title: Disturbed methylation at multiple imprinted loci in iPSCs derived from ICF1 patients during reprogramming

city: Naples

first: Ankit

last: Verma

state: Naples

address: Via Pietro Castellino, 111

country: Italy

institute: Institute of Genetics and Biophysics "A. Buzzati Traverso" (IGB-ABT)

type: Methylation profiling by genome tiling array

summary: DNA methylation perturbations have been a hallmark of Immunodeficiency, Centromeric instability, Facial anomalies type I (ICF1) syndrome. The early developmental abnormalities due to DNMT3B deficiency accounts for genome-wide methylation defects that have been addressed frequently in many studies. Biallelic hypomorphic DNMT3B mutations cause the ICF1 syndrome. We have investigated the role of DNMT3B in mediating methylation at imprinted loci by employing induced pluripotent stem cells (iPSC) lines derived from ICF1 patients and their derivative strains in which the DNMT3B mutations were corrected by CRISPR/Cas9 technology. For ICF1 syndrome, skin fibroblasts derived DNA were also included for comparison purposes. By employing a whole-genome array-based approach, we observed a strong hypomethylation at a subset of gDMRs and sDMRs. However, these did not fully recover methylation despite most of the other hypomethylated CpGs in the genome reverted to their epigenetic state in the corrected iPSC clones. Loss of imprinted methylation in the ICF iPSCs was accompanied by increased levels of histone H3K4 trimethylation and in some cases gene activation, which were only partially reversed upon correction. Overall our study provides a comprehensive analysis of the role of DNMT3B activity in genomic imprinting maintenance in human somatic cells and indicates a molecular mechanism explaining why imprinting defects are resistant to correction.

overall_design: DNA samples extracted from iPSCs were subjected to methylation analysis using Infinium MethylationEPIC 850K Bead Chip Array. We assessed the methylation levels at both genome-wide and imprinting control regions.

first: Varsha

last: Poondi Krishnan

first: Francesco

last: Cecere

first: Claudia

last: Angelini

first: Maria

last: Matarazzo

first: Andrea

last: Riccio


title: Fibroblasts-derived from Pluripotent Cells Harboring a Single Allele Knockout in Two Pluripotency Genes Exhibit DNA Methylation Abnormalities and Reprogramming Defects [BiSulfite-seq]

status: Public on Nov 27 2023

city: Jerusalem

first: Yosef

last: Buganim

address: Hadassah Medical School

department: Department of Developmental Biology and Cancer Research

country: Israel

institute: The Hebrew University

type: Methylation profiling by high throughput sequencing

summary: SGFP1 S2+/- and SGFP1 S2+/-;S4+/- MEFs show different methylation profiles and expose methylated genes specific to the SGFP1  S2+/-;S4+/- MEFs that are important for reprograming

overall_design: SGFP1 S2+/- and SGFP1 S2+/-;S4+/- MEFs were submitted to whole genome bisulfite sequencing.

first: Rachel

last: Lasry

first: Ofrah

last: Sabag

first: Ahmed

last: Radwan


title: Fibroblasts-derived from Pluripotent Cells Harboring a Single Allele Knockout in Two Pluripotency Genes Exhibit DNA Methylation Abnormalities and Reprogramming Defects

type: Expression profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Differential Gene Expression During Floral Transition in Pineapple

status: Public on Nov 22 2023

city: Kunming

first: Yun

last: Zheng

state: Yunnan

address: 452 Fengyuan Road

country: China

institute: Yunnan Agricultural University

type: Non-coding RNA profiling by high throughput sequencing

summary: Pineapple (Ananas comosus var. comosus) and ornamental bromeliads are commercially induced to flower by treatment with ethylene or its analogs. The apex is transformed from a vegetative to a floral meristem and shows morphological changes in 8 to 10 days, with flowers developing 8 to 10 weeks later. During eight sampling stages ranging from 6 hours to 8 days after treatment, 7,961 genes were found to exhibit differential expression (DEG) after the application of ethylene. In the first 3 days after treatment, there was little change in ethylene synthesis or in the early stages of the ethylene response. Subsequently, three ethylene response transcription factors (ERTF) were up-regulated and the potential gene targets were predicted to be the positive flowering regulator CONSTANS (CO), a WUSCHEL gene, two APETALA1/FRUTFULL (AP1/FUL) genes, an epidermal patterning gene and a jasmonic acid synthesis gene. We confirm that pineapple has lost the flowering repressor FLOWERING LOCUS C. At the initial stages, the SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) was not significantly involved in this transition. Another WUSCHEL gene and a PHD homeobox transcription factor, though not apparent direct targets of ERTF, were up-regulated within a day of treatment, their predicted targets being the up-regulated CO, auxin response factors, SQUAMOSA, and histone H3 genes with suppression of abscisic acid response genes. The FLOWERING LOCUS T (FT), TERMINAL FLOWER (TFL), AGAMOUS-like APETELAR (AP2) and SEPETALA (SEP) increased rapidly within 2 to 3 days after ethylene treatment. Two FT genes were up-regulated in the apex and not the leaf bases after treatment, suggesting that transport did not occur. These results indicated that the ethylene response in pineapple and possibly most bromeliads acts directly to promote the vegetative to flower transition via APETALA1/FRUITFULL (AP1/FUL) and its interaction with SPL, FT, TFL, SEP and AP2.

overall_design: A uniform plot with pineapple plants weighing 1.5 to 2 kg was selected in a commercial field (Cultivar 'MD1', PRI 73-50) at Dole Plantation, Wahiawa, Hawaii (21° 31’ 52.6 N; -158° 03’ 35.3 W) 6 weeks before commercial forcing. The plot was divided into three replications with two treatments (control and treated) and ~50 plants in each treatment block. Water (10 ml) as the control treatment or ethephon (50 mg ai in 10 mL) (Ethrel, Rhone-Poulenc, AG Company, North Carolina) as the flower induction treatment were injected into the center of the plant between 7:00 and 7:20 AM. At 8:30 to 8:45 am, three plants were harvested from each replication in the water control and ethephon-treated plants at each sampling time after treatment. Four grams of the "D" leaf base (most recently matured leaf) and the other leaves of the uprooted plants were trimmed in the field from the cut stem. The trimmed leaf bases and trimmed stem were chilled and immediately returned to the laboratory on ice at 10 AM for further processing. The bases of the trimmed leaves were removed from the apex of the stem to expose the stem apex, and <1 g of the apex was taken from each stem and frozen in liquid nitrogen. This initial sampling was completed by 2:00 PM and reported here as 6 hours after ethephon or water control treatment. Subsequent apex and ‘D’ leaf samples were taken 1, 2, 3, 4, 5, 6 and 8 days after treatment between 8:00 and 8:30 AM and processed by noon in the laboratory. Leaf bases were included as a control to allow comparison with non-apex tissue, since leaf primordia remained on the excised apex. All samples were stored at -80 ° C until ground into powder in liquid nitrogen for RNA extraction. Total RNA was extracted from the apex and leaf bases using the Qiagen RNeasy Plant Mini Kit (Qiagen, #74904) following the manufacturer's protocol. DNA was removed with the DNA-free DNA Removal Kit (Life Technologies, #AM1906M). Three biological replicates were sequenced for each sampling stage with three apices or leaf bases in each replication. Total RNA (2 µg) was used for the preparation of the mRNA-Seq library using the TruSeq Stranded mRNA LT kit (Illumina, USA) according to the manufacturer’s protocol. The size of the RNA-Seq library was evaluated by electrophoresis (1 μl of sample + 1 μl of loading dye 6X, 1% Agarose, TBE 1X Buffer, 30 minutes, 60V) and 1 μl of sample was used for quantification with a Qubit Fluorometer (Invitrogen, USA) using the DNA HS assay kit. The multiplexed pooled libraries were sequenced on Illumina HiSeq4000 with a 50 nt read length. Small RNA libraries were prepared from total RNA using the NEBNext Multiplex Small RNA Sample Kit for Illumina (E7300, NEB, Ipswich, MA) following the manufacturer’s protocol. The libraries were quantified by qPCR and sequenced in each lane with a read length of 50 nt.

first: Robert

middle: E

last: Paul


title: Partial in vivo reprogramming enables injury-free intestinal regeneration via autonomous Ptgs1 induction [RNAseq_IR]

status: Public on Nov 26 2023

city: Pohang

first: Somi

last: Kim

address: 47, Jigok-ro

department: Life Sciences

country: South Korea

institute: POSTECH

summary: Tissue regeneration after injury involves the dedifferentiation of somatic cells, a natural adaptive reprogramming that leads to the emergence of injury-responsive cells with fetal-like characteristics. However, there is no direct evidence that adaptive reprogramming involves a shared molecular mechanism with direct cellular reprogramming. Here, we induced dedifferentiation of intestinal epithelial cells using OSKM (Oct4, Sox2, Klf4, and c-Myc) in vivo. The OSKM-induced forced dedifferentiation showed similar molecular features of intestinal regeneration, including a transition from homeostatic cell types to injury-responsive-like cell types. These injury-responsive-like cells, sharing gene signatures of revival stem cells and atrophy-induced villus epithelial cells, actively assisted tissue regeneration following damage. In contrast to normal intestinal regeneration involving Ptgs2 induction, the OSKM promotes autonomous production of prostaglandin E2 via epithelial Ptgs1 expression. These results indicate prostaglandin synthesis is a common mechanism for intestinal regeneration, but involves a different enzyme when partial reprogramming is applied to the intestinal epithelium.

overall_design: We used intestinal organoids and irradiated the IR samples.

first: Jumee

first: Seung-Yeon

last: Lee

first: Hwa-Ryeon

first: Bon-Kyoung

last: Koo

first: Jong

middle: K

first: Hyuk-Jin

last: Cha


title: OSKM-induced mouse intestinal organoids [RNAseq_Dox]

summary: Tissue regeneration after injury is thought to involve the dedifferentiation of somatic cells, which is often understood as evidence for natural adaptative reprogramming in vivo. In the intestinal epithelium, acute tissue damage triggers the rapid emergence of injury-responsive cells with fetal-like characteristics, a sign of dedifferentiation. However, there is no direct evidence that tissue regeneration involves a shared molecular mechanism with direct cellular reprogramming. It is also not known whether dedifferentiation shares a single (de)differentiation pathway or consists of multiple parallel routes between adult and fetal states. Here, we induced dedifferentiation of intestinal epithelial cells by forced partial reprogramming in vivo using the “Yamanaka factors” (Oct4, Sox2, Klf4 and c-Myc: OSKM). The induced dedifferentiation showed shared molecular features of intestinal regeneration, with rapid transition from homeostatic intestinal cell types to injury-responsive-like cells sharing a common gene signature of revival stem cells and atrophy-induced villus epithelial cells. When applied to intestinal organoids, induced dedifferentiation allowed the organoids to adopt fetal characteristics ex vivo, suggesting a direct effect on the intestinal epithelium. In vivo, induced dedifferentiation promoted regeneration from acute tissue injury caused by ionizing radiation (IR) via the formation of injury-responsive-like cells. Taken together, in the intestinal epithelium, in vivo reprogramming shares the same molecular pathway as damage-induced tissue regeneration and facilitates the repair process.

overall_design: We used intestinal organoids from doxycycline(Dox)-inducible OSKM-expressing mice. Two different concentration of Dox was treated in the organoids to induce OSKM to different degrees. Two biological replicates were used.


title: Gene expression profiling of murine macrophages in response to infection with Cryptococcus neoformans yeast cells at distinct physiological states, including the dormant state known as viable but non-culturable (VBNC)

status: Public on Nov 23 2023

city: Paris

first: Hugo

last: Varet

address: 28 rue du Docteur Roux

country: France

institute: Institut Pasteur

summary: Macrophages play a pivotal role in determining the fate of invading Cryptococcus neoformans cells, and this outcome is highly dependent on how the pathogen modulates phagocyte activation and function. A common consequence of the C. neoformans-host interaction is the establishment of a latent infection, in which the yeast enters a dormant state. In this study, we investigated whether dormant cells could modulate murine bone marrow-derived macrophages (BMDMs) differently from non-dormant cells, thereby providing mechanistic insights into latent infection. To accomplish this, we examined the transcriptional responses of BMDMs upon in vitro infection with distinct physiological states of C. neoformans – exponential phase, stationary phase, or viable but non-culturable (VBNC; equivalent of dormant) cells – over a period of 6 hours. Our findings unveiled a relationship between the physiological state of the yeast and its impact on the transcriptome of BMDMs. Compared to non-dormant cells, particularly those in the logarithm phase, VBNC cells trigger a modest disruption in the host transcriptome, regulating a reduced number of genes, which clustered in far fewer Gene Ontology terms, many of which were unique to this specific condition. Nevertheless, VBNC cells retained the ability to regulate genes that are important for immune response, such as NLRP3 inflammasome-related genes. Our results suggest that VBNC cells retain the low immunostimulatory profile that is critical for the persistence of this fungal pathogen within the host.

overall_design: Comparative gene expression profiling analysis of mouse bone marrow-derived macrophages cultured alone (uninfected control; Uni) or co-cultured with logarithm-phase (E-Cn), stationary-phase (S-Cn), or dormant yeast cells (viable but non-culturable cells; D-Cn) of Cryptococcus neoformansstrain H99O.

first: Raffael

middle: J

last: de Castro

first: Aude

last: Sturny-Leclère

first: Christian

last: Hoffmann

first: Anamélia

middle: L

last: Bocca

first: Alexandre

last: Alanio


title: SEN1990 encodes a transcriptional regulator from Salmonella enterica serovar Enteritidis that modulates the expression of the SPI-17 encoded gene oafB

city: Santiago

first: Susan

last: Bueno

state: Región Metropolitana

address: Avda. Libertador Bernardo O’Higgins #340

department: Departamento de Genetica Molecular y Microbiología

country: Chile

institute: Pontificia Universidad Católica de Chile

summary: Excisable Genomic Islands (EGIs) are horizontally acquired genetic elements that harbor an array of genes with diverse functions. ROD21 is an EGI found integrated in the chromosome of Salmonella enterica serovar Enteritidis (Salmonella ser. Enteritidis).  While this island is known to be involved in the capacity of Salmonella ser. Enteritidis to cross the epithelial barrier and colonize sterile organs, the role for most of ROD21 genes remains unknown, and thus identification of their function is fundamental to understand the impact of this EGI on the bacterium pathogenicity. Therefore, in this work we used a bioinformatical approach to evaluate the function of ROD21-encoded genes and delve into the characterization of SEN1990, a gene encoding a putative DNA-binding protein. We characterized the predicted structure of SEN1990, finding that this protein contains a three-stranded winged helix-turn-helix (wHTH) DNA-binding domain. Additionally, we identified homologs of SEN1990 among other members of the EARL EGIs. Furthermore, we deleted SEN1990 in Salmonella ser. Enteritidis, finding no differences in the replication/maintenance of the excised ROD21, disconfirming the previous Refseq annotation of the protein. High-throughput RNA-sequencing was carried out to evaluate the effect of the SEN1990 absence on the bacterium global transcription. We found a downregulated expression of oafB, an SPI-17-encoded acetyltransferase involved in O-antigen modification, which was restored when the deletion of SEN1990 was complemented. Our findings suggests that SEN1990 encodes a wHTH domain-containing transcriptional activator that modulates the transcription of oafB from the SPI-17, suggesting a crosstalk between these pathogenicity islands and a possible new role of ROD21 in the pathogenesis of Salmonella ser. Enteritidis.

sample_organism: Salmonella enterica subsp. enterica serovar Enteritidis

overall_design: Salmonella ser. Enteritidis P125109 Wild-type and ΔSEN1990::frt strains were cultured overnight at 37°C in LB broth with agitation, and diluted in LB to OD600 0.01. The cultures were incubated at 37°C with agitation until OD600=0.6, at which samples were centrifugated at 8,000g for 8 min, the supernatant was discarded, and RNA was isolated using TRizol. Samples were further purified from salts using drop dialysis in MF-Millipore™ membranes (0.025µm pore diameter) against nuclease-free water for 4 h on ice. RNA-seq was performed as follows: Libraries were prepared using the Zymo-Seq RiboFree Total RNA Library Prep Kit and sequenced on an Illumina NovaSeq platform to a sequencing depth of at least 30 million read pairs (150 bp paired-end sequencing) per sample.

first: Guillermo

last: Hoppe-Elsholz

first: Alejandro

last: Piña-Iturbe

first: Alexis

middle: M

last: Kalergis


title: Differential gene expression during floral transition in pineapple.

city: Urbana

first: Dessiree

middle: Patricia

last: Zerpa Catanho

state: Illinois

address: 1201 W. Gregory Dr, 279 ERML

country: USA

institute: University of Illinois at Urbana-Champaign

overall_design: In this study, differential gene expression in Ananas comosus (cultivar 'MD2', PRI 73-50) was analyzed after ethylene treatment. Two different tissues ("D" leaf and stem apex) were collected at different times after ethylene treatment (1, 2, 3, 4, 5, 6 and 8 days after treatment) from treated and untreated "control" plants. Three replicates were sequenced.

first: Robert

last: Paull

first: Ray

last: Ming

first: Jennifer

last: Wai

first: Dessireé

last: Zerpa-Catanho


title: Lactobacillus reverses cesarean section-induced colitis susceptibility in offspring: A role in maintaining ILC3 homeostasis

city: Jinan

first: Yanan

last: Xia

state: Shandong Province

address: Wen Hua Xi Road

institute: Qilu Hospital of Shandong University

summary: The impact of microbial colonization during early life on immune system development and host health is well-established. Therefore,  we investigated whether alterations in the intestinal microbiota resulting from cesarean section (CS) would affect the colonic immune system.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for colons from adult ICR mice delivered vaginally or by CS. Both vaginal delivery (VD) and CS mice were treated with 3% DSS or normal water continuously for 8 days.

first: Yanqing

last: Li

first: Xiuli

last: Zuo


title: Integrated small RNA, mRNA and protein omics reveal a function of microRNAs orchestrating metabolic maturation of the developing human heart

city: Toronto

first: Paul

last: Delgado Olguín

state: Ontario

address: 686 Bay Street

country: Canada

institute: The Hospital for Sick Children


title: SF3B1 inhibitor pladienolide B induces pyroptosis of ovarian cancer cells

status: Public on Nov 25 2023

first: shourong

last: wang

address: No. 107, Wenhua West Road, Lixia District, Jinan City, Shandong Province

institute: Qilu Hospital，Shandong University

summary: In order to explore the effect of SF3B1 inhibitor, pladienolide B, on ovarian cancer cells, OVCAR8 cells were treated with DMSO or 2nM pladienolide B for 72h. Cells were collected and performed RNA sequencing.

overall_design: OVCAR8 cells were treated with DMSO or 2nM pladienolide B for 72h.Each group contains three replicates.

first: Shourong

last: Wang


title: Selective compartmentalization in condensin-depleted mitotic chromosomes [Hi-C]

city: Shenzhen

first: Haoyue

last: Zhang

address: Gaoke Innovation Center, Guangqiao Road, Guangming District, Shenzhen

institute: Shenzhen Bay Lab

summary: During mitosis, condensin activity is thought to disrupt interphase chromatin structures. Here, we utilize condensin-deficient mitotic chromosomes as a unique architectural platform to further investigate genome folding principles. Upon condensin loss, compartments progressively emerge in mitotic chromosomes. Euchromatin diverges into two different compartments A1 and A2, the former of which shows strong homotypic interactions while the latter exhibit reduced self-aggregation. Constitutive heterochromatin (B1) displays reduced level of compartmentalization and the normally inert facultative heterochromatin (B2) participates to compartmentalize the genome. Dynamically, A1 compartment is established remarkably fast with similarly efficient separation from B1 while reformation of B1 is delayed, implying that A1 self-attraction is the engine to compartmentalizalize the condensin-depleted mitotic chromosomes. Notified by the mitotic compartmentalization of B1 which lacks HP1 binding, we sought to explore the role of HP1 proteins in genome folding and demonstrat that HP1& are dispensible for chromatin structural restoration during cell divison. Our observations unveil delicate patterns and novel principles of genome compartmentalization that are otherwise hidden in interphase cells.

overall_design: G1E-ER4:SMC3-mAID-mCherry cells were treated with or without auxin (1mM) for 4 hours.

first: Fuhai

last: Liu

first: Gerd

last: Blobel


title: Human vascular smooth muscle cells display different gene expression patterns and response to EGF

city: Halle (Saale)

first: Virginie

last: Dubourg

address: Magdeburger Str. 6

department: Julius Bernstein Institute for Physiology

country: Germany

institute: Martin Luther University - Halle-Wittenberg

summary: RNAsequencing was performed for human primary aortic (HAoSMC) and cornary artery (HCASMC) smooth muscle cells from female donors, after incubation with or without EGF

overall_design: HAoSMC and HCASMC were incubated for 48h with 10 µg/L EGF (N=10-12)

first: Michael

last: Gekle

first: Gerald

last: Schwerdt

first: Barbara

last: Schreier

last: Kopf

first: Sigrid

last: Mildenberger

first: Ralf

middle: A

last: Benndorf


title: Selective compartmentalization in condensin-depleted mitotic chromosomes [ChIP-seq]

type: Genome binding/occupancy profiling by high throughput sequencing

overall_design: G1E-ER4:SMC2-mAID-mCherry cells were arrested to mitosis with nocodazole (200ng/ml) for 12 hours. Asynchronous cells were processed as controls


title: Partial in vivo reprogramming enables injury-free intestinal regeneration via autonomous Ptgs1 induction.

status: Public on Nov 24 2023

first: Somi

last: Kim


title: Integrated small RNA, mRNA and protein omics reveal a function of microRNAs orchestrating metabolic maturation of the developing human heart [smallRNAseq]

summary:
Background
As the fetal heart develops, cardiomyocyte proliferation potential decreases while fatty acid oxidative capacity increases, a highly regulated transition known as cardiac maturation. Small noncoding RNAs, such as microRNAs (miRNAs), contribute to the establishment and control of tissue-specific transcriptional programs. However, small RNA expression dynamics and genome wide miRNA regulatory networks controlling maturation of the human fetal heart remain poorly understood.
Results
Transcriptome profiling of small RNAs revealed the temporal expression patterns of miRNA, piRNA, circRNA, snoRNA, snRNA and tRNA in the developing human heart between 8 and 19 weeks of gestation. Our analysis revealed that miRNAs were the most dynamically expressed small RNA species throughout mid-gestation. Crossreferencing differentially expressed miRNAs and mRNAs predicted 6,200 mRNA targets, 2134 of which were upregulated and 4066 downregulated as gestation progresses. Moreover, we found that downregulated targets of upregulated miRNAs predominantly control cell cycle progression, while upregulated targets of downregulated miRNAs are linked to energy sensing and oxidative metabolism. Furthermore, integration of miRNA and mRNA profiles with proteomes and reporter metabolites revealed that proteins encoded in mRNA targets, and their associated metabolites, mediate fatty acid oxidation and are enriched as the heart develops.
Conclusions
This study revealed the small RNAome of the maturing human fetal heart. Furthermore, our findings suggest that coordinated activation and repression of miRNA expression throughout mid-gestation is essential to establish a dynamic miRNAmRNA-protein network that decreases  cardiomyocyte proliferation potential while increasing the oxidative capacity of the maturing human fetal heart.

overall_design: Transcriptome profiling of small RNAs (miRNA, piRNA, circRNA, snoRNA, snRNA, tRNA) using small RNA-seq for human fetal heart between 8 and 19 weeks of gestation.

first: Adar

last: Aharon-Yariv

first: Yaxu

first: Abdalla

last: Ahmed


title: Integrated small RNA, mRNA and protein omics reveal a function of microRNAs orchestrating metabolic maturation of the developing human heart [RNAseq]

overall_design: RNA-seq profiling of human fetal heart between 8 and 19 weeks of gestation.


title: Selective compartmentalization in condensin-depleted mitotic chromosomes [ChIP-seq II]


title: Transgene-free haematopoietic stem and progenitor cells from human induced pluripotent stem cells

first: Laurence

last: Guyonneau-Harmand

address: 27 rue Chaligny

institute: Etablissement Français du Sang

summary: Despite considerable efforts, the generation of vector-free hematopoietic stem cells (HSCs) from human induced pluripotent stem cells (hiPSCs) is not yet possible, preventing the development of cell therapies to cure hematologic diseases. Here, we report the generation of HSCs with multilineage and self-renewal potential from hiPSCs using a specific one-step procedure over a culture period of 17 days. After injection into immunocompromised mice, cells derived from hiPSCs settle in the bone marrow and form a robust multicellular hematopoietic population that can be serially transplanted. RNA sequencing of individual cells suggests that this repopulating activity likely relies on a hematopoietic population transcriptionally similar to HSCs derived from human embryonic aorta. Overall, our results should enable the generation of HSCs from hiPSC and will be instrumental to identify important regulators of HSC production during human ontogeny.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Embroid bodies (EB) derived from hiPS and cultivated until the chosen timepoints were dissociated in single cells and analysed by scRNAseq using the Chromium 10X v2 technology. Mouse bone marrow 20 weeks after engraftment of the EB cells was flushed and analysed  by scRNAseq using the Chromium 10X v3.1 technology

first: Olivier

last: Piau

first: Thierry

last: Jaffredo


title: Selective compartmentalization in condensin-depleted mitotic chromosomes [HiC-C]

overall_design: G1E-ER4:SMC2-mAID-mCherry cells were first arrested to prometaphase with nocodazole (200ng/ml) for 12 hours or 15 hours (for 8h release condition). Toward the end of mitotic arrest, cells were treated with auxin (1mM) for 0h (SMC2+), 0.5 hours, 1 hour, 4 hours or 8 hours respectively.


title: Human iPSCs are differentiated into the cell types constituting the BBB

city: Seoul

first: Haeryung

last: Lee

state: Seoul

address: Chungpa-dong, Yongsan-Gu, Yongsan-Gu

institute: Sookmyung Women's University

summary: We used a well-established protocol for each cell type for differentiating iPSCs into brain endothelial cells (iECs), pericytes (iPCs), and astrocytes (iASs), and then validated the identity of each differentiated cell using RNA sequencing.

overall_design: Comparative expression analysis of the differentially upregulated genes in the respective cells.

first: Jiyeon


title: Selective compartmentalization in condensin-depleted mitotic chromosomes

first: Haoyue

last: Zhang

overall_design: Refer to individual Series


title: Selective compartmentalization in condensin-depleted mitotic chromosomes [Hi-C II]

first: Fuhai

last: Liu

first: Gerd


title: Sequencing-based Functional Assays for Classification of BRCA2 Variants in Mouse ES Cells

status: Public on Nov 20 2023

city: Frederick

first: Shyam

last: Sharan

state: MD

address: Building 560, Room 32-33

institute: Mouse Cancer Genetics Program

type: Genome variation profiling by high throughput sequencing

summary: Sequencing of genes, such as BRCA1 and BRCA2, is recommended for individuals with a personal or family history of early onset and/or bilateral breast and/or ovarian cancer, or a history of male breast cancer. Such sequencing efforts have resulted in the identification of more than 6000 BRCA2 variants. The functional significance of most variants remains unknown; consequently, they are called variants of uncertain clinical significance (VUS). We have previously developed mouse embryonic stem cell (mESC)-based assays for functional classification of BRCA2 variants. While the assays are reliable, the experimental process is time-consuming. This has impeded the number of variants that can be examined at a time. To overcome this, we have now developed a next-generation sequencing (NGS)-based approach for functional evaluation of BRCA2 variants. In this approach, instead of using clonogenic or XTT-based cell survival assays, pools of mESC expressing 25-30 BRCA2 variants from a given exon are analyzed by NGS. We have also developed a statistical model that utilizes the NGS data to determine the probability of impact on function (PIF) for these variants. While CRISPR/Cas9-based high throughput methods have resulted in the functional characterization of thousands of variants, only a fraction of the variants are included in ClinVar. In contrast, our targeted approach, which involves generating specific variants, resulted in the functional evaluation of 223 variants that are all listed in ClinVar. Our functional classification of BRCA2 variants is concordant with the classification reported in ClinVar or reported by other well-established assays.

overall_design: Saturation genome editing (SGE) coupled with drug sensitivity assays of BRCA2

first: Sahu

last: Sounak

first: Biswas

last: Kajal


title: PTPMT1 is critical for both expansion and differentiation of activated CD8 T cells

status: Public on Nov 21 2023

city: Atlanta

first: Chao

last: Chen

state: GA

address: 1760 Haygood Drive NE,HSRB E362

department: Pediatrics

institute: Emory University

summary: To understand the function of PTPMT1 in CD8 T cells responses, we characterized the transcriptomes of activated CD8+ T cells from PTPMT1 wildtype and knockout mice

overall_design: CD8 T cells from PTPMT1 wildtype and knockout mice were isolated from spleen and lymph nodes. The purified CD8 T cells were activated by CD3/CD28 antibodies-coupled Dynabeads for 3 days in vitro, then the activated CD8 T cells were used for comparative gene expression profiling analysis by RNA-seq.

first: Cheng-kui

last: Qu


title: Comparative single-cell transcriptomic atlases reveal conserved and divergent features of drosophilid central brains

city: Suwon

first: Daehan

state: Gyeonggi

address: 2066 Seobu-ro

institute: Sungkyunkwan University

summary: To explore how brains change upon species evolution, we generated the first whole central brain comparative single-cell transcriptomic atlases of three closely-related but ecologically-distinct drosophilids: D. melanogaster, D. simulans and D. sechellia. D. melanogaster and D. simulans are cosmopolitan generalists, while the island endemic D. sechellia exhibits extreme niche specialism on the ripe noni fruit of the Morinda citrifolia shrub. The global cellular composition of central brains is well-conserved in the three Drosophila species, but we predicted a few cell types (perineurial glia, sNPF and Dh44 neurons) with divergent frequencies. Gene expression analysis revealed that distinct cell types within the central brain evolve at different rates and patterns; notably, several glial cell types exhibit the greatest divergence between species. Compared to D. melanogaster, the cellular composition and gene expression patterns of the central brain in D. sechellia displays greater deviation than those of D. simulans, indicating that the distinctive ecological specialization of D. sechellia is reflected in the structure and function of its brain. Gene expression changes in D. sechellia encompass metabolic and ecdysone signaling genes, indicative of adaptations to its novel ecological demands. Additional single-cell transcriptomic analysis on D. sechellia revealed genes and cell types responsive to noni juice supplementation, showing glial cells as key sites for both physiological and genetic adaptation to novel conditions. Our comparative transcriptomic atlases of drosophilid brains will provide an entry point to more broadly study the evolvability of nervous systems across and beyond the Drosophila genus.

sample_organism:
Drosophila melanogaster
Drosophila sechellia
Drosophila simulans

overall_design: Nuclei of Drosophila central brains were isolated using homogenization and FACS sorting and were analyzed by single-nucleus RNA-sequencing.

first: Lee

last: Daehan

first: Benton

last: Richard


title: Selective compartmentalization in condensin-depleted mitotic chromosomes [Hi-C I]

overall_design: G1E-ER4:HP1a-AID-mCherry/HP1b-FKBPF36V-GFP cells were arrested to prometaphase through nocodazole treatment for 8h. During the last 4 hours of nocodazole treatment, cells were treated with one of the following combinations of drugs: (i) 5-Ph-IAA(-)/dTag13(-); (ii) 5-Ph-IAA(+)/dTag13(-); (iii) 5-Ph-IAA(-)/dTag13(+) and (iv) 5-Ph-IAA(+)/dTag13(+). Cells were then allowed to be released from nocodazole and proceed toward the next cell cycle. After 1h and 6h of nocodazole release, early-G1 and late-G1 cells under different HP1 protein configurations were collected. Note, that drug treatment was maintained during the course of nocodazole release.

last: Blobel


title: Exercise-induced Crosstalk between Immune Cells and Adipocytes in Humans: Role of Oncostatin-M

city: Stockholm

first: Leonidas

middle: S

last: Lundell

state: Stockholm

address: Von Eulers Väg 4a

department: Physiology and Pharmacology

country: Sweden

institute: Karolinska Institute

summary: The discovery of novel exercise-regulated circulatory factors has fueled interest in the role of organ-crosstalk, especially between skeletal muscle and adipose tissue in mediating beneficial effects of exercise. We studied the adipose tissue transcriptome in men and women with normal glucose tolerance or type 2 diabetes following an acute exercise bout, revealing substantial exercise and time-dependent changes, and a sustained increase in inflammatory genes unique to Type 2 diabetes. We identify Oncostatin-M as one of the most upregulated adipose tissue secreted factors post exercise. In cultured human adipocytes, oncostatin-M enhanced MAPK signalling and regulates lipolysis. Oncostatin-M expression predominantly arises from adipose tissue immune cell fractions, while the corresponding receptors are expressed in adipocytes. Oncostatin-M expression is induced in cultured human Thp1 macrophages in response to exercise-like stimuli. Our results suggest immune cells, via secreted factors such as oncostatin-M, are important in crosstalk between skeletal muscle and adipose tissue during exercise, to regulate adipocyte metabolism and adaptation.

overall_design: Adipose tissue RNAseq from human participants with or without type 2 diabetes, before, immediately after, and 3 hours after exercise.

first: Lucile

middle: C

last: Dollet

first: Alexander

middle: V

last: Chibalin

first: Logan

last: Pendergrast

first: Nicola

last: Pillon

first: Thais

last: de Castro Barbosa

first: Daniel

middle: T

last: Cervone

first: Kenneth

last: Caidahl

first: Oksana

last: Dmytriyeva

first: Atul

last: Deshmukh

first: Romain

last: Barres

first: Mikael

last: Ryden

first: Harriet

last: Wallberg-Henriksson

first: Juleen

middle: R

last: Zierath

first: Anna

last: Krook


title: An atypical GdpP enzyme linking cyclic nucleotide metabolism to gene regulation in Mycoplasma bovis

city: Zhengzhou

first: Xifang

last: Zhu

state: Henan

address: No. 100, Science Avenue

institute: Zhengzhou University

summary: To better understand the role of MbovGdpP in cellular processes, the expression profiles of differentially expressed genes between M. bovis wild type strain HB0801 and MbovGdpP knock-out mutant were determined by RNA-sequencing (RNA-seq). Differential gene expression analysis identified up to 161 genes with significant changes in T6.290 (|log2 fold change| > 1.5 and p < 0.05), with 74 mRNA up-regulated and 87 down-regulated. Transcriptomic analyses revealed the biological importance of MbovGdpP.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for M. bovis wild type strain HB0801 and MbovGdpP knock-out mutant.

first: Eric

last: Baranowski

first: Xixi

last: Li

first: Aizhen

last: Guo


title: Single-cell analysis of CD4+ cytotoxic T lymphocytes in human oral squamous cell carcinoma

city: Fukuoka

first: Hu

last: Chen

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

department: Section of Oral and Maxillofacial Oncology, Division of Maxillofacial Diagnostic and Surgical Sciences, Faculty of Dental Science

country: Japan

institute: Kyushu University

summary: Cancer immunotherapy targeting CD8+ T cells has made remarkable progress, even for oral squamous cell carcinoma (OSCC), a heterogeneous epithelial tumor without a substantial increase in the overall survival rate over the past decade. However, the therapeutic effects remain limited due to therapy resistance. Thus, a more comprehensive understanding of the roles of CD4+ T cells and B cells is crucial for more robust development of cancer immunotherapy. In this study, we examined immune responses and effector functions of CD4+ T cells, CD8+ T cells and B cells infiltrating in OSCC lesions using single-cell RNA sequencing analysis, T cell receptor (TCR) and B cell receptor (BCR) repertoire sequencing analysis, and multi-color immunofluorescence staining. Finally, two Kaplan-Meier curves and several Cox proportional hazards models were constructed for the survival analysis. We observed expansion of CD4+ cytotoxic T lymphocytes (CTLs) expressing granzymes, which are reported to induce cell apoptosis, with a unique gene expression patterns. CD4+ CTLs also expressed CXCL13, which is a B cell chemoattractant. Cell-cell communication analysis and multi-color immunofluorescence staining demonstrated potential interactions between CD4+ CTLs and B cells, particularly IgD- CD27- double negative (DN) B cells. Expansion of CD4+ CTLs, DN B cells, and their contacts has been reported in T and B cell-activated diseases, including IgG4-related disease and COVID-19. Notably, we observed upregulation of several inhibitory receptor genes including CTLA-4 in CD4+ CTLs, which possibly dampened T and B cell activity. We next demonstrated comprehensive delineation of the potential for CD8+ T cell differentiation towards dysfunctional states. Furthermore, prognostic analysis revealed unfavorable outcomes of patients with a high proportion of CD4+ CTLs in OSCC lesions. Our study provides a dynamic landscape of lymphocytes and demonstrates a systemic investigation of CD4+ CTL effects infiltrating into OSCC lesions, which may share some pathogenesis reported in severe T and B cell-activated diseases such as autoimmune and infectious diseases.

overall_design: In this study, we examined immune responses and effector functions of CD4+ T cells, CD8+ T cells and B cells infiltrating in OSCC lesions via single-cell RNA sequencing analysis, T cell receptor (TCR) and B cell receptor (BCR) repertoire sequencing analysis from 10x chromium using samples from three OSCC patients.

first: Junsei

last: Sameshima

first: Shiho

last: Yokomizo

first: Tomoki

last: Sueyoshi

first: Haruki

last: Nagano

first: Yuka

last: Miyahara

first: Taiki

last: Sakamoto

first: Shinsuke

last: Fujii

first: Tamotsu

last: Kiyoshima

first: Thomas

last: Guy

first: Seiji

last: Nakamura

first: Masafumi

last: Moriyama

first: Naoki

last: Kaneko

first: Shintaro

last: Kawano


title: Chromatin priming elements direct tissue-specific gene activity prior to hematopoietic specification

city: Birmingham

first: Benjamin

last: Edginton-White

address: Medical School

department: ICGS

country: United Kingdom

institute: University of Birmingham

summary: Tissue-specific gene regulation during development involves the interplay between transcription factors and epigenetic regulators binding to enhancer and promoter elements. The pattern of active enhancers defines the cellular differentiation state. However, developmental gene activation involves a previous step called chromatin priming which is not fully understood. We recently developed a genome-wide functional assay that allowed us to functionally identify enhancer elements integrated in chromatin regulating each of five stages spanning the in vitro differentiation of embryonic stem cells to blood. We also measured global chromatin accessibility, histone modifications and transcription factor binding. The integration of these data identified and characterised cis-regulatory elements which become activated prior to the onset of gene expression, some of which are primed in a signalling-dependent fashion. Deletion of such a priming element leads to a delay in the upregulation of its associated gene in development. Our work uncovers the details of a complex network of regulatory interactions with the dynamics of early chromatin opening being at the heart of dynamic tissue-specific gene expression control.

overall_design: Targeted DNA sequencing of fragment libraries cloned into an enhancer reporter cassette in the HPRT locus of HM-1 ES cells and their differentiated progeny. Populations of cells were sorted for Venus-YFP reporter activity into negative, low, medium and high.

first: Bejamin

first: Alexander

last: Maytum

first: Peter

last: Keane

middle: N

last: Cockerill

first: Jean-Baptiste

last: Cazier

first: Constanze

last: Bonifer


title: Sordaria macrospora wild type and developmental mutant delta-asf1

city: Bochum

first: Minou

last: Nowrousian

address: Universitaetsstr. 150

department: Molecular and Cellular Botany

institute: Ruhr-University Bochum

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

relation:
SuperSeries of: GSE216629
SuperSeries of: GSE216632
SuperSeries of: GSE228529


title: ChIP-seq analysis of the Sordaria macrospora wild type and the developmental mutant delta-asf1

summary: Genome-wide distribtuion of histone H3K56ac (histone H3 acetylated at lysine 56) was analyzed in the wild type and the sterile mutant asf1. The asf1 gene encodes a conserved histone chaperone, and preliminary experimentes indicated changes in histone modifications in the mutant that were analyzed on a genome-wide basis in these experiments.

overall_design: The analysis was carried out with mycelia from the wild type and the delta-asf1 mutant. Two biologically independent replicates were obtained for each strain.

first: Jan

last: Breuer


title: Hypoxia activated TH-302 with VEGFR-2 blockade elicits G-MDSC accumulation in pancreatic cancer

city: Houston

first: Arthur

last: Liu

state: TX

address: 7455 Fannin Street

institute: UT MD Anderson Cancer Center

summary: Transcriptional profiling of bulk-tumor samples left untreated or treated with TH-302, VEGFR-2 blockade, or the combination

overall_design: Mice bearing 10-day preimplanted orthotopic pancreatic tumors were treated with 1 cycle of therapy (5x qd TH-302 and/or 2x q96h VEGFR-2 blockade) before resection. Tumors were processed into single-cell suspensions and 5 million cells were flash frozen and then processed for RNA-extraction

first: Michael

last: Curran


title: Effect of METTL3 knockdown on MCF10 breast cancer progression model

city: Greenville

first: Kyle

middle: D.

last: Mansfield

state: NC

address: 600 Moye Blvd, MS622

department: Biochemistry and Molecular Biology

institute: East Carolina University, Brody School of Medicine

summary: To investigate the cellular role of METTL3 in breast cancer progression, we established MCF10-A, MCF10-AT1, and MCF10-CA1H cell lines that had knockdown of METTL3 using CRISPR. We then performed gene expression profiling analysis using data obtained from RNA-seq of the various cell lines.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for METTL3 knockdown in MCF10-A, MCF10-AT1, and MCF10-CA1H cell lines.

first: Mohammed

middle: G

last: Dorgham

middle: D


title: Hi-C analysis of the Sordaria macrospora wild type and developmental mutant delta-asf1 [HiC]

summary: Chromatin organization of the wild type and delta-asf1 mutant of the filamentous ascomycete Sordaria macrospora was analyzed by Hi-C. The delta-asf1 mutant lacks the conserved histone chaperone ASF1 and is unable to produce sexual fruiting bodies. It was hypothesized that the lack of the histone chaperone ASF1 in the mutant might lead to differences in short- or long-distance interactions of chromatin. Furthermore, it was hypothesized that chromatin organization might change during sexual development in the wild type as a prerequisite for the drastic transcriptome changes during development that were observed in earlier studies.

overall_design: The analysis was carried out with mycelia from early developmental stages of the wild type and mutant (2 days for the wild type and 3 days for the slower-growing asf1 mutant to reach the same stage), and mycelia from later developmental stages of the wild type  (5 days). Two biologically independent replicates were obtained for each strain and time point.


title: Paediatric Glioma Histone H3.3 K27M/G34R Mutations Drive Abnormalities in Phase-Separated PML Nuclear Bodies.

status: Public on Nov 19 2023

city: Clayton

first: Hsiao Phin

middle: Joanna

last: Voon

state: Victoria

address: Monash University, Wellington Road

country: Australia

institute: Monash University

summary: Point mutations in histone variant H3.3 (H3.3 K27M, H3.3 G34R) and the H3.3-specific ATRX/DAXX chaperone complex are frequent events in paediatric gliomas. It is clear that H3.3 point mutations affect many chromatin modifications but the exact oncogenic mechanisms are an area of active investigation. Histone H3.3 has been previously linked to PML, a gene which is frequently mutated in Acute Promyelocytic Leukaemia. We find that H3.3 point mutations disrupt the formation of PML nuclear bodies and this prevents differentiation down glial lineages. Similar to PML-mutated Acute Promyelocytic Leukaemias, H3.3-mutated patient-derived gliomas cells are also sensitive to drugs which target PML bodies. We identify PML as a contributor to oncogenesis in H3.3-mutated gliomas and our results indicate that PML-targeting strategies may prove effective at treating H3.3-mutated paediatric gliomas.

overall_design: ChIP sequencing of H3.3 and PML in H3.3 K27M, H3.3 G34R, and ATRX KO/G34R mutated mouse ES cells. ChIP-sequencing of H3.3 in H3.3 K27M and H3.3 K27MKO corrected DIPG XIII and BT245 patient-derived glioma cells.

first: Hsiao

middle: P

first: Lee

middle: H

last: Wong


title: ChIP-seq analysis of the Sordaria macrospora wild type and the developmental mutant delta-asf1 [ChIP-seq]

summary: Genome-wide distribtuion of histone H3 and H3K27me3 (histone H3 tri-methylated at lysine 27) was analyzed in the wild type and the sterile mutant asf1. The asf1 gene encodes a conserved histone chaperone, and preliminary experimentes indicated changes in histone modifications in the mutant that were analyzed on a genome-wide basis in these experiments.

overall_design: The analysis was carried out with mycelia from the wild type and the delta-asf1 mutant. Two biologically independent replicates were obtained for each strain and antibody (against H3 or H3K27me3).


title: Mitigation of chromosome loss in clinical CRISPR-Cas9-engineered T cells [Pilot_Data]

status: Public on Nov 18 2023

city: Berkeley

first: Marena

middle: Isabelle

last: Trinidad

state: CA

address: 2151 Berkeley Way

institute: Innovative Genomics Institute

summary: CRISPR-Cas9 genome editing has enabled advanced T cell therapies, but occasional loss of the targeted chromosome remains a safety concern. To investigate whether Cas9-induced chromosome loss is a universal phenomenon and evaluate its clinical significance, we conducted a systematic analysis in primary human T cells. Arrayed and pooled CRISPR screens revealed that chromosome loss was generalizable across the genome and resulted in partial and entire loss of the chromosome, including in pre-clinical chimeric antigen receptor T cells. T cells with chromosome loss persisted for weeks in culture, implying the potential to interfere with clinical use. A modified cell manufacturing process, employed in our first-in-human clinical trial of Cas9-engineered T cells, dramatically reduced chromosome loss while largely preserving genome editing efficacy. Expression of p53 correlated with protection from chromosome loss observed in this protocol, suggesting both a mechanism and strategy for T cell engineering that mitigates this genotoxicity in the clinic.

overall_design: Primary human T cells were electroporated with Cas9 RNP (Cas9 protein + one of twelve different gRNAs) in an arrayed format. Genome edited T cells were barcoded using MULTI-seq before pooling and performing 10x Genomics 3' scRNA-seq.

first: Connor

last: Tsuchida

first: Nadav

last: Brandes

first: Raymund

last: Bueno

first: Thomas

last: Mazumder

first: Chun

middle: J

last: Ye

first: Jennifer

last: Doudna


title: Gene regulation of murine hematopoietic specification

first: Benjamin

last: Edginton-White


title: Loss of function of XBP1 splicing activity of IRE1a favors B cell tolerance breakdown

city: Zwijnaarde

first: Sophie

last: Janssens

address: Technologiepark 71

department: Inflammation Research Center

country: Belgium

institute: VIB/UGhent

summary: Tolerance breakdown leads to the development of autoimmune diseases in an increasing manner. B cells strongly contribute to the pathogenesis of these diseases, notably via the production of autoantibodies leading to cell depletion, receptor modulation and organ damages. However, the molecular mechanisms of B cell tolerance breakdown are badly known. Autoimmune diseases are frequently aggregated in families in two or more generations. The study of these familial forms could therefore help to better describe common biological mechanisms leading to B cell tolerance breakdown. By Whole-Exome Sequencing, we describe here in a multiplex family with several members presenting autoantibody-mediated autoimmunity a new heterozygous mutation (p.R594C) in ERN1 gene, coding for IRE1α. Using human cell lines and a knock-in (KI) transgenic mouse model, we showed that this mutation leads to a profound defect of IRE1α ribonuclease activity on XBP1 splicing. The KI mice developed a broad panel of autoantibodies, however in a subclinical manner. These results suggest that a decrease of XBP1s production could contribute to B cell tolerance breakdown and give new insights into the function of IRE1α which are important to consider for the development or IRE1α targeting strategies.

overall_design: We performed CITEseq to asses in an unbiased manner whether the R594C mutation in IRE1 affects the immune cell compartment.

first: Quentin

last: Reuschlé

first: Laurien

last: Van Heddegem

first: Clint

last: De Nolf

first: Pauline

last: Soulas-Sprauel


title: Intrinsically disordered regions of transcription factor encode multiple functions using interwoven sequence grammars (RNA-Seq)

city: Rehovot

first: Naama

last: Barkai

state: HaMerkaz

address: Herzl 234

department: Molecular Genetics

institute: Weizmann Institute of Science

summary: Intrinsically disordered regions (IDRs) are abundant within eukaryotic proteins, but their sequence-function relationship remains poorly understood. IDRs of transcription factors (TFs) can direct promoter selection and recruit coactivators, as exemplified by the budding-yeast TF- Msn2. To examine how low-complexity IDRs encode multiple functions, we compared genomic binding preferences, gene induction, and coactivator recruitment amongst a large set of designed Mns2-IDR mutants. We show that multiple regions across the >500AA IDR contribute to both functions. Yet, transcription activity was readily disrupted by variants having no consequences on Msn2 binding. Our data attribute this differential sensitivity to the integration of relaxed, composition-based code directing binding preferences with a more stringent, motif-based code controlling the recruitment of coactivators and transcription activity. Interwoven sequence grammar may present a general paradigm through which low-complexity IDRs encode multiple functions.

overall_design: The experimetns here are RNA-seq samples, collection of samples was performed at OD600 of 0.4 after treatment with 0.3 mM of H2O2 to induce stress, for controls we used the expresion profiling of non-genetically modified yeast strain (BY4741) or ΔMsn2

first: Vladimir

last: Mindel

first: Sagie

last: Brodsky

first: Aileen

last: Cohen

first: Wajd

last: Manadre

first: Felix

last: Jonas

first: Miri

last: Carmi


title: Mitigation of chromosome loss in clinical CRISPR-Cas9-engineered T cells [CART]

first: Marena

last: Trinidad

overall_design: Primary human T cells were electroporated with Cas9 RNP (Cas9 protein + one of three different gRNAs) in an arrayed format. Recombinant AAV6 encoding a chimeric antigen receptor (CAR) resulted in homology-directed repair. Genome edited T cells were barcoded using hashtag reagents before pooling and performing 10x Genomics 3' scRNA-seq.

first: Connor

last: Tsuchida

first: Nadav

last: Brandes

first: Raymund

last: Bueno

first: Byungjin

last: Hwang

first: Christopher

last: Chang

first: Yang

last: Sun

first: Justin

last: Eyquem


title: Effect of externally applied concave and convex curvature for 24 hours with continued duam SMAD inhibition (dSMADi) on human embryonic cell line H9 after 48 hours of dSMADi induction to attain early neuroepithelial features

first: Hyung-Min

last: Ahn

address: 145, Anam-ro, Seongbuk-gu

institute: Korea University

summary: To investigate the transcriptional change induced by externally applied concave and convex curvature, the former of which displayed tissue-level morphological change, human embryonic cell line H9 maintained for pluripotency with mTeSR1 for 72 hours and then induced to have early neuroepithelial features with N2B27 media containing 10 μM of SB431542 and 0.1 μM of LDN (dual SMAD inhibition) for 48 hours. Then along with the continued dual SMAD inhibition, plate curving cell culture of either concave or convex conformation for 24 hours was applied on the cell culture, at the end of which time point cell pellet was collected for bulk RNA sequencing.

overall_design: Triplicates of control (only without plate curving cell culture), concave curvature and convex curvature groups of human embryonic stem cell line-derived neuropithelium (hESC-NE) were prepared. Each two-member combination was compared to reveal differential expression.

first: Woong


title: Intrinsically disordered regions of the Msn2 transcription factor encode multiple functions using interwoven sequence grammars

first: Naama

overall_design: Refer to individual Series


title: Adult Dental Epithelial Stem Cell-Derived Organoids Deposit Hydroxylapatite Biomineral

first: Han-Sung

last: Jung

address: 50, Yonsei-ro, Seodaemun-gu

department: Dental School

institute: Yonsei University

summary: Ameloblasts are specialized cells derived from the dental epithelium that produce enamel, a hierarchically structured tissue comprised of highly elongated hydroxylapatite (OHAp) crystallites. Because enamel is acellular and has limited capacity to heal, regenerative technologies are highly desirable, yet have remained largely out of reach. This is in part due to the challenges of synthesizing crystallites with similar properties and assembling them in a mechanically robust structure. Herein, we demonstrate the ability to generate mineralizing dental epithelial organoids (DEOs) from adult dental epithelial stem cells (aDESCs) isolated from mouse incisor tissues. DEOs expressed ameloblast markers, could be maintained for more than five months (11 passages) in vitro in media containing modulators of Wnt, Egf, Bmp, Fgf and Notch signaling pathways, and were amenable to cryostorage. When implanted underneath murine kidney capsules, organoids produced OHAp crystallites similar in composition, size, and shape to mineralized dental tissues, including some enamel-like elongated crystals. DEOs are thus a powerful in vitro model for the development of the dental epithelium and enamel, and an important first step towards regrowth or replacement of enamel.

overall_design: Organoids were grown from aDESCs embedded in Matrigel using media supplemented with Wnt3a, R-spondin1, and Egf (minimal); minimal + Noggin (+N); +N + Fgf10 (+NF); or +NF + dibenzazepine (+NFD) for 14 days.

first: Hyun-Yi

last: Kim

first: Eun-Jung

first: Jong-Min

last: Lee


title: Mitigation of chromosome loss in clinical CRISPR-Cas9-engineered T cells [CROPSeq_Data]

overall_design: Primary human T cells were transduced with a lentiviral vector (CROP-seq) containing a unique Cas9 guide RNA and co-expressing GFP at an MOI <0.3. After FACS to isolate GFP+ T cells, Cas9 protein was electroporated. Genome edited T cells were barcoded using MULTI-seq before pooling and performing 10x Genomics 3' scRNA-seq. Along with the MULTI-seq barcodes and gene expression data, the sequence of the guide RNA was also sequenced.

first: Thomas

last: Mazumder

first: Chun

last: Ye

first: Jennifer

last: Doudna


title: Poly(ADP-ribosyl)ating enzymes coordinate changes in the expression of metabolic genes with developmental progression

status: Public on Nov 17 2023

city: Grand Forks

first: Alexei

last: Tulin

state: North Dakota

address: 501 N Columbia Rd Stop 9061

department: Epigenetics Department

institute: University of North Dakota

summary: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for PARG-EYFP in Drosophila melanogaster L3 larva puffstage 7-9.

overall_design: In total 4 samples: 2 biological replicates of L3 larva puffstage 7-9 w1118, y1 larvae used as control and 2 biological replicates of L3 larva puffstage 7-9 Parg27.1/Parg27.1; Pp{UAST::PARG-EYFP}, 69b-Gal4/TM2 larvae.

first: Bordet

last: Guillaume

first: Bamgbose

last: Gbolahan

first: Tulin

last: Alexei


title: The Effect of an Exopolysaccharide Probiotic Molecule from Bacillus subtilis on gene expression from four types of Breast Cancer Cells

city: Maywood

first: Clodia

last: Osipo

address: 2160 S. First Ave

institute: Loyola University Chicago

summary: Many well-known risk factors for breast cancer are associated with dysbiosis (an aberrant microbiome). However, how bacterial products modulate cancer are poorly understood. In this study, we investigated the effect of an exopolysaccharide (EPS) produced by the commensal bacterium Bacillus subtilis on breast cancer phenotypes. Although B. subtilis    is commonly included in probiotic preparations and its EPS protects against inflammatory diseases, it was virtually unknown whether B. subtilis-derived EPS affects cancer. This work investigated effects of EPS on breast cancer cells as a cancer model.  Short-term treatment with EPS inhibited proliferation of some breast cancer cells (T47D, MDA-MB-468, HCC1428, MDA-MB-453) while having little effect on others (MCF-7, MDA-MB-231, BT549, ZR-75-30). EPS induced G1/G0 cell cycle arrest of T47D cells while increasing apoptosis of MDA-MB-468 cells. EPS also enhanced aggressive phenotypes in T47D cells including cell migration and cancer stem cell survival. Long-term treatment with EPS (months) led to resistance in vitro and promoted tumor growth in immunocompromised mice. RNA-sequence analysis showed that EPS increased expression of pro-inflammatory pathways including STAT1 and NF-kB. IKKb and/or STAT1 signaling was necessary for EPS to modulate phenotypes of EPS sensitive breast cancer cells. These results demonstrate a multifaceted role for an EPS molecule secreted by the probiotic bacterium B. subtilis on breast cancer phenotypes and warrant future studies in immune competent mice and different cancer models.

overall_design: T47D cells (4x105), MCF-7 cells (1x106), MDA-MB-231 cells (2x105), or MDA-MB-468 cells (8x105) were plated in 10cm2 dishes overnight. The following day, cells at <70% confluence were treated with either 5μg/mL EPS or equal volume of PBS and incubated at 37°C for 24 hours.  Each condition was performed in 3 biological

first: Mai

last: Nguyen

first: Emila

last: Ma

first: Debra

last: Wyatt

first: Katherine

last: Kight


title: Intrinsically disordered regions of transcription factor encode multiple functions using interwoven sequence grammars (ChEC-seq)

first: Naama

last: Barkai

overall_design: ChEC-Seq experiments at OD600 of 4. 30 second Mnase activation. Repeats are included when existed. Presented strains with Med15-Mnase or Msn2-Mnase with sometimes intersecting genotypes of the Msn2 mutations

first: Vladimir

last: Mindel

first: Sagie

last: Brodsky

first: Aileen

last: Cohen

first: Wajd

last: Manadre

first: Felix

last: Jonas

first: Miri

last: Carmi


title: Impact of ERbeta on the ovarian transcriptome

city: Solna

first: Cecilia

last: Williams

address: Tomtebodav 23

department: Protein Science

institute: KTH Royal Institute of Technology

summary: To understand the mechanism of action of ERβ in the ovary, sequencing was performed on ovaries of WT and ERβKO mice.

overall_design: Ovary mRNA profiles of WT and ERβKO mice on a C57BL/6J background were generated by deep sequencing. 4-5 biological replicates were used.

first: Madeleine

last: Birgersson

first: Amena

last: Archer


title: Deciphering the endogenous cistrome and transcriptome of estrogen receptor beta in ovary

first: Rajitha

last: Indukuri

state: Sweden

address: Tomtebodavägen 23a, Solna

institute: Scilifelab

summary: ERβ is predominantly expressed in granulosa cells of the ovary, where its important for normal ovarian function and plays a vital role in folliculogenesis. However, the genome wide binding landscape of ERβ in the ovary was not determined and its interaction targets are unknown. To characterize the mechanism of ERβ, we performed ChIP-Seq using endogenous ERβ expression and mapped the cistrome of ERβ in the ovary using a validated antibody.

overall_design: ChIP-Seq was performed three times in the ovaries endogenously expressing ERβ. Binding sites present in at least two out of three replicates were considered for downstream analysis,


title: Fludioxonil-induced differential gene expression in Aspergillus fumigatus and its DtcsC and  Dskn7 mutants

status: Public on Nov 16 2023

city: Jena

first: Tongta

last: Sae-Ong

address: Adolf-Reichwein-Straße 23

department: Microbiome Dynamics

institute: Leibniz Institute for Natural Product Research and Infection Biology Hans Knöll Institute (Leibniz-HKI)

summary:
The antifungal compound fludioxonil targets the fungal specific group III hybrid histidin kinaseTcsC of A. fumigatus, which in turn activate the High Osmolarity Glycerol (HOG) pathway and  leads to drastic swelling of the fungal cells.
We investigated the different responses of the AfS35 (wild type), the completely resistant mutant strain DtcsC and the partial resistant mutant strain Dskn7 to fludioxonil.

overall_design: Comparative gene expression profiling of the A. fumigatus parental wild-type strain and the mutant strains DtcsC as well as Dskn7 after treatment with fludioxonil for 1 and 3 hours versus untreated controls

first: Sebastian

last: Schruefer

first: Annica

last: Pschibul

first: Sarah

last: Wah Wong

first: Thomas

last: Wolf

first: Sascha

last: Schäuble

first: Gianni

last: Panagiotou

first: Axel

last: Brakhage

first: Vishukumar

last: Aimanianda

first: Olaf

last: Kniemeyer

first: Frank

last: Ebel


title: Mitigation of chromosome loss in clinical CRISPR-Cas9-engineered T cells

first: Marena

last: Trinidad

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

first: Connor

last: Tsuchida

first: Nadav

last: Brandes

first: Raymund

last: Bueno

last: Mazumder

first: Chun

last: Ye

first: Jennifer

last: Doudna


title: Simultaneous selection of nanobodies for accessible epitopes on immune cells in the tumor microenvironment

city: Dallas

first: Todd

last: Aguilera

address: 2280 Inwood Rd

department: Molecular radiation biology

institute: UTSouthwestern- Medical Center

summary: In the rapidly advancing field of synthetic biology, there exists a critical need for technology to discover targeting moieties for therapeutic biologics. We are developing developed INSPIRE-seq, an approach that utilizes a nanobody library and next-generation sequencing to identify nanobodies selected for complex environments. INSPIRE-seq enables the parallel enrichment of immune cell-binding nanobodies that penetrate the tumor microenvironment. Clone enrichment and specificity vary varies across immune cell subtypes in the tumor, lymph node, and spleen. INSPIRE-seq identified a dendritic cell binding clone that binds PHB2. Single-cell RNA sequencing revealed a connection with cDC1s, and immunofluorescence confirmed nanobody-PHB2 colocalization along cell membranes. Structural modeling and docking studies assisted binding predictions and will guide nanobody selection. In this work, we demonstrate that INSPIRE-seq offers an unbiased approach to examine complex microenvironments and assist in the development of nanobodies, which could serve as active drugs, modified to become drugs, or used as targeting moieties. microenvironment, which can be distinct from   draining lymph nodes. To identify targets, we selected a clone enriched for dendritic cells that binds to   PHB2. Using single cell RNA sequencing, we observe PHB2 signaling is associated with activation in   cDC1’s. Immunofluorescence confirmed that the nanobody colocalizes with PHB2 in regions along the   cell membrane. Structural modeling with AlphaFold2 and antibody docking using Rosetta assist binding   site predictions, thus could be used to guide nanobody selection for future aims. This work shows that  INSPIRE-seq can interrogate complex microenvironments and may assist in developing therapeutics.

overall_design: CD45 magnetic microbeads enriched cells were isolated form Py8119 bearing mice (three mice per group) two hours after injection of either PBS, insertless phage display, CD45, DCs, or CD8 specific VHHs phage display libraries.

first: Thillai

last: Sekar

first: Eslam

last: Elghonaimy

first: Katy

middle: L

last: Swancutt

last: Diegeler

first: Isaac

last: Gonzalez

first: Cassandra

last: Hamilton

first: Peter

middle: Q

last: Leung

first: Jens

last: Meiler

first: Cristina

middle: E

last: Martina

first: Michael

last: Michael


title: A memory-like T follicular helper cell subset revealed by single-cell transcriptomic analysis

city: Beijing

first: Chen

last: Dong

address: Haidian District

department: Institute for Immunology and School of Medicine

institute: Tsinghua University


title: Overall avidity declines in TCR repertoires during latent CMV but not EBV infection

status: Public on Nov 15 2023

city: Lausanne

first: Sandra

last: Calderon

address: Génopode Building

department: Center for Integrative Genomics

country: Switzerland

institute: University of Lausanne

summary: The avidity of the T-cell receptor (TCR) for antigenic peptides presented by the MHC (pMHC) on cells is an essential parameter for efficient T cell-mediated immunity. Yet, whether the TCR-ligand avidity can drive the clonal evolution of virus antigen-specific CD8 T cells, and how this process is determined in latent Cytomegalovirus (CMV)- against Epstein-Barr virus (EBV)-mediated infection remains largely unknown. Here, we quantified monomeric TCR-pMHC dissociation rates on CMV- and EBV-specific individual TCR-alpha-beta clonotypes and polyclonal CD8 T cell populations in healthy donors over a follow-up time of 15-18 years. Within CMV/pp65-specific T cell repertoires, a progressive contraction of clonotypes with high TCR-pMHC avidity and low CD8 binding dependency was observed, leading to an overall avidity decline during long-term antigen exposure. We identified a unique transcriptional signature preferentially expressed by high-avidity CMV/pp65-specific T cell clonotypes, including the inhibitory receptor LILRB1. Interestingly, T cell clonotypes of high-avidity showed higher LILRB1 expression than the low-avidity ones and LILRB1 blockade moderately increased T cell proliferation. Similar findings were made for CD8 T cell repertoires specific for the CMV/IE-1 epitope. There was a gradual in vivo loss of high-avidity T cells with time for both CMV specificities, corresponding to virus-specific CD8 T cells expressing enhanced LILRB1 levels. In sharp contrast, the EBV/BMFL1-specific T cell clonal composition and distribution, once established, displayed an exceptional stability, unrelated to TCR-pMHC binding avidity or LILRB1 expression. Together, these findings reveal an overall long-term avidity decline of CMV- but not EBV-specific T cell clonal repertoires, highlighting the differing role played by TCR-ligand avidity over the course of these two latent herpesvirus infections. Our data suggest that the inhibitor receptor LILRB1 potentially restricts the clonal expansion of high-avidity CMV-specific T cell clonotypes during latent infection. We propose that the mechanisms regulating the long-term outcome of CMV- and EBV-specific memory CD8 T cell clonotypes in humans are distinct.

overall_design: Leukapheresis were collected from three HLA-A2-positive healthy individuals latently infected with the CMV virus (BCL1, BCL4, BCL6) in 2002 (i.e. time-point Tn). Blood samples from the same individuals were collected 15 years later (i.e. time-point Tn+15y). The following factors were assessed: Donor, time-point and CMV/pp65-specific T cell clonotypes of binding avidity (high vs. low).

first: Barbara

last: Couturaud

first: Bastien

last: Doix

first: Laura

last: Carretero-Iglesia

first: Mathilde

last: Allard

first: Sylvain

last: Pradervand

last: Hebeisen

first: Nathalie

last: Rufer


title: A time-resolved meta-analysis of consensus gene expression profiles during human T-cell activation

city: Leipzig

last: Rade

address: Perlickstr. 1

institute: Fraunhofer Institute for Cell Therapy and Immunology

summary: The coordinated transcriptional regulation of activated T-cells is based on a complex dynamic behavior of signaling networks. Given an external stimulus, T-cell gene expression is characterized by impulse and sustained patterns over the course. Here, we analyzed the temporal pattern of activation across different T-cell populations to develop consensus gene signatures for T-cell activation. We applied a meta-analysis of anti-CD3/CD28 induced CD4+ T-cell activation kinetics of publicly available transcriptome-wide time series using a random effects model. We used non-negative matrix factorization, an unsupervised deconvolution method, to infer changes in biological patterns over time. For verification and to further map a wider variety of the T-cell landscape, we performed a time series of transcriptome-wide RNA sequencing on activated Pan T-cells.

overall_design:
We performed a time-course experiment with anti-CD3/CD28 beads activated Pan T-cells from 4 healthy individuals. RNA-seq was performed before activation (0 hours) and 6, 12, 24, 48, 72 hours after activation. In addition, as a control, we sequenced Pan T-cells for the same time series without activation condition.
***Submitter statement concerning raw data availability: 'we do not have informed consent from the donors to publish the raw sequencing data.'***

first: Sebastian

last: Böhlen

first: Vanessa

last: Neuhaus

first: Dennis

last: Löffler

first: Conny

last: Blumert

first: Ulrike

last: Köhl

first: Susann

last: Dehmel

first: Katherina

last: Sewald

first: Kristin

last: Reiche


title: Ancestral gestational exposure to widely used neonicotinoid thiacloprid leads to global DNA methylation alterations in spermatozoa in three generations of male mice

city: Rennes

first: Fatima

last: SMAGULOVA

address: 9 av. Prof. Léon Bernard

institute: Irset - U1085


title: Ancestral gestational exposure to widely used neonicotinoid thiacloprid leads to global DNA methylation alterations in spermatozoa in three generations of male mice [RNA-Seq]

summary: Background: Neonicotinoids, a widely used class of insecticide that used is agriculture to control insect’s population, are serious public concern. Objectives: In this study, we examined the capacity of a neonicotinoid, thiacloprid (thia) to induce transgenerational effects in the male mice.

overall_design: Methods: The pregnant outbred Swiss female mice were exposed to thia at embryonic days E6.5 to E15.5 using “0”, and “6” mg/kg/day doses. We analyzed the gene expression in embryonic testis in first and third generation embryos.

last: Smagulova


title: Ancestral gestational exposure to widely used neonicotinoid thiacloprid leads to global DNA methylation alterations in spermatozoa in three generations of male mice [MEDIP-seq]

summary: Background: Neonicotinoids, a widely used class of insecticide that used is agriculture to control insect’s population, are serious public concern. Objectives: In this study, we examined the capacity of a neonicotinoid, thiacloprid (thia) to induce transgenerational effects in the male mice. Methods: The pregnant outbred Swiss female mice were exposed to thia at embryonic days E6.5 to E15.5 using “0”, and “6” mg/kg/day doses. The genome-wide analysis of DNA methylation analysis showed that differentially methylated regions (DMRs) are located at the promoters and enhancers of many important developmental genes. Notably, DMRs were largely overlapping the germ cell reprograming responsive gene (GRRs) and super-enhancer elements found to be active in ES cells, testis or brain.

overall_design:
MEDIP-seq of spermatozoa from first, second, and third generation adults.
Methylated DNA precipitation DNA-sequencing (MEDIP-seq) for methylated DNA in mouse spermatozoa was performed using 6 ug of DNA using EpiMark® Methylated DNA Enrichment Kit (Qiagen, E2600S) according to protocol provided by manufacture.


title: In situ spatial reconstruction of distinct normal and pathological cell populations within the human adrenal gland

city: Aurora

first: Neelanjan

last: Mukherjee

state: Colorado

address: 12800 E 19th Ave, RC1-South

department: Biochemistry and Molecular Genetics

institute: CU Anschutz

summary: The human adrenal gland consists of concentrically organized functionally distinct regions responsible for hormone production. Dysregulation of adrenocortical cell differentiation alters the proportion and organization of the functional zones of the adrenal cortex leading to disease. Current models of adrenocortical cell differentiation are based on mouse studies, but there are known organizational and functional differences between human and mouse adrenal glands. This study aimed to investigate the centripetal differentiation model in the human adrenal cortex and characterize aldosterone-producing micronodules (APMs) to better understand adrenal diseases such as primary aldosteronism. We applied spatially resolved in situ transcriptomics to human adrenal tissue sections from two individuals and identified distinct cell populations and their positional relationships. The results supported the centripetal differentiation model in humans, with cells progressing from the outer capsule to the zona glomerulosa, zona fasciculata, and zona reticularis. Additionally, we characterized two APMs in a 72-year-old female. Comparison with earlier APM transcriptomes indicated a subset of core genes, but also heterogeneity between APMs. The findings contribute to our understanding of normal and pathological cellular differentiation in the human adrenal cortex.

overall_design: Study consists of sectioned human donor adrenal glands processed for Visium Spatial Transcriptomics. There are a total of four samples, three from a 31 year old female and one from a 71 year old female.

first: Rui

last: Fu

first: Michael

last: Kaufman


title: Poly I:C-priming of adipose-derived mesenchymal stromal cells promotes a pro-tumorigenic phenotype in an immunocompetent mouse model of prostate cancer

status: Public on Nov 14 2023

city: West Lafayette

first: Sagar

middle: M

last: Utturkar

state: IN

address: 201 S University St

department: Institute for Cancer Research

institute: Purdue University

summary: While BM-MSC remain the most commonly studied MSC subtype and were the focus of original MSC polarization paradigm reports, MSC derived from other tissues may provide advantages for clinical use such as higher relative abundance and differential functional potential. In our previous work, we had identified phenotypical and functional consequences of TLR3 or TLR4 priming on ASC that partially resembled those previously reported for BM-MSC. In our assessment, LPS-priming of ASC rendered them immunostimulatory in immune killing assay coculture assays in the presence of human prostate adenocarcinoma cells and stimulated peripheral blood mononuclear cells. Understanding that tumorigenesis is a complex process involving many other tumor microenvironment components and other supportive tissues, we aimed to evaluate the consequences of TLR-priming in an immunocompetent model of androgen independent prostate cancer by assessing changes in tumor progression, gene expression and immune composition of tumors treated with TLR-primed ASC. The present work expands upon the impact of primed ASC on the prostate tumor microenvironment.

overall_design: human adipose-derived stem cells (ASCs) from a male Caucasian donor were cultured, incorporating lentiviral transduction to express renilla luciferase, and mouse prostate adenocarcinoma cells (TRAMP-C2) were modified for the study with Lv-Hras. Following expansion, ASCs were primed with Toll-like receptor agonists (Poly I:C or LPS). In an in vivo assessment, tumors were induced in immunecompetent C57/BL6 mice through the injection of TC2R cells with or without TLR-primed ASCs, and tumor growth was monitored. Tumor volume was calculated, and tissues were collected at specific milestones. RNA was subsequently isolated from the preserved tumors.

first: Cosette

last: Rivera-Cruz

first: Shreya

last: Kumar

first: Marxa

last: Figueiredo


title: A high-resolution transcriptomic and spatial atlas of cell types in the whole mouse brain

city: Seattle

first: Cindy

last: van Velthoven

state: WA

address: 615 Westlake Ave N

institute: Allen Institute

summary: The mammalian brain consists of millions to billions of cells that are organized into numerous cell types with specific spatial distribution patterns and structural and functional properties. An essential step towards understanding brain function is to obtain a parts list, i.e., a catalog of cell types, of the brain. Here, we report a comprehensive and high-resolution transcriptomic and spatial cell type atlas for the whole adult mouse brain. The cell type atlas was created based on the combination of two single-cell-level, whole-brain-scale datasets: a single-cell RNA-sequencing (scRNA-seq) dataset of ~7 million cells profiled (~4.0 million cells passing quality control), and a spatially resolved transcriptomic dataset of ~4.3 million cells using MERFISH. The atlas is hierarchically organized into four nested levels of classification: 34 classes, 338 subclasses, 1,201 supertypes and 5,322 clusters. We present a newly developed online platform, Allen Brain Cell (ABC) Atlas, to visualize the mouse whole brain cell type taxonomy and atlas along with the scRNA-seq and MERFISH data and metadata sets. We systematically analyzed the neuronal, non-neuronal, and immature neuronal cell types across the brain and identified a high degree of correspondence between transcriptomic identity and spatial specificity for each cell type. The results reveal unique features of cell type organization in different brain regions, in particular, a dichotomy between the dorsal and ventral parts of the brain: the dorsal part contains relatively fewer yet highly divergent neuronal types, whereas the ventral part contains more numerous neuronal types that are more closely related to each other. We also systematically characterized cell-type specific expression of neurotransmitters, neuropeptides, and transcription factors. The study uncovered extraordinary diversity and heterogeneity in neurotransmitter and neuropeptide expression and co-expression patterns in different cell types across the brain, suggesting they mediate myriad modes of intercellular communications. Finally, we found that transcription factors are major determinants of cell type classification in the adult mouse brain and identified a combinatorial transcription factor code that defines cell types across all parts of the brain. The whole-mouse-brain transcriptomic and spatial cell type atlas establishes a benchmark reference atlas and a foundational resource for deep and integrative investigations of cellular and circuit function, development, and evolution of the mammalian brain.

overall_design: Single cell RNA-seq using the 10x genomics v2 and v3 methods. Cells were isolated from whole mouse brain and each library represents a small dissection area. The dataset includes 299 10x genomics v2 libraries containing ~2.5 million cells and 482 10x genomics v3 libaries containing ~4.4 million cells.

first: Zizhen

last: Yao

last: Kunst

first: Meng

last: Zhang

first: Delissa

last: McMillen

first: Changkyu

last: Lee

first: Won

last: Jung

first: Jeff

last: Goldy

first: Aliya

last: Abdelhak

first: Matthew

last: Aitken

first: Katherine

last: Baker

first: Pamela

first: Eliza

last: Barkan

first: Darren

last: Bertagnolli

first: Ashwin

last: Bhandiwad

first: Cameron

last: Bielstein

first: Prajal

last: Bishwakarma

first: Jazmin

last: Campos

first: Daniel

last: Carey

first: Tamara

last: Casper

first: Anish

middle: B

last: Chakka

first: Rushil

last: Chakrabarty

first: Sakshi

last: Chavan

first: Min

last: Chen

last: Clark

first: Jennie

last: Close

first: Kirsten

last: Crichton

first: Scott

last: Daniel

first: Peter

last: DiValentin

first: Tim

last: Dolbeare

first: Lauren

last: Ellingwood

first: Elysha

last: Fiabane

first: Timothy

last: Fliss

first: James

last: Gee

last: Gerstenberger

first: Alexandra

last: Glandon

first: Jessica

last: Gloe

first: Joshua

last: Gould

last: Gray

first: Nathan

last: Guilford

first: Junitta

last: Guzman

last: Hirschstein

first: Windy

last: Ho

first: Mike

last: Huang

first: Madie

last: Hupp


title: ChIP-seq following CRISPRi perturbations to MYC enhancers in K562 cells

city: Stanford

first: Jesse

last: Engreitz

address: 240 Pasteur Drive

institute: Stanford University

summary: We previous identified enhancers that regulate MYC expression in K562 cells (Fulco et al. Science 2016). Here, we perturbed MYC enhancers with individual CRISPRi gRNAs and performed ChIP-seq to study the effects on chromatin state.

first: Elizabeth

last: Perez

first: Charles

last: Fulco

first: Rockwell

last: Anyoha


title: Sex Differences in the Neuronal Transcriptome and Synaptic Mitochondrial Function in Cerebral Cortex of a Multiple Sclerosis Model

status: Public on Nov 13 2023

city: Los Angeles

first: Yuichiro

last: Itoh

state: California

address: 635 Charles E. Young Drive South

department: Neurology

institute: UCLA

summary: Multiple sclerosis (MS) affects the cerebral cortex, inducing cortical atrophy and neuronal and synaptic pathology. Despite the fact that women are more susceptible to getting MS, men with MS have worse disability progression. Here, we address sex differences in neurodegenerative mechanisms focusing on the cerebral cortex using the MS model, chronic experimental autoimmune encephalomyelitis (EAE). RNA sequencing of neurons in cerebral cortex during EAE showed robust differential gene expression in male EAE mice compared to male healthy, age-matched, control mice. In contrast, there were few differences in female EAE mice compared to female controls. The most enriched differential gene expression pathways in male mice during EAE were mitochondrial dysfunction and oxidative phosphorylation. Mitochondrial morphology showed significant abnormalities in the cerebral cortex of EAE males, but not EAE females. Regarding function, synaptosomes isolated from cerebral cortex of male EAE mice demonstrated decreased oxygen consumption rates during respirometry assays. Together, cortical neuronal transcriptomics, mitochondrial morphology, and functional respirometry assays in synaptosomes revealed worse neurodegeneration in male EAE mice. This is consistent with worse neurodegeneration in MS men and reveals a model and a target to develop treatments to prevent cortical neurodegeneration and mitigate disability progression in MS men.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for cortex neuron RNAs

first: Noriko

first: Rhonda

last: Voskuhl


title: Too much of a good thing: Extended duration of gut microbiota depletion reverses protection from experimental autoimmune uveitis

city: Bethesda

first: Vijay

last: Nagarajan

state: Maryland

address: 10 Center Drive, 10/10N248

department: National Eye Institute

institute: National Institutes Of Health

summary: Microbial sequencing revealed progressive reduction of gut microbiota that showed some differences in the two ABX groups compared to untreated controls. Interestingly, duration of ABX was associated with a gradual disappearance of the CD4+ and CD4+CD8+ subset of gut intraepithelial lymphocytes (IELs). This IEL subset is microbiota-dependent and is absent in germ-free mice. Relative abundance of Lactobacillus reuteri correlated with frequencies of CD4+CD8+ IELs and reduced EAU. Notably, IELs in culture suppressed antigen-specific activation of autoreactive T cells.

overall_design: EAU-susceptible B10.RIII mice were treated orally with a broad-spectrum antibiotic cocktail starting at least 4-weeks (long-term) or 1-week (short-term) before IRBP161-180 immunization until the termination of the experiment. Gut microbiota were characterized by 16S amplicon sequencing and host gut immune elements were studied phenotypically and functionally.

first: Ryan

last: Salvador

first: Reiko

last: Horai

first: Amy

last: Zhang

first: Yingyos

last: Jittayasothorn

first: Jihong

last: Tang

first: Akriti

last: Gupta

first: Vijayaraj

first: Rachel

last: Caspi


title: The USP7-STAT3-granzyme-Par-1 axis regulates allergic inflammation by promoting differentiation of IL-5-producing Th2 cells

city: chiba city

first: Masahiro

last: Kiuchi

state: chiba

address: cyuoku inohana1-8-1

institute: Chiba univercity

summary: Uncontrolled type 2 immunity by type 2 helper T (Th2) cells causes intractable allergic diseases; however, whether the interaction of CD4+ T cells shapes the pathophysiology of allergic diseases remains unclear. We identified a subset of Th2 cells that produced the serine proteases granzyme A and B early in differentiation. Granzymes cleave protease-activated receptor (Par)-1 and induce phosphorylation of p38 mitogen-activated protein kinase (MAPK), resulting in the enhanced production of IL-5 and IL-13 in both mouse and human Th2 cells. Ubiquitin-specific protease 7 (USP7) regulates IL-4-induced phosphorylation of STAT3, resulting in granzyme production during Th2 cell differentiation. Genetic deletion of Usp7 or Gzma and pharmacological blockade of granzyme B ameliorated allergic airway inflammation. Furthermore, PAR-1+ and granzyme+ Th2 cells were colocalized in nasal polyps from patients with eosinophilic chronic rhinosinusitis. Thus, the USP7-STAT3-granzymes-Par-1 pathway is a potential therapeutic target for intractable allergic diseases.

overall_design: In this study, genomes were recovered from Th2 cells and sequenced; Th2 cell differentiation was performed in wild-type, Cre-ERT2 Usp7+/+, Cre-ERT2 Usp7fl/fl, Stat3fl/fl, and CD4-Cre Stat3fl/fl mice derived from Splenic naive CD4 T cells (CD44loCD62Lhi) were purified using a magnetic cell sorter (autoMACS; Miltenyi Biotec, Bergisch Gladbach, NRW, Germany). cultures of Th cells were performed as previously described. for Th2 cells, Naive CD4 T cells were cultured with IL-2 (25 U/mL), IL-4 (100 U/mL), anti-IFN-γ mAb, and CD28 mAb (1 μg/mL) in the presence of TCRβ mAb. For library preparation, libraries were prepared using Chromium Single Cell 3' Reagent Kits v3.1 according to the manufacturer's protocol (10X Genomics, Pleasanton, CA, USA). The prepared scRNA-seq libraries were sequenced using NovaSeq 6000 (Illumina) for 127 cycles (paired-end reads).

first: Jin

last: Kumagai

first: Kota

last: Kokubo

first: Hiroyuki

last: Yagyu

last: Nemoto

first: Kaori

last: Tsuji

first: Ken

last: Nagahata

first: Atsushi

last: Sasaki

first: Takahisa

last: Hishiya

first: Miki

last: Onoue

first: Rie

last: Shinmi

first: Yuri

last: Sonobe

first: Tomohisa

last: Iinuma

first: Syuji

last: Yonekura

first: Jun

last: Shinga

first: Toyoyuki

last: Hanazawa

first: Haruhiko

last: Koseki

first: Toshinori

last: Nakayama

first: Koutaro

last: Yokote

first: Kiyoshi

last: Hirahara


title: Loss of CFTR Function in Macrophages Alters the Cell Transcriptional Program and Delays Lung Resolution of Inflammation

city: New Orleans

first: Guoshun

last: Wang

state: LA

address: 533 Bolivar Street

department: Microbiology, Immunology and Parasitology

institute: Louisiana State University Health Sciences Center

summary: This study performed single-cell RNA sequencing (scRNA-seq) analyses on lung-lavaged cells from macrophage-specific cystic fibrosis (Mac-CF) mice and congenic wild-type (WT) mice that had been challeged with Pseudomonas aeruginsa (PsA) for different time points (1, 3 and 5 days). Differences in global gene transcription were interrogated and compared. Results demonstrate that CFTR loss of function in macrophages altered the cell transcriptional program that mostly affected mitochondrial respiration, immune responses, and cellular antioxidant system. Thus, loss of CFTR function in macrophages influences the cell homeostasis, leading to a dysregulated cellular response to infection that may exacerbate CF lung disease.

overall_design: Mac-CF and WT mice were challenged with PsA (2.0x10e7 CFU) for different time points (Days 1, 3 and 5).  Then, the assigned animals were sacrificed and their lungs lavaged with PBS (3x 0.8 ml). The lung-lavaged cells were collected and immediately processed for sc-RNA-seq.

first: Dianne

last: Wellems

first: Yawen

last: Hu


title: RNA-seq reveals that deletion of Rasgef1b affects gene expression under basal and LPS-induced conditions in macrophages

city: Jersey City

first: Thomas

middle: S

last: Postler

state: New Jersey

address: 95 Greene Street 5-018

department: Discovery Science

institute: International AIDS Vaccine Initiative

summary: Ras guanine exchange factor member 1b (RasGEF1b) of the RasGEF/CDC25 domain-containing family is preferentially expressed by macrophages. However, information is lacking about its role in macrophage function. In this study, we generated mice with ubiquitous deletion of Rasgef1b and used RNA-seq-based transcriptomics to compare the global gene expression in wild-type and knockout primary bone-marrow-derived macrophages under basal conditions and after lipopolysaccharide (LPS) treatment. Transcriptional filtering identified several uniquely differentially expressed genes (DEGs) with significant differences in the mRNA levels between wild-type and knockout macrophages. In total, 49 and 37 DEGs were identified at baseline and in LPS-activated macrophages, respectively. Distinct biological processes were significantly linked to downregulated genes at the basal condition only, and largely included chemotaxis, response to cytokines, and positive regulation of GTPase activity. Importantly, validation by RT-qPCR revealed that expression of the genes Ch25h, Serpinb2, and Ptchd1, which were identified as downregulated after LPS stimulation, was also decreased under basal conditions in the knock-out cells. This study provides a small collection of genes that shows relative expression changes affected by the absence of RasGEF1b in macrophages. Thus, we present the first evidence that RasGEF1b mediates the regulation of both steady-state and signal-dependent expression of genes and indicate that this GEF plays a role in the maintenance of the basal transcriptional level in macrophages. The data establish a resource that warrants further investigation to understand the physiological role of RasGEF1b.

overall_design: RNA-seq with polyadenylated RNA of bone-marrow-derived macrophages, with and without LPS stimulation for 4 h, isolated from wildtype or Rasgef1b knockout mice. All samples in biological triplicate.

first: Aristóbolo

last: Silva

first: Isadora

last: de Oliveira

first: Heliana

last: de Barros Fernandes

first: Sérgio

last: Lima

first: Felipe

last: Leão

first: Sankar

last: Ghosh

first: Maria

middle: C

last: Souza

first: Warrison

last: Andrade

first: Ricardo

last: Gazzinelli


title: Epigenetic-focused CRISPR/Cas9 screen identifies ASH2L as a regulator of glioblastoma cell survival

city: Istanbul

first: Hamzah

last: Syed

address: Rumelifeneri Yolu

department: School of Medicine

country: Turkey

institute: Koç University

summary: Glioblastoma is the most common and aggressive primary brain tumor with dismal prognosis, highlighting an urgent need for novel treatment strategies. In this study, we investigated epigenetic regulators of glioblastoma cell survival through CRISPR/Cas9 based genetic ablation screens using our customized sgRNA library EpiDoKOL, which targets critical functional domains of chromatin modifiers. Screens conducted in multiple cell lines revealed ASH2L, a histone lysine methyltransferase complex subunit, as a major regulator of glioblastoma cell viability.  ASH2L depletion led to cell cycle arrest and apoptosis. RNA sequencing and Cut&Run sequencing together identified a set of cell cycle regulatory genes, such as TRA2B, BARD1, KIF20B, ARID4A, SMARCC1 that were downregulated upon ASH2L depletion. Mass spectrometry analysis demonstrated the interaction partners of ASH2L in glioblastoma cell lines as COMPASS family members including SETD1A, SETD1B, MLL1 and MLL2, however glioblastoma cells had differential dependency to COMPASS members for survival. The growth of ASH2L-depleted glioblastoma cells was markedly slower than controls in orthotopic in vivo models. TCGA analysis showed high ASH2L expression in glioblastoma compared to low grade gliomas and immunohistochemical analysis revealed significant ASH2L expression in glioblastoma tissues, attesting to its clinical relevance. Therefore, high throughput, robust and affordable screens with focused libraries, such as EpiDoKOL, holds great promise to enable rapid discovery of novel epigenetic regulators of cancer cell survival, such as ASH2L. Together, we suggest that targeting ASH2L could serve as a new therapeutic opportunity for glioblastoma.

overall_design: For RNA sequencing experiment, gASH2L and gNT transduced U373 cells were collected 14days later as triplicates and RNA isolation was carried out to investigate the differentialiy expressed genes between two groups. For CRISPR screens, U373 and T98G cells were transduced with EpiDoKOL virus with low MOI (MOI=4) as 400 cell/sgRNA representation. Following transduction, initial time point samples were collected as single samples for both cell line. After Puromycin selection, cells were separated into two sets; Set1 was seeded to 6-well plates as 400 cell per guide representation and Set2 was seeded to 15 cm plates with 8000 cells per guide representation. Two sets were passaged for 12 days and at day30, final time point samples were collected as dublicates for each cell line.

first: Ezgi

last: Ozyerli-Goknar

middle: Y

last: Kala

first: Ali

last: Aksu

first: Ipek

last: Bulut

first: Ahmet

last: Cingöz

first: Sheik

last: Nuzamuddin

first: Martin

last: Biniossek

first: H

middle: T

last: Timmers

first: Fidan

last: Seker-Polat

first: Tunc

last: Morova

first: Can

last: Aztekin

first: Sonia

last: Kung

first: Nurcan

last: Tuncbag

first: Mehmet

last: Gonen

last: Philpott

first: Adam

last: Cribbs

first: Ceyda

last: Acilan

first: Nathan

middle: A

last: Lack

first: Tamer

last: Onder

first: Tugba

last: Bagci-Onder