title: Negative feedback control of neuronal activity by microglia

first: Ana

last: Badimon

state: NY

address: 1 Gustave L Levy PLace

department: Neuroscience

summary: Microglia, the brain resident macrophages, play a key role in the regulation of brain function by removing dying neurons, pruning non-functional synapses, and producing ligands that support neuronal survival. We have identified a new function of microglia as critical modulators of neuronal activity and associated behavioral responses in mice. We show that microglia respond to neuronal activation and that ablation of microglia amplifies and synchronizes the activity of neurons, leading to seizures. The suppressive impact of microglia on neuronal activation occurs in a highly localized fashion and depends on the ability of microglia to sense and catabolize extracellular ATP. ATP, which can be released upon neuronal activation by neurons and astrocytes, triggers the recruitment of microglia protrusions and is converted by the microglial ATP-hydrolyzing enzyme CD39 and CD73 into AMP and adenosine, a potent suppressor of neuronal activity. We show that the microglial sensing of ATP, the ensuing production of adenosine, and the adenosine-mediated control of neuronal response via the A1R are essential for the microglia-mediated regulation of neuronal activity and animal behavior. Our findings suggest that this microglia-driven negative feedback mechanism operates in a fashion similar to inhibitory neurons and plays an essential role in protecting the brain from excessive activation in health and diseases.

overall_design: Microglial or neuronal RNAs were isolated based on the eGFP-L10a expression of ribosomes. Single nuclei sequencing was performed on striatal nuclei using 10X technology.

first: Hayley

middle: J

last: Strassburger

first: Pinar

last: Ayata

first: Xinhong

last: Chen

first: Aditya

last: Nair

first: Ako

last: Ikegami

first: Philip

last: Hwang

first: Andrew

last: Chan

first: Steven

middle: M

last: Graves

first: Joseph

middle: O

last: Uweru

first: Carola

last: Ledderose

first: Munir

last: Gunes Kutlu

first: Michael

last: Wheeler

first: Anat

last: Khan

first: Masago

last: Ishikawa

first: Ying-Chih

last: Wang

first: Yong-Hwee

last: Loh

first: Jean

middle: X

last: Jiang

first: D

last: Surmeier

first: Simon

last: Robson

first: Wolfgang

middle: G

last: Junger

first: Robert

last: Sebra

first: Erin

last: Calipari

first: Paul

last: Kenny

first: Ukpong

last: Eyo

first: Marco

last: Colonna

first: Francisco

last: Quintana

first: Hiroaki

last: Wake

first: Viviana

last: Gradinaru

first: Anne

last: Schaefer


title: Prp19 is an independent prognostic marker and promotes neuroblastoma metastasis by regulating the Hippo-YAP signaling pathway [SK-N-AS]

status: Public on Aug 18 2020

city: Shanghai

first: Yuanxia

last: Cai

address: No.1665, Kongjiang Road, Yangpu District

country: China

institute: Xinhua Hospital

type: Expression profiling by high throughput sequencing

summary: This study first reveals that Prp19 is a potential oncogene in neuroblastoma, which is significantly correlated with poor prognosis and promote metastasis viaregulating Hippo-YAP pathway

overall_design: Prp19 knockdown was performed in neuroblastoma cell line SK-N-AS

first: Zhixiang

last: Wu


title: The Compendium of DNA-Binding Specificities of Transcription Factors in a Pathogenic Bacterium

city: Hong Kong

first: Jian

last: Yan

state: Hong Kong Special Administrative Region

address: 83 Tat Chee Avenue, Kowloon

department: Department of Biomedical Sciences

institute: City University of Hong Kong

type: Genome binding/occupancy profiling by high throughput sequencing

summary: The regulation of transcription is primarily exerted through transcription factors (TFs) binding to genomic DNA. Although molecular mechanisms of TFs have been studied individually for decades, a complete picture of binding profiles of all TFs and their precise targets in the genome are still lacking in the model pathogen Pseudomonas syringae. To this end, we performed a high-throughput systematic evolution of ligands by exponential enrichment (HT-SELEX) approach on all 301 annotated TFs in P. syringae. Robust enrichment of specific sequences was deduced to 118 SELEX motifs. We identified 12,464 interactions between 100 TFs and their target genes in the genome, for an individual TF ranging from 6 to 1481 sites. It showed that 90% TF binding was of dimeric site type, in which 85% with the head-to-head palindromic binding preference. To further explore the pathogenic mechanism of TFs in P. syringae, we mapped intricate networks of these TFs and their targets in the virulence-associated pathways, many of which were verified by orthologous methods such as ChIP-seq, EMSA, RT-qPCR and a reporter assay of promoter activity. By checking the enrichment of binding sites in pathways, we identified 25 virulence-associated master regulators of which 14 had never been characterized as TFs before. Overall, the present study provides a valuable resource for TF binding specificities in P. syringae and demonstrates a novel and an integrative analysis for seeking the virulence-associated TFs and its target genes. We expect that the results will significantly benefit future studies on the transcriptional regulation in P. syringae, and facilitate the design of drug targets to protect plants from attacks by relevant pathogens.

sample_organism: Pseudomonas savastanoi pv. phaseolicola 1448A

overall_design: In order to globally profile the DNA binding specificities of all TFs in P. syringae, we purified the proteins for all 301 annotated TFs in the genome and applied a high-throughput systematic evolution of ligands by exponential enrichment (HT-SELEX) approach.

first: Xin

last: Deng

first: Ligang

last: Fan

first: TingTing

last: Wang

first: Canfeng

last: Hua

first: Wenju

last: Sun


title: CRL5-dependent regulation of the small GTPAses ARL4C and ARF6 controls hippocampal morphogenesis

status: Public on Aug 17 2020

city: Davis

first: Sergi

last: Simo

state: CA

address: One Shields Avenue, 3402 Tupper Hall

department: Cell Biology and Human Anatomy

country: USA

institute: University of California Davis

summary: Purpose: Understanding the role of RBX2 during hippocampla development, pyramidal neuron migration and dendritogenesis.

overall_design: Comparison of  mRNA profiles from control (Rbx2 flox/flox) (Ctrl) and Rbx2 mutant (Rbx2 flox/flox; Nestin-Cre/+) (Rbx2cKO-Nes) telencephalons (n=3/genotype) at P1, using Illumina NextSeq 500 (Illumina, San Diego, CA) with paired-end 40bp reads.

first: Jisoo

middle: S

last: Han

first: Keiko

last: Hino

first: Wenzhe

last: Li

first: Raenier

middle: V

last: Reyes

first: Cesar

middle: P

last: Canales

first: Adam

last: Miltner

first: Yasmin

last: Haddadi

first: Junqing

first: Chao-Yin

last: Chen

first: Anna

last: La Torre

last: Simó


title: DNA mismatches reveal conformational penalties in protein-DNA recognition II

city: Durham

first: Raluca

last: Gordan

state: NC

address: 101 Science Dr, CIEMAS 2179

department: Center for Genomic and Computational Biology

institute: Duke University

summary: Transcription factors (TF) recognize specific genomic sequences to regulate complex gene expression programs. Although it is well established that TFs bind specific DNA sequences using a combination of base readout and shape recognition, some fundamental aspects of protein-DNA binding remain poorly understood. Many DNA-binding proteins induce changes in the DNA structure outside the intrinsic B-DNA envelope. However, how the energetic cost associated with distorting DNA contributes to recognition has proven difficult to study because the distorted DNA exists in low-abundance in the unbound ensemble. Here, we use a novel high-throughput assay called SaMBA (Saturation Mismatch-Binding Assay) to investigate the role of DNA conformational penalties in TF-DNA recognition. In SaMBA, mismatches are introduced to pre-induce DNA structural distortions much larger than those induced by changes in Watson-Crick sequence. Strikingly, approximately 10% of mismatches increased TF binding, and at least one mismatch was found that increased the binding affinity for each of 22 examined TFs. Mismatches also converted non-specific sites into high-affinity sites, and high-affinity sites into super-sites stronger than any known canonical binding site. Determination of high-resolution X-ray structures, combined with NMR measurements and structural analyses revealed that many of the mismatches that increase binding induce distortions similar to those induced by protein binding, thus pre-paying some of the energetic cost to deform the DNA. Our work indicates that conformational penalties are a major determinant of protein-DNA recognition, and reveals mechanisms by which mismatches can recruit TFs and thus modulate replication and repair activities in the cell.

overall_design: Saturation mismatch-binding assays (SaMBA) were performed for 22 eukaryotic transcription factors. Briefly, the SaMBA assays involved binding of tagged transcription factors to double-stranded Agilent microarrays in order to determine their binding specificity for putative DNA binding sites as well as sites containing all possible DNA mismatches (or mispairs). Each sequence of interest was represented on the array in 8-20 replicate spots. We report the signal intensity for each spot.


title: Uncovering the basis of protein-protein interaction specificity with a combinatorially complete library

city: Cambridge

first: Thuy-Lan

last: Lite

state: Massachusetts

address: 31 Ames Street

institute: Massachusetts Institute of Technology

summary: Protein-protein interaction specificity is often accomplished by molecular recognition and mediated by a small set of interfacial residues. However, the extent to which these residues serve positive specificity roles (encourage interaction with the cognate protein) or negative specificity roles (destabilize interaction with the non-cognate protein) is poorly understood. To systematically dissect these roles, we built a library of interface variants using a bacterial toxin-antitoxin system, Mesorhizobium opportunistum ParD3-ParE3, as a model. The ParD3 antitoxin is specific for its cognate toxin ParE3 and does not neutralize the non-cognate toxin ParE2. We built a saturation mutagenesis library at three key interface positions in ParD3 and measured the ability of each variant to neutralize ParE3 and ParE2 via a 10-hour bulk selection assay. A fitness score was assigned to each variant in the presence of each toxin based on the relative expansion of the variant during the experiment (between t = 0 and t = 600 min).

overall_design: Antitoxin variant frequency was measured for two toxins (M. opportunistum ParE3 and ParE2) upon toxin induction (t = 0) and after 10 hours of toxin and antitoxin library co-expression (t = 600). Two replicates were studied for each toxin strain. Selection in the presence of toxin expression was performed independently for each replicate.

first: Thúy-Lan

first: Robert

middle: A

last: Grant

first: Isabel

last: Nocedal

first: Monica

last: Guo

first: Michael

middle: T

last: Laub


title: Activation of AHR transcriptional activity upon treatment with indole-3-pyruvate

status: Public on Aug 19 2020

city: Heidelberg

first: Ahmed

last: Sadik

address: Im Neunheimer Feld 280

department: Metabolic Crosstalk in Cancer

country: Germany

institute: DKFZ

summary: Indole-3-pyruvate (I3P), an endogenous metabolite derived from tryptophan by gut microbiota and IL4I1 enzyme in humans can potentially activate the transcriptional activity of the Aryl Hydrocarbon receptor. Here we test this by stimulating AHR proficient U-87MG cells with I3P alone or in combination with the AHR antagonist SR1.

overall_design: We stimulated AHR proficient U-87MG cells with I3P alone or in combination with the AHR antagonist SR1.

first: Luis

last: Somarribas

first: Soumya

last: Mohapatra

first: Christiane

last: Opitz


title: Inhibition of MAN2A1 enhances tumor response to anti-PD-L1

first: jun

last: ge

address: 1239 Siping Road

department: School of Life Sciences and Technology

institute: Tongji University

summary: Immune checkpoint blockade (ICB) has shown remarkable efficacy, but in only a minority of cancer patients, suggesting the need to develop additional treatment strategies.We integrated transcriptional profiles of treatment-naïve human tumors and functional CRISPR screens to identify glycometabolism genes with immunomodulatory effects. We identified MAN2A1, encoding an enzyme in N-glycan maturation, as a key immunomodulatory gene. Analyses of public immune checkpoint blockade trial data also suggested a synergy between MAN2A1 inhibition and anti-PD-L1 treatment. Loss of Man2a1 in cancer cells increased their sensitivity to T cell-mediated killing. Man2a1 knockout enhanced response to anti-PD-L1 treatment and facilitated higher cytotoxic T cell infiltration in tumors under anti-PD-L1 treatment. Furthermore, a pharmacological inhibitor of MAN2A1, swainsonine, synergized with anti-PD-L1 in syngeneic melanoma tumor model, whereas each treatment alone had little effect.

overall_design: Wildtype or Man2a1 knockout B16F10 treated with IgG isotype control or anti-PD-L1 or swainsonine or the combination of anti-PD-L1 and swainsonine.

first: Sailing

last: Shi

first: Shengqing

last: Gu


title: Repression of Irs2 by let-7 miRNAs is essential for the homeostasis of the telencephalic neuroepithelium [miRNA-seq]

city: San Joan d'Alacant

first: Jose

last: Lopez-Atalaya

address: Av Ramon y Cajal s/n

department: Instituto Neurociencias

country: Spain

institute: CSIC-UMH

type: Non-coding RNA profiling by high throughput sequencing

summary: The structural integrity and cellular homeostasis of the embryonic stem cell niche are critical for normal tissue development. In the telencephalic neuroepithelium, this is controlled in part by cell adhesion molecules and regulators of progenitor cell lineage, but the specific orchestration of these processes remains unknown. Here we have studied the role of microRNAs in the embryonic telencephalon as key regulators of gene expression. By using the early recombiner Rx-Cre mouse, we demonstrate that miRNAs are essential to preserve the cellular homeostasis and structural integrity of the telencephalic neuroepithelium. We show that Rx-Cre;Dicerflox/flox mouse embryos have a severe disruption of the telencephalic apical junction belt, followed by invagination of the ventricular surface and formation of hyperproliferative rosettes. Transcriptomic analyses and functional experiments in vivo show that these defects result from upregulation of Irs2 upon loss of let-7 miRNAs, in an apoptosis-independent manner. Our results reveal an unprecedented relevance of miRNAs in early forebrain development, with potential mechanistic implications in pediatric brain cancer.

overall_design: The rostral-most region of the telencephalic primordium was dissected out from DicerF/F and RxCre-DicerF/F E11.5 embryos, and miRNA expression profiles were analysed by RNA sequencing (RNA-seq).

first: Rafael

last: Soler

first: Martina

last: Dori

first: Virginia

last: Fernandez

first: Victor

last: Borrell


title: PEARL-seq: A Photoaffinity Platform for the Analysis of Small Molecule-RNA Interactions

city: Waltham

first: Lee

last: Vandivier

state: MA

address: 830 Winter St

institute: Arrakis Therapeutics

summary: RNA is emerging as a valuable target for the development of novel therapeutic agents. The rational design of RNA-targeting small molecules, however, has been hampered by the relative lack of methods for the analysis of small molecule-RNA interactions. Here we present our efforts to develop such a platform using photoaffinity labeling. This technique, termed Photoaffinity Evaluation of RNA Ligation-Sequencing (PEARL-seq), enables the rapid identification of small molecule binding locations within their RNA targets and can provide information on ligand selectivity across multiple different RNAs. These data, when supplemented with small molecule SAR data and RNA probing data enables the construction of a computational model of the RNA-ligand structure, thereby enabling the rational design of novel RNA-targeted ligands.

sample_organism:
Homo sapiens
Synthetic plasmid

platform_organism:
Homo sapiens
Synthetic plasmid

overall_design: PEARL-seq, SHAPE-MaP and DMS-MaP analysis of the model RNA Aptamer21

first: Herschel

last: Mukherjee

first: J

middle: C

last: Blain

first: Donovan

middle: N

last: Chin

first: Jessica

middle: E

last: Friedman

first: Fei

last: Liu

first: Ashley

last: Maillet

first: Chao

last: Fang

first: Jenifer

middle: B

last: Kaplan

first: Jinxing

last: Li

first: David

last: Chenoweth

first: Allan

last: Christensen

first: Lars

middle: K

last: Petersen

first: Nils

last: Hansen

last: Barrera

first: Neil

last: Kubica

first: Gnanasambandam

last: Kumaravel

first: Jennifer

last: Petter


title: A small protein encoded by a putative long noncoding RNA inhibits proliferation and tumorigenicity in human colorectal cancer cells [RNA-Seq]

city: Bethesda

first: Lorinc

last: Pongor

state: MD

address: 37 Convent Dr,

department: DTB

institute: NIH

summary: This study on the initial characterization of FORCP demonstrates that FORCP is a novel, conserved small protein encoded by a mis-annotated lncRNA that regulates apoptosis and tumorigenicity in well-differentiated colorectal cancer cells.

overall_design: A total of four colon epithelial cell lines were sequenced for comparison of FORCP expression.

first: Ashish

last: Lal

first: Xiao

middle: L


title: A small protein encoded by a putative long noncoding RNA inhibits proliferation and tumorigenicity in human colorectal cancer cells [ChIP-Seq]


title: A small protein encoded by a putative long noncoding RNA inhibits proliferation and tumorigenicity in human colorectal cancer cells

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

overall_design: Refer to individual Series


title: Genetic screen for neuropathic pain study in importin alpha3 knock out mice

city: Rehovot

first: Mike

last: Fainzilber

address: 234 Herzl Street

department: Department of Biomolecular Sciences

country: Israel

institute: Weizmann Institute of Science

summary: Here we show that importin  alpha (α) 3 (KPNA4) controls pain responsiveness in peripheral sensory neurons. An importin α3 knockout mouse showed reduced responsiveness to noxious heat or chemical pain, as well as greater tolerance to neuropathic pain. Similar findings were obtained upon acute knockdown of importin α3 in peripheral sensory neurons. Transcriptome analyses and immunohistochemistry indicated that importin α 3 deficient neurons were impaired in nuclear import of c-Fos. Forty days after spared nerve injury acute down-regulation of importin α3, c-FOS, Jun and the dominat negative AAV9 viruses rescue the neuropathic pain phenotype.  In silico screens identified importin α3 deficiency mimicking drug leads that attenuated neuropathic pain and reduced c-Fos nuclear localization. Hence, perturbing c-Fos nuclear import by importin α3 can provide analgesia at peripheral loci in the nervous system

overall_design: Treatments to combat neuropathic pain using the importin alpha3-cFOS signalling

first: Letizia

last: Marvaldi


title: DNA mismatches reveal conformational penalties in protein-DNA recognition I

status: Public on Aug 16 2020

first: Raluca

last: Gordan

platform_organism:
Saccharomyces cerevisiae
Homo sapiens


title: Repression of Irs2 by let-7 miRNAs is essential for the homeostasis of the telencephalic neuroepithelium [RNA-seq]

first: Jose

last: Lopez-Atalaya

first: Rafael

last: Soler

first: Virginia

last: Fernandez

first: Victor

last: Borrell


title: DNA mismatches reveal conformational penalties in protein-DNA recognition

platform_organism:
Saccharomyces cerevisiae
Homo sapiens
synthetic construct


title: RNA Sequencing with de novo assembly of earthworm transcriptome by the treatment of lindane with different exposure and aging duration.

first: Ting

last: Xu

address: No. 1239 Road Siping

summary:
Purpose: We studied the potential effects of lindane aging in soil on model earthworm Eisenia fetida at the transcriptomic level.
Methods: transcriptomes of earthworms were generated by RNA sequencing using Illumina Hiseq Xten platform.
Results:  There were 1325 DE unigenes in G2 (1074 up and 251 down), 784 DE unigenes in G3 (457 up and 327 down), 3658 DE unigenes in G4 (2294 up and 1364 down), and 1511 DE unigenes in G5 (929 up and 582 down) compared with G1. Except in G2, lindane exposure caused significant impacts on cholinergic and GABAergic synaptic transmission in earthworms in other groups. The metabolic processes of ACh were also altered. It was of note that the term positive regulation of locomotion (GO:0040017) in G5 could be regarded as the phenotype of the interaction of two NTs.
Conclusions: We found that aging of lindane prior to exposure promoted the disrupting effects of lindane on earthworm neurotransmission.

overall_design: The effects of lindane on earthworm transcriptomes using different exposure and aging duration.

first: Juanjuan

last: Miao


title: GLI1+ mesenchymal stromal cells modulate epithelial metaplasia in lung fibrosis

city: San Francisco

first: Peng

last: Tien

address: 513 Parnassus Ave, Health Sciences East 1350

department: Department of Pulmonary Medicine

institute: University of California San Francisco

summary: A single-cell transcriptional analysis was performed on GLI1+ stromal cells from the adult murine lung during homeostasis and after fibrotic injury. The goal is to understand the role of GLI1+ stromal cells in lung fibrosis and repair. Whole adult murine lung tissue from two samples were separately dissociated to single cells and subjected to fluorescence activated cell sorting (FACS) to select all live GLI1+ cells. One sample was treated with bleomycin to induce fibrosis and the other was treated with saline as a control. The single cell RNA-sequencing library was  generated separately for the bleomycin and saline-treated samples. Cells were sequenced at a depth of ~70,000 reads/cell. We captured approximately 17,700 cells with a median of 2,400 genes detected per cell utilizing a droplet-based barcoding approach to capture single cells for RNA sequencing. After bleomycin-induced fibrosis, we identified a novel "myofibroblast" subset of GLI1 cells that contribute to injury and repair. Injured GLI1 cells also reveal dysregulation in key developmental pathways, including BMP signaling, that contribute to metaplastic repair after fibrotic injury.

overall_design: Two Gli1-CreERT2/R26R-YFP adult murine lungs  were analyzed. Both animals were injected with tamoxifen on five consecutive days to induce YFP expression in GLI1 cells. One week after tamoxifen induction, one lung was treated intranasally with bleomycin every 7 days for four weeks to induce fibrotic injury in the lung. The other lung was treated with saline at the same timepoints as a control. Seven days after the last treatment, the whole lung from each Gli1-CreERT2/R26R-YFP animal was separately dissociated to single cells, stained with DAPI, and subjected to FACS to select live GLI1+ cells. Doublets were excluded based on forward and side scatter, and live cells were selected based on DAPI fluorescent exclusion. Then the GLI1+ cells were sorted by endogenous YFP fluorescence. The live GLI1+ cells were prepared separately for the bleomycin and saline-treated samples using the 10X single cell instrument and v2 10X single cell kits according to standard protocol. Single cell sequencing of adult murine lung GLI1+ population with and without fibrotic injury was performed separately using the HiSeq2500.

first: Monica

last: Cassandras

first: Chaoqun

last: Wang

first: Tien

last: Peng


title: Long-term m5C methylome dynamics parallel phenotypic adaptation in the cyanobacterium Trichodesmium [RNA-seq]

city: Los Angeles

first: Eric

middle: Alva

last: Webb

address: 3616 Trousdale Parkway

institute: University of Southern California

summary: Our paper presents the results of a study in which we used whole genome bisulfite sequencing (WGBS) and RNA-Seq (i.e. transcriptomics) to examine the long-term epigenomic dynamics of an experimenta evolution study under high CO2 in the marine cyanobacterium Trichodesmium. We identify m5C methylated sites that rapidly change in response to short-term high CO2 exposure, which are then maintained for 4.5 years even after adaptation (i.e. trait canalization). After 7 years of CO2 selection, high-CO2 triggered methylation levels return to ancestral, low-CO2 levels, consistent with genetic assimilation theory and observations in eukaryotic model systems. These data suggest a potential role for m5C methylation in prokaryotic trait canalization and identify genetic assimilation as an evolutionary mechanism of potential biogeochemical importance under global change factors.

overall_design: Experimental design can be found at Walworth, N. G., Lee, M. D., Fu, F.-X., Hutchins, D. A., & Webb, E. A. (2016). Molecular and physiological evidence of genetic assimilation to high CO2 in the marine nitrogen fixer Trichodesmium. Proceedings of the National Academy of Sciences of the United States of America, 201605202–8. http://doi.org/10.1073/pnas.1605202113. For whole-genome bisulfite samples (WGBS), we sampled the founding ancestral population (T0). We then sampled WGBS biological replicates 4.5 years later (another time point near the time of Walworth et al. PNAS 2016) from the 380-selected cell lines (adapted to low-CO2), the 380s-to-750 cell lines (380-selected cell lines transferred to 750 µatm CO2 to 2 weeks), the 750-selected cell lines (adapted to high CO2), and the 750s-to-380 cell lines (750-selected cell lines transferred to 380 µatm CO2 for 2 weeks). Here, we also sequenced two biological replicate samples for RNA-Seq for all treatments at this time point (n=4 treatments). The 380-selected an 380s-to-750 raw reads can be found in another GEO project with accession number GSE86992. Within the GSE86992, the specific samples for the 380-selected and 380s-to-750 have sample accession numbers of GSM2318497, GSM2318498, GSM2318499, GSM2318500. The rest of the RNA-Seq samples and their metadata are below. Both these RNA samples and the GSE86992 samples were sampled simultaneously. Finally, we sampled for WGBS again 2.5 years later for both the 380-selected cell lines (adapted to low-CO2) and 750-selected cell lines (adapted to high CO2). All WGBS metadata is described in the other spreadsheet associated with this accession number

first: Nathan

last: Walworth


title: Essential gene profiles for human pluripotent stem cells identify uncharacterized genes and substrate dependencies [CRISPRseq]

status: Public on Aug 15 2020

city: Toronto

first: Jason

last: Moffat

state: ON

address: 686 Bay Street, Room 13.9705

department: Molecular Genetics

country: Canada

institute: University of Toronto

summary: Human pluripotent stem cells (hPSCs) provide an invaluable tool for modeling diseases and hold promise for regenerative medicine. For understanding pluripotency and lineage differentiation mechanisms, a critical first step involves systematically cataloging essential genes (EGs) that are indispensable for hPSC fitness, defined as cell reproduction in this study. To map essential genetic determinants of hPSC fitness, we performed genome-scale loss- of-function screens in an inducible Cas9 H1 hPSC line cultured on feeder cells and laminin to identify EGs. Among these, we found FOXH1 and VENTX, genes that encode transcription factors previously implicated in stem cell biology, as well as an uncharacterized gene, C22orf43/DRICH1. hPSC EGs are substantially different from other human model cell lines, and EGs in hPSCs are highly context dependent with respect to different growth substrates. Our CRISPR screens establish parameters for genome-wide screens in hPSCs, which will facilitate the characterization of unappreciated genetic regulators of hPSC biology.

overall_design: Genome-wide loss-of-function CRISPR screens in an inducible H1-iCas9 human pluripotent stem cell line grown on irradiated mouse embryonic fibroblasts (MEFs) as well laminin substrates.

first: Barbara

last: Mair

first: Jelena

last: Tomic

first: Sanna

last: Masud

first: Peter

last: Tonge

first: Alexander

last: Weiss

first: Matej

last: Usaj

first: Amy

middle: H

last: Tong

first: Jamie

middle: J

last: Kwan

first: Kevin

middle: R

last: Brown

first: Emily

last: Titus

first: Michael

last: Atkins

first: Katherin

last: Chan

first: Lise

last: Munsie

first: Andrea

last: Habsid

first: Hong

last: Han

first: Marion

last: Kennedy

first: Brenda

last: Cohen

first: Gordon

last: Keller


title: Repression of Irs2 by let-7 miRNAs is essential for the homeostasis of the telencephalic neuroepithelium

first: Jose

last: Lopez-Atalaya


title: Alteration of gene expression in cumulus cells reveals the role of ClpP in reproductive aging

city: Gainesville

first: Zongliang

last: Jiang

state: FL

address: 2033 Mowry Rd

department: Animal Sciences

institute: University of Florida

summary: We performed RNAseq analysis to determine the effect of ClpP-deletion on cumulus cells global gene expression profile in mature (3 month-old) and (6 month-old) mice. RNA sequencing revealed about 13,000 genes (FPKM > 0.1) expressed in cumulus cells. A total of 1025 genes were significantly differentially expressed  (p<0.05) in 3-month ClpP-/- cumulus cells compared to WT (548 up-regulated and 477 down-regulated). At 6-month, a total of 555 genes were significantly differentially expressed in ClpP-/- cumulus cells compared to WT (464 up-regulated and 91 down-regulated). In addition, 1012 and 1034 genes were significantly differentially expressed in both 6 month ClpP-/- and WT cumulus cells compared to younger ClpP-/- cumulus cells, respectively. In this study, we assessed the effect of ClpP global deletion on cumulus cells gene expression. Our findings provide new insight into the role of CLPP in the cumulus cells, and may help understand the potential mechanism of infertility and reproductive aging associated with ClpP-deficiency.

overall_design: Cumulus oophorus complexes (COCs) were collected from 3- and 6-month old ClpP knockout (ClpP-/-) and wild type (WT)  female mice (n=3 for each group) 48h after injection with 5IU pregnant mare serum gonadotropin (PMSG, Sigma). Cumulus cells were isolated  and 100 cells from each mouse were pooled for RNA sequencing analysis.

first: Tianren

last: Wang

first: Man

last: Zhang

first: Emre

last: Seli


title: AHR activation in glioblastoma cells [FICZ_KynA]

first: Ahmed

last: Sadik

summary: The objective of this study was to analyze AHR activation through aromatic amino acid metabolism. To this end, glioblastoma cells were exposed to aromatic amino acid derived metabolites and their ability to activate AHR was analysed. In addition, AHR activation was evaluated in glioblastoma cells expressing IL4I1, an aromatic amino acid degrading enzyme, with or without shRNA mediated knockdown of AHR.

overall_design: FICZ_KynA_U87_shC: expression profile of shCtrl U-87MG cells treated with 6-formylindolo(3,2-b)carbazole (FICZ) 100 nM or kynurenic acid (KynA) 50 μM for 24 h, was compared to expression profile of shCtrl U-87MG cells treated with DMSO (n = 4).

first: Luis

last: Somarribas

first: Soumya

last: Mohapatra

first: Christiane

last: Opitz


title: H3K27Ac of Ovarian Cancer Cell Lines

first: Michelle

middle: Renee

last: Jones

address: 8720 Alden Dv

department: Biomedical Sciences

institute: Cedars-Sinai Medical Center

summary: H3K27Ac was generated in a panel of 12 ovarian cancer cell lines to identify active regulatory elements in the genome.

overall_design: ChIP-Seq performed in 12 cells lines. Biological replicates were run for EFO27, VOA1056, Kuramochi.

first: Simon

last: Gayther


title: Inflammasomes within hyperactive dendritic cells stimulate T cell mediated anti-tumor immunity

status: Public on Aug 14 2020

first: Alex

last: Shalek

address: 45 Carleton St.

department: Institute of Medical Engineering and Science

institute: MIT

summary: Central to anti-tumor immunity are dendritic cells (DCs), which can stimulate long-lived  protective T cell responses. Recent studies have demonstrated that DCs can achieve a state of  hyperactivation, which is associated with inflammasome activities within living cells. Herein, we  report that hyperactive DCs have an enhanced ability to migrate to draining lymph nodes and  stimulate potent cytotoxic T lymphocyte (CTL) responses. This enhanced migratory activity is  dependent on the chemokine receptor CCR7 and is associated with a unique transcriptional  program that is not observed in conventionally activated or pyroptotic DCs. We discovered that  hyperactivating stimuli are uniquely capable of inducing durable CTL-mediated anti-tumor  immunity against tumors that are sensitive or resistant to PD-1 inhibition. These protective  responses are intrinsic to the cDC1 subset of DCs, depend on the inflammasome-dependent  cytokine IL-1β and enable tumor lysates to serve as immunogens. Therefore, hyperactive DCs  may diversify current approaches to cancer immunotherapy.

overall_design: RNA-seq readout of 3 biological replicates of induced FLT3 cDC1 or FLT3 cDC2 mouse cells treated with 'Nothing', 'LPS', 'LPS&PGPC', 'PGPC', 'LPS&Alum', and  'Alum'

first: Dania

last: Zhivaki

first: Francesco

last: Borriello

first: Ohn

last: Chow

first: Benjamin

last: Doran

first: Ira

last: Fleming

first: Derek

last: Theisen

first: Paris

last: Pallis

first: Caroline

last: Sokol

first: Ivan

last: Zanoni

first: Jonathan

last: Kagan


title: Cop1 wildtype and knockout ATAC-seq in BMDMs

city: South San Francisco

first: Rohit

last: Reja

address: 1 DNA Way

institute: Genentech

summary: The realization in the last decade that dysregulated microglia are intimately involved in Alzheimer’s disease (AD) pathogenesis has been a major advance. The precise mechanisms controlling pathogenic programs of microglia gene expression, however, remain poorly understood. The transcription factor (TF) c/EBPß is highly expressed in microglia and is known to regulate expression of pro-inflammatory genes. Notably, c/EBPß is upregulated in AD. Despite mounting evidence that the levels of this pivotal pro-inflammatory TF are tightly regulated, how this is achieved is unclear as alterations in its amounts are not reflected in transcript levels. Remarkably, we find that this TF is primarily regulated post-translationally. Here we show that the ubiquitin ligase Cop1 functions as a “brake” on microglial activation by targeting c/EBPß for ubiquitination and subsequent proteasomal mediated degradation. In the absence of Cop1, c/EBPß protein rapidly and dramatically accumulates leading to engagement of a potent pro-inflammatory and ApoE gene-expression program, evidenced by increased neurotoxicity in microglia-neuronal co-cultures. Antibody blocking studies revealed that the neurotoxicity was almost entirely attributable to complement. Unexpectedly, loss of a single allele of c/EBPß, rescued the pro-inflammatory phenotype underscoring a significant gene dosage effect. We also found that Cop1 deletion accelerated disease progression in a mouse model of tau-mediated neurodegeneration where elevated ApoE plays a deleterious role. Taken together these results point to c/EBPß as a potential therapeutic target for inflammation-driven neurodegeneration as the heterozygote animal is otherwise normal.

overall_design: ATACseq was performed in BMDMS on the following genotypes in three replicates: Cop1 wildtype and Cop1 knockout.

first: A

last: Ndoja

first: R


title: T-bet and STAT6 Coordinately Suppress the Development of IL-9-mediated Atopic Dermatitis-like Skin Inflammation in Mice

city: Chiba

first: Sohei

last: Makita

state: Chiba

address: 1-8-1 inohana chuo-ku

country: Japan

institute: Chiba university

summary: T-bet and STAT6 are critical factors for helper T cell differentiation in humans and mice. Additionally, polymorphisms in TBX21 (T-bet) and STAT6 are associated with the susceptibility of allergic diseases. However, precise mechanisms of the reciprocal regulation between T-bet and STAT6 in allergy remain unclear. To determine the reciprocal regulation in vivo, we investigated the phenotype of T-bet/STAT6 double-deficient (T-bet-/- STAT6-/-) mice. Unexpectedly, T-bet-/- STAT6-/- mice but not T-bet-/- mice or STAT6-/- mice spontaneously developed severe dermatitis. Not only eosinophils and mast cells but also CD4+ T cells infiltrated into the skin of T-bet-/- STAT6-/- mice. Adoptive transfer of CD4+ T cells of T-bet-/- STAT6-/- mice into SCID mice induced the accumulation of eosinophils and mast cells in the skin, while depletion of CD4+ T cells ameliorated the dermatitis in T-bet-/- STAT6-/- mice. Comprehensive transcriptome analyses revealed that IL-9 expression was enhanced in T-bet-/- STAT6-/- CD4+ T cells. Indeed, IL-9 neutralization ameliorated the dermatitis in T-bet-/- STAT6-/- mice. Importantly, T-bet-/- STAT6-/- CD4+ T cells expressed functional TSLP receptor and produced large amounts of IL-9 upon TSLP stimulation. These results indicate that T-bet and STAT6 suppress atopic dermatitis-like skin inflammation, possibly by inhibiting TSLP-dependent IL-9 production in CD4+ T cells.

overall_design: Total RNA of CD44+ CD4+ T cells in skin-draining LNs was isolated from WT mice, T-bet-/- mice, STAT6-/- mice, or T-bet-/- STAT6-/- mice and subjected to RNA-sequencing analysis.

first: S

first: T

last: Hiroaki

last: Iwata

first: H

last: Nakajima


title: Interphase-arrested Drosophila embryos activate zygotic gene expression and initiate Mid-Blastula Transition events at a low nuclear-cytoplasmic ratio

city: changsha

first: xiaoyun

last: lei

state: hunan

address: kaifu district, xiangya road

department: xiangya hospital

institute: central south university

summary: Externally deposited eggs begin development with an immense cytoplasm and a single overwhelmed nucleus. Rapid mitotic cycles restore normality as the ratio of nuclei to cytoplasm (N/C) increases. A threshold N/C has been widely proposed to activate zygotic genome transcription and onset of morphogenesis at the mid-blastula transition (MBT). To test whether a threshold N/C is required for these events, we blocked N/C-increase by downregulating cyclin/Cdk1 to arrest early cell cycles in Drosophila. Embryos that were arrested two cell cycles prior to the normal MBT activated widespread transcription of the zygotic genome including genes previously described as N/C-dependent. Zygotic transcription of these genes largely retained features of their regulation in space and time. Furthermore, zygotically regulated post-MBT events such as cellularization and gastrulation movements occurred in these cell-cycle-arrested embryos. These results are not compatible with models suggesting that these MBT events are directly coupled to N/C. Cyclin/Cdk1 activity normally declines in tight association with increasing N/C and is regulated by N/C. By experimentally promoting the decrease in cyclin/Cdk1, we uncoupled MBT from N/C increase, arguing that N/C guided down-regulation of cyclin/Cdk1 is sufficient for genome activation and MBT.

overall_design: 19 individual embryos

first: Isaac

last: Strong

first: Xiaoyun

last: Lei

first: Fang

last: Chen

first: Kai

last: Yuan

first: Patrick

last: O’Farrell


title: Transcriptome landscape of DNA-damage response at single-cell resolution

city: Ann Arbor

first: Jun Hee

last: Lee

state: MI

address: 109 Zina Pitcher Pl

institute: University of Michigan

summary:
Purpose: The goals of this study are to investigate how the transcriptome of colon cancer cells responds to genotoxic 5FU treatments utilizing single cell RNA-seq (scRNA-seq) technology.
Methods: The collection of the single cell transcriptomes obtained from three colon cancer cell lines, RKO (CRL-2577), HCT116 (CCL-247) and SW480 (CCL-228), was generated by Drop-seq method followed by sequencing through Illumina HiSeq-4000 High-Output system.
Results: A total of 10 datasets was generated for further analysis.

overall_design: A total of 10 Drop-seq experiments were run. Each experiment included three cell types (RKO, HCT116, SW480) with different 5-fluorouracil treatment which were equally mixed. Cells without drug treatment were included as controls. Controls were repeated four times and 10/50/200 μM 5FU treatment sets were repeated two times.

first: Sung

last: Park

first: Hyun

middle: M

last: Kang

first: Jun


title: COP1 depletion and LPS treatment of microglia in vitro

first: Rohit

summary: The realization in the last decade that dysregulated microglia are intimately involved in Alzheimer’s disease (AD) pathogenesis has been a major advance. The precise mechanisms controlling pathogenic programs of microglia gene expression, however, remain poorly understood. The transcription factor (TF) c/EBPß is highly expressed in microglia and is known to regulate expression of pro-inflammatory genes. Notably, c/EBPß is upregulated in AD. Despite mounting evidence that the levels of this pivotal pro-inflammatory TF are tightly regulated, how this is achieved is unclear as alterations in its levels are not reflected in transcript levels. Remarkably, we find that this TF is primarily regulated post-translationally. Here we show that the ubiquitin ligase Cop1 functions as a “brake” on microglial activation by targeting C/EBPß for ubiquitination and subsequent proteasomal mediated degradation. In the absence of Cop1, C/EBPß protein rapidly and dramatically accumulates leading to engagement of a potent pro-inflammatory and ApoE gene-expression program, evidenced by increased neurotoxicity in microglia-neuronal co-cultures. Antibody blocking studies revealed that the neurotoxicity was almost entirely attributable to complement. Unexpectedly, loss of a single allele of C/EBPß, rescued the pro-inflammatory phenotype underscoring a significant gene dosage effect. We also found that Cop1 deletion accelerated a mouse model of tau-mediated neurodegeneration where elevated ApoE plays a deleterious role.  Taken together these results point to C/EBPß as a potential therapeutic target for inflammation-driven neurodegeneration as the heterozygote animal is otherwise normal.

overall_design: RNA was extracted from wild type (WT_untreated), Cop1 KO microglia (Loxp_untreated), and wild type microglia treated with LPS (WT_LPS)


title: Cop1 wildtype and knock out ChIP-seq for CEBPb, H3K4me3 and H3K27ac in primary microglia

overall_design: ChIP-seq of CEBPb and 2 histone modifications (H3K4me3, H3K27ac) in microglia was performed on the following genotypes in two replicates: Cop1 wildtype and Cop1 knock out. We had one pooled input sample as control.

first: A


title: Cop1 wildtype and knock out ChIP-seq for CEBPb, H3K4me3 and H3K27ac in BMDMs

overall_design: ChIP-seq of CEBPb and 2 histone modifications (H3K4me3, H3K27ac ) was performed in BMDMs on the following genotypes in two replicates: Cop1 wildtype and Cop1 knock out. We had one pooled input sample as control.

first: R


title: Long term effects of COP1 deletion in microglia in vivo

overall_design: RNA was extracted from wild type (WT) and Cop1 KO microglia (Cop1KO) in vivo, >1.5 years post Cop1 deletion


title: DDX5 promotes oncogene C3 and FABP1 expressions and drives intestinal inflammation and tumorigenesis

city: San Diego

first: Wendy

last: Huang

address: 9500 Gilman Drive #0651

department: Cellular and Molecular Medicine

institute: University of California San Diego

summary: Tumorigenesis in different segments of the intestinal tract involves tissue-specific oncogenic drivers. In the colon, complement component 3 (C3) activation is a major contributor to inflammation and malignancies. By contrast, tumorigenesis in the small intestine involves fatty acid–binding protein 1. However, little is known of the upstream mechanisms driving their expressions in different segments of the intestinal tract. Here, we report that the RNA-binding protein DDX5 binds to the mRNA transcripts of C3 and fatty acid–binding protein 1 to augment their expressions posttranscriptionally. Knocking out DDX5 in epithelial cells protected mice from intestinal tumorigenesis and dextran sodium sulfate–induced col- itis. Identification of DDX5 as a common upstream regulator of tissue-specific oncogenic molecules provides an excellent therapeutic target for intestinal diseases.

overall_design: For steady state experiment, 8 week old littermates in wildtype or knockout genetic background were used for tissue harvest.

first: Nazia

last: Abbasi

first: Tianyun

last: Long

first: Yuxin

last: Li

first: Brian

last: Yee

first: Benjamin

middle: S

last: Cho

first: Juan

last: Hernandez

first: Evelyn

last: Ma

first: Parth

last: Patel

first: Debashis

last: Sahoo

first: Ibrahim

last: Sayed

first: Nissi

last: Varki

first: Soumita

last: Das

first: Pradipta

last: Ghosh

first: Gene

middle: W

last: Yeo


title: Gene expression profiling  in dorsolateral prostates of prepubertal and adult Sprague-Dawley rats dosed with Estradiol Benzoate, Estradiol and Testosterone

city: Jefferson

first: Vikrant

last: Vijay

state: AR

address: 3900 NCTR Road

department: Division of Systems Biology

institute: US FDA National Center for Toxicological Research

summary: Using rat prostate disease models that Prins and colleagues have developed (Ho et al., 2006 - PMID: 16740699), the purpose of the study is to identify gene(s) for diagnosing early stage of prostate diseases and monitoring the development of prostate diseases. To identify the genes, we analyzed whole genome expression profiling on the prostate from Sprague-Dawley rats dosed postnatally with estradiol benzoate, estradiol and testosterone at PNDs 30, 100 and 145.

overall_design: Sprague-Dawley male offspring were injected with 2.5 mg/kg body weight (BW) Estradiol Benzoate (EB) subcutaneously at PNDs 1, 3 and 5 (EB-treated group).  Male pups in the untreated (control) group were injected with vehicle. At PND 90, each group (the untreated or EB-treated group) were divided into two additional groups without or with additional hormone treatment [estradiol (E) and testosterone (T)]: control, T+E only, EB only and EB+T+E groups. Animals in the control and EB-only groups were implanted with three empty silastic tubing inserts, while animals in T+E only and EB+T+E groups were implanted with two silastic tubing inserts with T and one silastic tube insert with E. Prostates from PNDs 30, 100 and 145 male Sprague-Dawley rats dosed postnatally with EB, E and T were collected (n=3-4 per group). RNA were extracted and used for whole genome expression array (Agilent Technologies; 8 x 60K format).

first: Tao

last: Han

first: Noriko

last: Nakamura


title: Cholesterol pathway inhibition induces TGFβ signaling to promote basal differentiation  in pancreatic cancer

city: philadelphia

first: suraj

last: peri

state: PA

address: 333 cottman ave

department: biostatistics and bioinformatics

institute: Fox chase cancer center

summary: Oncogenic transformation alters the metabolism of cellular lipids to  sustain tumor growth. We define a reciprocal mechanism by which cholesterol metabolism  controls the formation and differentiation of pancreatic ductal adenocarcinoma (PDAC).  Disruption of distal cholesterol biosynthesis by conditional inactivation of Nsdhl, or treatment  with cholesterol-lowering statins caused murine pancreatic carcinomas induced by KrasG12D  expression and homozygous Trp53 loss to undergo a differentiation switch from a glandular to  basal (mesenchymal) phenotype. In parallel, PDACs from patients receiving statins had  enhanced mesenchymal features. Mechanistically, statins and NSDHL loss induced sterol  response element binding protein 1 (SREBP1), promoting autocrine transforming growth factor  beta (TGF) signaling, inducing epithelial-mesenchymal transition. Activation of promesenchymal  TGFβ effectors by cholesterol-lowering statins may select for poorly differentiated  carcinomas.

overall_design: For single-cell sequencing, KPPC and  KPPCN mice with advanced tumors, 3 of each genotype, were sacrificed 7-8 weeks of age.  Single-cell suspensions were isolated from minced tumors using Miltenyi Biotec GentleMacs  dissociator in gentleMACS C Tubes (#130-093-23) and mouse tumor tissue dissociation kit (#130-  096-730) as per the manufacturer’s instructions. Dead cells were removed by use of Dead Cell  removal microbeads (#130-090-101), hematopoeitic cells were depleted by CD45 MicroBeads  (#130-052-30). The Chromium controller was used to make single-cell droplet with GEM bead.  Single-cell suspensions were converted to barcoded scRNA-seq libraries by using the Chromium  Single Cell 3′ Library, Gel Bead & Multiplex Kit and Chip Kit V3 (10X Genomics, #PN-1000092),  18  loading an estimated 6,000 cells per library and following the manufacturer’s instructions.  Samples were processed using kits pertaining to the V3 barcoding chemistry of 10x Genomics.  For each replicate, all tumor samples were processed in parallel in the same thermal cycler. The  final libraries were profiled using the Bioanalyzer High Sensitivity DNA Kit (Agilent Technologies)  and quantified using the Qubit 2.0 (Thermal Fisher, #Q32851). Each single-cell RNA-seq library  was sequenced twice in two lanes of HiSeq 4000 (Illumina) to obtain single-end, 98 bp,  approximately 500 million reads per library.

first: Igor

last: Astsaturov

first: Suraj

last: Peri


title: Effects of COP1 deletion in microglia in vivo

first: Rohit

last: Reja

overall_design: RNA was extracted from wild type and Cop1 KO microglia in vivo, 2-3 post Cop1 deletion


title: Single cell RNA-seq of Cop1 knockout and wildtype microglia

overall_design: Whole intestines from E18.5 embryos (One E18.5 Mlkl -/- and one Casp8 C362A/C362A Mlkl -/- ) were dissociated into single cells and sequenced.

first: A

last: Ndoja

first: R


title: YB-1 inhibits proliferation of mesothelioma cells through multiple mechanisms

status: Public on Aug 13 2020

city: Concord, NSW

first: Kaitao

last: Lai

state: New South Wales

address: ANZAC Research Institute, Hospital Rd, Concord

department: Faculty of Medicine and Health

country: Australia

institute: Anzac Research Institute, The University Of Sydney

summary: Here we utilized an unbiased whole RNA-seq approach to characterize the transcriptional effects of YB-1 knockdown in three mesothelioma cell lines (MSTO, VMC23 and REN cells). Bioinformatic analysis showed that YB-1 knockdown regulated 150 common genes enriched for mitotic genes, integrins and extracellular matrix. However, each cell line also displayed a unique transcriptional signature, with differential deregulation of cell death, the cell cycle, and p53 signaling observed across the three cell lines.

overall_design: Next-generation RNAseq analysis of YB-1 knockdown across triplicate biological repeats in 3 mesothelioma cell lines

first: Andrew

last: Burgess

first: Thomas

last: Johnson


title: CDE-1 suppresses the production of risiRNA by coupling polyuridylation and degradation of 26S rRNA

city: Hefei

first: Shouhong

last: Guang

state: Anhui

address: 443 HuangShan Road

department: School of Life Sciences

institute: University of Science & Technology of China

summary: Antisense ribosomal siRNAs (risiRNAs) downregulate pre-rRNAs through the nuclear RNAi pathway in Caenorhabditis elegans. However, the biogenesis and regulation of risiRNAs remain obscure. Previously, we showed that 26S rRNAs are uridylated at the 3'-ends by an unknown terminal polyuridylation polymerase before the rRNAs are degraded by a 3' to 5' exoribonuclease SUSI-1(ceDIS3L2). There are three polyuridylation polymerases, CDE-1, PUP-2, and PUP-3, in C. elegans. Here, we found that CDE-1 is specifically involved in suppressing risiRNA production. CDE-1 localizes to perinuclear granules in the germline and uridylates both Argonaute-associated 22G-RNAs and 26S rRNAs at the 3'-ends. Immunoprecipitation followed by mass spectrometry (IP-MS) revealed that CDE-1 interacts with SUSI-1(ceDIS3L2). Consistent with those results, both CDE-1 and SUSI-1(ceDIS3L2) are required for the inheritance of RNAi. Therefore, this work identified a rRNA surveillance machinery of rRNAs that couples terminal polyuridylation and degradation.

overall_design: In this study, we compared total small RNAs from (1) control animals; (2) cde-1(tm936) mutant. And we also compared the NRDE-3 associated small RNAs in (3) control embryo; (4) cde-1(tm936) embryo mutant; and (5)eri-1(mg366) L3 animals (6)eri-1(mg366);cde-1(tm936) L3 mutant; and HRDE-1 associated small RNAs in (7) control young adult animals; (8) cde-1(tm936) young adult mutant;and WAGO-1 associated small RNAs in (9) control young adult animals; (10) cde-1(tm936) young adult mutant;we also compared 26S rRNAs 3' tail-seq  from (11,12) eri-1(mg366) embryo animals;(13,14)eri-1(mg366);cde-1(tm936) embryo animals and (15,16)eri-1(mg366);cde-1(tm936);cde-1::mCherry embryo animals, and compared 26S rRNAs 3' tail-seq  from (17,18) eri-1(mg366) L3 animals;(19,20)eri-1(mg366);cde-1(tm936) L3 animals and (21,22)eri-1(mg366);cde-1(tm936);cde-1::mCherry L3 animals.


title: Erythrophagocytosis drives anti-inflammatory programming of liver macrophages (Bulk RNA seq BMDMs)

city: Schlieren

first: Marc

last: Pfefferle

state: Schweiz

address: Wagistrasse 12

country: Switzerland

institute: University hospital Zürich (USZ)

summary: Under conditions of erythrolytic stress, which accompanies many disease states, macrophages play key roles in phagocytosing damaged RBCs and preventing the toxic effects of cell-free hemoglobin and heme to maintain homeostasis. Using a genetic mouse model of spherocytosis and single-cell RNA sequencing, we show that erythrolytic stress promotes expansion of a specific macrophage population in the liver  (which we named “erythrophagocytes”) expressing high levels of Marco and Hmox1 and low levels of MHC class II related genes with an anti-inflammatory gene expression signature. We confirmed the strong anti-inflammatory function of erythrophagocytes in two models of sterile inflammatory liver disease: anti-CD40 antibody-induced systemic inflammation syndrome with necrotizing hepatitis and diet-induced nonalcoholic fatty liver disease (NAFLD). The unique anti-inflammatory phenotype and function of erythrophagocytes was reproduced in vitro by heme-exposure of mouse macrophages, yielding a transcriptional profile that segregated heme-polarized from classical M1- and M2-polarized cells. The phenotype of anti-inflammatory erythrophagocytes coincided with NFE2L2/NRF2 driven gene expression and was abolished in Nfe2l2/Nrf2-deficient macrophages. Our findings point to a novel pathway that regulates macrophage functions to link RBC homeostasis and heme metabolism with innate immunity.

overall_design: Bone marrow derived macrophages (BMDMs) were differentiated into M1, M2 or erythrophagocyte-like macrophages and subjected to bulk RNA-sequencing

last: Pfefferlé

first: Giada

last: Ingoglia

first: Florence

last: Vallelian

first: Dominik

last: Schaer


title: Erythrophagocytosis drives anti-inflammatory programming of liver macrophages (Bulk RNA seq SPTA CD40 mac)

overall_design: Liver macrophages from three sph/sph mice and three littermate wild-type controls treated with intraperitoneal injection (i.p.) of agonistic CD40 antibody injection were isolated using anti-F4/80 antibody coated dynabeads and subjected to bulk RNA-seq. Liver macrophages from three sph/sph mice and three littermate wild-type controls with saline treatment were also subjected to bulk RNA-seq as controls.


title: Erythrophagocytosis drives anti-inflammatory programming of liver macrophages (Single-cell RNA-seq SPTA mac)

overall_design: Liver macrophages from a sph/sph mouse and its littermate wild-type control were isolated using anti-F4/80 antibody coated dynabeads and subjected to droplet-based single-RNA sequencing (10x genomics).


title: Erythrophagocytosis drives anti-inflammatory programming of liver macrophages

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

overall_design: Refer to individual Series


title: Erythrophagocytosis drives anti-inflammatory programming of liver macrophages (ATAC-seq)

summary: Under conditions of erythrolytic stress, which accompanies many disease states, macrophages play key roles in phagocytosing damaged RBCs and preventing the toxic effects of cell-free hemoglobin and heme to maintain homeostasis. Using a genetic mouse model of spherocytosis and single-cell RNA sequencing, we show that erythrolytic stress promotes expansion of a specific macrophage population in the liver  (which we named “erythrophagocytes”) expressing high levels of Marco and Hmox1 and low levels of MHC class II related genes with an anti-inflammatory gene expression signature. We confirmed the strong anti-inflammatory function of erythrophagocytes in two models of sterile inflammatory liver disease: anti-CD40 antibody-induced systemic inflammation syndrome with necrotizing hepatitis and diet-induced nonalcoholic fatty liver disease (NAFLD). The unique anti-inflammatory phenotype and function of erythrophagocytes was reproduced in vitro by heme-exposure of mouse macrophages, yielding a transcriptional profile that segregated heme-polarized from classical M1- and M2-polarized cells. Mapping transposase-accessible chromatin in single cells using sequencing (scATAC-seq) suggested NFE2L2/NRF2 as a critical driver of anti-inflammatory erythrophagocytes in the livers of hemolytic mice and heme-suppression of the inflammatory response was abolished in macrophages from Nfe2l2/Nrf2-deficient animals. Our findings point to a novel pathway that regulates macrophage functions to link RBC homeostasis and heme metabolism with innate immunity.

overall_design: Liver macrophages from a sph/sph mouse and its littermate wild-type control were isolated using anti-F4/80 antibody coated dynabeads and subjected to droplet-based single-cell ATAC(10x genomics).


title: Single cell RNAseq of human early stage lung adenocarcinoma with activating somatic KRAS mutations and associated normal lung tissues reveals alterations of key gene expression in alveolar type II pneumocytes

first: Linh

middle: My

last: Tran

state: California

department: Medicine - Pulmonary & Critical Care

institute: University of Los Angeles

summary: Mutant KRAS is the most common oncogenic driver of epithelial cancers. Nevertheless, the molecular changes induced by KRAS activation in primary epithelial cells remain unknown. Here, we determined transcriptional changes at single-cell resolution after KRAS activation in distal lung epithelial cell populations. We observed that activating somatic KRAS mutations had the most pronounced effect on alveolar type 2 (AT2) cells, one type of distal lung epithelial progenitor cells, whereas other distal lung epithelial cell types were similar to their wildtype KRAS counterparts. We found that tumor-associated AT2 cells highly expressed EPCAM and SFTPB, but had a significant reduction of STFPC and STFPD expression compared to their normal lung-associated counterparts.

overall_design:
All single-cell mRNA expression profiles were acquired from two patients diagnosed with lung adenocarcinoma (LUAD) carrying KRAS mutations. In brief, tumor lesion and associated distal normal lung tissues were collected from patients undergoing surgery without prior chemotherapy. Samples were dissociated into single cell suspension and subjected to single cell sequencing. Immune cells were filtered out from the results based on CD45 (PTPRC) expression utilizing in-silico sorting

***Please note that the raw data will be deposited in dbGaP due to patient privacy concerns.

first: Jane

last: Yanagawa

first: Eileen

last: Fung

first: Kostyantyn

last: Krysan

first: William

middle: D

last: Wallace

first: Gregory

last: Fishbein

first: Ashley

last: Prosper

first: Denise

last: Aberle

first: Steven

last: Dubinett


title: Gene expression profile of wild type, PiZ and PiZ/Chop-/- mouse livers at two different ages

city: Pozzuoli

first: Rossella

last: De Cegli

address: Via Campi Flegrei 34

country: Italy

institute: Tigem

summary: To investigate the role of CHOP in the pathogenesis of liver disease due to Z alpha-1 antitrypsin in juvenile and older mice, we performed Quant-seq experiments at 6 and 36 weeks of age in wild type, PiZ and PiZ/Chop-/- mouse livers

overall_design: total RNA was extracted from the livers of all experimental groups

first: R

first: S

last: Attanasio


title: ChIP-Seq of IRF4, BATF, and vIRF3 in BC-3 cells

city: Chicago

first: Eva

middle: Henriette

last: Gottwein

state: IL

address: 320 E Superior St., Tarry building, Room 6-752

department: Microbiology-Immunology

institute: Northwestern University

summary: The Kaposi's sarcoma-associated herpesviruses causes several cancers including Kaposi's sarcoma and primary effusion lymphoma (PEL). Our work reveals that the cellular transcription factors interferon regulatory factor 4 (IRF4) and basic leucine zipper ATF-like transcription factor (BATF), as well as the viral interferon regulatory factor 3 (vIRF3) are critical oncogenic dependencies specific to PEL cell lines. To understand the functions of these transcription factors, we performed ChIP-Seq on these proteins in BC-3 PEL cell line. Our results reveal that these oncogenic factors may cooperate to regulate the expression of the same mRNAs.


title: Fxr1 regulates sleep and synaptic homeostasis

first: Martin

last: Beaulieu

state: Ontario

address: 1 king's college circle room 4336

department: Pharmacology and Toxicology

institute: University of Toronto

summary: Comparison of ribosome associated RNA between Ctrl vs EW (or SD) and EW (or SD) vs EWFxr1 (or SDFxr1)

overall_design: GFP-Fxr1 (Fxr1) or GFP (Ctrl) was overexpressed in mPFC, than ribosome associated RNA was isolated from Control Sleep (S), Control Enforced Wake (EW) or EWFxr1 using RiboTag immunoprecipitation and subjected to RNAseq

first: Jivan

last: Khlghatyan

first: Jean-Martin


title: Paired Imaging and Sequencing of Protein-DNA Interactions in Single Human Cells Using microDamID

city: Berkeley

first: Nicolas

last: Altemose

state: CA

address: 327 Stanley Hall

department: Bioengineering

institute: University of California, Berkeley

summary: Genome regulation depends on carefully programmed protein-DNA interactions that maintain or alter gene expression states, often by influencing chromatin organization. Most studies of these interactions to date have relied on bulk methods, which in many systems cannot capture the dynamic single-cell nature of these interactions as they modulate cell states. One method allowing for sensitive single-cell mapping of protein-DNA interactions is DNA adenine methyltransferase identification (DamID), which records a protein’s DNA-binding history by methylating adenine bases in its vicinity, then selectively amplifies and sequences these methylated regions. These interaction sites can also be visualized using fluorescent proteins that bind to methyladenines. Here we combine these imaging and sequencing technologies in an integrated microfluidic platform (µDamID) that enables single-cell isolation, imaging, and sorting, followed by DamID. We apply this system to generate paired single-cell imaging and sequencing data from a human cell line, in which we map and validate interactions between DNA and nuclear lamina proteins, providing a measure of 3D chromatin organization and broad gene regulation patterns. µDamID provides the unique ability to compare paired imaging and sequencing data for each cell and between cells, enabling the joint analysis of the nuclear localization, sequence identity, and variability of protein-DNA interactions.

overall_design: Single-cell DamID to map lamina associated domains in HEK293T cells imaged on a microfluidic device.

first: Annie

last: Maslan

first: Aaron

last: Streets


title: miRNA expression in tracheal aspirates

status: Public on Aug 12 2020

city: Bloomington

first: Patricia

last: Silveyra

email: psilveyr@iu.edu, ptrcslvr@gmail.com

state: Indiana

department: Environmental and Occupational Health

institute: Indiana University Bloomington

summary: Extreme preterm infants are a growing population in neonatal intensive care units who carry a high mortality and morbidity.  Multiple factors play a role in preterm birth, resulting in major impact on organogenesis leading to complications including bronchopulmonary dysplasia (BPD).  The goal of this study was to identify biomarker signatures associated with prematurity and BPD.  We analyzed miRNA and mRNA profiles in tracheal aspirates (TAs) from 51 infants receiving invasive mechanical ventilation.  25 infants were extremely preterm and diagnosed with BPD, and 26 were term babies receiving invasive mechanical ventilation for elective procedures. We found specific mRNA-miRNA signatures in TAs that may serve as biomarkers for BPD pathogenesis, a consequence of extreme prematurity.

overall_design:
We extracted RNA  from 0.5 mL of tracheal aspirates from control neonates and premature babies with bronchopulmonary dysplasia using the Plasma/Serum RNA Purification Kit. The expression of 1,066 human miRNAs was quantified using the Human miRNome miScript® miRNA PCR Array (QIAGEN, MIHS-3216Z). Results (Ct values) were normalized to global means, and fold changes were calculated using the 2-ΔΔCT method.
control: 2,6,7,10,11,14,17,18,23,24,26,28,30,32,33,34,36,38,40,41,42,43,44,46,49,50
BPD: 1,3,4,5,8,9,12,13,15,16,19,20,21,22,25,27,29,31,35,37,39,45,47,48,51

first: Roopa

last: Siddaiah

first: Christiana

last: Oji-Mmuo

first: Nathalie

last: Fuentes

first: Deborah

last: Montes


title: AGO4 participates in germination during salinity

city: Coyoacan

first: V Miguel

last: Palomar

state: Mexico City

address: Circuito Exterior SN

department: Biochemistry

country: Mexico

institute: School of Chemistry, UNAM

type: Methylation profiling by high throughput sequencing

summary: In this work we studied the participation of RdDM pathway in response to salinity during germination, we found that the RdDM promotes germination while AGO4 null mutant negatively regulates the germination process.

overall_design: Col-0, ago4-2, ago4-3 and nrpe1-11 Arabidopsis germinating seeds under control and salinity conditions, each run was performed with a biological replicate

first: V

first: Alejandra

middle: A

last: Covarrubias


title: Coupled single cell RNA-seq and intracellular protein activity reveals immune-suppressive role of Trem2 in cancer II

city: Rehovot

first: Ido

last: Amit

address: 234 Herzl st.

department: Immunology

institute: Weizmann Institute of Science

summary: Cell function and activity is regulated through integration of signaling, epigenetic, transcriptional and metabolic pathways. Here we introduce INs-seq, an integrated technology for massively parallel recording of single-cell RNA-sequencing (scRNA-seq) and intracellular protein activity. We demonstrate the broad utility of INs-seq for reveling new immune subsets by profiling different intracellular signatures of immune signaling, transcription-factor combinations and metabolic activity. Comprehensive mapping of Arginase 1 expressing cells within tumor models, a metabolic immune signature of suppressive activity, discovers a novel Arg1+ Trem2+ regulatory myeloid cell (Mreg) and identifies markers, metabolic activity and pathways associated with these cells. Genetic ablation of Trem2 in mice inhibits the accumulation of intra-tumoral Mreg cells, leading to a marked decrease in dysfunctional CD8+ T cells and reduced tumor growth. This study establishes INs-seq as a broadly applicable technology for elucidating integrated transcriptional and intra-cellular maps, and identifies the molecular signature of myeloid suppressive cells in tumors.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: 9 different 10x experiments were done, with 20 samples in total. No repeats.

first: Yonatan

last: Katzenelenbogen

first: Fadi

last: Sheban

first: Adam

last: Yalin

last: Yofe

first: Dmitry

last: Svetlichnyy

first: Diego

last: Jaitin

first: Chamutal

last: Borenstein

first: Adi

last: Moshe

first: Hadas

last: Keren-Shaul

first: Merav

last: Cohen

first: Shuang-Yin

last: Wang

first: Baoguo

last: Li

first: Eyal

last: David

first: Tomer

last: Salame

first: Assaf

last: Weiner


title: Highly connected enhancer communities control lineage-determining genes in human mesenchymal stem cells

city: Odense M

first: Susanne

last: Mandrup

state: Fyn

address: Campusvej 55

department: Department of Biochemistry and Molecular Biologi

country: Denmark

institute: University of Southern Denmark

summary: Adipocyte differentiation is driven by waves of transcriptional regulators that reprogram the en-hancer landscape and change the wiring of the promoter interactome. Here, we establish enhancer-capture Hi-C to interrogate the extent and role of enhancer-to-enhancer interactions during adi-pocyte differentiation of human mesenchymal stem cells. We find that enhancers form an elaborate network that is highly dynamic during differentiation and tightly coupled to changes of enhancer activity. These interactions are important for enhancer function, since transcription factors at baited enhancers stabilize transcription factor binding at target enhancers, a phenomenon we term cross-interaction stabilization of transcription factors (CIST). Moreover, highly interconnected enhancers (HICE) act as integration hubs orchestrating differentiation by formation of insulated 3D enhancer communities, inside which, HICE, and other enhancers, converge on phenotypically important gene promoters. Collectively, these results indicate that dynamic enhancer interactions play a key role in regulation of enhancer function, and that HICE are important for both signal integration and compartmentalization of the genome.

overall_design: Genome-wide profiling of transcription factors using ChIP-seq, gene expression using RNA-seq and enhancer-anchored chromatin interactions using enhancer capture Hi-C during adipocyte differentiation of human mesenchymal stem cells.

first: Jesper

middle: G

last: Madsen


title: Erythrophagocytosis drives anti-inflammatory programming of liver macrophages [Agilent]

first: Marc

last: Pfefferle

overall_design: A two color common reference design was chosen with 3-5 independent biological replicates of each condition. Each experimental sample (Cy5 labeled) was hybridized against a non-treated reference sample (Cy3 labeled).

first: Christian

last: Schaer

last: Pfefferlé

first: Giada

last: Ingoglia

first: Dominik

first: Florence

last: Vallelian


title: Upper airway gene expression differentiates COVID-19 from other acute respiratory illnesses and reveals suppression of innate immune responses by SARS-CoV-2

city: San Francisco

first: Charles

last: Langelier

address: 499 Illinois St

department: Medicine

institute: University of California San Francisco

summary: We studied the host transcriptional response to SARS-CoV-2 by performing metagenomic sequencing of upper airway samples in 234 patients with COVID-19 (n=93), other viral (n=100) or non-viral (n=41) acute respiratory illnesses (ARIs). Compared to other viral ARIs, COVID-19 was characterized by a diminished innate immune response, with reduced expression of genes involved in toll-like receptor and interleukin signaling, chemokine binding, neutrophil degranulation and interactions with lymphoid cells. Patients with COVID-19 also exhibited significantly reduced proportions of neutrophils, macrophages, and increased proportions of goblet, dendritic and B-cells, compared to other viral ARIs. Using machine learning, we built 27-, 10- and 3-gene classifiers that differentiated COVID-19 from other acute respiratory illnesses with AUCs of 0.981, 0.954 and 0.885, respectively. Classifier performance was stable at low viral loads, suggesting utility in settings where direct detection of viral nucleic acid may be unsuccessful. Taken together, our results illuminate unique aspects of the host transcriptional response to SARS-CoV-2 in comparison to other respiratory viruses and demonstrate the feasibility of COVID-19 diagnostics based on patient gene expression.

overall_design:
Comparing host transcriptional response to SARS-CoV-2 infection versus response to other respiratory viral infection or non-viral acute respiratory infection.
**RAW DATA NOT PROVIDED DUE TO PATIENT PRIVACY CONCERNS**

first: Eran

last: Mick

first: Jack

last: Kamm


title: Genome-wide maps of macroH2A and H2B distribution in murine embryonic fibroblasts in dependence of Lsh.

city: Frederick

first: Scott

last: Durum

state: MD

address: Building 560, Room 31-71

department: CCR

institute: National Cancer Institute

summary: We report here the distribution of the histone variant macroH2A in murine embryonic fibroblasts derived fom Lsh-/- embryos or Lsh+/+ embryos day 14 gestation using ChIP-seq analysis. We find that macroH2A1 and macroH2A2 deposition are reduced in the absence of Lsh in contrast to H2B deposition. The study identifies Lsh as important regulator of genome wide macroH2A deposition.

overall_design: Examination of 2 different histone modifications in 2 cell types.

first: Kathrin

last: Muegge

first: Kai

last: Ni

first: Richard

last: Finney


title: Analysis of copy number variation (CNV) in human induced hepatic progenitor cells (hiHepPCs) by aCGH

city: Fukuoka

first: Atsushi

last: Suzuki

state: Fukuoka

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

department: Medical Institute of Bioregulation

institute: Kyushu University

type: Genome variation profiling by genome tiling array

summary: CNV comparison of human umvilical vein endothelial cells (HUVECs) and hiHepPCs generated from HUVECs transduced with defined transcription factors.

overall_design: aCGH experiments performed with 4 samples: hiHepPC (Cy5) versus HUVEC (Cy3)

first: Kanae

last: Matsuda-Ito


title: Genome-wide maps of chromatin modifications in KSHV-transformed primary effusion lymphoma cell lines

first: Eva

last: Gottwein

summary: The Kaposi's sarcoma-associated herpesviruses causes several cancers including Kaposi's sarcoma and primary effusion lymphoma (PEL). Our work reveals that the cellular transcription factors interferon regulatory factor 4 (IRF4) and basic leucine zipper ATF-like transcription factor (BATF), as well as the viral interferon regulatory factor 3 (vIRF3) are critical oncogenic dependencies specific to PEL cell lines. To understand the functions of these transcription factors, we performed ChIP-Seq on these proteins and other histone modifications in different PEL cell lines. Our results reveal that these oncogenic factors may cooperate to regulate the expression of the same mRNAs through promoter, enhancer, and superenhancer elements.

overall_design: ChIP-Seq of CTCF, and histone modifications in PEL cell lines


title: Transcriptome sequencing Data sets for human epithelial cells response during Francisella Tularensis infection.

status: Public on Aug 11 2020

city: Ness Ziona

first: Inbar

last: Cohen Gihon

address: Reuven 24

institute: Israel Institute For Biological Research

summary: The goal of this study is to determine the host response of human epithelial cells during infection with Francisella Tularensis. For this purpose, A549 human epithelial cell line was infected with Francisella tularensis spp. holarctica strain LVS for different times of infection, in duplicates. At different times post infection (0.5/1/3/6/12/24 hours post infection) cells were harvested and total RNA was extracted. RNA-seq libraries were constructed and sequencing of 100bp paired-end was performed on the Illumina NovaSeq 6000 system. Sequencing yielded about 22M reads per sample that were mapped to the human genome (Human: GRCh38) resulting with the identification of 21,066 transcripts. The expression of the infected samples was compared to mock sample, and RNA ratios were clustered using partitioning clustering. This approach allowed clustering of the cellular transcripts into 5 distinct classes based on similarities in temporal expression profiles. We next carried out GO term enrichment analysis for each of these five cluster. Our study represents the first detailed analysis of human epithelial response to Francisella tularensis infection, and provide a framework for comparative investigations of genes and mechanisms that may contribute to the infection.

overall_design: Examination of A549 cells at 6 different times post infection of Ft.+ uninfected sample in dupicates (=14 samples)

first: Adi

last: Bercovich-Kinori

first: Ma'ayan

last: Israeli

last: Cohen-Gihon

first: Ofir

first: Ofer

last: Cohen


title: Coupled single cell RNA-seq and intracellular protein activity reveals immune-suppressive role of Trem2 in cancer I

first: Ido

last: Amit

first: Yonatan

last: Katzenelenbogen

first: Fadi

last: Sheban

first: Adam

last: Yalin

last: Yofe

first: Dmitry

last: Svetlichnyy

first: Diego

last: Jaitin

first: Chamutal

last: Borenstein

last: Moshe

first: Hadas

last: Keren-Shaul

first: Merav

first: Shuang-Yin

last: Wang

first: Baoguo

last: Li

first: Eyal

last: David

first: Tomer

last: Salame

first: Assaf

last: Weiner


title: mRNA-Seq of BC-3 IRF4 KO, BATF KO, and vIRF3 KD

first: Eva

last: Gottwein

summary: The Kaposi's sarcoma-associated herpesviruses causes several cancers including Kaposi's sarcoma and primary effusion lymphoma (PEL). Our work reveals that the cellular transcription factors interferon regulatory factor 4 (IRF4) and basic leucine zipper ATF-like transcription factor (BATF), as well as the viral interferon regulatory factor 3 (vIRF3) are critical oncogenic dependencies specific to PEL cell lines. To understand the functions of these transcription factors, we performed mRNA-Seq on the PEL BC-3 cell lines that have been targeted for CRISPR/Cas9 IRF4 KO, BATF KO and MDM4 KO (as unrelated negative control) and CRISPRi-mediated vIRF3 KD. Our results reveal that these oncogenic factors may cooperate to regulate the expression of the same mRNAs.

overall_design: BC-3 Cas9 cells were infected with lentiviruses expressing sgRNAs against AAVS1, IRF4, BATF or MDM4. Cells were harvested after d3, d8 or d16 when gene KO affected cumulative live cell counts. BC-3 dCas9-KRAB cells were infected with lentiviruses expressing sgRNAs against AAVS1 and vIRF3 (guides i7 and i8). Cells were harvested after d4 when gene KO affected cumulative live cell counts.


title: Functional Interplay between Histone H2B ADP-Ribosylation and Phosphorylation Controls Adipogenesis

city: Dallas

first: W. Lee

last: Kraus

state: TX

address: 5323 Harry Hines Blvd.

institute: UT Southwestern Medical Center

summary: Although ADP-ribosylation of histones by PARP-1 has been linked to genotoxic stress responses, its role in physiological processes has remained elusive. We found that ADPribosylation of histone H2B-Glu35 inhibits the differentiation of adipocyte precursors, leading to a reduction of newly formed adipocytes in white adipose tissue. ADP-ribosylation of Glu35 by PARP-1 inhibits phosphorylation of adjacent H2B-Ser36, which is required for the proadipogenic gene expression program. It also inhibits adipogenesis in cultured cells and a mouse lineage tracing genetic model. The activity of PARP-1 on H2B requires NMNAT-1, a nuclear NAD+ synthase, which serves as a specificity factor to direct PARP-1 catalytic activity to Glu and Asp residues. Collectively, our results demonstrate a functional interplay between H2B-Glu35 ADP-ribosylation and H2B-Ser36 phosphorylation that controls adipogenesis and cancer cell proliferation.

overall_design: Using RNA-seq in 3T3-L1 cells ectopically expressing Wt or mutant histone H2B during a time course of differentiation.

first: Dan

last: Huang

first: Cristel

middle: V

last: Camacho

first: Rohit

last: Setlem

first: Keun

last: Ryu

first: Balaji

last: Parameswaran

first: Rana

last: Gupta

first: W


title: Transcriptome analysis of hiHepPCs in various culture conditions, human liver-derived hepatocytes, human liver-derived cholangiocytes, HUVECs, and HPBECs

first: Atsushi

last: Suzuki

summary:
Transcriptome analysis of human induced hepatic progenitor cells (hiHepPCs) in various culture conditions, human liver-derived hepatocytes, human liver-derived cholangiocytes, human umbilical vein endothelial cells (HUVECs), and human peripheral blood-derived endothelial cells (HPBECs)

We found that a specific combination of three transcription factors, FOXA3, HNF1A, and HNF6, could convert HUVECs and HPBECs into cells that closely resembled hepatic progenitor cells in vitro. These hiHepPCs were expandable in long-term culture and able to differentiate into hepatocytes and cholangiocytes in accordance with their culture conditions. We conducted RNA-seq analyses to investigate the characteristincs of hiHepPCs and their progenies, in addition to those of parental HUVECs, HPBECs, and human liver-derived cells.

overall_design: RNA-seq analyses for HUVECs (HUVEC-1 and HUVEC-2), HUVEC-derived hiHepPCs in monolayer (HUVEC-hiHepPC-2D-1, HUVEC-hiHepPC-2D-2, HUVEC-hiHepPC-2D-3, HUVEC-hiHepPC-2D-P16-1, HUVEC-hiHepPC-2D-P16-2, and HUVEC-hiHepPC-2D-P16-3), cell aggregate (HUVEC-hiHepPC-aggregate-1, HUVEC-hiHepPC-aggregate-2, and HUVEC-hiHepPC-aggregate-3), and spheroid cultures (HUVEC-hiHepPC-sheroid-1, HUVEC-hiHepPC-sheroid-2, and HUVEC-hiHepPC-sheroid-3), HPBECs (HPBEC-1 and HPBEC-2), HPBEC-derived hiHepPCs in monolayer (HPBEC-hiHepPC-2D-1 and HPBEC-hiHepPC-2D-2), cell aggregate (HPBEC-hiHepPC-aggregate-1 and HPBEC-hiHepPC-aggregate-2), and spheroid cultures (HPBEC-hiHepPC-spheroid-1 and HPBEC-hiHepPC-spheroid-2), human cholangiocyte-derived spheroids (human-cholangiocyte-spheroid-culture), and non-cultured human hepatocytes (human-hepatocyte-1 and human-hepatocyte-2).

first: Yasuyuki

last: Ohkawa

first: Kanae

last: Matsuda-Ito

first: Kenichi

last: Horisawa

first: Hiroki

last: Inada


title: Hhex promotes NK cell homeostatis by repressing BIM-dependent apoptosis

city: Clayton

first: Sebastian

last: Scheer

address: 15 Innovation Walk

institute: Monash University

summary: Hhex encodes a homeobox transcriptional regulator important for embryonic development and hematopoiesis. Hhex is highly expressed in NK cells and its germline deletion results in significant defects in lymphoid development, including NK cells. To determine if Hhex is intrinsically required throughout NK cell development or for NK cell function, we generated mice that specifically lack Hhex in NK cells. NK cell numbers was dramatically reduced, while NK cell differentiation, IL-15 response and function at the cellular level remained largely normal in the absence of Hhex. Increased IL-15 availability failed to fully reverse NK lymphopenia following conditional Hhex deletion, suggesting that Hhex regulates developmental pathways extrinsic to those dependent on IL-15. Gene expression and functional genetic approaches revealed that Hhex regulates NK cell survival by repressing expression of the key apoptotic mediator, BIM. These data implicates Hhex as a novel transcription factor essential for NK cell homeostasis and immunity.

overall_design: NK cells were enriched from C57BL/6 mice using the STEMCELL NK cell isolaton kit, cultured for 8 days in the presence of IL-15.


title: CDE-1 suppresses the production of risiRNA by coupling polyuridylation and degradation of rRNA

first: yun

last: wang

address: 443 HUANGSHAN ROAD

institute: University of Science and Technology of China

summary: Background: Modification of RNAs, particularly at the terminals, is critical for various essential cell processes; for example, uridylation is implicated in tumorigenesis, proliferation, stem cell maintenance, and immune defense against viruses and retrotransposons. Ribosomal RNAs can be regulated by antisense ribosomal siRNAs (risiRNAs), which downregulate pre-rRNAs through the nuclear RNAi pathway in Caenorhabditis elegans. However, the biogenesis and regulation of risiRNAs remain obscure. Previously, we showed that 26S rRNAs are uridylated at the 3'-ends by an unknown terminal polyuridylation polymerase before the rRNAs are degraded by a 3’ to 5’ exoribonuclease SUSI-1(ceDIS3L2).Results: Here, we found that CDE-1, one of the three C.elegans polyuridylation polymerases (PUPs), is specifically involved in suppressing risiRNA production. CDE-1 localizes to perinuclear granules in the germline and uridylates Argonaute-associated 22G-RNAs, 26S and 5.8S rRNAs at the 3’-ends. Immunoprecipitation followed by mass spectrometry (IP-MS) revealed that CDE-1 interacts with SUSI-1(ceDIS3L2). Consistent with these results, both CDE-1 and SUSI-1(ceDIS3L2) are required for the inheritance of RNAi.Conclusions: This work identified a rRNA surveillance machinery of rRNAs that couples terminal polyuridylation and degradation.

overall_design: In this study, we compared 26S and 5.8S rRNAs 3' tail-seq  from (1) eri-1(mg366) embryo animals;(13,14)eri-1(mg366);cde-1(tm936) embryo animals and (15,16)eri-1(mg366);cde-1(tm936);cde-1::mCherry embryo animals, and compared 26S rRNAs 3' tail-seq  from (17,18) eri-1(mg366) L3 animals;(19,20)eri-1(mg366);cde-1(tm936) L3 animals and (21,22)eri-1(mg366);cde-1(tm936);cde-1::mCherry L3 animals.

first: Shouhong

last: Guang


title: Three-way combinatorial CRISPR screen for analyzing interactions among druggable targets

city: Pokfulam, Hong Kong

first: Alan

last: Wong

address: 21 Sassoon Road

department: School of Biomedical Sciences

country: Hong Kong

institute: The University of Hong Kong

summary: We present a CRISPR-based multi-gene knockout screening system and new toolkits for extensible assembly of barcoded high-order combinatorial guide RNA libraries en masse. We apply this system for systematically identifying not only pairwise but also three-way synergistic therapeutic target combinations, and successfully validated double and triple combination regimens for suppression on cancer cell growth and protection against Parkinson’s disease-associated toxicity. This system overcomes the practical challenges to experiment on a large number of high-order genetic and drug combinations, and is applicable for uncovering the rare synergistic interactions between druggable targets.

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
synthetic construct

overall_design: Performed combinatorial CRISPR-mediated gene knockout screens in ovarian cancer and Parkinson's disease cell models.

first: Peng

last: Zhou

first: Becky

last: Chan

first: Yuk

last: Wan

first: Chaya

middle: T

last: Yuen


title: Resolving fate and transcriptome of hematopoietic stem cell clones

city: Nanjing

first: Xi

last: Wang

address: Longmian Avenue 101, Jiangning District

institute: Nanjing Medical University

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

overall_design: Refer to individual Series


title: Transcriptional profiling of pH-dependent cell cycle progression

city: Zürich

first: Reinhard

last: Dechant

address: Otto-Stern-Weg 3

department: Institute of Biochemistry

institute: ETH Zürich

summary: Cells have evolved a complex network of signaling cascades that mediate cellular adaptation to availability of nutrients, growth factors or stress. In these networks, cellular information flow is mostly mediated by posttranslational modifications, most notably phosphorylation, or signaling molecules such as GTPases. However, genetic analysis has also implicated a number of proton transporters in regulating cellular signaling processes. Here, we report the transcriptional response of starved cells upon stimulation with serum and glucose upon inhibition of Sodium Hydrogen Exchangers and compare these responses to inhibition of cycle progression with the p300/CBP inhibitor C646.

overall_design: The gene expression profile of starved cells and starved cells stimulated with FCS and glucose for 4 hours in the presence of Dimethyl Amiliride (DMA, 100µM),  C646  (10µM) or DMSO only, was compared by Illumina sequencing.


title: Atf-6 regulates lifespan through ER-mitochondrial calcium homeostasis

city: Quechee

first: Michael

last: Steinbaugh

state: VT

address: PO Box 1520

institute: Acid Genomics

summary: Functional crosstalk between organelles is critical for maintaining cellular homeostasis. Individually, dysfunction of both endoplasmic reticulum (ER) and mitochondria have been linked to cellular and organismal aging, but little is known about how mechanisms of inter-organelle communication might be targeted to extended longevity. The metazoan unfolded protein response (UPR) maintains ER health through a variety of mechanisms beyond its canonical role in proteostasis, including calcium storage and lipid metabolism. Here we provide evidence that in C. elegans, inhibition of the conserved UPR mediator, activating transcription factor (atf)-6 increases lifespan via modulation of calcium homeostasis and signaling to the mitochondria. Loss of atf-6 confers long life via downregulation of the ER calcium buffering protein, calreticulin. Function of the ER calcium release channel, the inositol triphosphate receptor (IP3R/itr-1), is required for atf-6 mutant longevity while a gain-of-function IP3R/itr-1 mutation is sufficient to extend lifespan. IP3R dysfunction leads to altered mitochondrial behavior and hyperfused morphology, which is sufficient to suppress long life in atf-6 mutants. Highlighting a novel and direct role for this inter-organelle coordination of calcium in longevity, the mitochondrial calcium import channel, mcu-1, is also required for atf-6 mutant longevity. Altogether this study reveals the importance of organellar coordination of calcium handling in determining the quality of aging, and highlights calcium homeostasis as a critical output for the UPR and atf-6 in particular.

overall_design: RNA-seq analysis of N2 (wild-type) and atf-6 strains.

first: Kristopher

last: Burkewitz

first: Sneha

last: Dutta

first: Charlotte

last: Kelley

first: Erin

last: Cram

first: William

middle: B

last: Mair


title: Bulk RNA seq of intestinal immune cells from naïve, LCMV-Arm and LCMV-Cl13 infected mice

city: Toronto

first: David

last: Brooks

address: 610 University Avenue

department: Princess Margaret Cancer Centre

institute: University Health Network

summary: We report that the gastrointestinal tract (GIT) is a previously unappreciated long-term reservoir of chronic LCMV-Cl13 infection, and, chronic viral replication in the GIT has a profound effect on the local immune compartment as well as the development of subsequent immune responses. CD45+ immune cells were sorted and analyzed by RNAseq from the small and large intestines of naive mice, or mice infected 30 days prior with LCMV-Arm (acute virus) or LCMV-Cl13 (chronic virus). We show that GIT immune cells from acute and chronically infected mice differ substantially from naive mice; chronically infected mice show increases in genetic pathways involved in T cell activation and killing, inflammasome activation and interferon signaling; chronically infected mice show reduced expression of genes involved in T cell memory and antigen presenting cell activation; and chronic infection induces metabolic changes unique to the small but not large intestinal immune compartment.

overall_design: 3 naïve, 3 LCMV-Armstrong (acute) infected and 3 LCMV-Cl13 (chronic) infected small (SI) and large (LI) intestinal samples of sorted CD45+ cells


title: Coupled single cell RNA-seq and intracellular protein activity reveals immune-suppressive role of Trem2 in cancer

first: Ido

last: Amit


title: The transcription factor Scleraxis differentially regulates gene expression in tenocytes isolated at different developmental stages

city: Herts

first: Deborah

middle: Jane

last: Guest

address: Hawkshead Lane, North Mymms, Hatfield

department: Deptartment of Clinical Sciences and Services

country: United Kingdom

institute: Royal Veterinary College

summary:
Purpose:- RNA sequencing was performed on adult and fetal equine tenocytes expressing a specific short hairpin RNA against scleraxis (shSCX) versus a non-target (NT) scrambled control
Methods:-   mRNA profiles of four independent biological lines of adult and fetal NT and shSCX expressing tenocytes were generated by RNA sequencing, using Illumina NovaSeq6000. The sequence reads that passed quality filters were analyzed using the pseudoaligner Salmon and differential expression quantified using DESeq2. Gene ontology analysis was then conducted using Panther. Pathway analysis was conducted using GeneAnalytics. Network analysis was conducted using the STRING plugin in the Cytoscape app.
Results:- SCX knockdown had a larger effect on gene expression in fetal tenocytes, effecting 477 genes in comparison to the 183 genes effected in adult tenocytes, indicating that SCX-dependent processes may differ in these two developmental stages.

overall_design: mRNA profiles of four independant lines of adult and fetal NT and shSCX expressing equine tenocytes were generated by RNA sequencing, using Illumina NovaSeq6000.

first: Yasmin

middle: Z

last: Paterson

first: Naomi

last: Evans

first: Shohei

last: Kan

first: Adam

last: Cribbs

first: Frances

last: Henson


title: Diacylglycerol Kinase 1 and Associated Lipid Mediators Modulate Rice Root Architecture

city: St. Louis

first: Xuemin

state: Missouri

address: 975 North Warson Road

institute: Donald Danforth Plant Science Center

summary: Through transcriptomic profiling, three auxin-related genes (IAA24, PIN1C, and YUC5), one GA-related gene (GA20ox2), one cell wall metabolic process gene (G4OM1), and one defense response gene (DRP-RG3) were found, which may have vital association to illuminate the function of OsDGK1.

overall_design: Total RNA samples were extracted from seven-day-old soil-grown root tissue of WT-DJ, dgk1-1, and OE2. Each line contains three biological repetitions.

first: Shu

last: Yuan


title: NKT cells co-expressing a GD2-specific chimeric antigen receptor and IL-15 mediate regression of refractory metastatic neuroblastoma

status: Public on Aug 10 2020

city: Houston

first: Leonid

last: Metelitsa

state: Texas

address: One Baylor Plaza

institute: Baylor College of Medicine

summary: Despite remarkable progress in B cell malignancies, T cells expressing chimeric antigen receptors (CARs) remain ineffective in solid tumors in part because T cells do not traffic effectively to or survive in tumor tissues. We engineered Vα24-invariant natural killer T cells (NKTs), which intrinsically home to tumor sites, to co-express a GD2-specific CAR with interleukin (IL)15 and evaluated their therapeutic efficacy in children with relapsed/resistant neuroblastoma (NB)(NCT03294954). The first three patients received a single infusion of 3x10e6 autologous CAR-NKTs per square meter of body surface area and tolerated the treatment well. CAR-NKTs expanded in vivo, localized to the tumor, and in one patient induced a near-complete regression of bone metastatic lesions.

overall_design: Single-cell RNA-seq of pre-infusion CAR-NKTs from three cancer patients.

first: Andras

last: Heczey

first: Amy

last: Courtney

first: Antonino

last: Montalbano

first: Simon

last: Robinson

first: Ka

last: Liu

first: Mingmei

last: Li

first: Nisha

last: Ghatwai

first: Olga

last: Dakhova

first: Bin

first: Tali

last: Raveh

first: Erica

last: Di Pierro

first: Barbara

last: Savoldo

first: Gianpietro

last: Dotti


title: Viral manipulation of functionally distinct interneurons in mice, non-human primates and humans

city: Cambridge

first: Kathryn

last: Allaway

state: MA

address: 75 Ames St

department: Stanley Center for Psychiatric Disease

institute: Broad Institute

summary: Recent success in identifying gene regulatory elements in the context of recombinant adeno-associated virus vectors have enabled cell type-restricted gene expression. However, within the cerebral cortex these tools are presently limited to broad classes of neurons. To overcome this limitation, we developed a strategy that led to the identification of multiple novel enhancers to target functionally distinct neuronal subtypes. By investigating the regulatory landscape of the disease gene Scn1a, we identified enhancers that target the breadth of its expression, including two selective for parvalbumin and vasoactive intestinal peptide cortical interneurons. Demonstrating the functional utility of these elements, we found that the PV-specific enhancer allowed for the selective targeting and manipulation of fast-spiking cortical interneurons across species, from mice to humans.

overall_design: scATAC library generated from Dlx6a-Cre;flox-Sun1-eGFP mouse primary visual cortex (V1) at P28 on the 10X Chromium  platform.

first: Giuseppe

last: Saldi

first: Jordane

last: Dimidschstein


title: RNAseq of NRVMs infected with AdCryABR120G by the manipulation of Ube2v1 expression

city: Cinicnnati

first: Katherine

middle: E

last: Yutzey

state: OH

address: 240 Albert Sabin Way

department: Divison of Molecular Cardiovascular Biology

institute: Cinicnnati Children's Hospital Medical Center

summary: Neonatal rat ventricle myocytes (NRVMs) were infected with CryABR120G adenovirus. Knockdown or overexpression of Ube2v1 was performed by siUbe2v1 transfection or AdUbe2v1 infection in CryABR120G-expressing cells. Total RNA was extracted from NRVMs of four groups: CTR (untreated control), R120G (AdCryABR120G infection), RSIV1 (AdCryABR120G infection with siUbe2v1 transfection), and RADV1 (coinfection with AdCryABR120G and AdUbe2v1), in triplicate, for gene expression profiling.

overall_design: Total RNA from NRVMs in triplicate per group was pooled for deep RNA sequencing

last: Xu

first: Jeffrey

last: Robbins