title: Human Microglial State Dynamics in Alzheimer's Disease Progression [RNA-seq]

summary:
In this study, we derived iPS-derived microglia-like cells (iMGLs) from a healthy donor (Sample: Control) CRISPR engineered to carry a dead CAS9 fused to the transcriptional repressor KRAB (dCAS9-KRAB) and treated these cultures with pre-formed amyloid beta 1-42 fibrils.
We then performed gene expression profiling analysis using data obtained from RNA-seq of treated and untreated cells at 4 different time points.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for amyloid fibril treated cells at baseline and following treatment.

first: Matheus

last: Victor


title: Effect of COPI depletion on senescence model systems

status: Public on Sep 28 2023

city: London

first: LMS

last: Bioinformatics Core

address: Hammersmith Hospital Campus, Du Cane Road

department: Bioinformatics Core

country: United Kingdom

institute: MRC London Institute of Medical Sciences

type: Expression profiling by high throughput sequencing

summary:
To examine effects of COPI depletion on senescence transcriptional signatures we generated cell lines (IMR90 or IMR90 ER:RAS cells) with inducible 2 COPI shRNAs or control shRNAs and induced to senesce with either treatment with Bleomycin or 4OHT treatment.
Transcriptional analysis was performed 3 days after induction of doxycycline inducible harpins and 10 days after senescence induction.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for IMR90/IMR90 ER:RAS cells and its KD derivatives (shRNA Control, shCOPB2_1, shCOPB2_2).

first: Domhnall

last: McHugh

first: Jesus

last: Gil

first: Sanjay

last: Khadayate


title: TFEB drives mTORC1 hyperactivation and kidney disease in Tuberous Sclerosis Complex

city: Boston

first: Damir

last: Khabibullin

state: MA

address: 20 Shattuck St

country: USA

institute: Brigham and Women's Hospital

summary: Tuberous Sclerosis Complex (TSC) is caused by germline TSC1 or TSC2 mutations, leading to hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1) and tumors in multiple organs including the brain, heart, lung (lymphangioleiomyomatosis), and kidney (angiomyolipoma and renal cell carcinoma). Previously, we found that TFEB is constitutively active in models of TSC. To determine the impact of TFEB in vivo, we generated two novel mouse models of TSC, resulting in premature death, in which kidney pathology was the primary phenotype. RNA sequencing revealed that lysosomal and proteasomal gene pathways were the most highly upregulated in the TSC2-deficient kidneys. Knockout of TFEB rescued both kidney pathology and overall survival in both models, indicating that TFEB is the primary driver of renal disease in TSC. Importantly, mTORC1 activity, which was elevated in the TSC2 knockout kidneys, was normalized by TFEB knockout.  Knockdown of Rheb or treatment of TSC2-deficient cells with Rapamycin paradoxically increases TFEB phosphorylation at the mTORC1-site (S211) and relocalizes TFEB from the nucleus to the cytoplasm via a Rag-dependent mechanism. Accordingly, treatment of TSC2 knockout mice with Rapamycin normalized lysosomal gene expression, similar to TFEB knockout, suggesting that the beneficial effects of Rapamycin in TSC are TFEB-dependent. These results change the view of the mechanisms leading to mTORC1 hyperactivation in TSC and may lead to novel therapeutic avenues for the treatment of TSC.

overall_design: To investigate the effects of Tfeb inactivation in the setting of Tsc2 deficiency in vivo we created two distinct mouse models: whole-body tamoxifen inducible CaggCre model and kidney-specific KSPCre, both in BL/6 background. Main comparison groups in both models included control mice (group 1), mice with inactivation of Tsc2 (group 2), mice with inactivation of Tfeb (group 3) and mice with inactivation of both Tsc2 and Tfeb (group 4). Gene expression changes were analyzed using RNA-sequencing in three biological replicates per experimental group in both models. Kidneys from CaggCre groups were harvested at P30; kidneys from KSPCre groups were harvested at P50. In addition, gene expression changes induced by pharmacologic inhibition of mTOR versus Tfeb inactivation were assesed in KSPCre model using 6 separate groups and 3 biological replicates per group: control mice treated with vehicle control (group 1); control mice treated with Rapamycin (1mg/kg/3 times per week, starting at P25 for a total of 11 injections) (group 2); mice with inactivation of Tsc2 treated with Vehicle control (group 3); mice with inactivation of Tsc2 treated with Rapamycin as in group 2 (group 4); mice with inactivation of both Tsc2 and Tfeb treated with Vehicle control (group 5); mice with inactivation of both Tsc2 and Tfeb treated with Rapamycin as in group 2 (group 6).

first: Nicola

last: Alesi

first: Dean

last: Rosenthal

first: Elie

last: Akl

first: Elizabeth

last: Henske


title: Human Microglial State Dynamics in Alzheimer's Disease Progression [scRNA-seq]

city: Cambridge

last: Sun

address: 455 Main St

department: EECS

institute: Whitehead Institute

summary: In this study, we derived iPS-derived microglia-like cells (iMGLs) from a healthy donor (Sample: Control) CRISPR engineered to carry a dead CAS9 fused to the transcriptional repressor KRAB (dCAS9-KRAB) and treated these cultures with the pro-inflammatory stimulant LPS (Sample: LPS).

overall_design: in vitro transcribed sgRNAs were delivered via lipofectamine  targeting HIF1A, FOXO3 and FOXP2 (Sample: LPS_CRISPRiA and B).


title: Effect of NMT inhibition on a model of NALFD in mice

summary:
To examine the effects of NMTi treatment on progression of NAFLD model, mice on chow or high-fat diet were treated with vehicle or additionally on high-fat diet treated with DDD86481.
Transcriptional analysis was performed after 19 weeks of high-fat diet with monthly treatments with 3 rounds of NMT inhibitor treatment.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for Vehicle treated Chow and High-fat diet mice compared to NMTi treated mice.


title: Ribosome profiling reveals the fine-tuned response of Escherichia coli to acid stress

city: Freiburg

first: Rick

last: Gelhausen

state: Baden Württemberg

address: Georges-Köhler-Allee 106

department: Department of Computer Science

country: Germany

institute: Albert-Ludwigs-University Freiburg

summary: The response to acidity is crucial for neutralophilic bacteria. Escherichia coli has a well characterized regulatory network to induce multiple defense mechanisms against excess of protons. Nevertheless, systemic studies of the transcriptional and translational reprogramming of E. coli to different acidic strengths have not yet been performed. Here, we used ribosome profiling and mRNA sequencing to determine the response of E. coli to pH 7.6, 5.8 and 4.4. Data were analyzed using the high-throughput HRIBO pipeline and previously undetected adaptations of E. coli to acid stress were found including up-regulation of glycerol catabolism and siderophore production, down-regulation of many membrane proteins and regulation by the transcriptional regulators YdeO, MhpR, IscR, and YdcI. Several examples of differential transcriptional and translational regulation of genes were identified as well as potential novel small open reading frames. These results expand the acid resistance network and provide new insights into the fine-tuned response of E. coli.

sample_organism: Escherichia coli str. K-12 substr. MG1655

overall_design: Comparison of genome-wide mRNA levels and translational activity at pH 7.6, 5.8 and 4.4 in LB medium.

first: Kilian

last: Schumacher

first: Rolf

last: Backofen

first: Willow

last: Kion-Crosby

first: Lars

last: Barquist

first: Kirsten

last: Jung


title: Combining targeting of the cancer-metabolome with cancer-associated stress antigens impacts transcriptomic heterogeneity, dynamics, and efficacy of engineered T-cells

status: Public on Sep 27 2023

city: Utrecht

first: Peter

last: Brazda

address: Heidelberglaan 100

department: Center for Translational Immunology

country: Netherlands

institute: University Medical Center Utrecht

summary: The majority of cancers cannot be efficiently targeted with engineered T cell strategies. In this line, we explored how and whether γδTCR-mediated cancer metabolome targeting can be combined with the attack of cancer-associated stress antigens, like NKG2D-ligands, in order to overcome limited targeting of many tumors, and at the same time skew heterogeneous transcriptomic signatures of engineered immune cells towards more active, and less exhausted phenotypes.Single cell transcriptomic analysis revealed that only NKG2D-4-1BBCD28TM chimera reprogrammed TEGs significantly, by skewing CD4+ TEG heterogeneity towards more adhesive, proliferative, cytotoxic, and less exhausted transcriptional signatures.

overall_design: TEGs (T cells engineered with defined gamma delta TCRs), 'CD28 chimeras' (TEGs expressing the NKG2D-CD28wt chimera), '41BB chimeras' (TEGs expressing the NKG2D-4-1BBCD28TM chimera),  '103_41BB chimeras'  (TEGs expressing the 103-4-1BB chimera)  were co-cultured together with RPMI 8226 cells in the presence of 10 µM PAM during 48h. The '_t' label marks samples that originated from a shared donor.   After incubation, cells were sorted by expression of γδTCR+CD4+ or γδTCR+CD8+ (pan-γδTCR-PE, clone IMMU510; CD4-PB, clone RPA-T4; CD8-PerCP Cy5.5, RPA-T8) into 384-well PCR plates using ARIA II. Cells were sequenced according to the SORT-seq method based on CEL-Seq2, and libraries were sequenced with paired end sequencing on Nextseq 500.

first: Patricia

last: Hernández-López

first: Eline

last: van Diest

first: Sabine

last: Heijhuurs

first: Inez

last: Johanna

first: Angelo

last: Meringa

first: Mara

middle: J

last: Nicolasen

first: Thomas

middle: A

last: Kluiver

first: Rosemary

last: Millen

first: Effrosyni

last: Karaiskaki

first: Trudy

last: Straetemans

first: Hans

last: Clevers

first: Remco

last: de Bree

first: Hendrik

middle: G

last: Stunnenberg

first: Weng

middle: C

last: Peng

first: Zsolt

last: Sebestyén

first: Dennis

middle: X

last: Beringer

first: Jürgen

last: Kuball


title: Enhancer somatic mutations with functional impact in lung cancers identified by leveraging the tissue-specific enhancer-target genes regulatory network [ChIP-seq]

city: Milan

first: Francesco

last: Ferrari

address: Via Adamello 16

country: Italy

institute: IFOM ETS, the AIRC Institute of Molecular Oncology

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Enhancers are non-coding genomic features that regulate the transcriptional output of their target genes. Their dysregulation has been associated with various diseases including cancer. However, identification and characterisation of non-coding mutations that are relevant for tumorigenesis and prognosis remain a major challenge. Here we present the detection and characterisation of enhancer mutations in a genome-wide analysis of three lung cancer subtypes. We carefully define lung tissue-specific enhancers across the genome by integrating novel experimental data and reference public datasets of epigenomic signatures from eight lung cell lines and primary tissue. We observe that in lung cancer, the mutation burden at enhancers and exons is similar, whereas their corresponding mutation signatures show stark differences. Our results indicate that recurrent mutations in individual enhancers significantly impact the expression of target genes with potential relevance for prognosis. We also demonstrate that the genes enriched for mutated enhancers recurrently converge on the regulation of key biological process and pathways.

overall_design: Genomewide lung specific enhancer profiling using epigenomic signatures

first: Judith

middle: M

last: Hariprakash


title: Enhancer somatic mutations with functional impact in lung cancers identified by leveraging the tissue-specific enhancer-target genes regulatory network

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

overall_design: Refer to individual Series


title: In vivo macrophage engineering reshapes the tumor microenvironment leading to eradication of liver metastases

first: Stefano

last: Beretta

address: Via Olgettina, 60

institute: San Raffaele Telethon Institute for Gene Therapy


title: In vivo macrophage engineering reshapes the tumor microenvironment leading to eradication of liver metastases [Visium]

summary: Liver metastases are associated with poor response to current pharmacological treatments, including immunotherapy. We describe a lentiviral vector (LV) platform to selectively engineer liver macrophages, including Kupffer cells and tumor-associated macrophages (TAMs), to deliver type I interferon (IFNα) to liver metastases. Gene-based IFNα delivery delays the growth of colorectal and pancreatic ductal adenocarcinoma liver metastases in mice. Response to IFNα is associated with TAM immune activation, enhanced MHC-II-restricted antigen presentation and reduced exhaustion of CD8+ T cells. Conversely, increased IL-10 signaling, expansion of Eomes CD4+ T cells, a cell type displaying features of type I regulatory T (Tr1) cells, and CTLA-4 expression are associated with resistance to therapy. Targeting regulatory T cell functions by combinatorial CTLA-4 immune checkpoint blockade and IFNα LV delivery expands tumor-reactive T cells, attaining complete response in most mice. These findings support a promising therapeutic strategy with feasible translation to patients with unmet medical need.

overall_design: Spatial transcriptomics to investigate, within the metastatic liver, the areas of preferential IFNa signaling on three distinct cohorts: (1) Control: Control LV-treated mice, (2) Partial responder: IFNa LV-treated mice with reduced tumor volume as compared to Control and, (3) Resistant: IFNa LV-treated mice with tumor volume similar to Control.

last: Kerzel

first: Giovanna

last: Giacca

first: Ivan

last: Merelli

first: Luigi

last: Naldini

first: Mario

middle: L

last: Squadrito


title: Integrin α3-dependent gene expression in Th17 cells generated during experimental autoimmune encephalomyelitis

city: Durham

first: Maria

last: Ciofani

state: NC

address: 207 Research Drive, 128 Jones Building

department: Integrative Immunobiology

institute: Duke University School of Medicine

summary: To understand the global gene expression changes in Itga3-deficient Th17 cells, we performed RNA-seq with fate-mapped Th17 cells isolated from inguinal lymph nodes of EAE-induced mice at day 7 post EAE induction.

overall_design: To acquire samples from the same host, naïve 2D2 CD4+ T cells isolated from Il17aCre/WT R26Zsgreen/WT (WTIl17a R26ZG) (CD45.1+) or Itga3fl/fl Il17aCre/WT R26Zsgreen/WT (KOIl17a R26ZG) mice were mixed at a 1:1 ratio then adoptively transferred Tcra-/- mice. On the following day, EAE was induced. ZG+ cells were FACS-sorted from iLN cells. 10 iLNs were pooled into one replicate, and three total replicates were prepared for each genotype.

first: Eunchong

last: Park

first: Alejandro

last: Barrera


title: Spatiotemporal modeling of chemoresistance evolution in breast tumors uncovers dependencies on SLC38A7 and SLC46A1

city: Ottawa

first: Julie

last: St-Pierre

state: Ontario

address: 451 Smyth Rd

department: Biochemistry, Microbiology and Immunology

country: Canada

institute: University of Ottawa

summary: In solid tumors, drug concentrations decrease with distance from blood vessels; however, the cellular adaptations accompanying gradated exposure of cancer cells to drugs are largely unknown. We thus profiled the changes in gene expression through a gradient of concentrations for two anthracyclines commonly used to treat breast cancer, doxorubicin and epirubicin.

overall_design: MCF7 cells were exposed to incremental doses of doxorubicin or epirubicin (1,5,10,15,25,50, and 100 nM). Control cells for each drug were kept in culture with DMSO. Three biological replicates are available for each dose, for each drug.

first: Yannick

last: Audet-Delage


title: In vivo macrophage engineering reshapes the tumor microenvironment leading to eradication of liver metastases [scRNA-seq]

overall_design: Single-cell transcriptomics on metastatic liver in three distinct cohorts: (1) Control: Control LV-treated mice, (2) Partial responder: IFNa LV-treated mice with reduced tumor volume as compared to Control and, (3) Resistant: IFNa LV-treated mice with tumor volume similar to Control.

first: Thomas


title: Uncoupling the distinct functions of HP1 proteins during heterochromatin establishment and maintenance

status: Public on Sep 26 2023

city: Waltham

first: Melissa

last: Seman

address: 415 South St

institute: Brandeis University

summary: H3K9 methylation (H3K9me) marks transcriptionally silent genomic regions called heterochromatin. A conserved class of HP1 proteins are critically required to establish and maintain heterochromatin. HP1 proteins bind to H3K9me, recruit factors that promote heterochromatin formation, and oligomerize to form phase-separated condensates. We do not understand how HP1 protein binding to heterochromatin establishes and maintains transcriptional silencing. Here, we demonstrate that the S.pombe HP1 homolog, Swi6, can be completely bypassed to establish silencing at ectopic and endogenous loci when an H3K4 methyltransferase, Set1 and an H3K14 acetyltransferase, Mst2 are deleted. Deleting Set1 and Mst2 enhances Clr4 enzymatic activity, leading to higher H3K9me levels and increased spreading. In contrast, Swi6 and its capacity to oligomerize were indispensable during epigenetic maintenance. Our results demonstrate the role of HP1 proteins in regulating histone modification crosstalk during establishment and identifies a genetically separable function in maintaining epigenetic memory.

overall_design: Chromatin immunoprecipitation sequencing (ChIP-seq) for H3K9me2 and H3K9me3

first: Alexander

last: Levashkevich

first: Ajay

last: Larkin

first: Fengting

last: Huang

first: Kaushik

last: Ragunathan


title: Effect of NMTi on senescence model systems

first: LMS

last: Bioinformatics Core

summary:
To examine effects of NMT inhibition on senescence transcriptional signatures we treated IMR90 ER:RAS cells with either IMP1088 (300nM) or DDD86481 (1.5uM) for 72h 7 days after induction with 4-OHT treatment.
Transcriptional analysis was performed 3 days after drug treatment and 10 days after senescence induction.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for IMR90 ER:RAS cells and its drug treatments.

first: Domhnall

last: McHugh

first: Jesus

last: Gil

first: Sanjay

last: Khadayate


title: Transcriptional heterogeneity overcomes super-enhancer disrupting drug combinations in multiple myeloma [RNA-seq]

status: Public on Sep 25 2023

city: Atlanta

first: Benjamin

last: Barwick

state: GA

address: 1365 Clifton Rd. NE WCI-C 4th Floor Benches 36-37

department: Hematology and Medical Oncology

institute: Emory University

summary: Multiple myeloma (MM) is a malignancy that is often driven by MYC and that is sustained by IRF4, which are upregulated by super-enhancers.  IKZF1 and IKZF3 bind to super-enhancers and can be degraded using immunomodulatory imide drugs (IMiDs).  Successful IMiD responses downregulate MYC and IRF4; however, this fails in IMiD-resistant cells.  MYC and IRF4 downregulation can also be achieved in IMiD-resistant tumors using inhibitors of BET and EP300 transcriptional coactivator proteins; however, in vivo these drugs have a narrow therapeutic window.  By combining IMiDs with EP300 inhibition, we demonstrate greater downregulation of MYC and IRF4, synergistic killing of myeloma in vitro and in vivo, and an increased therapeutic window.  Interestingly, this potent combination failed where MYC and IRF4 expression was maintained by high levels of the AP-1 factor BATF.  Our results identify an effective drug combination and a previously unrecognized mechanism of IMiD resistance.

overall_design: To determine the transcriptional changes induced by the ImmunoModulatory imide Drug (IMiD) pomalidomide (POM) and the EP300 inhibitor GNE-781 (G781) alone or in combination on multiple myeloma JJN3 and KMS11 cells with (+BATF) or with out (+eGFP) BATF overexpression.

first: Seth

last: Welsh

first: P

last: Bergsagel


title: A molecular switch governs mitochondrial metabolism and cellular senescence during aging

city: Hong Kong

first: Tom

last: Cheung

address: Clear Water Bay, Kowloon

department: Division of Life Science

country: Hong Kong

institute: Hong Kong University of Science and Technology


title: Plant structural and storage glucans trigger distinct transcriptional responses that modulate the motility of Xanthomonas pathogens

city: Campinas

first: Joaquim

last: Martins Junior

state: SP

address: Rua Giuseppe Maximo Scolfaro 10000

department: Brazilian Biorenewables National Laboratory LNBR

country: Brazil

institute: Brazilian Center for Research in Energy and Materials CNPEM

summary: Some phytopathogens are outfitted with a broad and diverse repertoire of enzymatic systems that enable the breakdown and utilization of host polysaccharides as a source of carbon, energy, and stimuli. However, the functional assignment of these enzymatic systems and the influence of their products on modulating pathogen behavior during host colonization is yet poorly comprehended. In this study, we performed RNA-seq analyses to provide a comprehensive, genome-wide view of the transcriptional response of the model phytopathogen Xanthomonas citri pv. citri 306 (hereafter X. citri 306) to cellobiose, a component of structural β-1,4-glucans (majorly cellulose), and storage α-glucans, seeking to better understand how they are assimilated and the impacts of their sensing on bacterial behavior and physiology. Structural β-1,4-glucans and storage α-glucans (starch) are spatially discretized in the plant cell, therefore representing spatiotemporal references for the bacterium during host colonization. Combining transcriptional and genome mining analyses with gene knockout and cell motility assays, we show that X. citri 306 harbors molecular systems for the breakdown and assimilation of these carbohydrates, revealing that cellobiose upregulates genes related to flagellum assembly and type IV pili, inducing a higher-motility state. In contrast, starch suppresses genes related to chemotaxis, flagellum assembly and biofilm dispersion, decreasing motility. Taken together, these results unravel that besides using structural β-glucans and storage α-glucans as sources of carbon and energy, Xanthomonas bacteria also sense them, adapting its metabolism and controlling transitions between higher and lower motility states for successful host colonization.

relation:
Reanalysis of: GSM4825435
Reanalysis of: GSM4825436
Reanalysis of: GSM4825437
Reanalysis of: GSM4825438
Reanalysis of: GSM4825439
Reanalysis of: GSM4825440

overall_design: mRNA profiles of  X. citri grown on minimal media XVM2m containing 0.05% (w/v) cellobiose or starch instead of fructose and sucrose relative to the media XVM2m containing glucose as a reference.

first: Isabela

last: Bonfim

first: Douglas

last: Paixão

first: Gabriela

middle: F

last: Persinoti

first: Priscila

middle: O

last: Giuseppe

first: Mario

middle: T

last: Murakami


title: Transcriptional heterogeneity overcomes super-enhancer disrupting drug combinations in multiple myeloma [ChIP-seq]

overall_design: To determine binding sites of IRF4 with and without BATF2 overexpression

first: P Leif


title: Genome wide mRNA targets of CPEB4 in muscle stem cells

summary: We performed CPEB4 RIP-seq on freshly isolated muscle stem cells. We found that CPEB4 associated genes are enriched in metabolic relevant pathways such as some mitochondrial protein coding genes

overall_design: Two biological replicates of CPEB4 RIP-seq compared with two sets of IgG RNA-seq. BGI DNBSEQ-G400 Sequencer. PE100 sequencing set

middle: H

last: CHEUNG

first: Wenshu

last: ZENG


title: Selective DNA-PK inhibition enhances chemotherapy and ionizing radiation activity in soft-tissue sarcomas

city: Bordeaux

first: Amandine

last: Crombe

address: 229 cours de l'Argonne

country: France

institute: Institut Bergonié

summary: Purpose: Patients with advanced soft-tissue sarcomas (STSs) exhibit a poor prognosis and have few therapeutic options. DNA-dependent protein kinase catalytic subunit (DNA-PK) is a multifunctional serine—threonine protein kinase that plays a crucial role in DNA double-strand damage repair via nonhomologous end joining (NHEJ). Experimental design: To investigate the therapeutic potential of DNA-PK targeting in STS, we first evaluated the prognostic value of DNA-PK expression in two large cohorts of patients with STS. We then used the potent and selective DNA-PK inhibitor AZD7648 compound to investigate the antitumor effect of the pharmacological inhibition of DNA-PK in vitro via MTT, apoptosis, cell cycle, and proliferation assays. In vivo studies were performed with patient-derived xenograft models to evaluate the effects of AZD7648 in combination with chemotherapy or ionizing radiation on tumor growth. The mechanisms of sensitivity and resistance to DNA-PK inhibition were investigated by using a genome-wide CRISPR-Cas9 positive screen. Results: DNA-PK overexpression is significantly associated with poor prognosis in patients with sarcomas. Selective pharmacological inhibition of DNA-PK strongly synergizes  with radiation- and doxorubicin-based regimen in sarcoma models. By using a genome-wide CRISPR-Cas9 positive screen, we identified genes involved in sensitivity to DNA-PK inhibition. Conclusion: DNA-PK inhibition deserves clinical investigation to improve response to current therapies in patients with sarcoma

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
synthetic construct

overall_design: For the CRISPR screen, we utilized the human knockout pooled library GeCKO V2 (Addgene 1000000048), consisting of 122,411 sgRNAs targeting 19,050 genes. In this experiment, 240 million IB114-cas9 cells were infected at a multiplicity of infection (MOI) of 0.3 to achieve a 500X coverage. The cells were then selected for three days at 0.75µg/mL of Puromycin. For the DNAPK sensitivity screen, we treated 30.106  cells with DMSO, and an equal number of cells (30.106) were treated with 40µM of DNAPK inhibitor. The cells were subjected to treatment for 72 hours before collecting them for DNA extraction. Upon comparing the treated cells to the control, the survival rate was approximately 20%. Genomic DNA was extracted using the DNeasy Blood and Tissue kit (Qiagen ID: 69504) following the manufacturer's protocol. The entire genomic DNA was sequenced by Nucleus Biotech. SgRNA was amplified, and Next-Generation Sequencing (NGS) was performed on the Illumina NextSeq platform, targeting 60 million reads.

first: Audrey

last: Laroche-Clary

first: Coralie

last: Josensi


title: Enhancer somatic mutations with functional impact in lung cancers identified by leveraging the tissue-specific enhancer-target genes regulatory network [ATAC-seq]

first: Francesco

last: Ferrari

first: Judith

last: Hariprakash


title: Endogenous B cell TACI-Short-APRIL signaling

status: Public on Sep 22 2023

city: New York

first: Miguel

last: Fribourg

state: NY

address: 1 Gustave Levy Pl

department: Medicine

institute: Icahn School of Medicine at Mount Sinai

summary: Here we show that TACI-S increases its expression with maturation and colocalizes in the intracellular compartment with the ligand APRIL. APRIL downregulation in human B cells, eliminated class switch. These studies suggest the importance of intracellular APRIL together with TACI-S in maintaining humoral immunity.

overall_design: TACI isoforms, long and short, are coexpressed in human B cells.To analyze independently the functional effects of TACI isoforms, we transduced each one  in human BJAB B cell line and analyzed their transcriptional program in resting condition

first: Yolanda

last: Carmona


title: Pax7 ablation model enables evaluation of stem cell niche formation in the absence of mouse Pax7 cell competition

city: Irvine

first: Michael

last: Hicks

state: CA

address: 845 Health Sciences Road, Room 3101

institute: University of California, Irvine

summary: Sequencing files, and processed data correspond to Figure 3 of Regenerating Human Skeletal Muscle Forms an Emerging Niche In Vivo to Support PAX7 cells. Data contains single nucleus RNA seq from Pax7 ablated mdx-NSG mice, control mdx-NSG mice, with or without an injury. These mice offer superior engraftment by human skeletal muscle stem and progenitor cells. Pax7 ablation model enables evaluation of human stem cell niche formation in the absence of mouse Pax7 cell competition. Included in this dataset is everything required to run independent analysis. We have included raw Fastq files and processed files that are ready to be uploaded into Loupe Browser or R-Studio/Seurat V3. To summarize Cloupe Files can be uploaded to Loupe Browers and the BC_Matrix.H5 files can be uploaded directly to R. Furthermore, the RDS files are processed R-program files that can be immediately uploaded into R for analysis. We have included an example R script for users to follow and a few PDFs of excepted UMAPs using all combined files at PCA 35.

overall_design:
Hicks and Pyle crossed mdx-NSG mice to Rosa-DTX and Pax7-Cre mice to generate homozygous, congenic strains. Mice were then crossed to generate F1 heterozygous mdx-NSG Pax7-DTX mice that resulted in >80% mouse satellite cell ablation after tamoxifen treatment. 12-week-old female litter mate mdx-NSG Pax7-DTX mice were split into the following groups:
AP1. Pax7 wildtype, No injury
AP2. Pax7 ablation, No injury
AP3. Pax7 wildtype, Injury
AP4. Pax7 ablation, Injury
To generate data, four groups of mice were either given tamoxifen chow or normal chow for 7 days, at which time mice were given an injury with Barium Chloride (BaCl2) or non-injured. To induce Pax7 ablation, 400mg/kg tamoxifen citrate chow (Envigo) was given to mice for 7 days. Pax7 wildtype were given standard chow. To induce injury, mice were then injected intramuscularly in the TA with 1.2% BaCl2 diluted in 50uL 0.9% NaCl Saline. All mice were fed standard chow following injury. Mice were sacrificed at 8 days post injury. Control, non-injured mice were sacrificed at the same time point so that all mice were age-matched. Nuclei were immediately from freshly dissected skeletal muscle isolated following a modified 10X Genomics Frankenstine protocol. All nuclei were collected using the On-Chip Flow Cell gentle sorter to increase nuclei purity from muscle debris. Nuclei were immediately taken for processing using 10X Genomics library construction and sequencing on an Illumina Nova6000. The provided Metrics Summary describes the number of nuclei and sequencing reads for each group. Fastq files were aligned CellRanger and processed using Seurat.

first: April

last: Pyle


title: A single-cell atlas of the peripheral immune response in patients with influenza A virus infection

status: Public on Sep 21 2023

city: Hefei

first: Yin

last: Zhang

state: Anhui

address: No. 218 Jixi Rd

country: China

institute: First Affiliated Hospital of Anhui Medical University

summary: Influenza A virus (IAV) continues to be a critical global health problem, especially for vulnerable populations such as children and pregnant women. While certain circulating cell subsets are thought to differentially modulate the immune response to IAV infection, the nature and function of these subsets is unclear. Our findings provide a comprehensive understanding of the cellular subtypes and molecular characteristics of PBMCs across different populations with IAV infection. By revealing significant differences in immune responses to IAV among these populations, our study could aid in the development of immune intervention measures to control IAV and improve patient outcomes.

overall_design: We performed single-cell RNA sequencing (scRNA-seq) on peripheral blood mononuclear cells (PBMCs) isolated from healthy controls (HC), pregnant healthy controls (PHC), infected adults (IA), infected pregnant women (PI), and infected children (IC) using the Singleron Matrix® platform.

first: Lu

last: Zong

first: Ying

last: Huang

first: Yuanhong

last: Xu


title: PD-1 defines a distinct, functional, tissue-adapted state in Vδ1+ T cells with implications for cancer immunotherapy

first: Shraddha

last: Kamdar

address: Guys Hospital,Great Maze Pond, London Bridge

department: Peter Gorer Department of Immunobiology

institute: King’s College London

summary: Checkpoint inhibition (CPI), particularly that targeting the inhibitory co-receptor, programmed cell death protein (PD-1), has transformed cancer care. Although CPI can de repress cancer antigen-specific αβ T cells that ordinarily show PD-1-dependent exhaustion, it can also be efficacious against cancers evading αβ T cell recognition. In such settings γδ T cells have been implicated, but the immunological significance of PD-1 expression by tissue associated human Vδ1+ cells remains uncharacterised. Here we demonstrate that a transcriptional signature of intratumoral Vδ1+ cells predicts response to anti-PD-1 in patients with melanomas of relatively low neoantigen load. Moreover, by employing a  protocol yielding substantial numbers of human skin γδ cells, we show that PD-1+ Vδ1+ cells display a transcriptomic programme of tissue-residence, survival/self-renewal, and functional competence distinct from the canonical exhaustion programme of co-located PD- 1+ CD8+ αβ T cells. Indeed, skin PD-1+ Vδ1+ cells retained effector responses to T cell receptor signalling that were inhibitable by PD-1 engagement and partially derepressed by CPI. The biological and therapeutic implications are both discussed.

overall_design: In the study presented here, 785 human genes were profiled using NanoString's nCounter Immune Exhaustion Panel in FACS sorted PD1+ CD8 T cells (n=6 biological replicates/group), PD1- CD8 T cells (n=9 biological replicates/group), Vd1+ PD1+ gamma delta T cells (n=8 biological replicates/group) and Vd1+ PD1- gamma delta T cells (n=8 biological replicates/group) obtained from human skin samples cultured for a total of 6 weeks in human IL-2 and IL-15 (3 weeks 'on grid' isolation phase and 3 weeks 'off grid' expansion phase. See growth protocol described in line 77 for details).

first: Daniel

last: Davies

first: Richard

last: Woolf

first: Iva

last: Zlatareva

first: Maria

last: Iannitto

first: Cienne

last: Morton

first: Yasmin

last: Haque

first: Hannah

last: Martin

first: Olga

last: O'Neill

first: Oliver

last: Nussbaumer

first: Adrian

last: Hayday

last: Wu


title: Spatial RNA sequencing analysis confirm ACTC1+ myofibers support a transplanted SMPC pool

first: Michael

last: Hicks

summary: Spatial transcriptomics analysis of three different experiments (describe below) identify regenerating ACTC1+ myofibers as a supportive cell type for transplanted SMPCs. Data correspond to Figures 6-7 and Extended Data Figures 8-10 in Hicks et al., "Regenerating Human Skeletal Muscle Forms an Emerging Niche In Vivo to Support PAX7 cells".

overall_design:
Experiment 1: Spatial RNAseq of fetal skeletal muscle tissue. Fetal human week 20 quadriceps, hamstring, and gastrocnemius skeletal muscle were sectioned onto slides and prepared for spatial RNAseq analysis.  Morphology markers included ACTC1+ myofibers and DAPI+ACTC1- cells adjacent to myofibers, which would likely represent a large population of PAX7 cells.  A third group DAPI+ACTC1- cells away from myofibers was also sequencing which likely represents connective tissues, tenocyotes, or other non-myogenic tissue.  This set of tissues was iniatially used as a positive control while optimizing spatial RNA sequencing parameters that would later be used for the engraftment experiments.
Experiment 2: Spatial RNAseq of engrafted fetal SMPCs in vivo. Primary fetal SMPCs were FACS-enriched from fetal week 18 skeletal muscle using CD45-CD235a-ERBB3+NGFR+. 500,000 SMPCs were then transplantated into mdx-DBA/2-NSG PAX7-knockout mice. Prior to transplantation, mice were subjected to BaCl2 injury to tibialis anterior muscle, and adminstered tamoxifen to inducibly ablate endogenous Pax7+ cells. Mice were terminated and had their tibialis anteriors sectioned after 2months, 4months, and a final group was subjected to a BaCl2 reinjury at the 2month mark but collected and sectioned at the 4month mark. These tissue samples were then processed for spatial RNAseq accordingly.
Experiment 3: Spatial RNAseq of engrafted genetically modified hPSC-derived SMPCs in vivo with and without AP1903 treatment. H9 hPSCs were genetically edited to inducibly ablate ACTC1+ cells by inserting an ACTC1-iCasp9 into the 3' UTR of ACTC1. After isolating a clonal knock-in line, the hPSCs was differentiated to skeletal muscle in vitro, and SMPCs were enriched using flow cytrometry HNK1-ERBB3+NGFR+. The SMPCs were expanded in vitro for 3 days and 1,000,000 SMPCs were then transplantated into mdx-DBA/2-NSG PAX7-knockout mice. and before transplantation into PAX7-Cre mice. Prior to transplantation, mice were subjected to BaCl2 injury to tibialis anterior muscle, and adminstered tamoxifen to inducibly ablate endogenous Pax7+ cells. After transplantation, mice were treated with the AP1903 by IP injections starting at two weeks, and continued every two weeks, throughout the 90 days to ablate ACTC1+ myofibers. Control mice were treated with 0.1% DMSO in saline.  After 90 days tissues were sectioned and tissue samples were processed for Spatial RNAseq.

first: Ben

last: Clock

last: Pyle


title: Simultaneous estimation of gene regulatory network structure and RNA kinetics from single cell gene expression

first: Christopher

last: Jackson

address: 101 Avenue of the Americas

department: Center for the Genomics of Neurodegenerative Disease

institute: New York Genome Center

summary: Cells respond to environmental and developmental stimuli by changing their transcriptomes through both regulation of transcription rate and regulated mRNA decay. These biophysical properties determine the transcriptional state of a cell, but measuring them requires metabolic RNA labeling (e.g. 4-thiouracil pulse-chase) to separate RNA decay from synthesis rates. We approach this problem by sequencing individual Saccharomyces cerevisiae cell transcriptomes by continuously sampling from a population without metabolic labeling.  Using this continuous-sampling system, we measure expression in 180,000 individual cells both prior to and in response to rapamycin treatment. The rates of change for each transcript can be calculated on a per-cell basis to give smooth, biologically relevant, estimates of RNA velocity. We then train deep learning models to use this transcriptomic and velocity information to make time-dependent predictions about RNA biophysics, and to infer causal regulatory relationships between transcription factors and their genes.

overall_design: To minimize the interval between observed data points, we continually pump culture into excess saturated ammonium sulfate to collect cells, fixing transcriptomes at the time of collection. We use this system to quantify the transcriptomic response to rapamycin on a continuous basis, collecting 173,361 cells across 8 defined 10-minute time intervals, from two biological replicate experiments. Samples were collected continuously into 50 mL conicals containing 20 mL RNAlater. Sample collection conicals were changed every 10 minutes and the collected samples were placed on ice. After 20 minutes, 200 ng/mL rapamycin was added to the stirring culture. Sampling continued for 60 additional minutes, yielding 2 10-minute sample pools without rapamycin treatment and 6 10-minute sample pools with rapamycin treatment.

first: Ina

last: Suresh

first: Angela

last: Hickey

first: David

last: Gresham


title: Impact of myeloid cell specific deletion of EGFR on cardiac Cd11b+ non-myocyte gene expression

city: 3601 Spruce Street

first: Yan

last: Zhou

state: PA

address: 333 Cottman Ave

department: Bioinformatics

institute: Fox Chase Cancer Center

summary: Epidermal Growth Factor Receptor (EGFR) is a critical factor regulating numerous aspects of cell biology. Though it is widely recognized as an oncogene, EGFR has been implicated in cardiovascular health, and has been shown to have unique cell specific roles in the heart. Macrophages and other myeloid cells play key roles in normal cardiac physiology and post injury pathological outcomes. Myeloid cell EGFR has been implicated in disease, though its role in the normal or failing heart has yet to be established. Here, we crossed EGFR floxed mice with LysM Cre transgenics to specifically delete EGFR in Cd11b+ myeloid cells. We then isolated non-cardiomyocytes and subject them to Cd11b microbeads and subsequent column concentration to obtain cardiac Cd11b+ non-myocyte myeloid cells. We used samples from floxed EGFR controls, and EGFR specific myeloid knockouts. In comparisson to control, myeloid EGFR knockouts had 763 differential transcripts when consdering p adjusted less than 0.05 and log2foldchange greater than absolute value 1.5.

overall_design: To generate 1 biological sample, roughly 500 mg of heart tissue (5-6 hearts) was digested in 12 mls of RPMI media using 450 u/ml of Collagenase II and 60 u/ml of hyaluronidase. Non-myocyte cells were then collected, washed with PBS and subject to Miltenyi "Debris Removal Kit" to separate cells from Debris. Fresh cells were then washed in PBS and subject to Miltenyi "Cd11b Microbeads" and "MiniMacs Separator" to concentrate Cardiac Cd11b+ non-myocyte myeloid cells. In total, three biological replicates were analyzed for each of the 2 genotypes.

first: Douglas

last: Tilley


title: Amino acid intake strategies define pluripotent cell states

first: Lydia

last: Finley

address: 430 E 67th St

department: Cell Biology Program

institute: Memorial Sloan Kettering Cancer Center

summary: Mammalian preimplantation development is associated with marked metabolic robustness, and embryos can develop under a wide variety of nutrient conditions, including even the complete absence of soluble amino acids. Here we show that mouse embryonic stem cells (ESCs) capture the unique metabolic state of preimplantation embryos and proliferate in the absence of several essential amino acids. Amino acid independence is enabled by constitutive uptake of exogenous protein through macropinocytosis, alongside a robust lysosomal digestive system. Following transition to more committed states, ESCs reduce digestion of extracellular protein and instead become reliant on exogenous amino acids. Accordingly, amino acid withdrawal selects for ESCs that mimic the preimplantation epiblast. More broadly, we find that all lineages of preimplantation blastocysts exhibit constitutive macropinocytic protein uptake and digestion. Taken together, these results highlight exogenous protein uptake and digestion as an intrinsic feature of preimplantation development and provide insight into the catabolic strategies that enable embryos to sustain viability before implantation.

overall_design: RNA-seq of mouse embryonic stem cells expressing a tamoxifen-responsive version of murine Tfe3. Tamoxifen induces nuclear translocation of Tfe3.

first: Pavlina

middle: K

last: Todorova

first: Katrina

middle: I

last: Paras

middle: W


title: APS1 and celiac patients reveal autoimmune amelogenesis imperfecta

city: Rehovot

first: Jakub

last: Abramson

address: 234 Herzl st.

department: Department of Immunology

country: Israel

institute: Weizmann Institute of Science

summary: To investigate the dependence of amelogenesis imperfecta-related gene expression and ameloblast-related gene expression on Aire in the thymus, mTEChi from Aire+/+ and Aire-/- were sorted and subjected to bulk RNAseq.

overall_design: mRNA profiles of mature medullary thymic epithelial cells (mTEChi) from young (4.5 weeks old)  NOD.Aire-/- mice ,were generated by deep sequencing, using the MARSseq method.

first: Abramson

last: Jakub

first: Gruper

last: Yael


title: Single-cell RNA sequencing reveals the heterogeneity of IL-10 producing regulatory B cells in lupus-prone mice

status: Public on Sep 20 2023

city: Charlottesville

first: Loren

last: Erickson

state: Virginia

address: 345 Crispell Drive

department: Department of Microbiology, Immunology, and Cancer Biology

institute: University of Virginia

summary: B cells have both pathogenic and protective roles in autoimmune disease, including systemic lupus erythematosus (SLE). Deficiencies in the number or immunosuppressive function of IL-10 producing regulatory B cells (Bregs) can cause exacerbated autoimmune inflammation. However, the exact role of Bregs in lupus pathogenesis has not been elucidated. We carried out gene expression by scRNA-seq to characterize differences in splenic Breg subsets and molecular profiles through stages of disease progression in lupus-prone mice.

overall_design: Splenic CD19+ IL-10 producing B cells (Bregs) were isolated from female MRL/lpr mice at the pre-disease (6-8 weeks) and active-disease (10-12 weeks) stages and analyzed by scRNA-seq

first: Razan

last: Alajoleen

first: Xin

last: Luo

first: Andrea

middle: R

last: Daamen

first: Peter

middle: E

last: Lipsky


title: Detection of queuosine and queuosine precursors in tRNAs by direct RNA sequencing

status: Public on Sep 19 2023

city: Berlin

first: Yu

last: Sun

address: Philippstr. 13

department: Institut für Biologie

institute: Humboldt-Universität zu Berlin

summary: Queuosine (Q) is a complex tRNA modification found at position 34 of four tRNAs with a GUN anticodon, and it regulates the translational efficiency and fidelity of the respective codons that differ at the Wobble position. In bacteria, the biosynthesis of Q involves two precursors, preQ0 and preQ1, whereas eukaryotes directly obtain Q from bacterial sources. The study of queuosine has been challenging due to the limited availability of high-throughput methods for its detection and analysis. Here, we have employed direct RNA sequencing using nanopore technology to detect the modification of tRNAs with Q and Q precursors. These modifications were detected with high accuracy on synthetic tRNAs as well as on tRNAs extracted from Schizosaccharomyces pombe and Escherichia coli by comparing unmodified to modified tRNAs using the tool JACUSA2. Furthermore, we present an improved protocol for the alignment of raw sequence reads that gives high specificity and recall for tRNAs ex cellulo that, by nature, carry multiple modifications. Altogether, our results show that such 7-deazaguanine-derivatives are readily detectable using direct RNA sequencing. This advancement opens up new possibilities for investigating these modifications in native tRNAs, furthering our understanding of their biological function.

sample_organism:
Escherichia coli
Schizosaccharomyces pombe
synthetic construct

overall_design: To investigate the detection of Q by direct RNA nanopore sequencing, in vitro-transcribed tRNAAsp, tRNAAsn, tRNAHis, and tRNATyr with or without Q-modification were subjected to direct RNA sequencing (nanopore). Total tRNA purified from S. pombe, either unmodified or Q-modified in vivo, was sequenced, and the experiment was conducted with three biological samples per condition. Additionally, total tRNA from E. coli wt, queA∆, queF∆, and tgt∆ was subject to sequencing to investigate the detection of Q and Q precursors preQ1 and preQ0. We then analyzed the error rates of each samples using data generated from nanopore sequencing using JACUSA2.

first: Ann

last: Ehrenhofer-Murray


title: Effect of EZH2 inhibition on NUT carcinoma gene expression over time

city: boston

first: Chris

last: French

address: 75 francis st.

department: Pathology

institute: BWH

summary: To determine roughly the time point at which most transcriptional changes occur in response to tazemetostat (taz, aka EPZ-6438 (EPZ)) treatment of NUT carcinoma cells, we performed RNAseq over a time course.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for PER-403 and 10-15 NUT carcinoma cells treated with taz for 36h, 72h, 96h, and 144h.

first: Christopher

first: Dora

last: Huang


title: EMA2 is required for target-directed scnRNA degradation.

status: Public on Sep 18 2023

city: Montpellier

first: Kazufumi

last: Mochizuki

address: 141 rue de la Cardonille

institute: Institute of Human Genetics, CNRS

type: Non-coding RNA profiling by high throughput sequencing

summary: Piwi-associated small RNAs of the ciliated protozoan Tetrahymena uses such mechanism to induce heterochromatin formation followed by programmed DNA elimination. In this process, target-directed small RNA degradation (TDSD) of small RNAs complementary to the somatic genome enables small RNAs to specifically target the germline-limited sequences for DNA elimination. The data here indicate that the SUMO E3 ligase Ema2 is required for the accumulation of long non-coding RNAs (lncRNAs) from the somatic genome and thus for TDSD.

overall_design: High-throughput sequencing and analyses of small RNAs were performed as described (Mutazono et al 2019). The 2020 version of the MAC genome assembly (Sheng et al. 2020) was obtained from Tetrahymena Genome Database and fragmented into 10 kb pieces. Each 10 kb fragment that contains longer than 3 kb mappable sequence was used as a MDS tile (total 10,235 tiles). Each Type-A IES (Noto et al. 2015) was used as an IES tile (total of 4,691 tiles). Normalized numbers (RPKM [reads per kilobase of unique sequences per million]) of 26- to 32-nt small RNAs that uniquely matched one of the MDS and IES tiles were counted.

first: Salman

last: Shehzada

first: Tomoko

last: Noto


title: Diffrential expression of urinary bladder cells microRNAs ( miRs) in cyclophosphamide induced cystitis

city: Memphis

first: Udai

middle: P

last: Singh

state: TN

address: 881 Madison Avenue

department: Pharmacy

institute: University of Tennessee Health Science Center

summary: During cyclophosphamide (CYP) induced ctstitis progression differential expression of microRNAs (miRs) play a role in urinary bladder inflammation and detrusor function. We identified eight UBs miRs that are known to be involved in various other bladder pathologies which exhibited altered expression in CYP mice and e predicted to have a role in inflammation and smooth muscle function (miR-34c-5p, -34b-3p, -212-3p, -449a-5p, -21a-3p, -376b-3p, -376b-5p and -409-5p).

overall_design: We observed several UBs miRs including miR-34c-5p, miR-21a-3p, miR-34b-3p miR-212-3p, miR-449a-5p, miR-376b-3p, miR-376b-5p, and miR-409-5p, expression in CYP-induced as compared to control, suggesting that IC may result from altered miRs expression mediated CYP induction.

first: Mousumi

last: Mandal

first: Ahmed

last: Rakib

first: M Dennis

last: Leo

first: Md Abdullah

last: Al Mamun


title: Identification of Malat1-chromatin interaction by ChIRP-seq

city: Houston

first: Dhiraj

last: Kumar

state: Texas

address: 1881 East Road

department: Cancer Biology

institute: The University of Texas MD Anderson Cancer Center

summary: We performed Malat1 pulldown using biotin labeled Stellaris Oligo in 4T1-TGL WT cells.

overall_design: The enrichment of Malat1 was confirmed from pulldown samples using qRT-PCR and sent for sequencing at MDACC Sequencing Core Facilities.

first: Filippo

last: Giancotti

first: Chang-Jiun

last: Wu


title: Identification of the role of tumor intrinsic Malat1 in regulation of immune microenvironment [scRNA-seq]

summary: We have injected 4T1-TGL breast cancer cells into Balbc mice through tail vein. In order to see immune cells infiltration, we have isolated CD45+ cells from these mice lungs at different time points by FACS. These isolated cells were further subjected for scRNA-seq. For base line we have isolated CD45+ cells from Naive balbc mice.

overall_design:
Lung CD45+ cells were isolated for scRNA-seq and sent for sequencing at MDACC Sequencing Core Facilities. The sample name was annotated below.
SC78_1: Naïve
SC78_2: WT-D10
SC78_3: KO-D10
SC78_6:WT-D20
SC78_7: KO-D20
SC78_9: WT-D30
SC78_10: KO-D30


title: Adolescent social housing protects against adult emotional and cognitive deficits and alters the PFC and NAc transcriptome in male and female C57BL/6J mice

city: Richmond

first: JENNIFER

last: WOLSTENHOLME

state: VA

address: 1217 E Marshall Street, KMSB Rm 536

institute: Virginia Commonwealth University

summary: Adolescence is a critical period in cognitive and emotional development, characterized by high levels of social interaction and increases in risk-taking behavior including binge drinking. Adolescent exposure to social stress and binge ethanol have individually been associated with the development of social, emotional, and cognitive deficits, as well as increased risk for alcohol use disorder. Disruption of cortical development by early life social stress and/or binge drinking may partly underlie these enduring emotional, cognitive, and behavioral effects. The study goal is to implement a novel neighbor housing environment to identify the effects of adolescent neighbor housing and/or binge ethanol drinking on (1) a battery of emotional and cognitive tasks (2) adult ethanol drinking behavior, and (3) the nucleus accumbens and prefrontal cortex transcriptome. Adolescent male and female C57BL/6J mice were single or neighbor housed with or without access to intermittent ethanol. One cohort underwent behavioral testing during adulthood to determine social preference, expression of anxiety-like behavior, cognitive performance, and patterns of ethanol intake. The second cohort was sacrificed in late adolescence and brain tissue was used for transcriptomics analysis. As adults, single housed mice displayed decreased social interaction, deficits in the novel object recognition task, and increased anxiety-like behavior, relative to neighbor-housed mice. There was no effect of housing condition on adolescent or adult ethanol consumption. Adolescent ethanol exposure did not alter adult ethanol intake. Transcriptomics analysis revealed that adolescent housing condition and ethanol exposure resulted in differential expression of genes related to synaptic plasticity in the nucleus accumbens and genes related to methylation, the extracellular matrix and inflammation in the prefrontal cortex. The behavioral results indicate that social interaction during adolescence via the neighbor housing model may protect against emotional, social, and cognitive deficits. In addition, the transcriptomics results suggest that these behavioral alterations may be mediated in part by dysregulation of transcription in the frontal cortex or the nucleus accumbens

overall_design: Adolescent male and female C57BL/6J mice were single or neighbor housed (on PND 26) with or without access to intermittent ethanol from PND 29-52. PFC and Nac were harvested on PND 57 and RNA was isolated for Affy chip hybridization.

first: Jyoti

last: Lodha

first: Emily

last: Brocato

first: McKensie

last: Nash

first: Madison

last: Marcus

first: A

last: Pais

first: Alex

middle: B

first: Michael

last: Miles

first: Jennifer

last: Wolstenholme


title: Role of lncRNA in Dormancy and Metastatic Reactivation

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

overall_design: Refer to individual Series


title: Unique territorial and sub-chromosomal organization revealed in the holocentric moth Bombyx mori [Hi-C head]

status: Public on Sep 17 2023

city: Paris

first: Héloïse

last: Muller

address: 26 rue d'Ulm

institute: Institut Curie

summary: Genome organization in diverse eukaryotes follows conserved principles including the formation of chromosome territories (CT), chromatin compartments, and TADs. Here, we describe the 3D architecture of holocentric chromosomes in the silkworm Bombyx mori. At the genome-wide scale, B. mori chromosomes are highly territorial lacking any visible trans contact pattern. At the chromosomal scale, B. mori chromosomes segregate into three chromatin compartments: an active A and an inactive B as described in other eukaryotes, and a third type, X, with a unique contact pattern. Compartment X is strongly enriched for short-range interactions and depleted of long-range interactions, hosts a specific combination of genetic and epigenetic features and localizes towards the periphery of CT. Biophysical simulations reveal the necessity of the combined effects of affinity-based compartmentalization and activity-based loop extrusion to lead to the unique interaction patterns observed. Our analyses contribute to our understanding how chromosomes fold highlighting the evolutionary plasticity of 3D genome organization.

overall_design: Hi-C and ChIP-seq and RNA-seq data from Bombyx mori p50 reference strain embryos post-diapause. ChIP-seq experimments are all performed in duplicates. Hi-C have been done at several embryonic time points: 2h, 24h and 48h after dipause release and on adults heads. RNA-seq has been perfomred at 24h after diapause release

first: Ines

last: Drinnenberg

first: José

last: Gil Jr


title: Unique territorial and sub-chromosomal organization revealed in the holocentric moth Bombyx mori [RNA-seq]

last: Gil Jr.


title: Transcriptional heterogeneity overcomes super-enhancer disrupting drug combinations in multiple myeloma [ATAC-seq]

city: Atlanta

first: Benjamin

last: Barwick

address: 1365 Clifton Rd. NE WCI-C 4th Floor Benches 36-37

institute: Emory University

summary: Multiple myeloma (MM) is a malignancy that is often driven by MYC and that is sustained by IRF4, which are upregulated by super-enhancers.  IKZF1 and IKZF3 bind to super-enhancers and can be degraded using immunomodulatory imide drugs (IMiDs).  Successful IMiD responses downregulate MYC and IRF4; however, this fails in IMiD-resistant cells.  MYC and IRF4 downregulation can also be achieved in IMiD-resistant tumors using inhibitors of BET and EP300 transcriptional coactivator proteins; however, in vivo these drugs have a narrow therapeutic window.  By combining IMiDs with EP300 inhibition, we demonstrate greater downregulation of MYC and IRF4, synergistic killing of myeloma in vitro and in vivo, and an increased therapeutic window.  Interestingly, this potent combination failed where MYC and IRF4 expression was maintained by high levels of the AP-1 factor BATF.  Our results identify an effective drug combination and a previously unrecognized mechanism of IMiD resistance.

overall_design: To determine the chromatin accesibility changes induced by the ImmunoModulatory imide Drug (IMiD) pomalidomide (POM) and the EP300 inhibitor GNE-781 (G781) alone or in combination on multiple myeloma JJN3 and KMS11 cells with (+BATF) or with out (+eGFP) BATF overexpression.

first: Seth

middle: J

last: Welsh

first: P

last: Bergsagel


title: Single-cell multi-omic analysis of the vestibular schwannoma ecosystem uncovers a nerve injury-like state [RNA-seq-invitro]

status: Public on Sep 15 2023

city: St Louis

first: Bhuvic

last: Patel

state: MO

address: 660 S. Euclid Ave

department: Neurosurgery

institute: Washington University in St. Louis School of Medicine

summary:
Vestibular schwannomas (VS) are benign tumors that lead to significant neurologic and otologic morbidity. How VS heterogeneity and the tumor microenvironment (TME) contribute to the pathogenesis of these tumors remains poorly understood. We performed scRNA-seq on 15 VS samples, with paired scATAC-seq (n=6)  and exome sequencing (n=12). We identified diverse Schwann cell (SC), stromal, and immune populations in the VS TME and found that repair-like and MHC-II antigen presenting subtype SCs are associated with increased myeloid cell infiltrate, implicating a nerve injury-like process. Deconvolution of RNA-expression data from 195 tumors revealed Injury-like tumors are associated with larger tumor size, and scATAC-seq identified transcription factors associated with nerve repair among SCs from Injury-like tumors. Ligand-receptor analysis and in vitro experiments suggested that SCs recruit monocytes. Our study indicates that Injury-like SCs may cause tumor growth via myeloid cell recruitment and identifies molecular pathways that may be targeted to prevent tumor progression.

This SubSeries is part of a SuperSeries (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE216784).

overall_design: Bulk RNA-seq was performed on immortalized human Schwann cells. There are no experimental groups, controls or references.

first: Thomas

last: Barrett

first: Saad

last: Khan

first: Aldrin

last: Yim

first: Sangami

last: Pugazenthi

first: Tatenda

last: Mahlokozera

first: Gregory

last: Zipfel

first: Jacques

middle: A

last: Herzog

last: Chicoine

first: Cameron

last: Wick

first: Nedim

last: Durakovic

first: Joshua

last: Osbun

first: Matthew

last: Shew

first: Akash

first: Craig

last: Buchman

first: Allegra

last: Petti

first: Sidharth

middle: V

last: Puram

first: Albert

last: Kim


title: piggyBac-Mediated Genomic Integration of Linear dsDNA-Based Library for Deep Mutational Scanning in Mammalian Cells

status: Public on Oct 05 2023

city: Tianjin

first: Shuai

last: Li

state: Tianjin

address: Huanhuxi Road

institute: Tianjin Tumor Hospital

summary: Deep mutational scanning (DMS) makes it possible to perform massively parallel quantification of the relationship between genetic variants and phenotypes of interest. However, the difficulties in introducing large variant libraries into mammalian cells greatly hinder DMS under physiological states. Here we developed two novel strategies for DMS library construction in mammalian cells, namely ‘piggyBac-in-vitro ligation’ and ‘piggyBac-in-vitro ligation-PCR’. For the first strategy, we took the ‘in-vitro ligation’ approach to prepare high-diversity linear dsDNAs, and integrate them into the mammalian genome with a piggyBac transposon system. For the second strategy, we further added a PCR step using the in-vitro ligation dsDNAs as templates, for the construction of high-content genome-integrated libraries via large-scale transfection. Both strategies could successfully establish genome-integrated EGFP-chromophore randomized libraries in HEK293T cells and enrich the green fluorescence-chromophore amino acid sequences. And we further identified a novel transcriptional activator peptide with the ‘piggyBac-in-vitro ligation-PCR’ strategy. Our novel strategies greatly facilitate the construction of large variant DMS library in mammalian cells, and may have great application potential in the future.

overall_design: To introduce large variant libraries into the mammalian genome in a relatively simple way for DMS study, we revised our previously developed plasmid-independent library construction strategy called ‘in-vitro ligation’, a method that achieves transient library expression in mammalian cells via transfection of double-stranded DNAs (dsDNAs) with intact expression cassette which is obtained by ligation of PCR-generated dsDNAs containing degenerate codons. We sought to improve the ‘in-vitro ligation’ strategy by: (1) employing a piggyBac transposon system to integrate the in-vitro ligation dsDNAs into the mammalian genome, and (2) boosting the preparation yield of full-length in-vitro ligation dsDNAs via PCR reaction, thus facilitating large-scale transfection. Here we describe these improved strategies, which we refer to as ‘piggyBac-in-vitro ligation’ and ‘piggyBac-in-vitro ligation-PCR’ strategies, and validate their applications via the identification of green fluorescence-chromophore amino acid sequences and also a novel transcriptional activator peptide.

first: Weijun

last: Su

first: Yi

last: Wang

first: Yanjie

last: Zhao

first: Yifan


title: Unique territorial and sub-chromosomal organization revealed in the holocentric moth Bombyx mori [ChIP-seq]

first: Héloïse

last: Muller

first: Ines

last: Drinnenberg

first: José

last: Gil Jr


title: Single-cell multi-omic analysis of the vestibular schwannoma ecosystem uncovers a nerve injury-like state [scRNA-seq]

first: Bhuvic

last: Patel

summary:
Vestibular schwannomas (VS) are benign tumors that lead to significant neurologic and otologic morbidity. How VS heterogeneity and the tumor microenvironment (TME) contribute to the pathogenesis of these tumors remains poorly understood. We performed scRNA-seq on 15 VS samples, with paired scATAC-seq (n=6)  and exome sequencing (n=12). We identified diverse Schwann cell (SC), stromal, and immune populations in the VS TME and found that repair-like and MHC-II antigen presenting subtype SCs are associated with increased myeloid cell infiltrate, implicating a nerve injury-like process. Deconvolution of RNA-expression data from 195 tumors revealed Injury-like tumors are associated with larger tumor size, and scATAC-seq identified transcription factors associated with nerve repair among SCs from Injury-like tumors. Ligand-receptor analysis and in vitro experiments suggested that SCs recruit monocytes. Our study indicates that Injury-like SCs may cause tumor growth via myeloid cell recruitment and identifies molecular pathways that may be targeted to prevent tumor progression.

This SubSeries is part of a SuperSeries (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE216784). Matching whole exome sequencing data is available via NCBI dbGaP (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA981645/).

overall_design: Single cell RNA-seq data was performed on sporadic vestibular schwannoma tumors. There are no experimental groups, controls or references.

first: Thomas

last: Barrett

first: Aldrin

last: Yim

first: Saad

last: Khan

first: Sangami

last: Pugazenthi

first: Tatenda

last: Mahlokozera

first: Gregory

last: Zipfel

first: Jacques

last: Herzog

first: Michael

first: Cameron

middle: C


title: Integrated single-cell analysis of preleukemic mouse models

city: Cambridge

first: Bertie

last: Gottgens

address: Jeffrey Cheah Biomedical Centre

institute: University of Cambridge

summary: Hematological malignancies develop through acquisition of mutations and clonal expansion in hematopoietic stem and progenitor cells (HSPCs). However, how individual mutations perturb the entire hematopoietic system towards malignant phenotypes remains elusive. Here, we have performed single cell RNA sequencing of bone marrow HSPCs from 8 different mutant mouse models to study mutation-specific preleukemic perturbations.

overall_design: Single cell RNA sequencing of lineage negative / c-Kit positive (LK) cells for 38 animals from eight different mutant mouse models and their wild-type littermates

first: Tomoya

last: Isobe

first: Nicola

last: Wilson

first: Berthold

last: Göttgens

first: Rebecca

last: Hannah


title: Single-cell multi-omic analysis of the vestibular schwannoma ecosystem uncovers a nerve injury-like state [scATAC-seq]

overall_design: Single-cell ATAC-seq data was performed on sporadic vestibular schwannoma tumors. There are no experimental groups, controls or references.

last: Chicoine

last: Wick

first: Nedim

last: Durakovic

first: Joshua

last: Osbun

first: Matthew

last: Shew

first: Akash

first: Craig

last: Buchman

first: Allegra

last: Petti

first: Sidharth

last: Puram

first: Albert

last: Kim


title: Single-cell multi-omic analysis of the vestibular schwannoma ecosystem uncovers a nerve injury-like state [bulk RNA-seq]

first: Bhuvic

last: Patel

overall_design: Bulk RNA-seq was performed on sporadic vestibular schwannoma tumors. There are no experimental groups, controls or references.

first: Thomas

last: Barrett

first: Saad

last: Khan

first: Aldrin

last: Yim

first: Sangami

last: Pugazenthi

first: Tatenda

last: Mahlokozera

first: Gregory

last: Petti.


title: Formation of Müller glia-derived progenitor cells in retinas depleted of microglia

status: Public on Sep 14 2023

city: Columbus

first: Heithem

last: El-Hodiri

state: OH

address: 333 W 10th Ave

institute: Ohio State University College of Medicine

summary: Recent studies have demonstrated the complex coordination of pro-inflammatory signaling and reactive microglia/macrophage on the formation Müller glial-derived progenitor cells (MGPCs) intheretinas offish, birds and mice.We generated scRNA-seq libraries to identify transcriptional changes in Müller glia (MG) that result from the depletion of microglia from the chick retina. We found significant changes in different networks of genes in MG in normal and damaged retinas when the microglia are ablated. We identified a failure of MG to upregulate Wnt-ligands, Heparin binding epidermal growth factor (HBEGF), Fibroblast growth factor (FGF), retinoic acid receptors and genes related to Notch-signaling. Inhibition of GSK3β, to simulate Wnt-signaling, failed to rescue the deficit in formation of proliferating MGPCs in damaged retinas missing microglia. By comparison, application of HBEGF or FGF2 completely rescued the formation of proliferating MGPCs in microglia-depleted retinas. Similarly, injection of a small molecule inhibitor to Smad3 or agonist to retinoic acid receptors partially rescued the formation of proliferating MGPCs in microglia-depleted damaged retinas. According to scRNA-seq libraries, patterns of expression of ligands, receptors, signal transducers and/or processing enzymes to cell-signaling via HBEGF, FGF, retinoic acid and TGFβ are rapidly and transiently upregulated by MG after neuronal damage, consistent with important roles for these cell-signaling pathways in regulating the formation of MGPCs. We conclude that quiescent and activated microglia have a significant impact upon the transcriptomic profile of MG. We conclude that signals produced by reactive microglia in damaged retinas stimulate MG to upregulate cell signaling through HBEGF, FGF and retinoic acid, and downregulate signaling through TGFβ/Smad3 to promote the reprogramming on MG into proliferating MGPCs.

overall_design: To investigate the mechanisms underlying the failure of Muller glia-derived progenitor cell formation in chick retinas when the microglia/macrophage have been ablated, we compared RNA expression profiles amongst Muller glia and microglia from control or damaged retinas (with NMDA), with or without microglia ablation (with clodronate).

middle: M

first: Isabella

last: Palazzo

first: Warren

last: Campbell

first: Andy

last: Fischer


title: CUT&RUN profiling of histone H3K27ac and H3K27me3 localization after pharmacological inhibition of EZH2 and BET proteins

first: Kyle

last: Eagen

state: TX

address: 1 Baylor Plaza

department: Department of Molecular and Cellular Biology

institute: Baylor College of Medicine

summary: NUT carcinoma (NC), an aggressive carcinoma, is driven by the BRD4-NUT fusion oncoprotein. BRD4, a BET protein, binds to chromatin through its two bromodomains, and when fused to NUT forms very large super-enhancers, termed megadomains. Targeting BRD4-NUT with BET bromodomain inhibitors (BETi) are a promising treatment, but limited as monotherapy. To identify additional dependencies in NC, we performed a genetic rescue screen in NC cells depleted of BRD4-NUT and identified EZH2 as a top correlated hit. Indeed, inhibition of EZH2 using the clinical compound, tazemetostat (taz), potently blocked growth of NC cells, and when combined with BETi was highly synergistic. Epigenetic and transcriptomic analysis revealed that taz reversed the EZH2-specific H3K27me3 silencing mark, and restored expression of multiple tumor suppressor genes while having no effect on megadomain-associated genes. CDKN2A was identified as the only amongst all taz-derepressed genes to confer resistance to taz in a CRISPR-CAS9 screen. In pre-clinical models, combined taz and BETi synergistically blocked growth and prolonged survival of NC-xenografted mice, with all mice cured in one cohort.

overall_design: CUT&RUN to assess histone H3K27ac and H3K27me3 localization after tazemetostat (EPZ-6438; EZH2 inhibitor) alone or tazemetostate combined with ABBV-075 (BET inhibitor) treatment of 10-15 and PER-403 cells, two NUT carcinoma cell lines.

first: Catherine

last: Hawkins

first: Christopher

last: Ponne

first: Quan

last: Le

first: Madeleine

last: Lemieux

last: French


title: Single-cell multi-omic analysis of the vestibular schwannoma ecosystem uncovers a nerve injury-like state

last: Patel

summary:
Vestibular schwannomas (VS) are benign tumors that lead to significant neurologic and otologic morbidity. How VS heterogeneity and the tumor microenvironment (TME) contribute to the pathogenesis of these tumors remains poorly understood. We performed scRNA-seq on 15 VS samples, with paired scATAC-seq (n=6)  and exome sequencing (n=12). We identified diverse Schwann cell (SC), stromal, and immune populations in the VS TME and found that repair-like and MHC-II antigen presenting subtype SCs are associated with increased myeloid cell infiltrate, implicating a nerve injury-like process. Deconvolution of RNA-expression data from 195 tumors revealed Injury-like tumors are associated with larger tumor size, and scATAC-seq identified transcription factors associated with nerve repair among SCs from Injury-like tumors. Ligand-receptor analysis and in vitro experiments suggested that SCs recruit monocytes. Our study indicates that Injury-like SCs may cause tumor growth via myeloid cell recruitment and identifies molecular pathways that may be targeted to prevent tumor progression.

This SuperSeries is composed of the SubSeries listed below. Matching whole exome sequencing data is available via NCBI dbGaP (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA981645/).


title: Discrete chromatin alterations and deregulated gene expression upon PROTAC-induced rapid loss of the trithorax protein ASH2L [ChIP-seq(H3K27me3) second]

status: Public on Sep 12 2023

city: Aachen

first: Mirna

last: Barsoum

state: North Rhine Westphalia

address: Pauwelsstrasse 30

department: Institute of Biochemistry and Molecular Biology

institute: Uniklinik RWTH Aachen

summary: The trithorax protein ASH2L is essential for organismal and tissue development and for cell proliferation. ASH2L is a subunit of KMT2/COMPASS methyltransferase complexes that catalyze the methylation of histone H3 lysine 4 (H3K4). Tri- and mono-methylation of H3K4 (H3K27me3 and H3K27me3) are associated with active promoters and enhancers, respectively. The molecular relevance of these modifications is not fully understood. We have used mouse embryo cells with a PROTAC-sensitive, degradable ASH2L to assess the functional consequences of KMT2 complex inactivation. The rapid loss of ASH2L resulted in a sequential alterations of histone marks at promoters, first a decrease of H3K27me3, then an increase of H3K27me3, and a decrease of H3K27me3 during the first 16 hrs, while an increase in H3K27me3 was very slow. These consequences were most prominent at CpG island promoters within a window of ±1 kb of the transcription start sites. Despite the the rapid loss of ASH2L, the effect on transcription in the first 8 hrs was minimal. This was accompanied with an alterations in gene expression and associated proliferation stop and cell cycle arrest. These findings suggest an order series of events upon loss of ASH2L that requires considerable amount of time to unfold.

overall_design: We have generated a conditional knockout mouse, in which we can delete exon 4 of Ash2l. This prevents production  of Ash2l protein, however due to the long half-life of Ash2l, it requires days before the protein is depleted. From these animals we established mouse embryo fibroblasts (MEF). Upon knockout of the endogenous alleles, the cells stop proliferating and enter senescence. We have now introduced into these cells a construct that expresses an FKBP-ASH2L fusion protein. Upon knockout of the endogenous alleles, the MEF cells proliferate dependent on FKBP-ASH2L. We find that this fusion protein is sensitive to PROTACs (Proteolysis Targeting Chimeras) and is degraded by more than 99% within 30 minutes after addition  of a PROTAC.

first: Roksaneh

last: Sayadi Boroujeni

first: Alexander

last: Stenzel

first: Philip

last: Bussmann

first: Juliane

last: Lüscher-Firzlaff

first: Bernhard

last: Lüscher


title: Discrete chromatin alterations and deregulated gene expression upon PROTAC-induced rapid loss of the trithorax protein ASH2L [ChIP-seq(Ash2l)]

summary: The trithorax protein ASH2L is essential for organismal and tissue development and for cell proliferation. ASH2L is a subunit of KMT2/COMPASS methyltransferase complexes that catalyze the methylation of histone H3 lysine 4 (H3K4). Tri- and mono-methylation of H3K4 (Ash2l and H3K4me1) are associated with active promoters and enhancers, respectively. The molecular relevance of these modifications is not fully understood. We have used mouse embryo cells with a PROTAC-sensitive, degradable ASH2L to assess the functional consequences of KMT2 complex inactivation. The rapid loss of ASH2L resulted in a sequential alterations of histone marks at promoters, first a decrease of Ash2l, then an increase of H3K4me1, and a decrease of H3K27ac during the first 16 hrs, while an increase in H3K27me3 was very slow. These consequences were most prominent at CpG island promoters within a window of ±1 kb of the transcription start sites. Despite the the rapid loss of ASH2L, the effect on transcription in the first 8 hrs was minimal. This was accompanied with an alterations in gene expression and associated proliferation stop and cell cycle arrest. These findings suggest an order series of events upon loss of ASH2L that requires considerable amount of time to unfold.


title: Discrete chromatin alterations and deregulated gene expression upon PROTAC-induced rapid loss of the trithorax protein ASH2L [ChIP-seq(H3K27me3) first]


title: Combinatorial single-cell profiling of major chromatin types with MAbID

city: Utrecht

first: Robin H.

last: van der Weide

state: Utrecht

address: Uppsalalaan 8

institute: Hubrecht Institute

summary: Gene expression programs result from the collective activity of numerous regulatory factors. Studying their cooperative mode of action is imperative to understand gene regulation, but simultaneously measuring these factors within one sample has been challenging. Here, we introduce MAbID, a method for combinatorial genomic profiling of histone modifications and chromatin-binding proteins. MAbID employs antibody-DNA conjugates to insert genomic barcodes at sites of epitope occupancy, enabling  combined incubation of multiple antibodies to reveal the genomic distributions of many epigenetic markers simultaneously. We used MAbID to profile all major chromatin types and multiplexed measurements without loss of individual data quality. Moreover, we obtained joint measurements of six epitopes in single cells of mouse bone marrow and during mouse in vitro differentiation, capturing associated changes in multifactorial chromatin states. Thus, MAbID holds the potential to gain unique insights into the interplay between gene regulatory mechanisms, especially for low-input samples and in single cells.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Epigenomic profiling using antibody-conjugates in K562, mESC, early differentiating mNPC, and bone-marrow samples.

first: Silke

last: Lochs

first: Robin

first: Kim

middle: L

last: de Luca

first: Tessy

last: Korthout

first: Ramada

last: van Beek

first: Hiroshi

last: Kimura

first: Jop

last: Kind


title: Discrete chromatin alterations and deregulated gene expression upon PROTAC-induced rapid loss of the trithorax protein ASH2L [ChIP-seq(H3K4me3)]

summary: The trithorax protein ASH2L is essential for organismal and tissue development and for cell proliferation. ASH2L is a subunit of KMT2/COMPASS methyltransferase complexes that catalyze the methylation of histone H3 lysine 4 (H3K4). Tri- and mono-methylation of H3K4 (H3K4me3 and H3K4me1) are associated with active promoters and enhancers, respectively. The molecular relevance of these modifications is not fully understood. We have used mouse embryo cells with a PROTAC-sensitive, degradable ASH2L to assess the functional consequences of KMT2 complex inactivation. The rapid loss of ASH2L resulted in a sequential alterations of histone marks at promoters, first a decrease of H3K4me3, then an increase of H3K4me1, and a decrease of H3K27ac during the first 16 hrs, while an increase in H3K27me3 was very slow. These consequences were most prominent at CpG island promoters within a window of ±1 kb of the transcription start sites. Despite the the rapid loss of ASH2L, the effect on transcription in the first 8 hrs was minimal. This was accompanied with an alterations in gene expression and associated proliferation stop and cell cycle arrest. These findings suggest an order series of events upon loss of ASH2L that requires considerable amount of time to unfold.


title: Discrete chromatin alterations and deregulated gene expression upon PROTAC-induced rapid loss of the trithorax protein ASH2L [ATAC-Seq]


title: Discrete chromatin alterations and deregulated gene expression upon PROTAC-induced rapid loss of the trithorax protein ASH2L [ChIP-seq(H3K4me1)]

summary: The trithorax protein ASH2L is essential for organismal and tissue development and for cell proliferation. ASH2L is a subunit of KMT2/COMPASS methyltransferase complexes that catalyze the methylation of histone H3 lysine 4 (H3K4). Tri- and mono-methylation of H3K4 (H3K4me1 and H3K4me1) are associated with active promoters and enhancers, respectively. The molecular relevance of these modifications is not fully understood. We have used mouse embryo cells with a PROTAC-sensitive, degradable ASH2L to assess the functional consequences of KMT2 complex inactivation. The rapid loss of ASH2L resulted in a sequential alterations of histone marks at promoters, first a decrease of H3K4me1, then an increase of H3K4me1, and a decrease of H3K27ac during the first 16 hrs, while an increase in H3K27me3 was very slow. These consequences were most prominent at CpG island promoters within a window of ±1 kb of the transcription start sites. Despite the the rapid loss of ASH2L, the effect on transcription in the first 8 hrs was minimal. This was accompanied with an alterations in gene expression and associated proliferation stop and cell cycle arrest. These findings suggest an order series of events upon loss of ASH2L that requires considerable amount of time to unfold.


title: Discrete chromatin alterations and deregulated gene expression upon PROTAC-induced rapid loss of the trithorax protein ASH2L [ChIP-seq(H3K27ac)]

summary: The trithorax protein ASH2L is essential for organismal and tissue development and for cell proliferation. ASH2L is a subunit of KMT2/COMPASS methyltransferase complexes that catalyze the methylation of histone H3 lysine 4 (H3K4). Tri- and mono-methylation of H3K4 (H3K27ac and H3K27ac) are associated with active promoters and enhancers, respectively. The molecular relevance of these modifications is not fully understood. We have used mouse embryo cells with a PROTAC-sensitive, degradable ASH2L to assess the functional consequences of KMT2 complex inactivation. The rapid loss of ASH2L resulted in a sequential alterations of histone marks at promoters, first a decrease of H3K27ac, then an increase of H3K27ac, and a decrease of H3K27ac during the first 16 hrs, while an increase in H3K27me3 was very slow. These consequences were most prominent at CpG island promoters within a window of ±1 kb of the transcription start sites. Despite the the rapid loss of ASH2L, the effect on transcription in the first 8 hrs was minimal. This was accompanied with an alterations in gene expression and associated proliferation stop and cell cycle arrest. These findings suggest an order series of events upon loss of ASH2L that requires considerable amount of time to unfold.


title: Discrete chromatin alterations and deregulated gene expression upon PROTAC-induced rapid loss of the trithorax protein ASH2L [Click-it]


title: Real-Time qPCR Array of miRNAs in Leishmania-infected THP-1 macrophages

status: Public on Sep 11 2023

city: Ribeirão Preto

middle: Cristina Ribeiro

last: Fernandes

address: Av. Bandeirantes, 3900

institute: University of São Paulo

summary: Leishmania amazonensis is a protozoan that primarily infects macrophages and causes cutaneous leishmaniasis in humans. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at posttranscriptional levels. Previous work demonstrated changes in miRNA profile of host cells favoring parasite survivel. Thus, here we demonstrate that human macrophages upregulate several miRNAs on the initial time points of infection, including the hsa-miR-372, hsa-miR-373, and hsa-miR-520d, which present the same seed. Further functional analysis demonstrated that inhibition of the miR-372 impaired Leishmania survival in THP-1 macrophages and the effect was further enhanced with combinatorial inhibition of the miR-372/373/520d family, pointing to a cooperative mechanism. Our study demonstrated miRNA-dependent modulation of polyamines production, establishing permissive conditions for intracellular parasite survival.Our findings suggest that the miR-372/373/520d family may represent a potential target for the development of new therapeutic strategies against cutaneous leishmaniasis.

overall_design:
qPCR array for miRNA expression profile. Differentiated THP-1 macrophages where either infected with Leishmania amazonensis promastigotes (MOI 5) or left non-infected as indicated and miRNAs were quantified after 4 or 24 hours of infection (N=4 for each group).
qPCR array was performed using the Qiagen Human Inflammatory Response & Autoimmunity, RT-PCR Array platform and all data were analyzed using QIAGEN's web-based software (https://geneglobe.qiagen.com/us/analyze). Unfortunately, the resulting analysis table does not give the normalized values for each sample, and instead provides averaged normalized value per group.

first: J

first: S

last: Muxel

first: L

last: Floeter-Winter


title: Sequential deregulation of histone marks, chromatin accessibility and gene expression in response to PROTAC-induced degradation of ASH2L

first: Mirna

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


title: Maternal antibiotic exposure enhances neonatal ILC2 responses and aggravates allergic airway inflammation in adults [ATAC-seq]

status: Public on Sep 08 2023

first: Jie

last: Zhou

address: No22.Qixiangtai Rd.,Heping Dist,Tianjin P.R. China

institute: Tianjin Medical University

summary: Disruption of circadian rhythm during pregnancy produced adverse health outcomes in offspring. However, the role of maternal circadian rhythms in infants’ immunity and their susceptibility to inflammation remains poorly understood. Here we reported that disruption of circadian rhythms in pregnant mice profoundly aggravated the severity of neonatal inflammatory disorders, including necrotizing enterocolitis (NEC) and sepsis. The diminished production of maternal-derived docosahexaenoic acid (DHA) and the impaired immunosuppressive function of myeloid-derived suppressor cells (MDSCs) in neonates played a dominant role in this process. Mechanistically, DHA enhanced the immunosuppressive function of neonatal MDSCs viaPPARγ mediated mitochondrial oxidative phosphorylation. Transfer of MDSCs or perinatal supplementation of DHA relieved neonatal inflammation induced by maternal rhythms disruption. These observations revealed an important role of maternal circadian rhythms in the control of neonatal inflammation via metabolic reprograming of myeloid cells.

overall_design: A total of 50000 lung ILC2s (prenatal antibiotic exposure group and control group) were collected  in 1.5 mL RNase-free tubes  for ATAC-sequencing

first: Haixu

last: Xu

first: Zhaohai

last: Cui

first: Lijuan

last: Zhang


title: LncRNA Malat1 suppresses pyroptosis and T cell-mediated killing of incipient metastatic cells [RNA-seq_4T1]

first: Dhiraj

last: Kumar

summary: Malat1 was KO using paired gRNA CRISPR-cas9 system from 4T1 cells.

overall_design:
Total RNA was isolated using the Maxwell instrument from 4T1 WT or Malat1 KO cells and sent to BGI for RNA-seq. The sample name was annotated below.
1. 4T1_Ctrl-1: 4T1-Ctrl-1
2. 4T1_Ctrl-2: 4T1-Ctrl-2
3. 4T1_Ctrl-3: 4T1-Ctrl-3
4. 4T1_Malat1_KO1-1: 4T1-KO#3-1
5. 4T1_Malat1_KO1-2: 4T1-KO#3-2
6. 4T1_Malat1_KO1-6: 4T1-KO#3-3
7. 4T1_Malat1_KO2-1: 4T1-KO#43-1
8. 4T1_Malat1_KO2-2: 4T1-KO#43-2
9. 4T1_Malat1_KO2-1: 4T1-KO#43-3

first: Filippo

last: Giancotti


title: Increased hyaluronan by naked mole-rat HAS2 extends healthspan in mice

city: Los Angeles

first: Steve

last: Horvath

address: 695 Charles E. Young Drive South, Box 708822

department: Human Genetics

institute: University of California, Los Angeles

type:
Expression profiling by array
Methylation profiling by array

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

platform_organism:
Homo sapiens
Mus musculus
Rattus norvegicus

overall_design: The data includes 19 Blood of Mus musculus, 20 Kidney of Mus musculus, 19 Liver of Mus musculus, 20 Muscle of Mus musculus, 20 Skin of Mus musculus

first: Vera

last: Gorbunova


title: Single-cell transcriptomic atlas reveals tissue architecture and deciphers pathological reprogramming during retinal ischemia in Macaca fascicularis

status: Public on Sep 06 2023

city: Shanghai

first: Li

last: Lin

address: No. 639 manufacturing bureau road, Huangpu District, Shanghai

institute: Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine

summary: Acute retinal arterial ischemia diseases (ARAIDs) are ocular emergencies that require immediate intervention within a restricted therapeutic window to prevent blindness. Howbeit, the underlying molecular mechanisms contributing to the pathogenesis of ARAIDs remain enigmatic. Herein, this study aims to present the single cell RNA sequencing (scRNA-seq) atlas of ischemic alterations in the primate retina as a preliminary endeavor in understanding the molecular complexities of ARAIDs. In our study, it was determined that rods experienced a significant metabolic reprogramming during retina ischemia, resulting in compromised mitochondrial functions. Furthermore, we observed a noteworthy transcriptional alteration in the activation of microglia induced by ischemia. The targeted correction of the proinflammatory cytokine CXCL8 effectively suppresses microglia M1 polarization in retinal ischemia, ultimately reducing the pro-inflammatory transformation. In addition, our study has observed the apoptotic inclination of endothelial cells and the heightened interaction with microglia, which signifies the influence of microglia in disrupting the blood barrier during the initial phase. In conclusion, our research has successfully identified and described the pathological alterations occurring in several significant cell types during a short period of ischemia. These observations provide valuable insights for ameliorating retinal damage and promoting the restoration of vision.

overall_design: The ophthalmic artery of one macaca fascicularis was ligated for six hours, another macaca fascicularis underwent the same operation without ligation. The retinas of two macaca fascicularis were isolated immediately and digested into single cell suspension for scRNS-seq.


title: Spatiotemporal understanding of healthy and disordered early pregnancy governed by immune-featured stromal cells

city: Beijing

first: Min

last: Yang

state: Beijing

address: No. 5, yiheyuan road, haidian district,

institute: Peking University

overall_design: Refer to individual Series


title: Live tracking of basal stem cells of the epidermis during growth, homeostasis, and injury response in zebrafish

city: Yokohama

first: Zhengcheng

last: Liu

state: Kanagawa

address: Midori-ku, Nagatsuta-cho, 4259

department: School of Life Science and Technology

country: Japan

institute: Tokyo Institute of Technology

summary: Basal stem cells of the epidermis continuously differentiate into keratinocytes and replenish themselves via self-renewal to maintain skin homeostasis. Numerous studies have attempted to reveal how basal cells undergo differentiation or self-renewal; however, this has been hampered by a lack of robust basal cell markers and analytical platforms that allow single-cell tracking. Here, we report integrin b4 (itgb4) is a useful marker for basal cell labelling, irrespective of the body region, stage, and regenerative status. We employed Cre-loxP recombination in combination with live cell tracking of single basal clones in the caudal fin and investigated the embryonic origin and behaviour of basal cells during fish growth and homeostasis. Although most basal cells, including those in fins, became quiescent in the adult stage, genetic cell ablation showed that basal cells were reactivated to either self-renew or differentiate, depending on the injured cell type. Our findings provide a platform for quantitative in vivo imaging of basal stem cells applicable at wider stages and under various conditions and reveal unanticipated basal cell dynamism in response to distinct wound signals.

overall_design:
To investigate whether itgb4+ epidermal basal precursors already possess a basal cell-like identity immediately after fate segregation at 1 day post fertilization (dpf), we isolated itgb4+ basal precursors, krt4+ periderm cells from zebrafish embryos at 1 dpf, and adult itgb4+ basal cells from zebrafish caudal fin by fluorescence-activated cell sorting.
We then performed gene expression profiling analysis using data obtained from RNA-seq of 3 different cells at 2 time points.
Comparative gene expression profiling analysis of RNA seq data for embryonic basal cell precursors, embryonic periderm, and adult basal cells.

first: Yidan

last: Meng

first: Ayu

last: Ishikura

first: Atsushi

last: Kawakami


title: Single cell transcriptomic atlas of human plasmacytoid dendritic cells reveals a distinct cytotoxic-like subset in HIV-1 infected individuals.

status: Public on Sep 05 2023

city: Aarhus

first: Lamin

last: Cham

address: Skybe

department: Infectious Disease

country: Denmark

institute: Aarhus University Hospital

summary: To understand how the transcriptome of pDCs is impacted by HIV-1 infection and exogenous stimulation, we sorted pDCs from people with HIV-1 (PWH) on antiretroviral treatment before and after toll-like receptor 9 (TLR9) agonist treatment, as well as from healthy controls, and performed single-cell (sc)-RNA sequencing. We generated a transcriptomic atlas of over 112,000 pDCs. Our cluster analysis revealed an inducible cytotoxic-like pDC cluster characterized by high expression of both antiviral and cytotoxic genes. We further reported that this cytotoxic-like pDC cluster is distinct from NK and T cells. This observation was further supported by our cell-cell communication analysis revealing that cytotoxic-like pDCs exhibit similar incoming and outgoing cellular communicating signals as other pDCs. In addition to the cytotoxic-like pDC cluster, we also characterized two homogenous pDC clusters (pDC1 and pDC2) and an exhausted pDC cluster. In conclusion, our findings provide a comprehensive pDC transcriptomic atlas and open a new scope on the mechanisms of regulation and function of cytotoxic-like pDCs in HIV and other infections.

first: Ole

middle: S

last: Sogaard


title: Expression data from SFZ cells and costal chondrocytes

city: Suita

first: Tomohiko

last: Murakami

state: OSAKA

address: 1-8 Yamada-Oka

institute: Osaka University

summary: To understand difference between SFZ cells and costal chondrocytes, total RNAs from SFZ cells and costal chondrocytes were analyzed by microarray.

overall_design: Superficial zone cells of articular cartilage (SFZ) were prepared from femur and tibial joint of postnatal 4days ICR mice. Costal chondrocytes were prepared from ribs of postnatal 4days ICR mice. Total RNAs were analyzed by microarray using an Affymetrix Mouse Genome 430 2.0 Array.