title: Antagonism between H3K27me3 and genome-lamina association drives atypical spatial genome organization in the totipotent embryo [scDamID]

first: Jop

last: Kind

summary: In this study, we profiled genome-Nuclear Lamina (NL) contacts during the first stages of mouse embryonic development. We discovered a remarkable cell-to-cell variability in NL-contacts at the 2-cell stage, which is particularly strong on the paternal allele. We additionally obtained single-cell profiles for H3K27me3, H3K9me3 and DNA accessibility at this stage, but did not observe the same large-scale variability. The variability in NL-contacts did not appear to affect the transcription of underlying genes. Comparing NL-contact profiles with diverse histone modification profiles showed that large regions of typical NL-contacts are lost and instead are enriched for H3K27me3 during early development. To investigate the relationship between H3K27me3 and NL association, we used a conditional EED KO mouse model, which results in an absence of H3K27me3 during oocyte development and the early embryo. Profiling NL-contacts at the 2-cell stage in this system revealed that regions enriched with H3K27me3 in WT regain NL association in the EED maternal KO. In addition, the loss of H3K27me3 resulted in more similar NL association profiles on the maternal and paternal allele. Together, these results suggest that H3K27me3 antagonizes NL association and that the non-canonical broad H3K27me3 domains present in the early embryo may be responsible for the early-embryo specific loss of NL associations in these regions.

first: Isabel

last: Geirreiro

first: Franka

last: Rang

first: Yumiko

last: Kitazawa

first: Femke

last: Groenveld

first: Ramada

last: van Beek

first: Silke

last: Lochs

first: Ellen

last: Boele

first: Antoine

last: Peters


title: Antagonism between H3K27me3 and genome-lamina association drives atypical spatial genome organization in the totipotent embryo

status: Public on Aug 09 2024

city: Utrecht

address: Uppsalalaan 8

country: Netherlands

institute: Hubrecht Institute

type: Genome binding/occupancy profiling by high throughput sequencing

type: Expression profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Endogenous glucocorticoids are required for normal macrophage activation and gastric Helicobacter pylori immunity [ATAC-seq]

status: Public on Aug 19 2024

city: Morgantown

first: Gangqing

last: Hu

state: West Virginia

address: 64 Medical Center Drive

department: MicroBiology, Immunology, and Cell Biology

country: USA

institute: West Virginia University

summary: Glucocorticoids are steroid hormones well-known for their potent anti-inflammatory effects. However, their immunomodulatory properties are multifaceted. Increasing evidence suggests that glucocorticoid signaling promotes effective immunity and that disruption of glucocorticoid signaling impairs immune function. In this study, we conditionally deleted the glucocorticoid receptor (GR) in the myeloid lineage using theLysM-Credriver (myGRKO). We examined the impact on macrophage activation and gastric immune responses toHelicobacter pylori, the best-known risk factor of gastric cancer. Our results indicate that compared to WT, GRKO macrophages exhibited higher expression of proinflammatory genes in steroid-free conditions. However, when challenged in vivo, GRKO macrophages exhibited aberrant chromatin landscapes and impaired proinflammatory gene expression profiles. Moreover, gastric colonization withHelicobacterrevealed impaired gastric immune responses and reduced T cell recruitment in myGRKO mice. As a result, myGRKO mice were protected from atrophic gastritis and pyloric metaplasia development. These results demonstrate a dual role for glucocorticoid signaling in preparing macrophages to respond to bacterial infection but limiting their pathogenic activation. In addition, our results support that macrophages are critical for gastric anti-Helicobacterimmunity.

overall_design: Assays for chromatin accessibility (ATAC-seq) of freshly isolated thioglycollate-induced peritoneal macrophages isolated from vehicle-treated WT mice, vehicle-treated mice with Nr3c1 deletion with the Lysm Cre driver or the same genotypes treated in vivo for 3 hours with Helicobacter pylori. There are 3-4 replicates per group.

first: Stuti

last: Khadka

first: Sebastian

middle: A

last: Dziadowicz

first: Xiaojiang

last: Xu

first: Lei

last: Wang

first: Jonathan

middle: T

last: Busada


title: ATF6 promotes colorectal cancer growth and stemness by regulating the Wnt pathway II

status: Public on Aug 08 2024

city: South San Francisco

first: Matt

last: Grimmer

state: CA

address: 455 E Grand Ave.

department: Oncology Bioinformatics

institute: Genentech

summary: The unfolded protein response maintains endoplasmic reticulum (ER) homeostasis by sensing protein-folding stress and orchestrating cellular adaptation via the ER-transmembrane proteins IRE1, PERK and ATF6. Malignant cells can co-opt IRE1 and PERK to sustain growth; however, the importance of ATF6 in cancer remains poorly deciphered. We observed elevated ATF6 transcriptional activity in several cancers including colorectal carcinoma (CRC). Genetic silencing or small molecule inhibition of ATF6 blocked cell cycle progression and reduced viability of several human CRC cell lines in vitro and disrupted tumor progression in vivo. Unexpectedly, ATF6 interference disabled Wnt and Myc signaling and reduced stemness. ATF6 inhibition attenuated growth of organoids derived from malignant but not normal human intestinal tissue, decreasing Wnt-pathway activity and driving cellular differentiation. Wnt-surrogate agonism in a Wnt-dependent CRC organoid restored pathway activity and rescued growth under ATF6 blockade. Our findings identify ATF6 as an unexpected facilitator of oncogenic Wnt signaling in CRC.

overall_design: We identified ATF6, a latent transcription factor involved in the stress response pathway, as a regulator of cellular state in colon cancer. More specifically, genetic interference with ATF6 resulted in colon cancer cell growth inhibition. Bulk RNA-seq data indicated that this process was imparted through loss of stem cell function and a concomitant increase in cellular differentiation. We developed a small molecule inhibitor of ATF6 that primarily acted by promoting ATF6 degradation. This compound displayed cell growth inhibition in various CRC models but had limited impact in normal human colon organoids. To further investigate the impact of ATF6 on cellular state, chromatin accessibility, and cellular differentiation, we performed snRNA analysis contrasting a control arm and SMI arm in 2 technical replicates per group.

first: Jeffrey

middle: J

last: Rodvold

first: Matthew

middle: R

first: Karen

last: Ruiz

first: Scot

last: Marsters

first: Ioanna

last: Oikonomidi

first: Eileen

middle: I

last: Tan-Aristy

first: Victoria

middle: C

last: Pham

first: Tamal

last: Sarkar

middle: M

last: Harnoss

first: Whitney

last: Shatz-Binder

first: Zora

last: Modrusan

first: Thomas

middle: D

last: Wu

first: Jennie

last: Lill

first: Elisia

last: Villemure

first: Joachim

last: Rudolph

first: Felipe

last: de Sousa e Melo

first: Avi

last: Ashkenazi


title: Single-cell time-resolved multi-omics reveal apoptotic and ferroptotic heterogeneity during foam cell formation

status: Public on Aug 10 2024

city: Shanghai

first: Yiwen

last: Wang

address: 2005, Songhu Road, Yangpu District

country: China

institute: Fudan University

summary: Macrophage-derived foam cell plays a pivotal role in the plaque formation and rupture during the progression of atherosclerosis. Foam cells are destined to divergent cell fate and functions in response to external stimuli based on their internal states, which however is hidden in the traditional studies based on population of cells. Herein, we used time-resolved and single-cell multi-omics to investigate the macrophage heterogeneity along foam cell formation. Dynamic metabolome and lipidome outlined the dual regulating axis of inflammation and ferroptosis. Single cell metabolomics and lipidomics further demonstrated a macrophage continuum featuring a differed susceptibility to apoptosis and ferroptosis. Using single-cell transcriptomic profiling, we verified the divergent cell fate toward apoptosis or ferroptosis. Therefore, the molecular choreography underlying the divergent cell fate during foam cell formation was revealed, which is of high significance for the understanding of the pathogenesis of atherosclerosis and development of new drug targets.

overall_design: One PBMC-derived late-stage foam cell.

first: Ling

last: Lin

first: Liang

last: Qiao


title: Less Deformable Erythrocyte Subpopulations Biomechanically Induce Endothelial Inflammation in Sickle Cell Disease

city: Cartersville

first: Marina

last: Michaud

state: GA

address: 28 Cass Station Dr NW

department: School of Medicine

institute: Emory University

summary: Sickle cell disease (SCD) is canonically characterized by reduced red blood cell (RBC) deformability leading to microvascular obstruction and inflammation. While the biophysical properties of sickle RBCs are known to influence SCD vasculopathy, the contribution of poor RBC deformability to endothelial dysfunction has yet to be fully explored. Leveraging interrelated in vitro and in silico approaches, we introduce a new paradigm of SCD vasculopathy in which poorly deformable sickle RBCs directly cause endothelial dysfunction via mechanotransduction, where endothelial cells sense and pathophysiologically respond to aberrant physical forces independently of microvascular obstruction, adhesion, or hemolysis. We demonstrate that perfusion of sickle RBCs or pharmacologically-dehydrated healthy RBCs into small venule-sized “endothelialized” microfluidics leads to pathologic physical interactions with endothelial cells that directly induce inflammatory pathways. Using a combination of computational simulations and large venule-sized endothelialized microfluidics, we observed that perfusion of heterogeneous sickle RBC subpopulations of varying deformability, as well as suspensions of dehydrated normal RBCs admixed with normal RBCs leads to aberrant margination of the less-deformable RBC subpopulations towards the vessel walls, causing localized, increased shear stress. Increased wall stress is dependent on the degree of subpopulation heterogeneity and oxygen tension and leads to inflammatory endothelial gene expression via mechanotransductive pathways. Our multifaceted approach demonstrates that the presence of sickle RBCs with reduced deformability leads directly to pathological physical (i.e., direct collisions and/or compressive forces) and shear-mediated interactions with endothelial cells and induces an inflammatory response, thereby elucidating the ubiquity of vascular dysfunction in SCD.

overall_design: Single-cell RNA sequencing (scRNA-seq) was performed on human umbilical vein endothelial cells (HUVECs) collected after exposure to healthy red blood cells (hRBCs) or a binary suspension of 5% pharmacologically-dehydrated RBCs (pdRBCs) in hRBCs, using a four-channel 50 µm microfluidic device. To mimic the decreased deformability of sickle RBCs, hRBCs were pharmacologically-dehydrated with nystatin and high sucrose washes according to established practices to create RBCs with an increased mean corpuscular hemoglobin concentration of 40-42 g/dL.  Following endothelialization of the microfluidic channels, the hRBC or 5% pdRBCs were perfused according to our established protocol.  After endothelial cells were collected from the microfluidic devices and resuspended in media, the suspension was passed through 70 and 40 μm cell strainers and centrifuged at 500xg at 4°C for 10 minutes. For RBC lysis, ACK buffer was added. For immediate single-cell capture, the cells were pelleted at 400xg at 4°C for 5 minutes and resuspended in PBS containing 1.0% BSA to get a concentration of 500 cells/μl. For single-cell RNA sequencing (scRNA-seq), barcoded gel bead-in-emulsions (GEMs) were generated from single-cell suspensions with gel beads and reverse transcription (RT) mix on the Chromium Controller, following the 10x Genomics workflow. Following RT and cDNA amplification, the Chromium Next GEM 5’reagent kit v2 (10x Genomics) was used to generate 5’ gene expression libraries. Gene expression libraries were prepared using the Chromium Next GEM 5’reagent kits v2 (10x Genomics). GEMs were generated and barcoded by loading single-cell suspensions along with gel beads and reverse transcription master mix onto Chromium Next GEM Chip K and running on the 10x Genomics chromium controller. Following reverse transcription, the cDNA was amplified and used to generate 5’ gene expression libraries. The cDNA and gene expression libraries were quantified using a Qubit 3.0 fluorometer, and quality was assessed using HS DNA chips and 2100 Bioanalyzer (Agilent Technologies). Sequencing was performed using massively parallel sequencing on the Illumina Novaseq S4 platform, targeting ~50,000 reads/cell to capture the expression of ~1000–2000 transcripts/cell.

first: Christina

last: Caruso

first: Beena

last: Thomas

first: Manoj

last: Bhasin

first: Wilbur

last: Lam


title: Systems analysis of miR-199a/b-5p and multiple miR-199a/b-5p targets during chondrogenesis

city: NEWCASTLE UPON TYNE

first: David

last: Young

address: Centre For Life

country: United Kingdom

institute: Newcastle University

summary: Changes in chondrocyte gene expression can contribute to the development of osteoarthritis (OA), and so recognition of the regulative processes during chondrogenesis can lead to a better understanding of OA. microRNAs (miRNAs) are key regulators of gene expression in chondrocytes/OA and we have used a combined experimental, bioinformatic, and systems biology approach to explore the multiple miRNA-mRNA interactions that regulate chondrogenesis. A longitudinal chondrogenesis bioinformatic analysis identified paralogues miR-199a-5p and miR-199b-5p as pro-chondrogenic regulators. Experimental work demonstrated alteration of miR-199a-5p or miR-199b-5p expression led to significant inverse modulation of key chondrogenic genes and extracellular matrix production. miR-199a/b-5p targetsFZD6, ITGA3andCAV1were identified by inhibition experiments and verified as direct targets by luciferase assay. The experimental work was used to generate and parameterize a multi-miRNA 14-day chondrogenesis kinetic model to be used as a repository for the experimental work and as a resource for further investigation of this system. This is the first multi-miRNA model of a chondrogenesis-based system, and highlights the complex relationships between regulatory miRNAs, and their target mRNAs.

overall_design: To investigate the effects of miR199-5p on mesenchymal stromal cell differentiation, cells were transfected prior to differentiation with inhibitors of miR-1995p.

first: Matt

last: Barter

first: Jamie

last: Soul

first: Daryl

last: Shanley

first: Krutik

last: Patel


title: Pathway Preferential Estrogens prevent hepatosteatosis due to ovariectomy and high-fat diets

city: Urbana

first: Zeynep

last: Madak Erdogan

state: IL

address: 1201 S Goodwin Ave

department: FSHN

institute: UIUC

summary: Pathway preferential estrogen 1 (PaPE-1) is a novel estrogen receptor ligand that has been shown to favorably affect metabolic tissues without adverse effects on reproductive tissues. In this study, we have examined the effects of PaPE-1 on metabolic health, in particular, examin-ing its effects on the liver transcriptome and on plasma metabolites in two different mouse mod-els: diet-induced obesity (DIO) and leptin-deficient (ob/ob) mice. PaPE-1 significantly decreased liver weight and lipid accumulation in both DIO and ob/ob models and lowered the expression of genes associated with fatty acid metabolism and collagen deposition. Overall, our findings support a beneficial metabolic role for PaPE-1 and suggest that PaPE-1 may protect postmenopausal women from fatty liver disease without increasing reproductive cancer risk.

overall_design: RNA-seq

last: Madak-Erdogan


title: LAP2alpha facilitates myogenic gene expression by preventing spreading of nucleoplasmic lamin A/C to active chromatin regions

city: Vienna

first: Roland

last: Foisner

address: Dr.-Bohr-Gasse 9

department: Center for Medical Biochemistry

country: Austria

institute: Max Perutz Labs, Medical University of Vienna

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

This research was funded in whole or in part by the Austrian Science Fund (FWF) to Roland Foisner:

P32512-B/DOI:10.55776/P36503
https://www.doi.org/10.55776/P36503

P36503-B/DOI:10.55776/P32512
https://www.doi.org/10.55776/P32512


title: RNA sequestration in P-bodies sustains myeloid leukaemia

first: Thomas

last: Eder

address: Veterinaerplatz 1

department: Department for Biological Sciences and Pathobiology

institute: University of Veterinary Medicine, Vienna

summary:
Post-transcriptional mechanisms are fundamental safeguards of progenitor
cell identity and are often dysregulated in cancer. Here, we identified regulators
of P-bodies as crucial vulnerabilities in acute myeloid leukaemia (AML) through
genome-wide CRISPR screens in normal and malignant haematopoietic
progenitors. We found that leukaemia cells harbour aberrantly elevated
numbers of P-bodies and show that P-body assembly is crucial for initiation and
maintenance of AML. Notably, P-body loss had little effect upon homoeostatic
haematopoiesis but impacted regenerative haematopoiesis. Molecular
characterization of P-bodies purified from human AML cells unveiled their
critical role in sequestering messenger RNAs encoding potent tumour
suppressors from the translational machinery. P-body dissolution promoted
translation of these mRNAs, which in turn rewired gene expression and
chromatin architecture in leukaemia cells. Collectively, our findings highlight
the contrasting and unique roles of RNA sequestration in P-bodies during tissue
homoeostasis and oncogenesis. These insights open potential avenues for
understanding myeloid leukaemia and potential therapeutic interventions.

overall_design: CUT&Tag of shDDX6 and shCTRL plus IgG controls

first: Ludovica

last: Proietti

first: Gabriele

last: Manhart

first: Bernhard

last: Alber

first: Florian

last: Grebien


title: LAP2alpha facilitates myogenic gene expression by preventing spreading of nucleoplasmic lamin A/C to active chromatin regions (RNA-Seq)

summary:
A-type lamins form a filamentous meshwork beneath the nuclear membrane that anchors large heterochromatic chromatin domains at the nuclear envelope. They also exist as a more dynamic, non-filamentous pool in the nuclear interior, where they interact with gene-rich euchromatin. Lamin-associated polypeptide 2 alpha (LAP2alpha) binds to and maintains the nucleoplasmic lamin pool, but the functional significance of this interaction is poorly understood. Here we investigate the genome-wide chromatin association of A-type lamins during myogenic differentiation in the presence and absence of LAP2alpha.  We find that A-type lamins are significantly reorganized on chromatin upon depletion of LAP2alpha, spreading towards active chromatin and accumulating at regions of active H3K27ac and H3K4me3 histone marks close to the genes whose expression is impaired in the absence of LAP2alpha. This reorganization of A-type lamins on chromatin is accompanied by depletion of the active chromatin mark H3K27ac and a significant delay of myogenic differentiation. Thus, the interplay of lamins and LAP2alpha is crucial for proper positioning of intranuclear lamins on chromatin to allow normal myogenic differentiation.

This research was funded in whole or in part by the Austrian Science Fund (FWF) to Roland Foisner:

P32512-B/DOI:10.55776/P36503
https://www.doi.org/10.55776/P36503

P36503-B/DOI:10.55776/P32512
https://www.doi.org/10.55776/P32512

overall_design: Gene expression profiling by RNA-sequencing of proliferating (Prol) and differentiating wildtype and LAP2alpha knockout immortalized myoblasts (D0: start of differentiation; D2: day 2 after induction of differentiation). LAP2alpha knockout myoblasts were created using the CRISPR-Cas9 system.

first: Simona

last: Ferraioli

first: Fatih

last: Sarigol

first: Celine

last: Prakash

first: Daria

last: Filipczak

first: Nana

last: Naetar


title: RAS-mutant AML LSCs originate from GMPs and drive clinical resistance to BH3 mimetics [ATAC-Seq]

city: New York

first: BiNGS

last: Core

address: 1470 Madison Ave

institute: Icahn School of Medicine at Mount Sinai

summary: Cancer driver mutations often show distinct temporal acquisition patterns, but the biological basis for this, if any, remains unknown. RAS mutations occur invariably late in the course of acute myeloid leukemia (AML), upon progression or relapsed/refractory disease1-6. Here, by employing synthetic leukemogenesis in human cells, we first show that RAS mutations are obligatory late events that need to succeed earlier cooperating mutations. We provide the mechanistic explanation for this in a requirement for mutant RAS to specifically transform committed progenitors of the myelomonocytic lineage (granulocyte-monocyte progenitors, GMPs) harboring previously acquired driver mutations, revealing that advanced leukemic clones originate from a different cell type than more ancestral clones. Furthermore, we demonstrate that RAS-mutant leukemia stem cells (LSCs) give rise to monocytic disease, as frequently observed in patients with poor responses to treatment with the BCL2 inhibitor drug Venetoclax (VEN). We show that this is because RAS-mutant LSCs, in contrast to RAS-WT LSCs, have altered BCL2 family gene expression profiles and are resistant to VEN, driving clinical resistance and relapse with monocytic features. Our findings demonstrate that a specific genetic driver by imposing a specific LSC target cell restriction shapes the non-genetic cellular hierarchy of AML and critically impacts therapeutic outcomes in patients.

overall_design: Synthetic leukemogenesis models in human iPSC-HSPCs and primary CB HSPCs

first: Eirini

last: Papapetrou


title: LAP2alpha facilitates myogenic gene expression by preventing spreading of nucleoplasmic lamin A/C to active chromatin regions (ChIP-Seq)

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for A-type lamins, LAP2alpha and H3K27ac and H3K4me3 histone marks in wildtype and LAP2alpha knockout immortalized myoblasts in the proliferating (Prol) and early differentiating state (D2: day 2 after induction of differentiation).


title: Function and Mechanism of NAT10-mediated N4-acetylcytidine modification in osteosarcoma

city: Guangzhou

first: Yutong

last: Zou

address: No.58, Zhongshan Rd.2

institute: The First Affiliated Hospital of Sun Yat-sen University


title: Small proteome of Methanosarcina mazei: Insights from Ribo-seq and peptidomics under different nitrogen conditions

city: Freiburg

first: Rick

last: Gelhausen

state: Baden Württemberg

address: Georges-Köhler-Allee 106

department: Department of Computer Science

country: Germany

institute: Albert-Ludwigs-University Freiburg

summary: We used ribosome profiling (Ribo-seq) to generate a translatome map for the archaeon M. mazei under two growth conditions. The analysis of the datasets allowed to generate a high confidence inventory of small proteins in the model archaeon M. mazei and their regulation under nitrogen limiting conditions.

overall_design: Ribo-seq and RNA-seq experiments to study the transcriptome and the translatome of M. mazei in two growth conditions.

first: Britta

last: Jordan

first: Muhammad

last: Tufail

first: Lydia

last: Hadjeras

first: Liam

last: Cassidy

first: Tim

last: Habenicht

first: Miriam

last: Gutt

first: Rolf

last: Backofen

first: Andreas

last: Tholey

first: Cynthia

last: Sharma

first: Ruth

last: Schmitz


title: LTBR-mediated immunomodulation of the tumor microenvironment promotes anti-tumor responses

city: Tarrytown

first: Wei-Yi

last: Cheng

state: NY

address: 777 Old Saw Mill River Rd

institute: Regeneron Pharmaceuticals, Inc.

summary: The presence of high endothelial venules (HEV) and tertiary lymphoid structures (TLS) in solid tumors is correlated with favorable prognosis in many cancer types and has been associated with better treatment responses to immune-checkpoint blockade (ICB). However, the molecular mechanisms underlying intratumoral HEV and TLS formation and their contribution to anti-tumor responses remain unclear. Lymphotoxin beta receptor (LTBR) signaling is a critical regulator of lymph node organogenesis and when combined with antiangiogenic and ICB treatment can augment tumor-associated HEV formation. Here we demonstrate that LTBR signaling modulates the tumor microenvironment via multiple mechanisms to promote anti-tumor T cell responses. Systemic activation of the LTBR pathway via agonistic antibody treatment induced tumor-specific HEV formation, upregulated the expression of TLS-related chemokines, and enhanced dendritic cell (DC) and T cell infiltration and activation in syngeneic tumor models. In vitro studies confirmed direct effects of LTBR agonism on DC activation and maturation and associated DC- mediated T cell activation. Single agent LTBR agonist treatment inhibited syngeneic tumor growth in a CD8+ T cell- and HEV-dependent manner; and the LTBR agonist enhanced anti-tumor effects of anti-PD-1 and CAR T cell therapies, respectively. An in vivo tumor screen for TLS-inducing cytokines revealed that the combination of LTBR agonism and lymphotoxin alpha (LT⍺) expression promoted robust intratumoral TLS induction and enhanced tumor responses to anti-CTLA-4 treatment. Collectively, this study highlights crucial functions of LTBR signaling in modulating the tumor microenvironment and inform future HEV/TLS- based strategies for cancer treatments.

overall_design: The GFP-expressing tumor cells were subcutaneously implanted in BALB/c mice. The tumor-bearing mice were treated with 3 doses of isotype control or LTBR agonist antibody at 2 mg/kg, every other day. There are 4 treatment groups: "Isotype control mAb treated", "LTBR agonist mAb treated", "Isotype control mAb treated + LTa expression", "LTBR agonist mAb treated + LTa expression". At the end of study, 5 tumors per treatment group (N=5) were harvested, dissociated, stained by endothelial cell marker CD31 and immune cell marker CD45, and sorted by flow cytometry to collect the immune cells (CD45+CD31-GFP-), endothelial cells (CD31+CD45-GFP-) and stromal cells (CD45-CD31-GFP-) for scRNA sequencing.

first: Disi

last: An

first: Guoying

last: Chen

first: Katja

last: Mohrs

first: Christina

last: Adler

first: Namita

last: Gupta

first: Gurinder

middle: S

last: Atwal

first: David

last: DiLillo

first: Christopher

last: Daly

first: John

last: Lin

first: Frank

last: Kuhnert


title: Antagonism between H3K27me3 and genome-lamina association drives atypical spatial genome organization in the totipotent embryo [scDam&T-seq]

first: Jop

last: Kind

overall_design: Profiling Nuclear Lamina (NL) contacts, H3K27me3, H3K9me3 and accessibility in mouse preimplantation embryos using scDamID and scDam&T-seq.

first: Isabel

last: Geirreiro

first: Franka

last: Rang

first: Yumiko

last: Kitazawa

first: Femke

last: Groenveld

first: Ramada

last: van Beek

first: Silke

last: Lochs

first: Ellen

last: Boele

first: Antoine

middle: H

last: Peters


title: Adipocyte associated glucocorticoid signaling regulates normal fibroblast function which is lost in inflammatory arthritis [fibroblast_bulk RNAseq]

city: Boston

first: Heather

last: Faust

state: MA

address: 60 Fenwood Road

institute: Brigham and Women's Hopsital

summary: Fibroblasts play critical roles in tissue homeostasis, but in pathologic states can drive fibrosis, inflammation, and tissue destruction. In the joint synovium, fibroblasts provide homeostatic maintenance and lubrication. Little is known about what regulates the homeostatic functions of fibroblasts in healthy conditions. We performed RNA sequencing of healthy human synovial tissue and identified a fibroblast gene expression program characterized by enhanced fatty acid metabolism and lipid transport. We found that fat-conditioned media reproduces key aspects of the lipid-related gene signature in cultured fibroblasts. Fractionation and mass spectrometry identified cortisol in driving the healthy fibroblast phenotype, confirmed using glucocorticoid receptor gene (NR3C1) deleted cells. Depletion of synovial adipocytes in mice resulted in loss of the healthy fibroblast phenotype and revealed adipocytes as a major contributor to active cortisol generation via Hsd11β1 expression. Cortisol signaling in fibroblasts mitigated matrix remodeling induced by TNFα- and TGFβ, while stimulation with these cytokines repressed cortisol signaling and adipogenesis. Together, these findings demonstrate the importance of adipocytes and cortisol signaling in driving the healthy synovial fibroblast state that is lost in disease.

overall_design:
Comparative gene expression profiling analysis of bulk RNA-seq data for  cultured human synovial fibroblasts stimulated with cortisol, fat conditioned media, TNFa, TGFB, mifepristone (GC antagonist) or a combination of these stimulations for 4 or 22 hours.
*** Submitter declares that human raw data are being deposited to dbGaP***

first: Michael

middle: B

last: Brenner


title: Adipocyte associated glucocorticoid signaling regulates normal fibroblast function which is lost in inflammatory arthritis [adipose_scRNAseq]

overall_design: Comparative gene expression profiling analysis of single cell RNA-seq data for healthy human adipose tissue fibroblasts.


title: Endometrial Cancer Susceptibility Gene CTCF is Essential for Progesterone Dependent Uterine Maturation

city: RTP

first: Franco

last: Demayo

state: NC

address: 111 TW Alexander Drive

institute: NIEHS

summary: Receptors for estrogen and progesterone frequently interact, via Cohesin/CTCF loop extrusion,  at enhancers distal from regulated genes; CTCF is decreased or mutated in >20% of human endometrial tumors, indicating its importance in uterine homeostasis. To better understand how CTCF-mediated enhancer-gene interactions impact endometrial development and function, the Ctcf gene was selectively deleted in female reproductive tissues of mice. Prepubertal Ctcfd/d uterine tissue exhibited normal morphology, with a significant reduction in the number of uterine glands compared to those without Ctcf deletion (Ctcff/f mice). Post-pubertal Ctcfd/d uteri were hypoplastic with significant reduction in both the size of the endometrial stroma and number of glands. Transcriptional profiling revealed increased expression of stem cell molecules Lif, EOMES and Lgr5, and enhanced inflammation pathways following Ctcf deletion. Analysis of the response of the uterus to steroid hormone stimulation showed that the response of the uterus to estrogen was not impacted, but that CTCF deletion affects a subset of progesterone responsive genes. This finding indicates 1) mediators of P signaling remain functional following Ctcf deletion and 2) certain P regulated genes are sensitive to Ctcf deletion, suggesting they depend on gene-enhancer interactions that require CTCF. The P responsive genes altered by CTCF ablation included Ihh, Fst and Errfi1, as well as other genes involved in the regulation of critical  processes including hormone response, transcription, and inhibition of tumor generation. Overall, our findings reveal that uterine Ctcf plays a key role in P dependent expression of uterine genes underlying optimal post pubertal uterine development.

overall_design: RNAseq was done

first: Sylvia

last: Hewitt

first: Artiom

last: Gruzdev

first: Cynthia

last: Willson

first: San-Pin

last: Wu

middle: P

last: Lydon

first: Niels

last: Galjart

first: Francesco

last: DeMayo


title: Biofilm matrix regulation by Candida glabrata Zap1 under acidic conditions: transcriptomic and proteomic analyses

city: Aveiro

first: Ana

last: Soares

address: Campus de Santiago

country: Portugal

institute: University of Aveiro

summary: Transcriptional profiling of Candida glabrata zap1Δ and zap1Δ::ZAP1 strains, comparing cells grown under planktonic and biofilm-inducing conditions. Goal was to analyse the effect of ZAP1 deletion in the transcriptomic profile of C. glabrata biofilm cells (in comparision to planktonic cells), under acidic conditions, in order to study the function of the Zap1 transcription factor in C. glabrata biofilm matrix production.

overall_design: Candida glabrata zap1Δ and zap1Δ::ZAP1 strains were cultivated in RPMI at pH 4 for 24 h under planktonic and biofilm-inducing conditions, and then the RNA was extracted from the cells and hybridised in a species-specific DNA microarray for C. glabrata (biofilm vs. planktonic)

first: Bruna

middle: F

last: Gonçalves

first: Diana

last: Pires

first: Liliana

last: Fernandes

first: Miguel

last: Pacheco

first: Tiago

last: Ferreira

first: Hugo

last: Osório

first: Mariana

last: Henriques

first: Sónia

last: Silva


first: Thomas

last: Eder

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Genome-wide CRISPR/Cas9 screen of murine leukemia cells, translatome (polysome profiling) and transcriptomic (RNA-seq) interactors of human acute myeloid leukemia cell lines

first: Ludovica

last: Proietti

first: Gabriele

last: Manhart

first: Bernhard

last: Alber

first: FlorianGrebien

last: Grebien


title: A rewiring of the earliest immune events leading to T-cell mediated disease following intestinal microbial infection in a PINK1KO mouse model of Parkinson’s disease

city: Montreal

first: Jo

middle: Anne

last: Stratton

state: Quebec

address: 3801 University street

department: Neurology and Neurosurgery

country: Canada

institute: McGill University

summary: Parkinson’s disease (PD) is characterized by a protracted period of non-motor symptoms, including gastrointestinal (GI) dysfunction, which can precede the development of cardinal motor dysfunction by decades. This long prodrome of disease is highly suggestive of immune cell involvement in the initiation of disease, but currently the field lacks robust modeling systems to study such mechanisms. The leading hypotheses for the origin of PD, known as the Braak’s hypothesis, states that pathology is first initiated in the GI tract due to environmental triggers, such as pathogens that enter the GI tract in genetically predisposed individuals. Our group has developed GI-targeted pathogen-induced PD mouse modeling systems (in PINK1 KO mice with gram negative bacterial infections) and found that T cells are a major player in driving PD-like motor symptoms at late stages following infection. Herein, we now map the initiating immune events at the site of infection at the earliest stages with the goal of shedding light on the earliest mechanisms triggering T cell-mediated pathological processes relevant to PD. Using unbiased single cell sequencing, we demonstrate that myeloid cells are the earliest dysregulated immune cell type in PINK1 KO infected mice (at 1-week post-infection) followed by a dysregulated T cell response shortly after (at 2 weeks post-infection). We find that these myeloid cells have an enhanced proinflammatory profile, are more mature, and develop enhanced capacity for antigen presentation. Using unbiased prediction analysis, our data suggests that cytotoxic T cells and myeloid cells are particularly poised for interacting with each other, and we identify possible direct cell-cell interaction pathways that might be implicated. How or if these early aberrant immune responses play a key role in initiating PD autoimmunity is the subject of further investigation.

overall_design: Transcriptomic analysis of colonic lamina propria cells in PINK1 KO mice compared to wild type following intestinal microbial infection

first: Recinto

last: Sherilyn Junelle

first: Alexandra

last: Kazanova

first: Hicham

last: Bessaiah

first: Brendan

last: Cordeiro

first: Sriparna

last: Mukherjee

first: Shobina

last: Premachandran

first: Adam

last: MacDonald

first: Jessica

last: Pei

first: Alexis

last: Allot

first: Elia

last: Afanasiev

first: Moein

last: Yaqubi

first: Michel

last: Desjardins

first: Heidi

last: McBride

first: Louis-Eric

last: Trudeau

first: Samantha

last: Gruenhei


title: Structural perturbation of chromatin domains with multiple developmental regulators can severely impact gene regulation and development [RNA-seq-brain]

status: Public on Aug 07 2024

city: Bethesda

first: Pedro

last: Rocha

state: Maryland

address: 6 Center Drive Building 6B 2B220

department: NICHD

institute: National Institutes of Health

summary: Chromatin domain boundaries delimited by CTCF motifs can restrict the range of enhancer action. However, disruption of domain structure often results in mild gene dysregulation and thus predicting the impact of boundary rearrangements on animal development remains challenging. Here, we tested whether structural perturbation of a chromatin domain with multiple developmental regulators can result in more acute gene dysregulation and severe developmental phenotypes. We targeted clusters of CTCF motifs in a domain of the mouse genome containing three FGF ligand genes¬—Fgf3, Fgf4, and Fgf15—that regulate several developmental processes¬. Deletion of the 23.9kb cluster that defines the centromeric boundary of this domain¬ resulted in ectopic interactions of the FGF genes with enhancers located across the deleted boundary that are active in the developing brain. This caused strong induction of FGF expression and perinatal lethality with encephalocele and orofacial cleft phenotypes. Heterozygous boundary deletion was sufficient to cause these fully penetrant phenotypes, and strikingly, loss of a single CTCF motif within the cluster also recapitulated ectopic FGF expression and caused encephalocele. However, such phenotypic sensitivity to perturbation of domain structure did not extend to all CTCF clusters of this domain, nor to all developmental processes controlled by these three FGF genes¬—for example, the ability to undergo lineage specification in the blastocyst and pre-implantation development were not affected. By tracing the impact of different chromosomal rearrangements throughout mouse development, our work starts to uncover the determinants of phenotypic robustness and sensitivity to perturbation of chromatin boundaries.  Our data show how small sequence variants at certain domain boundaries can have a surprisingly outsized effect and must be considered as potential sources of gene dysregulation during development and disease.

overall_design: RNA sequencing was performed in the following tissues: forebrain and midbrain of E11.5 embryos. For each line homozygous embryos and WT littermates were processed using trizol reagent.After confirming that the RNA integrity number for each sample was above 8, libraries were prepared using TruSeq Stranded mRNA prep kit with PolyA purification and sequenced on Novaseq6000.

first: Shreeta

last: Chakraborty

first: Alicia

last: Evans

first: Ryan

middle: K

last: Dale


title: DNA methylation arrays: The Interferon γ Pathway Enhances Pluripotency and X-Chromosome Reactivation in iPSC Reprogramming

city: Barcelona

first: Mercedes

last: Barrero

address: Dr. Aiguader, 88

country: Spain

institute: Centre for Genomic Regulation

summary: To investigate how IFNγ treatment during early stages of reprogramming could affect DNA methylation levels and its link with X-chromosome reactivation, we analyzed 5mC and 5hmC levels in cells undergoing reprogramming, in the presence or absence of IFNγ treatment, using mouse DNA methylation arrays.

overall_design: Mouse DNA methylation arrays for cells undergoing reprogramming at days 5 and 7, in the presence or absence of IFNγ. Analysis of 5mC and 5hmC levels.

first: Bernhard

last: Payer

first: José

middle: L

last: Sardina

first: Anna

middle: V

last: López-Rubio

first: Aleksey

last: Lazarenkov


title: RNA-seq: The Interferon γ Pathway Enhances Pluripotency and X-Chromosome Reactivation in iPSC Reprogramming

summary: In order to identify trasncriptomic changes happening upon the activation of the IFNγ pathway during pluripotency acquisition, female NPCs were induced for reprogramming in the presence or absence of IFNγ for the first five days of the process. RNA-sequencing was performed at days 2, 5 and 7 in control and treated samples. ESCs and NPCs were also included in the study as controls.

overall_design: RNA-seq of NPCs, ESCs and cells undergoing reprogramming at days 2, 5 and 7, in the presence or absence of IFNγ.


title: Targeting axonal guidance dependencies in glioblastoma with ROBO1 CAR T cells [Genome-wide CRISPR-Cas9]

city: Toronto

first: Jason

last: Moffat

state: Ontario

address: 686 Bay Street

department: Genetics and Genome Biology

institute: Hospital for Sick Children

summary: Resistance to genotoxic therapies and tumor recurrence are hallmarks of glioblastoma (GBM), an aggressive brain tumor. Here, we explore functional drivers of post-treatment recurrent GBM. By conducting genome-wide CRISPR-Cas9 knockout screens in patient-derived GBM models, we uncover distinct genetic dependencies in recurrent tumor cells absent in their patient-matched primary predecessors, accompanied by increased mutational burden and differential transcript and protein expression. These analyses map a multilayered genetic response to drive tumor recurrence, identifying protein tyrosine phosphatase 4A2 (PTP4A2) as a novel modulator of self-renewal, proliferation and tumorigenicity at GBM recurrence. Genetic perturbation or small molecule inhibition of PTP4A2 activity represses axon guidance activity through a dephosphorylation axis with roundabout guidance receptor 1 (ROBO1), exploiting a functional dependency on ROBO signaling. Importantly, engineered anti-ROBO1 single-domain antibodies mimic effects of PTP4A2 inhibition. Since a pan-PTP4A inhibitor was limited by poor penetrance across the blood brain barrier (BBB) in vivo, a second-generation chimeric antigen receptor (CAR)-T cell therapy was engineered against ROBO1 that elicits specific and potent anti-tumor responses in vivo. A single dose of anti-ROBO1 CAR-T cells doubles median survival in patient-derived xenograft (PDX) models of recurrent glioblastoma, and also eradicates tumors in ~50% of mice in PDX models of adult lung-to-brain metastases and pediatric relapsed medulloblastoma. We conclude that functional reprogramming drives tumorigenicity and dependence on a multi-targetable PTP4A-ROBO1 signaling axis at GBM recurrence, with potential in other malignant brain tumors.

overall_design: Genome-wide CRISPR-Cas9 screens were performed in primary and recurrent glioblastoma cell lines using the TKOv3 CRISPR library.  Dropout profiles were compared to establish primary and recurrent cellular dependencies.

first: Chirayu

last: Chokshi

first: Benjamin

last: Brakel

first: Kevin

last: Brown

first: Sheila

last: Singh


title: CRISPR Screen: The Interferon γ Pathway Enhances Pluripotency and X-Chromosome Reactivation in iPSC Reprogramming

summary: To investigate the genes and pathways involved in X-chromosome reactivation and pluripotency acquisition, we generated a whole-genome CRISPR knockout screen during female NPC reprogramming into iPSCs. We analyzed the gRNA abundance in NPCs and three different reprogramming populations: non-pluripotent, early pluripotent and late pluripotent. Pair-wise comparisons of gRNA frequencies in different populations were done to identify genes and pathways involved in cell survival in reprogramming conditions, colony formation, early pluripotency acquisition and late pluripotency/X-chromosome reactivation.

overall_design: DNA sequencing of CRISPR-gRNA libraries in NPCs and cells undergoing reprogramming with different pluripotency and X-chromosome status.


title: Targeted Amplicon Sequencing: The Interferon γ Pathway Enhances Pluripotency and X-Chromosome Reactivation in iPSC Reprogramming

summary: Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) requires activation of the pluripotency network and resetting of the epigenome by erasing the epigenetic memory of the somatic state. In female mouse cells, a critical epigenetic reprogramming step is the reactivation of the inactive X chromosome. Despite its importance, a systematic understanding of the regulatory networks linking pluripotency and X-reactivation is missing. Here we reveal important pathways for pluripotency acquisition and X-reactivation using a genome-wide CRISPR screen during neural precursor to iPSC reprogramming. In particular, we discover that activation of the interferon γ (IFNγ) pathway early during reprogramming accelerates pluripotency acquisition and X-reactivation. IFNγ stimulates STAT3 signaling and the pluripotency network and leads to enhanced TET-mediated DNA demethylation, which consequently boosts X-reactivation. We therefore gain a mechanistic understanding of the role of IFNγ in reprogramming and X-reactivation and provide a comprehensive resource of the molecular networks involved in these processes.

overall_design: A custom amplicon oxidative BS-sequencing protocol was developed to assess the allele-specific hydroxymethylation status of regions surrounding several Differential (hydroxy)Methylated CpGs -D(h)MP- in escapee and X-reactivating gene promoters identified at days 5 or 7 of reprogramming by DNA methylation arrays. The validation was done for day 5 reprogramming samples in the presence or absence of IFNγ with the aim to calculate 5hmC percentages in an allele-specific manner. Regions to be analyzed were selected to include species-specific SNPs and at least a D(h)MP while maintaining an amplicon size of 200-500 bp. Paired oxidative Bisulfite (OxBS) and the mock-oxidation reaction (Bisulfite BS) treated DNA from day 5 reprogramming samples were used. Next Generation Sequencing was performed using MiSeq (300bp, single-end).

first: Enrique

last: Blanco

last: Luis Sardina


title: Structural perturbation of chromatin domains with multiple developmental regulators can severely impact gene regulation and development [RNA-seq-blastocyst]

overall_design: RNA sequencing was done in single blastocysts, by isolating RNA using Dynabeads mRNA DIRECT Purification Kit as previously described (Huffman et al., 2012). Blastocysts at E4.5 were flushed out of the uterine horns from super-ovulated females using M2 media and transferred into 50 µl of pre-warmed lysis buffer (100mM Tris-HCl pH-7.5, 500mM LiCl, 10mM EDTA pH-8, 1%LiDS, 5mM DTT) in DNA low binding PCR tubes. Lysed embryos were stored in -20C and processed within a few weeks. Dynabeads Oligo(dT)25 mRNA isolation beads were warmed to room temperature for 30 mins and rinsed with 100 µl of lysis buffer by vortexing continuously for 5 mins. 10 µl of bead suspension was used per embryo lysate. The poly A tail of mRNA was allowed to anneal to beads by mixing the embryo lysates and bead suspension in a vortexer for 5 mins at low speed, followed by 5 mins incubation without shaking. Tubes were briefly spun and placed in a magnetic stand to collect the clear supernatant, which was used for DNA isolation using 2X SPRI beads to determine the genotype of each embryo. mRNA bead complexes were washed twice using Wash Buffer A and twice with Wash Buffer B by vortexing for 5 mins each. cDNA and library prep were then processed using an adaptation of the smartseq2 protocol (Picelli et al., 2014; Satija et al., 2015; Trombetta et al., 2014). Buffers were removed and beads annealed with mRNA was resuspended in 12.5µl of RNA suspension mix containing 20U/µl SUPERase-In RNase inhibitor (Thermo Fisher, #AM2694), 10mM each dNTP Mix (New England Biolabs, #N0447L) and 100µM polydT oligonucleotide primer (custom sequence ‘AGACGTGTGCTCTTCCGATCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN’ synthesized by Integrated DNA Technologies). Bead suspension is heated at 75°C for 5mins to denature the RNA and supernatant is transferred into a new RNase free 0.2ml PCR tube.  To each sample tube 7.5µl of first strand reverse transcription mix with 100mM DTT, 5X SuperScript IV Buffer, SuperScriptTM IV Reverse Transcriptase (Thermo Fisher, #18090050), 20U/µl SUPERase-In RNase inhibitor (Thermo Fisher, #AM2694), 100µM of template switching oligo with 3G’s and an adaptor sequence same as polydT oligonucleotide (custom sequence ‘AGACGTGTGCTCTTCCGATCTNNNNNrGrGrG’, Integrated DNA Technologies) was added and incubated at 50°C for 60 mins to prepare the first strand of cDNA, followed by 85°C for 5mins to inactivate the enzyme. The first strand of cDNA is bound by the template switching oligo and a complementary sequence to the template switching oligo was synthesized, where a PCR oligo is hybridized (custom sequence ‘AGACGTGTGCTCTTCCGATCT’, Integrated DNA Technologies) and amplified. PCR cDNA amplification was done for 16 cycles (98°C for 15 secs, 67°C for 20 secs and 72°C for 10 mins) in a thermal cycler with KAPA Hifi HotStart ReadyMix (Roche, #9420398001). Pre-amplication cDNA mix was purified with 0.8X SPRI beads and quality of cDNA was assessed with a D5000 Screen Tape assay (Agilent, #5067-5588) using Agilent 4150 Tapestation. cDNA mix was diluted to 0.2ng/µl and used for library preparation as described earlier (Trombetta et al., 2014) using Nextera XT DNA Library Prep Kit (Illumina, #FC-131-1024). Briefly, in a 384 well plate, 1 µl of cDNA, 2µl of Tagment DNA Buffer and 1µl of Amplicon Tagment Mix was mixed in ice and tagmentation was done in a thermocycler at 55°C for 10 mins. Reaction was neutralized with 1 µl of Neutralize Tagment Buffer, incubated at room temperature for 5 mins and transferred to ice. Amplification PCR was set up with 3µl of Nextera PCR Mastermix and 2µl of each adaptor from Illumina DNA/RNA UD Indexes Set A (Illumina, #20027213) per sample. Tagmented DNA was incubated at 72°C for 3 mins, 95°C for 30 secs and 12 cycles of 95°C for 10 secs, 55°C for 30 secs, 72°C for 1 min and a final extension at 72°C for 5 mins. Library DNA was purified with 0.9X SPRI and quality was assessed using D1000 Screen Tape assay (Agilent, #5067-5585) before sequencing.

middle: A


title: Transcription factor Ikzf1 associates with Foxp3 to repress gene expression in Treg cells and limit autoimmunity and anti-tumor immunity

city: Suita

first: Kenji

last: Ichiyama

state: Osaka

address: 3-1 Yamadaoka

country: Japan

institute: Osaka University

summary: To investigate the role of IkE5 in Treg cell biology, we established IkE5-deficient Treg cells from Foxp3-Cre IkE5 floxed mice. We then performed Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for several transcription factors and epigenetic regulators as well as the histone modification H3K27ac in wild-type and IkE5-deficient Treg cells.

overall_design: Comparative binding peak analysis of ChIP-seq data for wild-type and IkE5-deficient Treg cells.


summary: To investigate the role of IkE5 in Treg cell biology, we established IkE5-deficient Treg cells from Foxp3-Cre IkE5 floxed mice. We then performed assay for transposase-accessible chromatin (ATAC)-seq to evaluate the chromatin accessibility in wild-type and IkE5-deficient Treg cells.

overall_design: Comparative binding peak analysis of ATAC-seq data for wild-type and IkE5-deficient Treg cells.


summary: To investigate the role of IkE5 in Treg cell biology, we established IkE5-deficient Treg cells from Foxp3-Cre IkE5 floxed mice. We then performed gene expression profiling analysis using data obtained from bulk RNA-seq of wild-type and IkE5-deficient Treg cells.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for wild-type and IkE5-deficient Treg cells.


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

overall_design: Refer to individual Series


title: ATF6 promotes colorectal cancer growth and stemness by regulating the Wnt pathway

status: Public on Aug 06 2024

city: South San Francisco

first: Matt

last: Grimmer

address: 455 E Grand Ave.

institute: Genentech

summary: The unfolded protein response maintains endoplasmic reticulum (ER) homeostasis by sensing protein-folding stress and orchestrating cellular adaptation via the ER-transmembrane proteins IRE1, PERK and ATF6. Malignant cells can co-opt IRE1 and PERK to sustain growth; however, the importance of ATF6 in cancer remains poorly deciphered. We observed elevated ATF6 transcriptional activity in several cancers including colorectal carcinoma (CRC). Genetic silencing or small molecule inhibition of ATF6 blocked cell cycle progression and reduced viability of several human CRC cell lines in vitro and disrupted tumor progression in vivo. Unexpectedly, ATF6 interference disabled Wnt and Myc signaling and reduced stemness. ATF6 inhibition attenuated growth of organoids derived from malignant but not normal human intestinal tissue, decreasing Wnt-pathway activity and driving cellular differentiation. Wnt-surrogate agonism in a Wnt-dependent CRC organoid restored pathway activity and rescued growth under ATF6 blockade. Our findings identify ATF6 as an unexpected facilitator of oncogenic Wnt signaling in CRC.

overall_design: We observed that conditional shRNA silencing of ATF6 curbed the growth of CCK81 and Colo201 human cancer cell lines. RNA-seq analysis was used to reveal ATF6-dependent cellular transcripts that contribute to cancer cell growth and survival by harvesting RNA from ATF6-silenced or non-targeting control cells after 2 and 4 days. Each condition was harvested in biological triplicate. Sample lines analyzed included both pooled (shATF6) and single cell clones (shATF6.1, shATF6.2).

first: Jeffrey

last: Rodvold

first: Matthew

first: Karen

last: Ruiz

first: Scot

last: Marsters

first: Ioanna

last: Oikonomidi

first: Eileen

last: Tan-Aristy

first: Victoria

last: Pham

first: Tamal

last: Sarkar

first: Jonathan

last: Harnoss

first: Whitney

last: Shatz-Binder

first: Zora

last: Modrusan

first: Thomas

last: Wu

first: Jennie

last: Lill

first: Elisia

last: Villemure

first: Joachim

last: Rudolph

first: Felipe

last: de Sousa e Melo


title: Effect of Lhx2 in K562 cells

city: Setagaya-ku

first: Kenji

last: Kitajima

state: Tokyo

address: 2–1-6 Kamikitazawa

department: Stem Cell Project

institute: Tokyo Metropolitan Institute of Medical Science

summary: Lhx2 is a LIM-homeobox transcription factor which could induce hematopoietic stem cell-like cells from mouse embryonic stem cells and induced pluripotent stem cells. However, the effects of Lhx2 overexpression in the human chronic myeloid leukemia cell line K562 remains unknown. Therefore we carried out Lhx2 overexpression in K562 cells.

overall_design: The overexpression of Lhx2 was performed using a retroviral vector carrying IRES-EGFP. Empty vector (EGFP) and the Lhx2 expression vector (Lhx2) were transduced into K562 cells and cultured for 2 days in RPMI/10%FCS. Then, EGFP+ transduced cells were isolated and cultured for an additional 3 days in RPMI/10%FCS.

first: K

first: Y

last: Nishito


title: Landscape of super-enhancer-associated lncRNAs in hepatocellular carcinoma

first: Li

last: Peng

address: No. 107 Yanjiang West Road

institute: Sun Yat-Sen Memorial Hospital


title: Landscape of super-enhancer-associated lncRNAs in hepatocellular carcinoma [ChIP-seq]

summary: Super-enhancer (SE) plays a vital role in the determination of cell identity and cell fate through the control of cell- or tissue-specific gene regulation. Upregulated expression of coding genes is frequently associated with SE. However, the transcription dysregulation driven by SE, from long non-coding RNAs (lncRNAs) viewpoint, remains unclear. Here, SE-associated lncRNAs in HCC were comprehensively screened.

overall_design: Examination of histone modification in HCC cells.

first: Xiao-Qing

last: Yuan


title: The chromatin loop release factor WAPL regulates EBV latency status by restricting LMP production

first: Benjamin

last: Gewurz

address: 181 Longwood Avenue, Channing Laboratory, 8th Floor

institute: Brigham and Women's Hospital

summary: Epstein-Barr virus (EBV) is a ubiquitous human pathogen that is etiologically linked to several cancers and has been connected to multiple sclerosis. EBV employs a series of latency programs, including latency III, latency II, and latency I, to persistently colonize the B-cell compartment. Each of these programs is associated with human malignancies. However, our understanding remains incomplete of how these distinct latency programs are epigenetically restricted. While regulation of the chromatin structure of the EBV genome by cohesin and CTCF contributes to genome regulation, the function of the cohesin release factor Wings Apart-Like Protein Homolog (WAPL) had not previously been understood. In this study, we employ RNA-seq combined with immunofluorescence analysis to determine that loss of WAPL leads to aberrant expression of the oncogenic latent membrane proteins LMP1 and LMP2A in latency I Burkitt lymphoma cells. Through a combination of Hi-C, Hi-ChIP, and ChIP-qPCR, we uncover that WAPL loss causes alterations in looping across the EBV genome and specifically induces formation of loops between the LMP promoter and the oriLyt enhancers. We propose that EBV coopts WAPL to maintain a strict latency I state and, without WAPL, leaky expression of the LMP proteins occurs.

overall_design: HiC sequencing of EBV genome in WAPL WT and KO cell lines

first: Laura

last: Murray-Nerger

first: Davide

last: Maestri

first: Xiang

last: Liu

first: Zhixuan

last: Li

first: Nina

last: Auld

first: Italo

last: Tempera

first: Mingxiang

last: Teng

middle: E


title: CTCF ChIPseq of mouse uterus

first: Franco

last: Demayo

summary: Receptors for estrogen and progesterone frequently interact, via Cohesin/CTCF loop extrusion,  at enhancers distal from regulated genes; CTCF is decreased or mutated in >20% of human endometrial tumors, indicating its importance in uterine homeostasis. To better understand how CTCF-mediated enhancer-gene interactions impact endometrial development and function, the Ctcf gene was selectively deleted in female reproductive tissues of mice. Prepubertal Ctcfd/d uterine tissue exhibited normal morphology, with a significant reduction in the number of uterine glands compared to those without Ctcf deletion (Ctcff/f mice). Post-pubertal Ctcfd/d uteri were hypoplastic with significant reduction in both the size of the endometrial stroma and number of glands. Transcriptional profiling revealed increased expression of stem cell molecules Lif, EOMES and Lgr5, and enhanced inflammation pathways following Ctcf deletion. Analysis of the response of the uterus to steroid hormone stimulation showed that the response of the uterus to estrogen was not impacted, but that CTCF deletion affects a subset of progesterone responsive genes. This finding indicates 1) mediators of P signaling remain functional following Ctcf deletion and 2) certain P regulated genes are sensitive to Ctcf deletion, suggesting they depend on gene-enhancer interactions that require CTCF. The P responsive genes altered by CTCF ablation included Ihh, Fst and Errfi1, as well as other genes involved in the regulation of critical  processes including hormone response, transcription, and inhibition of tumor generation. Overall, our findings reveal that uterine Ctcf plays a key role in P dependent expression of uterine genes underlying optimal post pubertal uterine development.

overall_design: CTCF ChIPseq was done on ovariectomized adult mouse uterus from animale treated for 1 hour with sesame oil vehicle or with 1 mg progesterone

first: Sylvia

last: Hewitt

first: Artiom

last: Gruzdev

first: Cynthia

middle: J

last: Willson

first: San-Pin

last: Wu

first: John

last: Lydon

first: Niels

last: Galjart

first: Francesco

last: DeMayo


title: Aldehydes alter TGF-β signaling and induce obesity and cancer

city: Manhasset

first: Lopa

last: Mishra

address: 350 Community Drive

institute: The Feinstein Institutes for Medical Research & Cold Spring Harbor Laboratory

summary: Obesity and fatty liver diseases-metabolic dysfunction-associated steatotic liver disease (MASLD and MASH) affect over a third of the global population and are exacerbated in individuals with reduced functional aldehyde dehydrogenase 2 (ALDH2), observed in approximately 560 million people. Current treatment to prevent disease progression to cancer remains inadequate, requiring innovative approaches. We observe that Aldh2-/- and Aldh2-/-Sptbn1+/- (ASKO) mice develop phenotypes of human Metabolic Syndrome (MetS) and MASH with altered lipid metabolism and TGF-β signaling, leading to pro-fibrotic and pro-oncogenic phenotypes, which is restored to normal with siRNA to SPTBN1. Significantly, therapeutic inhibition of SPTBN1 blocks MASH and fibrosis in a human 3D MASH model. This study identifies SPTBN1 as a critical regulator of the functional phenotype of toxic aldehyde-induced MASH and a potential therapeutic target.

overall_design: Using unbiased transcriptome methods snRNA-seq and RNA-seq to characterize liver metabolism alterations in mice (WT, Sptbn1+/-, Aldh2-/-, Aldh2-/-Sptbn1+/-) under normal chow diet. Using bulk RNA-seq analysis of human 3D MASH coculture cells to explore the therapeutic effects of targeting Sptbn1.

first: Xiaochun

last: Yang

first: Krishanu

last: Bhowmick

first: Shuyun

last: Rao

first: Xiyan

last: Xiang


title: Hydrogel biomaterials that stiffen and soften on demand reveal that skeletal muscle stem cells harbor a mechanical memory

city: Philadelphia

first: Christopher

last: Madl

state: PA

address: 3231 Walnut St

department: Materials Science and Engineering

institute: University of Pennsylvania

summary: Muscle stem cells (MuSCs) are specialized cells that reside in adult skeletal muscle poised to repair muscle tissue. The ability of MuSCs to regenerate damaged tissues declines markedly with aging and in diseases such as Duchenne muscular dystrophy, but the underlying causes of MuSC dysfunction remain poorly understood. Both aging and disease result in dramatic increases in the stiffness of the muscle tissue microenvironment from fibrosis.  MuSCs are known to lose their regenerative potential if cultured on stiff plastic substrates. We sought to determine if muscle stem cells harbor a memory of their past microenvironment and if it can be overcome. We tested MuSCs in situ using dynamic hydrogel biomaterials that soften or stiffen on demand in response to light and found that freshly isolated MuSCs develop a persistent memory of substrate stiffness characterized by loss of proliferative progenitors within the first three days of culture on stiff substrates. MuSCs cultured on soft hydrogels had altered cytoskeletal organization and activity of Rho and Rac GTPase and YAP mechanotransduction pathways compared to those on stiff hydrogels. Pharmacologic inhibition identified RhoA activation as responsible for the mechanical memory phenotype, and single cell RNA sequencing revealed a molecular signature of the mechanical memory. These studies highlight that microenvironmental stiffness regulates MuSC fate and leads to MuSC dysfunction that is not readily reversed by changing stiffness. Our results suggest that stiffness can be circumvented by targeting downstream signaling pathways to overcome stem cell dysfunction in aged and disease states with aberrant fibrotic tissue mechanics.

overall_design: Primary skeletal muscle stem cells were isolated from the hindlimbs of young (2 mo) or aged (>24 mo) C57BL/6 mice and were subsequently cultured on poly(ethylene glycol) hydrogels with either 12 kPa or 40 kPa stiffness for 1, 3, or 7 days. A subset of cells were treated with rhosin, a RhoA inhibitor, for three days prior to collection. Cells were enzymatically detached from the hydrogels and processed for single cell sequencing according to the Parse Biosciences Evercode WT version 1 protocol.

first: Helen

last: Blau


title: USP8 coordinates ESCRT-mediated membrane repair, xenophagy, and oxidative stress responses during Mycobacterium tuberculosis infection

city: St Louis

first: Pallavi

last: Chandra

state: MO

address: 660 S Euclid Ave

department: Department of Medicine, Div of Infectious Diseases

institute: Washington University School of Medicine

summary: We show that USP8 colocalizes with intracellular Mtb, recognizes phagosomal membrane damage, and is required for ESCRT-dependent membrane repair. Furthermore, USP8 regulates the NRF2-dependent antioxidant signature. Taken together, our study shows a central role of USP8 in promoting intracellular growth of Mtb by coordinating phagosomal membrane repair, ubiquitin-driven selective autophagy, and the oxidative stress response.

overall_design: BMDMs were seeded at E6 cells per well in a 6-well -plate and transfected with scrambled or Usp8 siRNA at 50nM. 2 days later the samples were either not infected or infected with H37Rv at MOI 5 for 24 and 72 h. RNA was prepared using the Direct-Zol RNA Mini-Prep kit. Each sample had 6 replicates.

first: Jennifer

last: Philips


title: Elucidating the mechanisms underlying H3K18la contributes to NSCLC progression

city: Zhengzhou

first: Cai

last: Zhang

address: 1#, Jianshedong Road

institute: The First Affiliated Hospital of Zhengzhou University

summary: Histone lactylation has been served as a novel epigenetic modification that directly stimulates gene transcription from chromatin. In order to study the regulatory mechanisms of H3K18la in non-small cell lung cancer (NSCLC), we performed ChIP-Seq to identify its target genes in NCI-H1299 cell lines.

overall_design: ChIP-Seq with anti-H3K18la in NCI-H1299 cells

first: Lu

last: Zheng


title: Structural perturbation of chromatin domains with multiple developmental regulators can severely impact gene regulation and development

first: Pedro

last: Rocha


title: The chromatin loop release factor WAPL regulates EBV latency status by restricting LMP production [HiChIP]

first: Benjamin

last: Gewurz

summary: Epstein-Barr virus (EBV) is a ubiquitous human pathogen that is etiologically linked to several cancers and has been connected to multiple sclerosis. EBV persists in its human hosts through a biphasic replication cycle that depends on three latency stages. Understanding the host mechanisms that contribute to the tight regulation of viral gene expression in the discrete states of latency III, latency II, and latency I is important for developing therapeutic approaches for treating EBV positive cancers. Regulation of the chromatin structure of the EBV genome by cohesin and CTCF plays a role in this latency type switching. However, the function of the cohesin release factor WAPL had not previously been understood. In this study, we employ RNA-seq combined with immunofluorescence analysis to determine that the cohesin release factor WAPL plays a role specifically in inhibiting the expression of the oncogenic latent membrane proteins LMP-1 and LMP-2A. Through a combination of Hi-ChIP and ChIP-qPCR, we uncover that WAPL loss alters the looping interactions within the EBV genome and alters the histone modifications present at the LMP promoter. We propose that EBV coopts WAPL to maintain a latency I state and, without WAPL, leaky expression of the LMP proteins occurs.

overall_design: Hi-ChIP was performed as previously descibed. WAPL was knocked out in MUTU I cells via CRISPR-Cas9 or an sgControl guide RNA was used. ChIP for H3K27Ac was performed.

first: Laura

last: Murray-Nerger

first: Davide

last: Maestri

first: Xiang

last: Liu

first: Zhixuan

last: Li

first: Nina

middle: R

last: Auld

first: Italo

last: Tempera

first: Mingxiang

last: Teng


title: Highly sensitive mapping of type II topoisomerase DNA cleavage sites with SHAN-seq

first: Ian

last: Morgan

state: MD

address: 50 Center Dr.

institute: National Heart Lung and Blood Institute

summary: Type II topoisomerases (topos) are a ubiquitous and essential class of enzymes that form transient enzyme-bound double-stranded breaks on DNA called cleavage complexes. The location and frequency of these cleavage complexes on DNA is important for cellular function, genomic stability, and a number of clinically important anticancer and antibacterial drugs, e.g., quinolones. We developed a simple high-accuracy end-sequencing (SHAN-seq) method to sensitively map type II topo cleavage complexes on DNA in vitro. Using SHAN-seq, we detected Escherichia coli gyrase and topoisomerase IV cleavage complexes at hundreds of sites on supercoiled pBR322 DNA, approximately one site every ten bp, with frequencies that varied by two-to-three orders of magnitude. These sites included previously identified sites and 20-50 fold more new sites. We show that the location and frequency of cleavage complexes at these sites are enzyme-specific and vary substantially in the presence of the quinolone, ciprofloxacin, but not with DNA supercoil chirality, i.e., negative vs. positive supercoiling. SHAN-seq’s exquisite sensitivity provides an unprecedented single-nucleotide resolution view of the distribution of gyrase and topoisomerase IV cleavage complexes on DNA. Moreover, the discovery that these enzymes can cleave DNA at orders of magnitude more sites than the relatively few previously known sites resolves the apparent paradox of how these enzymes resolve topological problems throughout the genome.

overall_design: Supercoiled pBR322 DNA was cleaved with E. coli gyrase or topoisomerase IV and spiked with three DNA oligonucleotides. The DNA was subject to standard DNA-seq library preparation to label the enzyme-cleaved ends, fragmented, and subject to a second round of standard DNA-seq library preparation to label the fragmented ends. The labelled DNA was pair-end sequenced and mapped to the plasmid and spike-in oligonucleotide reference sequences. The reads from the topo-cleaved plasmid DNA ends were normalized by the reads from the spike-in ends and were used to determine the gyrase or topo IV cleavage levels at each position on the plasmid.

first: Shannon

last: Mckie

first: Rachel

last: Kim

first: Yeonee

last: Seol

first: Gabor

last: Harami

first: Anthony

last: Maxwell

first: Keir

last: Neuman


title: Blood-Generating Heart-Forming Organoids (BG-HFOs) recapitulate co-development of the hematopoietic system and the embryonic heart

city: Hannover

first: Robert

last: Zweigerdt

state: Germany

address: Carl-Neuberg-Str. 1

department: LEBAO

institute: Medizinische Hochschule Hannover (MHH)

summary: Hematopoietic stem cells (HSCs) derive from the hemogenic endothelium of the dorsal aorta, which develops in spatial and functional proximity to the heart anlagen in the human embryo. Despite the biomedical importance of HSCs, their in vitro induction and niche-like stabilization in a proper developmental context is challenging and poorly established. The underlying reasons include the poorly understood signals controlling HSC formation and the lack of unequivocal markers specifying their stages of differentiation and maturation.  Our recently established human pluripotent stem cell (hPSC)-derived heart-forming organoids (HFOs) represent complex, well-patterned multi-tissue structures recapitulating key aspects of heart, foregut and vasculature development. Modulating HFO development by the stage-dependent supplementation of hemato-endothelial factors, we here report the generation of blood-generating-HFOs (BG-HFOs). While maintaining their well-patterned heart anlagen, BG-HFOs comprise a dense, mesenchyme-embedded endothelial layer giving rise to hematopoietic cells thereby resembling – in vitro - aspects of the intra-embryonic region giving rise to definitive hematopoiesis in vivo. Single cell-RNA sequencing of BG-HFOs revealed a gene expression signature of hematopoietic progenitor cells (HPCs) as well as patterns resembling hematopoietic derivatives such as erythroid, megakaryocytic and myeloid cells. Notably, BG-HFO-derived CD34pos CD43pos and CD34neg CD43pos cells display erythroid and myeloid potential in colony forming unit (CFU) assays, while CD34pos CD43neg HPCs retain lymphoid potential, observed via generation of T-cell progenitors in artificial thymic organoid (ATO)-based assays. This suggests presence of different multipotent HPCs in BG-HFOs and a potential transient downregulation of CD43 in lymphoid-restricted HPCs. Together, BG-HFOs represent the first human model of cardiac, endothelial and multipotent-hematopoietic cell co-development. The study promotes research on the multi-tissue interactions of hematopoiesis in vitro, overcoming limitations of such studies in the human embryo.

overall_design: BG-HFOs were dissociated on day 14 of differentiation and relative gene expression was analyzed via single cell RNA sequencing. The gene expression analysis revealed the cell composition of the organoids.

first: Miriana

last: Dardano

first: Lika

last: Drakhlis


title: Single-cell  transcription and chromatin accessbility profiles of IDH mutant gliomas

first: Jingyi

last: Wu

address: 360 Longwood Ave

department: Department of Cancer Biology

institute: Dana-Farber Cancer Institute

summary: we use single cell RNA-seq and ATAC-seq to understand the cell states and epigenetic states of IDH mutant gliomas, including the condition with DNMT1 inhibitor treatment

overall_design:
Primary IDH mutant glioma samples  are collected after surgery and used for the establishment of organoid. Dissociated tumors are used for scRNA-seq and scATA-seq assays from 10x genomics.

**'Submitter declares that the raw data will be deposited in dbGaP due to patient privacy concerns.***

first: L

middle: N

last: Castro

first: Mario

last: Suva

first: Bradley

last: Bernstein


title: "The Wild West of Spike-in Normalization": Benchmarking the ChIP-seq spike-in normalization method

status: Public on Aug 05 2024

city: La Jolla

first: Alon

last: Goren

address: 9375 Gilman Dr

department: Department of Medicine

institute: University of California San Diego

summary: Spike-in normalization is a powerful approach to assess global changes in data obtained from genomic mapping of DNA-associated proteins by methods such as ChIP-sequencing (ChIP-seq) or CUT&RUN. While multiple spike-in methods provide detailed documentation, the implementation of these approaches often omit critical quality control steps and veer from the established procedures. Here, we show that proper application of spike-in normalization can increase quantification accuracy across a spectrum of conditions. Next, we outline how misuse of spike-in approaches can create erroneous biological interpretations and provide guidelines to minimize pitfalls when applying this approach to normalize data from protein-DNA interaction results.

overall_design: Building on ChIP-Rx benchmarking of creating a titration of epitope levels, we carry out a similar titration experiment of histone acetylation. Mitotically-arrested cells (low H3K9ac) were mixed with unsynchronized cells (high H3K9ac) in six ratios (0/100, 5/95, 25/75, 50/50, 75/25, 100/0). To all samples, a constant amount of two spike-in species chromatin (D. melanogaster and S. cerevisiae) were added. All replicates are technical replicates, deriving from the same pool of input chromatin. We next examine the operation limits of the approach over a large variation in the ratio of spike-in and target chromatin. We measure mitotic ChIP-seq H3K9ac signal in TSA or DMSO treated cells, varying spike-in amount from 1-10000x. Mitotically arrested cells were treated with either TSA (increasing acetylation) or DMSO control. For each set of treatments, spike-in was varied over five orders of magnitude (0.00025 to 2.5x spike-in/target ratio). Input samples ("input") contain the sheared chromatin that was not used for ChIP and serve as a control to identify non-specific signal.

first: Lauren

last: Patel

first: Yuwei

last: Cao

first: Eric

last: Mendenhall

first: Chris

last: Benner


title: YAP1 status defines two intrinsic subtypes of LCNEC with distinct molecular features and therapeutic vulnerabilities

city: Houston

first: Carl

last: Gay

state: TX

address: 1515 Holcombe Blvd

department: Thoracic-Head & Neck Med Onc

institute: The University of Texas MD Anderson Cancer Center

summary: Purpose: Large cell neuroendocrine carcinoma (LCNEC) is a high-grade neuroendocrine malignancy that, like the more common small cell lung cancer (SCLC), is associated with an absence of druggable oncogenic driver mutations, a clinically aggressive disease course, and dismal prognosis. In contrast to SCLC, however, there is little evidence to guide optimal treatment strategies which are often adapted from SCLC and non-small cell lung cancer (NSCLC) approaches. While there have been some efforts to describe the molecular landscape of LCNEC, to date there are few links between distinct biologic phenotypes of LCNEC and therapeutic vulnerabilities.  Experimental design: To better define the biology of LCNEC, we analyzed cell line and patient genomic data and performed immunohistochemistry and single-cell (sc)RNAseq of core needle biopsies from LCNEC patients and preclinical models. Results: Here, we demonstrate that the presence or absence of YAP1 distinguishes two subsets of LCNEC. The YAP1-high subset is mesenchymal and inflamed and characterized, alongside TP53 mutations, by co-occurring alterations in CDKN2A/B and SMARCA4. Therapeutically, the YAP1-high subset demonstrates vulnerability to MEK and AXL targeting strategies, including a novel preclinical AXL CAR-T cell, as well as predicted vulnerability to SMARCA2 degraders and CDK4/6 inhibitors. Meanwhile, the YAP1-low subset is epithelial and immune-cold and more commonly features TP53 and RB1 co-mutations, similar to those observed in pure SCLC. Notably, the YAP1-low subset is also characterized by expression of SCLC subtype-defining transcription factors - especially ASCL1 and NEUROD1 - and, as expected given its transcriptional similarities to SCLC, exhibits putative vulnerabilities reminiscent of SCLC, including Delta-like ligand 3 (DLL3) and CD56 targeting, as with novel preclinical DLL3 and CD56 CAR T-cells, and DNA damage repair (DDR) inhibition.  Conclusion: YAP1 defines distinct subsets of LCNEC with unique biology. These findings highlight the potential for YAP1 to guide personalized treatment strategies for LCNEC.

overall_design: Six patient derived xenograft tumors and six patient biopsies with a diagnosis of LCNEC or mixed histology, including LCNEC, were collected and analyzed by single-cell RNAseq.

first: Allison

last: Stewart

first: Yuanxin

last: Xi


title: XOL-1 regulates developmental timing by modulating the H3K9 landscape in C. elegans early embryos

city: Ann Arbor

first: Gyorgyi

last: Csankovszki

state: MI

address: 1105 N University Ave

department: MCDB

institute: University of Michigan

summary: Sex determination in the nematode C. elegans is controlled by the master regulator XOL-1 during embryogenesis. Expression of xol-1 is dependent on the ratio of X chromosomes and autosomes, which differs between XX hermaphrodites and XO males. In males, xol-1 is highly expressed and in hermaphrodites, xol-1 is expressed at very low levels. XOL-1 activity is known to be critical for the proper development of C. elegans males, but its low expression was considered to be of minimal importance in the development of hermaphrodite embryos. Our study reveals that XOL-1 plays an important role as a regulator of developmental timing during hermaphrodite embryogenesis. Using a combination of imaging and bioinformatics techniques, we found that hermaphrodite embryos have an accelerated rate of cell division, as well as a more developmentally advanced transcriptional program when xol-1 is lost. Further analyses reveal that XOL-1 is responsible for regulating the timing of initiation of dosage compensation on the X chromosomes, and the appropriate expression of sex-biased transcriptional programs in hermaphrodites. We found that xol-1 mutant embryos overexpress the H3K9 methyltransferase met-2 and have an altered H3K9me landscape. Some of these effects of the loss of xol-1 gene were reversed by the loss of met-2. These findings demonstrate that XOL-1 plays an important role as a developmental regulator in embryos of both sexes, and that MET-2 acts as a downstream effector of XOL-1 activity in hermaphrodites.

overall_design: RNA-seq profiles of early embryo and L1 larval stage C. elegans samples. The following genotypes are included: N2 Bristol strain (wild type), TY4403 him-8 (e1489) IV and WM458 xol-1 (ne4472) X. Genotype and developmental stage for each sample are listed separately.

first: Eshna

last: Jash

first: Anati Alyaa

last: Azhar

first: Hector

last: Mendoza

first: Zoey

last: Tan

first: Halle

last: Escher

first: Dalia

last: Kaufman

first: Györgyi


title: Pseudoknot RNA degraders – directed and irreversible degradation of SARS-CoV-2 genome as an antiviral strategy

city: Cambridge

first: Muxin

last: Gu

email: muxingu@gmail.com, mg445@cam.ac.uk

address: Jeffrey Cheah Biomedical Centre, Puddicombe Way

department: Haematology

institute: University of Cambridge

summary: We demonstrate that the pseudoknot binder MTDB selectively degrades coronaviral pseudoknots in vitro, in cells and in a SARS-CoV-2 infection mouse model, which means it has great potential to be used as an anti-SARS-CoV-2 therapeutic.

sample_organism: Severe acute respiratory syndrome coronavirus 2

overall_design: Oxford Nanopore sequencing of SARS-CoV-2 RNA

first: Sigitas

last: Mikutis

first: Maria

last: Rebelo

first: Eliza

last: Yankova

first: Cong

last: Tang

first: Madoka

last: Hazemi

first: Ana

last: Coelho

last: Eleftheriou

first: George

last: Vassiliou

first: Pedro

last: Simas

first: Francisco

last: Corzana

first: Konstantinos

last: Tzelepis

first: Gonçalo

last: Bernardes


title: Disclosing potential therapeutic targets associated with Osimertinib resistance in non-small cell lung cancer cell line H1975

city: Yinchuan

first: Xuexia

last: Tong

address: No. 804 Shenglijie

institute: General Hospital of Ningxia Medical University

summary: Osimertinib, a third-generation epidermal growth factor receptor tyrosine kinase inhibitor, is a highly effective and valuable treatment option for advanced non-small cell lung cancer (NSCLC) patients with EGFR mutations, like T790M. However, acquired resistance ultimately limits its clinical application. In this study, we constructed H1975/OSI cell lines and utilized potentially complementary transcriptomic and proteomic techniques, which may provide insight into the intricately complex molecular mechanisms, to reveal potential therapeutic targets associated with Osimertinib resistance.  Uni-omics and multi-omics analyses were conducted on the transcriptomic and proteomic (4D label-free) expression profiles, which involved differential expression analysis, GO functional annotation and KEGG pathway enrichment analysis, correlation analysis of transcription factors, PPI network and Cytoscape analysis. We highlighted multiple signaling pathways and eleven hub-genes, including NOP56, DDX21, PDCD11, CCNB1, TOP21, KPNA2, DDX5, EFTUD2, BRCA1, LMNB1 and HIF1A, that may be the key to overcoming resistance. Further validation of these targets and molecules in preclinical and clinical studies could lead to the development of new and more effective treatment options for H1975-resistant patients.

overall_design: H1975/OSI cell line was induced in vitro through intermittent induction. Cell activity was measured using cell counting kit-8 assay. Uni-omics and multi-omics analyses were conducted on the transcriptomic and proteomic (4D label-free) expression profiles, which involved differential expression analysis, GO functional annotation and KEGG pathway enrichment analysis, correlation analysis of transcription factors, PPI network and Cytoscape analysis.

first: Fang

last: Li


title: Specific requirement of the p84/p110γ complex of PI3Kγ for antibody-activated, inducible cross-presentation in murine type 2 DCs

status: Public on Aug 04 2024

city: Paris

first: Despoina

last: Koumantou

address: 16 rue Henri Huchard

country: France

institute: INSERM U1149

summary: Cross-presentation by MHCI is optimally efficient in type 1 dendritic cells (DC) due to their high capacity for antigen processing. However, through specific pathways, other DCs, such as type 2 DCs and inflammatory DCs (iDCs) can also cross-present antigens. FcγR-mediated uptake by type 2 DC and iDC subsets mediates antibody-dependent cross-presentation and activation of CD8+ T cell responses. Here, we identify an important role for the p84 regulatory subunit of PI3Kγ in mediating efficient cross-presentation of exogenous antigens in otherwise inefficient cross-presenting cells, such as type 2 DCs and GM-CSF-derived iDCs. We show that FcγR-mediated cross-presentation on type 2 and iDCs depends on the enzymatic activity of the p84/p110γ complex of PI3Kγ, which controls the activity of the NADPH oxidase NOX2 and ROS production in murine spleen type 2 DCs and GM-CSF-derived iDCs. In contrast, p84/p110γ is largely dispensable for cross-presentation by type 1 DCs. These findings suggest that PI3Kγ-targeted therapies, currently considered for oncological practice, may interfere with the ability of type 2 DCs and iDCs to cross-present antigens contained in immune complexes.

overall_design: To investigate differential expression of genes in conventional dendritic cells, we performed bulk RNA sequencing in murine splenic WT type 1 and type 2 DCs. We also analysed murine splenic type 2 DCs from mice that are KO for each one of the regulatory subunits of PI3Kγ, p84 and p101. The dendritic cells were coming from mice either in steady state or euthanized 5 hours after intraperitoneal injections of Ova-anti Ova immune complexes.

first: Saveanu

last: Loredana

first: Koumantou

last: Despoina

first: Bourdely

last: Pierre

first: Lameiras

last: Sonia


title: Histone marks identify novel transcription factors that parse CAR-T cell subset-of-origin, clinical potential and expansion [CUT&RUN]

city: Seattle

first: Ye

last: Zheng

state: WA

address: 1100 Fairview ave N

institute: Fred Hutchinson Cancer Center

summary: Chimeric antigen receptor-modified T cell (CAR-T) immunotherapy has revolutionized the treatment of blood cancers. Parsing the genetic underpinnings of T cell precursor quality and subsequent CAR-T efficacy is challenging. RNA-seq informs infused CAR-T state, but the nature of dynamic transcription during activation hinders identification of transiently or minimally expressed genes, such as transcription factors, and over-emphasizes effector and metabolism genes. We investigated whether analyses of transcriptionally repressive and permissive histone methylation marks reveal associations with CAR-T potential beyond what is seen by transcriptomic analysis. We assessed human CD8+ T cell naïve, central and effector memory subsets that form the substrate of CAR-T cell products, and CAR-T cells derived from these subsets. We extended these observations into the clinic, by examining CAR-T products from a clinical trial of lymphoma patients (NCT01865617). We report that histone marks provide a rich dataset for identification of genes not apparent by conventional transcriptomics. Histone marks improved identification of T cell subsets, CAR-T manufactured from these subsets, and CAR-T manufactured from central memory cells from healthy donors and patients. Using this discriminative approach, we controlled for clinical factors and identified a factor, KLF7, associated with CAR-T cell expansion in patients. Epigenomic methods are an orthogonal, robust and wide-reaching approach for the assessment of T cell immunotherapeutic quality.

overall_design: To investigate the relationship of transcriptomic output and histone marks in CD8+ T cell subsets we performed RNA-seq analyses and CUT&RUN analyses of transcriptionally repressive H3K27me3 and permissive H3K4me2 histone marks on sorted healthy donor (HD) CD8+ naïve, central memory (CM), effector memory (EM) and CM-enriched CD8+ CAR-T cells from a clinical trial (NCT01865617). Quality check (QC) metrics were satisfied for RNA-seq samples and data (6 HD, 3 T cell subsets/HD) and CUT&RUN data (4 HD, 3 T cell subsets/HD) and all 15 patients from NCT01865617. We compare and contrast RNA-seq and CUT&RUN using a custom bioinformatic platform (https://github.com/yezhengSTAT/CAR-T_Histone).

first: Salvatore

last: Fiorenza

first: Janaki

last: Purushe

first: Jay

last: Sarthy

first: Derek

last: Janssens

first: Alyssa

last: Sheih

first: Erik

last: Kimble

first: Delaney

last: Kirchmeier

first: Tinh-Doan

last: Phi

first: Jordan

last: Gauthier

first: Alexandre

last: Hirayama

first: Stanley

last: Riddell

first: Qian

last: Wu

first: Raphael

last: Gottardo

first: David

last: Maloney

first: Jean

last: Yang

first: Steven

last: Henikoff

first: Cameron

last: Turtle


title: Histone marks identify novel transcription factors that parse CAR-T cell subset-of-origin, clinical potential and expansion [RNA-seq]

first: Ye

last: Zheng

first: Salvatore

last: Fiorenza


title: Disruption of the intellectual disability-linked gene Hs6st2 in mice decreases heparan sulfate 6-O-sulfation in the brain and impairs memory

city: Old Westbury

first: Ying-Tao

last: Zhao

address: Riland building, Room 024 Northern Boulevard, P.O. Box 8000

department: Biomedical Sciences

institute: New York Institute of Technology

summary: Heparan sulfate (HS) is a linear polysaccharide that plays a key role in cellular signaling networks. HS functions are regulated by its 6-O-sulfation, which is catalyzed by HS 6-O-sulfotransferases (HS6STs). Although mutations in HS6ST2 cause intellectual disability in human patients, the role and molecular mechanisms of HS6ST2 in the adult mammalian brain remain unknown. Here we found that the brain specifically expresses the long isoform of Hs6st2, which encodes a protein that retains the sulfotransferase domain and acquires a novel sub-structure. To determine the role of Hs6st2 in the brain, we carried out a series of behavioral and molecular assessments on the Hs6st2 knockout mice. We found that Hs6st2 knockout mice exhibit high body weight, hyperactivity, and memory-related deficits. To determine the molecular mechanisms underlying these deficits, we carried out strong anion exchange-high performance liquid chromatography and RNA sequencing. We found that knockout of Hs6st2 decreases HS 6-O-sulfation levels in the brain and impairs transcriptome in the hippocampus. We also found that the transcriptome changes are enriched in genes involved in ribosome and protein translation pathways, which are likely due to the downregulation of the fibroblast growth factor signaling. Together, our study demonstrates the role and molecular mechanisms of Hs6st2 in the adult mammalian brain, which provides new insights into the role of HS in brain health and disease.

overall_design: In this study, we analyzed the splicing patterns of Hs6st2 in different mouse tissues and investigated the protein structures of the long and short Hs6st2 isoforms using machine learning-based predictions. We also used cryo-recovery approach to re-generate the Hs6st2 knockout mice (KO) and carried out a series of behavioral and molecular assessments, including behavioral tests, strong anion exchange-high performance liquid chromatography (SAX-HPLC), and RNA sequencing (RNA-seq).

first: Sohyun

last: Moon

first: Jerry


title: Vagal Sensory Neuron-Derived FGF3 Regulates Insulin Secretion and Energy Balance

summary: The vagus nerve has long been associated with the regulation of energy balance. However, the identity of the molecules mediating these effects remain largely unknown. Here, we used RNA sequencing to interrogate the molecular repertoire of the mouse vagal afferents. We found that Fgf3 mRNA is upregulated in the jugular-nodose ganglia under acute insulin resistance. Together, we provide evidence for efferent-like roles of the vagal afferents and identify Fgf3 as a novel vagal sensory-derived metabolic hormone that regulates insulin secretion and energy expenditure.

overall_design: Systemic and vagal sensory overexpression of Fgf3 increased insulin secretion, improved glucose excursion, and enhanced energy expenditure and physical activity. Selective overexpression of Fgf3 in pancreas-projecting vagal afferents recapitulated similar favorable metabolic outcomes. Conversely, mice lacking Fgf3 specifically in the vagal afferents innervating the pancreas exhibited impaired glucose clearance and blunted glucose-stimulated insulin release in high-fat diet settings.

first: Azeddine

last: Tahiri

middle: Y

first: Abdelfattah

last: El Ouaamari


title: Genome-wide CRISPR/Cas9 library screening identified OGDH as a regulator of disease progress and HMAs-resistance in MDS by reprogramming glutamine metabolism

city: Suzhou

first: Jiaqian

last: Qi

state: Jiangsu

address: No.188 Shizi Road

institute: The First Affiliated Hospital of Soochow University

summary: Resistance of hypomethylating agents (HMAs) remains a challenge in myelodysplastic neoplasms (MDS), leading to a poor prognosis. Altered metabolism is also reported to be closely associated with drug resistance in hematological malignancies. Here, we conducted a genome-wide CRISPR/Cas9 library screening to identify candidate genes involved in drug sensitivity.

overall_design: Library: Use a genome-wide CRISPR/Cas9 knockout library (e.g., GeCKO v2.0) containing sgRNAs targeting all known genes. Lentiviral Packaging: Produce lentivirus containing the CRISPR library by co-transfecting HEK293T cells with the library and packaging plasmids.Experimental Design Using SKM-1 Cell Line for Decitabine Treatment and Sequencing Analysis at Day 0, Day 7, and Day 14. The NEBNext® Ultra™ DNA Library Prep Kit for Illumina® to prepare them for next-generation sequencing (NGS) on an Illumina HiSeq platform.

first: Xiaoyan

last: Xu

first: Depei

first: Yue

last: Han


title: The chromatin loop release factor WAPL regulates EBV latency status by restricting LMP production [RNA-seq]

city: Boston

first: Benjamin

last: Gewurz

address: 181 Longwood Avenue, Channing Laboratory, 8th Floor

institute: Brigham and Women's Hospital

overall_design: WAPL sgRNA or a control sgRNA transduced into Cas9 expressing cells. WPL was knocked out using CRISPR-Cas9. Cells were collected, and polyA-enrichment was performed prior to library preparation and sequencing.

first: Laura

last: Murray-Nerger

first: Davide

last: Maestri

first: Xiang

last: Liu

first: Zhixuan

last: Li

first: Nina

last: Auld

first: Italo

last: Tempera

first: Mingxiang

last: Teng


title: Transcriptome analysis in JPH203-treated HUVEC

city: Mitaka

first: Jun-ichi

last: Suehiro

address: 6-20-2, Shinkawa

department: Department of Pharmacology and Toxicology

institute: Kyorin University School of Medicine

summary: We performed transcriptome analysis using DNA microarray in JPH203-treated HUVEC to elucidate endothelial cell response to LAT1 inhibition.

overall_design: Total 2 samples were derived from HUVEC in EGM2 culture media with 100µM Captisol or JPH203 for 48 hours.

first: Jun-chi

first: Youichiro

last: Wada

first: Hiroyuki

last: Sakurai


title: SETDB1 suppresses interferon responses and NK cell-mediated immunosurveillance specifically in monocytic AML: H3K9me3 ChIP-seq of cSAM leukemia cells with Setdb1 depletion [ChIP-seq]

status: Public on Aug 03 2024

city: Tokyo

first: Yu-Hsuan

last: Chang

address: 4-6-1 Shirokanedai, Minato-ku

institute: The University of Tokyo

summary: Monocytic acute myeloid leukemia (AML) responds poorly to current treatments, including venetoclax-based therapy. We conducted in vivo and in vitro CRISPR/Cas9 library screenings using a mouse monocytic AML model, and identified SETDB1 and its binding partners (ATF7IP and TRIM33) as crucial tumor promoters in vivo. The growth-inhibitory effect of Setdb1 depletion in vivo was mainly dependent on NK cell-mediated cytotoxicity. Mechanistically, SETDB1 depletion upregulated interferon-stimulated genes and NKG2D ligands through demethylation of histone H3 Lys9 at the monocyte-specific enhancer regions, thereby enhancing their immunogenicity to NK cells and intrinsic apoptosis. Importantly, these effects were not observed in non-monocytic leukemia cells. We also identified the expression of MNDA and its murine counterpart Ifi203 as biomarkers to predict the sensitivity of each AML to SETDB1 depletion. Our study highlights the critical and selective role of SETDB1 in monocytic AML and underscores its potential as a therapeutic target for current unmet needs.

overall_design: Examination of H3K9me3 levels among coding genes and transposable elements in cSAM leukemia cells (transformed by SETBP1 and ASXL1 mutations) with Setdb1 depletion. cSAM cells were transduced with sgRNA (coexpress puromycin) plasmids and performed chromatin immunoprecipitation DNA-sequencing (ChIP-seq) after 1 µg/ml puromycin selection for 4 days.

first: Susumu

last: Goyama


title: SETDB1 suppresses interferon responses and NK cell-mediated immunosurveillance specifically in monocytic AML: CRISPR/Cas9 screens on cSAM leukemia cells

overall_design: To identify epigenetic regulators that play essential roles in vivo, we performed CRISPR/Cas9 library screens using mouse AML cells transformed by SETBP1 and ASXL1 mutations (cSAM: combined expression of SETBP1 and ASXL1 Mutations12) and a pooled single guide RNA (sgRNA) library targeting 640 epigenetic genes (3 sgRNAs per gene). The amplification and virus production were performed by the Broad institute using standard protocols (https://portals.broadinstitute.org/gpp/public/resources/protocols). Viruses were tittered and optimal virus concentrations allowing for 10 - 20% infection were used. 1E07 cSAM cells were transduced with the sgRNA library. After 24 hours, puromycin (1 µg/ml) was added for selection. After 2 days, 20 million cells were frozen at −80 °C for genomic DNA extraction and deep-sequencing. The remaining cells were transplanted into sublethally irradiated (5.25 Gy) C57BL/6 mice (2 or 5E06 cells per mouse) or cultured in 6-well plates (2E06 cells per well). For the in vivo transplantation assay, bone marrow cells were harvested from the moribund leukemic mice, and GFP+ leukemia cells were sorted using BD FACSAriaIII (BD biosciences). For the in vitro culture, cSAM cells were passaged every 2 or 3 days, over 14-day incubation period. DNA was extracted from each sample using the Blood and Cell Culture DNA Maxi kit (QIAGEN) according to the manufacturer’s instructions.