title: Human fetal testis exposed to THC and/or CBD

city: Rennes

first: Frédéric

last: Chalmel

state: France

address: 9 avenue du Pr. Léon Bernard

department: Physiology and physiopathology of the urogenital tract

institute: Inserm U1085-Irset

summary: Background: The  regular use of cannabis by young men has been associated with an increased incidence of testicular germ cell tumors (TGCT). TGCT, the most common cancer in young adults, is believed to arise from an alteration of testicular fetal germ cells differentiation during development, with a possible subsequent environmental trigger (eg drugs or other chemicals) during puberty or adulthood leading to cancer. Cannabis consumption by pregnant women is currently increasing worldwide, and legalization for its recreational and therapeutic purposes is debated in numerous countries. In this context, we aimed to determine whether cannabis exposure can affect development of the fetal testis.   Methods: Since phytocannabinoids act on an endogenous system called the endocannabinoid system (ECS), we first investigated these signaling pathways in the human fetal testis, from 6 to 17 developmental weeks. We next investigated the effects of the two main components of cannabis, (−)-Δ9-trans-tetrahydrocannabinol (THC) and cannabidiol (CBD), on the human fetal testis ex vivo.  Results: We show the presence of two key endocannabinoids, 2-arachidonylglycerol (2-AG) and anandamide (AEA), albeit with lower levels. The human fetal testis also expressed several enzymes and receptors of this signaling pathway. Human fetal testicular explants collected from first trimester were exposed to CBD, THC or CBD/THC [ratio 1:1] at concentrations ranging from 10-7 to 10-5M during 72h to 14 days. Phytocannabinoids treatments affected fetal testicular cell proliferation and viability, as well as testosterone secretion by Leydig cells and AMH secretion by Sertoli cells. Transcriptomic analysis performed by BRB-seq on exposed versus unexposed fetal testis explants showed 187 differentially expressed genes (DEGs), some of which involved in toxic substances response and steroid synthesis.   Conclusions: Our study provides the first evidence of the presence of ECS in human fetal testis and support a potential adverse effect of cannabis consumption in pregnant women on the male reproductive function development.

overall_design:
Samples consist in Bulk RNA coming from Embryonic human testis from 8-10 or 10-12 gestational week treated with CBD, THC and a mixture of CBD and THC (ratio 1: 1) at 10-5M and their respective control, DMSO for CBD, EtOH for THC or DMSO/EtOH for CBD/THC. For each six  conditions, 10 replicates are analyse so in total 120 samples.
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**Raw data will be submitted to EGA**

first: Juliette

last: Dochez-Arnault

first: Bertrand

last: Evrard

first: Aurélie

last: Lardenois

first: Aurore

last: Gely-Pernot


title: Symmetric inheritance of parental histones governs epigenome maintenance and stem cell identity [scRNA-seq]

status: Public on Jun 15 2023

city: Copenhagen

first: Anja

last: Groth

address: Blegdamsvej 3B

country: Denmark

institute: Novo Nordisk Foundation Center for Protein Research

type: Expression profiling by high throughput sequencing

summary: Modified parental histones are segregated symmetrically to daughter DNA strands during replication and inherited through mitosis. How this may sustain the epigenome and cell identity remains unknown. Here, we show that transmission of histone-based information during replication maintains epigenome fidelity and embryonic stem cell plasticity. Asymmetric segregation of parental histones H3-H4 in MCM2-2A mutants compromised mitotic inheritance of histone modifications and globally altered the epigenome. This included widespread spurious deposition of repressive modifications, suggesting elevated epigenetic noise. Moreover, H3K9me3 loss at repeats caused de-repression and H3K27me3 redistribution across bivalent promoters correlated with misexpression of developmental genes. MCM2-2A mutation challenged dynamic transitions in cellular states across the cell cycle, enhancing naïve pluripotency and reducing lineage priming in G1. Further, developmental competence was diminished, correlating with impaired exit from pluripotency. Collectively, this argues that epigenetic inheritance of histone modifications maintains a correctly balanced and dynamic chromatin landscape able to support mammalian differentiation.

overall_design: Multiplexed scRNAseq study of WT, MCM2-2A (2 biological replicates, 2 technical replicates) mutant in histone recycling and rescued cell line (MCM2-R, 1 biological replicate) by reversing the mutation back to the WT genotype by genome editing. Additional sample information can be found in Supplementary Table 1.

first: Alice

last: Wenger

first: Alva

last: Biran

first: Nicolas

last: Alcaraz

first: Alba

last: Redó-Riveiro

first: Annika

middle: C

last: Sell

first: Robert

last: Krautz

first: Valentin

last: Flury

first: Nazaret

last: Reverón-Gómez

first: Christian

last: Vaagenso

first: Victor

last: Solis-Mezarino

first: Moritz

last: Völker-Albert

first: Axel

last: Imhof

first: Robin

last: Andersson

first: Joshua

middle: M

last: Brickman


title: Analysis of gene expression influenced by miR-27b in Huh-7 cells.

city: Osaka

first: Hiroyuki

last: Mizuguchi

address: Suita Yamadaoka 1-6

country: Japan

institute: Osaka university

summary: To claryfy the genes regulated by miR-27b in human hepatic cells, whole human genome microarrays were employed to analyze gene expression.

overall_design: mRNA expression 48 hours after introducing miR-27b into Huh-7 cells were analyzed.

first: Tsutomu

last: Imaizumi

first: Eiko

last: Sakai


title: RNA seq in CD8T cells

city: columbus

first: Ruoning

last: Wang

state: OH

address: 700 Children's Drive

country: USA

institute: Nationwide children's hospital

summary:
Purpose: we evaluated the T cell mediated anti tumor immune response under asparagine restriction
Methods: Total RNA was isolated according to the RNeasy Mini Kit (Qiagen) and treated with DNase I. RNA quality was assessed using the Agilent 2100 Bioanalyzer and RNA Nano Chip kit (Agilent Technologies, CA). RNA-seq libraries were generated using TruSeq Stranded Total RNA with Ribo-Zero Globin Complete kit (Illumina, CA). Approximately 60–80 million paired-end 150 bp reads were generated per sample using Illumina HiSeq 4000 platform
Results: We performed gene expression profiling analysis using data obtained from RNA-seq of T cells cultured in presence and absence of asparagine at 3 different time points and found that asparagine restriction induces biphasic response in T cell.

overall_design: mRNA profiles of CD8 T cells were generated by deep sequencing, using Illumina HiSeq 4000.

first: JNRashida

last: Gnanaprakasam


title: RBFOX2 recognizes N6-methyladenosine to suppress transcription and block myeloid differentiation

city: Shanghai

first: Xiaoyang

last: Dou

address: 320 Yue Yang Road

country: China

institute: CAS Center for Excellence in Molecular Cell Science, CAS

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Symmetric inheritance of parental histones governs epigenome maintenance and stem cell identity [SCAR-seq]

first: Anja

last: Groth

overall_design: SCAR-seq (Sister Chromatids After Replication) time-series measuring histone partition of H3K27me3, H3K4me3 and H3K27ac in wildtype (WT) and MCM2 histone-binding mutant (MCM2-2A) mouse embryonic stem cells (WT cells, 2 MCM2-2A clones, 2 replicates each, stranded input controls). Crosslinked SCAR-seq measuring partition of SUZ12 and H3K27me3 in WT cells and 2 MCM2-2A clones (2 replicates each, stranded input controls). SCAR-seq measuring histone partition of H3K27me3 (parental histones) and H4K20me0 (new histones) in WT cells, 2 Pole4 KO clones, 1 MCM2-2A clone and 1 MCM2-R clone in nascent chromatin (2 replicates each, stranded input controls). Additional sample information can be found in Supplementary Table 1.

first: Alice

last: Wenger

first: Alva

last: Biran

first: Nicolas

last: Alcaraz

first: Alba

last: Redó-Riveiro


title: Symmetric inheritance of parental histones governs epigenome maintenance and stem cell identity

overall_design: This SuperSeries is composed of the SubSeries listed below. Refer to individual Series.


title: RNAseq of LSK cells from knock-in mice with Calr frameshifts

city: Fukushima

first: Kazuhiko

last: Ikeda

state: Fukushima

address: 1 Hikariga-oka

department: Blood Transfusion and Transplantation Immunology

institute: Fukushima Medical University

summary: Gene expression of hematopoietic stem/progenitor cells from mice with frameshifted Calr was examined.

overall_design: LSK cells were sorted from 2 types of Calr-frameshift KI mice (ins2 mice and del10 mice), and gene expression was compared.

first: Keiji

last: Minakawa


title: Ribosomal RNA 2’-O-methylation dynamics during development and differentiation impact cell fate decisions.

first: Anders

middle: Henrik

last: Lund

address: Ole Maaløes Vej 5

institute: University of Copenhagen,BRIC

summary: Variable rRNA 2'-O-me contributes to ribosome heterogeneity. We show that the rRNA 2'-O-me profile is dynamic in a tissue-specific manner during the directed differentiation of human embryonic stem cells (hESCs). 2'-O-me at position 26S:3904 transiently decreases at the neural progenitor (NPC) stage during neurogenesis. Knock-out of SNORD52, the guide of 28S:3904 2'-O-me, results in hESCs shifting to a NPC identity. We report that the lack of 28S:3904 methylation leads to an increased binding to the FMRP protein and translation of WNT pathway members.

overall_design: Ribosome profiling of H9 and H9SNORD52 knockout cells. H9 cells have rRNA 28S:Um3904 methylation whereas H9SNORD52 knockout cells do not have 28S:Um3904 methylation.

first: Sophia

middle: J

last: Häfner

first: Kübra

last: Altinel

middle: H


title: Symmetric inheritance of parental histones governs epigenome maintenance and stem cell identity [ChIP-seq]

overall_design: ChIP-seq for H3K27me3, H3K4me3, H3K9me3 and H3K27ac in WT cells and 3 MCM2-2A clones (2 replicates each, input controls). ChIP-seq for SUZ12 in WT cells and 2 MCM2-2A clones (3 replicates each, input controls). ChIP-seq for H3K27me3, H3K4me3 and H3K9me3 in WT cells, 1 MCM2-2A clone and 1 matching MCM2-R clone (3 replicates each, input controls). Additional sample information can be found in Supplementary Table 1.

first: Annika

last: Sell

first: Robert

last: Krautz

first: Valentin

last: Flury

first: Nazaret

last: Reverón-Gómez

first: Victor

last: Solis-Mezarino

first: Moritz

last: Völker-Albert

first: Axel

last: Imhof

first: Robin

last: Andersson

first: Joshua

last: Brickman


title: Symmetric inheritance of parental histones governs epigenome maintenance and stem cell identity [RNA-seq]

first: Anja

last: Groth

overall_design: Total RNA-seq of 8 WT vs. 8 MCM2-2A clones (2-6 replicates each; rRNA-depleted) and WT vs.3 POLE4-KO clones (3 biological replicates each; rRNA-depleted). Rescue experiment including WT, MCM2-2A and the corresponding MCM2-R in which the mutation was reverted (4 biological replicates; rRNA-depleted). Additional sample information can be found in Supplementary Table 1

first: Alice

last: Wenger

first: Alva

last: Biran


title: ERK1/2 signaling is necessary for definitive endoderm formation

city: Singapore

first: Adrian

middle: Kee Keong

last: Teo

state: Singapore

address: 61 Biopolis Drive, Proteos

department: Stem Cells and Diabetes Laboratory

country: Singapore

institute: IMCB, A*STAR

summary:
Purpose: To compare gene expressions of undifferentiated cells and differentiated cells treated with basal media, basal media + FGF2, basal media + SU5402 and bsal media + ERKiII.
Methods:For differentiation, hPSCs was set up 2 days before initiation of DE at Day 0 (D0). On D0, media was replaced with using RPMI/2%B27, Activin, CHIR99021, LY294002 +/- FGF2, SU5402 or TCS ERKi. The cells were harvested on D3. Cells were cultured in triplicates for each condition, total RNA extraction was performed and 1000 ng of purified RNA was sent for RNA-seq. Genes that are differentially expressed were analyzed by comparing RNA expression at different conditions.
Results: Principal component analysis confirmed that our replicate samples clustered together, indicating good reproducibility. ACLY and ACLYF30 samples clustered close to each other but were non-overlapping, suggesting hish similarity in their cellular identities.  Contrastingly, ACLYSU and ACLYERKi samples are found isolated from each other and also from undifferentiated hPSCs (UD), ACLY and ACLYF30 samples, indicating that the inhibition of FGFR or ERK1/2 had a huge impact on redirecting DE cell fate despite the presence of DE-inducing conditions.
Conclusions: Blocking of FGFR with SU5402 and ERK2 signalling with ERKiII perturb cell cycle and cell fate specification even in the presence of definitive endoderm-inducing conditions.

overall_design: 3 replicates per condition - undifferentiated hESCs, D3 ACLY, D3 ACLYF30, D3 ACLYSU, D3 ACLYERKi

first: Hwee

last: Lau

first: Nur

middle: S

last: Amirruddin

first: Larry

last: Loo

first: Vidhya

last: Gomathi Krishnan

first: Shawn

last: Hoon

middle: K


title: Psychedelics Reopen the Social Reward Learning Critical Period

status: Public on Jun 14 2023

city: Baltimore

first: Loyal

middle: A

last: Goff

state: MD

address: 733 N. Broadway Avenue

department: Institute of Genetic Medicine

institute: Johns Hopkins University

summary: Psychedelics are a broad class of drugs defined by their ability to induce an altered state of consciousness. These drugs have been used for millennia in both spiritual and medicinal contexts, and a number of recent clinical successes have spurred a renewed interest in developing psychedelic therapies. Nevertheless, a unifying mechanism that can account for these shared phenomenological and therapeutic properties remains unknown. Here we demonstrate in mice that the ability to reopen the social reward learning critical period is a shared property across psychedelics. Interestingly, the time course of critical period reopening is proportional to the duration of acute subjective effects reported in humans. Furthermore, the ability to reinstate social reward learning in adulthood is paralleled by metaplastic restoration of oxytocin mediated long-term depression (OT-LTD) in the Nucleus Accumbens (NAc). Finally, identification of differentially expressed genes in the ‘open state’ versus ‘closed state’, provides evidence that reorganization of the extracellular matrix (ECM) is a common downstream mechanism underlying psychedelic mediated critical period reopening. Together these results have significant implications for the implementation of psychedelics in clinical practice, as well as the design of novel compounds for the treatment of neuropsychiatric disease.

overall_design: Bulk RNA-Seq analysis of mouse Nucleus Accumbens (NAc) at select time points after treatment with LSD, Ketamine, MDMA, or cocaine or saline controls. We analyzed the gene expression data set between conditions where the critical period is in the open state (i.e. 48 hours and two weeks post LSD treatment, 48 hours post ketamine treatment, 48 hours post MDMA treatment) versus conditions where the critical period remains in or returns to the closed state (i.e. 48 hours and two weeks post saline treatment, 48 hours and two weeks post cocaine treatment, and two weeks post ketamine treatment).

first: Romain

last: Nardou

first: Edward

last: Sawyer

first: Young

last: Song

first: Makenzie

last: Wilkinson

first: Yasmin

last: Padovan-Hernandez

first: Júnia

last: Lara de Deus

first: Noelle

last: Wright

first: Carine

last: Lama

first: Sehr

last: Faltin

first: Genevieve

middle: L

last: Stein-O’Brien

first: Gül

last: Dölen


title: Spontaneous EBV-reactivation during in vitro B-cell differentiation: a model for polymorphic EBV-driven lymphoproliferations studied with targeted single cell expression analysis

city: York

first: Matthew

middle: Anthony

last: Care

state: North Yorkshire

address: Wentworth Way

department: Department of Biology

country: United Kingdom

institute: The University of York

summary: Epstein Barr Virus (EBV) driven B-cell neoplasms arise from reactivation of latently infected B-cells. In a subset of patients EBV drives a polymorphous lymphoproliferative disorder (LPD) in which B-cell differentiation is retained. Spontaneous EBV reactivation following B-cell mitogen stimulation provides a potential model of polymorphic EBV-driven LPD. We have developed an in vitro model of plasma cell (PC) differentiation from peripheral blood memory B-cells. To assess the frequency and phenotypes of EBV-associated populations derived during differentiation we analyzed 8 differentiations at the PC stage, with a targeted single cell gene expression panel. We identified subpopulations of EBV-gene expressing cells with PC and/or B-cell expression features in differentiations from all tested donors. EBV-associated cells varied in frequency ranging from 3-28% of cells. Most EBV-associated cells expressed PC genes such as XBP1 or MZB1 and in all samples these included a quiescent PC fraction lacking cell cycle gene expression. With increasing EBV-associated cells, populations with B-cell features became prominent, co-expressing germinal centre (GC) and activated B-cell gene patterns. The presence of highly proliferative EBV-associated cells was linked to retained MS4A1/CD20 expression and IGHM and IGHD co-expression, while IGHM only and class-switched cells were enriched in quiescent PC fractions. Thus, patterns of gene expression in primary EBV reactivation are consistent with recently proposed models relating EBV-mediated transformation in lymphoblastoid cell lines to features of GC B-cells, and suggest a particular association between spontaneously developing EBV-expansions and IgM+ IgD+ non-switched B-cells.

overall_design: Single cell experiments were carried out using the BD Rhapsody Express Single-cell Analysis System. Cryopreserved cells were thawed and dead cells removed by lymphoprep density gradient centrifugation. Cells were enumerated on a hemocytometer. The cells from individual donors were labelled with a unique sample tag identifier using the BD Single-Cell Multiplexing Kit. Donors (n=8) were pooled in equal ratios, with the aim of loading 4000-5000 cells per donor onto a cartridge. Donors 1-4 and 5-8 were run on independent cartridges using a custom designed 701 gene panel.

first: Reuben

last: Tooze

first: Sophie

last: Stephenson


title: Inhibition of protein translation under matrix-deprivation stress in breast cancer cells

city: Bengaluru

first: Annapoorni

last: Rangarajan

state: Karnataka

address: DBG, Biological Sciences Division, IISc, CV Raman Road

department: Developmental Biology and Genetics

country: India

institute: Indian Institute of Science

summary: Matrix-deprivation stress leads to cell-death by anoikis, whereas overcoming anoikis is critical for cancer metastasis. Work from our lab and others has identified a crucial role for the cellular energy sensor AMPK in anoikis-resistance, highlighting a key role for metabolic reprogramming in stress survival. Protein synthesis is a major energy-consuming process that is tightly regulated under stress. Although an increase in protein synthesis in AMPK-depleted experimentally-transformed MEFs has been associated with anoikis, the status and regulation of protein translation in epithelial-origin cancer cells facing matrix-detachment remains largely unknown. Our study shows that protein translation is mechanistically abrogated at both initiation and elongation stages by the activation of the unfolded protein response (UPR) pathway and inactivation of elongation factor eEF2, respectively. Additionally, we show inhibition of the mTORC1 pathway known for regulation of canonical protein synthesis. We further functionally assay this inhibition using SUnSET assay, which demonstrates repression of global protein synthesis in MDA-MB-231 and MCF7 breast cancer cells when subjected to matrix-deprivation. In order to gauge the translational status of matrix-deprived cancer cells, we undertook polysome profiling. Our data revealed reduced but continuous mRNA translation under matrix-deprivation stress. An integrated analysis of transcriptomic and proteomic data further identifies novel targets that may aid cellular adaptations to matrix-deprivation stress and can be explored for therapeutic intervention.

overall_design: To investigate global gene expression changes that occur following matrix-deprivation in MDA-MB-231 breast cancer cell line

first: Neha

last: Deshpande

first: Sai

last: Balaji

first: Shweta

last: Warrier

first: Adithya

last: Chedere

first: Shraddha

last: Mohanty


title: Paxbp1 is indispensable for the survival of CD4 and CD8 double-positive thymocytes

city: Shenzhen

first: Wen

middle: Ting

last: Li

address: No,1120,LianHua Road,FuTian District

institute: Shenzhen PKU-HKUST Medical Center

summary: The lifespan of double-positive (DP) thymocytes is critical for intrathymic development and shaping the peripheral T cell repertoire. However, the molecular mechanisms that control DP thymocyte survival remain poorly understood. Paxbp1 is a conserved nuclear protein that has been reported to play important roles in cell growth and development. Its high expression in T cells suggests a possible role in T cell development. Here, we observed that deletion of Paxbp1 resulted in thymic atrophy in mice lacking Paxbp1 in the early stages of T cell development. Conditional loss of Paxbp1 resulted in fewer CD4+CD8+ DP T cells, CD4 and CD8 single positive (SP) T cells in the thymus, and fewer T cells in the periphery. Meanwhile, Paxbp1 deficiency had limited effects on the CD4-CD8- double negative (DN) or immature single-positive (ISP) cell populations. Instead, we observed a significant increase in the susceptibility of Paxbp1-deficient DP thymocytes to apoptosis. Consistent with this, RNA-Seq analysis revealed a significant enrichment of the apoptotic pathway within differentially expressed genes in Paxbp1-deficient DP cells compared to control DP cells. Together, our results suggest a new function for Paxbp1, which is an important mediator of DP thymocyte survival and critical for proper thymic development.

overall_design: Paxbp1-deficient DP thymocytes are highly sensitive to apoptosis. In order to determine whether disregulation of a genetic pathway could explain the increased apoptosis of Paxbp1 cKO DP thymocytes, we performed high-throughput RNA sequencing (RNA-Seq) to compare gene transcription between control and Paxbp1-deficient DP cells.

first: Wenting

first: Xiaofan

last: Chen

first: Cong

last: Huang

first: Bo

last: Yu


title: Separation of transcriptional repressor and activator functions in HDAC3 [RNA-Seq]

city: Stockholm

first: Mattias

last: Mannervik

address: Svante Arrhenius väg 20C

country: Sweden

institute: Stockholm University

summary: The histone deacetylase HDAC3 is associated with the NCoR/SMRT co-repressor complex and its canonical function is in transcriptional repression, but it can also activate transcription. Here we show that the repressor and activator functions of HDAC3 can be genetically separated in Drosophila. A lysine substitution in the N-terminus (K26A) disrupts its catalytic activity and activator function, whereas a combination of substitutions (HEBI) abrogating the interaction with SMRTER enhance repressor activity beyond wild-type in the early embryo. We conclude that the critical functions of HDAC3 in embryo development involve catalytic-dependent gene activation and non-enzymatic repression by several mechanisms, including tethering of loci to the nuclear periphery.

overall_design: Analysis of gene expression by RNA-seq from HDAC3 shRNA, shRNA-resistant rescueWT, rescueY303F, rescueK26A, rescueHEBI and Control (tub-Gal4/+) embryos in 4 replicates.

first: M

last: Tang

first: I

last: Regadas

first: S

last: Belikov


title: Zmym2 is essential for methylation of germline genes and active transposons in embryogenesis

city: Montreal

first: William

middle: A.

last: Pastor

state: Quebec

address: 3655 Promenade Sir-William-Osler

department: Biochemistry

country: Canada

institute: McGill University

type: Methylation profiling by high throughput sequencing


title: Alteration of target cleavage patterns and off-target reduction of antisense oligonucleotides incorporating 2-N-carbamoyl- or (2-pyridyl)guanine

city: Yokohama

first: Takayuki

last: Kanagawa

address: Midori-ku Nagatsuta-cho

institute: Tokyo Institute of Technology


title: Effect of miR-122 overexpression of mRNA distribution on the polyribosomes

city: Dusseldorf

first: Mirco

last: Castoldi

state: No allergies

address: Moorenstr. 5

country: Germany

institute: University Hospital Dusseldorf

summary:
The aim of this study was to identify at genome-wide level, the effect of miR-122 on the distribution of its responsive genes on the polyribosomes. For this purpose human hepatoma cells Huh-7 were transfected for 48 hrs with LNA-based miR-122 inhibitors (122-PM) or miR-122 mimics (122-MIM).
Following the treatment with cycloheximide, cells were lysed and layered on a 10-50% sucrose gradient. Following ultracetrifigation the polyribosomes were separated in 12 different fractions

overall_design: The individual polyribosomal fractions of cells transfected with either miR-122 mimics (122-MIM) or antagomiRs (122-PM) were then combined to generate polyribosomal pools of decreasing densities [i.e., P1 = Heavy (fractions A1-A4), P2 = Middle (fractions A5-A8), and P3 = Light (fractions A9-A12)]. These six different RNA pools (i.e., three densities for two conditions for two different experiments) were hybridized to a total of 12 Affymetrix microarrays.


title: Alteration of target cleavage patterns and off-target reduction of antisense oligonucleotides incorporating 2-N-carbamoyl- or (2-pyridyl)guanine [Rluc]

summary: We performed microarrays to evaluate the gene expression changes after 24 h of antisense oligonucleotide transfection.

overall_design: Gene expression in HeLa cells were measured 24 hours after treatment of guanine 2-amino group modified ASOs.  All experiments were performed independenlty three times.

first: Aya

last: Koyoma

first: Yoshiaki

last: Masaki

first: Kohji

last: Seio


title: Separation of transcriptional repressor and activator functions in HDAC3 [CUT&TAG]

overall_design: Analysis of HDAC3 genome occupancy by CUT&Tag in w1118 and HDAC3 shRNA embryos.


title: Alteration of target cleavage patterns and off-target reduction of antisense oligonucleotides incorporating 2-N-carbamoyl- or (2-pyridyl)guanine [MALAT]


title: Excitatory amino acid transporter 1 supports adult hippocampal neural stem cell self-renewal.

city: Columbus

first: Elizabeth

middle: Diana

last: Kirby

address: 1835 Neil Ave, 55 Psycholgy

department: Psychology

institute: The Ohio State University

summary: Within the adult mammalian dentate gyrus (DG) of the hippocampus, glutamate stimulates neural stem cell (NSC) self-renewing proliferation, providing a link between adult neurogenesis and local circuit activity. Here, we show that glutamate-induced self-renewal of adult DG NSCs requires glutamate transport via excitatory amino acid transporter 1 (EAAT1) to stimulate lipogenesis. Loss of EAAT1 prevented glutamate-induced self-renewing proliferation of NSCs in vitro and in vivo, with little role evident for canonical glutamate receptors. Transcriptomics and further pathway manipulation revealed that glutamate simulation of NSCs relied on EAAT1 transport-stimulated lipogenesis, likely secondary to intracellular Ca2+ release and glutamate metabolism. Our findings demonstrate a critical, direct role for EAAT1 in stimulating NSCs to support neurogenesis in adulthood, thereby providing insights into a non-canonical mechanism by which NSCs sense and respond their niche.

overall_design: NSCs of passage 4 were plated 75,000 cells per well in a 12 well plate and treated with 1 µM TFB-TBOA or vehicle for 15 minutes prior to the addition of 100 µM glutamate or vehicle (n = 3 wells per treatment). After 48 hours, cells were harvested with Accutase (StemCell technologies) and a subset (approximately 75,000 cells) was pelleted prior to RNA extraction using the NucleoSpin RNA Plus XS kit (Takara) following manufacturer protocol. Assessment of RNA quality with Qubit RNA HS assay kit (Invitrogen) revealed RIN values of at least 8 for all samples.

first: Joshua

middle: D

last: Rieskamp


title: Intradermal skin test with mRNA vaccines as a surrogate marker of T cell immunity in immunosuppressed patients

city: Epalinges

first: Mathilde

last: Foglierini-Perez

address: Chemin de Boveresses 155

country: Switzerland

institute: CHUV

summary: The role of T cell immunity in protection against COVID-19 in immunocompromised patients (ICp) who failed to mount serological responses remains ill-defined. Intradermal skin test (IDT) with mRNA vaccines may represent a simple, reliable and affordable tool to measure T cell response in seronegative patients.We compared anti-SARS-CoV-2 antibodies and cellular responses in vaccinated ICp (n=58), healthy seronegative naive controls (NC, n=8), and healthy seropositive vaccinated controls (VC, n=32) by Luminex, spike-induced IFN-γ ELIPSOT and an IDT 3 to 6 months after vaccination. ICp regrouped 18 transplant recipients, 33 individuals with autoimmune diseases, and eight patients with primary immunodeficiencies. In three vaccinated volunteers, we performed a skin biopsy 24h after IDT and single-cell RNAseq of the skin-infiltrating CD45+ cells. Twenty-five percent of seronegative NC had a positive ELIPSOT (2/8) and IDT (1/4), compared to 95% (20/21) and 93% (28/30) in seropositive VC, respectively. Single-cell RNA seq data of positive IDT consistently showed a mixed population of helper and cytotoxic T cells composed of memory T cells in 87%. The TCR repertoire of infiltrating skin lymphocytes revealed 18/1218 clonotypes with known specificities against SARS-CoV-2, among which six were spike-specific. Seronegative ICp with positive Elispot and IDT were in the majority treated with B cell-depleting reagents only, while those with negative IDT were all transplant recipients.  Our results indicate that local reaction to IDR is mainly composed of memory T cells and includes SARS-CoV-2-specific T cells, opening the perspective to use IDT as a correlate of protection in immunosuppressed patients.

overall_design: In three vaccinated volunteers, we performed a skin biopsy 24h after IDT and single-cell RNAseq of the skin-infiltrating CD45+ cells (10X genomics: 5'GEX and TCR )

first: Benedict

last: Fallet

last: Foglierini

first: Raphael

last: Porret

first: Ana

last: Alcaraz

first: Christophe

last: Sauvage

last: Jenelten

first: Teofila

last: Caplanusi

first: Michel

last: Gilliet

first: Laurent

last: Perez

first: Alexandre

last: Harrari

first: Sara

last: Bobisse

last: Gottardo

first: Giuseppe

last: Pantaleo

first: Yannick

last: Muller


title: Transcriptomic landscape of early hair follicle and epidermal development

city: Helsinki

first: Marja

last: Mikkola

address: P.O.B 56 (Viikinkaari 5)

country: Finland

institute: University of Helsinki

overall_design: Refer to individual Series


title: Zmym2 is essential for methylation of germline genes and active transposons in embryogenesis [RNA-seq]

first: William

last: Pastor

summary: ZMYM2 is a transcriptional repressor whose role in development is largely unexplored. We found that Zmym2-/- mice show embryonic lethality by E10.5. Molecular characterization of Zmym2-/- embryos revealed two distinct defects. First, they fail to undergo DNA methylation and silencing of germline gene promoters, resulting in widespread upregulation of germline genes. Second, they fail to methylate and silence the evolutionarily youngest and most active LINE element subclasses in mice. This results in ubiquitous overexpression of LINE1 protein as well as aberrant expression of transposon-gene fusion transcripts. Interaction and colocalization data indicate that ZMYM2 homes to germline genes via binding to the non-canonical polycomb complex PRC1.6 and to transposons via the TRIM28 complex. ZMYM2-/- human embryonic stem cells also show aberrant upregulation and demethylation of young LINE elements, indicating a conserved role in repression of active transposons. ZMYM2 is thus an important new factor in DNA methylation patterning in early embryogenesis.

overall_design: Comparitive gene expression analysis of RNA-seq data for wild-type and Zmym2 knockout E8.5 mouse whole embryos

first: Deepak

last: Saini

first: Adda-Lee

last: Graham-Paquin

first: Maxime

last: Bouchard


title: Transcriptomic landscape of early hair follicle and epidermal development [RNA-seq]

summary: In-depth transcriptional analysis of mature hair placode and interfollicular epithelium using bulk RNA-seq method.

overall_design: 7 samples pairs of placode-enriched and IFE populations isolated from E14.5 wild type dorsal skin epithelium.

first: Ana-Marija

last: Sulic

first: Rishi

last: Roy

first: Jukka

last: Jernvall

first: Irma

last: Thesleff


title: Transcriptomic landscape of early hair follicle and epidermal development [scRNA-seq]

summary: In-depth transcriptional analysis of mature hair placode and interfollicular epithelium using scRNA-seq method.

overall_design: Whole dorsal skin epithelium scRNAseq was performed on one wild type E14.5 replicate, and placode-enriched on two wild type E14.5 biological replicates.


title: Zmym2 is essential for methylation of germline genes and active transposons in embryogenesis [ChIP-seq]

overall_design: Genome binding/occupancy profiling by high throughput sequencing between non-target control and Zmym2KO mESCs (grown in either Serum+lif conditions or as embryoid bodies)


title: Zmym2 is essential for methylation of germline genes and active transposons in embryogenesis [WGBS-seq]

overall_design: Methylation profiling by high throughput sequencing for wild-type and Zmym2 knockout E8.5 mouse whole embryos


title: Single-cell RNA sequencing identifies regulators of differentiation and nutritional cues in Drosophila female germ cells

city: Guangzhou

first: Zhipeng

last: Sun

state: GuangDong

address: Wushan Distict

institute: South China Agricultural University

summary: Drosophila ovarian germline stem cells (GSCs) are a powerful model for stem cell research. However, due to the scarcity of GSCs in ovarian tissue, it is difficult to obtain the transcriptional profile of GSCs and identify novel GSC markers. In this study, we took advantage of single cell RNA sequencing (scRNA-seq) to profile the germline cells and somatic cells in wild type Drosophila ovary. We then performed an in vivo RNAi screen and network analysis to identify genes that are involved in the early stages of germ cell differentiation. We identified 33 genes with limited expression during early germ cell development and identified 19 genes that potentially regulate germ cell differentiation. Among these, an uncharacterized gene, which we named eggplant (eggpl), that is highly expressed in GSC and downregulated in early daughter cells. Upon RNAi knockdown of eggpl, we observed an increase in germ cell proliferation, an accumulation of cysts in the early mitotic (2- and 4-cell) stages and an increase in overall ovary size compared to control when flies were maintained on a standard diet. In flies fed a rich yeast diet, the expression of eggpl was significantly lower and the effects of eggpl RNAi were suppressed, suggesting that downregulation of eggpl may link nutritional status to germ cell proliferation and differentiation. We also found that the matrix metalloproteases, Mmp1 and Mmp2 as well as the tissue inhibitor of metalloproteases (Timp) are additional regulators of eggpl. Collectively, this study provides new insight into the signaling networks that regulate early germ cell development and identifies eggpl as a key player in this process.

overall_design: The whole ovarian cells were isolated  according to 10× Genomics recommendations. Based on the scRNA-seq data, we generated an altas of ovarian cell types, and identified the germline stem cell cluster. Then, a series of experimental validations and novel genes screening related to GSC identity. Among these GSC specific genes, we further explore the function of  an uncharacteristic gene namely eggpl using genetic approaches

first: Todd

middle: G

last: Nystul

first: Guohua

last: Zhong


title: The Siglec-sialic acid-axis is a target for innate immunotherapy of glioblastoma

city: Basel

first: DBM Bioinformatics

last: Core Facility

state: BS

address: Hebelstrasse 20

department: Departement of Biomedicine

institute: University of Basel

summary: Recently, ‘don’t eat me’-signals like CD47 have emerged as novel innate immune checkpoints, enabling cancer cells to evade clearance by phagocytes such as microglia (MG) or monocyte-derived cells (MdCs). Here, we aim at defining the role of inhibitory Siglec-9 in human and its mouse homologue Siglec-E in innate-centered immunotherapy against GBM. TCGA RNA-sequencing data revealed a significant correlation between high expression of immunoinhibitory SIGLEC9 and poor survival in GBM patients (log-rank p = 0.02). Using a CT-2A orthotopic GBM mouse model with MG-specific spatio-temporal deletion of Siglece (Sall1CreERT2 x Sigleceflox), we observed high MG-proliferation upon Siglece knockout (Ki-67+ MG 14.8% in Creneg vs. 34.9% in Crepos, p < 0.0001) accompanied by an enhanced microglial GBM-cell uptake (5.6% in Creneg vs. 12.3% in Crepos, p < 0.001). By extending the Siglece knockout to the MdC compartment (Cx3cr1CreERT2 x Sigleceflox) we observed a significantly prolonged survival in the Crepos population (21d in Creneg vs. 27d post-tumor injection in Crepos, p = 0.018), which could be further promoted by combining Siglece knockout with CD47 blockade (11% long-term remission in Crepos + anti-CD47). Proteomics analysis revealed increased antigen processing and presentation capabilities of SigleceKO MdCs which was confirmed by ex-vivo OT-1 cross-presentation assays. This increased T cell priming upon MdC SigleceKO was further boosted by addition of anti-PD1 antibody to the SigleceKO + anti-CD47 combination. Resulting in 23% of animals experiencing long-term remission in the triple treatment arm, even after tumor re-challenge. Genetic targeting of sialic acids, the ligand for Siglec receptors, on CT-2A cells (GNE-KO), resulted in increased GBM-cell phagocytosis by MG and MdCs and less exhausted tumor-infiltrating CD8+ T cells (14.8% in WT vs. 5.9% in GNE-KO, p = 0.003). In a translational approach, we are currently testing anti-Siglec-9 treatment regimens in patient GBM explants, cultured for 5 days in perfusion bioreactors.

overall_design: 10x 5'end scRNA-seq analysis of sorted CD45+/CD11B+ cells from induced glioblatoma tumors in 3 WT mice and 4 mice with induced microglia-specific KO of Siglece.

first: Philip

last: Schmassmann

first: Gregor

last: Hutter

first: Julien

last: Roux


title: Gene expression profile of non-hepatocyte cells of the liver of wild-type and Alb/MDA-9 mice

city: Richmond

first: Wright Center for Clinical and Translational Research

last: Bioinformatics Core

state: VA

address: 203 E Cary St

institute: Virginia Commonwealth University

summary: We created a mouse line with hepatocyte-specific overexpression of the oncogene MDA-9/SDCBP (Alb/MDA-9). We wanted to check how overexpression of MDA-9 in hepatocytes affect gene expression in surrounding non-parenchymal cells. We isolated non-hepatocyte cells from WT and Alb/MDA-9 mice and performed scRNA-seq in these samples.

overall_design: Non-hepatocyte cells from livers of WT and Alb/MDA-9 mice were harvested by differential centrifugation and scRNA-seq was performed using 10x Genomics platform.

first: Devanand

last: Sarkar

first: Debashri

last: Manna

first: Rachel

last: Mendoza

first: Jinze

last: Liu

first: Xufeng

last: Qu


title: Multiplexed Identification of Cytotoxic Cell assay (PAINTKiller assay)

first: Tongjin

last: WU

address: MD6, 14 Medical Drive

department: Institute for Health Innovation and Technology

institute: National University of Singapore

summary: Cell mediated cytotoxicity plays important roles in host immune defence and cancer immunosurveillance. Current technologies that study cell cytotoxicity are target cell centric and lack the capability to identify and isolate cytotoxic effector cells. We developed the Multiplexed Identification of Cytotoxic Cell assay (PAINTKiller), a novel flow cytometry compatible method for direct identification and sorting of cytotoxic effector cells. We demonstrate that this technique can accurately identify cytotoxic effector cells which can be isolated, expanded and retained its superior cytotoxic capability. This technique can also be easily combined with other flow cytometry complementary assays, cell sorting and sequencing. The results indicate that PAINTKiller assay will be a powerful tool to uncover the determinants of functional immune response and will be of significant interest for cell therapy manufacturing workflow.

overall_design: Human primary NK cells were enriched from PBMCs using EasySep™ Human NK Cell Enrichment Kit (STEMCELL Technologies, 19055) and expanded for 9-10 days using ImmunoCult™ NK Cell Expansion Kit (STEMCELL Technologies, 100-0711). For sequencing, expanded NK cells were modified with anti-CFSE capture antibody before co-cultured with or without CFSE-stained K562 at a ratio of 3:1. After 4 hours, cells were collected and pre-blocked by Fc-blocking reagent (Biolegend, 422301). Samples were stained with anti-FITC-PE (Southern Biotech, cat no: 6400-09) while concurrently barcoded with β2M-targeting hashtag before pooled and stained with a cocktail of TotalSeq-A antibodies targeting PE, CD3, CD56, CD16, CD57, CCR7, CD45RA, CD107a, CD69, NKG2D, NKp44, and PD-1. Cells were washed with cold PBS/2% FBS and re-suspended in PBS before loading on the Chromium Controller (10x Genomics).

last: Wu

first: Lih Feng

last: Cheow


title: Development of an AAV-LK03 variant with improved liver lobular biodistribution in the humanised FRG model

city: Westmead

first: Suzanne

last: Scott

state: NSW

address: 214 Hawkesbury Rd

country: Australia

institute: Children's Medical Research Institute

summary: AAV variants may differ in their tropism for human vs mouse hepatocytes or portal vs central hepatocytes.  We examined the engraftment of human hepatocytes in the FRG mouse model and the transduction of hepatocytes in this model using single nuclear RNA-seq.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Single nuclear RNA-seq of human hepatocytes engrafted into FRG mouse liver, including pre- and 5 months post-engrafted, as well as AAV-treated samples

first: Marti

last: Cabanes-Creus

first: Renina

last: Gale Navarro

first: Rodrigo

last: Carlessi

first: Ramon

last: Roca-Pinilla

first: Elena

last: Denisenko

first: Grober

last: Baltazar

first: Sophia

last: Liao

first: Erhua

last: Zhu

first: Laurence

middle: O

last: Wilson

first: Alistair

last: Forrest

first: Ian

middle: E

last: Alexander

first: Nina

last: Tirnitz-Parker

first: Leszek

last: Lisowski


title: Single-Cell Gene Expression Analysis of First Trimester Placenta Samples Linked to Idiopathic Recurrent Pregnancy Loss

city: Kansas City

first: Boryana

last: Koseva

state: MO

address: 2420 Pershing Road

department: CMRI

institute: Children's Mercy Hospital

summary: The extravillous trophoblast (EVT) cell lineage is a key feature of placentation and critical for spiral artery remodeling and successful pregnancy. Our knowledge of transcriptional regulation driving EVT cell development is limited. Here, we mapped the transcriptome and epigenome landscape as well as chromatin interactions of human trophoblast stem (TS) cells and their transition into the differentiated EVT cell lineage. Chromatin accessibility in intergenic regions was more extensive in EVT cells than in TS cells in the stem state, which is consistent with increased enhancer-driven gene regulation. Using reference epigenome annotation, we noted that 18% of the chromatin landscape in EVT cells was uncharted.  We linked regulatory regions to their cognate target genes and characterized the three-dimensional organization of the TS cell functional genome by high throughput chromosome conformation capture. Integration of chromatin accessibility, long-range chromatin interactions, transcriptomic, and transcription factor binding motif enrichment enabled identification of transcription factors and regulatory mechanisms associated with EVT cell lineage development. Subsequent analyses elucidated functional roles for   TFAP2C, SNAI1 and EPAS1 in the regulation of EVT cell lineage development.  EPAS1 was identified as an upstream regulator of key EVT cell transcription factors, including ASCL2  and SNAI1 and was together with its targets linked to diseases of pregnancy, including  idiopathic recurrent pregnancy loss and low birth weight. Collectively, we have revealed activation of a dynamic regulatory network that provides a framework for understanding EVT cell specification in trophoblast cell lineage development and human placentation.

overall_design: Villous tissue was isolated from the product of conception derived from patients with idopathic recurrent pregnancy loss (RPL) and analyzed using scRNAseq.

first: Elin

last: Grundberg

first: Emily

last: Wesley

first: Warren

last: Cheung

first: Courtney

last: Marsh


title: Antisense Oligonucleotide Rescue of CGG Expansion-Dependent FMR1 Mis-Splicing in Fragile X Syndrome Restores FMRP

city: Worcester

first: Sneha

last: Shah

state: Massachusetts

address: 373 Biotech II, Suite 204, Plantation Street

department: Molecular Medicine

institute: UMASS CHAN MEDICAL SCHOOL

summary: Aberrant alternative splicing of mRNAs results in dysregulated gene expression in multiple neurological disorders. Here we show that hundreds of mRNAs are incorrectly expressed and spliced in white blood cells and brain tissue of individuals with fragile X syndrome (FXS). Surprisingly, the FMR1 (Fragile X Messenger Ribonucleoprotein 1) gene is transcribed in >70% of the FXS tissues. In all FMR1 expressing FXS tissues, FMR1 RNA itself is mis-spliced in a CGG expansion-dependent manner to generate the little-known FMR1-217 RNA isoform, which is comprised of FMR1 exon 1 and a pseudo-exon in intron 1. FMR1-217 is also expressed in FXS premutation carrier-derived skin fibroblasts and brain tissue. We show that in cells aberrantly expressing mis-spliced FMR1, antisense oligonucleotide (ASO) treatment reduces FMR1-217, rescues full-length FMR1 RNA, and restores FMRP (Fragile X Messenger RibonucleoProtein) to normal levels. Notably, FMR1 gene reactivation in transcriptionally silent FXS cells using 5-aza-2′-deoxycytidine (5-AzadC), which prevents DNA methylation, increases FMR1-217 RNA levels but not FMRP. ASO treatment of cells prior to 5-AzadC application rescues full-length FMR1 expression and restores FMRP. These findings indicate that mis-regulated RNA processing events in blood could serve as potent biomarkers for FXS and that in those individuals expressing FMR1-217, ASO treatment may offer a new therapeutic approach to mitigate the disorder.

overall_design: Comparative gene expression profiling analysis of Chip-seq data for two FXS male patients and three typically developing males

first: Kevin

last: Sharp

first: Jonathan

last: Watts

first: Elizabeth

last: Berry-Kravis

first: Joel

last: Richter


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


overall_design: Comparative gene expression profiling analysis of RNA-seq data for ten FXS male patients and seven typically developing males

first: Sithara

middle: R

last: Ponny

last: Lee


title: Dietary intake of xylose impacts the transcriptome and proteome of tissues involved in xylose metabolism in swine

status: Public on Jun 13 2023

city: Ames

first: James

last: Koltes

state: Iowa

address: 1221 Kildee Hall

department: Department of Animal Science

institute: Iowa State University

summary:
We investigated the transcriptomics and proteomics profiling between different xylose supplementation diets (0% xylose-free vs. 8% xylose (8 g D-xylose/kg)) in swine.
We used RNA-seq and proteomics data from kidney, jejunum, liver, and muscle from two replicates of 12 crossbred gilts each to provide 24 animals (12 per dietary 100 treatment; L337 × Camborough, PIC Inc., Hendersonville, TN).

overall_design: We performed differential gene expression and proteomics analyses between different xylose diets using 24 swine animals (12 control vs. 12 xylose treatment) from 4 tissues (kidney, jejunum, liver, and muscle)

first: Nichole

middle: F

last: Huntley

first: Marcela

middle: M

last: De Souza

first: Matthew

last: Schulte

first: Hamid

last: Beiki

first: Andressa

last: de Lima

first: Abigail

last: Jantzi

first: Steven

last: Lonergan

first: Elisabeth

first: John

last: Patience


title: The  opposing impacts of dietary methionine restriction on tumor growth in immunodeficient and immunocompetent mice

city: RTP

first: Xiaojiang

last: Xu

state: North Carolina

address: 111 T. W. Alexander Dr.,

institute: NIEHS

summary: Methionine, a sulfur-containing essential amino acid, is a key component of dietary proteins important for protein synthesis, sulfur metabolism, antioxidant defense, and signaling. However, the role of methionine in cancer progression remains inconclusive. On one hand, dietary methionine restriction is known to repress cancer growth and improve cancer therapy in xenografted tumors. On the other hand, methionine is also critical for T cell activation and differentiation, making it a potential tumor suppression nutrient by enhancing T cell-mediated anti-tumor immunity. Here we investigated the interaction between dietary methionine, immune cells, and cancer cells by allografting CT26.WT mouse colon carcinoma cells into immunocompetent Balb/c mice or immunodeficient NSG mice, then analyzed how dietary methionine contents affect their growth. Our results show that dietary methionine restriction suppresses tumor growth in immunodeficient NSG mice but promotes tumor progression in immunocompetentt Balb/c mice.

overall_design: Total RNA was extracted from allografted CT26.WT tumors isolated from mice fed with either a high methionine diet containing 0.86% methionine (0.86%) or a methionine restricted diet containing 0.172% methionine (0.172%). All RNA-seq libraries were prepared with the TruSeq Stranded/Ribo kit (Illumina, San Diego, CA) and sequenced using the single-end 76bp protocol at about 500 million reads per library using the NextSeq 500 platform (Illumina) per the manufacturer’s protocol.

first: Xiaoling

last: Li

first: Ming

last: Ji


title: Dietary methionine restriction impairs anti-tumor immunity through gut microbiota

summary: Dietary methionine restriction represses growth and improves therapeutic responses in several pre-clinical settings. However, how this dietary intervention impacts cancer progression in the context of the immune system is unknown. Here we analyzed the CD45+ immune cells from the small intestine of control (CTRL) diet or methionine-restricted (MR) diet fed tumor-free C57BL/6J donor mice and tumor-bearing Apc <min+/-> recipient mice transplanated with feces from these diet-fed tumor-free C57BL/6J mice by scRNA-seq. Our analysis indicate that fecal microbes from methionine-restricted tumor-free C57BL/6J mice are sufficient to represss T cell activation in the small intestine of Apc <min+/-> mice.

overall_design: CD45+ immune cells purified from the small intestine of diet-fed C57BL6/J donor mice and fecal transplanted Apc<min+/-> mice, with 2 mice per sample, were counted and examined for viability with trypan blue staining using a TC-20 cell counter (Bio-Rad). Approximately 10,000 live cells with above 70% viability were loaded into the Single Cell Chip to generate single cell emulsion in Chromium Controller (10x Genomics, 120263) with Chromium Single Cell 3’ Library & Gel Bead Kit v3.1 (10x Genomics, 1000268). Reverse transcription of mRNA and cDNA amplification were carried out following the manufacture’s instruction (10x Genomics, 1000268). The amplified cDNA was further fragmented to construct NGS libraries. The libraries were then sequenced by the NIEHS Epigenomics and DNA Sequencing Core Laboratory with the parameters recommended in the manufacture’s instruction.


title: Effect of childhood ionizing radiation exposure and calorie restriction on NASH-like pathological disorder induction and hepatocellular carcinoma development in mouse model

city: Chiba

first: Kazuhiro

last: DAINO

address: 4-9-1 Anagawa, Inage-ku

institute: National Institutes for Quantum and Radiological Science and Technology

summary: To investigate gene expression patterns in mouse hepatocytes induced by ionizing radiation and calorie restriction.

overall_design: Gene expression pattens were analyzed in mouse hepatocytes after childhood ionizing radiation and adult-onset calorie restriction.

first: Yi

last: Shang

first: Takamitsu

last: Morioka

last: Daino

first: Mayumi

last: Nishimura

first: Shizuko

last: Kakinuma


title: NFI transcription factors regulate zonal olfactory receptor expression

city: New York

first: Elizaveta

last: Bashkirova

state: NY

address: 3227 Broadway

institute: Columbia University

summary: Expression of one out of > 1000 olfactory receptor (OR) genes is stochastic, yet organized into anatomic "zones" along the dorsoventral axis of the olfactory epithelium. Here we profile the gene expression, chromatin state and nuclear architecture of olfactory sensory neurons and olfactory progenitors of different zones. We discovered that zonal OR expression is specified by two processes: low-level polygenic transcription of OR genes in olfactory progenitors, as wells as the chromatin structure and nuclear architecture during olfactory differentiation. Specifically, in cells of any zonal segment, polygenic transcription in olfactory progenitors defines the OR repertoire eligible for OR choice as the cells mature, while an increased level of heterochromatin and chromatin compaction represses ectopic more dorsally expressed ORs.  We find that this process is regulated by the NFI family of transcription factors that are expressed in a graded fashion in the olfactory epithelium. Conditional deletion of NFI A, B, and X results in a transformation of the chromatin state and nucellar architecture and an accompanying shift in the preferred OR repertoire.

overall_design: Several types of data are included: RNA-seq profiles the expression differences between GBC, INP, iOSN and mOSN cells from zone1 (dorsal most), zone2/3 (dorsomedial) and zone4/5 (ventral most). RNA-seq data also profiles gene expression in the olfactory epithelium upon conditional NFI A, B and X deletion either early (in olfactory progenitors) or late (in mature OSNs). RNA-seq also profiles gene expression changes in mature OSNs and olfactory progenitors from zone5 (the ventral most zone) in NFI A,B,X deletion, NFI A,B deletion, NFIX deletion, and NFI A,B,X heterozygotes. Single-cell RNA-seq in cells from zone1/2 and zones 3/4/5 profiles gene expression in single cells.  Spatial transcriptomics shows the shift in the position of olfactory receptor expression within the olfactory epithelium upon Nfi A,B,X deletion. Native ChIP-seq for H3K9me3 and H3K79me3 profiles deposition of these chromatin marks during differentiation (in GBC, INP, iOSN and mOSN cells), in OSNs from different zones (zone1, zone2/3, zone4/5 and Olfr1507+ expressing zone5 mOSNs), as well as in zone4/5 cells upon NFI A,B,X deletrion. Hi-C in mOSNs from zone1 and zone4/5, iOSNs from zone1 and zone4/5, and cells from NFI knockout and wt zone4/5 shows differences in nuclear architecture in cells from different zones, and changes upon NFI A,B,X knockout. Dip-C in zone1 and zone4/5 mOSNs shows nuclear architecture of single cells. Hi-C in OSNs expressing a transgenic knock in teto-Olfr17 allele and Gng8-tTA driver shows nuclear architecture in cells that received a pulse of Olfr17 during the progenitor stage.

first: Stavros

last: Lomvardas

first: Nell

last: Klimpert

first: Alexander

last: Fleischmann


title: Transcriptomic profile of 3D spheroids developed from Astyanax Liver-derived cells - SFL and CFL.

first: Huzaifa

last: Hassan

institute: Stowers Institute for Medical Research

summary: Liver-derived cells from the surface and cave-adapted morphs of Astyanax mexicanus are valuable in vitro resources to explore the metabolism of these unique fish. However, 2D cultures have not yet fully mimicked the metabolic profile of the fish liver. Also, 3D culturing can modulate the transcriptomic profile of cells when compared to its 2D counterpart. Hence, to widen the range of metabolic pathways that can be depicted in vitro, we cultured the liver-derived SFL and CFL into 3D spheroids. We 3D cultured the cells at various cell seeding densities for 4 weeks and characterized the resultant transcriptome. The 3D cultured SFL and CFL cells indeed depicted a wider range of metabolic pathways as compared to the 2D culture. Further, the 3D spheroids also showed surface and cave-specific responses, making the spheroids an exciting system to study cave adaptation. Taken together, SFL and CFL spheroids prove to be a promising model for overall understanding of altered metabolism in Astyanax mexicanus.

overall_design: We cultured SFL and CFL cells in 3D spheroids of varying densities, 10, 25k, 50k and 100k. Thereafter, we collected the total RNA for bulk-RNAseq and further analysis of the transcriptome.

first: Tathagata

last: Biswas

first: Naresh

last: Rajendran

first: Chongbei

last: Zhao

first: Nicolas

last: Rohner


title: Effect of BAF disruption on chromatin accessibility in activated CD8+ T cells [ATAC-seq]

city: San Diego

first: Bryan

last: McDonald

state: CA

address: 10010 N Torrey Pines Rd

institute: Salk Institute for Biological Studies

summary:
To investigate the role of cBAF in regulation of Tbet binding, polyclonal naive CD8 T cells were stimulated for 48h with anti-CD3, CD28, and hIL-2.
Cells were then treated with DMSO, ACBI1, or BRM014 for 6 hours, and treated with recombinant mouse IL-12 for the last two hours of inhibitor treatment. Chromatin accessibility profiling was performed via ATAC-seq.

overall_design: ATAC-seq on WT CD8+ T cells activated with anti-CD3, anti-CD28, and hIL-2 for 48h in vitro.

first: Brent

last: Chick


title: Effect of BAF disruption on T-bet binding in activated CD8+ T cells [ChIP-seq]

summary:
To investigate the role of cBAF in regulation of Tbet binding, polyclonal naive CD8 T cells were stimulated for 48h with anti-CD3, CD28, and hIL-2.
Cells were then treated with DMSO, ACBI1, or BRM014 for 6 hours, and treated with recombinant mouse IL-12 for the last two hours of inhibitor treatment. ChIP-seq against T-bet was then performed.

overall_design: T-bet ChIP-seq on WT CD8+ T cells activated with anti-CD3, anti-CD28, and hIL-2 for 48h in vitro with or without IL-12, ACBI-1, or BRM014.

first: Nasiha

last: Ahmed


title: Astroglial Hmgb1 regulates postnatal astrocyte morphogenesis and cerebrovascular maturation

city: Ottawa

first: Ziying

last: Liu

state: ON

address: 1200 Montreal Road

institute: National Research Council, Canada

summary: Astrocytes are intimately linked with brain vessels, a relationship that is critical for neuronal health and function. However, key astroglial molecular factors that drive these physical and functional associations during postnatal brain development have not yet been identified. We characterized structural and transcriptional changes in mouse cortical astrocytes and microvessels during the first two postnatal weeks and found that high-mobility group box 1 (Hmgb1), normally upregulated with injury and involved in adult cerebrovascular repair, was highly expressed in astrocytes at birth to then decreased rapidly. Astrocyte-selective ablation of Hmgb1 in newborn mice affected astrocyte morphology and endfoot placement, induced disruption of endfoot proteins connexin43 and aquaporin-4, induced transcriptional changes in astrocytes, and profoundly altered endothelial cell ultrastructure. While lack of astroglial Hmgb1 did not affect the blood-brain barrier or angiogenesis postnatally, it impaired neurovascular coupling and behavior in adult mice. These findings identify astroglial Hmgb1 as a key player in postnatal gliovascular maturation.

overall_design: 16  RNA samples were used for RNA sequencing (4 mutant endothelial cell samples and 4 mutant astrocytes cell samples were isolated from 4 mutant animals; 4 wild type endothelial cell samples and 4 wild type astrocytes cell samples were isolated from 4 wild type animals).

first: Moises

last: Freitas-Andrade

first: Qing

middle: Y

first: Sonia

last: Leclerc

first: Youlian

last: Pan

first: Baptiste

last: Lacoste


title: Age-related brainstem degeneration through microRNA modulation in mice

city: Kamakura

first: Satoshi

last: Kondo

state: Kanagawa

address: Tebiro 6-10-1

department: New Projects Development Division

institute: Toray Industries,Inc.

summary: The specific miRNAs were altered in the brain of senescense-accerelated mouse prone 8 (SAMP8) compered with age-matched senescence-accerelated mouse resistant 1 (SAMR1).

overall_design: The mice brains were divided into the hippocampus, brainstem, and cerebral cortex, and the miRNA expression profiling were evaluated in the three brain regions.

first: K

last: Rie

first: M

last: Tsutomu


title: Effect of Arid1a deletion in antiviral effector CD8+ T cells. [RNA-seq]

summary:
To investigate the roll of ARID1A in the regulation of effector CD8+ T cell responses, we crossed GzmB-Cre P14 Thy1.1 mice with Arid1a-fl/fl mice to generate Arid1a Het and KO mice.
We then performed gene expression profiling analysis of WT, Het, and KO cells in vivo at d3, d5, and d8 post-LCMV Armstrong infection.

overall_design: RNA-seq gene expression profiling analysis for WT, Arid1a Het, and Arid1a KO P14 CD8+ T cells at d3 (total cells) and d8 (TE, EEC, and MP cell subsets) following LCMV Armstrong infection.


title: The 3D chromatin landscape of rhabdomyosarcoma [ChIP-Seq]

first: Benjamin

last: Stanton

state: Ohio

address: 700 Children's Drive

department: Center for Childhood Cancer

institute: Nationwide Children's Hospital

summary: Rhabdomyosarcoma (RMS) is a pediatric soft tissue cancer with no precision therapy available for affected patients. We hypothesized that with a general paucity of known mutations in RMS, chromatin structural driving mechanisms are essential for tumor proliferation. Thus, we carried out high-depth in situ Hi-C in representative cell lines and patient-derived xenografts to understand chromatin architecture in each major RMS subtype. We report a comprehensive 3D chromatin structural analysis and characterization of fusion-positive (FP-RMS) and fusion-negative rhabdomyosarcoma (FN-RMS). We have generated spike-in in situ Hi-C chromatin interaction maps for the commonest FP-RMS and FN-RMS cell lines, and compared our data with patient derived xenograft (PDX) models. In our studies we uncover common and distinct structural elements in large Mb-scale chromatin compartments, tumor-essential genes within variable topologically associating domains, and unique patterns of structural variation. This study enables a comprehensive resource for contextualizing gene regulation events in RMS, and high-depth chromatin interactivity maps for identification of functionally critical chromatin domains in this tumor.

overall_design: Genomic localization of CTCF, MYOD1, and H3K27ac, and H3K9me3 in rhabdomyosarcoma cell lines by ChIP-seq

first: Meng

last: Wang

first: Prethish

last: Sreenivas

last: Sunkel

first: Myron

last: Ignatius

middle: Z


title: Effect of Arid1a deletion on chromatin accessibility in antiviral effector CD8+ T cells. [Cut & Run]

summary:
To investigate the role of ARID1A in the regulation of transcription factor binding and histone modifications in effector CD8+ T cell responses, we crossed GzmB-Cre P14 Thy1.1 mice with Arid1a-fl/fl mice to generate Arid1a KO mice.
We then performed CUT&RUN against ARID1A, BATF, ETS, Tbet, H3K27ac, H3K27me3 in WT, Arid1a KO, or Tbx21 (Tbet) KO cells in vivo at d0, 48h in vitro activated, d5, and d8 post-LCMV Armstrong infection.

overall_design: Transcription factor and histone modification profiling analysis by CUT&RUN for WT, Arid1a KO, and Tbx21 (Tbet) KO P14 CD8+ T cells at d0 (naive), 48h in vitro activated, d5 (total cells), d8 (TE, EEC, and MP cell subsets) following LCMV Armstrong infection.

first: McDonald

last: Bryan

first: Chick

last: Brent


title: Loss of the mitochondrial protein Abcb10 results in altered arginine metabolism in MEL and K562 cells and nutrient stress signaling through ATF4

city: Salt Lake City

first: W. Zac

last: Stephens

state: UT

address: 15 N. Medical Drive, EEJMRB 4208, EEJMRB 4208

department: Pathology

institute: University of Utah

summary: Abcb10 is a mitochondrial membrane protein involved in hemoglobinization of red cells. Abcb10 topology and ATPase domain localization suggest it exports a substrate, likely biliverdin (Shum et al Sci Transl Med 13, (2021), PMC8300486), out of mitochondria that is necessary for hemoglobinization. In this study we generated Abcb10 deletion cell lines in both mouse (Murine Erythroleukemia (MEL) and human erythroid precursor (Human Myelogenous Leukemia K562) cells to better understand the consequences of Abcb10 loss. Loss of Abcb10 resulted in an inability to hemoglobinize upon differentiation in both K562 and MEL cells with reduced heme and intermediate porphyrins and decreased levels of Aminolevulinic acid synthase 2 activity. Metabolomic and transcriptional analysis revealed that Abcb10 loss gave rise to decreased cellular arginine levels, increased transcripts for cationic and neutral amino acid transporters with reduced levels of the citrulline to arginine converting enzymes Argininosuccinate synthetase and Argininosuccinate lyase. The reduced arginine levels in Abcb10 null cells gave rise to decreased proliferative capacity and excess arginine supplementation improved both Abcb10 null proliferation and hemoglobinization upon differentiation. Abcb10 null cells showed increased phosphorylation of Eukaryotic Translation Initiation Factor 2 Subunit Alpha, increased expression of nutrient sensing transcription factor ATF4 and downstream targets DNA damage inducible transcript 3 (Chop) and ChaC Glutathione Specific Gamma-Glutamylcyclotransferase 1 (Chac1) and Arginyl-tRNA Synthetase 1 (Rars). These results suggest that when the Abcb10 substrate is trapped in the mitochondria, the nutrient sensing machinery is turned on remodeling transcription to block protein synthesis necessary for proliferation and hemoglobin biosynthesis in erythroid models.

overall_design: RNAseq expression profiling of WT MEL cells and Abcb10 knockout MEL cells differentiated for 0, 2 and 5 days in 1.5% DMSO.

first: Diane

middle: H

last: Ward

first: W

first: Chris

last: Stubben


title: Effect of Arid1a deletion on chromatin accessibility in antiviral effector CD8+ T cells. [ATAC-Seq]

first: Bryan

last: McDonald

summary:
To investigate the role of ARID1A in the regulation of effector CD8+ T cell responses, we crossed GzmB-Cre P14 Thy1.1 mice with Arid1a-fl/fl mice to generate Arid1a Het and KO mice.
We then performed chromatin accessibility profiling analysis by ATAC-seq in WT, Het, and KO cells in vivo at d0, 48h in vitro activated, d3, d5, and d8 post-LCMV Armstrong infection.

overall_design: Chromatin accessibility profiling analysis by ATAC-seq for WT, Arid1a Het, and Arid1a KO P14 CD8+ T cells at d0 (naive), 48h in vitro activated, d3 (total cells), d5 (total cells) and d8 (TE, EEC, and MP cell subsets) following LCMV Armstrong infection.


title: Cell-cell interactome of the hematopoietic niche and its changes in acute myeloid leukemia

city: Galway

first: Eva

last: Szegezdi

address: University Road

country: Ireland

institute: University of Galway

summary: Acute myeloid leukemia (AML) is an aggressive cancer which causes the rapid proliferation of immature myeloid-lineage blood cells (myeloblasts) in the bone marrow. These leukemic cells receive trophic signals from the bone marrow micro-environment (BMM) in which they reside, that are essential to the initiation and maintenance of the disease. Because this is a complex and heterogeneous tissue, many aspects of this environment remain poorly characterised due to experimental hurdles. Here we performed single-cell RNA-sequencing of bone marrow aspirates from 10 AML patients across multiple key timepoints in disease progression (diagnosis, post-treatment, relapse).

overall_design: Longitudinally-collected BM aspirates were obtained for 10 AML patients from the time of diagnosis, post-treatment/remission and at relapse from The Finnish Haematology Registry and Clinical Biobank. scRNAseq was performed  on the mononuclear cell fraction using 10X Genomics Single Cell 3’ Solution, version 3.1 Libraries were sequenced an illumina NovaSeq 6000 with goal minimum sequencing depth of 50,000 reads/cell.

first: Sarah

last: Ennis

first: Alessandra

last: Conforte

first: Tatiana

last: Cichocka

first: Sukhraj

middle: P

last: Dhami

first: Pamela

last: Nicholson

first: Philippe

last: Krebs

first: Pilib

last: Ó Broin


title: The 3D chromatin landscape of rhabdomyosarcoma [HiC]

city: Columbus

first: Benjamin

last: Stanton

overall_design: Human rhabdomyosarcoma (RMS) cell lines RD, SMS-CTR, Rh30 and Rh41 cells were spiked in with 20% mouse myoblasts (C2C12) and fixed with Formaldehyde. Mouse chromatin reads were used as reference to normalize the RMS samples. Two independent biological replicates for each cell line were combined informatically to get contact maps with 30 million valid, long-range cis contacts.

first: Meng

last: Wang

first: Prethish

last: Sreenivas


title: Parallel use of human stem cell lung and heart models provide insights for SARS-CoV-2 treatment

city: Parkville

first: Michael

last: See

state: VIC

address: Royal Children's Hospital, 50 Flemington Rd

institute: MCRI

summary: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) primarily infects the respiratory tract, but pulmonary and cardiac complications occur in severe coronavirus disease 2019 (COVID-19). To elucidate molecular mechanisms in the lung and heart, we conducted paired experiments in human stem cell-derived lung alveolar type II (AT2) epithelial cell and cardiac cultures infected with SARS-CoV-2. With CRISPR-Cas9-mediated knockout of ACE2, we demonstrated that angiotensin-converting enzyme 2 (ACE2) was essential for SARSCoV-2 infection of both cell types but that further processing in lung cells required TMPRSS2, while cardiac cells required the endosomal pathway. Host responses were significantly different; transcriptome profiling and phosphoproteomics responses depended strongly on the cell type. We identified several antiviral compounds with distinct antiviral and toxicity profiles in lung AT2 and cardiac cells, highlighting the importance of using several relevant cell types for evaluation of antiviral drugs. Our data provide new insights into rational Q2 drug combinations for effective treatment of a virus that affects multiple organ systems.

overall_design: Comparative gene expression profiling of SARS-CoV-2 infected lung AT2 cells and cardiac cultures was performed by inoculating cultures with 10^4 TCID50 of SARS-CoV-2 (VIC01 isolate). Supernatant samples were harvested at 0-, 1- and 3-days post-infection for RNA extraction and RNA sequencing.

first: Rajeev

last: Rudraraju

first: Matthew

middle: J

last: Gartner

first: Jessica

last: Neil

first: Beth

last: Stout

first: Joseph

last: Chen

first: Elise

last: Needham

first: Charley

last: Mackenzie-Kludas

first: Leo

last: Lee

first: Mingyang

first: Hayley

last: Pointer

first: Kathy

last: Karavendzas

first: Dad

last: Abu-Bonsrah

first: Damien

last: Drew

first: Abbas

last: Salavaty

first: Peter

last: Currie

first: Wai-Hong

last: Tham

first: Sean

last: Humphrey

first: Mirana

last: Ramialison

first: David

last: Elliott

first: Enzo

last: Porrello

first: Jose

last: Polo

first: Kanta

last: Subbarao


title: The active form of Mef2c promotes maturation of induced cardiomyocytes

city: Shinjuku-ku

first: Hidenori

last: Kojima

state: Tokyo

address: 5 Shinanomachi

department: Cardiology

institute: Keio University School of Medicine

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

overall_design: Refer to individual Series


title: Effect of Trp53 mutations on mouse oviductal epithelial cell spheroids

city: London

first: Gabriel

last: DiMattia

state: ONTARIO

address: 750 Base Line Rd E

institute: Western University

summary: To identify mutant p53-dependent pathway alterations that are enhancing spheroid survival properties in mouse oviductal epithelial (OVE) cell lines, we generated OVE4 and OVE16 cell lines with either Trp53 deletion or stable expression of p53R175H. RNA-seq was performed on RNA from OVE spheroids to determine differential gene expression. Gene set enrichment analysis was performed to indentify enriched pathways in parental OVE spheroids compared to spheroids with Trp53 deletion or expression of p53R175H

overall_design: Total RNA was isolated from 3 biological replicates of OVE4 and OVE16 spheroids and their Trp53 mutation-containing derivatives (OVE4-Trp53ko, OVE4-p53R175H, OVE16-Trp53ko, OVE16-p53R175H). Differential gene expression was analyzed by RNA-seq. Pathway analyzis was perfomed by gene set enrichment analysis using the Hallmarks gene sets from the Molecular Signatures Database.

first: Jacob

last: Haagsma

first: Bart

last: Kolendowski

first: Adrian

last: Buensuceso

first: Yudith

last: Valdes

first: Trevor

last: Shepherd


title: The active form of Mef2c promotes maturation of induced cardiomyocytes with p300 in direct cardiac reprogramming

summary:
Direct cardiac reprogramming converts fibroblasts into induced cardiomyocytes (iCMs) with the minimal combination of transcription factors, Gata4 (G), Mef2c (M), and Tbx5 (T). However, the induction of functional mature iCMs is inefficient and the mechanisms remain elusive.
Mef2c is a central transcription factor in direct cardiac reprogramming. We investigated the effect of Mef2c isoforms(M1, M2, M6) and transcriptional activity (M2TAD) on cardiac reprogramming on cardiac reprogramming. Then, we found that the active form of Mef2c evoked epigenetic remodeling cooperating with p300 and promoted the maturation of iCMs.

overall_design: We analyzed global gene expression levels in GT/M1-, GT/M2-, GT/M6-, and GT/M2TAD-transduced MEFs at 1 week by microarray (Clariom S Array, Mouse, Affymetrix) according to the manufacturer’s instructions. Prior to analyses, all data were normalized by using a Single Space Transformation and Robust Multichip Analysis (SST-RMA) algorithm with Affymetrix Expression Console software version1.4.

first: Taketaro

last: Sadahiro

first: Masaki

last: Ieda


title: Mef2c/p300 Promotes Maturation of Induced Cardiomyocytes

city: Shinjuku

address: 35 Shinanomachi

summary: Cardiac transcription factors (TFs) directly reprogram fibroblasts into induced cardiomyocytes (iCMs), where Mef2c acts as a pioneer factor with Gata4 and Tbx5 (GT). However, generation of functional and mature iCMs is inefficient and molecular mechanisms underlying this process remains largely unknown. Here we found that transduction of transcriptionally activated Mef2c via fusion of the powerful MyoD transactivation domain increased generation of beating iCMs by 30-fold in combination with GT.

overall_design: MEFs were transduced with the retrovirus (Gata4, Mef2cM2, Mef2c_TAD, Tbx5) mixtures as indicated. Cells were collected after 4 weeks. Hearts were harvested E12.5 and 8 weeks old for bulk RNA-seq.


title: DNA Repair Function Scores for 2172 Variants in the BRCA1 Amino-Terminus

status: Public on Jun 12 2023

first: Jeffrey

last: Parvin

state: OH

address: 460 West 12th Ave

department: Department of Biomedical Informatics

institute: The Ohio State University

summary: Single nucleotide variants are the most frequent type of sequence changes detected in the genome and these are frequently variants of uncertain significance (VUS). VUS are changes in DNA for which disease risk association is unknown. Thus, methods that classify the functional impact of a VUS can be used as evidence for variant interpretation. In the case of the breast and ovarian cancer specific tumor suppressor protein, BRCA1, pathogenic missense variants frequently score as loss of function in an assay for homology-directed repair (HDR) of DNA double-strand breaks. We previously published functional results using a multiplexed assay for 1056 amino acid substitutions residues 2-192 in the amino terminus of BRCA1. In this study, we have re-assessed the data from this multiplexed assay using an improved analysis pipeline. These new analysis methods yield functional scores for more variants in the first 192 amino acids of BRCA1, plus we report new results for BRCA1 amino acid residues 193-302. We now present the functional classification of 2172 BRCA1 variants in BRCA1 residues 2-302 using the multiplexed HDR assay. Comparison of the functional determinations of the missense variants with clinically known benign or pathogenic variants indicated 93% sensitivity and 100% specificity for this assay. The results from BRCA1 variants tested in this assay are a resource for clinical geneticists for evidence to evaluate VUS in BRCA1.

overall_design: In this study, we have re-analyzed the BRCA1 amino-terminus (DOI: 10.1016/j.ajhg.2018.07.016) for the functional impact of single missense substitutions using an updated analysis protocol. The samples provided below are the sequencing data for BRCA1 amino acid residues 193-302, as well as the barcode map (.tsv) used to map the variants.

first: Mariame

last: Diabate

first: Muhtadi

last: Islam

first: Gregory

last: Nagy

first: Tapahsama

last: Banerjee

first: Shruti

last: Dhar

first: Nahum

last: Smith

first: Aleksandra

middle: I

last: Adamovich

first: Lea

last: Starita


title: Canonical BAF complex activity shapes the enhancer landscape that licenses CD8+ T cell effector and memory fates

first: Bryan

last: McDonald


title: Evidence of a role for CutRS and actinorhodin in the secretion stress response in Streptomyces coelicolor M145

city: Norwich

first: Ainsley

last: Beaton

address: Norwich Research Park

institute: John Innes Centre

summary: CutRS was the first two-component system to be identified in Streptomyces species and is highly conserved in this genus. It was reported >25 years ago that deletion of cutRS increases the production of the antibiotic actinorhodin in Streptomyces coelicolor but despite this early work the function of CutRS has remained enigmatic until now. Here we show that deletion of cutRS upregulates the production of the actinorhodin biosynthetic enzymes up to 260-fold in the cutRS mutant, explaining the increase in actinorhodin production. However, while ChIP-seq identified 86 CutR binding sites in S. coelicolor none of these are in the actinorhodin BGC, so the effect is indirect. The directly regulated CutR targets identified in this study are implicated in extracellular protein folding, including two of the four highly conserved the HtrA-family foldases, HtrA3 and HtrB and a putative VKOR enzyme which is predicted to recycle DsbA following its catalysis of disulphide bond formation in secreted proteins. We thus tentatively propose a role for CutRS in sensing and responding to protein misfolding outside the cell. Since actinorhodin can oxidise cysteine residues and induce disulphide bond formation in proteins its over production in the ∆cutRS mutant may be a response to protein misfolding on the extracellular face of the membrane.

overall_design: ChIP-seq was carried out to determine the DNA binding sites of the response regulator CutR, part of the two component system CutRS, in Streptomyces coelicolor. This is a conserved two component system found in all member of the genus. CutRS knock out mutants show a different phenotype in the presence of glucose compared to the wild type - and so to understand this further these expirments were carried out on samples grown on DNA (no glucose) and DNAD (with glucose). Being analysed in this experiment is DNA binding of the response regulator CutR in S. coelicolor. S. coelicolor ΔcutRS containing pSS170-Cterm3xFLAG-CutRS (CutS and FLAG-tagged CutR under the native promotor) and S. coelicolor ΔcutRS containing pSS170-Cterm3x_vnz_FLAG-CutRS (CutS and FLAG-tagged CutR from S. venezuelae under the native promotor) grown in the presence and absence of glucose. This was compared to S. coelicolor WT (no FLAG) grown in the same way and each was carried out and analysed in duplicate.

first: Thomas

middle: C

last: McLean

first: Carlo

last: de Oliveira Martins

first: Govind

last: Chandra

first: Barrie

last: Wilkinson

first: Matthew

last: Hutchings


title: The 3D chromatin landscape of rhabdomyosarcoma

first: Benjamin

last: Stanton


title: Effect of hematopoietic-specific heterozygous loss of Dnmt3a on the molecular pathophysiology of colitis-associated colon cancer (CAC) in a mouse model

city: Gainesville

first: Alberto

last: Riva

state: FL

address: 2033 Mowry Rd

department: ICBR

institute: University of Florida

summary: To model clonal hematopoiesis driven by DNMT3A loss-of-function, bone marrow chimerae with Dnmt3a(+/-) (experiment) or Dnmt3a(+/+) (control) were established in C57BL6/J wt mice, followed by standard CAC induction by AOM/DSS

overall_design: Colon tumors were dissected, and whole tumor transcriptomes were compared by RNA-seq (polyA) between Dnmt3a(+/-)-CH (n=4) and control Dnmt3a(+/+)-transplanted (n=3) animals  from two independent experiments

first: Olga

last: Guryanova


title: MOF-mediated histone H4 lysine 16 acetylation governs mitochondrial and ciliary functions by controlling promoter interactions (scATAC-Seq)

city: Chicago

first: Rui

last: Yi

state: IL

address: 303 E. Chicago Avenue

institute: Northwestern University

summary: Histone H4 lysine 16 acetylation (H4K16Ac), governed by the histone acetyltransferase (HAT) MOF, orchestrates critical functions in gene expression regulation and chromatin interaction. In this study, we show that conditional genetic deletion of Mof but not Kansl1, the essential component of the NSL complex, causes severe defects during murine skin development. Single-cell and bulk RNA-seq, in combination with MOF ChIP-seq, reveal that numerous MOF targeted and downregulated genes are highly enriched in mitochondria and cilia. Genetic deletion of Uqcrq, an essential subunit for electron transport chain Complex III, recapitulates the defects observed in MOF cKO. Single-cell ATAC-seq reveals that MOF targeted genes are controlled prominently through promoter interactions.

overall_design: scATAC-Seq

first: Dongmei

last: Wang

first: Haimin

last: Li

first: Navdeep

last: Chandel

first: Yali

last: Dou


title: MOF-mediated histone H4 lysine 16 acetylation governs mitochondrial and ciliary functions by controlling promoter interactions (RNA-Seq)

overall_design: Poly(A) enriched whole transcriptome RNAseq on epithelial cells isolated from E16.5 coontrol and MOF/K14Cre cKO mice or P0.5 control and Kansl1/K14Cre cKO mice.


title: Aging disrupts gene expression timing during muscle regeneration

city: Boulder

first: Jesse

middle: Vigoda

last: Kurland

state: Colorado

address: 1925 Colorado Avenue

department: MCDB

institute: University of Colorado Boulder

summary: Skeletal muscle function and regenerative capacity decline during aging, yet factors driving these changes are incompletely understood. Muscle regeneration requires temporally coordinated transcriptional programs to drive myogenic stem cells to activate, proliferate, fuse to form myofibers, and to mature myonuclei, restoring muscle function after injury. We assessed global changes in myogenic transcription programs distinguishing muscle regeneration in aged mice from young mice by comparing pseudotime trajectories from single-nucleus RNA sequencing of myogenic nuclei. Aging-specific differences in coordinating myogenic transcription programs necessary for restoring muscle function occur following muscle injury, likely contributing to compromised regeneration in aged mice. Differences in pseudotime alignment of myogenic nuclei when comparing aged to young mice via Dynamic Time-Warping revealed pseudotemporal differences becoming progressively more severe as regeneration proceeds. Disruptions in timing of myogenic gene expression programs may contribute to incomplete skeletal muscle regeneration and declines in muscle function as organisms age.

overall_design: 5x snRNA-seq libraries were prepared from injured and uninjured, aged and adult, murine TA muscle. Uninjured samples were done in replicate. 4 dpi and 7 dpi samples were done in singlets from aged and adult mice.

first: Bradley

last: Olwin

first: Alicia

last: Cutler


title: Next Generation Sequencing of ribosome-associated RNA from mouse skeletal muscle endothelial cells after 4 week treatment with vehicle or 17β-estradiol

city: Dallas

first: Philip

last: Shaul

state: TX

address: 5323 Harry Hines Blvd.

department: Pediatrics

institute: UT Southwestern

summary: The study evaluated the effects of estradiol treatment on gene translation in mouse skeletal muscle microvascular endothelial cells.  This was accomplished by isolating ribosomes via immunoprecipitation of a tagged ribosomal protein, followed by deep sequencing of the ribosome-associated RNA (in triplicate).  The sequence reads that passed quality filters were analyzed using GO analysis.

overall_design: Skeletal muscle microvascular endothelial cell ribosome-associated RNA profiles were generated on female mice treated with control or 17β-estradiol for 4 weeks

first: Anastasia

last: Sacharidou

middle: W


title: Increased hyaluronan by naked mole-rat HAS2 improves healthspan in mice [microbiome]

city: Rochester

first: Fathima

last: Athar

state: New York

address: 500 Joseph C Wilson Blvd.

department: Biology

institute: University of Rochester

summary: Abundant high molecular weight hyaluronic acid (HMW-HA) contributes to cancer resistance and possibly longevity of the longest-lived rodent, the naked mole-rat1,2. To study whether the benefits of increased HMW-HA could be transferred to other animal species, we generated a transgenic mouse overexpressing naked mole-rat hyaluronic acid synthase 2 gene (nmrHAS2). nmrHAS2 mice showed increase in hyaluronan levels in several tissues, and lower incidence of spontaneous and induced cancer, extended lifespan and improved healthspan. The transcriptome signature of nmrHAS2 mice shifted towards that of longer-lived species. The most striking change observed in nmrHAS2 mice was attenuated inflammation across multiple tissues. HMW-HA reduced inflammation via several pathways including direct immunoregulatory effect on immune cells, protection from oxidative stress, and improved gut barrier function during aging. These findings demonstrate that the longevity mechanism that evolved in the naked mole-rat can be exploited to other species, and open new avenues for using HMW-HA to improve lifespan and healthspan.

overall_design: The gut microbiome undergoes changes during aging and gut dysbiosis can further contribute to systemic inflammation. Since the old nmrHAS2 mice showed a heathier gut, we compared the microbial composition between 7- and 24-months old nmrHAS2 and control mice by sequencing 16S rDNA.

first: Zhihui

last: Zhang

first: Andrei

last: Seluanov

first: Vera

last: Gorbunova


title: Endometrial transcriptomic profiles in patients with severe IUA and normal control

city: Nanjing

first: Simin

last: Yao

address: 321 Zhongshan Road

institute: The Affiliated Drum Tower Hosipital, Medical School, Nanjing University

summary: Intrauterine adhesion (IUA) is characterized by endometrial fibrosis with partial or complete uterine cavity obliteration. IUA patients often undergo amenorrhea/ hypomenorrhea, abdominal pain, infertility, early pregnancy loss or severe placental complications. In this work, endometria samples were collected from severe IUA and control patients. RNA-seq was performed to identify differences between IUA and normal control and investigate the mechanisms of IUA development.

overall_design: Endometria samples from 7 severe IUA patients (Ba) and 7 normal controls (Con)

last: Hu


title: Metronomic administration of topotecan alone and in combination with docetaxel inhibits epithelial-mesenchymal transition in aggressive variant prostate cancers

city: AUBURN

first: Amit

middle: Kumar

last: Mitra

state: AL

address: HARRISON COLLEGE OF PHARMACY

department: DRUG DISCOVERY AND DEVELOPMENT

institute: AUBURN UNIVERSITY

summary: To identify molecular signatures and their potential to serve as novel biomarkers for monitoring treatment efficacy and disease progression response to treatment efficacy and disease progression

overall_design: Effects of METRO-TOPO and CONV-TOPO exposure for six weeks on ARLow mCRPC/NEPC PC-3 tumor model (3D spheroid) were assessed using RNAseq.

first: Taraswi

last: Mitra Ghosh

first: Robert

last: Arnold

middle: K


title: MOF-mediated histone H4 lysine 16 acetylation governs mitochondrial and ciliary functions by controlling promoter interactions (scRNA-Seq)

first: Rui

last: Yi

overall_design: scRNA-Seq

first: Dongmei

last: Wang

last: Li


title: Pharmacological YAP activation promotes regenerative repair of cutaneous wounds.

status: Public on Jun 11 2023

city: La Jolla

first: Edyta

last: Grzelak

address: 10550 N. Torrey Pines Road, SR202

department: Chemistry

institute: The Scripps Research Institute

summary: Chronic cutaneous wounds remain a persistent unmet medical need that decrease expectancy and quality of life. Here, we report that topical application of PY-60, a small molecule activator of the transcriptional coactivator YAP, promotes regenerative repair of cutaneous wounds in pig and human models. Pharmacological YAP activation enacts a reversible pro-proliferative transcriptional program in keratinocytes and dermal cells that results in accelerated re-epithelization and regranulation of the wound bed. These results demonstrate that transient topical administration of a YAP activating agent may represent a generalizable therapeutic approach to treating chronic wounds.

overall_design: Comparisons of the transcriptomes of primary human keratinocytes treated with PY-60 vs. DMSO.

first: Michael

last: Bollong


title: Effect of depletion of UBL5 on gene expression in hepatocellular carcinoma cell lines HepG2 and Huh7

city: Richmond

first: Xianjun

last: Fang

state: Virginia

address: 1101 E. Marshall Street, Sanger Hal, Sanger Hall, Room 2-016D

institute: Virginia Commonwealth university

summary:
To determine the effects of UBL5 on genome-wide gene expression, we used UBL5 siRNA to knock down UBL5 in HepG2 and Huh7 cells.
We then performed gene expression profiling analysis using data obtained from RNA-seq.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for Control siRNA and UBL5 siRNA KD in HepG2 and Huh7 cells.

first: Wei

first: Bei

first: Jinze

last: Liu


title: DNA methylation profiling to determine the primary sites of metastatic cancers using formalin-fixed paraffin-embedded tissues [wgbs_set]

city: Shanghai

first: Leiqin

address: 320 Yueyang Road

institute: CAS Center for Excellence in Molecular Cell Science (CEMCS)

summary: Accurate identification of the primary site of metastatic cancer is critical to guide the subsequent treatments. There is a significant portion of patients whose primary sites are initially classified as uncertain, and 3-9% of cancer patients are diagnosed with cancer of unknown primary (CUP) even after comprehensive diagnostic workups. Yet, a widely accepted molecular test is still not available. Here, we presented the combination of a novel DNA methylation sequencing-based method and an algorithm to predict the tissues of origin for metastatic cancers. The assay applied degraded DNA from formalin-fixed, paraffin-embedded (FFPE) tissues to generate reduced represent bisulfite sequencing libraries (FFPE-RRBS). Comparable DNA methylation metrics were obtained for the paired fresh frozen (FF) RRBS and FFPE-RRBS libraries and the FFPE-RRBS libraries of matched primary and metastatic cancer tissues. We generated and systemically evaluated 28 molecular classifiers built on four methylation evaluation methods and seven machine-learning approaches from a training data set of 498 primary cancer patients. Of those classifiers, the beta values-based (mean methylation) linear support vector (BELIVE) performed the best, achieving overall accuracies of 81-95% for identifying the primary sites of 215 metastatic cancer patients by utilizing the top-k predictions (k=1, 2, 3). The prediction accuracies ranged from 92% to 98% for 4702 patients with primary tumors in a cross-validation cohort. Lastly, BELIVE successfully identified the tissues of origin for approximately 81-93% of cases in a cohort of 68 patients initially diagnosed with CUP.

overall_design:
We sequenced RRBS libraries from 503 samples of primary tumor, 218 samples of metastatic tumor, and 68 samples of CUP patients.The assay was conducted using genomic DNA purified either from 10-20 mg of FF tissues or five to eight 5-10 µm FFPE tissue sections. Board-certified pathologists checked one H&E stained slide to ensure that tumor cells accounted for 10% or more cell population and that the necrosis area was less than 50%.
NOTE FROM SUBMITTER: Raw data (FASTQ files) are controlled to protect patient privacy.

first: Shutao

last: He


title: Human femoral atheroma exhibit inflammation-resolving myeloid and lymphoid bias compared with carotid atheroma

first: Matthew

last: Feinstein

address: 303 E Chicago Ave, Tarry 12-723

summary: Pathologic and clinical investigations suggest that femoral artery plaques are less inflammatory than other vascular beds, including carotid arteries. However, limited data exist regarding comparative immune landscapes and inflammatory polarization that may underlie such differences in femoral versus carotid plaque.

overall_design:
Femoral or carotid artery plaques obtained from endarterectomy of patients at Northwestern Medicine were processed into single-cell suspensions and underwent single-cell ribonucleic acid sequencing (scRNA-seq). ScRNA-seq was performed on a total of 65,920 leukocytes from 13 individuals undergoing femoral (N=9; 35265 CD45+ cells) or carotid (N=4; 30655 cells) endarterectomy. For scRNA-seq analyses, suspensions were then enriched for live cells and CD45+ cells. Dead cells were removed with a dead cell removal kit (Miltenyi Biotec 130-090-101 or Stem Cell Technologies 17899) according to manufacturer’s instructions. Suspensions were then enriched for immune cells with CD45 positive selection using a CD45+ enrichment kit (Miltenyi, 130-045-801) and cells were counted. The CD45+ enriched single cell suspensions of plaque were converted to barcoded scRNA-seq libraries using the Chromium Single Cell 3ʹ Library, Gel Bead, and Chip Kit from 10X Genomics. The CD45+ enriched single cell suspensions of plaque were converted to barcoded scRNA-seq libraries using the Chromium Single Cell 3ʹ Library, Gel Bead, and Chip G from 10X Genomics. The Chromium Single Cell 3’ V3.1 Reagent (10x Genomics, PN-1000286) kit was used to prepare scRNA-seq libraries. Reverse transcription, barcoding, complementary DNA amplification and purification for library preparation were performed according to the manufacturer’s instructions. Sequencing was performed on a NovaSeq 6000 platform with Read 1 of 28nt and Read 2 of 90bp (Illumina). Sequencing reads were demultiplexed and aligned to the human GRCh38 transcriptome using the CellRanger V3 software (10X Genomics). Filtering, unsupervised clustering, differential expression and additional analysis were completed using Seurat V4 and ClusterProfiler packages for R. To remain consistent with the institutionally approved protocol and due to identifiability concerns, data are shared here down to the level of gene counts tables/matrices.
*** Raw data is not included. Data are provided down to the level of de-identified gene lists and counts tables to be compliant with the institutionally approved human subjects protocol used to collect the samples used in this study. ***

first: Joshua

last: Slysz

first: Arjun

last: Sinha

last: DeBerge

first: Shalini

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