title: Non-coding RNAs shape cortical neurons developmental trajectories

first: Julien

last: Prados

address: Rue Michel Servet 1

department: SCMU

institute: University of Geneva

summary: A hallmark of cortical evolution is the high dynamic subventricular zone (SVZ) expansion, where basal progenitors (BPs) amplify and neuronal transcriptional programs unfold. How non-coding molecular factors such as microRNAs influence these developmental trajectories and regulate the acquisition of cortical type identities is largely unknown. Here we demonstrate that miR-137 and miR-122 regulate the positioning and identity features of superficial layer cortical neurons by acting at distinct steps of their developmental trajectories. MiR-137 sustains basal progenitor amplification by reverting their neurogenic commitment and inducing high proliferative state upregulating Cd63 and inhibiting Myt1l. Cd63 is an extra-cellular matrix (ECM) receptor which interacts with b3- and 1-integrin pathways to promote proliferation, while Myt1l is a transcription factor that promotes and sustains neuronal fate. The BPs amplification by miR-137 is converted in the promotion of intracortical projecting neuron (ICPN) identity and L2/3 expansion. As opposed to miR-137, miR-122 acts postmitotically, affecting the bioelectrical properties, the calcium and cytoskeleton dynamics of newborn neurons as well as their transcriptional program, leading to a persistent molecular immaturity across time. Overall, these findings reveal that miR-137 and miR-122 are key regulators of the developmental trajectory of cortical neurons across evolution.

overall_design: Investigation of the non-coding RNAs miR-122 and miR-137 overexpression on progenitor and neuron transcriptional programs.   psil-miR122, psil-miR137 or psil-scramble (control) plasmids were in utero electroporated together with pUB-GFP or pUB-Tomato plasmid in mouse embryos at embryonic day (E) 14.5, and cells collected at E15.5, E17.5 or postnatal day (P) 7 using fluorescence activated cell sorting (FACS).   For comparison with layer 4 and layer 2/3 control cell molecular identities, pUB-GFP/Tomato plasmid was in utero electroporated at E14 and E15.5 respectively and cells collected at P3 and P7 using FACS.   Collected cells were then processed for single-cell transcriptomics.  psil-miR122 was in utero electroporated together with pUB-GFP plasmid in mouse embryos at E14.5; superficial and deep layers were microdissected at P7 and cells collected using FACS before being processed for bulk RNA sequencing.

first: Ugo

last: Tomasello

first: Esther

last: Klingler

first: Mathieu

last: Niquille

first: Nandkishor

last: Mule

first: Laura

last: de Vevey

first: Antonio

last: da Silva Santinha

first: Victor

last: Borrell

first: Alexandre

last: Dayer

first: Denis

last: Jabudon


title: Higher-order chromatin organization defines PR and PAX2 binding to regulate endometrial cancer cell gene expression (ATACseq)

status: Public on Mar 09 2022

city: buenos aires

first: Patricia

last: Saragüeta

state: CABA

address: Vuelta de Obligado 2490

country: Argentina

institute: IByME

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Estrogen (E2) and Progesterone (Pg) via their specific receptors, ER and PR respectively, are major determinants in the development and progression of endometrial malignancies. We have studied how E2 and the synthetic progestin R5020 affect genomic function in Ishikawa endometrial cancer cells. Using ChIPseq in cells exposed to the corresponding hormones, we identified cell specific binding sites for ER (ERbs) and PR (PRbs), mostly binding to independent sites and both adjacent to PAXbs. Long-range interactions (HiC) showed enrichment of PRbs and PAXbs, which we call progestin control regions (PgCRs) inside TADs with differentially progestin-regulated genes. Effects of hormone treatments on gene expression were detected by RNAseq. PgCRs correlate with open chromatin independently of hormonal stimuli. In summary, endometrial response to progestins in differentiated endometrial tumor cells results in part from binding of PR to compartmentalized PgCRs in hormone-independent open chromatin, which include binding of partner transcription factors, in particular PAX2.

overall_design: Endometrial adenocarcinoma Ishikawa cells were treated with R5020 10nM or E2 10nM for different times and submitted to ATACseq.

first: Alejandro

last: La Greca


title: Single cell analysis informs efficient reprogramming of human somatic cells to dendritic cells type 1

status: Public on Mar 08 2022

city: Lund

first: Evelyn

last: Halitzki

address: Sölvegatan 17

country: Sweden

institute: Lund University

summary: Type-1 dendritic cells (DC1s) are critical cellular mediators of anti-tumor immunity, as their ability to prime CD8+ T-cells is crucial for response to checkpoint blockade and adoptive T-cell transfer. Vaccination strategies to harness this unique DC subset for immunotherapy have been limited by their rarity in peripheral blood and lack of homogeneous alternative cell sources. We have previously identified PU.1, IRF8 and BATF3 transcription factors (TF) as sufficient to induce DC1 program in mouse fibroblasts but reprogramming efficiency in human cells was low. Here, we investigated single-cell transcriptional dynamics during human DC1 reprogramming. Human induced DC1s (hiDC1s) generated from embryonic fibroblasts acquire global DC1 transcriptional profile and activate antigen presentation signatures. Interestingly, other DC subsets are not induced at the single cell level. We extracted gene modules associated with successful reprogramming and identified inflammatory signaling and the DC1 reprogramming TF network as key drivers of the process. Combining inflammatory cytokine signaling with TF constitutive overexpression lead to improved reprogramming efficiency by 190-fold. HiDC1s acquire the ability to uptake dead cells, respond to stimuli, secrete inflammatory cytokines and perform antigen presentation. Remarkably, intra-tumoral vaccination in mouse models increased infiltration of antigen-specific CD8+ T-cells, promoted a T-cell cytotoxic profile and conferred protection against tumor growth. Finally, we demonstrated efficient hiDC1 generation from human adult somatic cells including dermal fibroblasts and mesenchymal stem cells with a xeno-free protocol. These findings provide insights into human DC1 specification and reprogramming and represent a platform for generating patient-specific DC1s, a long-sought DC subset for cancer immunotherapy.

overall_design: Examination of global occupancy for PU.1, IRF8 and BATF3 together or individually at day 2 of cDC1 reprogramming in HDFs.

first: Fábio

middle: F

last: Rosa

first: Cristiana

last: Pires

first: Ilia

last: Kurochkin

first: Carlos-Filipe

last: Pereira


title: The NFkB protein RELA/p65 binds predominantly transcript introns and exons

city: Dundee

first: Dimitrinka

last: Ditsova

state: Scotland

address: 64 Atholl Street

department: Division of Gene Regulation and Expression

country: United Kingdom

institute: University of Dundee

summary: We investigated the ability of the NFkB protein p65 to bind RNA and what its target transcrips were. In this CLIP-seq experiment we demonstrated that p65 is capable of binding RNA and it has a preference to introns and exons, suggesting it binds unspliced RNA.

overall_design: p65 and IgG seCLIP (single-end crosslinking and immunoprecipitation) libraries were prepared from Jurkat cells. Cells were UV cross-linked at 254 nM with an energy setting of 400 mJ/cm2. IP, RNA isolation and library preparation was performed as described by Van Nostrand et al, 2018. Lysates were treated with RNase I diluted 1:150. IPs were performed with 7.5µg anti-p65 Santa Cruz antibody sc-8008 or 7.5µg mouse IgG. The chromatin was sonicated in Bioruptor (Diagenode) at low power for 5 min (30 s on/off). Antibodies were tethered to protein G magnetic beads for 1h at room temperature and afterwards, the lysates were incubated with the beads. The next day 2% of lysate+bead was set aside as input. Library prep proceeded as described in Van Nostrand et al, 2018. RNA was purified using Zymo RNA Clean and Concentrator-5. DNA libraries were extracted from agarose gels in the end using a gel extraction kit (QIAGEN). Libraries were quantified using D1000 Agligent TapeStation.

first: Victoria

middle: H

last: Cowling


title: Protein Kinase C Establishes the Signaling Boundary Between Mesodermal and Extraembryonic Lineages in Human Embryonic Stem Cell Differentiation

city: Macau

first: Guokai

last: Chen

address: University of Macau Avenida da Universidade Taipa, Macau, China

country: China

institute: University of Macau

summary: The interplay among mitogenic signaling pathways is crucial for proper embryogenesis. These pathways collaboratively act through intracellular master regulators to determine specific cell fates. Identifying the master regulators is critical to understanding embryogenesis and to developing new applications of pluripotent stem cells. In this report, we demonstrate protein kinase C (PKC) as an intrinsic master switch between embryonic and extraembryonic cell fates in the differentiation of human pluripotent stem cells (hPSCs). PKCs are essential to inducing the extraembryonic lineage downstream of various mitogenic modulators. PKC-alpha (PKCα) suppresses BMP4-induced mesoderm differentiation, and PKC-delta (PKCδ) is required for extraembryonic trophoblast cell fate. PKC activation overrides mesoderm induction conditions and leads to extraembryonic fate. In contrast, PKC inhibition leads to β-catenin activation, switching cell fate from extraembryonic to mesoderm lineages. This study establishes PKC as a central player directing the segregation of extraembryonic and embryonic lineages. The manipulation of intrinsic PKC activity could greatly enhance cell differentiation under mitogenic regulation in stem cell applications.

overall_design: Total RNA obtained from H1 hESC cultured in E6 medium supplemented with 20 ng/ml BMP4, 100ng/ml FGF2, 5 μM GF109203X, 3 mM 2DG, 100 nM LDN193189, 5 μM CHIR99021, 50 nM TPA, 1 μM PD0325901, 10 μM SB431542 and 5 μM DAPT. Samples were collected after 6 days.


title: Analysis of estrogen-induced mRNA in endometrial cancer cells with forced expression of ER

city: Hirosaki

first: Kayo

last: Horie

address: 66-1 Hon-cho

country: Japan

institute: Hirosaki university

summary: We used the endometrial adenocarcinoma cell line in which the ERα cDNA gene was transfected and forcedly expressed ER. Then the factors induced by estrogen (Estradiol: E2) were measured by microarray.

overall_design: Ishikawa cells were transfected ERα cDNA gene. After 24hr hours , E2 was added at a concentration of 1X10-7 M or the same amount of ethanol was added to the control group. RNA was recovered from cells 24h, 48h and 72h after E2 was added, and comprehensive analysis was performed by microarray.


title: p65

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

overall_design: Refer to individual Series


title: Response of MET mutant or WT cells to HGF and/or Cabozantinib treatment

city: Vancouver

first: William

last: Lockwood

state: British Columbia

address: 675 West 10th Avenue

department: Integrative Oncology

country: Canada

institute: BC Cancer

summary: Targeted therapies for MET exon 14-skipping (METΔex14)-driven lung cancers have generated some promising results but response rates remain below that seen for other kinase-driven cancers. One strategy for improving treatment outcomes is to employ rational combination therapies to enhance the suppression of tumour growth and delay or prevent the emergence of resistance. To this end, we profiled the transcriptomes of MET-addicted lung tumours and cell lines and identified the RAS-mitogen-activated protein kinase (MAPK) pathway as a critical effector required for METΔex14-dependent growth. Ectopic expression MET in an isogenic cell line model showed that overexpression of the mutant METΔex14 receptor led to higher levels of MAPK phosphorylation and nuclear import, resulting in increased expression and phosphorylation of nuclear MAPK targets. In comparison, other known MET effectors were unaffected. Inhibition of this pathway by KRAS knockdown in MET-addicted cells in vitro led to decreased viability in only the METΔex14-expressing cells. Conversely, decoupling RAS-MAPK axis, but not other effector pathways, from MET activity via the introduction of constitutively active mutants conferred resistance to MET inhibitors in vitro. Our results suggest that aberrant hyperactivity of the MET receptor caused by the exon 14-skipping mutation does not uniformly upregulate all known downstream effectors, rather gaining a predilection for aberrantly activating and subsequently relying on the RAS-MAPK pathway. These findings provide a rationale for the co-targeting of the RAS-MAPK pathway alongside MET to prolong therapeutic response and circumvent resistance to improve patient survival.

overall_design: Three MET-addicted cancer cell lines (H1993, Hs746T, and H596) were treated with either 1) 0.1% DMSO, 2) 0.1% DMSO + 25ng/mL HGF, or 3)10μM Cabozantinib + 25ng/mL HGF. Following a 6 hour incubation with the indicated treatment, RNA was harvested for expression analysis using Affymetrix arrays. In all, three biological replicates were analyzed for each gene under the three treatment conditions for each cell line, for a total of 27 samples.

middle: W

first: Daniel

last: Lu


title: RNase A treatment increases the recruitment of p65 to DNA

summary: We investigated the ability of the NFkB protein p65 to bind DNA after RNA digestion. In this ChIP-seq experiment, we investigated the genome-wide binding profiles of p65 in Jurkat cells. We demonstrated that p65 binding to promoters and promoter proximal regions is enhanced by digestion of RNA with RNase A.

overall_design: p65 ChIP (chromatin immunoprecipitation) libraries were prepared from Jurkat cells. Three experimental conditions were used for cells: DMSO, 200ng/ml PMA and 300ng/ml ionomycin (3h) stimulated and stimulated and 2mg/ml PureLink Rnase A treated. Cells were cross-linked with 1% parafolmaldehyde for 10 minutes and glycine was added to quench the reaction. The cell lysates were prepared as described (Varshney et al., 2018). The chromatin was sonicated in Bioruptor (Diagenode) at high power for 40 min (30 s on/off). 40 micrograms of the sonicated chromatin were used for each IP. 5% of each extract used in the IPs were taken as inputs.For p65, 7.5ug of anti-p65 antibody or rabbit polyclonal IgG antibody were added to samples and incubated overnight at 4oC, before incubation with 30ul Protein G Dynabeads (Life Technologies) for an additional 2h. Beads were eluted as desribed by Varshney et al before Proteinase K treatment and reverse crosslinking at 42oC overnight. Eluted DNA was purified by QIAquick PCR purification kit (QIAGEN).


title: H3K9 acetylation in the promoter-proximal region in high-fat diet fed mouse liver

city: Indianapolis

first: Núria

last: Morral

state: IN

address: 975 West Walnut, IB130

department: Medical and Molecular Genetics

country: USA

institute: Indiana University School of Medicine

type: Expression profiling by high throughput sequencing

summary: Genome-wide analysis of H3K9ac in the liver of mice fed a control or a high-fat diet, and asked whether this histone mark associates with changes in gene expression. We found that over 70% of RNAPII peaks in promoter-proximal regions overlapped with H3K9ac, consistent with a role of H3K9ac in the regulation of transcription. When comparing high-fat with control diet, approximately 17% of the differentially expressed genes were associated with changes in H3K9ac in their promoters, showing a strong correlation between changes in H3K9ac signal and gene expression. Overall, our data indicate that in response to a high-fat diet, dysregulated gene expression of a subset of genes may be attributable to changes in transcription elongation driven by H3K9ac. Our results point at an added mechanism of gene regulation that may be important in the development of metabolic diseases. Citation: Morral N, Liu S, Conteh AM, Chu X, Wang Y, Dong XC, Liu Y, Linnemann AK, Wan J. Aberrant gene expression induced by a high fat diet is linked to H3K9 acetylation in the promoter-proximal region. Biochim Biophys Acta Gene Regul Mech. 2021 Mar;1864(3):194691. doi: 10.1016/j.bbagrm.2021.194691. Epub 2021 Feb 6. PMID: 33556624; PMCID: PMC7933127.

overall_design: Mice (n=4) were fed a high-fat (HFD, 60% kcal fat) or a control diet (CD, 18% kcal fat) for 10 weeks. Sonicated chromatin from liver tissue was immunoprecipitated with an antibody to H3K9ac, and DNA was subjected to massive parallel sequencing. H3K9ac is associated with transcription activity, and peaks were expected to overlap with areas of open chromatin. To identify these areas, ATAC-seq was carried out in control diet-fed livers.To investigate the relationship between H3K9ac signal and gene expression levels transcript abundance was determined by RNA-seq (polyA).


title: ATACseq and RNAseq of mouse CD4 T cells

city: columbus

first: Ruoning

last: Wang

state: OH

address: 700 Children's Drive

institute: Nationwide children's hospital


title: RNA seq of mouse CD4 T cells

summary:
Purpose: To gain more mechanistic insights into the effects of SDHB deletion on T cells, we performed RNAseq in activated WT and SDHB cKO T cells.
Methods: Total RNA was isolated according to the RNeasy Mini Kit (Qiagen) and treated with DNase I. RNA quality was assessed using the Agilent 2100 Bioanalyzer and RNA Nano Chip kit (Agilent Technologies, CA). RNA-seq libraries were generated using TruSeq Stranded Total RNA with Ribo-Zero Globin Complete kit (Illumina, CA). Approximately 60–80 million paired-end 150 bp reads were generated per sample using Illumina HiSeq 4000 platform
Results: RNA-seq revealed an enriched pro-inflammatory gene signature in SDHB cKO T cells after activation
Conclusions: SDH inactivation results in a pro-inflammatory gene signature in T cells after activation

overall_design: mRNA profiles of activated WT and SDHB KO CD4 T cells

first: Benjamin

last: Sunkel


title: Combinatorial tumor suppressor inactivation efficiently initiates lung adenocarcinoma with therapeutic vulnerabilities

city: Stanford

first: Gabor

last: Boross

state: CA

address: 450 Serra Mall

institute: Stanford University

summary: Here, using a quantitative autochthonous mouse model system and performing iterative in vivo functional screens (mainly with programmable multiplexed CRISPR/Cas9-induced genotypes via ultra-deep sequencing of DNA barcoded tumors: Tuba-seq), we uncover genetic and biochemical changes that enable efficient lung tumor initiation in the absence of oncogene alterations. Through the generation of hundreds of diverse combinatorial tumor suppressor alterations, we demonstrate that inactivation of suppressors of the RAS/MAPK and PI3K pathways allows for stepwise and efficient acquisition of growth advantage that can drive the development of oncogene-negative lung adenocarcinoma. Furthermore, we demonstrate that oncogene-negative tumors with activated RAS/MAPK and PI3K pathways are vulnerable to pharmacological inhibition of these signaling axes.

overall_design: Ultra-deep sequencing of DNA barcode amplicon and RNA-seq

first: Maryam

last: Yousefi

first: Christopher

last: Murray

first: Carly

last: Weiss

first: Hongchen

last: Cai

first: Laura

last: Andrejka

first: Leo

first: Min

middle: K

last: Tsai

first: Wen-Yang

last: Lin

first: Emily

middle: L

last: Ashkin

first: Caterina

middle: I

last: Colón

first: Pauline

last: Chu

first: Su-Kit

last: Chew

first: Pegah

last: Yakhchalian

first: Charles

last: Swanton

first: Christian

middle: A

last: Kunder

first: Dmitri

last: Petrov

first: Monte

middle: M

last: Winslow


title: Patient-specific MDS-RS iPSCs define the mis-spliced transcript repertoire and chromatin landscape of SF3B1-mutant HSPCs

city: New York

first: Eirini

middle: P.

last: Papapetrou

address: One Gustave L. Levy Place Box 1044A

institute: Icahn School of Medicine at Mount Sinai

summary: SF3B1 K700E is the most frequent mutation in myelodysplastic syndrome (MDS), but the mechanisms by which it drives MDS pathogenesis remain unclear. We derived a panel of 18 genetically matched SF3B1 K700E- and SF3B1 WT-induced pluripotent stem cell (iPSC) lines from patients with MDS with ring sideroblasts (MDS-RS) harboring isolated SF3B1 K700E mutations and performed RNA and ATAC sequencing in purified CD34+/CD45+ hematopoietic stem/progenitor cells (HSPCs) derived from them. We developed a novel computational framework integrating splicing with transcript usage and gene expression analyses and derived a SF3B1 K700E splicing signature consisting of 59 splicing events linked to 34 genes, which associates with the SF3B1 mutational status of primary MDS patient cells. The chromatin landscape of SF3B1 K700E HSPCs showed increased priming toward the megakaryocyte- erythroid lineage. Transcription factor motifs enriched in chromatin regions more accessible in SF3B1 K700E cells included, unexpectedly, motifs of the TEAD family. TEAD expression and transcriptional activity were upregulated in SF3B1-mutant iPSC-HSPCs, in support of a Hippo pathway-independent role of TEAD as a potential novel transcriptional regulator of SF3B1 K700E cells. This study provides a comprehensive characterization of the transcriptional and chromatin landscape of SF3B1 K700E HSPCs and nominates novel mis-spliced genes and transcriptional programs with putative roles in MDS-RS disease biology.

overall_design: 16 RNA-seq and 15 ATAC-seq iPSC lines; 2-3 SF3B1 WT and 2-3 SF3B1 K700E replicates per patient

first: Georgios

last: Asimomitis

first: André

last: Deslauriers

first: Elli

last: Papaemmanuil


title: RNA-seq of mouse mammary basal cells sorted from G0 and G1.

city: Irvine

first: Lupe

last: Gutierrez

address: 825 Health Sciences Rd D288

institute: University of California, Irvine

summary: Molecular/cellular events that associate with epithelial-to-mesenchymal transition (EMT) are linked to acquisition of cancer stem cell traits, but whether EMT mechanisms regulate tissue epithelial cell fate and stem cell activity in vivo remains ambiguous. Using single-cell RNA sequencing, we detect heterogeneous EMT gene expression in stem cell-containing mammary basal/myoepithelial cells that normally possess both epithelial and smooth muscle characteristics. We show that EMT-inducing transcription factor Zeb1 is required within the mammary epithelium for ductal branching morphogenesis and regeneration, and that it promotes basal cell fate and regulates stem cell proliferation dynamics. We provide genetic and molecular evidence for EMT-dependent and -independent mechanisms of Zeb1 function, and show that Zeb1 cooperates with YAP to directly activate targets that inhibit Wnt signaling or control cell cycle. Together, our findings underscore Zeb1-mediated EMT control in governing mammary basal cell fate and proliferative activity, but with an interesting turn that involves mechanisms beyond EMT.

overall_design: RNA-seq experiments were performed using mouse mammary basal cells to identify genes that are differentially expressed between G0 and G1.

first: Xing

last: Dai

first: Yingying

last: Han

first: Alvaro

last: Villareal-Ponce

first: Guadalupe


title: Blockade of intrinsic PD-1 promotes survival, chemo resistance and tumor growth in human colon cancer

city: Napoli

first: Luigi

last: Portella

address: M. Semmola

department: Research Department

country: Italy

institute: Istituto Nazionale Tumori di Napoli - IRCCS fondazione "Pascale"

summary: Human colon cancer cells HT29, HCT116, LoVo robustly expressed PD-1. PD-1 signaling significantly decreased proliferation and promoted apoptosis in PD1+human colon cancer cells. The human anti-PD-1, Nivolumab (NIVO) ptomoted proliferation through pERK/pAKT signaling, reduced apoptosis and protected PD-1+ cells from Chemo/Radiotherapy (CRT). In vivo, NIVO promoted HT29 tumor growth reducing Oxaliplatin (OX) efficacy. As opposite to colon cancer cells, PD-1 signaling protects melanoma cells, thus NIVO treated human colon versus melanoma cancer cells, PES43 were evaluated for RNAseq. Among the commonly affected genes, opposite regulation was revealed between HT29 and HCT116 colon versus PES43 melanoma cells. BATF2, DRAM1, FXYD3, IFIT3, MT-TN, TNFRSF11A were upregulated in PES43 and downregulated in HT29 and HCT116 while CLK1, DCAF13, DNAJC2, MTHFD1L, PRPF3, PSMD7, SCFD1 were downregulated in PES43 and upregulated in HT29 and HCT116. DEGs were significantly enriched in the functional categories of the interferon pathway, innate immune, cytokine-mediated signaling pathway, neutrophil activation, immune effector process, granulocyte activation and cellular nitrogen compound metabolic process. Moreover, intrinsic PD-1 was expressed in 11/48 (22.9%) primary colorectal cancer and associated with tumor dimension (pT). Thus, PD-1 inhibition through NIVO protects human colon cancer cells and activate tumor survival pathways

overall_design: mRNA profile Nivolumab (NIVO), soluble PD-L1 (sPDL-1) and NIVO+sPDL-1-treated human colorectal cancer cells HCT116 and HT29 and human melanoma cells PES43 for 6h and 24h

first: Caterina

last: Ieranò

first: Dario

last: Righelli

first: Stefania

last: Scala


title: ATAC seq of mouse CD4 T cells

first: Ruoning

last: Wang

summary:
Purpose: To gain more mechanistic insights into the effects of SDHB deletion on T cells, we performed ATAC seq in activated WT and SDHB cKO T cells.
Methods: 5x104 cells per experiment were first washed with RSB buffer and gently permeabilized with RSB lysis buffer  on ice. Cells were suspended in 50 uL of tagmentation master mix prepared from Illumina Tagment DNA TDE1 Enzyme and Buffer Kit components (#20034197), and transposition was performed for 30 minutes at 37°C. Tagmented DNA fragments were isolated using Qiagen MinElute PCR Purification columns prior to library amplification. ATAC-seq libraries were amplified with barcoded Nextera primers for 14 cycles, and excess primers were removed by size selection with AMPure XP beads. Libraries were sequenced on the HiSeq4000 platform running in PEx150bp mode.
Results: ATAC-seq revealed distinctive patterns of DNA accessibility that are associated with genomic loci of genes involved in T cell activation and inflammation in activated WT and SDHB cKO CD4 T cells
Conclusions: DNA accessibility that is associated with genomic loci of inflammatory genes and transcription factors that regulate inflammatory genes in CD4 T cells

overall_design: ATAC seq of naïve and activated WT and SDHB KO CD4 T cells

first: Benjamin

last: Sunkel


title: The RNA Helicase DHX16 Provides Specific Viral RNA for RIG-I to Trigger Innate Antiviral Immunity

city: Galveston

first: Adam

last: Hage

state: Texas

address: 301 University Blvd

department: Microbiology and Immunology

institute: University of Texas Medical Branch at Galveston

summary: Type-I interferon (IFN-I) is essential to establish antiviral innate immunity. Unanchored (or free) polyubiquitin (poly-Ub) has been shown to regulate IFN-I responses, however few unanchored poly-Ub interactors are known. To identify factors regulated by unanchored poly-Ub in a physiological setting, we developed an approach to isolate unanchored poly-Ub from lung tissue and identified the DEAH-box RNA helicase DHX16 as a factor that enhances IFN-I production. Silencing of DHX16 in cells and in vivo diminished IFN-I responses against influenza virus. DHX16-dependent IFN-I production requires RIG-I and unanchored K48-poly-Ub synthesized by the E3 Ub ligase TRIM6. DHX16 recognizes a signal in influenza segments that undergo splicing and requires its intact RNA helicase motif for direct, high-affinity interactions with specific viral RNAs. Our study establishes DHX16 as a potentiator of RIG-I-mediated IFN-I production and recognizes a mechanism for activation of antiviral immunity requiring unanchored poly-Ub and the viral RNA recognition activity of DHX16

overall_design: Next Generation Sequencing of human A549 lung cells treated with either non-targeting control or DHX16 targeting siRNA and either mock or influenza A virus infected (MOI=1) for 24 hours.

first: Preeti

last: Bharaj

first: Sarah

last: van Tol

first: Maria

last: Giraldo

first: Leopoldo

last: Aguilera-Aguirre

first: Xuping

last: Xie

first: Steven

middle: G

last: Widen

first: Hong

last: Moulton

first: Jeffrey

middle: R

last: Johnson

first: Nevan

middle: J

last: Krogan

first: Adolfo

last: García-Sastre

first: Pei-Yong

last: Shi

first: Alexander

middle: N

last: Freiberg

first: Ricardo

last: Rajsbaum


title: Colon cancer cells after escape of late apoptosis

city: Geneva

first: Celine

last: Delucinge

address: 1 rue Michel Servet

department: iGE3 Genomics Platform

country: Switzerland


title: Colon cancer cells after escape of late apoptosis [exp_2]

summary: In order to explore the potential role of escape of late apoptosis in metastasis, we induced apoptosis and subsequently inhibited the death process in a primary culture of colon cancer cells. Here we wanted to explore the transcriptome of cells treated with the conditioned medium of these surviving cells. Thes cells have been treated for 48h with conditionned media previous RNA extraction and RNAseq.

overall_design: Primary colon cancer cells (CC14) were treated with conditioned media (CM) from cells treated with STS and 6h after with DIDS and Q-VD_Oph.  After 48h of CM treatments , cells were harvested and total RNA extracted using TRIzol Reagent (Ambion) following the manufacturer's protocol. Each condition was independently processed in triplicates and the libraries were prepared using Illimina TruSeq protocols. The samples were sequenced on a HiSeq Illumina 2500 machine with 50bp reads followed by standard analyses.

first: Ariel

last: Ruiz i Altaba

first: Arwen

last: Conod


title: Colon cancer cells after escape of late apoptosis [exp_1]

summary: In order to explore the potential role of escape of late apoptosis in metastasis, we induced apoptosis and subsequently inhibited the death process in a primary culture of colon cancer cells. Here we wanted to explore the transcriptome of such "undead" cells  7 days after apoptosis induction.

overall_design: Primary colon cancer cells (CC14) were treated with STS and 6h after with DIDS and Q-VD_Oph. 7 days after these treatments, cells were harvested and total RNA extracted using TRIzol Reagent (Ambion) following the manufacturer's protocol. Each condition was independently processed in triplicates and the libraries were prepared using Illimina TruSeq protocols. The samples were sequenced on a HiSeq Illumina 4000 machine with 50bp reads followed by standard analyses.

last: Altaba


title: 10x Genomics VDJ seqeuncing of IOMAgl germinal center B cells

status: Public on Mar 07 2022

first: Michel

last: Nussenzweig

state: NY

address: 1230 York Ave

institute: Rockefeller University

summary: This experiment was performed to identify somatic hypermutations in germinal center B cells of a mouse expressing the inferred germline of IOMA BCR after and a 5 step sequential immunization regimen.

overall_design: This experiment was performed to identify somatic hypermutations in germinal center B cells of mouse HP3, homozyogous for the IghIOMAiGL and IgkIOMAiGL transgenes expressing the inferrred germline of IOMA after and a 5 step immunization regimen. The mice were immunized sequentially with mi3 nanoparticles. First primed with IGT2-mi3 and sequentially boosted with IGT1-mi3 (at week 5), 426c degly2 D279N-mi3 (week 10) , and twice with mosaic8-mi3 (week 15 and 20). HP3 was sacrified in week 23 and spleen and mesenteric lymph nodes were processed and stored in LN2. Then Germinal center B cells were sroted as live, singlet, lineage dump-, B220+, CD95+ CD38- lymphocytes.

first: Harald

last: Hartweger

first: Victor

last: Ramos

middle: C

first: Harry

middle: B

last: Gristick

first: Pamela

last: Bjorkman


title: Preclinical efficacy and safety study of encapsulated human proliferating hepatocyte organoids in the treatment of liver failure

city: Shanghai

first: Lijian

last: Hui

address: yueyang road 320

department: SIBCB

institute: CEMCS, CAS

summary:
Purpose: The goals of this study are to analyze the NGS-derived  transcriptome profiling (RNA-seq)  between the normal, control  and experimental group after extended hepatectomy.
Results:  All sequencing reads from RNA-seq were mapped to the mouse reference genome (GRCm38.84) using hisat2-2.10 (Kim et al., 2015). FPKM (fragments per kilobase of exon per million fragments mapped) values were calculated by Cufflinks v.2.2.1 (Trapnell et al., 2012) using default parameters for gene expression levels. We used htseq-count (Anders et al., 2015) to count reads on genes. Differential expression analysis was performed using DESeq2 (R package, (Love et al., 2014)). Genes were considered differentially expressed if FPKM >=1 in at least one sample and |fold change| >= 2, adjusted p-value < 0.05. The gene ontology analyses was performed using Metascape v3.5 (http://metascape.org/gp/index.html, (Zhou et al., 2019)). Principal component analysis was performed with whole genes using the R package (function: prcomp()).
Conclusions: Our study represents the first detailed analysis of liver transcriptomes after extended hepatectomy, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.

overall_design:
Methods: Liver mRNA profiles from normal control mice (10 weeks old), EMC-treated control mice (2 days after extended hepatectomy) and  eLO- treated mice (2 days after extended hepatectomy and 14 days after extended hepatectomy) were generated by deep sequencing, in duplicate using Illumina NovaSeq 6000. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation was performed using SYBR Green assays.
liver tissue mRNA profiles of normal control mice (10 weeks old), EMC-treated control mice (2 days after extended hepatectomy) and  eLO- treated mice (2 days after extended hepatectomy and 14 days after extended hepatectomy)

first: Xiang

last: Yuan

first: Baihua

last: Wu


title: A Novel Role for Nucleolin in Splice Site Selection

city: Jerusalem

first: Ruth

last: Sperling

address: The Edmond J. Safra Campus - Givat Ram

department: Department of Genetics

country: Israel

institute: The Hebrew University of Jerusalem

summary: In search for nuclear partners of initiator-tRNA, previously shown to be involved in a quality control of splice site selection, we identified nucleolin (NCL) as specifically and directly bound to it in the nucleus and not in the cytoplasm (using affinity purification of UV crosslinked nuclear initiator-tRNA followed by mass spectrometry). To further explore the role of NCL in splice site selection, we knocked down NCL. We report the activation of 399 latent splice sites upon NCL knockdown, as revealed by RNA deep sequencing of siRNA treated NCL compared to control siRNA. Our study identifies NCL as the first protein component of this quality control mechanism of splice site selection. Our study thus establishes an experimental strategy and computational pipeline that could be used to identify other components of this quality control mechanism.

overall_design:
Examination of latent splicing activation in 2 samples with NCL knockdown by siRNA against NCL compared to 2 paired samples with control siRNA.
Note: Equal contribution made by: K. Shefer, A. Boulos, V. Gotea, M. Arafat

first: Kinneret

last: Shefer

first: Ayub

last: Boulos

first: Valer

last: Gotea

first: Maram

last: Arafat

first: Yair

last: Ben Chaim

first: Aya

last: Muharram

first: Sara

last: Isaac

first: Amir

last: Eden

first: Joseph

first: Laura

last: Elnitski


title: The ACF chromatin remodeling complex is essential for Polycomb repression

city: Eugene

first: Hideki

last: Tanizawa

state: OR

address: 1370 Franklin Blvd

department: Institute of Molecular Biology

institute: University of Oregon

type: Methylation profiling by high throughput sequencing

summary: Establishing and maintaining appropriate gene repression is critical for the health and development of multicellular organisms. Histone H3 lysine 27 (H3K27) methylation is a chromatin modification associated with repressed facultative heterochromatin, but the mechanism of repression remains unclear. We used a forward genetic approach to identify novel genes involved in transcriptional silencing of H3K27 methylated chromatin in the filamentous fungus, Neurospora crassa. We found crf4-1Iswi and crf4-2Itc1/Acf1 are required for repression of a subset of H3K27 methylated genes and that their protein products interact to form an ACF-like chromatin remodeling complex. This N. crassa ACF-like complex is promiscuous, yet can target facultative heterochromatin, where it is responsible for pulling the +1 nucleosome in the 5’ direction.    H3K27 methylated genes that are up-regulated when crf4-1Iswi or crf4-2Itc1/Acf1 are deleted show a downstream shift of the +1 nucleosome suggesting that proper nucleosome positioning is important for repression of facultative heterochromatin.

overall_design: MNase-seq, H3K27 methylation ChIP-seq, RNA-seq and DamID with Neurospora crassa.

first: Elizabeth

middle: T

last: Wiles

first: Colleen

last: Mumford

first: Kevin

last: McNaught

first: Eric

middle: U

last: Selker


title: Zebrafish establish female germ cell identity by advancing cell proliferation and meiosis

city: Taipei

first: Bon-chu

last: Chung

state: Taiwan

address: 128 Academia Road, Section 2, Nankang, Taipei 115, Taiwan

country: Taiwan

institute: Academia Sinica

summary: In this study, we utilize the RNA-seq of germ cell to uncover the early germ cell development. To obtain fish with defined sex, we crossed domesticated zebrafish with the Nadia strain that has a female-dominant W segment. These fish were placed on a ziwi:GFP background to facilitate sorting of fluorescent germ cells for transcriptomic analysis. We analyzed the transcriptomes of germ cells at 10 to 14 days postfertilization (dpf), when sex dimorphic changes started to appear.

overall_design: Germ cells mRNA of 10-,12- and 14-day post fertilization in W-hybrid (ZW*Tupfel long-fin) and Z-hybrid (ZZ*Tupfel long-fin)

first: You-Jiun

last: Pan


title: Activating STING1-dependent immune signaling in TP53 mutant and wild-type acute myeloid leukemia

city: Baltimore

first: Michael

middle: Jonathan

last: Topper

state: MD

address: 1650 Orleans St., Suite 530

department: Oncology

institute: Johns Hopkins University

summary: In the present study we show that in the subset of acute myeloid leukemias (AML) with mutations in TP53, associated with poor prognosis, DNMTis, important drugs for treatment of AML, enable expression of ERVs and IFN and inflammasome signaling in a STING1 dependent manner.  We previously reported in solid tumors that PARPis combined with DNMTis induce an IFN/inflammasome response that is dependent on STING1 and is mechanistically linked to generation of a homologous recombination defect (HRD). We now show that STING1 activity is actually increased in TP53 mutant compared with WT TP53 AML. Moreover, in TP53 mutant AML, STING1-dependent IFN/inflammatory signaling is increased by DNMTi treatment, whereas in AMLs with wild-type (WT) TP53, DNMTis alone have no effect. However, while combining DNMTis with PARPIs now increases IFN/inflammatory gene expression in WT TP53 AML cells, signaling induced in TP53 mutant AML is still several-fold higher.   Notably, induction of HRD in both TP53 mutant and WT AMLs follows the pattern of STING1-dependent IFN and inflammatory signaling we have observed with drug treatments.  These findings increase our understanding of the mechanisms that underlie DNMTi+PARPi treatment, and also DNMTi combinations with immune therapies, and a personalized approach, stratifying by p53 status, for use of such therapies, including potential immune activation of STING1 in AML and other cancers.

overall_design: Treatment induced gene expression in MOLM14 cells was measured at 72 hours after exposure to 10nM Decitabine and/or 5nM Talazoparib.


title: Transcriptome of single and double pannexin1-knock out zebrafish lines

city: Toronto

first: Georg

last: Zoidl

state: Ontario

address: 4700 Keele Street

department: Psychology

institute: York University

summary: Pannexin 1 (Panx1) forms ATP-permeable membrane channels that play roles in the nervous system. The analysis of roles in both standard and pathological conditions benefits from a model organism with rapid development and early onset of behaviors. Such a model was developed by ablating the zebrafish panx1a gene using TALEN technology. Here, RNA-seq data of 6dpf wild type and knock-out zebrafish is reported which have been generated by gene editing of exon 4 of the panx1a gene.

overall_design: Paired end sequencing of TL-control, panx1a knock-out, panx1b knock-out and double knock-out

first: Nickie

last: Safarian


city: Oxford

first: Robin

last: Choudhury

address: L6 West Wing, John Radcliffe Hospital, Headley Way

department: Cardiovascular Medicine, Radcliffe Department of Medicine

institute: University of Oxford

summary: Purpose: RNA-sequencing of peripheral blood neutrophils from STEMI and NSTEMI patients at the time of presentation versus a control sample obtained from the same patients 1 month post-AMI to determine differentially expressed genes and pathways.

overall_design: RNA-sequencing of peripheral blood neutrophils from STEMI and NSTEMI patients at presentation and 1 month post-AMI.

first: Naveed

last: Akbar

first: Adam

last: Braithwaite

middle: P


title: Single Cell Transcriptomic Analysis of Popliteal Lymphatic Vessels and Peripheral Veins with Smooth Muscle Cell Enrichment in TNF-Tg vs WT Mice

city: Rochester

first: H. Mark

last: Kenney

state: New York

address: 601 Elmwood Ave, Box 665

department: Center for Musculoskeletal Research

institute: University of Rochester Medical Center

summary: Lymphatic dysfunction exists in tumor necrosis factor transgenic (TNF-Tg) mice and rheumatoid arthritis (RA) patients. While joint-draining TNF-Tg popliteal lymphatic vessels (PLVs) have deficits in contractility during end-stage arthritis, the nature of lymphatic muscle cells (LMCs) and their TNF altered transcriptome remain unknown. Thus, we performed single-cell RNA-sequencing (scRNAseq) on TNF-Tg vs wild-type peripheral vasculature. We utilized strategic enrichment for smooth muscle cells using a Cspg4-Cre;tdTomato reporter model, and through post-hoc analysis we validated the ability to enrich for these tdTomato+ populations based on scatter alone. As such, we evaluated changes in LMC populations vs vascular smooth muscle cells (VSMCs), and dynamic changes in peri-vascular immune cell populations during chronic inflammatory-erosive arthritis.

overall_design: Four total single-cell RNA-seq samples are provided. Two samples are replicates from Cspg4-Cre;tdTomato mice with cell enrichment for tdTomato+ cells (tdT1 and tdT2). Two samples derive from age-matched (9-months-old) WT and TNF-Tg, where the WT serves as control to the TNF-Tg mice with chronic inflammatory-erosive arthritis. The WT and TNF-Tg samples were sorted based on scatter alone. Each sample consists of cell isolates derived from n = 3 mice pooled together, and all mice were on a C57BL6 background.

first: Schwarz

last: Edward

first: Kenney

last: H. Mark

first: Wu

last: Chia-Lung


title: RNA-Seq analysis to compare myeloid cells from PD-L1-LysMCre mice with littermate Wild-type controls

first: Hao

last: Zuo

address: P&S 7-401, 630 WEST 168th STREET

institute: Columbia University

summary: Programmed death-ligand 1 (PD-L1) is predominantly expressed in the antigen-presenting cells (APCs) that are originated and abundant in bone marrow. The roles of PD-L1 in bone cell differentiation and cancer bone metastasis remain unclear. Here we show that PD-L1 antibody or PD-L1 conditional knockout in the hematopoietic or myeloid lineage suppresses osteoclast differentiation in vitro and in vivo. Bone metastases of breast cancer and melanoma are diminished by PD-L1 antibody or PD-L1 deletion in the myeloid lineage. Transcriptional profiling of bone marrow cells reveals that PD-L1 deletion in the myeloid cells up-regulates immune stimulatory genes, leading to increased macrophage M1 polarization, decreased M2 polarization, enhanced IFN? signaling, and elevated T cell recruitment and activation. All these alterations result in heightened anti-tumor immunity in the cancer microenvironment. Our findings support PD-L1 antibody as a potent therapy for bone metastasis of breast cancer and melanoma by simultaneously suppressing osteoclast and enhancing immunity.

overall_design: Examination of mRNA profiles of myeloid cells from mice with PD-L1 wild-type (WT) or lysozyme-Cre (LysMCre) mediated PD-L1 conditional knockout by deep sequencing, in biological triplicate, using Illumina HiSeq 2500

first: Yihong

last: Wan


title: Assessment of zearalenone-induced cell survival and global gene regulation in mouse TM4 Sertoli cells

city: St-Hyacinthe

first: Younes

last: Chorfi

state: Québec

address: 3200 Sicotte

institute: Université de Montreal

summary: Zearalenone (ZEA) is a non-steroidal xenoestrogen mycotoxin produced by many Fusarium fungal species, which are common contaminants of cereal crops destined for worldwide human and animal consumption. ZEA has been linked to various male reproduction problems including decreased fertility potential. In this study, the direct impact of ZEA on the immature Sertoli TM4 cell line was evaluated. Results showed that high concentration of ZEA increase reactive oxygen species via the activation of MAPK signaling. Transcriptome analysis was performed on TM4 cell line treated with ZEA and genes involved in sex differentiation (Fgfr2, Igf1, Notch1, Sox9) and extracellular matrix (ECM) formation (Ctgf, Fam20a, Fbn1, Mmp9, Postn, Sparcl1, Spp1) were identified at the center of the functional protein association network suggesting that ZEA could be detrimental to the early steps of Sertoli cell differentiation.

overall_design: 12 samples : 6 controls and 6 treated with Zearalenone

first: Christian

last: Savard

first: Sadaf

last: Gawhary

first: Alexandre

last: Boyer


title: NDUFS4 deficiency in female and male inguinal white fat

city: Seoul

first: Kyung-Mi

last: Choi

address: 1 Gwanak-ro, Gwanak-gu

country: South Korea

institute: Seoul National University

summary: We compared gene expression between high fat diet (HFD)-fed Adipo-NDUFS4 knockout (KO) and WT mice in inguinal white adipose tissue (iWAT or scWAT).

overall_design: Transcriptome profiles of iWAT of Adipo-NDUFS4 KO and WT fed with HFD and raised at room temperature.

first: John

last: Yoon


title: Lepr+ mesenchymal cells sense diet to modulate intestinal stem cells via the Leptin-Igf1-Igf1r axis

city: Beijing

first: min

last: Deng

address: Beijing HaiDian District, Mingyuanxilu No.2

institute: China Agricultural University

summary: We demonstrated that Lepr+ mesenchymal cells surround intestinal crypts where ISCs and transit-amplifying (TA) cells localize. The abundance of these cells increased upon administration of a high-fat diet (HFD) but dramatically decreased upon fasting. Depletion of Lepr+ mesenchymal cells resulted in fewer ISCs, compromised architecture of crypt-villi axis and impaired intestinal regeneration. Furthermore, Lepr+ cell-derived Igf1 has been identified as an important effector that promotes the proliferation of ISCs and TA cells. Deletion of Igf1 in Lepr+ cells partially recapitulated Lepr+ cell-ablated intestinal phenotypes during both homeostasis and regeneration. Overall, Lepr+ mesenchymal cells sense diet alteration and function as a novel niche for ISCs via the stromal Igf1 - epithelial Igf1r axis, which is critical for intestinal homeostasis and regeneration. These findings revealed that Lepr+ mesenchymal cells are an important mediator that links diet to ISC function and might provide a novel therapeutic target for gut diseases.

overall_design: Examination of the whole mesenchymal cells and Lepr+ cells in intestine.

first: Min

middle: F

last: Guerrero-Juarez

first: Xiaole

last: Sheng

first: Jiuzhi

last: Xu

first: Xu

last: Yang

first: Guilin

last: Li

first: Bing

last: Zhang

first: Xiaowei

last: Liu

first: Kaichun

last: Wu

first: Fazheng

last: Ren

first: Maksim

middle: V

last: Plikus

first: Zhengquan

last: Yu

first: Cong

last: Lv


title: Multi-omic rejuvenation of naturally aged tissues by a single cycle of transient reprogramming

city: Sabadell

first: Antoni

last: Berenguer-LLergo

state: Barcelona

address: Parc del Taulí, 1

department: A8G2 Group - Rheumatology Service

country: Spain

institute: Parc Taulí Research and Innovation Institute (I3PT)

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

overall_design: Refer to individual Series


title: LSD1 Facilitates Euchromatic Origin Firing and Regulates Replication Timing [ChIP-seq]

first: Xiaohan

address: 38 Xueyuan road

department: Biochemistry and Biophysics

institute: Peking Unversity Health Science Center

summary: The chromatin-based rules governing the selection and activation of replication origins remain to be elucidated. It is believed that DNA replication initiates from open chromatin domains, thus replication origins residing in regulatory elements that are located at open and active chromatin. However, we report here that lysine specific demethylase 1 (LSD1), which biochemically catalyzes H3K4me1/2 demethylation to favor chromatin condensation, interacts with the DNA replication machinery. We found that LSD1 level peaks in early S phase. We demonstrated that LSD1 promotes DNA replication by facilitating origin firing in euchromatic regions and through regulating replication timing. Indeed, euchromatic zones enriched in H3K4me2 are the preferred sites for pre-RC binding in early replication. Remarkably, LSD1 deficiency leads to a genome-wide switch from early to late in replication timing. We showed that LSD1-promoted DNA replication is mechanistically linked to the loading of TICRR (TopBP1-Interacting Checkpoint and Replication Regulator) onto the pre-RC and subsequent recruitment of the initiator Cdc45 during origin firing. Together, these results reveal an unexpected role for LSD1 in euchromatic origin firing and replication timing.

overall_design: Examination of dynamic H3K4me2 on chromatin at the onset of DNA replication in HeLa cell; Examination of LSD1 and CDC45 distribution on chromatin in the early S phase in HeLa cells.

first: Yue

last: Wang

first: Yunchao

last: Huang

first: Xinhua

first: Yu


title: Transcriptome analysis of acute graft-versus-host disease in children

city: Cincinnati

first: Rebekah

last: Karns

address: 3333 Burnet Ave

department: Gastroenterology, Hepatology, & Nutrition

institute: Cincinnati Children's Hospital Medical Center

summary: Steroid-refractory gastrointestinal graft-versus-host disease (GI GVHD) is a major barrier to successful hematopoietic stem cell transplant (HSCT).  Poor understanding of the pathophysiology of GI GVHD contributes to continued poor outcomes and high mortality rates.  We therefore obtained rectosigmoidal mucosal biopsies from post-HSCT patients with GI GVHD and submitted them to RNA-sequencing in order to transcriptomally characterize GI GVHD.  These were compared to patients undergoing endoscopy for routine clinical indications.  Using single end, 50bp reads processed using Kallisto using genome annotations from Gencode v24 with transcripts per million as the output. We included 14,239 trancsripts in our analysis, where we compared GVHD vs non-GVHD with significance defined as FDR<0.05 and FC 1.5 using R package DESeq2.  We identified 164 key genes, of which 141 were upregulated in GVHD, and were ontologically related to microbial response, key immune effectors, and cell migration/chemotaxis. Down-regulated genes were related to nutrient metabolism.  Additionally, we performed WGCNA that highlighted ERK as a key upregulated pathway in GI GVHD.

overall_design: RNA-seq of rectosigmoidal mucosal biospies obtained from post-HSCT patients with GI GVHD and non-GVHD controls.

first: Pooja

last: Khandelwal


title: LSD1 Facilitates Euchromatic Origin Firing and Regulates Replication Timing [Repli-seq]

overall_design: Examination of genome-wide replication profile after LSD1 knockdown, overexpression or inhibition.


title: Temporal and Quantitative Transcriptomic Differences Define Sexual Dimorphism in Murine Post Natal Bone Aging

city: Boston

first: Boston University

last: Microarray and Sequencing Resource

state: MA

address: 72 East Concord Street, E631

department: Microarray and Sequencing Resource

institute: Boston University

summary: Time is a central element to development, pathology and aging of the skeleton. Since the transcriptome is a representation of the phenome, we hypothesized that characterization of the sex-specific, temporal transcriptomic differences in male and female bones over an 18 month period would be informative to the underlying molecular processes that lead to postnatal sexual dimorphism. Sex-specific gene expression regardless of age was primarily associated with connective tissues. Temporal clustering of the transcriptome identified two skeletal tissue-associated, sex-specific patterns of gene expression. One set of genes, associated with skeletal patterning and morphology and showed peak expression earlier in females. The second set of genes, associated with coupled remodeling had quantitatively higher expression in females and exhibited a broad peak between 3 to 12 months, concurrent with the animals’ reproductive period. Results of phenome-level structural assessments of the tibia and vertebrae, and in vivo and in vitro analysis of cells having osteogenic potential, were consistent with the existence of functionally unique, skeletogenic cell populations that are separately responsible for appositional growth and intramedullary bone cell functions.

overall_design: A total of 80 male and female Brd2wt/wt and Brd2wt/lo animals from BUMC were sacrificed at 3, 6, 9, 12, and 18 months (n=4 per combination of timepoint, sex, and genotype).  An additional set of 4 female Brdwt/wt animals from Jackson Laboratories (JAX) was sacrificed at 3 months as a control group.

first: Louis

last: Gerstenfeld


title: Transcriptional profiles of inguinal fat of HIV-infected and healthy control patients.

city: Ikoma

first: Hitomi

last: Takada

state: Nara

address: 8916-5 Takayama-cho

department: Graduate School of Science and Technology

institute: Nara Institute of Science and Technology

summary: Although the survival rate for patients infected with human immunodeﬁciency virus type 1 (HIV-1) has dramatically improved due to recent advancements in antiretroviral therapy, the mechanism of pathogenesis of HIV-associated lipodystrophy remains poorly understood. To elucidate the pathophysiological mechanism of disease, we analyzed gene expression profiles of inguinal adipose tissues from HIV-infected and healthy conrol patients. Genetic analysis revealed that fat tissue from patients with HIV typically had increased immune activation, mitochondrial toxicity, chronic inflammation, progressive fibrosis and adipocyte dysfunction including insulin resistance, inhibited adipocyte differentiation and accelerated apoptosis. Of note, triglyceride synthesis and lipolysis were inhibited in adipose tissue from patients with HIV.

overall_design: cDNA was synthesized from total RNA isolated from inguinal adipose tissues of two HIV-infected patients and two healthy control patients and hybridized on Agilent SurePrint G3 Human GE 8 × 60k Microarray Ver3.0.

first: Takanobu

last: Mashiko

first: Akira

last: Kurisaki

first: Kotaro

last: Yoshimura


title: Multi-omic rejuvenation of naturally aged tissues by a single cycle of transient reprogramming [RRBS]

city: Cambridge

first: Felix

last: Krueger

address: Granta Park

department: Bioinformatics

institute: Altos Labs

summary: The expression of the pluripotency factors OCT4, SOX2, KLF4 and MYC (OSKM) can transform somatic differentiated cells into pluripotent stem cells in a process known as reprogramming. Transient OSKM expression perturbs cellular homeostasis in a reversible manner and cycles of such incomplete reprogramming rejuvenate features of aging in cells and progeroid mice. However, little is known about the mechanisms involved. Here, we have studied changes in the DNA methylome, transcriptome and metabolome in naturally aged mice subject to a single period of transient OSKM expression in all tissues. We found that this manipulation is sufficient to rejuvenate a substantial fraction of the DNA methylation changes in gene regulatory elements that occur upon aging in the pancreas, liver and spleen. Similarly, we observed rejuvenation at the transcriptomic level, especially regarding biological processes known to change during aging. Finally, a subset of serum metabolites normally altered with aging were restored to young levels upon reprogramming. These observations indicate that a single period of OSKM expression can drive epigenetic, transcriptomic and metabolomic changes towards a younger configuration in multiple tissues and in the serum.

overall_design: Reduced representation bisulfite sequencing of pancreas, spleen and liver after a single cycle of transient reprogramming.  Each sample represents tissue from a single mouse.

first: Dafni

last: Chondronasiou

first: Diljeet

last: Gill

first: Lluc

last: Mosteiro

first: Rocio

last: Urdinguio

first: Sylvere

last: Durand

first: Fanny

last: Aprahamian

first: Nitharsshini

last: Nirmalathasan

first: Antonio

last: Berenguer

first: Maria

last: Abad

first: Daniel

middle: E

last: Martin-Herranz

first: Camille

middle: S

last: Otto-Attolini

first: Neus

last: Prats

first: Guido

last: Kroemer

first: Mario

last: Fraga

first: Wolf

last: Reik

first: Manuel

last: Serrano


title: Long noncoding RNA LSINCT5 is up-regulated and related to  progression in esophageal squamous cell carcinoma

city: Shijiazhuang

first: Li

last: Jing

state: Hebei

address: NO12 Jiankang Road

department: Department of Medical Oncology

institute: the Fourth Hospital of Hebei Medical University

summary: We aim to evaluate the expression and putative role of LSINCT5 on progression of ESCC. The expression of LSINCT5 was first examined in ESCC cell lines using RT-qPCR method. Using next-generation RNA-Seq technology, the differential gene expression before and after the knockdown of LSINCT5 in ESCC was analyzed and functionally annotated.

overall_design: To explore the putative functional role of LSINCT5 in ESCC, we performed differential gene expression analysis of the mRNAs from ESCC cells before and after knockdown of LSINCT5 by transfection with siRNA using next-generation RNA-Seq technology and functional annotation to identify pathways of interest.

first: Xiang-hong

last: Zhang


title: LSD1 Facilitates Euchromatic Origin Firing and Regulates Replication Timing [RNA-seq]

first: Xiaohan

last: Yang

overall_design: Transcriptome analysis after LSD1 knockdown.

first: Yue

last: Wang

first: Yunchao

last: Huang

first: Xinhua

last: Liu

first: Yu


title: Circular RNA signatures of human healing and non-healing wounds

status: Public on Mar 06 2022

city: Stockholm

first: Ning

last: Xu

address: CMM L8:02

institute: Karolinska Institutet

summary:
In an RNAseq analysis, we have identified the circular RNAs cCHST15 and cTNFRSF21 with high levels in venous ulcers compared to acute wounds or skin.
The biological function of these two circular RNAs in human epidermal keratinocytes during wound repair has not been studied.
To study the genes regulated by cCHST15 or cTNFRSF21 , we transfected siRNA targeting the diagnostic junctions of either cCHST15 or cTNFRSF21  into human primary epidermal keratinocytes to knockdown cCHST15 or cTNFRSF21  expression, respectively.
We performed a global transcriptome analysis of keratinocytes upon circRNA knockdown using Affymetrix arrays.

overall_design: Gene expression profiling of keratinocytes transfected with either siRNA control (n = 3) or si-cCHST15 (n = 3) or si-cTNFRSF21 (n =3) for 24 hours was carried out by using human Clariom™ S assay (ThermoFisher Scientific) at the Bioinformatics and Expression Analysis (BEA) core facility at Karolinska Institutet.

middle: X

last: Landén

first: Zhuang

middle: A

last: Toma


title: Expression data from HER2+ breast cancer cell lines.

city: Valencia

first: Pilar

last: Eroles

address: Menendez Pelayo acc4

institute: INCLIVA

summary:
miRNA expression is often disregulated in cancer pathways, such as progression of resistance.
We used microarrays in order to determine the differences in miRNA expression between breast cancer cell lines and their resistant pairs.

overall_design: We analysed two different cell lines (BT474 and SKBR3) compared to their induced resistant cell lines (BT474r and SKBR3r). Each of the four cell lines was analysed by triplicate.

first: Paula

last: Cabello


title: PLK inhibitors identified by high content phenotypic screening promote maturation of human iPSC-derived cardiomyocytes

city: Shanghai

first: Mengying

last: Feng

address: no. 67 Chifeng road, Yangpu district

department: School of Life Sciences and Technology

institute: Tongji University

summary: We performed RNA-Seq analysis of human pluripotent stem cells derived cardiomyocytes (hPSC-CMs) which were treated with Centrinone or Volasertib for 14 days to investigate the moleclular mechanism of inhibiting L-type calcium channel (LTCC) to promote cardiomyocyte proliferation.

overall_design: RNA-Seq analysis of NM treated NRVCs and hPSC-CMs for 12 hours, 3 replicates per group.

first: Hongjie

first: Ke

last: Wei


title: Neutrophil activation phenotypes in healthy subjects

city: Durham

first: Grace

middle: M

last: Lee

state: North Carolina

address: Box 3486, DUMC

institute: Duke University

summary: RNA was extracted from neutrophils from healthy subjects whose neutrophil function has been extensively characterized for RNA sequencing.

overall_design: RNA was extracted from 107 neutrophils from 4 “low” and 3 ”high” subjects for RNA sequencing

first: Stacey

last: Maskarinec

first: Margaret

last: McKelvy

first: Halie

last: Hotchkiss

first: Madelaine

last: Duarte

first: Bechtler

last: Addison

first: Sanjay

last: Khandelwal

first: Gowthami

last: Arepally


title: LSD1 Facilitates Euchromatic Origin Firing and Regulates Replication Timing

first: Xiaohan

last: Yang


title: Differential gene expression in leukemic blasts and leukemic stem cells (LSC) sorted from pediatric AML patients compared to control myeloblasts and hematopoietic stem cells (HSC) sorted from cord blood

city: Gent

first: Barbara

last: Depreter

state: Not in US

address: C. Heymanslaan 10

department: Internal Medicine and Pediatrics

country: Belgium

institute: University of Ghent

summary:
Purpose: Currently, only  few reports address the molecular abnormalities of LSC compared to HSC in pedAML. Identifying LSC aberrations is crucial to tackle the high relapse rate and to develop therapeutic targeting strategies for LSC elimination, while ensuring salvage of normal HSCs.
Methods: Blasts and stem cells were separated based on CD38 expression (positive and negative, respectively). Leukemic blasts (n=4), LSCs (n=3) and lymphocytes (n=4) were sorted from 4 de novo pedAML patients (two FLT3-ITD, two FLT3 WT). As control, CD34+CD38+ (n=3) and CD34+CD38- (n=2) cells were sorted from cord blood (CB). RNA was extracted using the miRNeasy Mini Kit (Qiagen) in combination with on-column DNase I digestion (RNase-Free DNase set, Qiagen). Mean RIN of all sorted fractions was 9.3 (95% CI 9.1-9.5). All sorted cell fractions (n=15) were profiled on a custom 8x60K human Gene expression micro-array, containing probes for all human protein-coding genes with lncRNA content based on LNCipedia 2.13 (Biogazelle), as follows: 20 ng RNA was pre-amplified using the Complete Whole Transcriptome Amplification Kit (Sigma-Aldrich). Amplified RNA was subsequently labelled using the Genomic DNA ULS Labeling Kit (Agilent) and hybridized to the array in combination with CGH blocking to reduce background signaling. Micro-arrays were analyzed using an Agilent micro-array scanner and Feature Extraction software (v12.0). Probe intensities were background subtracted, quantile-normalised and log2-based probe intensities were calculated.

overall_design: To examine differential gene expression between LSC-HSC and leukemic blasts-control myeloblasts within pediatric AML.

first: Tim

last: Lammens

first: Jan

last: Philippé


title: Gene expression data from wildtype and Grin1 deleted bone marrow megakaryocytes

city: Canberra

first: James

middle: Iain

last: Hearn

state: ACT

address: 131 Garran Rd

department: Division of Genome Sciences and Cancer

country: Australia

institute: Australian National University

summary:
N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated cation channels with high calcium permeability. Their activity supports megakaryocytes proliferation and maturation, however their downstream effects on the megakaryocyte transcriptome remain poorly elucidated.
We used Clariom S Pico Arrays to profile the transcriptome changes caused by loss of Grin1 in mouse bone marrow megakaryocytes.

overall_design: Conditional deletion of Grin1, the gene encoding the obligate NMDAR subunit GluN1, in mouse megakaryocytes was directed by the Pf4 promoter using the Cre-LoxP system. This was achieved by breeding Pf4-Cre with Grin1-loxP/loxP mice. Bone marrow megakaryocytes were isolated by magnetic bead isolation, and total RNA isolated before transcriptome changes were profiled by RNA microarray technology.

middle: I

first: Taryn

last: Green

first: Colin

middle: L

last: Hisey

first: Markus

last: Bender

first: Emma

last: Josefsson

first: Nicholas

last: Knowlton

first: Juliane

last: Baumann

first: Raewyn

last: Poulsen

first: Stefan

middle: K

last: Bohlander

first: Maggie

last: Kalev-Zylinska


title: Transcriptomic and epigenetic profiling of the pancreata from old mice after transient OSKM activation and recovery

first: Antoni

last: Berenguer-LLergo

summary: OSKM activation can restore age-associated features in vitro, and its cyclic transient expression can extend lifespan in progeroid mice. We found that a single period of transient OSKM activation in old mice reconfigures its transcriptome and methylome towards youthful profile

overall_design: Pancreas samples coming from young control (13 weeks), old control (55 weeks) and old (55 weeks) mice after transient OSKM activation and recovery for 2 weeks were collected and analysed.

first: Dafni

last: Chondronasiou

first: Manuel

last: Serrano


title: An alternatively spliced p62 isoform promotes breast cancer chemoresistance

city: Hefei

first: Hao

last: Wang

state: Anhui

address: 81 Meishan Road

institute: Anhui Medical University

summary: Chemoresistance remains a major obstacle to the successful treatment of breast cancer. Especially, more than 80% of cases cannot achieve pathological complete response (pCR) in patients who received neoadjuvant chemotherapy (NAC). Understanding the mechanisms involved in chemoresistance can guide the development of efficient therapies in patients with breast cancer. Herein, we identified a novel p62 isoform with a short 3′UTR (p62-SU, 662-nt) that is associated with chemoresistance by RNA-sequence and verified by qRT-PCR, 3′RACE, and northern blot in breast cancer cells and tissue specimens. Furthermore, enforced expression of p62-SU dramatically promoted the ability of proliferation, migration, invasion, and chemoresistance compared with p62 isoform with a long/full-length 3′UTR (p62-LU, 1485-nt) in vivo and in vitro. Mechanistically, we revealed that CPSF1 could regulate the 3′UTR shorting of p62 by alternative polyadenylation and then enhanced chemoresistance in breast cancer cells. In addition, we found that p62-SU escaped the repression of miR-124-3p and promoted the ability of p62-SU to produce more protein and, subsequently, p62-dependent chemoresistance. Together, our data suggest the p62-SU, generated by CPSF1, plays an essential role in the regulation of breast cancer chemoresistance through CPSF1-p62-miR-124-3p signaling.

overall_design: RNA sequencing data from 10 pre- and post-NAC breast cancer sample pairs.

first: Zhengsheng

last: Wu

first: Qianying

last: Guo


title: Human IFN-λ3 and IFN-λ4 show distinct molecular phenotypes in macrophages

city: Kalyani

first: Sreedhar

last: Chinnaswamy

state: WB

country: India

institute: National Institute of Biomedical Genomics

summary: CD14+ cells were used to obtain M1 or M2-MDMs in presence of recombinant human IFN-λ3 or IFN-λ4.  Briefly, four Caucasian donors (n=4; one female and three males) in between 31-41 y of age, were used to obtain highly homogenous CD14+ cells; these were purchased from a commercial source. The CD14+ cells were incubated with GM-CSF (for M1-MDM) or M-CSF (for M2-MDM) in presence or absence of IFN-λ3 (at 50 ng/ml) or IFN-λ4 (at 6 μg/ml) for six days, and then the cells were fully washed off any residual growth factors or IFNs.  After six days of differentiation, the macrophages were matured with LPS for 24 h, then the total cellular RNA was isolated and RNA-seq was performed at QuickBiology, Psadena, CA, USA Summary of the study: Type III interferons or IFN-λs have emerging roles beyond antiviral defense at barrier surfaces. IFN-λ3 and IFN-λ4 are genetically associated with several infectious and inflammatory diseases; however, strong linkage at the IFNL locus circumvents a clearer understanding of their differential influence on the disease phenotypes. Comparative studies to examine the effect of IFN-λ3 and IFN-λ4 on immune cells are lacking. In this study, we used human CD14+ monocytes differentiated into macrophages (MDMs) in presence of IFN-λ3 or IFN-λ4 under M1 or M2 conditions, and after maturation with lipopolysaccharides, to perform RNA-seq analysis in order to understand the changes in molecular phenotypes that may have been induced by the two IFN-λs. Firstly, we found that IFN-λ4 can induce several genes, both unique and common to those induced by IFN-λ3, especially in M1-MDMs; our analysis showed that close to 870 genes (in both cell types) were reciprocally regulated by IFN-λ3 and IFN-λ4. Secondly, the induction of differentially expressed gene pattern suggested that IFN-λ3 has a stronger effect on M2-MDMs compared to M1-MDMs while the reverse is true for IFN-λ4.

overall_design:
Sequecing RNA from MDMs

Grant ID: IA/I/17/1/503122
Grant title: Functional interrogation of interferon lambda 4 (IFN-L4) in human health and disease
Funding agency: DBT/Wellcome Trust India Alliance Fellowship


title: Balancing MAPKs and AKT signaling pathway from compensated to decompensated state: a critical mechanism of Lingguizhugan decoction in the treatment of heart failure

city: Urumqi

first: Zhang

middle: Qing

last: hua

state: Xinjiang

address: Liudao Bay Street

institute: The Second Affiliated Hospital of Xinjiang Medical University

summary: Heart failure (HF), a major health concern worldwide, with high morbidity and mortality, renders the urgent need for new treatments. Lingguizhugan decoction (LD), a classic Chinese formula, has been clinically used to treat HF. However, the molecular mechanisms involved are not fully elucidated. In this study, we first used the experimental model of transverse aortic constriction (TAC)-induced HF in C57BL/6J mice to evaluate the therapeutic efficacy of LD. Our results showed that LD exerted protective effects against TAC-induced HF by alleviating cardiac dysfunction and cardiac dilation, which might be associated with LD’s effects on down-regulating CH-related gene and protein expression. To further reveal the potential molecular mechanisms of LD in the treatment of HF, we utilized the network pharmacology to predict the core targets and potential pathways and further utilized the transcriptomics to provide changed gene profile in the heart tissue. Our results suggested that cardiac hypertrophy signaling including AKT and MAPKs signaling pathways were significantly enriched, indicating that these pathways may be the major regulatory for LD on HF treatment. Furthermore, the protein levels in AKT-GSK3β/mTOR and MAPKs pathway were determined in both compensated and decompensated states. LD inhibited the MAPKs activation, especially, reducing phosphorylated levels of p38 from the compensated state and phosphorylated levels of ERK from the decompensated state, and the latter might depend on the regulation of AMPK by LD. Moreover, LD exerts a dual role in regulating AKT-GSK3β/mTOR/P70S6K signaling pathway, characterized by active effects in the compensated state, and inhibitory effects in the decompensated state. The regulatory effect of LD on mTOR might rely on activating AKT in the compensatory state, while might be jointly determined by AKT and AMPKαin the decompensated state. Finally, by using UPLC-QE-MS/MS analysis, we detected 62 compounds in LD extract, and found 19 of which were detected in plasma of rats. Among them, 17 bioactive compounds likely regulate AKT-GSK3β/mTOR and MAPKs signaling pathway by using bioinformatics prediction. Taken together, our study showed that LD did have a better therapeutic effect on HF via dynamically balancing AKT-GSK3β/mTOR/P70S6K pathway and MAPKs pathway. Moreover, our study also provides possible bioactive compounds responsible for LD treatment on HF.

overall_design: 9 samples in total, including Sham1, Sham2, Sham3,TAC1,TAC2,TAC3,LD1,LD2,LD3

first: Yao

last: Chen

first: Lin

last: Li

first: Cunyu

last: Hu

first: Xin

last: Zhao

first: Peng

last: Zhang

first: Yanxu

last: Chang

first: Ye

last: Shang

first: Yafeng

last: Pang

first: Weiqiang

last: Qian

first: Xianzhe

last: Qiu

first: Hongxia

first: Yuhong


title: EZHIP / CXorf67 mimics oncohistones to inhibit PRC2 in PFA ependymoma

city: Heidelberg

first: Marcel

last: Kool

address: Im Neuenheimer Feld 280

department: Pediatric Neurooncology

country: Germany

institute: German Cancer Research Center DKFZ

summary:
Expression of CXorf67 is an oncogenic mechanism that drives H3K27 hypomethylation in PFA ependymomas by mimicking K27M mutated oncohistones.
Background: Posterior fossa A (PFA) ependymomas comprise one out of nine molecular groups of ependymoma. PFA tumors are mainly diagnosed in infants and young children, show a poor prognosis and are characterized by a lack of the repressive histone H3 lysine 27 trimethylation (H3K27me3) mark. Recently, we reported CXorf67 overexpression as hallmark of PFA ependymoma and showed that CXorf67 can interact with EZH2 thereby inhibiting polycomb repressive complex 2 (PRC2), but the mechanism of action remained unclear. Methods: We performed mass spectrometry (MS) and peptide modelling analyses to identify the functional domain of CXorf67 responsible for binding and inhibition of EZH2. Our findings were validated by immunocytochemistry, western blot and methyltransferase assays. Results: We find that the inhibitory mechanism of CXorf67 is similar as in diffuse midline gliomas harboring H3K27M mutations. A small, highly conserved peptide sequence located in the C-terminal region of CXorf67 mimics the sequence of K27M mutated histones and binds to the SET domain of EZH2. This interaction blocks EZH2 methyltransferase activity and inhibits PRC2 function causing de-repression of PRC2 target genes including genes involved in neurodevelopment. Conclusions: Expression of CXorf67 is an oncogenic mechanism that drives H3K27 hypomethylation in PFA tumors by mimicking K27M mutated histones. Disrupting the interaction between CXorf67 and EZH2 may serve as a novel targeted therapy for PFA tumors but also for other tumors that overexpress CXorf67. Based on its function, we have renamed CXorf67 into EZH Inhibitory Protein (EZHIP).

overall_design: Total RNAs isolated from HEK293T cells transduced with lentiviral constructs expressing either full-length EZHIP/CXorf67 or truncated versions (N-terminal part, Middle part, or C-terminal part) of CXorf67 /EZHIP were analyzed on human Affymetrix 133plus2.0 arrays

first: Jens-Martin

last: Hübner


title: ATP7B Gene Therapy of Autologous Reprogrammed Hepatocytes Alleviates Copper Accumulation in a Murine Model of Wilson’s Disease

status: Public on Mar 05 2022

city: Duarte

first: Cai

last: Hongxia

state: California

address: 1500E Duarte Rd

institute: City of hope Medical Center

summary:
Purpose：Wilson's disease (WD) is a rare hereditary disorder due to ATP7B gene mutation, causing pathologic copper storage mainly in the liver and neurological systems. Hepatocyte transplantation showed therapeutic potential, however, this strategy is often hindered by a shortage of quality donor cells and by allogeneic immune rejection. Here we evaluate the function and efficacy of autologous reprogrammed, ATP7B gene-restored hepatocytes using a murine model of WD
Mathods and Results: By reprogramming hepatocytes from ATP7B-/- mice with small molecules, sufficient liver progenitor cells (LPCs) are harvested. After lentivirus-mediated miniATP7B gene transfection and re-differentiation, functional LPC-ATP7B-Heps are developed. RNA-seq data show that compared with LPC-GFP-Heps with enrichment of genes mainly in pathways of oxidative stress and cell apoptosis, in LPC-ATP7B-Heps under high copper stress pathways for copper ion binding and cell proliferation are enriched. LPC-ATP7B-Heps transplantation into ATP7B-/- mice alleviates deposition of excess liver copper with its associated inflammation and fibrosis, comparable to those observed using normal primary hepatocytes at four months after transplantation.
Conclusion: We establish the autologous reprogrammed, ATP7B gene restored LPC-ATP7B-Heps and further transplantation demonstrate alleviated copper accumulation in WD mice.

overall_design: 3 groups of mRNA profiles of reporgrammed, ATP7B or GFP lentivirus transfected hepatocytes from ATP7B-/- mice and reprogrammed, GFP transfected hepatocytes from wildtype mice. All cells underwent copper stress in vitro.

last: Cai

first: Xiaoping

last: Wang


title: Next Generation Sequencing of TGS24-treated human breast cancer cell line

city: Kolkata

first: Subhrangsu

last: Chatterjee

state: West Bengal

address: Kankurgachi

institute: Bose Institute

summary: We report the differential gene expression of TGS24-treated breast cancer cells

overall_design: Examination of MAPK12 pathway gene expression upon TGS24-treated breast cancer cells


title: Effect of BCDIN3D knock-out on gene expression in Chronic Myelogenous Leukemia (CML) Hap1 cells.

city: Austin

first: Blerta

last: Xhemalce

state: Texas

address: 2500 Speedway

institute: University of Texas at Austin

type: Non-coding RNA profiling by high throughput sequencing

summary: WT and BCDIN3D knocked-out Hap1 cells were analysed by RNA- and microRNA-Seq to uncover the effect of BCDIN3D on specific microRNAs and their messenger RNA targets.

overall_design: Hap1 WT and BCDIN3D knock-out cells were grown for 48h in IMDM+10%FBS+PSQ. RNA and protein was purified using the Norgen RNA/protein purification plus kit. Two biological replicates were tested. The same total RNA of RIN 10 was used for RNA-Seq and microRNA-Seq.

first: Nathan

last: Abell


title: AKT/ERK-mediated activation of CDK7-MED1-AR axis drives resistance to pan BET bromodomain inhibitor ZEN-3694 in advanced prostate cancer [Exome-Seq]

city: Philadelphia

first: Irfan

last: Asangani

state: PA

address: 421 Curie Blvd

department: Cancer Biology

institute: University of Pennsylvania

summary: Prostate cancer cells were cultured in the presence of the drug Zenith-3694 for a period of 16-20 weeks to develop drug-resistant cells and subjected to Exome sequencing.

overall_design: Zenith-3694 resistant Pca cells were subjected to Exome sequencing along with their parental counterparts. Briefly, DNA was extracted from the below-mentioned cells and 1ug of total DNA was used for library preparation using Illumina Nextera Flex Enrichment kit and IDT exome capture probes. Sequencing run was performed using NextSeq mid output 2x150 kit.

first: Ramakrishnan

last: Natesan

first: Shweta

last: Aras


title: RNAseq assessment of early IL-33-mediated signaling on donor T cells in mourse GVHD model

city: Pittsburgh

first: Heth

last: Turnquist

address: 200 Lothrop Street , E1554 BST

department: Surgery

institute: University Of Pittsburgh

summary: To understand pathways that IL-33 is controlling early after allogeneic hematopoietic cell transplantation (AlloHCT), we performed RNAseq on sorted splenic B6 ST2fl/fl and ST2WT donor CD4+ T cells co-transferred into the same BALB/c recipient on d5 post-alloHCT

overall_design: B6 CD45.1+ (ST2WT) and B6 CD45.2+ CD4-CrexR26-LSL-YFPxSt2fl/fl (ST2fl/fl) donor splenic CD4+ T cells were enriched from CD90.1+ B6 or H2-Kd BALB/c recipient mice (n=4/group) using negative depletion with Dynabeads (Life Technologies). The cells were stained for CD3, CD4, CD90.1, H2-Kd CD45.1, CD45.2 and sorted for ST2WT and ST2fl/fl CD4+ populations on a FACSAria II, directly into SmartSeq low-input RNA kit lysis buffer for library prep and RNA sequencing. Libraries were prepared from isolated RNA using Nextera XT DNA library prep kits, and RNA sequencing was performed on Illumina NextSeq500 by the Health Sciences Sequencing Core at the University of Pittsburgh. Raw sequencing data were processed using CLC Genomics Workbench 20.0.3 (QIAGEN Inc., https://digitalinsights.qiagen.com) for quality control and aligned to the Mus musculus genome version GRCm38.p6. Reads assigned to each gene underwent TMM normalization and differential expression analysis was performed using edgeR within CLC Genomics to compare ST2WT versus ST2fl/fl donor T cells. The top differentially expressed genes were filtered by adjusted p-value q<0.05 and fold-change greater than 1.5 for subsequent downstream pathway analysis. The relative expression shown in heatmaps was calculated as the fragments per kilobase exon per million mapped reads value for each sample divided by the mean expression of that gene in all samples per each experiment. GSEA from the Broad Institute (http://www.broad.mit.edu/gsea) was used to calculate the enrichment of genes in each set.

first: Gaelen

last: Dwyer


title: AKT/ERK-mediated activation of CDK7-MED1-AR axis drives resistance to pan BET bromodomain inhibitor ZEN-3694 in advanced prostate cancer

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

overall_design: Refer to individual Series


title: A resilient mutualistic interaction between cucumber mosaic virus and its natural host Arabidopsis halleri to adapt to an environmental change

city: Kusatsu, Shiga

first: Midori

last: Tabara

address: 1-1-1, Noji-Higashi

institute: Ritsumeikan University

summary: A pseudometallophyte Arabidopsis halleri is frequently found to be infected with cucumber mosaic virus (CMV) in its natural habitat. The purpose of this study is to elucidate the effect of CMV infection on its natural hosts. The CMV(Ho) strain isolated from A. halleri was inoculated into clonal A. halleri plants that were obtained from runners of mother plants, and the pathosystem consisting of CMV(Ho) and its natural host A. halleri was established.In low heavy metal environment, the CMV(Ho) infection caused growth retardation in the above-ground part (stems and leaves) of host plants, and thereby conferred strong drought tolerance on host plants. On the other hand, in high heavy metal environment, which simulates a natural habitat for A halleri, the CMV(Ho) infection did not cause any symptoms to host plants and conferred mild drought tolerance. And the result of transcriptome analysis suggests that CMV(Ho) is recognized as a symbiont rather than a pathogen by its host plant. These results indicate a resilient mutualistic interaction between CMV(Ho) and its natural host A. halleri to adapt to an environmental change.

overall_design: RNA-seq is perfomed using three CMV-free, three CMV-infected, three  CMV-free zinc excess and three CMV-infected zinc excess leaves of Arabidopsis halleri.

first: Shimpei

last: Uraguchi

first: Izumi

last: Watanabe

first: Hideki

last: Takahashi

first: Toshiyuki

last: Fukuhara


title: AKT/ERK-mediated activation of CDK7-MED1-AR axis drives resistance to pan BET bromodomain inhibitor ZEN-3694 in advanced prostate cancer [RNA-Seq]

summary: Prostate cancer cells were cultured in the presence of the drug Zenith-3694 for a period of 16-20 weeks to develop drug-resistant cells and subjected to RNA sequencing.

overall_design: Comparison of three different prostate cancer cell lines (VCAP, LNCAP, 22RV1)  upon acquiring drug resistance.

first: Mohammed

last: Alhusayan


title: RNA high-throughput sequencing for Lupinus micranthus Guss

city: Lanzhou

first: Chai

last: Shujie

address: No.1 Yingmen Village, Anning District, Lanzhou city

institute: Gansu Agricultural University

summary: RNAseq for 18 Lupinus micranthus Guss samples

overall_design: Examination of 2 group (American vs Iceland seeds) with three different treatments (no treatment, PEG drought for 24 hours, PEG drought for 72 hours) in 18 samples.

first: ShuJie

last: Chai

first: Wenke

last: Dong

first: Huiling

last: Ma


title: Recruitment of transcriptional effectors by Cas9 creates cis regulatory elements and demonstrates distance-dependent transcriptional regulation

city: Haifa

first: Noam

last: Kaplan

address: Efron 1

institute: Technion - Israel Institute of Technology

summary: It is essential to regulate the expression of genes, such as those encoding the proteins of the cardiac sarcomere. This regulation is often mediated by cis regulatory elements termed enhancers and repressors that recruit transcription factors to gene-distal sites. However, the relationship between transcription factors recruitment to gene-distant sites and the regulation of gene expression is not fully understood. Specifically, it is unclear if such recruitment to any genomic site is sufficient to form an enhancer or repressor at the site, and what is the relationship between the cis regulatory element’s position and its ability to control the transcription of distant genes. Using dead Cas9 to recruit either viral or endogenous transcription factor activation domains, we demonstrate that targeting 'naïve' genomic sites lacking open chromatin or active enhancer marks is sufficient to alter the chromatin signature of the target site, the distant gene promoter, and significantly induce the distant gene expression, even across chromatin insulating loci. The magnitude of induction is affected by the distance between the activation site and the cognate gene in a non-linear manner. Dead Cas9 mediated recruitment of repression domains behaves similarly to activation in that targeting of non-regulatory regions could repress gene expression with a nonlinear distance dependence and across chromatin insulating loci. These findings expand the models of enhancer generation and function by showing that an arbitrary genomic site can become a regulatory element and interact epigenetically and transcriptionally with a distant promoter. They also provide new fundamental insights into the rules governing gene expression.

overall_design: One Rat2 CRL-1764 Hi-C sample was sequenced

first: Jubran

last: Boulos

first: Itai

last: Ehrlich

first: Nili

last: Avidan

first: Omer

last: Shkedi

first: Lilac

last: Haimovich-Caspi

first: Izhak

last: Kehat


title: In Vivo Transcriptomic Profiling using Cell Encapsulation Identifies Effector Pathways of Systemic Aging

city: Lausanne

first: Jerome

last: Feige

address: EPFL Innovation Park, Building H

department: Musculo-Skeletal Health

institute: Nestle Institute Of Health Sciences

summary: Sustained exposure to a young systemic environment rejuvenates aged organisms and promotes cellular function. However, due to the intrinsic complexity of tissues it remains challenging to pinpoint niche-independent effects of circulating factors on specific cell populations. Here we describe a method for the encapsulation of human and mouse skeletal muscle progenitors in diffusible polyethersulfone hollow fiber capsules that can be used to profile systemic aging in vivo independent of heterogeneous short-range tissue interactions. We observed that circulating long-range signaling factors in the old systemic environment lead to an activation of Myc and E2F transcription factors, induce senescence and suppress myogenic differentiation. Importantly, in vitro profiling using young and old serum in 2D culture does not capture all pathways deregulated in encapsulated cells in aged mice. Thus, in vivo transcriptomic profiling using cell encapsulation allows for the characterization of effector pathways of systemic aging with unparalleled accuracy.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: QuantSeq from encapsulated mouse myoblast cells which subcutaneously implanted into young and aged mice for ten days and human myoblast cell which cultivated in vitro and exposed to the young and aged serum for four and ten days.

first: Omid

last: Mashinchian

first: Xiaotong

last: Hong

first: Eugenia

last: Migliavacca

first: Gregory

last: Lefebvre

first: Christophe

last: Boss

first: Filippo

last: De Franceschi

first: Joris

last: Michaud

first: Emmeran

last: Le Moal

first: Jasmin

last: Collerette-Tremblay

first: Joan

last: Isern

first: Sylviane

last: Metairon

first: Frederic

last: Raymond

first: Patrick

last: Descombes

first: Nicolas

last: Bouche

first: Pura

last: Muñoz-Cánoves

middle: N

first: C

last: Bentzinger


title: Deciphering the Molecular Signature of Human Hyalocytes in Relation to Other Innate Immune Cell Populations

city: Freiburg

first: Clemens

last: Lange

address: Kilianstraße 5

department: Klinik für Augenheilkunde

institute: Uniklinik Freiburg

summary: Purpose: The present study compares the transcriptional profiles of human hyalo-cytes, retinal microglia as well as classical, intermediate and non-classical mono-cytes in a pairwise analysis.   Methods: Using fluorescence-activated cell sorting, hyalocytes and retinal microglia were isolated from the vitreous or retinal tissue of enucleated eyes due to melano-ma of the iris, ciliary body or choroid. In addition, classical, intermediate and non-classical monocytes were isolated from whole blood. RNA sequencing was per-formed and the transcriptional profiles of all five immune cell populations were ana-lyzed using bioinformatic analysis.  Results: Pairwise analysis indicated distinct differences between hyalocytes and all three subsets of monocytes, whereas a high degree of similarity was apparent when compared to retinal microglia, with comparable expression levels of established MG markers such asTREM2, P2RY12 and TMEM119. Cluster analysis based on signa-ture genes of yolk sac-derived cells as well as hematopoietic stem and progenitor cells indicated that in contrast to monocytes, human hyalocytes and retinal microglia share a common origin in the yolk sac. Among the top expressed genes in hyalo-cytes, SPP1, CD74 and C3 were significantly upregulated when compared to mon-ocytes. Despite the high level of similarity, genes such as TNF, CCL3 and CXCL8 were significantly upregulated in hyalocytes when compared to retinal microglia. In addition, ten hyalocyte-specific genes were identified, among them FOS, DUSP1 and EGR2.   Conclusions: This study provides detailed insights into the molecular profile of iso-lated human hyalocytes and, for the first time, human retinal microglia. The expres-sion profile of hyalocytes is similar to that of retinal microglia and indicates that hy-alocytes are, contrary to the prevailing notion, yolk sac-derived cells. The hyalocyte-specific markers identified in this study will contribute in the future to deciphering the different roles of this fascinating cell population in health and diseases of the vitre-oretinal interface.

overall_design: RNA sequencing of human hyalocytes (n=5), retinal microglia (n=5) as well as classical (n=6), intermediate (n=5) and non-classical (n=5) monocytes.

first: Julian

last: Wolf

first: Stefaniya

last: Boneva

first: Hansjürgen

last: Agostini

first: Günther

last: Schlunck

first: Anja

last: Schlecht


title: Impaired reorganization of centrosome structure underlies human congenital dilated cardiomyopathy

city: Baltimore

first: Matthew

last: Miyamoto

state: MD

address: 670 W Baltimore St

department: Cardiology

institute: University of Maryland Medical Center

summary: During cardiomyocyte (CM) maturation, the centrosome undergoes a dramatic structural reorganization whereby certain components, previously localized at the centrioles, become localized to the nuclear envelope. Here, we generated a scRNA-seq data set of iPSC-CMs and identified impaired CM maturation as a key dysregulated process underlying a case of congenital dilated cardiomyopathy

overall_design: We sampled human differentiation day 35 iPSC-CMs from a congenital dilated cardiomyopathy iPSC line and a control iPSC line.

first: Young

middle: W

last: Chun

first: Charles

middle: C

last: Hong


title: Effect of the primed exposure of Diesel CAST model aerosol in air-liquid interface BEAS-2B cells exposed to native birch pollen

city: Munich

first: Joana

last: Candeias

address: Biedersteinerstr. 29

department: Center Allergy and Environment (ZAUM)

institute: Technical University Munich & Helmholtz Center Munich

summary: BEAS-2B cells, at air-liquid interface, were exposed to Diesel CAST model aerosol in an vitro exposure system and, later, to native birch pollen using a Pollen Sedimentation Chamber stabilished before. The same exposure was performed, without the primed anthropogenic exposure. The goal was to understand the effect of pre-exposure to a model diesel aerosol in allergic sensitization, using a model stabilished that mimics a real life exposure as closer as possible.

overall_design: For the combined exposure, BEAS-2B, at air-liquid interface, were exposed to Diesel CAST model aerosol for 2 hours and after 24 hours exposed to 4 mg whole birch pollen for different incubation times (2h25 min, 6h50 min and 24h). The cells were seeded in 24mm Transwells 48 hours before exposure and kept in air lift conditions 24h before start. Effect of combined expousre were referenced against cells exposed to clean air and pollen. For the pollen exposure alone, BEAS-2B, also at air-liquid interface, were exposed to 4 mg birch pollen for the indicated times. Here, effects of birch whole pollen were referenced against cells exposed to air.  Three replicates were performed for each incubation time.

first: Elias

middle: J

last: Zimmermann

first: Christoph

last: Bisig

first: Nadine

last: Gawlitta

first: Sebastian

last: Oeder

first: Thomas

last: Groeger

first: Ralf

first: Carsten

middle: B

last: Schmidt-Weber

first: Jeroen

last: Buters


title: Whole transcriptome sequencing of E18 mouse cortex

city: Beijing

first: Wei

last: Song

address: 1 Beichen West Road, Chaoyang District

institute: Beijing Institutes of Life Science, CAS

summary: We observed Csmd3-deficient mice exhibited growth retardation, atypical learning and memory behaviors. Futher, cortical neurons from mice lacking Csmd3 showed abnormal development. To identified the role of Csmd3 in cortex develepement, we performed whole transcriptome sequencing on embryonic 18 days cortex from Csmd3-/- and wild type mice. As a result, a total of 2133 differentially expressed genes (DEGs) were identified, including 1400 upregulated genes and 733 downregulated genes. Downregulated DEGs were mainly related to synaptic functions, while the upregulated DEGs were involved in multiple progresses related to cell-to-cell communication.  In terms of cell type preference as referring to the single cell sequencing data in mouse neocortex at E18, downregulated DEGs were exlusively enriched into neurons and upregulated DEGs were involved with multiple cell types. Our study is the first detailed analyses of Csmd3 in development mouse cortex, identifing Csmd3 plays a pivotal role in synaptic organazation and transmission, also has an effect on glia-vascular inter-cellular communication.

overall_design: Total mRNA profiles of E18 WT, Csmd3-/- cortex by deep sequencing using Illumina NovaSeq 6000.

first: Quan

last: Li


title: Transposon sequencing for pathway-specific unipolar polysaccharide biosynthesis screen in Agrobacterium tumefaciens C58

city: Bloomington

first: Clay

last: Fuqua

state: IN

address: 1001 E. Third Street

department: Biology

institute: Indiana University

summary: Performed en masse sequencing of transposon insertions among A. tumefaciens mutants deficient in unipolar polysaccharide (UPP) production in two different genetic backgrounds, each one specific for one of the two chemical species of UPP (UPPGlcN and UPPGalN)

overall_design: Screening strains overproduce UPP polysaccharide due to elevated cyclic diguanylate monophosphate (cdGMP, because of deletion of pruA ATU_RS05580 regulator) and are also genetically disabled for production of multiple polysacccharides: cellulose, succinoglycan, Beta 1,2-glucans and Beta 1,3-glucans).  Mutants were isolated in this back ground with either the uppY (ATU_RS02370) or the uppW (ATU_RS11495) polysaccharide polymerase genes also deleted.

first: Maureen

last: Onyeziri