title: Subclonal evolution revealed by single-cell profiles of expressed mutations identifies adaptation mechanisms to immunotherapy in melanoma [scRNA-seq]

status: Public on Oct 25 2024

city: Bethesda

first: Maxwell

last: Lee

state: MD

address: Building 37, Room 5120

department: CCR

institute: National Cancer Institute, NIH

summary: Intratumoral heterogeneity (ITH) arises from distinct subclonal expansion following genetic or epigenetic alterations, and profoundly influences how tumors response to their immune microenvironment. Tumor progression trees based on single-cell mutational profiles have made it possible to trace subclonal evolution; however, conventional tree-building methods can incorporate only a limited number of cells and mutations, restricting their application to larger single-cell data. To investigate the effect of ITH on the therapeutic response of melanoma, we have developed Trisicell (https://trisicell.rtfd.io), a computational toolkit for scalable inference of mutational ITH through assessment of single-cell genomic variant data. By applying Trisicell to genetically matched mouse melanoma datasets, we found that expressed mutations are sufficient to effectively drive subclonal evolution. On single-cell, full-length RNA sequencing data of mouse melanoma from preclinical immune checkpoint blockade (ICB) studies, the analysis showed that the subtree-seeding mutations in the trees identified distinct subclones associated with a specific developmental state and neural crest lineage markers. Using the tree to trace cell lineages, we found that neoantigens depleted by ICB were predominantly expressed in minor subclones, suggesting that post-treatment recurrence is driven by immunoediting. Moreover, these neoantigens were enriched with those derived from frameshift mutations and mutated nuclear genes. Importantly, recurrently mutated genes in ICB-responding human melanoma exhibited the same features. We next used Trisicell to analyze single-cell, full-length RNA data of brain metastases (BM) from melanoma patients treated with ICB, and discovered that relapsing BM from ICB-responding patients exhibited subclones that also expressed a higher fraction of frameshift mutations and were associated with elevated levels of infiltrated T cells. Notably, they also exhibited more mutated HLA genes and expressed high level of the novel immune checkpoint HLA-G, a putative local immunosuppressive mechanism. In summary, applying Trisicell to single-cell transcriptomic analyses allowed us to identify novel features of ICB-responding melanoma neoantigens and distinct mechanisms for adapting to immunotherapy at different sites. These results have important implications for both melanoma evolution and target identification for immunotherapies, including ICB and cancer vaccines. This is the first study to trace subclonal evolution of neoantigens at single-cell resolution, demonstrating that Trisicell and our datasets represent important resources for the field.

overall_design: 24 clonal sublines were generated from parental mouse melanoma line B2905. For each subline, 8 cells were sorted, and cDNA library was generated from each cell by Smart-seq2 protocol. The library were then submitted for sequencing.

first: Farid

middle: R

last: Mehrabadi

first: Eva

last: Pérez-Guijarro

first: Kerrie

last: Marie

first: Salem

last: Malikic

first: Erfan

last: Azer

first: Howard

last: Yang

first: Charli

last: Gruen

first: Chih

last: Wu

first: Welles

last: Robinson

first: Huaitian

last: Liu

first: Can

last: Kizilkale

first: Michael

last: Kelly

first: Christina

last: Marcelus

first: Cari

last: Smith

first: Sung

last: Chin

first: Jessica

last: Ebersole

first: Sandra

last: Burkett

first: Aydin

last: Buluc

first: Naema

last: Nayyar

first: Philipp

last: Hähnel

first: Magali

last: de Sauvage

first: Albert

first: Samuel

last: Markson

first: Ryan

last: Sullivan

first: Priscilla

middle: K

last: Brastianos

first: Funda

last: Ergun

first: Glenn

last: Merlino

first: Chi-Ping

last: Day

first: S

last: Sahinalp


title: Subclonal evolution revealed by single-cell profiles of expressed mutations identifies adaptation mechanisms to immunotherapy in melanoma.

status: Public on Oct 27 2024

first: Maxwell

last: Lee

country: USA

type: Expression profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Tet3 regulates terminal cell differentiation at the metabolic level

status: Public on Oct 22 2024

city: Muenster

first: Marcos

middle: J.

last: Araúzo-Bravo

address: Rogentstrasse

department: Cell and Developmental Biology

country: Germany

institute: Max Planck Institute for Molecular Biomedicine

summary: To study the role of tet3 and 5-hydroxymethylation in mouse intestine homeostasis our objectives are: To determine the cell types that show a 5hmC reduction level in the absence of Tet3. Identify the role of Tet3 on transcriptional regulation in the small intestinal epithelium. Define the physiological functions impaired in Tet3 -/- small intestine epithelium.

overall_design: Single cell RNA-seq analysis of control and Tet3 knockout mice. The Control data is split in 6 fastq files.  The Tet3 knockout data is split into 6 fastq files.

first: Isabel

last: Mulet

first: Daniela

last: Gerovska

first: Guangming

last: Wu

middle: J

first: Natalia

last: Tapia


title: Microglia Depletion Reduces Human Neuronal APOE4-Driven Pathologies in a Chimeric Alzheimer’s Disease Model

city: San Francisco

first: Nuo

last: Chen

state: California

address: 1650 Owens St

department: GIND

institute: Gladstone Institutes

summary: Despite strong evidence supporting the involvement of both apolipoprotein E4 (APOE4) and microglia in Alzheimer’s Disease (AD) pathogenesis, the effects of microglia on neuronal APOE4-driven AD pathogenesis remain elusive. Here, we examined such effects utilizing microglial depletion in a chimeric model with human neurons in mouse hippocampus. Specifically, we transplanted homozygous APOE4, isogenic APOE3, and APOE-knockout (APOE-KO) induced pluripotent stem cell (iPSC)-derived human neurons into the hippocampus of human APOE3 or APOE4 knock-in mice, and depleted microglia in half the chimeric mice. We found that both neuronal APOE and microglial presence were important for the formation of Aβ and tau pathologies in an APOE isoform-dependent manner (APOE4 > APOE3). Single-cell RNA-sequencing analysis identified two pro-inflammatory microglial subtypes with high MHC-II gene expression that are enriched in chimeric mice with human APOE4 neuron transplants. These findings highlight the concerted roles of neuronal APOE, especially APOE4, and microglia in AD pathogenesis.

overall_design: Single-cell RNA sequencing on isolated microglia from hippocampi of chimeric mice with human neurons transplanted into mouse hippocampus. Specifically, homozygous APOE4 (hE4), isogenic APOE3 (hE3), and APOE-knockout (hEKO) induced pluripotent stem cell (iPSC)-derived human neurons were transplanted into the hippocampus of human APOE3 or APOE4 knock-in (E3KI and E4KI) mice. The five groups of mice are: E4KI, hE4-E4KI, E3KI, hE3-E3KI, and hEKO-E4KI.

first: Rao

last: Antara

first: Chen

last: Nuo

first: Hao

last: Yanxia

first: Yadong

last: Huang


title: Extracellular Vesicles Derived from Inflammatory Cardiomyocytes Mediate Macrophage Polarization and Alleviate Myocarditis via p38 MAPK Pathway

status: Public on Oct 23 2024

city: Jinan

first: Yingnan

last: You

address: Jingwuweiliu Road

country: China

institute: Shandong provincial hospital

summary: EVs derived from lipopolysaccharide （LPS）-stimulated cardiomyocytes （EVLPS） exhibit anti-inflammatory effects on macrophages and reduce cardiac inflammation in mice.To investigate  gene expression changes induced by EVLPS treatment in inflammatory RAW264.7 using next-generation sequencing，we performed RNAseq.

overall_design: mRNA profiles of LPS-stimulated RAW264.7 treated with either EVLPS or PBS were generated by deep sequencing, in quadruplicate

first: Yanjie

last: Jiang

first: Bo

last: Han


title: Targeting pyruvate metabolism generates distinct CD8+ T cell responses to gammaherpesvirus and B lymphoma

status: Public on Oct 16 2024

city: Lebanon

first: Edward

last: Usherwood

state: NH

address: Rubin Building, Level 7, 710-53

department: Microbiology and Immunology

institute: Geisel School of Medicine at Dartmouth College

summary: T cells rely on different metabolic pathways to differentiate into effector or memory cells, and metabolic intervention is a promising strategy to optimize T cell function for immunotherapy. Pyruvate dehydrogenase (PDH) is a nexus between glycolytic and mitochondrial metabolism, regulating pyruvate conversion either to lactate or acetyl-CoA. Here, we retrovirally transduced pyruvate dehydrogenase kinase 1 (PDK1) or pyruvate dehydrogenase phosphatase 1 (PDP1), which control PDH activity, into CD8+ T cells to test effects on T cell function. Although PDK1 and PDP1 were expected to influence PDH in opposing directions, by several criteria they induced similar changes relative to control T cells. Seahorse metabolic flux assays showed both groups exhibited increased glycolysis and oxidative phosphorylation. Both groups had improved primary and memory recall responses following infection with murine gammaherpesvirus-68 (MHV-68). However, metabolomics using labeled fuels indicated differential usage of key fuels by metabolic pathways. Importantly, after B cell lymphoma challenge the engineered CD8+ T cells produced smaller effector populations in the tumor, resulting in larger tumors and poorer survival compared with control cells. Overall, this study indicates that PDK1 and PDP1 both enhance metabolic capacity, but the context of the antigenic challenge significantly influences the consequences for T cell function.

overall_design: To investigate the transcriptional changes with the modified PDH phosphorylation status in CD8+ T cells, we retrovirally transduced mouse CD8+ T cells with empty vector, PDK1 or PDP1.

first: Taewook

last: Kang

first: Owen

middle: M

last: Wilkins


title: Light induced targeting enables proteomics on endogenous condensates

status: Public on Oct 18 2024

city: Freiburg

first: Choongman

last: Lee

address: Stuebeweg 51

institute: Max Planck Institute of Immunobiology and Epigenetics

summary: Endogenous condensates with transient constituents are notoriously difficult to study with common biological assays like mass-spectrometry and other proteomics profiling. Here we report a method for light-induced targeting of endogenous condensates (LiTEC) in living cells. LiTEC combines the identification of molecular zip codes that target the endogenous condensates with optogenetics to enable controlled and reversible partitioning of an arbitrary cargo, such as enzymes commonly used in proteomics, into the condensate in a blue light dependent manner. We demonstrate a proof of concept by combining LiTEC with proximity-based biotinylation (BioID) and uncover putative components of transcriptional condensates in mouse embryonic stem cells. Our approach opens the road to genome-wide functional studies of endogenous condensates.

overall_design: To test the effect of LiTEC on transcription, global RNA-seq was performed with and without blue-light exposure (30 min, ~3.42 mW/cm^2) on wildtype mESCs and LiTEC mESCs. To check how transcription changes over time, RNAs were extracted on 0 hour, 1 hour and 4 hour after 30 min of blue light exposure (or without blue light exposure)

first: Laura

last: Arrigoni

first: Ward

last: Deboutte

first: Ibrahim

last: Cisse


title: The CIC::DUX4 oncoprotein maintains DNA integrity through direct regulation of the catalytic subunit of DNA polymerase epsilon (POLE).

first: Ross

last: Okimoto

address: 513 Parnassus Ave

institute: University of California, San Francisco

summary: Transcription factor (TF) fusion oncoproteins represent cancer specific alterations that arise from chromosomal rearrangements.  Through target gene recognition, TF fusions can disseminate transcriptional responses that collectively work to drive tumorigenesis. Thus, identifying the molecular targets that operate as a disease driving network can potentially uncover key actionable dependencies. We have taken this strategy to dissect the underlying biological mechanism by which CIC::DUX4, a fusion oncoprotein associated with dismal outcomes, drives sarcomagenesis. We and others have defined a CIC::DUX4 fusion mediated network that dysregulates cell-cycle and DNA replication checkpoints. Specifically, CIC::DUX4-mediated CCNE1 upregulation compromises the G1/S transition leading to high DNA replication stress and conferring a dependence on the G2/M checkpoint kinase, WEE1. Thus, WEE1 provides a molecular brake to enable effective DNA repair prior to mitotic entry. Importantly, the mechanism by which CIC::DUX4 regulates DNA repair remains unknown. Here we show that the catalytic subunit of DNA polymerase epsilon (POLE) is essential for DNA integrity and cellular division in CIC::DUX4 sarcoma. Mechanistically, POLE loss increases DNA damage and induces p21-mediated cellular senescence to limit CIC:DUX4 mediated tumor growth in vitro and tumor formation in vivo. Altogether, we credential POLE as a CIC::DUX4 target and further characterize a functional network through which CIC::DUX4 operates to drive tumor progression and survival.

overall_design: To examine the regulatory role of CIC::DUX4 on POLE expression, we performed RNA interference (RNAi) to silence POLE in NCC-CDS1_X1_C1 patient-derived sarcoma cells. To understand how POLE regulates cellular senescence in CIC::DUX4 sarcoma cells, we conducted transcriptional profiling via RNA-Seq in POLE-deficient and POLE replete NCC_CDS1_X1_C1 cells.

first: Zeinab

last: Kosibaty

first: Cuyler

last: Luck

middle: A


title: Comprehensive identification of coronary artery disease-associated variants regulating vascular smooth muscle cell gene expression [CUT&RUN]

status: Public on Oct 14 2024

city: Charlottesville

first: Nicolas

last: Barbera

state: Virginia

address: 1335 Lee Street

department: Department of Genome Sciences

institute: University of Virginia

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Coronary artery disease (CAD) is a complex disorder with genetic and environmental influences. Genome-wide association studies (GWAS) have identified over 300 genomic loci associated with disease risk. However, identifying the functional variants within these loci has been limited in large part due to linkage disequilibrium. This represents a critical step in understanding the molecular mechanisms underlying disease risk. To identify and prioritize candidate causal CAD-associated variants, we performed lentivirus-based massively parallel reporter assays (lentiMPRAs) in primary vascular smooth muscle cells (SMCs), which play significant roles in atherosclerosis, the underlying cause of CAD. We tested 25892 CAD-associated variants for their allele-specific enhancer activity in quiescent and proliferative SMCs, modeling healthy and disease conditions. We identified 122 candidate variants showing significant enhancer activity and differences in reporter gene expression between risk and non-risk alleles. We also identified 23 variants showing condition-biased allelic imbalance and 41 variants showing sex-biased allelic imbalance. We further functionally characterized 25892 variants by performing CUT&RUN assays to identify variants in enhancer and promoter regions of SMCs. By integrating the results of these experiments, we identified a credible set of 49 CAD-associated variants in functionally relevant regions. Furthermore, by comparing these identified variants with our previously obtained expression quantitative trait loci (eQTL) data, 27 of these 49 variants were associated with SMC gene expression levels. Finally, we performed CRISPRi experiments on 8 variants comprising 9 variant-gene pairs, rs35976034 (MAP1S), rs4888409 (CFDP1), rs73193808 (MAP3K7CL), rs67631072 (INPP5B/FHL3), rs1651285 (SNHG18), rs17293632 (SMAD3), rs2238792 (ARVCF), rs4627080 (NRIP3), to confirm their regulatory potential of nearby gene expression. Taken together, our results comprehensively fine-map the causal variants that confer increased risk of CAD through their effects on vascular smooth muscle cells.

overall_design: Cleavage-under-targets-&-release-using-nuclease (CUT&RUN) for histone modifications H3K4me3, H3K27ac, and H2BK20ac in primary vascular smooth muscle cells in standard and serum-free media

first: Alexia

last: Wallace

first: Noah

last: Perry

first: Lily

last: Lei

first: Hester

last: den Ruijter

first: Mete

last: Civelek


title: Spatial-Mux-seq: Multiplexed spatial mapping of chromatin features, transcriptome, and proteins at tissue scale

city: Philadelphia

first: Liran

last: Mao

state: PA

address: 422 Curie Boulevard

department: Perelman School of Medicine

institute: University of Pennsylvania

summary: we present a multi-modal spatial technology that allows simultaneous profiling of four different modalities, including multi epigenomic modalities (any combination of two histone modifications and open chromatin at the same time), whole transcriptome, and a panel of proteins at tissue scale and cellular level in a spatially resolved manner.

overall_design: Spatially resolved multiple modalities sequencing in mouse tissues.

first: Yanxiang

last: Deng

first: Pengfei

last: Guo


title: Comprehensive identification of coronary artery disease-associated variants regulating vascular smooth muscle cell gene expression [lentiMPRA]

overall_design: lentiMPRA were performed on 6 vascular smooth muscle cell lines in two different culture conditions, testing 25892 CAD-associated variants for enhancer activity and allelic imbalance.


title: Autoimmune CD4 T cells maintain function and survive in chronic autoimmunity by restricting terminal differentiation (NOD)

status: Public on Oct 12 2024

city: Salt Lake City

first: Maria

last: Bettini

state: Utah

address: University of Utah

department: Pathology

institute: University of Utah

summary: The mechanisms behind survival of autoimmune CD4 T cells during chronic stimulation are unclear. Examining early time-points during T cell priming showed that activation of autoimmune CD4 T cells in the absence of infectious signals allowed maintenance of TCF1 expression, albeit at reduced levels. Tcf7 locus was epigenetically modified in circulating autoimmune CD4 T cells, suggesting a pre-programmed de novo methylation of the locus in early stages of autoimmune CD4 T cell differentiation which mirrored the epigenetic profile of recently recruited CD4 CD62L+ T cells in the tissue.  Collectively, the data presented here show that the unique environment during autoimmune CD4 T cell priming allows T cells to finetune TCF1 expression to maintain long-term survival and function.

overall_design: Single cell ATACseq/RNAseq analysis was performed using 10X Multiome kit. TCR+ cells were sorted from non-draining lymph nodes and pancreatic islets of two 10-week-old NOD female mice

first: Nouf

last: Aljobaily

first: Denise

last: Allard

first: Bryant

last: Perkins

first: Arielle

last: Raugh

first: Tessa

last: Galland

first: Yi

last: Jing

first: W. Zac

last: Stephens

first: Matthew

middle: L

first: J. Scott

last: Hale


title: Gene expression analysis of prostate cancer cells resistant to CDK7 inhibitors

city: London

first: Simak

last: Ali

state: London

address: 1/F ICTEM, Hammersmith Hospital

department: Cancer

country: United Kingdom

institute: Imperial College London

summary: CDK7 has emerged as a cancer target because of its pivotal roles in cell cycle progression and transcription. Several small molecule CDK7 inhibitors (CDK7i) are now in clinical evaluation. Identifying patients most likely to respond to treatment and early detection of tumour evolution towards resistance, are necessary for optimal implementation of cancer therapies. Continuous culturing of prostate cancer cells with Samuraciclib, an ATP-competitive/non-covalent CDK7i, led to outgrowth of resistant cells. RNA-seq expression analysis was undertaken to identify changes in gene expression, revealing acquistion of a mutation, c.289G>A in the CDK7 gene, causing the p.Asp97Asn change.

overall_design: To model acquired resistance in cancer, we cultured 22Rv1 prostate cancer cells in culture medium containing 500nM of Samuraciclib for 4 months. To investigate transcriptomic changes in Samuraciclib-resistant cells, RNA-seq was performed using 6 bioreplicates of RNA prepared from 22Rv1 Samuraciclib-resistant cells (SAMRES) and parental 22Rv1 cells (SAMSEN).

first: Laki

last: Buluwela

first: Chun-Fui

last: Lai


title: The evolution of expression divergence in wild C. elegans

city: Atlanta

first: Avery

middle: Davis

last: Bell

state: GA

address: North Avenue

department: School of Biological Sciences

institute: Georgia Institute of Technology

summary: To examine the ways in which gene expression varies in natural populations and its relationship to genetic divergence, we estimated allele-specific expression in the offspring of multiple wild C. elegans strains crossed with the laboratory reference strain N2. Allele-specific expression analyses are uniquely sensitively able to identify cis regulatory changes, and coupled with analyses of differential expression between parental strains and parents and offspring can determine the regulatory pattern and inheritance mode of gene expression across the genome. We chose 7 strains that represented a range of divergence from N2 and spanned the species tree to cross with N2: EG4348; DL238; CB4856 (the classical ‘Hawaiian’ strain); ECA722; QX1211; and ECA701 and XZ1516 (two extremely diverged strains). This study sheds light on the relationship of genetic and expression divergence, the global patterns of inheritance and regulatory mode of genes in C. elegans, and the factors that differentiate genes with expression divergence from those with stabler expression.

overall_design: We performed RNA-sequencing on experimentally matched young (day 1) adult Caenorhabditis elegans from multiple natural genetic backgrounds (wild strains) and their hybrid offspring. Specifically, we RNA sequenced  three replicates each of 7 wild C. elegans strains (EG4348, DL238, CB4856, ECA722, QX1211, ECA701, XZ1516), the laboratory reference strain N2, and the F1 offspring of crosses between each of the wild strain and N2.

last: Davis Bell

first: Francisco

last: Valencia

first: Annalise

middle: B

last: Paaby


title: Elevated DNA Damage without signs of aging in the short-sleeping Mexican Cavefish

status: Public on Oct 13 2024

city: Tallahassee

first: Evan

last: Lloyd

state: FL

address: 107 Chieftain Way

institute: Florida State University

summary: Dysregulation of sleep has widespread health consequences and represents an enormous health burden. Short-sleeping individuals are predisposed to the effects of neurodegeneration, suggesting a critical role for sleep in the maintenance of neuronal health. While the effects of sleep on cellular function are not completely understood, growing evidence has identified an association between sleep loss and DNA damage, raising the possibility that sleep facilitates efficient DNA repair. The Mexican tetra fish,Astyanax mexicanusprovides a model to investigate the evolutionary basis for changes in sleep and the consequences of sleep loss. Multiple cave-adapted populations of these fish have evolved to sleep for substantially less time compared to surface populations of the same species without identifiable impacts on healthspan or longevity. To investigate whether the evolved sleep loss is associated with DNA damage and cellular stress, we compared the DNA Damage Response (DDR) and oxidative stress levels betweenA. mexicanuspopulations. We measured markers of chronic sleep loss and discovered elevated levels of the DNA damage marker γH2AX in the brain, and increased oxidative stress in the gut of cavefish, consistent with chronic sleep deprivation. Notably, we found that acute UV-induced DNA damage elicited an increase in sleep in surface fish but not in cavefish. On a transcriptional level, only the surface fish activated the photoreactivation repair pathway following UV damage. These findings suggest a reduction of the DDR in cavefish compared to surface fish that coincides with elevated DNA damage in cavefish. To examine DDR pathways at a cellular level, we created an embryonic fibroblast cell line from the two populations ofA. mexicanus. We observed that both the DDR and DNA repair were diminished in the cavefish cells, corroborating thein vivofindings and suggesting that the acute response to DNA damage is lost in cavefish. To investigate the long-term impact of these changes, we compared the transcriptome in the brain and gut of aged surface fish and cavefish. Strikingly, many genes that are differentially expressed between young and old surface fish do not transcriptionally vary by age in cavefish. Taken together, these findings suggest that have developed resilience to sleep loss, despite possessing cellular hallmarks of chronic sleep deprivation.

overall_design: To characterize the transcriptomic profiles of surface fish embryonic fibroblast cells (SEF) and Pachón embryonic fibroblast cells (PEF), we generated bulk RNA sequencing data from 3 replicates from similar cell passage numbers.

first: Fanning

last: Xia

first: Kinsley

last: Moore

first: Carolina

last: Zertuche

first: Aakriti

last: Rastogi

first: Rob

last: Kozol

first: Olga

last: Kenzior

first: Wesley

last: Warren

first: Lior

last: Appelbaum

first: Rachel

last: L Moran

first: Chongbei

last: Zhao

first: Erik

last: Duboue

first: Nicolas

last: Rohner

first: Alex

last: C Keene


title: Intracellular expression of a chimeric antigen reverses resistance to cancer immunotherapy

city: Kyoto

first: kenji

last: Chamoto

state: Kyoto

address: Yoshida Konoe-cho, Sakyo-ku

department: Department of Immunology and Genomic Medicine

country: Japan

institute: Graduate School of Medicine, Kyoto University

summary: The interaction of frameshift mutation-derived cancer neoantigens and cancer immunotherapy remains unknown. We found that live cell adjuvant or cDNA transfection in the muscle, which express MHC-class I and class II-restricted non-self-peptides, generated broad-spectrum anti-tumor immunity. Such chimeric peptides did not need to be tumor-neoantigens, but must be in a single chain (complete T cell antigen: CTA). Long product of frameshift mutation frequently contained CTA and the colon cancer patients with the long frameshift products (>120 amino acids) showed a good prognosis. Mechanistically, live cell adjuvant expressing CTA strengthened crosstalk between dendritic cells and CD8+ T cells in a CD4+ T cells-dependent manner. This cross talk suppressed CD8+ T cell exhaustion and produced stem-cell like progenitor CD8+ T cells in vivo. Combination of the live cell adjuvant conversed unresponsive tumors to responsive to PD-1 blockade therapy. Together, our findings provide a new broad-spectrum cancer immunotherapy, and clarify the type of frameshift neoantigens controlling the efficacy.

overall_design: A total of 4 single cell rna seqence samples, including a pair of tumor-infiltrating CD8+ T cells(CD8 TILs), and a pair of draining lymph node single cells(DLN). Treatment group were MC38 tumor-bearing mice (control),and MC38 tumor-bearing mice receive alive adjuvant Bpmel-SIY-OVAII immunization.(SIY-OVAII)

first: Rongsheng

last: Zhang

first: Yaguchi

last: Tomonori

first: Tasuku

last: Honjo

first: Kenji


title: Autoimmune CD4 T cells maintain function and survive in chronic autoimmunity by restricting terminal differentiation

first: Maria

last: Bettini

overall_design: pancreatic islets, spleen+non-draining lymph nodes were harvested from BDC2.5 transferred cells into congenically labelled NOD mice. BDC2.5 CD4+Vb4 cells were sorted and subjected to 10X multiome scATAC/Gene expression sequncing

first: Nouf

last: Aljobaily

first: Denise

last: Allard

first: Bryant

last: Perkins

first: Arielle

last: Raugh

first: Tessa

last: Galland

first: Yi

last: Jing

first: W. Zac

last: Stephens

first: Matthew

first: J. Scott

last: Hale


title: Effects of aging on gene expression in multiple tissues in surface and cave Astyanax mexicanus

first: Evan

last: Lloyd

summary: Dysregulation of sleep has widespread health consequences and represents an enormous health burden. Short-sleeping individuals are predisposed to the effects of neurodegeneration, suggesting a critical role for sleep in the maintenance of neuronal health. While the effects of sleep on cellular function are not completely understood, growing evidence has identified an association between sleep loss and DNA damage, raising the possibility that sleep facilitates efficient DNA repair. The Mexican tetra fish, Astyanax mexicanus provides a model to investigate the evolutionary basis for changes in sleep and the consequences of sleep loss. Multiple cave-adapted populations of these fish have evolved to sleep for substantially less time compared to surface populations of the same species without identifiable impacts on healthspan or longevity. To investigate whether the evolved sleep loss is associated with DNA damage and aging, we compared the transcriptional response to aging between surface and cave morphs of A. mexicanus

overall_design: To investigate differences in the transcriptional response to aging, we dissected the whole brain, liver, heart, gut, and a portion of muscle, from young (~1 year) and aged (7-8 year) surface and cave fish, extracted the RNA from each sample, and performed gene expression analysis.

first: Lloyd

last: Evan

first: Xia

last: Fanning

first: Moore

last: Kinsley

first: Zertuche

last: Carolina

first: Rastogi

last: Aakriti

first: Kozol

last: Rob

first: Kenzior

last: Olga

first: Warren

last: Wesley

first: Appelbaum

last: Lior

first: Moran

last: Rachel L.

first: Zhao

last: Chongbei

first: Duboue

last: Erik

first: Rohner

last: Nicholas

first: Keene

last: Alex C.


title: Dynamicdosage compensation of transposable elementsin mammals

status: Public on Feb 12 2025

city: Boston

first: Chunyao

last: Wei

state: MA

address: 185 Cambridge St.

department: Molecular Biology

institute: Massachusetts General Hospital

summary: In mammals, X-linked dosage compensation involves two processes: X-inactivation to balance X chromosome dosage between males and females, and X-hyperactivation to achieve X-autosome balance. Transposable elements (TEs) are repetitive mobile DNA sequences and major sources of X chromosome dosage. Current studies in X-linked dosage compensation mainly focus on genes, with TEs still remaining elusive. Here we use “So-Smart-Seq” to comprehensively determine the allelic dynamics of TEs in the context of imprinted and random X-chromosome inactivation (XCI). We find that chromosomal loci and genetic backgrounds significantly impact TE silencing, and reveal putative roles of SINEs in shaping 3D chromatin architectures. We show remarkable difference in TE silencing between two forms of XCI and discover that X-hyperactivation is absent in TEs. Our data provide deep insight for TE studies and greatly contribute to complete understanding of X-chromosome wide dosage compensation.

sample_organism:
Mus musculus
Mus musculus castaneus

platform_organism:
Mus musculus
Mus musculus castaneus

overall_design:
For the third-party data reanalysis, 188 GSM samples (GSM7980593-7980780) from GSE168455 were reanalyzed. Allelic expression of transposable elements during imprinted XCI was extracted from data GSE168455 that reveal full allelic transcriptome of single preimplantation embryos at various stages from WT and Xist mutant mouse. The allelic ratio between two X chromosome was calculated for each TE family.
To acquire allelic expression dynamics of transposable elements in random XCI, allelic bulk RNAseq was performed in differentiating TST ES cells, a mus/cas hybrid ES cell line where X chromosome from mus allele is progressively silenced during random XCI. Expression of transposable elements were determined  and the allelic ratio between two X chromosome was calculated for each TE family.

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The table below lists GEO accessions reused/reanalyzed for this study.
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first: Barry

last: Kesner

first: Jeannie

middle: T

last: Lee


title: Therapeutic benefits of maintaining CDK4/6 inhibitors and incorporating CDK2 inhibitors beyond progression in breast cancer

status: Public on Oct 10 2024

city: New York

first: Jessica

middle: Sarah

last: Armand

state: NY

department: Pathology and Cell Biology

institute: Columbia University Irving Medical Center

summary: The combination of CDK4/6 inhibitors (CDK4/6i) and endocrine therapy (ET) has significantly improved outcomes for patients with hormone receptor-positive (HR+) metastatic breast cancer. However, most patients eventually develop resistance, leading to treatment discontinuation. The therapeutic benefits of maintaining CDK4/6i or transitioning to CDK2 inhibitors (CDK2i) beyond disease progression remain unclear. Here, we show that maintaining CDK4/6i and ET or combining them with CDK2i effectively suppresses the growth of drug-resistant HR+ breast cancer cells by prolonging G1-phase progression. CDK4/6i maintenance triggers a non-canonical pathway for Rb inactivation through post-translational degradation, resulting in attenuated E2F activity and slowed G1 progression. ET further augments this effect by inhibiting c-Myc-mediated E2F amplification. Although the maintenance of CDK4/6i and ET outperforms CDK2i with ET, the triple combination of CDK4/6i, CDK2i, and ET most effectively suppresses both E2F activity and tumor growth. Moreover, while overexpression of both cyclin E and A can promote resistance to the CDK4/6i and CDK2i combination, cyclin E plays a more pivotal role in developing resistance. These findings highlight the potential of sustained CDK4/6i therapy and the incorporation of CDK2i to mitigate resistance in HR+ breast cancer.

overall_design: To gain insight into differences in gene expression upon introduction of CDK2i after dual resistance, palbociclib (CDK4/6i) +fulvestrant (ET) resistant MCF-7 cells were either discontinued from treatment, maintained in palbociclib + fulvestrant, switched to INX-315 (CDK2i) + fulvestrant, or switched to palbociclib + INX-315 + fulvestrant for 20 days then submitted to bulk RNA sequencing.

middle: S

first: Heewon

last: Yang


title: Effects of UV-B radiation on whole-body gene expression of surface and cave Astyanax mexicanus

first: Evan

last: Lloyd

summary: Dysregulation of sleep has widespread health consequences and represents an enormous health burden. Short-sleeping individuals are predisposed to the effects of neurodegeneration, suggesting a critical role for sleep in the maintenance of neuronal health. While the effects of sleep on cellular function are not completely understood, growing evidence has identified an association between sleep loss and DNA damage, raising the possibility that sleep facilitates efficient DNA repair. The Mexican tetra fish, Astyanax mexicanus provides a model to investigate the evolutionary basis for changes in sleep and the consequences of sleep loss. Multiple cave-adapted populations of these fish have evolved to sleep for substantially less time compared to surface populations of the same species without identifiable impacts on healthspan or longevity. To investigate whether the evolved sleep loss is associated with DNA damage and cellular stress, we compared the DNA Damage Response (DDR) between A. mexicanus populations.

overall_design: To investigate differences in the DNA damage response, we subjected surface and cave larvae, at 6 days post-fertilization, to 60 seconds of UV-B radiation, or 60 seconds of white light as control. We then pooled individuals in groups of 4, extracted the RNA from each pooled sample, and performed gene expression analysis.

first: Fanning

last: Xia

first: Kinsley

last: Moore

first: Carolina

last: Zertuche

first: Aakriti

last: Rastogi

first: Rob

last: Kozol

first: Olga

last: Kenzior

first: Wesley

last: Warren

first: Lior

last: Appelbaum

first: Rachel

last: Moran

first: Chongbei

last: Zhao

first: Erik

last: Duboue

first: Rhoner

first: Alex

middle: C

last: Keene


title: Trans-generational adaptation to maternal climate through hormone transport in plants [bulk RNA-seq]

status: Public on Oct 11 2024

city: Norwich

first: William

last: Bezodis

address: Norwich Research Park, Colney Lane

department: Crop Genetics

institute: John Innes Centre

summary: Whether and how organisms can inherit environmental information from their parents is a major question in evolutionary theory. Plants have evolved to link reproductive development to seasonal environmental cues and seed dormancy is highly contingent on the environmental temperature during seed set, although the mechanism by which seeds acquire seasonal timing information is unclear. Here we show that loss of maternal like heterochromatin protein 1 (LHP1) causes an inability of progeny seeds to sense temperature and that this is linked mechanistically to reduced ABA levels in seeds and hyperaccumulation of free nitrate. Remarkably, single cell transcriptomics reveals that in both maternal fruit and seed tissues, the effect of small changes in temperature closely phenocopies the lhp1 mutant phenotype and ABA biosensor imaging reveal large fluxes of maternal ABA into seeds is modulated by temperature cues. We show that temperature activates ABA production in leaves and that maternal ABA is necessary and sufficient for progeny seeds to acquire seed dormancy. Thus, we reveal that the climate experience of mother plants causes adaptation of progeny behaviour via hormone transport during seed set.

overall_design: siliques were dissected to separate fruit tissue and seed tissue which were sequenced seperately. Lhp1-6 and WT Col-0 were grown at 16°CD/14°CN.

first: Xiaochao

last: Chen

first: Steven

last: Penfield


title: Bile acid synthesis impedes tumor-specific T cell responses during liver cancer.

status: Public on Oct 09 2024

city: La Jolla

first: Kathryn

last: Lande

state: CA

address: 10010 N Torrey Pines Rd

department: IGC

institute: Salk Institute

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

overall_design: Refer to individual Series


title: Covalent Targeting of Splicing in T Cells

first: Jahan

last: Rahman

state: New York

address: 417 E 68th St

department: Center for Hematologic Malignancies

institute: Memorial Sloan Kettering

summary: Despite significant interest in therapeutic targeting of splicing, few chemical probes are available for the proteins involved in splicing. Here, we show that elaborated stereoisomeric acrylamide chemical probe EV96 and its analogues lead to a selective T cell state-dependent loss of interleukin 2-inducible T cell kinase (ITK) by targeting one of the core splicing factors SF3B1. Mechanistic investigations suggest that the state-dependency stems from a combination of differential protein turnover rates and availability of functional mRNA pools that can be depleted due to extensive alternative splicing. We further introduce a comprehensive list of proteins involved in splicing and leverage both cysteine- and protein-directed activity-based protein profiling (ABPP) data with electrophilic scout fragments to demonstrate covalent ligandability for many classes of splicing factors and splicing regulators in primary human T cells. Taken together, our findings show how chemical perturbation of splicing can lead to immune state-dependent changes in protein expression and provide evidence for the broad potential to target splicing factors with covalent chemistry.

overall_design: Expanded primary human T cells were pelleted (600 g, 5 min, 4 °C) and re-suspended in fresh T cell media at 2 x 106 cells/mL for compound treatment. T cells were treated with WX-02-23 (5 µM), WX-02-43 (5 µM), PladB (10 nM)) or DMSO control in 6-well plates (2 mL/well, 4 x 106 cells/treatment) with or without CD3/CD28 stimulation for 8 h. For treatment conditions that required T cell activation, plates were pre-coated with 1.5 mL PBS containing CD3 (5 µg/mL, BioXCell) and CD28 (2 µg/mL, BioXCell) overnight at 4 °C or for 2 h at 37 °C and washed with PBS (3 mL, 3 times) prior to treatment.

first: Kevin

last: Scott

first: Hiroyuki

last: Kojima

first: Nathalie

last: Ropek

first: Charles

middle: D

first: Tiffany

last: Zhang

first: Simon

last: Hogg

first: Henry

last: Sanford

first: Caroline

last: Webster

first: Xiaoyu

first: Bruno

last: Melillo

first: Benjamin

middle: F

last: Cravatt

first: Jiankun

last: Lyu

first: Omar

last: Abdel-Wahab

first: Ekaterina

middle: V

last: Vinogradova


title: Cavefish

first: Evan

last: Lloyd


title: Autoimmune CD4 T cells maintain function and survive in chronic autoimmunity by restricting terminal differentiation (whole genome enzymatic methylation sequencing)

first: Maria

last: Bettini

type: Methylation profiling by high throughput sequencing

overall_design: ndLN and pancreatic islets were subjected to whole genome enzymatic methylation sequencing

first: Nouf

last: Aljobaily

first: Denise

last: Allard

first: Bryant

last: Perkins

first: Arielle

last: Raugh

first: Tessa

last: Galland

first: Yi

last: Jing

first: W. Zac

last: Stephens

first: Matthew

first: J. Scott

last: Hale


title: Bile acid synthesis impedes tumor-specific T cell responses during liver cancer [RNA-seq2]

first: Kathryn

last: Lande

summary: The tumor's metabolic landscape significantly influences anti-tumor immunity, but it remains unclear how metabolic pathways commonly active in the organ of tumor-origin influence immunosurveillance. This study focused on the liver and identified accumulation of primary conjugated and secondary bile acids (BAs) as metabolic features associated with hepatocellular carcinoma and liver cancer models. Inhibiting conjugated BA synthesis through BAAT-deletion in hepatocytes enhanced tumor-specific T cell responses, reduced growth, and sensitized tumors to immune checkpoint blockade. BAs regulated CD8+ T cells differently; primary BAs like TCDCA induced oxidative stress and secondary BAs like LCA impaired T cell function through ER stress, which was countered by UDCA. Indeed, dietary UDCA provision suppressed tumor progression. These findings demonstrate how manipulating organ-specific metabolites affects antitumor immunity and modifying BA synthesis or dietary intake could enhance immunotherapy in liver cancer.

overall_design:
To investigate the effect of LCA treatment on wild type and Nur77 KO CD8+ T cells
Gene expression profiling analysis using data obtained from RNA-seq of T Cells treated with LCA.

first: Siva Karthik

last: Varanasi

first: Susan

last: Kaech


title: Bile acid synthesis impedes tumor-specific T cell responses during liver cancer [CITE-seq]

overall_design: Single-cell RNA CITE-seq of immune cells from tumor brearing livers from naïve (mock infected), AAV-sgSCR and AAV-sgBAAT were performed at Day 30 post infection. Each sample is a pool of 3 tumor samples.


title: Bile acid synthesis impedes tumor-specific T cell responses during liver cancer [RNA-seq1]

overall_design:
To investigate the effect of bile acid treatment on T Cell gene expression.
Gene expression profiling analysis using data obtained from RNA-seq of T Cells treated with different bile acids.in vitro activated CD8+ T cells  were exposed with different bile acids for 24 hours following with RNA was isolated using Qiagen RNA isoaltion kit.


title: Comprehensive profiling of migratory primordial germ cells reveals niche-specific differences in non-canonical Wnt and Nodal-Lefty signaling in anterior vs posterior migrants

status: Public on Oct 08 2024

city: San Francisco, CA

first: Rebecca

middle: Garrett

last: Jaszczak

address: 35 Medical Center Way, Room 913D

institute: UCSF

summary: Mammalian primordial germ cells (PGCs) migrate asynchronously through the embryonic hindgut and dorsal mesentery to reach the gonads. To characterize transcriptional heterogeneity of migrating PGCs and their niches, we performed single-cell RNA sequencing of 13,262 mouse PGCs and 7,868 surrounding somatic cells during migration (E9.5, E10.5, E11.5) and in anterior versus posterior locations to enrich for leading and lagging migrants. Analysis of PGCs by position revealed dynamic gene expression changes between faster or earlier migrants in the anterior and slower or later migrants in the posterior at E9.5. At E10.5, we found that anterior PGCs upregulate Nodal transcriptional targets including Lefty1/2 and validated this LEFTY1/2 upregulation via whole-mount immunofluorescence staining. This positional and temporal atlas of mouse PGCs supports the idea that niche interactions along the migratory route elicit changes in proliferation, actin dynamics, pluripotency and epigenetic reprogramming.

overall_design: We pooled sorted GFP+ germ cells and an equal amount of GFP- somatic cells from each timepoint and anatomical location. We loaded counted cells onto a 10X V2 chip to create a single cell emulsion. For E9.5 Anterior, we loaded 3400 cells, and for E9.5 Posterior, 2800 cells. For E10.5 Anterior, we loaded two technical replicates totaling 9300 cells. For E10.5 Posterior, we loaded two technical replicates totaling 11200 cells. For the two E11.5 biological replicates, we loaded 15,000 and 10,000 cells, respectively. Library creation was done in house following 10X ChromiumTM Single Cell 3' Reagent Kits v2 User Guide Rev A. E9.5 and E11.5 libraries were sequenced on HiSeq 4000 (Illumina) with paired-end sequencing parameters: Read1, 98 cycles; Index1, 14 cycles; Index2, 8 cycles; and Read2, 10 cycles. E10.5 libraries were sequenced on Novaseq (Illumina) with paired-end sequencing parameters: Read1, 150 cycles; i7 Index, 8 cycles; i5 index, 0 cycles; and Read2, 150 cycles.

middle: G

first: Jay

last: Zussmann

first: Diana

last: Laird


title: Cathepsin L as a dual-target to mitigate muscle wasting while enhancing anti-tumor efficacy of anti-PD-L1 [Bulk2]

city: Busan

first: Haeseung

last: Lee

address: 63 Beon-gil 2, Busandaehag-ro, Geumjeong-gu

department: College of Pharmacy

country: South Korea

institute: Pusan National University

summary: Immune checkpoint inhibitors (ICIs) have revolutionized cancer therapy; however, their use is frequently associated with immune-related adverse events (irAEs). In this study, anti-PD-L1 therapy exacerbates muscle wasting in tumor-bearing male mice despite its anti-tumor efficacy, accompanied by an accumulation of CD8+ T cells in muscle. Single-cell RNA sequencing identifies these cells as tissue-resident memory-like CD49a+ CD8+ T cells. While CD8+ T cell depletion prevents muscle wasting, it compromises the anti-tumor efficacy of anti-PD-L1. To resolve this paradox, we identify cathepsin L (CTSL) as a dual-target capable of suppressing both tumor progression and CD8+ T cell-mediated muscle wasting, through integrative transcriptomic analysis. Pharmacological inhibition of CTSL not only mitigates anti-PD-L1-induced muscle wasting but also further suppresses tumor growth, potentially via downregulation of BNIP3. Here, we show that CTSL is a dual-action target to uncouple anti-tumor efficacy from muscle-specific irAEs, offering a strategy to improve clinical outcomes of ICIs.

overall_design: To identify effective therapeutic targets for both cachexia and muscle-related immune-related adverse events (irAEs), we generated RNA-seq data from muscles in a tumor bearing mouse model treated with IgG, anti-PD-L1, and anti-CD8a

first: Nayoung

last: Song


title: Selective Mitochondrial Respiratory Complex I Subunit Deficiency Causes Tumor Immunogenicity

status: Public on Oct 05 2024

first: Tevis

last: Vitale

state: Massachusetts

address: 50 Industrial Drive, LC6201

institute: Dana-Farber Cancer Institute

summary: Targeting of specific metabolic pathways in tumor cells has the potential to sensitize them to immune-mediated attack. Here we provide evidence for a specific means of mitochondrial respiratory Complex I (CI) inhibition that improves tumor immunogenicity and sensitivity to immune checkpoint blockade (ICB). Targeted genetic deletion of the CI subunits Ndufs4 and Ndufs6, but not other subunits, induces an immune-dependent tumor growth attenuation in mouse melanoma models. We show that deletion of Ndufs4 induces expression of the transcription factor Nlrc5 and genes in the MHC class-I antigen presentation and processing pathway. This induction of MHC-related genes is driven by an accumulation of pyruvate dehydrogenase-dependent mitochondrial acetyl-CoA downstream of CI subunit deletion. This work provides a novel functional modality by which selective CI inhibition restricts tumor growth, suggesting that specific targeting of Ndufs4, or related CI subunits, increases T-cell mediated immunity and sensitivity to ICB.

overall_design: To investeigate the transcriptional changes that lead to the observed immunogenicity of the sgNdufs4 cells, we performed gene expression profiling using data obtained from RNA-seq of the sgControl and sgNdufs4 cell lines under standard culture conditions.

first: Jiaxin

last: Liang

first: Xixi

last: Zhang

first: Thomas

last: Jackson

first: Deyang

last: Yu

first: Mark

last: Jedrychowski

first: Steve

middle: P

last: Gygi

first: Hans

middle: R

last: Widlund

first: Kai

middle: W

last: Wucherpfennig

first: Pere

last: Puigserver


title: Decapping activators Edc3 and Scd6 act redundantly with Dhh1 in nutrient-replete cells to post-transcriptionally repress starvation-induced pathways [Ribo and RNA-seq]

city: Bethesda

first: Alan

last: Hinnebusch

address: 6 Center Drive, Building 6, Room 233

department: NICHD

institute: NIH

summary: Degradation of many yeast mRNAs involves decapping by Dcp1:Dcp2. Previous studies on decapping activators Edc3 and Scd6 suggested limited roles in promoting mRNA decay in yeast cells. RNA-seq analysis of mutants lacking one or both proteins reveals that Scd6 and Edc3 have largely redundant activities in targeting numerous mRNAs for degradation, which are masked in the single mutants. These transcripts are frequently targeted by decapping activators Dhh1 and Pat1 and the evidence suggests that Scd6/Edc3 act interchangeably to recruit Dhh1 to Dcp2 independently of Pat1. Ribosome profiling shows that redundancy between Scd6 and Edc3 and their functional interactions with Dhh1 and Pat1 extends to translational repression of particular transcripts, including a cohort of poorly translated mRNAs displaying interdependent regulation by all four factors. Scd6/Edc3 also participate with Dhh1/Pat1 in post-transcriptional repression of proteins required for respiration and catabolism of non-optimal carbon sources, which are normally expressed only in limiting glucose. Simultaneously eliminating Scd6/Edc3 increases mitochondrial membrane potential and elevates metabolites of the tricarboxylic acid and glyoxylate cycles critical for growth at low glucose levels. Thus, Scd6/Edc3 act redundantly, in parallel with Dhh1 and in cooperation with Pat1, to adjust gene expression to nutrient availability by controlling mRNA decapping and decay.

overall_design: Ribosome profile and RNA-sequencing studies include total 16 samples, 8 for ribosome profiling and 8 for total RNA-sequencing from duplicate cultures of WT, edc3∆, scd6∆, and edc3∆scd6∆ yeast strains. The RNA-Seq samples had ERCC Spike-In added to the samples.  Yeast strains were grown in YPD to OD600 = 0.6~0.8 at 30˚C, then flash frozen.  Ribosome protected or total RNA libraries were prepared from yeast cells and sequenced.

first: Rakesh

last: Kumar

first: Fan

first: Shreyas

last: Niphadkar

first: Chisom

last: Onu

first: Anil

middle: K

last: Vijjamarri

first: Miriam

last: Greenberg

first: Sunil

last: Laxman


title: Decapping activators Edc3 and Scd6 act redundantly with Dhh1 in nutrient-replete cells to post-transcriptionally repress starvation-induced pathways [ChIP-seq]

overall_design: ChIP-Seq study includes 6 samples.  Triplicates of each wild type and edc3∆scd6∆ yeast strain.


title: Cattle Cleavage Under Targets & Release Using Nuclease (CUT&RUN)  for CENP-A locus identification

status: Public on Oct 02 2024

city: Moscow

first: Brenda

last: Murdoch

state: Idaho

address: 875 Perimeter Dr.

department: Department of Animal, Veterinary & Food Sciences

institute: University of Idaho

summary: CUT&RUN experiment was carried out on bovine semeimbranosous muscle satellite cells to identify the locus of the accumulation of the centromeric protein A (CENP-A) on the cattle Y-chromosome. This was carried out to support the annotation of the DNA signal comprising tandem array of a centromeric satellite signal which characterizes the centromere on the Y-chromosome.

overall_design: To identify the locus of CENP-A signal accumulation with the pull down assay from the bovine satellite cells, the GeneTex CENP-A antibody with catalog number GTX13939 was used in this study. The antibody is monoclonal and of the isotype IgG1

first: Temitayo

middle: A

last: Olagunju

first: Benjamin

last: Rosen

first: Holly

last: Neibergs

first: Gabrielle

middle: M

last: Becker

first: Kimberly

last: Davenport

first: Christine

last: Elsik

first: Tracy

last: Hadfield

first: Sergey

last: Koren

first: Kristen

last: Kuhn

first: Arang

last: Rhie

first: Katie

last: Shira

first: Amy

last: Skibiel

first: Morgan

last: Stegemiller

first: Jacob

last: Thorne

first: Patricia

last: Villamediana

first: Noelle

middle: E

last: Cockett

first: Timothy

last: Smith


title: Cathepsin L as a dual-target to mitigate muscle wasting while enhancing anti-tumor efficacy of anti-PD-L1 [Bulk1]

status: Public on Oct 01 2024

first: Haeseung

last: Lee

overall_design: To identify effective therapeutic targets for both cachexia and muscle-related immune-related adverse events (irAEs), we generated RNA-seq data from lung and muscles in a tumor bearing mouse model.

first: Nayoung

last: Song


title: Effect of p.E475G mutation in the PNPT1 gene on 293T global transcriptome

status: Public on Oct 07 2024

city: Milano

first: Federico

last: Zambelli

address: Via Giovanni Celoria 26

department: Department of Biosciences

country: Italy

institute: University of Milan

summary: We genereted via genome editing 293T cell lines with the heterozygous and homozygous nucleotide substitution c.1424A>G in the PNPT1 gene (namely EK81 and EK81.8, respectively), causing the amino acidic variation p.E475G, and studied their transcriptomes before and after 6 hours of treatment with 1 µM staurosporine to induce apoptosis

overall_design: Comparative RNA expression analysis of RNA-seq data for 293T cells and its derivatives EK81 and EK81.8

first: Federica

last: Briani

first: Roberto

last: Pizzoccheri

first: Giulio

last: Pavesi

first: Tania

last: Camboni


title: The six unique histone deacetylase (HDAC) 1 complexes vary greatly in abundance and utilize distinct molecular assemblies

city: Leicester

first: Shaun

last: Cowley

address: Henry Wellcome Building, Lancaster Road

department: Molecular and Cell Biology

institute: University of Leicester

summary: Histone deacetylase 1 and 2 (HDAC1/2) are highly related enzymes that that regulate histone acetylation levels in all cells, as catalytic and structural components of six unique multi-protein complexes: SIN3, NuRD, CoREST, MIDAC, MIER1 and RERE. Co-immunoprecipitation of HDAC1-Flag followed by mass-spectrometry revealed that 92% of the HDAC1 in mouse embryonic stem cells resides in 3 complexes, NuRD (49%), CoREST (28%) and SIN3 (15%).  To delineate the function of these different biochemical entities we compared binary structures of the MTA1:HDAC1 and MIDAC:HDAC1 to identify conserved residues on the surface of HDAC1 that would potentially perturb one or more complexes. A single mutation, Y48E, disrupts binding to all complexes with the exception of SIN3. Remarkably, rescue experiments performed with HDAC1-Y48E in HDAC1/2 double-knockout cells, showed that retention of SIN3 binding alone is sufficient for cell viability. Gene expression and histone acetylation patterns were perturbed in both Y48E and a second mutant cell line, HDAC1-E63R, indicating that cells require a full repertoire of the HDAC1/2 complexes to regulate their transcriptome appropriately. Comparative analysis of SIN3/HDAC1 and MTA1/HDAC1 structures confirmed the differential modes of HDAC1 recruitment, with the surface including Y48 bound only by ELM2/SANT domain containing complexes. We provide novel molecular insights into the abundance, co-factors and assemblies of this crucial family of chromatin modifying machines.

overall_design: To investigate the functions of the distinct HDAC1 corepressor containing complexes we stably expressed a variety of HDAC1-Flag constructs (WT, Y48E, E63R) in Hdac1/2 conditional double knockout ESCs. With the aim of disrupting complex specific interactions between HDAC1 and its binding partners. Following deletion of endogenous Hdac1/2 we performed bulk RNA-seq to determine the consequences of the Y48E and E63R mutations on gene expression in ESCs relative to the WT HDAC1 construct.

first: Ahmad

last: Alshehri

first: India

last: Baker

first: David

last: English

first: Trong

last: Khoa Pham

first: Louise

last: Fairall

first: Mark

middle: O

last: Collins

first: John

last: Schwabe


title: Proximity-dependent labeling identifies dendritic cells that prime the antitumor CD4+ T cell response

status: Public on Oct 04 2024

first: Tiago

last: Castro

address: 1230 York Avenue

institute: The Rockefeller University

summary: Dendritic cells (DCs) are uniquely capable of transporting tumoral antigens to tumor-draining lymph nodes (tdLNs), and also interact with effector T cells within the tumors themselves, mediating both natural antitumor immunity and the response to checkpoint blockade immunotherapy. Using LIPSTIC (Labeling Immune Partnerships by SorTagging Intercellular Contacts)-based single-cell transcriptomics, we identify individual DCs capable of presenting antigen to CD4+ T cells in the tdLN as well as inside tumors. These represent only a fraction of all DCs present and display a distinctive activated phenotype that in the LN includes production of cytokine IL-27, required for efficient T cell priming and tumor rejection. Tumor progression results in loss of effective priming of naïve CD4+ T cells, downstream of transcriptional changes in DCs that are manifested already when they arrive at the tdLN, which can be rescued by CTLA4 checkpoint blockade.

overall_design: Proximity-dependent labeling and subsequent RNA sequencing (Plate-based scRNA-seq, 10x Genomics, and bulk RNA-seq) were used to analyze dendritic cells (DCs) from tumor-draining lymph nodes (tdLNs) and tumor microenvironments (TME) in mice. The dataset includes RNA profiles of DCs involved in tumor-specific CD4+ T cell interactions, captured via photoactivation and LIPSTIC labeling, under steady-state and checkpoint blockade conditions.

first: Aleksey

last: Chudnovskiy

first: Sandra

last: Nakandakari-Higa

first: Ang

last: Cui

first: Chia-Hao

last: Lin

first: Moshe

last: Sade-Feldman

first: Brooke

last: Phillips

first: Juhee

last: Pae

first: Luka

last: Mesin

first: Juliana

last: Bortolatto

first: Lawrence

last: Schweitzer

first: Giulia

last: Pasqual

first: Li-Fan

last: Lu

first: Nir

last: Hacohen

first: Gabriel

last: Victora


title: Combining multiple stressors unexpectedly blocks bacterial migration and growth

status: Public on Sep 30 2024

city: Urbana

first: Joseph

last: Sanfilippo

state: IL

address: 600 S Matthews Ave

institute: University of Illinois

summary: In nature, organisms experience combinations of stressors. However, laboratory studies use batch cultures, which simplify reality and focus on population-level responses to individual stressors. In recent years, bacterial stress responses have been examined with single-cell resolution using microfluidics. Here, we use a microfluidic approach to simultaneously provide a physical stressor (shear flow) and a chemical stressor (H2O2) to the human pathogen Pseudomonas aeruginosa. By treating cells with levels of flow and H2O2 that commonly co-occur in human host tissues, we discover that previous reports significantly overestimate the H2O2 levels required to block bacterial growth. Specifically, we establish that flow increases H2O2 effectiveness 50-fold, explaining why previous studies lacking flow required much higher concentrations. Using natural H2O2 levels, we identify the core H2O2 regulon, characterize OxyR-mediated dynamic regulation, and demonstrate that multiple H2O2 scavenging systems have redundant roles. By examining single-cell behavior, we serendipitously discover that the combined effects of H2O2 and flow block pilus-driven surface migration. Thus, our results counter previous studies and reveal that natural levels of H2O2 and flow synergize to restrict bacterial motility and survival. By studying two stressors at once, our research highlights the limitations of oversimplifying nature and demonstrates that physical and chemical stress can combine to yield unpredictable effects.

overall_design: We performed RNA seqiencing on P. aeruginosa cells 5 minutes after 8 µM H2O2 exposure. Cells with no H2O2 treatment served as control.

first: Anuradha

last: Sharma

first: Alexander

last: Shuppara

first: Gilberto

last: Padron


title: Young and Aged Striatal Astrocyte MERFISH Panel

status: Public on Oct 03 2024

city: Los Angeles

first: Kay

last: Linker

address: 10833 Le Conte Avenue, 53-263 CHS

department: Physiology

institute: UCLA

summary: Given that the striatum is a large, multifunctional nucleus, we next aimed to understand where  astrocyte subtypes exist in the striatum at a single-cell resolution using MERFISH. We reproducibly identified the seven astrocyte subtypes, some of which were dorsal (A1, A7), medial (A3, A6) or ventral dominant (A4).

overall_design: MERFISH was performed according to Vizgen's instructions. Data was initally processed using MERLIN, and cell segmentation completed with Vizgen software.

first: Baljit

last: Khakh


title: Mouse Striatum 18 months of age

summary: The striatum is a functionally diverse brain region. We investigated aged astrocyte and glia diversity within this brain region.

overall_design: To investigate the functional heterogeneity of astrocytes from a defined brain region, we performed single cell RNA-seq of striatal cells from aged mice 18 months old

first: Linker

last: Kay

first: Khakh

last: Baljit


title: Genotyping of hundreds of individuals from Kazakh population in Kazakhstan

city: Astana

first: Ulykbek

last: Kairov

address: Kabanbay batyr ave.53

department: Center for Life Sciences

country: Kazakhstan

institute: National Laboratory Astana, Nazarbayev University

summary: Genotyping of hundreds of healthy individuals with Kazakh ethnicity as a reference cohort for further genetic investigations

overall_design: Control samples without any inherited and severe diseases


title: Abberant neuronal Wnt activation causes defective TDP43 localization

status: Public on Sep 27 2024

city: Baltimore

first: Shanthini

last: Sockanathan

state: Maryland

address: PCTB1004, 725 North Wolfe Street

department: Neuroscience

institute: The Johns Hopkins University School of Medicine

summary: Conserved neuropathologies across multiple neurodegenerative diseases have identified shared causal pathways for neurodegeneration. One pathway involves the RNA-binding protein TDP-43, whose nuclear exclusion and cytoplasmic accumulation are established drivers of neurodegeneration. TDP-43 mislocalization is attributed to defective nucleocytoplasmic transport (NCT), but the underlying mechanisms are unclear. The vertebrate-specific six-transmembrane enzyme, Glycerophosphodiester phosphodiesterase 2 (GDE2 or GDPD5), cleaves the glycosylphosphatidylinositol (GPI)-anchor that tethers some proteins to the membrane. Here, we show that loss of GDE2 leads to aberrant sustained activation of canonical Wnt signaling in adult neurons, which is sufficient to cause NCT and nuclear pore abnormalities and the nuclear exclusion of TDP-43. GDE2 disruption coincides with TDP-43 nuclear exclusion in postmortem tissue from patients with Amyotrophic Lateral Sclerosis (ALS), and neuronal Wnt signaling is erroneously activated in Drosophila and induced human spinal neuronal (iSN) models of ALS. Wnt knockdown in Drosophila ALS models mitigates cell death, while pharmacological inhibition of Wnt activation in iSNs from ALS patients rescues mRNA levels of known TDP-43 splicing targets. Our study identifies GDE2 as a critical regulator of Wnt signaling in adult neurons and highlights Wnt pathway activation as a previously unappreciated mechanism contributing to NCT and TDP-43 abnormalities in disease.

overall_design: We carried out RNA-seq comapring cortices from 19-month WT and Gde2KO animals to evaluate splicing defects due to TDP43 pathology. To look for pathways initiating changes in TDP43 trafficking, we performed RNAseq analyzing cortices from 4-month old WT and Gde2KO animals.

last: Zhang

first: Loyal

last: Goff


title: Phlpp1 regulates endochondral ossification via the chondrocyte phospho-proteome

city: Rochester

first: Alexandra

last: Krivonos

state: MN

address: 200 1st St SW

department: Quantitative Health Sciences

institute: Mayo Clinic

summary: Appendicular growth and bone mass acquisition are controlled by a variety of growth factors, hormones, and mechanical forces in a dynamic process called endochondral ossification. Chondrocytes in the growth plate must proliferate and undergo hypertrophy to drive growth plate expansion and lay the template for bone. Pleckstrin homology (PH) domain and leucine rich repeat phosphatase 1 and 2 (Phlpp1 and Phlpp2) are protein phosphatases that regulate intracellular signaling cascades through posttranslational modification of AKT, PKC, and S6K, among others. Phlpp1 controls chondrocyte proliferation and survival and germline deletion of Phlpp1 suppresses bone lengthening. Here, we demonstrate that Phlpp2 does not regulate endochondral ossification. Phlpp2-/- mice are phenotypically indistinguishable from their wildtype (WT) littermates, with similar bone length, bone mass, and growth plate dynamics. By contrast, Phlpp1/2-/- mice are phenotypically indistinguishable from Phlpp1-/- mice. Deletion of Phlpp1 or Phlpp2 had moderate effects on the chondrocyte transcriptome and proteome compared to WT control cells. By contrast, Phlpp1-/- and Phlpp1/2-/- chondrocytes had significantly altered phospho-proteomes compared to WT and Phlpp2-/- chondrocytes. Data integration via multiomics analysis revealed that RAF-MEK-ERK signaling was altered in cells lacking Phlpp1. In conclusion, these data demonstrate that Phlpp1, but not Phlpp2, regulates endochondral ossification via the chondrocyte phospho-proteome.

overall_design: Chondrocytes were isolated from five-day-old female wildtype (WT), Phlpp1-/-, Phlpp2-/-, and Phlpp1/2-/- mice (n=3 per genotype) and RNA was submitted for bulk RNA-Sequencing.

first: Samantha

last: Weaver

first: Jennifer

middle: J

last: Westendorf


title: Compensatory electron transfer from the ubiquinol pool circumvents cytochrome c oxidase deficiency and restores effector and memory T cell function

first: Peter

last: McGuire

state: MD

address: 10 Center Dr, Building 10, Room 7N260A

department: NHGRI

summary: The ultimate goal of immunometabolism is to modulate metabolic pathways to enhance immunity. Here, we investigate the potential of an alternative oxidase (AOX) to counteract cytochrome c oxidase (COX) deficiency in T cells. COX is vital for oxidative phosphorylation (OXPHOS), and its deficiency leads to redox imbalances, impaired metabolism, reactive oxygen species (ROS), and apoptosis, resulting in T cell immunodeficiency. We introduced an AOX into COX-deficient T cells, which revealed a normalization of genes involved in redox balance, apoptosis, and metabolic pathways. Mitochondrial function, glycolysis and TCA cycle function were also restored. AOX-enhanced T cells showed improved activation, proliferation, differentiation, and memory formation, resulting in better immune responses against viral challenges. These findings suggest that targeting the ubiquinol pool could be a promising therapeutic strategy for enhancing T cell function in conditions characterized by COX dysfunction and compromised immune responses.

overall_design:
This study includes read counts from two RNAseq experiments. In the first experiment, RNA was derived from T cells from four groups of mice on a C57Bl/6J background: wildtype (WT), Cox10fl/fl;Cd4-Cre (TCox10-/-), Rosa26-targeted Ciona intestinalis alternative oxidase expressing (Aox), and TCox10-/-/Aox double mutants. The goal of this experiment was to evaluate the ability of AOX to compensate for COX deficiencies in T cells. In the second experiment, RNA is derived from T cells from two groups of mice on a C57BL/6-Tg(TcraTcrb)1100Mjb/J (OT-1) background: wildtype (WT_IL15), and TCox10-/-/Aox double mutants (TCox10-/-/Aox_IL15). The goal of this experiment was to evaluate the ability of AOX to compensate for COX deficiencies in memory T cells.

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Submitter states that missing raw files are due to file loss.
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first: Tatiana

middle: N

last: Tarasenko

first: Emily

middle: B

last: Warren

first: Amanda

middle: L

last: Fuchs

first: Bharati

last: Singh

first: Jose

last: Marin Franco


title: Role of Piezo1-mediated mechanotransduction in osteocyte differentiation

status: Public on Sep 20 2024

first: Yizhong

last: Hu

address: 190 Longwood Ave.

institute: Harvard School of Dental Medicine

summary: Mechanical force is a fundamental regulator of bone development and homeostasis. Mechanosensitive osteocytes are the most abundant bone cells that form interconnected dendrites to respond to mechanical stimuli and interact with the bone-forming osteoblasts and the bone-remodeling osteoclasts. However, the molecular mechanisms underlying osteocyte maturation and dendrite formation remain unclear. By generating a Piezo1 "knock-out" osteocyte cell line, we identified a key role of Piezo1-mediated mechanotransduction in osteocyte differentiation. QRT-PCR analysis revealed delayed osteocyte differentiation in the Piezo1 KO cells relative to WT cells. By performing bulk RNA sequencing of WT and Piezo1 KO OCY454 cells at early (D1), intermediate (D14), and late (D28) stages of differentiation allowed for the identification of key signaling pathways in driving normal osteocyte dfiferentiation, as well as  those regulated by Piezo1 mechanotransduction.

overall_design: To mechanistically investigate the role of Piezo1 in osteocyte differentiation, we generated a Piezo1 "knock-out"  (KO) osteocyte cell line using CRISPR-Cas9 gene editing. Exon 19 through exon 21 from the OCY454 cell line were deleted, confirmed by sanger sequencing, qRT-PCR, western blot, and immunofluroescent staining. WT and Piezo1 KO OCY454 cells were differentiated for 1 (basal), 14, and 28 days at 37C and collected for bulk RNA-sequencing analysis. Two replicates were performed for each group.

first: Yu

last: Jin

first: Yuchen

last: Liu

first: Yingzi

last: Yang


title: Single cell gene expression profile for normal adult canine neuroretina

city: Philadelphia

first: Raghavi

last: Sudharsan

address: 3900 Delancey Street

institute: University of Pennsylvania

summary: Retinal single cell gene expression profile from two male dogs at 40 weeks of age.

overall_design: Retinal punches were collected from canine neuroretina and dissociated into single cells for scRNAseq analysis.

first: William

last: Beltran


title: Beta cell-specific PAK1 enrichment ameliorates diet-induced glucose intolerance by promoting insulin biogenesis and minimizing beta cell apoptosis

status: Public on Sep 25 2024

city: Duarte

first: Debbie

middle: C.

last: Thurmond

state: California

address: 1500 E Duarte Rd

institute: City of Hope

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

overall_design: Refer to individual Series


title: The MicroRNAmiR-223constrains Colitis-associated tumorigenesis by limiting myeloid cell infiltration and Chemokine expression

city: Maynooth

first: Eoin

last: McNamee

state: Co Kildare

address: Rm 2.19 Bioscience bldg

department: Biology

country: Ireland

institute: Maynooth University

summary: Aberrant intestinal inflammation plays a critical role in the development of colitis-associated colorectal cancer (CAC), yet the mechanisms behind this progression are not clearly defined. While altered microRNA (miRNA) expression is observed in CAC, it is unclear how myeloid-specific microRNA’s impact on the inflammatory process that underpins the continuum from Ulcerative colitis (UC) to tumorigenesis. Here we used the Azoxymethane-Dextran Sodium Sulfate (AOM-DSS) (10mg/kg) model of CAC on miR-223-/y and WT mice, with an overall aim of identifying differences in their expression profiles following AOM/DSS treatment.

overall_design: To investigate the effects of miR-223 on the development of colitis associated colorectal cancer, we used miR-223-/y and C57BL/6J mice treated with azoxymethane(AOM)/dextran sodium sulfate(DSS) (10mg/kg). Total RNA was extracted from colonic tissue and gene expression analysis was performed on RNA-seq results.

first: Eóin

first: Ciara

last: Flynn

first: Gary

last: Markey

first: Viola

last: Dengler

first: Charlotte

last: Farrelly

first: Holger

last: Eltzschig

first: Joanne

middle: C

last: Masterson

first: Glenn

middle: T

last: Furuta


title: Gene expression changes induced by SMPD1 or GPR64 silencing in A673 Ewing Sarcoma cells

city: San Antonio

first: Yidong

last: Chen

state: Texas

address: 8403 Floyd Curl Drive, MSC 7784

department: Population Health Sciences

institute: UT Health Science Center at San Antonio

summary: We used RNA-sequencing to compare the gene expression changes induced by SMPD1 silencing and GPR64 silnecing.

overall_design: A673 cells were were transfected with either scrambled-siRNA or SMPD1-siRNA or GPR64-siRNA for 48 hours. Transfection was carried out using Lipofectamine™ RNAiMAX Transfection Reagent (Thermo Fisher) according to the manufacturer’s protocol. After 48 hours, cells were harvested and total RNA was extracted using TRIzol reagent (Invitrogen). For whole-genome transcriptome profiling, four libraries were generated from total RNA sequencing.

first: Kruthi

last: Suvarna

first: Panneerselvam

last: Jayabal

first: Yuzuru

last: Shiio

first: Zhao

last: Lai


title: Enhancer activation from transposable elements in extrachromosomal DNA

city: Stanford

first: Matthew

last: Jones

address: 500 Main Street (Building 76)

institute: Massachusetts Institute of Technology

summary: Extrachromosomal DNA (ecDNA) is a hallmark of aggressive cancer, contributing to both oncogene amplification and tumor heterogeneity. Here, we used Hi-C, super-resolution imaging, and long-read sequencing to explore the nuclear architecture of MYC-amplified ecDNA in colorectal cancer cells. Intriguingly, we observed frequent spatial proximity between ecDNA and 68 repetitive elements which we called ecDNA-interacting elements or EIEs. To characterize a potential regulatory role of EIEs, we focused on a fragment of the L1M4a1#LINE/L1 which we found to be co-amplified with MYC on ecDNA, gaining enhancer-associated chromatin marks in contrast to its normally silenced state. This EIE, in particular, existed as a naturally occurring structural variant upstream of MYC, gaining oncogenic potential in the transcriptionally permissive ecDNA environment.  This EIE sequence is sufficient to enhance MYC expression and is required for cancer cell fitness. These findings suggest that silent repetitive genomic elements can be reactivated on ecDNA, leading to functional cooption and amplification. Repeat element activation on ecDNA represents a mechanism of accelerated evolution and tumor heterogeneity and may have diagnostic and therapeutic potential.

overall_design: To interrogate the conformational state of circular extrachromosomal DNA (ecDNA), we performed Hi-C on COLO 320DM colorectal cancer cells.

first: Katerina

last: Kraft

first: Sedona

last: Murphy

first: Quanming

last: Shi

first: Aarohi

last: Bhargava-Shah

first: Christy

last: Luong

first: King

last: Hung

first: Britney

middle: H

last: He

first: Rui

last: Li

first: Seung

last: Park

first: Natasha

last: Weiser

first: Jens

last: Luebeck

first: Vineet

last: Bafna

first: Jef

middle: D

last: Boeke

first: Paul

middle: S

last: Mischel

first: Alistair

last: Boettiger

first: Howard

middle: Y

last: Chang


title: Methylation profiling of plasma cell-free DNA in pediatric brain tumor patients

city: Chicago

first: Shejuan

last: An

address: 303 East Superior Street

institute: Northwestern University

summary: Using cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq), we analyzed methylation profiles in 93 plasma samples from 77 pediatric brain tumor patients and 16 non-neoplastic patients. Binomial GLMnet classifiers of tumor and tumor subtypes were built in training sets, and performance was evaluated in test sets. The methylation profiles from plasma cfMeDIP-seq discriminated tumor from non-tumor patients with 0.83 accuracy (precision = 0.93, sensitivity = 0.86, and specificity = 0.67). Among major tumor subtypes vs. nontumors, circumscribed astrocytic glioma showed an accuracy of 0.86 (precision = 0.88, sensitivity = 0.88, and specificity = 0.83), and glioneuronal tumors had an accuracy of 0.83 (precision, sensitivity, and specificity = 0.83). Circumscribed astrocytic glioma and glioneuronal tumors could be discriminated from other tumor subtypes with 0.79 and 0.82 accuracy, respectively.

overall_design: We analyzed methylation profiles in 93 plasma samples from 77 pediatric brain tumor patients and 16 non-neoplastic patients. Binomial GLMnet classifiers of tumor and tumor subtypes were built in training sets, and performance was evaluated in test sets.

first: Craig

last: Horbinski

first: Kathleen

last: McCortney

first: Jordain

last: Walshon

first: Kaethe

last: Leonard

first: Matthew

last: McCord

first: Michael

last: DeCuypere


title: Single-cell sequencing of human induced pluripotent stem cell-derived ventral midbrain astrocytes

status: Public on Sep 24 2024

city: Cardiff

first: Zongze

summary: Regional specificity of stem cell-derived astrocytes is believed to be an important prerequisite for their applications in disease modelling and cell-based therapies. The regional identity of these astrocytes is often defined by the positional characteristics of their antecedent, stem cell-derived neural progenitors patterned to a fate of interest, with the assumption that the positional specification is to be preserved by the derived astrocytes. Using a human iPSC line designed for tracing midbrain floor plate derivatives, here we show that lineage composition of the derived astrocytes is not a faithful recapitulation of the founder progenitor population, as demonstrated by the loss of floor plate differentiated progeny in the final astrocyte products. Using deep single cell RNA sequencing, we identified distinct transcriptomic signatures of midbrain floor plate-derived astrocytes. Our study highlights the need for rigorous characterisation of PSC-derived regional astrocytes and provides a valuable resource for assessing midbrain floor plate-derived human astrocytes.

overall_design: Human induced pluripotent stem cell-derived astrocytes and neurons isolated for CD49f by fluorescence activated cell sorting or unisolated were analysed using single cell RNA sequencing.

first: Lucia

middle: F

last: Cardo

first: Michal

last: Rokicki

first: Caleb

last: Webber

first: Meng


title: Analysis of rectal transcriptome in ulcerative colitis (UC) patients who received tofacitinib

city: New York

first: Kyungku

last: Jang

address: 430 East 29th Street

department: Microbiology

institute: New York University

summary: The gold of this study is to identify genes that differentially expressed in rectum of ulcerative colitis patients who are responisve to tofacitinib compared with the patients who are refractory to the drug. This study may help to identify preditive markers to determine tofacitinib responders prior to the treatment.

overall_design: We enrolled 48 UC patients for this project and collected rectum pinch biospies during colonoscopy at the baseline (week 0, before tofacitinib treatment) and end of study (week 24, after tofacitinib treatment). RNA-seq was performed using RNA isolated from the biopsies .

first: Kyung

middle: K

first: Defne

last: Ercelen

first: Chaoting

last: Zhou

first: Jordan

last: Axelrad

first: Ken

last: Cadwell


title: Beta cell-specific PAK1 enrichment ameliorates diet-induced glucose intolerance by promoting insulin biogenesis and minimizing beta cell apoptosis [ChIP-Seq]

first: Debbie

last: Thurmond

summary: Aims/ hypothesis: p21 (Cdc42/Rac1) activated Kinase 1 (PAK1) is depleted in type 2 diabetic human islets compared to non-diabetic (ND) human islets, and acute PAK1 restoration to type 2 diabetic human islets can restore insulin secretory function ex vivo. We hypothesized that beta cell specific PAK1 enrichment in vivo carries the capacity to mitigate high-fat diet-induced glucose intolerance by increasing the functional beta cell mass. Methods: Type 2 diabetic or ND human islets expressing exogenous PAK1 specifically in beta cells were used for bulk RNA-sequencing (RNA-seq). Human EndoC-

overall_design: Human EndoC-H1 cells overexpressing myc-tagged PAK1 were used for chromatin immunoprecipitation (ChIP) and ChIP-sequencing (ChIP-seq).

first: Miwon

last: Ahn

first: Sangeeta

last: Dhawan

first: Erika

last: McCown

first: Pablo

middle: A

last: Garcia

first: Supriyo

last: Bhattacharya

first: Roland

last: Stein


title: Self-Organized Generation Of Hepatic Cord Like Organoids (HCLOs) With Zonal Diversity (ChIPseq)

status: Public on Sep 18 2024

city: Cincinnati

first: Hasan Al

last: Reza

state: OH

address: 3333 Burnet Ave

department: Developmental Biology

institute: CCHMC

summary: To investigate the efect of extracellular bilirubin and intracellular ascorbate on the development of zonal hepatocytes in human liver organoids.

overall_design: We performed EP300 ChIPseq on Hepatic Cord Like Orgnaoids (HCLOs) including Pericentral Organoids (PCO) and Periportal Organoids (PPO) to elucidate the epigenetic regulation behind zonal gene expression.

first: Hasan

first: Takanori

last: Takebe


title: Regulation of gene expression by Hcm1 in chronic LiCl stress

city: Worcester

first: Jennifer

last: Benanti

address: 364 Plantation St., LRB 525

department: Molecular, Cell and Cancer Biology

institute: University of Massachusetts Medical School

summary: Gene expression was quantified in S. cerevisiae cells after 40 hours of LiCl treatment or control conditions. Cells expressing wild type (WT) Hcm1, a stable Hcm1 mutant (Hcm1-3N), or a consitutitvely active, phosphomimetic mutant (Hcm1-8E)  were compared to allow the identification of genes whose expression depends upon dynamic phosphorylation of Hcm1.  We found that dynamic phosphorylation of Hcm1 is required for expression of Hcm1 target genes in LiCl stress.

overall_design: Gene expression was measured in cells expressing wild type (WT) Hcm1, Hcm1-3N (3N) or Hcm1-8E (8E). Each strain was innoculated into both control medium and medium containing 150mM LiCl. After 24 hours of growth, saturated cultures were diluted into the same growth media. Samples were collected after an additional 16 hours (40 hours total of total growth) when cells were in logarithmic phase. Biological triplicate experiments were performed.

first: Michelle

middle: M

last: Conti

first: Rui

last: Li

first: Lihua

last: Zhu


title: Self-Organized Generation Of Hepatic Cord Like Organoids (HCLOs) With Zonal Diversity (RNA-seq)

summary: To investigate the efect of extracellular bilirubin and intracellular ascorbate on the development of zonal hepatocytes in human liver organoids

overall_design: We performed RNAseq on control HLOs compared to HLOs treated with extracellular bilirubin and genetically modified mGULO HLOs that express intracellular ascorbate

first: Hasa


title: Self-Organized Generation Of Hepatic Cord Like Organoids (HCLOs) With Zonal Diversity (snRNAseq)

overall_design: We performed snRNAseq on Hepatic Cord Like Orgnaoids (HCLOs) including Pericentral Organoids (PCO) and Periportal Organoids (PPO).


title: Beta cell-specific PAK1 enrichment ameliorates diet-induced glucose intolerance by promoting insulin biogenesis and minimizing beta cell apoptosis [RNA-Seq]

overall_design:
Type 2 diabetic or ND human islets expressing exogenous PAK1 specifically in beta cells were used for bulk RNA-sequencing (RNA-seq), to investigate differential gene expression.
Islets from ND and type 2 diabetes human donors were transduced at 100 MOI with Ad-RIP Ctrl or Ad-RIP-hPAK1 for 1 hr at 37 degrees C. Transduced islets were then washed twice and incubated for 48 hrs in CMRL medium.


title: Systematic perturbations of SETD2, NSD1, NSD2, NSD3 and ASH1L reveal their distinct contributions to H3K36 methylation

city: Montreal

first: Jacek

last: Majewski

state: Quebec

address: 740 Dr. Penfield

department: Human Genetics

country: Canada

institute: McGill University

summary: Methylation of histone 3 lysine 36 (H3K36me) has emerged as an essential epigenetic component for the faithful regulation of gene expression. Despite its importance in development, disease, and cancer, how the molecular agents collectively shape the genome-wide deposition of H3K36me is unclear. Here, we use mouse mesenchymal stem cells to comprehensively perturb the components of the H3K36me deposition machinery and infer the activities of the five most prominent players: SETD2, NSD1, NSD2, NSD3, and ASH1L. We find that H3K36me2 is the most abundant of the three methylation states and that it is predominantly deposited at intergenic regions by NSD1, and in part by NSD2. In contrast, H3K36me1/3 are most abundant within exons and are positively correlated with gene expression. We demonstrate that while SETD2 is responsible for depositing most H3K36me3, it also deposits H3K36me2 within transcribed genes. Additionally, loss of SETD2 results in an increase of exonic H3K36me1, suggesting that other H3K36 methyltransferases may prime gene bodies with lower methylation states ahead of transcription. Through a reductive approach, we uncover the distribution patterns of NSD3- and ASH1L-catalyzed H3K36me2. While NSD1/2 establish broad intergenic H3K36me2 domains, NSD3 deposits H3K36me2 peaks centered on active promoter and enhancer regions. Meanwhile, the activity of ASH1L is restricted to regulatory elements of developmentally relevant genes, and our analyses implicate PBX2 as a potential recruitment factor. Overall, our study provides new insights into the regulation of H3K36me and helps to consolidate the wealth of previous observations in the context of structured analyses.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-Seq) for histone modifications H3K36me1, H3K36me2 and H3K36me3 as well as transposase-accessible chromatin with sequencing (ATAC-Seq) in C3H10T1/2 mouse mesenchymal stem cells.

first: Gerald

last: Shipman

first: Reinnier

last: Padilla

first: Cynthia

last: Horth

first: Bo

last: Hu

first: Eric

last: Bareke

first: Francisca

last: Vitorino

first: Joanna

last: Gongora

first: Benjamin

first: Chao

last: Lu


title: LSK immunophenotype hematopoietic stem cells in Dapp1 knockout mice

city: 重庆Chongqing

first: Zeyu

last: Fu

state: 重庆Chongqing

address: 高滩岩正街

department: College of Preventive Medicine

institute: AMU

summary: To investigate the impact of perturbing key differentiation driver genes on hematopoietic stem cell fate decision, we employed a Dapp1 gene knockout approach. This allowed us to observe changes in the subpopulations of hematopoietic stem cells at the LSK level in mice. Additionally, we assessed alterations in their fate decision and lineage differentiation tendencies

overall_design:
We conducted an in-depth single-cell RNA sequencing study using the 10X Genomics platform to analyze mouse bone marrow LSK (Lin^⁻Sca-1^⁺c-Kit^⁺) cells. Starting with ethically euthanized mice, we extracted femurs and tibias to harvest the bone marrow. The marrow cells were flushed out using a syringe with PBS containing 2% FBS to create a single-cell suspension. This suspension was filtered through a 40 µm cell strainer to eliminate debris and cell clumps, ensuring a pure population of cells for analysis. To isolate our target LSK cells, we employed fluorescence-activated cell sorting (FACS), selecting for Lineage-negative, Sca-1-positive, and c-Kit-positive cells. We assessed the viability of these cells using trypan blue staining and adjusted the concentration to approximately 1,000 cells/µL, optimizing conditions for the 10X Genomics Chromium Controller. We then loaded the purified single-cell suspension, along with the necessary reagents and gel beads, into a 10X Genomics Single Cell 3' Chip. Running the chip in the Chromium Controller, we encapsulated individual cells into Gel Bead-In-Emulsions (GEMs), enabling the barcoding of mRNA from each cell during reverse transcription within the GEMs. Following emulsion breakage, we purified the barcoded cDNA and performed PCR amplification. The amplified cDNA was evaluated for quality and quantity using an Agilent Bioanalyzer to ensure downstream sequencing success. Constructing sequencing libraries from the high-quality cDNA, we followed the 10X Genomics protocol meticulously. These libraries were then sequenced on an Illumina NovaSeq platform, adhering to the recommended read lengths and depths to achieve comprehensive single-cell transcriptional coverage.
The sequencing data generated were processed using the 10X Genomics Cell Ranger pipeline, which aligned reads and produced a gene expression matrix for each cell. For data analysis, we utilized various bioinformatics tools, prominently featuring our novel scRL (single-cell Reinforcement Learning) framework. This advanced analytical approach allowed us to identify distinct cell populations and gene expression patterns with high precision. Most critically, employing scRL enabled us to obtain quantitative indicators of the strength of cell lineage differentiation fate decisions—a level of insight not achievable with existing methods.

first: Song

last: Wang

first: Shilei

last: Chen

first: Junping


title: PolySUMOylation of PCNA and Rad52 restricts centromeric recombination in fission yeast

status: Public on Sep 15 2024

city: Wroclaw

first: Karol

last: Kramarz

address: Kanonia 6/8

department: Faculty of Biological Sciences

country: Poland

institute: University of Wrocław

summary: SUMOylation, a conserved post-translational modification in eukaryotes, regulates protein function, localization, and stability. However, the role of SUMO chains in genome maintenance is still emerging. Using Schizosaccharomyces pombe, we show that loss of SUMO chains results in spontaneous replication stress, DNA damage, and elevated centromeric recombination. To investigate SUMO-dependent interactome at the sites of Rad52 repair, we used split-SUMO-ID proteomics approach. It allowed analysis of local SUMOylation content at the Rad52 repair sites, and enabled identification of the essential replication factor PCNA. We found that SUMO chain-modified PCNA antagonizes Rad8-mediated PCNA polyubiquitination, modulating the choice of post-replication repair pathways at stalled forks within centromeres. In the absence of polySUMOylation, excessive PCNA polyubiquitination drives elevated recombination at centromeres. Artificial tethering of a SUMO chain to Rad52 suppresses this defect. Our findings uncover an essential role for SUMO chains in centromere maintenance by modulating DNA repair pathway choice under endogenous replication stress.

overall_design:
Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for CFP-Cnp1 histone variant (human CENP-A) from fission yeast Schizosaccharomyces pombe.

Please note that additional data processing was carried out to remove background more stringently for the GSM8506073, GSM8506075, and GSM8506077 ChIP-seq samples. The processed data files and data processing description have been updated on May 29, 2025.

first: Katarzyna

last: Markowska

first: Ireneusz

last: Litwin

first: Julia

last: Kończak

first: Dorota

last: Misiorna

first: Aleksandra

last: Broczkowska

first: Paulina

last: Tomaszewska

first: Michał

last: Tracz

first: Mika

last: Haenen


title: An alveolus lung on a chip model of Mycobacterium fortuitum lung infection

city: Dallas

first: Victoria

middle: Anette

last: Ektnitphong

state: TX

address: 5323 Harry Hines Blvd.

department: Internal Medicine

institute: UTSW Medical Center

summary: Lung disease due to non-tuberculous mycobacteria (NTM) is rising in incidence. While both 2D cell culture and animal models exist for NTM infections, a major knowledge gap is the early responses of human alveolar and innate immune cells to NTM within the human lung microenvironment. Here we describe development of a humanized, 3D, alveolus lung-on-a-chip (ALoC) model of Mycobacterium fortuitum infection that incorporates only primary human cells such as pulmonary vascular endothelial cells in a vascular channel, and type I and II alveolar cells and monocyte-derived macrophages in an alveolar channel along an air-liquid interface. M. fortuitum introduced into the alveolar channel primarily infected macrophages, with rare bacteria inside alveolar cells. Bulk-RNA sequencing of infected chips revealed marked upregulation of transcripts for cytokines, chemokines and secreted protease inhibitors (SERPINs). Our results demonstrate how a humanized ALoC system can identify critical early immune and epithelial responses to M. fortuitum infection. We envision potential application of the ALoC to other NTM and for studies of new antibiotics

overall_design: Alveolar lung-on-a-chips (ALoCs) that were seeded with primary human alveolar epithelial cells & human monocyte-derived macrophages in the apical channel and human microvascular pulmonary endothelial cells in the basal channel were either infected with M. fortuitum at an MOI of ~1 to the epithelial cells or left uninfected.   24 hours post-infection, control and infected chips were lysed with TriZol and RNA was isolated using phenol & the Qiagen RNeasy Mini Kit.  We performed bulk RNA-sequencing of the apical channel on uninfected and infected chips 24 hours after infection.

first: Beatriz

last: Dias

first: Priscila

last: Campos

first: Michael

last: Shiloh


title: Scaled and efficient derivation of loss-of-function alleles in risk genes for neurodevelopmental and psychiatric disorders in human iPSCs

status: Public on Sep 17 2024

city: Evanston

first: Jubao

last: Duan

address: 1001 University Place

department: Center for Psychiatric Genetics

institute: NorthShore University HealthSystem

summary: Translating genetic findings for neurodevelopmental and psychiatric disorders (NPD) into actionable disease biology would benefit from large-scale and unbiased functional studies of NPD genes. Leveraging the cytosine base editing (CBE) system, here we developed a pipeline for clonal loss-of-function (LoF) allele mutagenesis in human induced pluripotent stem cells (hiPSCs) by introducing premature stop-codons (iSTOP) that lead to mRNA nonsense-mediated-decay (NMD) or protein truncation. We tested the pipeline for 23 NPD genes on 3 hiPSC lines and achieved highly reproducible, efficient iSTOP editing in 22 NPD genes. Using RNAseq, we confirmed their pluripotency, absence of chromosomal abnormalities, and NMD. Interestingly, for three schizophrenia risk genes (SETD1A, TRIO, CUL1), despite the high efficiency of base editing, we only obtained heterozygous LoF alleles, suggesting their essential roles for cell growth. We replicated the reported neural phenotypes of SHANK3-haploinsufficiency and found CUL1-LoF reduced neurite branches and synaptic puncta density. This iSTOP pipeline enables a scaled and efficient LoF mutagenesis of NPD genes, yielding an invaluable shareable resource.

overall_design: As part of the SSPsyGene Consortium, our Assay and Data Generation Center (ADGC) for the Model of iPSC-derived Neurons for NPD (MiNND) aims to employ the CBE editor-based iSTOP approach to generate isogenic hiPSC lines carrying LoF alleles for about 150-200 NPD genes on multiple donor genetic backgrounds.  Here, leveraging an improved reporter gene editing enrichment system that can substantially increase the CBE iSTOP editing efficiency in hiPSC, we established a semi-automated pipeline for parallel and efficient clonal LoF mutagenesis of a large number of genes. We tested the workflow on 23 NPD genes with 3 donor hiPSC lines (KOLF2.2J, CW20107, MGS_CD14). We obtained high and reproducible iSTOP editing efficiency across all three hiPSC lines. We systematically characterized the engineered isogenic iSTOP hiPSC lines for pluripotency, karyotyping, neuron differentiation capacity, and the expected NMD and LoF.

first: Siwei

last: Zhang

first: Zhiping

middle: P

last: Pang


title: Self-Organized Generation Of Hepatic Cord Like Organoids (HCLOs) With Zonal Diversity

first: Hasan Al

last: Reza

overall_design: Refer to individual Series