学院上海分子细胞科学卓越创新中心

overall_design: Examination of transcriptome in two SSCs

first: Ying

first: Meng

first: Xi

last: Zhang


title: Straightforward extraction of high-quality RNA from glyoxal-fixed cells facilitates transcriptome analysis after cell staining and sorting by flow cytometry

status: Public on Sep 30 2020

first: Felix

last: Krueger

address: Granta Park

department: Bioinformatics

institute: Altos Labs

summary: We have developed a methodology for isolation of high-quality RNA from cells that are fixed, stained and sorted by flow cytometry that allows routine transcriptomic analysis of highly purified cell populations and single cells. This method, essentially developed by modifying existing staining and sorting protocols, involves fixation of cells with glyoxal, an alternative fixative to commonly used formaldehyde, followed by methanol permeabilization, a 2-step primary and secondary antibody staining and sorting by flow cytometry. The advantage of using glyoxal is that it does not crosslink RNA to proteins nor form stable RNA adducts, ensuring that RNA remains accessible and amenable to enzymatic manipulation after glyoxal fixation. The dataset comprising mRNA seq libraries from unprocessed or fixed and stained human cancer cells demonstrate that RNA recovered from glyoxal-fixed cells does not retain sufficient glyoxal adducts to impair reverse transcription, and also reveal very few differentially expressed genes between the 2 groups. The dataset derived from fixed and stained cells that were sorted into CCNB1 positive or negative fractions show the applicability of this method for sorting highly purified cell fractions as the CCNB1 positive fractions show strong enrichment for G2-phase cells according to the GO analysis.

overall_design: RNA-seq libraries from COLO205 and MCF7 human cancer cells that were unprocessed or fixed and stained for CCNB1, and from MCF7 cells that were stained and flow sorted into CCNB1 positive and CCNB1 negative fractions.

first: Prasanna

last: Channathodiyil

first: Jonathan

last: Houseley


title: Epithelial cells in 2D and 3D culture exhibit significant differences in long-range genomic interactions [RNA-Seq]

first: Xin

address: 800 Dong Chuan Rd.

department: School of Biomedical Engineering

institute: Shanghai Jiaotong University

summary: Recent studies have characterized the genomic structures of many eukaryotic cells, often with a focus on their relation to gene expression. So far, these studies have largely only investigated cells grown in 2D culture, although the transcriptomes of 3D cultured cells are generally closer to their in vivo phenotype. To examine the effects of spatial constraints on chromosome conformation, we investigated the genomic architecture of mouse hepatocytes grown in 2D and 3D cultures using in situ Hi-C. Our results reveal significant differences in long-range genomic interactions, notably in compartment identity and strength as well as in TAD-TAD interactions, but only minor differences at the TAD level. RNA-seq analysis reveals an up-regulation in the 3D cultured cells of those genes involved in physiological hepatocyte functions. We find that these genes are associated with only a subset of the structural changes, suggesting that the differences in genomic structure are indeed critically important for transcriptional regulation but also that there are major structural differences owing to other functions than gene expression. Overall, our results indicate that growth in 3D significantly alters longer-range genomic interactions, which may be consequential for a subset of genes that are important for the physiological functioning of the cell.

overall_design: The genome-wide interactome were generated using in-situ Hi-C method in both ltwo-dimensional (2D) and three-dimensional (3D) cultured AML12 cells which were arrest at G1/S

first: X

first: Q

last: Sun

first: Z

last: Shao


title: Epithelial cells in 2D and 3D culture exhibit significant differences in long-range genomic interactions


title: Werner Helicase Prevents Cell Death in Cancers with Microsatellite Instability (WGS)


title: Werner Helicase Prevents Cell Death in Cancers with Microsatellite Instability (ChIP-Seq)


title: Systematic Analysis of Transcription Factors Binding to Noncoding Variants

city: La Jolla

first: Yunjiang

last: Qiu

state: CA

address: 9500 Gilman Drive

institute: University of California, San Diego

summary: A large number of sequence variants have been linked to complex human traits and diseases, but deciphering their biological functions is still challenging since most of them reside in the noncoding DNA. To fill this gap, we have systematically assessed the binding of 270 human transcription factors (TF) to 95,886 noncoding variants in the human genome using an ultra-high-throughput multiplex protein-DNA binding assay, termed SNP evaluation by Systematic Evolution of Ligands by EXponential enrichment (SNP-SELEX). The resulting 828 million measurements of TF-DNA interactions enable estimation of the relative affinity of these TFs to each variant in vitro and allow for evaluation of the current methods to predict the impact of noncoding variants on TF binding. We show that the Position Weight Matrices (PWMs) of most TFs lack sufficient predictive power, while the Support Vector Machine (SVM) combined with the gapped k-mer representation show much improved performance, when assessed on results from independent SNP-SELEX experiments involving a new set of 61,020 sequence variants. We report highly predictive models for 94 human TFs and demonstrate their utility in genome-wide association studies (GWAS) and understanding of the molecular pathways involved in diverse human traits and diseases.

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
synthetic construct

overall_design: 768 experiments with HT-SELEX for six SELEX cycles to measure allelic TF binding for 95,886 SNPs. TF ChIP-seq and RNA-seq were performed in HepG2 cells to validate allelic TF binding. STARR-seq experiments were performed to identify SNPs that affect enhancer activity in HepG2 and HEK293 cells with three replicates. In situ Hi-C experiments were performed in HepG2 cells and human islets to identify target genes of SNPs.

first: Jian

last: Yan

first: André

last: Ribeiro dos Santos

first: Paola

last: Benaglio

first: Bing

last: Ren


title: Epithelial cells in 2D and 3D culture exhibit significant differences in long-range genomic interactions [Hi-C]


title: Werner Helicase Prevents Cell Death in Cancers with Microsatellite Instability

first: Andre

overall_design: Refer to individual Series


title: Metagenomic sequencing and 16S rRNA sequence of gut microbiota associated with autism spectrum disorders patients

city: Nanjing City

first: Xingyin

state: Jiangsu Province

address: Long Mian Street No.1

institute: Nanjing Medical University


title: Transient ACE Inhibition Suppresses Future Fibrogenic Capacity and Heterogeneity of Cardiac Fibroblast Subpopulations

city: Phoenix

first: Taben

middle: Mary

last: Hale

state: AZ

address: 425 N 5th St

department: Basic Medical Sciences

institute: University of Arizona

summary: Transient angiotensin converting enzyme (ACE) inhibition in spontaneously hypertensive rats (SHR) protects against future injury-induced cardiac inflammation, fibrosis, and dysfunction. Here, we used single cell RNA sequencing (scRNA-Seq) to test the hypothesis that transient ACE inhibitor treatment would induce a persistent shift in hypertensive cardiac fibroblast subpopulations. Adult male SHRs (11 weeks old) were treated for 2 weeks with an ACE inhibitor, enalapril (30mg/kg/day, p.o.), or water (control) followed by a 2-week washout period (n=7/group). Cardiac fibroblasts were isolated from the left ventricle and subjected to scRNA-Seq. Nine clusters of fibroblasts (numbered 0-8) were identified with 98% of cells clustering into subsets 0-6. Transient treatment produced significant changes within and across clusters. Cluster 1 depicted the highly fibrogenic gene profile, with cluster 6 serving as a gateway to cluster 1. Transient ACE inhibition depleted the gateway and expanded cluster 0, which was the least fibrogenic profile. Moreover, within cluster 1 fibroblasts, ACE inhibition reduced expression of individual fibrosis genes (e.g. COL1A1, COL3A1, and FN1; all p<0.05). Clusters 2-5 reflected proliferative, moderately fibrogenic, translationally active, and lowly inflammatory subsets of fibroblasts, all of which exhibit attenuated fibrogenic gene expression after transient ACE inhibition. In conclusion, transient ACE inhibition shifts cardiac fibroblast subpopulations and degree of activation resulting in an overall reduced fibrogenic phenotype.

overall_design: Single cell RNA-sequencing of cardiac fibroblasts from SHR with and without prior transient ACE inhibitor treatment.

first: Alexandra

last: Garvin

first: Matthew

middle: D

last: De Both

first: Joshua

last: Talboom

first: Merry

last: Lindsey

middle: J

last: Huentelman


title: MicroRNA Regulation of Oxidative Stress, Proliferation, and Neural Tube Closure

status: Public on Sep 24 2020

city: Houston

first: Brittney

last: Lozzi

state: TX

address: 1 Baylor Plaza

department: Cell & Gene Therapy

institute: Baylor College of Medicine

summary: single-cell sequencing of the mouse cranial region at E8.25 (the start of neural tube closure, at E9.5 (the end of neural tube closure, and of a miR-302 knockout embryo at E9.5 (example of neural tube closure defrect).

overall_design: The cranial region anterior to the otic placode was dissected for wildtype embryos at E8.25 and E9.5 and for a miR-302 knockout embryo at E9.5. Cells were dissociated with papain, quenched with FBS. Filtered and resuspended in 30ul of DMEM +10%FBS. Cell suspension was put into 10x genomics V3 chemistry for single cell 3' sequencing.

first: Rachel

last: Keuls


title: Metagenomic sequencing of gut microbiota associated with autism spectrum disorders patients

last: Liu

summary: Here we report metagenomic sequencing data in gut microbiota of  autism spectrum disorders (ASD) compared with healthy volunteers (30 for ASD children and 30 for healthy controls, respectively).  The genes changed in autistic subjects involved 1,312,364 analytes that compare to 1,335,835 analytes in healthy controls.   The number of taxa in autistic subjects were significantly increased as compared to the healthy controls based on the phylum and genus level (P = 0.001). However, the number of species were significantly decreased in autistic subjects (P = 0.001).

overall_design:
Microbial DNA from fecal samples [healthy control (n=30), ASD patients (n=30)] were collected. A total amount of 1μg DNA per sample was used as input material for the DNA sample preparations. The DNA sample was fragmented by sonication to a size of 350 bp, then end-polished, A-tailed, and ligated with the full-length adaptor for Illumina sequencing with further PCR amplification.

Subject IDs (e.g. A3) in this study represent the same individual with identical id in GSE113690.

first: Xuhua

last: Mao

first: Qisha

first: Zhou

last: Dan


title: Deficiency of Serotonin Receptor 6 (5-HT6R) or/and its constitutive activity leads to the reduced neurogenesis and the depression-like behavior

status: Public on Sep 21 2020

city: shanghai

first: wang

last: ying

address: 320 yueyang road

institute: shanghai instiutes for biological science chinese acadeema of science

summary: Here, we reported that the genetic knock-out of 5-HT6R or specific knock-down of the receptor constitutive activity led to the depression-like behavior in mice. This was likely due to the impaired hippocampal neurogenesis and progressive loss of the neural stem cell (NSC) pool. Furthermore, the critical role of receptor and constitutive activity was confirmed in human NSC function with genetic and chemical modulation, and were well reproduced in human cerebral organoids. The following mechanistic study revealed that cAMP-CREB signaling was involved in the 5-HT6R regulation and its constitutive activity keeps the basal cAMP level.

overall_design: Human neural stem cell mRNA profiles of treated by vehicle/st1936/sb271046 were generated by deep sequencing, in duplicate, using  Illumina Hiseq platform.

first: Huiying

last: Qiao


title: Genome-wide maps of chromatin state in pluripotent and lineage-committed cells.

status: Public on Sep 23 2020

city: Jerusalem

first: Adi

last: Alajem

address: Givat Ram Campus

department: Biological Chemistry

institute: The Hebrew University of Jerusalem

summary: We applied sequential ChIP-bisulfite-sequencing for H3K4me1 and H3K27ac in mouse embryonic stem cells (ESCs). By obtaining over ~4*10^9  bases of sequence from chromatin immunoprecipitated DNA which was bisulfite treated, we identified enhancers that were differentially methylated between these two marks. We found a global gain of CpG methylation, primarily in H3K4me1-marked nucleosomes during mouse ESC differentiation. This gain occurred largely in enhancer regions that regulate genes critical for differentiation. We showed that higher levels of DNA methylation in H3K4me1 comparing to H3K27ac indicates cellular heterogeneity of enhancer states. We then used single-cell RNA-seq profiles to show that this heterogeneity correlates with gene expression during ESC differentiation. Furthermore, we showed that heterogeneity of enhancer methylation correlates with transcription start site methylation. Our results provide insights into enhancer-based functional variation in complex biological systems.

overall_design: Examination of H3K4me1 and H3K27ac DNA that was sequentially bisulfite treated in embryonic stem cells and differentiated cells

first: Shlomtzion

last: Lahav

first: Sofia

last: Ratgauzer

first: Hava

last: Roth

first: Danny

last: Bavli

first: Alex

last: Motzik

first: Itay

last: Peled

first: Oren

last: Ram


title: Novel Immune Modulators Enhance Caenorhabditis elegans Resistance to Multiple Pathogens

status: Public on Sep 19 2020

first: Natalia

last: Kirienko

address: 6100 Main St. MS140

department: BioSciences

institute: Rice University

summary: Traditional treatments for bacterial infection have focused upon directly inhibiting growth of the pathogen. However, an equally important determinant of infection outcome is the host defense response. We previously performed a high-throughput chemical screen to identify small molecules that rescued the nematode Caenorhabditis elegans from infection by Pseudomonas aeruginosa. Over 20 of the hits stimulated host defense gene expression. During in-depth studies of five such molecules using microarray analysis, bioinformatic clustering, and RNAi knockdown of candidate gene targets, we identified PMK-1/p38 MAPK and SKN-1/Nrf2 as two key pathways modulated by these hits. Interestingly, the molecules studied did not depend on a single pathway for ameliorating P. aeruginosa pathogenesis in liquid-based assay, but did rely on the PMK-1/p38 MAPK pathway during a colonization-based infection assay on agar. A subset of these molecules was also protective against Enterococcus faecalis and Staphylococcus aureus. In general, the compounds showed little toxicity against mammalian cells or worms, consistent with their identification in a phenotypic, high-content screen. These molecules possess significant potential for use as tools to study innate immune processes

overall_design: There are 18 samples total that comprise three biological replicates of N2 young adult animals exposed to LK32, LK34, LK35, LK38, LK56, or DMSO control


title: Fecal Exosomes treatment of B6 mice for 14 days

city: LOUISVILLE

first: Anil

last: Kumar

state: Kentucky

address: 505 S Hancock ST, 333 CTRB

department: Microbiology & Immunology

institute: University of Louisville

summary:
Fecal Exosomes from RCD and HFD (isolated from 12 months of their respective diets and purified by sucrose gradient) were orally gavaged to B6 mice for 14 days while they fed HFD. These exosomes were characterized for CD63+A33 duol positivity.
After 14 days, liver tissue were harvested. RNA was isolated and sent to invitorgen for Affymetrix array. The purpose of the experiment was to see which set of genes go up or down after these exosomes treatment and their link to metabolic syndrome.

overall_design: We used affymetric array to see differential gene expression after the fecal exosomes treatment in B6 mice.

first: Yun

last: Teng

first: Huang-Ge

last: Zhang


title: Abnormal neocortex arealization and Sotos-like syndrome-associated behavior in Setd2 mutant mice

first: Lichao

last: Xu

overall_design: H3K36me3 ChIP-seq profiles of E13.5 control and Emx1-Cre;Setd2 fl/fl mice cortex were generated by deep sequencing, in two replicates, using Illumina Hi-seq X Ten.

first: Yue

last: Zheng

first: Xuejing

last: Li

first: Andi

last: Wang

first: Dawei

last: Huo

first: Qinglan

first: Shikang

first: Zhiyuan

last: Luo

first: Ying

last: Liu

first: Fuqiang

first: Xudong

last: Wu

first: Min

first: Yan

last: Zhou


title: Iron deficiency response of interactions between Brachypodium and Pseudomonas fluorescens SBW25 under controlled growth conditions.

status: Public on Sep 22 2020

city: Corvallis, OR

first: Rene

last: Boiteau

state: OR

address: 104 CEOAS Administration Building

institute: Oregon State University

summary: The goal of this study was to evaluate the molecular mechanisms by which Brachypodium distachyon grown with and without Pseudomonas fluorescens (P. fluorescens) strain SBW25 respond to Fe deprivation. Fe deprivation induced Brachypodium secretion of phytosiderophores and reduced biomass production while inoculation with P. fluorescens resulted in alterations of extracellular metabolite abundances. Results provide insight into the role of iron in interactions between a host plant and root associated bacteria.

sample_organism:
Pseudomonas fluorescens
Brachypodium distachyon

overall_design: Examination of transcriptional profiles from Brachypodium distachyon BD21 and Pseudomonas fluorescens SBW25 under sterile vs inoculated with and without Fe amendment.

first: Lye

last: Markillie

first: Hugh

last: Mitchell